Methods in Enzymology 480 - Glycobiology

METHODS IN ENZYMOLOGY Editors-in-Chief

JOHN N. ABELSON AND MELVIN I. SIMON Division of Biology California Institute of Technology Pasadena, California Founding Editors

SIDNEY P. COLOWICK AND NATHAN O. KAPLAN

Academic Press is an imprint of Elsevier 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 32 Jamestown Road, London NW1 7BY, UK First edition 2010 Copyright # 2010, Elsevier Inc. All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: permissions@ elsevier.com. Alternatively you can submit your request online by visiting the Elsevier web site at http://elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made For information on all Academic Press publications visit our website at elsevierdirect.com ISBN: 978-0-12-380999-5 ISSN: 0076-6879 Printed and bound in United States of America 10 11 12 10 9 8 7 6 5 4 3 2 1

CONTRIBUTORS

Karen L. Abbott Complex Carbohydrate Research Center, University of Georgia Cancer Center, Athens, Georgia, USA Markus Aebi Institute of Microbiology, Department of Biology, ETH Zu¨rich, Zu¨rich, Switzerland Yassir Ahmed Centre for Glycobiology, School of Biological Sciences, University of Liverpool, Liverpool, United Kingdom Katsumi Ajisaka Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Akiha-ku, Niigata, Japan Kazuhiro Aoki Complex Carbohydrate Research Center, The University of Georgia, Athens, Georgia, USA Tatyana Y. Belenkaya Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA Silvia Bleuler-Martinez Institute of Microbiology, Department of Biology, ETH Zu¨rich, Zu¨rich, Switzerland Alex Butschi Institute of Molecular Life Sciences, University of Zu¨rich, Zu¨rich, Switzerland Dianne Cooper The William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London, London, United Kingdom Diego O. Croci Laboratorio de Inmunopatologı´a, Instituto de Biologı´a y Medicina Experimental, Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas, Buenos Aires, Argentina

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Contributors

Richard D. Cummings Department of Biochemistry, The Glycomics Center, Emory University School of Medicine, Atlanta, Georgia, USA Cristina De Castro Universita` di Napoli Federico II, Dipartimento di Chimica Organica e Biochimica, Complesso Universitario Monte Santangelo, Via Cynthia, Napoli, Italy Michael Demetriou Department of Neurology, and Department of Microbiology and Molecular Genetics; Institute for Immunology, University of California, Irvine, California, USA Kurt Drickamer Division of Molecular Biosciences, Department of Life Sciences, Imperial College, Biochemistry Building, London, United Kingdom Arye Elfenbein Dartmouth Medical School, Hanover, NH, USA Tomohiko Fukuda Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, Aoba-ku, Sendai Miyagi, Japan Kiyoshi Furukawa Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Japan Koichi Furukawa Department of Biochemistry II, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, Japan Mattia Garbani Institute of Microbiology, Department of Biology, ETH Zu¨rich, Zu¨rich, Switzerland Teunis B. H. Geijtenbeek Center of Infection and Immunity Amsterdam, and Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Hildegard Geyer Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University Giessen, Giessen, Germany Rudolf Geyer Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University Giessen, Giessen, Germany

Contributors

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Ani Grigorian Department of Neurology, and Institute for Immunology, University of California, Irvine, California, USA Sonja I. Gringhuis Center of Infection and Immunity Amsterdam, and Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Jianguo Gu Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, Aoba-ku, Sendai Miyagi, Japan Scott E. Guimond Centre for Glycobiology, School of Biological Sciences, University of Liverpool, Liverpool, United Kingdom Robert S. Haltiwanger Department of Biochemistry and Cell Biology, Institute for Cell and Developmental Biology, Stony Brook University, Stony Brook, New York, USA Michael O. Hengartner Institute of Molecular Life Sciences, University of Zu¨rich, Zu¨rich, Switzerland Yoshio Hirabayashi Laboratory for Molecular Membrane Neuroscience, Brain Science Institute, RIKEN, Japan Otto Holst Division of Structural Biochemistry, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Borstel, Germany Nobuko Hosokawa Department of Molecular and Cellular Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan Juan M. Ilarregui Laboratorio de Inmunopatologı´a, Instituto de Biologı´a y Medicina Experimental, Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas, Buenos Aires, Argentina Tomoya Isaji Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, Aoba-ku, Sendai Miyagi, Japan Hamed Jafar-Nejad Brown Foundation Institute of Molecular Medicine (IMM), Department of Biochemistry & Molecular Biology, The University of Texas Health Science Center at Houston, Houston, Texas, USA

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Contributors

¨nzler Markus Ku Institute of Microbiology, Department of Biology, ETH Zu¨rich, Zu¨rich, Switzerland Yukiko Kamiya Institute for Molecular Science and Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, and Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan Yoshinobu Kariya Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, Aoba-ku, Sendai Miyagi, Japan Koichi Kato Institute for Molecular Science and Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, and Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya; The Glycoscience Institute, Ochanomizu University, Tokyo, Japan Taroh Kinoshita Research Institute for Microbial Diseases, and WPI Immunology Frontier Research Center, Osaka University, Suita, Osaka, Japan Ayako Kohyama-Koganeya Laboratory for Molecular Membrane Neuroscience, Brain Science Institute, RIKEN, Japan ¨thy Peter Lu Institute of Microbiology, Department of Biology, ETH Zu¨rich, Zu¨rich, Switzerland Tom V. Lee Brown Foundation Institute of Molecular Medicine (IMM), Department of Biochemistry & Molecular Biology, The University of Texas Health Science Center at Houston, Houston, Texas, USA Hassan Lemjabbar-Alaoui Department of Anatomy, University of California, San Francisco, California, USA Christina Leonhard-Melief Department of Biochemistry and Cell Biology, Institute for Cell and Developmental Biology, Stony Brook University, Stony Brook, New York, USA Paik Gee Lim Division of Molecular Biosciences, Department of Life Sciences, Imperial College, Biochemistry Building, London, United Kingdom Xinhua Lin State Key Laboratory of Biomembrane and Membrane Biotechnology, and Key Laboratory of Stem Cell, Institute of Zoology, Chinese Academy of Sciences, Beijing, China; Division of Developmental Biology, Cincinnati Children’s

Contributors

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Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA Yusuke Maeda Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, and PRESTO, Japan Science and Technology Agency, Saitama, Japan Rebecca L. Miller Centre for Glycobiology, School of Biological Sciences, University of Liverpool, Liverpool, United Kingdom Tatsuo Miyazaki Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Akiha-ku, Niigata, Japan Antonio Molinaro Universita` di Napoli Federico II, Dipartimento di Chimica Organica e Biochimica, Complesso Universitario Monte Santangelo, Via Cynthia, Napoli, Italy Shoko Nishihara Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, Hachioji, Tokyo, Japan Tetsuya Okajima Department of Biochemistry II, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, Japan Michel Ouellet Laboratory of Human Immuno-Retrovirology, Research Centre for Infectious Diseases, Faculty of Medicine, Laval University, Quebec, Canada Michelangelo Parrilli Universita` di Napoli Federico II, Dipartimento di Chimica Organica e Biochimica, Complesso Universitario Monte Santangelo, Via Cynthia, Napoli, Italy Mauro Perretti The William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London, London, United Kingdom Susana A. Pesoa Laboratorio de Inmunopatologı´a, Instituto de Biologı´a y Medicina Experimental, Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas, Buenos Aires, Argentina J. Michael Pierce Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA

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Contributors

Zoi Pipriou Division of Molecular Biosciences, Department of Life Sciences, Imperial College, Biochemistry Building, London, United Kingdom Alex S. Powlesland Division of Molecular Biosciences, Department of Life Sciences, Imperial College, Biochemistry Building, London, United Kingdom Tania M. Puvirajesinghe Centre for Glycobiology, School of Biological Sciences, University of Liverpool, Liverpool, United Kingdom Adria´n Quintero-Martinez Division of Molecular Biosciences, Department of Life Sciences, Imperial College, Biochemistry Building, London, United Kingdom Gabriel A. Rabinovich Laboratorio de Inmunopatologı´a, Instituto de Biologı´a y Medicina Experimental, Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas, and Departamento de Quı´mica Biolo´gica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina Steven D. Rosen Department of Anatomy, University of California, San Francisco, California, USA Yuta Sakaidani Department of Biochemistry II, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, Japan Sachiko Sato Glycobiology and Bioimaging Laboratory, Research Centre for Infectious Diseases, Faculty of Medicine, Laval University, Quebec, Canada Takeshi Sato Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Japan Michael Simons Section of Cardiovascular Medicine, Departments of Internal Medicine and Cell Biology, Yale University School of Medicine, New Haven, Connecticut, USA David F. Smith Department of Biochemistry, The Glycomics Center, Emory University School of Medicine, Atlanta, Georgia, USA Xuezheng Song Department of Biochemistry, The Glycomics Center, Emory University School of Medicine, Atlanta, Georgia, USA

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Christian St-Pierre Glycobiology and Bioimaging Laboratory, Research Centre for Infectious Diseases, Faculty of Medicine, Laval University, Quebec, Canada Weston B. Struwe National Institute for Bioprocessing Research and Training, Dublin-Oxford Glycobiology Group, Conway Institute for Biomolecular and Biomedical Sciences, University College Dublin, Belfield, Dublin, Ireland Hideyuki Takeuchi Department of Biochemistry and Cell Biology, Institute of Cell and Developmental Biology, Stony Brook University, Stony Brook, New York, USA Naoyuki Taniguchi Department of Disease Glycomics Laboratory, The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, and Disease Glycomics Team, RIKEN Advanced Science Institute, Wako, Saitama, Japan Maureen E. Taylor Division of Molecular Biosciences, Department of Life Sciences, Imperial College, Biochemistry Building, London, United Kingdom Michael Tiemeyer Complex Carbohydrate Research Center, and Department of Biochemistry and Molecular Biology, The University of Georgia, Athens, Georgia, USA Michel J. Tremblay Laboratory of Human Immuno-Retrovirology, Research Centre for Infectious Diseases, Faculty of Medicine, Laval University, Quebec, Canada Jeremy E. Turnbull Centre for Glycobiology, School of Biological Sciences, University of Liverpool, Liverpool, United Kingdom Kenji Uchimura Section of Pathophysiology and Neurobiology, National Center for Geriatrics and Gerontology, Aichi, Japan Irma van Die Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands Toin H. van Kuppevelt Department of Biochemistry 280, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands Caroline M. W. van Stijn Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands

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Contributors

Charles E. Warren Department of Biochemistry and Molecular Biology and Program in Genetics, University of New Hampshire, Durham, NH, USA Yihui Wu State Key Laboratory of Biomembrane and Membrane Biotechnology, and Key Laboratory of Stem Cell, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Qingsong Xu Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, Aoba-ku, Sendai Miyagi, Japan

PREFACE

In 2006, we published three volumes in the Methods in Enzymology dedicated to the field of Glycobiology as follows: Glycobiology (Volume 415), Glycomics (Volume 416), and Functional Glycomics (Volume 417). We have seen the tremendous progress in the Glycobiology field since then. In particular, an explosive progress was made in immunology, neuroglycobiology, glycomics, signal transduction, and many other disciplines, examining each unique system and employing new technology. The Academic Press kindly gave another opportunity to update the introduction of new methods to a large variety of readers who would like to contribute to the advancement of Glycosciences. In the current series of Methods in Enzymology, Glycomics (Volume 478), Functional Glycomics (Volume 479), and Glycobiology (Volume 480), have been dedicated to disseminate information on the methods in determining the biological roles of carbohydrates, thanks to Academic Press Manager, particularly to Ms. Zoe Kruze and Ms. Delsy Retchagar. The third volume (Volume 480) covers proteoglycan function, infection, immunity, and carbohydrate-binding proteins, including galectin, and new development, including O-glycosylation in Notch and related signaling. The accompanying volume Glycomics (Volume 478) covers glycomics revealed by mass spectrometric analysis, carbohydrate-binding proteins, and chemical glycobiology, including protein–carbohydrate interaction, synthetic carbohydrate chemistry, and identification of carbohydrate-binding protein by carbohydrate mimicry peptides. On the other hand, Functional Glycomics (Volume 479) covers new development in glycosciences, including functional studies of glycosylation in stem cells, functions revealed by gene knockout mouse, glycan defects in muscular dystrophy, and tumor formation. I tried to bring as new development as possible of these expanding fields in these books, and I believe that we have a collection of outstanding contributors who have expertise in their respective fields. I believe that this book will be useful to a wide variety of readers from graduate students, researchers in academic, and industry, to those who would like to teach glycobiology and glycosciences at various levels. We hope that this book will contribute to further explosive progress in glycosciences and glycobiology. MINORU FUKUDA xxiii

METHODS IN ENZYMOLOGY

VOLUME I. Preparation and Assay of Enzymes Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME II. Preparation and Assay of Enzymes Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME III. Preparation and Assay of Substrates Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME IV. Special Techniques for the Enzymologist Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME V. Preparation and Assay of Enzymes Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME VI. Preparation and Assay of Enzymes (Continued) Preparation and Assay of Substrates Special Techniques Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME VII. Cumulative Subject Index Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME VIII. Complex Carbohydrates Edited by ELIZABETH F. NEUFELD AND VICTOR GINSBURG VOLUME IX. Carbohydrate Metabolism Edited by WILLIS A. WOOD VOLUME X. Oxidation and Phosphorylation Edited by RONALD W. ESTABROOK AND MAYNARD E. PULLMAN VOLUME XI. Enzyme Structure Edited by C. H. W. HIRS VOLUME XII. Nucleic Acids (Parts A and B) Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME XIII. Citric Acid Cycle Edited by J. M. LOWENSTEIN VOLUME XIV. Lipids Edited by J. M. LOWENSTEIN VOLUME XV. Steroids and Terpenoids Edited by RAYMOND B. CLAYTON xxv

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VOLUME XVI. Fast Reactions Edited by KENNETH KUSTIN VOLUME XVII. Metabolism of Amino Acids and Amines (Parts A and B) Edited by HERBERT TABOR AND CELIA WHITE TABOR VOLUME XVIII. Vitamins and Coenzymes (Parts A, B, and C) Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT VOLUME XIX. Proteolytic Enzymes Edited by GERTRUDE E. PERLMANN AND LASZLO LORAND VOLUME XX. Nucleic Acids and Protein Synthesis (Part C) Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME XXI. Nucleic Acids (Part D) Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME XXII. Enzyme Purification and Related Techniques Edited by WILLIAM B. JAKOBY VOLUME XXIII. Photosynthesis (Part A) Edited by ANTHONY SAN PIETRO VOLUME XXIV. Photosynthesis and Nitrogen Fixation (Part B) Edited by ANTHONY SAN PIETRO VOLUME XXV. Enzyme Structure (Part B) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XXVI. Enzyme Structure (Part C) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XXVII. Enzyme Structure (Part D) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XXVIII. Complex Carbohydrates (Part B) Edited by VICTOR GINSBURG VOLUME XXIX. Nucleic Acids and Protein Synthesis (Part E) Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME XXX. Nucleic Acids and Protein Synthesis (Part F) Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME XXXI. Biomembranes (Part A) Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME XXXII. Biomembranes (Part B) Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME XXXIII. Cumulative Subject Index Volumes I-XXX Edited by MARTHA G. DENNIS AND EDWARD A. DENNIS VOLUME XXXIV. Affinity Techniques (Enzyme Purification: Part B) Edited by WILLIAM B. JAKOBY AND MEIR WILCHEK

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VOLUME XXXV. Lipids (Part B) Edited by JOHN M. LOWENSTEIN VOLUME XXXVI. Hormone Action (Part A: Steroid Hormones) Edited by BERT W. O’MALLEY AND JOEL G. HARDMAN VOLUME XXXVII. Hormone Action (Part B: Peptide Hormones) Edited by BERT W. O’MALLEY AND JOEL G. HARDMAN VOLUME XXXVIII. Hormone Action (Part C: Cyclic Nucleotides) Edited by JOEL G. HARDMAN AND BERT W. O’MALLEY VOLUME XXXIX. Hormone Action (Part D: Isolated Cells, Tissues, and Organ Systems) Edited by JOEL G. HARDMAN AND BERT W. O’MALLEY VOLUME XL. Hormone Action (Part E: Nuclear Structure and Function) Edited by BERT W. O’MALLEY AND JOEL G. HARDMAN VOLUME XLI. Carbohydrate Metabolism (Part B) Edited by W. A. WOOD VOLUME XLII. Carbohydrate Metabolism (Part C) Edited by W. A. WOOD VOLUME XLIII. Antibiotics Edited by JOHN H. HASH VOLUME XLIV. Immobilized Enzymes Edited by KLAUS MOSBACH VOLUME XLV. Proteolytic Enzymes (Part B) Edited by LASZLO LORAND VOLUME XLVI. Affinity Labeling Edited by WILLIAM B. JAKOBY AND MEIR WILCHEK VOLUME XLVII. Enzyme Structure (Part E) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XLVIII. Enzyme Structure (Part F) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XLIX. Enzyme Structure (Part G) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME L. Complex Carbohydrates (Part C) Edited by VICTOR GINSBURG VOLUME LI. Purine and Pyrimidine Nucleotide Metabolism Edited by PATRICIA A. HOFFEE AND MARY ELLEN JONES VOLUME LII. Biomembranes (Part C: Biological Oxidations) Edited by SIDNEY FLEISCHER AND LESTER PACKER

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VOLUME LIII. Biomembranes (Part D: Biological Oxidations) Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LIV. Biomembranes (Part E: Biological Oxidations) Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LV. Biomembranes (Part F: Bioenergetics) Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LVI. Biomembranes (Part G: Bioenergetics) Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LVII. Bioluminescence and Chemiluminescence Edited by MARLENE A. DELUCA VOLUME LVIII. Cell Culture Edited by WILLIAM B. JAKOBY AND IRA PASTAN VOLUME LIX. Nucleic Acids and Protein Synthesis (Part G) Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME LX. Nucleic Acids and Protein Synthesis (Part H) Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME 61. Enzyme Structure (Part H) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 62. Vitamins and Coenzymes (Part D) Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT VOLUME 63. Enzyme Kinetics and Mechanism (Part A: Initial Rate and Inhibitor Methods) Edited by DANIEL L. PURICH VOLUME 64. Enzyme Kinetics and Mechanism (Part B: Isotopic Probes and Complex Enzyme Systems) Edited by DANIEL L. PURICH VOLUME 65. Nucleic Acids (Part I) Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME 66. Vitamins and Coenzymes (Part E) Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT VOLUME 67. Vitamins and Coenzymes (Part F) Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT VOLUME 68. Recombinant DNA Edited by RAY WU VOLUME 69. Photosynthesis and Nitrogen Fixation (Part C) Edited by ANTHONY SAN PIETRO VOLUME 70. Immunochemical Techniques (Part A) Edited by HELEN VAN VUNAKIS AND JOHN J. LANGONE

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VOLUME 71. Lipids (Part C) Edited by JOHN M. LOWENSTEIN VOLUME 72. Lipids (Part D) Edited by JOHN M. LOWENSTEIN VOLUME 73. Immunochemical Techniques (Part B) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 74. Immunochemical Techniques (Part C) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 75. Cumulative Subject Index Volumes XXXI, XXXII, XXXIV–LX Edited by EDWARD A. DENNIS AND MARTHA G. DENNIS VOLUME 76. Hemoglobins Edited by ERALDO ANTONINI, LUIGI ROSSI-BERNARDI, AND EMILIA CHIANCONE VOLUME 77. Detoxication and Drug Metabolism Edited by WILLIAM B. JAKOBY VOLUME 78. Interferons (Part A) Edited by SIDNEY PESTKA VOLUME 79. Interferons (Part B) Edited by SIDNEY PESTKA VOLUME 80. Proteolytic Enzymes (Part C) Edited by LASZLO LORAND VOLUME 81. Biomembranes (Part H: Visual Pigments and Purple Membranes, I) Edited by LESTER PACKER VOLUME 82. Structural and Contractile Proteins (Part A: Extracellular Matrix) Edited by LEON W. CUNNINGHAM AND DIXIE W. FREDERIKSEN VOLUME 83. Complex Carbohydrates (Part D) Edited by VICTOR GINSBURG VOLUME 84. Immunochemical Techniques (Part D: Selected Immunoassays) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 85. Structural and Contractile Proteins (Part B: The Contractile Apparatus and the Cytoskeleton) Edited by DIXIE W. FREDERIKSEN AND LEON W. CUNNINGHAM VOLUME 86. Prostaglandins and Arachidonate Metabolites Edited by WILLIAM E. M. LANDS AND WILLIAM L. SMITH VOLUME 87. Enzyme Kinetics and Mechanism (Part C: Intermediates, Stereo-chemistry, and Rate Studies) Edited by DANIEL L. PURICH VOLUME 88. Biomembranes (Part I: Visual Pigments and Purple Membranes, II) Edited by LESTER PACKER

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VOLUME 89. Carbohydrate Metabolism (Part D) Edited by WILLIS A. WOOD VOLUME 90. Carbohydrate Metabolism (Part E) Edited by WILLIS A. WOOD VOLUME 91. Enzyme Structure (Part I) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 92. Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 93. Immunochemical Techniques (Part F: Conventional Antibodies, Fc Receptors, and Cytotoxicity) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 94. Polyamines Edited by HERBERT TABOR AND CELIA WHITE TABOR VOLUME 95. Cumulative Subject Index Volumes 61–74, 76–80 Edited by EDWARD A. DENNIS AND MARTHA G. DENNIS VOLUME 96. Biomembranes [Part J: Membrane Biogenesis: Assembly and Targeting (General Methods; Eukaryotes)] Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 97. Biomembranes [Part K: Membrane Biogenesis: Assembly and Targeting (Prokaryotes, Mitochondria, and Chloroplasts)] Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 98. Biomembranes (Part L: Membrane Biogenesis: Processing and Recycling) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 99. Hormone Action (Part F: Protein Kinases) Edited by JACKIE D. CORBIN AND JOEL G. HARDMAN VOLUME 100. Recombinant DNA (Part B) Edited by RAY WU, LAWRENCE GROSSMAN, AND KIVIE MOLDAVE VOLUME 101. Recombinant DNA (Part C) Edited by RAY WU, LAWRENCE GROSSMAN, AND KIVIE MOLDAVE VOLUME 102. Hormone Action (Part G: Calmodulin and Calcium-Binding Proteins) Edited by ANTHONY R. MEANS AND BERT W. O’MALLEY VOLUME 103. Hormone Action (Part H: Neuroendocrine Peptides) Edited by P. MICHAEL CONN VOLUME 104. Enzyme Purification and Related Techniques (Part C) Edited by WILLIAM B. JAKOBY

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VOLUME 105. Oxygen Radicals in Biological Systems Edited by LESTER PACKER VOLUME 106. Posttranslational Modifications (Part A) Edited by FINN WOLD AND KIVIE MOLDAVE VOLUME 107. Posttranslational Modifications (Part B) Edited by FINN WOLD AND KIVIE MOLDAVE VOLUME 108. Immunochemical Techniques (Part G: Separation and Characterization of Lymphoid Cells) Edited by GIOVANNI DI SABATO, JOHN J. LANGONE, AND HELEN VAN VUNAKIS VOLUME 109. Hormone Action (Part I: Peptide Hormones) Edited by LUTZ BIRNBAUMER AND BERT W. O’MALLEY VOLUME 110. Steroids and Isoprenoids (Part A) Edited by JOHN H. LAW AND HANS C. RILLING VOLUME 111. Steroids and Isoprenoids (Part B) Edited by JOHN H. LAW AND HANS C. RILLING VOLUME 112. Drug and Enzyme Targeting (Part A) Edited by KENNETH J. WIDDER AND RALPH GREEN VOLUME 113. Glutamate, Glutamine, Glutathione, and Related Compounds Edited by ALTON MEISTER VOLUME 114. Diffraction Methods for Biological Macromolecules (Part A) Edited by HAROLD W. WYCKOFF, C. H. W. HIRS, AND SERGE N. TIMASHEFF VOLUME 115. Diffraction Methods for Biological Macromolecules (Part B) Edited by HAROLD W. WYCKOFF, C. H. W. HIRS, AND SERGE N. TIMASHEFF VOLUME 116. Immunochemical Techniques (Part H: Effectors and Mediators of Lymphoid Cell Functions) Edited by GIOVANNI DI SABATO, JOHN J. LANGONE, AND HELEN VAN VUNAKIS VOLUME 117. Enzyme Structure (Part J) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 118. Plant Molecular Biology Edited by ARTHUR WEISSBACH AND HERBERT WEISSBACH VOLUME 119. Interferons (Part C) Edited by SIDNEY PESTKA VOLUME 120. Cumulative Subject Index Volumes 81–94, 96–101 VOLUME 121. Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 122. Vitamins and Coenzymes (Part G) Edited by FRANK CHYTIL AND DONALD B. MCCORMICK

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VOLUME 123. Vitamins and Coenzymes (Part H) Edited by FRANK CHYTIL AND DONALD B. MCCORMICK VOLUME 124. Hormone Action (Part J: Neuroendocrine Peptides) Edited by P. MICHAEL CONN VOLUME 125. Biomembranes (Part M: Transport in Bacteria, Mitochondria, and Chloroplasts: General Approaches and Transport Systems) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 126. Biomembranes (Part N: Transport in Bacteria, Mitochondria, and Chloroplasts: Protonmotive Force) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 127. Biomembranes (Part O: Protons and Water: Structure and Translocation) Edited by LESTER PACKER VOLUME 128. Plasma Lipoproteins (Part A: Preparation, Structure, and Molecular Biology) Edited by JERE P. SEGREST AND JOHN J. ALBERS VOLUME 129. Plasma Lipoproteins (Part B: Characterization, Cell Biology, and Metabolism) Edited by JOHN J. ALBERS AND JERE P. SEGREST VOLUME 130. Enzyme Structure (Part K) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 131. Enzyme Structure (Part L) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 132. Immunochemical Techniques (Part J: Phagocytosis and Cell-Mediated Cytotoxicity) Edited by GIOVANNI DI SABATO AND JOHANNES EVERSE VOLUME 133. Bioluminescence and Chemiluminescence (Part B) Edited by MARLENE DELUCA AND WILLIAM D. MCELROY VOLUME 134. Structural and Contractile Proteins (Part C: The Contractile Apparatus and the Cytoskeleton) Edited by RICHARD B. VALLEE VOLUME 135. Immobilized Enzymes and Cells (Part B) Edited by KLAUS MOSBACH VOLUME 136. Immobilized Enzymes and Cells (Part C) Edited by KLAUS MOSBACH VOLUME 137. Immobilized Enzymes and Cells (Part D) Edited by KLAUS MOSBACH VOLUME 138. Complex Carbohydrates (Part E) Edited by VICTOR GINSBURG

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VOLUME 139. Cellular Regulators (Part A: Calcium- and Calmodulin-Binding Proteins) Edited by ANTHONY R. MEANS AND P. MICHAEL CONN VOLUME 140. Cumulative Subject Index Volumes 102–119, 121–134 VOLUME 141. Cellular Regulators (Part B: Calcium and Lipids) Edited by P. MICHAEL CONN AND ANTHONY R. MEANS VOLUME 142. Metabolism of Aromatic Amino Acids and Amines Edited by SEYMOUR KAUFMAN VOLUME 143. Sulfur and Sulfur Amino Acids Edited by WILLIAM B. JAKOBY AND OWEN GRIFFITH VOLUME 144. Structural and Contractile Proteins (Part D: Extracellular Matrix) Edited by LEON W. CUNNINGHAM VOLUME 145. Structural and Contractile Proteins (Part E: Extracellular Matrix) Edited by LEON W. CUNNINGHAM VOLUME 146. Peptide Growth Factors (Part A) Edited by DAVID BARNES AND DAVID A. SIRBASKU VOLUME 147. Peptide Growth Factors (Part B) Edited by DAVID BARNES AND DAVID A. SIRBASKU VOLUME 148. Plant Cell Membranes Edited by LESTER PACKER AND ROLAND DOUCE VOLUME 149. Drug and Enzyme Targeting (Part B) Edited by RALPH GREEN AND KENNETH J. WIDDER VOLUME 150. Immunochemical Techniques (Part K: In Vitro Models of B and T Cell Functions and Lymphoid Cell Receptors) Edited by GIOVANNI DI SABATO VOLUME 151. Molecular Genetics of Mammalian Cells Edited by MICHAEL M. GOTTESMAN VOLUME 152. Guide to Molecular Cloning Techniques Edited by SHELBY L. BERGER AND ALAN R. KIMMEL VOLUME 153. Recombinant DNA (Part D) Edited by RAY WU AND LAWRENCE GROSSMAN VOLUME 154. Recombinant DNA (Part E) Edited by RAY WU AND LAWRENCE GROSSMAN VOLUME 155. Recombinant DNA (Part F) Edited by RAY WU VOLUME 156. Biomembranes (Part P: ATP-Driven Pumps and Related Transport: The Na, K-Pump) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER

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VOLUME 157. Biomembranes (Part Q: ATP-Driven Pumps and Related Transport: Calcium, Proton, and Potassium Pumps) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 158. Metalloproteins (Part A) Edited by JAMES F. RIORDAN AND BERT L. VALLEE VOLUME 159. Initiation and Termination of Cyclic Nucleotide Action Edited by JACKIE D. CORBIN AND ROGER A. JOHNSON VOLUME 160. Biomass (Part A: Cellulose and Hemicellulose) Edited by WILLIS A. WOOD AND SCOTT T. KELLOGG VOLUME 161. Biomass (Part B: Lignin, Pectin, and Chitin) Edited by WILLIS A. WOOD AND SCOTT T. KELLOGG VOLUME 162. Immunochemical Techniques (Part L: Chemotaxis and Inflammation) Edited by GIOVANNI DI SABATO VOLUME 163. Immunochemical Techniques (Part M: Chemotaxis and Inflammation) Edited by GIOVANNI DI SABATO VOLUME 164. Ribosomes Edited by HARRY F. NOLLER, JR., AND KIVIE MOLDAVE VOLUME 165. Microbial Toxins: Tools for Enzymology Edited by SIDNEY HARSHMAN VOLUME 166. Branched-Chain Amino Acids Edited by ROBERT HARRIS AND JOHN R. SOKATCH VOLUME 167. Cyanobacteria Edited by LESTER PACKER AND ALEXANDER N. GLAZER VOLUME 168. Hormone Action (Part K: Neuroendocrine Peptides) Edited by P. MICHAEL CONN VOLUME 169. Platelets: Receptors, Adhesion, Secretion (Part A) Edited by JACEK HAWIGER VOLUME 170. Nucleosomes Edited by PAUL M. WASSARMAN AND ROGER D. KORNBERG VOLUME 171. Biomembranes (Part R: Transport Theory: Cells and Model Membranes) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 172. Biomembranes (Part S: Transport: Membrane Isolation and Characterization) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER

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VOLUME 173. Biomembranes [Part T: Cellular and Subcellular Transport: Eukaryotic (Nonepithelial) Cells] Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 174. Biomembranes [Part U: Cellular and Subcellular Transport: Eukaryotic (Nonepithelial) Cells] Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 175. Cumulative Subject Index Volumes 135–139, 141–167 VOLUME 176. Nuclear Magnetic Resonance (Part A: Spectral Techniques and Dynamics) Edited by NORMAN J. OPPENHEIMER AND THOMAS L. JAMES VOLUME 177. Nuclear Magnetic Resonance (Part B: Structure and Mechanism) Edited by NORMAN J. OPPENHEIMER AND THOMAS L. JAMES VOLUME 178. Antibodies, Antigens, and Molecular Mimicry Edited by JOHN J. LANGONE VOLUME 179. Complex Carbohydrates (Part F) Edited by VICTOR GINSBURG VOLUME 180. RNA Processing (Part A: General Methods) Edited by JAMES E. DAHLBERG AND JOHN N. ABELSON VOLUME 181. RNA Processing (Part B: Specific Methods) Edited by JAMES E. DAHLBERG AND JOHN N. ABELSON VOLUME 182. Guide to Protein Purification Edited by MURRAY P. DEUTSCHER VOLUME 183. Molecular Evolution: Computer Analysis of Protein and Nucleic Acid Sequences Edited by RUSSELL F. DOOLITTLE VOLUME 184. Avidin-Biotin Technology Edited by MEIR WILCHEK AND EDWARD A. BAYER VOLUME 185. Gene Expression Technology Edited by DAVID V. GOEDDEL VOLUME 186. Oxygen Radicals in Biological Systems (Part B: Oxygen Radicals and Antioxidants) Edited by LESTER PACKER AND ALEXANDER N. GLAZER VOLUME 187. Arachidonate Related Lipid Mediators Edited by ROBERT C. MURPHY AND FRANK A. FITZPATRICK VOLUME 188. Hydrocarbons and Methylotrophy Edited by MARY E. LIDSTROM VOLUME 189. Retinoids (Part A: Molecular and Metabolic Aspects) Edited by LESTER PACKER

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VOLUME 190. Retinoids (Part B: Cell Differentiation and Clinical Applications) Edited by LESTER PACKER VOLUME 191. Biomembranes (Part V: Cellular and Subcellular Transport: Epithelial Cells) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 192. Biomembranes (Part W: Cellular and Subcellular Transport: Epithelial Cells) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 193. Mass Spectrometry Edited by JAMES A. MCCLOSKEY VOLUME 194. Guide to Yeast Genetics and Molecular Biology Edited by CHRISTINE GUTHRIE AND GERALD R. FINK VOLUME 195. Adenylyl Cyclase, G Proteins, and Guanylyl Cyclase Edited by ROGER A. JOHNSON AND JACKIE D. CORBIN VOLUME 196. Molecular Motors and the Cytoskeleton Edited by RICHARD B. VALLEE VOLUME 197. Phospholipases Edited by EDWARD A. DENNIS VOLUME 198. Peptide Growth Factors (Part C) Edited by DAVID BARNES, J. P. MATHER, AND GORDON H. SATO VOLUME 199. Cumulative Subject Index Volumes 168–174, 176–194 VOLUME 200. Protein Phosphorylation (Part A: Protein Kinases: Assays, Purification, Antibodies, Functional Analysis, Cloning, and Expression) Edited by TONY HUNTER AND BARTHOLOMEW M. SEFTON VOLUME 201. Protein Phosphorylation (Part B: Analysis of Protein Phosphorylation, Protein Kinase Inhibitors, and Protein Phosphatases) Edited by TONY HUNTER AND BARTHOLOMEW M. SEFTON VOLUME 202. Molecular Design and Modeling: Concepts and Applications (Part A: Proteins, Peptides, and Enzymes) Edited by JOHN J. LANGONE VOLUME 203. Molecular Design and Modeling: Concepts and Applications (Part B: Antibodies and Antigens, Nucleic Acids, Polysaccharides, and Drugs) Edited by JOHN J. LANGONE VOLUME 204. Bacterial Genetic Systems Edited by JEFFREY H. MILLER VOLUME 205. Metallobiochemistry (Part B: Metallothionein and Related Molecules) Edited by JAMES F. RIORDAN AND BERT L. VALLEE

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VOLUME 206. Cytochrome P450 Edited by MICHAEL R. WATERMAN AND ERIC F. JOHNSON VOLUME 207. Ion Channels Edited by BERNARDO RUDY AND LINDA E. IVERSON VOLUME 208. Protein–DNA Interactions Edited by ROBERT T. SAUER VOLUME 209. Phospholipid Biosynthesis Edited by EDWARD A. DENNIS AND DENNIS E. VANCE VOLUME 210. Numerical Computer Methods Edited by LUDWIG BRAND AND MICHAEL L. JOHNSON VOLUME 211. DNA Structures (Part A: Synthesis and Physical Analysis of DNA) Edited by DAVID M. J. LILLEY AND JAMES E. DAHLBERG VOLUME 212. DNA Structures (Part B: Chemical and Electrophoretic Analysis of DNA) Edited by DAVID M. J. LILLEY AND JAMES E. DAHLBERG VOLUME 213. Carotenoids (Part A: Chemistry, Separation, Quantitation, and Antioxidation) Edited by LESTER PACKER VOLUME 214. Carotenoids (Part B: Metabolism, Genetics, and Biosynthesis) Edited by LESTER PACKER VOLUME 215. Platelets: Receptors, Adhesion, Secretion (Part B) Edited by JACEK J. HAWIGER VOLUME 216. Recombinant DNA (Part G) Edited by RAY WU VOLUME 217. Recombinant DNA (Part H) Edited by RAY WU VOLUME 218. Recombinant DNA (Part I) Edited by RAY WU VOLUME 219. Reconstitution of Intracellular Transport Edited by JAMES E. ROTHMAN VOLUME 220. Membrane Fusion Techniques (Part A) Edited by NEJAT DU¨ZGU¨NES VOLUME 221. Membrane Fusion Techniques (Part B) Edited by NEJAT DU¨ZGU¨NES VOLUME 222. Proteolytic Enzymes in Coagulation, Fibrinolysis, and Complement Activation (Part A: Mammalian Blood Coagulation Factors and Inhibitors) Edited by LASZLO LORAND AND KENNETH G. MANN

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VOLUME 223. Proteolytic Enzymes in Coagulation, Fibrinolysis, and Complement Activation (Part B: Complement Activation, Fibrinolysis, and Nonmammalian Blood Coagulation Factors) Edited by LASZLO LORAND AND KENNETH G. MANN VOLUME 224. Molecular Evolution: Producing the Biochemical Data Edited by ELIZABETH ANNE ZIMMER, THOMAS J. WHITE, REBECCA L. CANN, AND ALLAN C. WILSON VOLUME 225. Guide to Techniques in Mouse Development Edited by PAUL M. WASSARMAN AND MELVIN L. DEPAMPHILIS VOLUME 226. Metallobiochemistry (Part C: Spectroscopic and Physical Methods for Probing Metal Ion Environments in Metalloenzymes and Metalloproteins) Edited by JAMES F. RIORDAN AND BERT L. VALLEE VOLUME 227. Metallobiochemistry (Part D: Physical and Spectroscopic Methods for Probing Metal Ion Environments in Metalloproteins) Edited by JAMES F. RIORDAN AND BERT L. VALLEE VOLUME 228. Aqueous Two-Phase Systems Edited by HARRY WALTER AND GO¨TE JOHANSSON VOLUME 229. Cumulative Subject Index Volumes 195–198, 200–227 VOLUME 230. Guide to Techniques in Glycobiology Edited by WILLIAM J. LENNARZ AND GERALD W. HART VOLUME 231. Hemoglobins (Part B: Biochemical and Analytical Methods) Edited by JOHANNES EVERSE, KIM D. VANDEGRIFF, AND ROBERT M. WINSLOW VOLUME 232. Hemoglobins (Part C: Biophysical Methods) Edited by JOHANNES EVERSE, KIM D. VANDEGRIFF, AND ROBERT M. WINSLOW VOLUME 233. Oxygen Radicals in Biological Systems (Part C) Edited by LESTER PACKER VOLUME 234. Oxygen Radicals in Biological Systems (Part D) Edited by LESTER PACKER VOLUME 235. Bacterial Pathogenesis (Part A: Identification and Regulation of Virulence Factors) Edited by VIRGINIA L. CLARK AND PATRIK M. BAVOIL VOLUME 236. Bacterial Pathogenesis (Part B: Integration of Pathogenic Bacteria with Host Cells) Edited by VIRGINIA L. CLARK AND PATRIK M. BAVOIL VOLUME 237. Heterotrimeric G Proteins Edited by RAVI IYENGAR VOLUME 238. Heterotrimeric G-Protein Effectors Edited by RAVI IYENGAR

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VOLUME 239. Nuclear Magnetic Resonance (Part C) Edited by THOMAS L. JAMES AND NORMAN J. OPPENHEIMER VOLUME 240. Numerical Computer Methods (Part B) Edited by MICHAEL L. JOHNSON AND LUDWIG BRAND VOLUME 241. Retroviral Proteases Edited by LAWRENCE C. KUO AND JULES A. SHAFER VOLUME 242. Neoglycoconjugates (Part A) Edited by Y. C. LEE AND REIKO T. LEE VOLUME 243. Inorganic Microbial Sulfur Metabolism Edited by HARRY D. PECK, JR., AND JEAN LEGALL VOLUME 244. Proteolytic Enzymes: Serine and Cysteine Peptidases Edited by ALAN J. BARRETT VOLUME 245. Extracellular Matrix Components Edited by E. RUOSLAHTI AND E. ENGVALL VOLUME 246. Biochemical Spectroscopy Edited by KENNETH SAUER VOLUME 247. Neoglycoconjugates (Part B: Biomedical Applications) Edited by Y. C. LEE AND REIKO T. LEE VOLUME 248. Proteolytic Enzymes: Aspartic and Metallo Peptidases Edited by ALAN J. BARRETT VOLUME 249. Enzyme Kinetics and Mechanism (Part D: Developments in Enzyme Dynamics) Edited by DANIEL L. PURICH VOLUME 250. Lipid Modifications of Proteins Edited by PATRICK J. CASEY AND JANICE E. BUSS VOLUME 251. Biothiols (Part A: Monothiols and Dithiols, Protein Thiols, and Thiyl Radicals) Edited by LESTER PACKER VOLUME 252. Biothiols (Part B: Glutathione and Thioredoxin; Thiols in Signal Transduction and Gene Regulation) Edited by LESTER PACKER VOLUME 253. Adhesion of Microbial Pathogens Edited by RON J. DOYLE AND ITZHAK OFEK VOLUME 254. Oncogene Techniques Edited by PETER K. VOGT AND INDER M. VERMA VOLUME 255. Small GTPases and Their Regulators (Part A: Ras Family) Edited by W. E. BALCH, CHANNING J. DER, AND ALAN HALL

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VOLUME 256. Small GTPases and Their Regulators (Part B: Rho Family) Edited by W. E. BALCH, CHANNING J. DER, AND ALAN HALL VOLUME 257. Small GTPases and Their Regulators (Part C: Proteins Involved in Transport) Edited by W. E. BALCH, CHANNING J. DER, AND ALAN HALL VOLUME 258. Redox-Active Amino Acids in Biology Edited by JUDITH P. KLINMAN VOLUME 259. Energetics of Biological Macromolecules Edited by MICHAEL L. JOHNSON AND GARY K. ACKERS VOLUME 260. Mitochondrial Biogenesis and Genetics (Part A) Edited by GIUSEPPE M. ATTARDI AND ANNE CHOMYN VOLUME 261. Nuclear Magnetic Resonance and Nucleic Acids Edited by THOMAS L. JAMES VOLUME 262. DNA Replication Edited by JUDITH L. CAMPBELL VOLUME 263. Plasma Lipoproteins (Part C: Quantitation) Edited by WILLIAM A. BRADLEY, SANDRA H. GIANTURCO, AND JERE P. SEGREST VOLUME 264. Mitochondrial Biogenesis and Genetics (Part B) Edited by GIUSEPPE M. ATTARDI AND ANNE CHOMYN VOLUME 265. Cumulative Subject Index Volumes 228, 230–262 VOLUME 266. Computer Methods for Macromolecular Sequence Analysis Edited by RUSSELL F. DOOLITTLE VOLUME 267. Combinatorial Chemistry Edited by JOHN N. ABELSON VOLUME 268. Nitric Oxide (Part A: Sources and Detection of NO; NO Synthase) Edited by LESTER PACKER VOLUME 269. Nitric Oxide (Part B: Physiological and Pathological Processes) Edited by LESTER PACKER VOLUME 270. High Resolution Separation and Analysis of Biological Macromolecules (Part A: Fundamentals) Edited by BARRY L. KARGER AND WILLIAM S. HANCOCK VOLUME 271. High Resolution Separation and Analysis of Biological Macromolecules (Part B: Applications) Edited by BARRY L. KARGER AND WILLIAM S. HANCOCK VOLUME 272. Cytochrome P450 (Part B) Edited by ERIC F. JOHNSON AND MICHAEL R. WATERMAN VOLUME 273. RNA Polymerase and Associated Factors (Part A) Edited by SANKAR ADHYA

Methods in Enzymology

VOLUME 274. RNA Polymerase and Associated Factors (Part B) Edited by SANKAR ADHYA VOLUME 275. Viral Polymerases and Related Proteins Edited by LAWRENCE C. KUO, DAVID B. OLSEN, AND STEVEN S. CARROLL VOLUME 276. Macromolecular Crystallography (Part A) Edited by CHARLES W. CARTER, JR., AND ROBERT M. SWEET VOLUME 277. Macromolecular Crystallography (Part B) Edited by CHARLES W. CARTER, JR., AND ROBERT M. SWEET VOLUME 278. Fluorescence Spectroscopy Edited by LUDWIG BRAND AND MICHAEL L. JOHNSON VOLUME 279. Vitamins and Coenzymes (Part I) Edited by DONALD B. MCCORMICK, JOHN W. SUTTIE, AND CONRAD WAGNER VOLUME 280. Vitamins and Coenzymes (Part J) Edited by DONALD B. MCCORMICK, JOHN W. SUTTIE, AND CONRAD WAGNER VOLUME 281. Vitamins and Coenzymes (Part K) Edited by DONALD B. MCCORMICK, JOHN W. SUTTIE, AND CONRAD WAGNER VOLUME 282. Vitamins and Coenzymes (Part L) Edited by DONALD B. MCCORMICK, JOHN W. SUTTIE, AND CONRAD WAGNER VOLUME 283. Cell Cycle Control Edited by WILLIAM G. DUNPHY VOLUME 284. Lipases (Part A: Biotechnology) Edited by BYRON RUBIN AND EDWARD A. DENNIS VOLUME 285. Cumulative Subject Index Volumes 263, 264, 266–284, 286–289 VOLUME 286. Lipases (Part B: Enzyme Characterization and Utilization) Edited by BYRON RUBIN AND EDWARD A. DENNIS VOLUME 287. Chemokines Edited by RICHARD HORUK VOLUME 288. Chemokine Receptors Edited by RICHARD HORUK VOLUME 289. Solid Phase Peptide Synthesis Edited by GREGG B. FIELDS VOLUME 290. Molecular Chaperones Edited by GEORGE H. LORIMER AND THOMAS BALDWIN VOLUME 291. Caged Compounds Edited by GERARD MARRIOTT VOLUME 292. ABC Transporters: Biochemical, Cellular, and Molecular Aspects Edited by SURESH V. AMBUDKAR AND MICHAEL M. GOTTESMAN

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VOLUME 293. Ion Channels (Part B) Edited by P. MICHAEL CONN VOLUME 294. Ion Channels (Part C) Edited by P. MICHAEL CONN VOLUME 295. Energetics of Biological Macromolecules (Part B) Edited by GARY K. ACKERS AND MICHAEL L. JOHNSON VOLUME 296. Neurotransmitter Transporters Edited by SUSAN G. AMARA VOLUME 297. Photosynthesis: Molecular Biology of Energy Capture Edited by LEE MCINTOSH VOLUME 298. Molecular Motors and the Cytoskeleton (Part B) Edited by RICHARD B. VALLEE VOLUME 299. Oxidants and Antioxidants (Part A) Edited by LESTER PACKER VOLUME 300. Oxidants and Antioxidants (Part B) Edited by LESTER PACKER VOLUME 301. Nitric Oxide: Biological and Antioxidant Activities (Part C) Edited by LESTER PACKER VOLUME 302. Green Fluorescent Protein Edited by P. MICHAEL CONN VOLUME 303. cDNA Preparation and Display Edited by SHERMAN M. WEISSMAN VOLUME 304. Chromatin Edited by PAUL M. WASSARMAN AND ALAN P. WOLFFE VOLUME 305. Bioluminescence and Chemiluminescence (Part C) Edited by THOMAS O. BALDWIN AND MIRIAM M. ZIEGLER VOLUME 306. Expression of Recombinant Genes in Eukaryotic Systems Edited by JOSEPH C. GLORIOSO AND MARTIN C. SCHMIDT VOLUME 307. Confocal Microscopy Edited by P. MICHAEL CONN VOLUME 308. Enzyme Kinetics and Mechanism (Part E: Energetics of Enzyme Catalysis) Edited by DANIEL L. PURICH AND VERN L. SCHRAMM VOLUME 309. Amyloid, Prions, and Other Protein Aggregates Edited by RONALD WETZEL VOLUME 310. Biofilms Edited by RON J. DOYLE

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VOLUME 311. Sphingolipid Metabolism and Cell Signaling (Part A) Edited by ALFRED H. MERRILL, JR., AND YUSUF A. HANNUN VOLUME 312. Sphingolipid Metabolism and Cell Signaling (Part B) Edited by ALFRED H. MERRILL, JR., AND YUSUF A. HANNUN VOLUME 313. Antisense Technology (Part A: General Methods, Methods of Delivery, and RNA Studies) Edited by M. IAN PHILLIPS VOLUME 314. Antisense Technology (Part B: Applications) Edited by M. IAN PHILLIPS VOLUME 315. Vertebrate Phototransduction and the Visual Cycle (Part A) Edited by KRZYSZTOF PALCZEWSKI VOLUME 316. Vertebrate Phototransduction and the Visual Cycle (Part B) Edited by KRZYSZTOF PALCZEWSKI VOLUME 317. RNA–Ligand Interactions (Part A: Structural Biology Methods) Edited by DANIEL W. CELANDER AND JOHN N. ABELSON VOLUME 318. RNA–Ligand Interactions (Part B: Molecular Biology Methods) Edited by DANIEL W. CELANDER AND JOHN N. ABELSON VOLUME 319. Singlet Oxygen, UV-A, and Ozone Edited by LESTER PACKER AND HELMUT SIES VOLUME 320. Cumulative Subject Index Volumes 290–319 VOLUME 321. Numerical Computer Methods (Part C) Edited by MICHAEL L. JOHNSON AND LUDWIG BRAND VOLUME 322. Apoptosis Edited by JOHN C. REED VOLUME 323. Energetics of Biological Macromolecules (Part C) Edited by MICHAEL L. JOHNSON AND GARY K. ACKERS VOLUME 324. Branched-Chain Amino Acids (Part B) Edited by ROBERT A. HARRIS AND JOHN R. SOKATCH VOLUME 325. Regulators and Effectors of Small GTPases (Part D: Rho Family) Edited by W. E. BALCH, CHANNING J. DER, AND ALAN HALL VOLUME 326. Applications of Chimeric Genes and Hybrid Proteins (Part A: Gene Expression and Protein Purification) Edited by JEREMY THORNER, SCOTT D. EMR, AND JOHN N. ABELSON VOLUME 327. Applications of Chimeric Genes and Hybrid Proteins (Part B: Cell Biology and Physiology) Edited by JEREMY THORNER, SCOTT D. EMR, AND JOHN N. ABELSON

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VOLUME 328. Applications of Chimeric Genes and Hybrid Proteins (Part C: Protein–Protein Interactions and Genomics) Edited by JEREMY THORNER, SCOTT D. EMR, AND JOHN N. ABELSON VOLUME 329. Regulators and Effectors of Small GTPases (Part E: GTPases Involved in Vesicular Traffic) Edited by W. E. BALCH, CHANNING J. DER, AND ALAN HALL VOLUME 330. Hyperthermophilic Enzymes (Part A) Edited by MICHAEL W. W. ADAMS AND ROBERT M. KELLY VOLUME 331. Hyperthermophilic Enzymes (Part B) Edited by MICHAEL W. W. ADAMS AND ROBERT M. KELLY VOLUME 332. Regulators and Effectors of Small GTPases (Part F: Ras Family I) Edited by W. E. BALCH, CHANNING J. DER, AND ALAN HALL VOLUME 333. Regulators and Effectors of Small GTPases (Part G: Ras Family II) Edited by W. E. BALCH, CHANNING J. DER, AND ALAN HALL VOLUME 334. Hyperthermophilic Enzymes (Part C) Edited by MICHAEL W. W. ADAMS AND ROBERT M. KELLY VOLUME 335. Flavonoids and Other Polyphenols Edited by LESTER PACKER VOLUME 336. Microbial Growth in Biofilms (Part A: Developmental and Molecular Biological Aspects) Edited by RON J. DOYLE VOLUME 337. Microbial Growth in Biofilms (Part B: Special Environments and Physicochemical Aspects) Edited by RON J. DOYLE VOLUME 338. Nuclear Magnetic Resonance of Biological Macromolecules (Part A) Edited by THOMAS L. JAMES, VOLKER DO¨TSCH, AND ULI SCHMITZ VOLUME 339. Nuclear Magnetic Resonance of Biological Macromolecules (Part B) Edited by THOMAS L. JAMES, VOLKER DO¨TSCH, AND ULI SCHMITZ VOLUME 340. Drug–Nucleic Acid Interactions Edited by JONATHAN B. CHAIRES AND MICHAEL J. WARING VOLUME 341. Ribonucleases (Part A) Edited by ALLEN W. NICHOLSON VOLUME 342. Ribonucleases (Part B) Edited by ALLEN W. NICHOLSON VOLUME 343. G Protein Pathways (Part A: Receptors) Edited by RAVI IYENGAR AND JOHN D. HILDEBRANDT VOLUME 344. G Protein Pathways (Part B: G Proteins and Their Regulators) Edited by RAVI IYENGAR AND JOHN D. HILDEBRANDT

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VOLUME 345. G Protein Pathways (Part C: Effector Mechanisms) Edited by RAVI IYENGAR AND JOHN D. HILDEBRANDT VOLUME 346. Gene Therapy Methods Edited by M. IAN PHILLIPS VOLUME 347. Protein Sensors and Reactive Oxygen Species (Part A: Selenoproteins and Thioredoxin) Edited by HELMUT SIES AND LESTER PACKER VOLUME 348. Protein Sensors and Reactive Oxygen Species (Part B: Thiol Enzymes and Proteins) Edited by HELMUT SIES AND LESTER PACKER VOLUME 349. Superoxide Dismutase Edited by LESTER PACKER VOLUME 350. Guide to Yeast Genetics and Molecular and Cell Biology (Part B) Edited by CHRISTINE GUTHRIE AND GERALD R. FINK VOLUME 351. Guide to Yeast Genetics and Molecular and Cell Biology (Part C) Edited by CHRISTINE GUTHRIE AND GERALD R. FINK VOLUME 352. Redox Cell Biology and Genetics (Part A) Edited by CHANDAN K. SEN AND LESTER PACKER VOLUME 353. Redox Cell Biology and Genetics (Part B) Edited by CHANDAN K. SEN AND LESTER PACKER VOLUME 354. Enzyme Kinetics and Mechanisms (Part F: Detection and Characterization of Enzyme Reaction Intermediates) Edited by DANIEL L. PURICH VOLUME 355. Cumulative Subject Index Volumes 321–354 VOLUME 356. Laser Capture Microscopy and Microdissection Edited by P. MICHAEL CONN VOLUME 357. Cytochrome P450, Part C Edited by ERIC F. JOHNSON AND MICHAEL R. WATERMAN VOLUME 358. Bacterial Pathogenesis (Part C: Identification, Regulation, and Function of Virulence Factors) Edited by VIRGINIA L. CLARK AND PATRIK M. BAVOIL VOLUME 359. Nitric Oxide (Part D) Edited by ENRIQUE CADENAS AND LESTER PACKER VOLUME 360. Biophotonics (Part A) Edited by GERARD MARRIOTT AND IAN PARKER VOLUME 361. Biophotonics (Part B) Edited by GERARD MARRIOTT AND IAN PARKER

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Auxiliary and Autonomous Proteoglycan Signaling Networks Arye Elfenbein* and Michael Simons† Contents 1. 2. 3. 4. 5.

Overview of Proteoglycan Structure, Nomenclature, and Function Growth Factor Signaling Integrin Interactions Autonomous Signaling Proteoglycan Downregulation: Endocytosis and Ectodomain Shedding 6. Cell Adhesion 7. Migration 8. Experimental Procedures 8.1. Immunoblotting for heparan sulfate proteoglycans 8.2. Proteoglycan chimera oligomerization 8.3. Qualitative and quantitative endocytosis assays 8.4. Rac1 pull-down assay 8.5. Multicellular migration assay 8.6. Two-chambered migration assay (adhesion and migration) Acknowledgments References

4 5 6 6 9 10 14 17 17 19 21 24 26 27 28 29

Abstract Proteoglycans represent a structurally heterogeneous family of proteins that typically undergo extensive posttranslational modification with sulfated sugar chains. Although historically believed to affect signaling pathways exclusively as growth factor coreceptors, proteoglycans are now understood to initiate and modulate signal transduction cascades independently of other receptors. From within the extracellular matrix, proteoglycans are able to shield protein growth factors from circulating proteases and establish gradients that guide cell migration. Extracellular proteoglycans are also critical in the maintenance of growth factor stores and are thus instrumental in modulating paracrine signaling. * Dartmouth Medical School, Hanover, NH, USA Section of Cardiovascular Medicine, Departments of Internal Medicine and Cell Biology, Yale University School of Medicine, New Haven, Connecticut, USA

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Methods in Enzymology, Volume 480 ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)80001-1

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2010 Elsevier Inc. All rights reserved.

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At the cell membrane, proteoglycans stabilize ligand–receptor interactions, creating potentiated ternary signaling complexes that regulate cell proliferation, endocytosis, migration, growth factor sensitivity, and matrix adhesion. In some cases, proteoglycans are able to independently activate various signaling cascades, attenuate the signaling of growth factors, or orchestrate multimeric intracellular signaling complexes. Signaling between cells is also modulated by proteoglycan activity at the cell membrane, as exemplified by the proteoglycan requirement for effective synaptogenesis between neurons. Finally, proteoglycans are able to regulate signaling from intracellular compartments, particularly in the context of storage granule formation and maintenance. These proteoglycans are also major determinants of exocytic vesicle fate and other vesicular trafficking pathways. In contrast to the mechanisms underlying classical ligand–receptor signaling, proteoglycan signaling is frequently characterized by ligand promiscuity and low-affinity binding; likewise, these events commonly do not exhibit the same degree of reliance on intermolecular structure or charge configurations as other ligand–receptor interactions. Such unique features often defy conventional mechanisms of signal transduction, and present unique challenges to the study of their indispensable roles within cell signaling networks.

1. Overview of Proteoglycan Structure, Nomenclature, and Function The myriad contributions made by proteoglycans to physiological processes such as cellular proliferation, migration, immunity, and matrix assembly are largely owed to the structural diversity within this molecular superfamily. Originally characterized by their ability to facilitate ligand– receptor interactions and confer structural stability upon tissues, proteoglycans have more recently been shown to hold profound influence over cell physiology by independently engaging in the initiation and modulation of numerous cell signaling networks (Schaefer and Schaefer, 2010). Proteoglycans consist of a protein core to which linear, negatively charged polysaccharide chains known as glycosaminoglycans (GAGs) are covalently linked. GAGs are comprised of disaccharide repeats and vary with respect to their sulfation patterns. GAGs are categorized by the structure of their disaccharide chain sequences; among the GAG subtypes are heparan sulfates, chondroitin sulfates, keratin sulfates, and dermatan sulfates. These structurally distinct chains confer different binding properties and signaling capabilities upon the nascent core proteins to which they become attached (Kjelle´n and Lindahl, 1991). Some protein cores, such as those of neuropilins and betaglycan/transforming growth factor beta receptor III, may be expressed without the addition of GAG chains (Mythreye and Blobe, 2009), whereas those of

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others, such as the syndecans, are not found on the cell surface without GAG modifications (Tkachenko et al., 2005). Furthermore, certain proteoglycans are capable of undergoing modification by multiple GAG types, while other proteoglycans are limited in the type of GAG that may become attached. Tissue distribution of proteoglycans also varies significantly among types. While some (including syndecan 4) are ubiquitously expressed, others are only found within particular tissues, as in the cases of neurocan, exclusively expressed in the nervous system (Rauch et al., 2001). The numerous cellular localization patterns of proteoglycans likewise reflect their functional versatility; these molecules may perform their various roles in signaling, vesicular trafficking and structural support from the cell membrane, intracellular compartments, or the extracellular matrix.

2. Growth Factor Signaling The range of disaccharide modifications, protein core structures, and expression patterns across different tissues results in a high degree of structural variability and functional versatility within the proteoglycan family. Negative charges carried by proteoglycan GAG chains also facilitate ionic interactions with growth factors, a diverse family of soluble proteins that induce cellular responses such as migration, proliferation, and endocytosis once they bind membrane receptors and induce their dimerization (Bernfield et al., 1999; Murakami et al., 2008; Ornitz et al., 1992). This process of receptor activation results in subsequent receptor phosphorylation on intracellular residues, thus communicating the external stimulatory impulse toward the cytosol, where protein components are mobilized to initiate various parallel signaling cascades. Proteoglycans influence growth factor signal transduction in several capacities. On account of their ability to directly bind growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF), proteoglycans in the extracellular matrix are able to maintain highconcentration stores of growth factors that would otherwise be diluted by passive diffusion. These proteoglycans also facilitate the formation of growth factor gradients and prevent their premature degradation (Gru¨nert et al., 2008; Saksela et al., 1988). At the cell membrane, proteoglycans are able to stabilize interactions between growth factor ligands and their receptors, thus potentiating the resultant intracellular signaling events. In certain contexts, proteoglycans are required for effective signaling to occur, as with syndecans and the fibroblast growth factor receptors (FGFRs) (Yayon et al., 1991); in others they serve to inhibit signaling, as shown with betaglycan’s ability to act as a coreceptor with type II activin receptor for the binding of inhibin (Lewis et al., 2000).

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In contrast to the high degree of specificity generally required for receptor– ligand interactions, proteoglycan coreceptors typically exhibit a lower affinity for growth factors (Nugent and Iozzo, 2000). The specificity of these proteoglycan–growth factor interactions has comprised the basis for intense research, with variable results reported for different systems (Ashikari-Hada et al., 2004; Guimond and Turnbull, 1999; Kreuger et al., 2006). Although the structural features and charge conformations required to create proteoglycan– protein specificity remain undefined, it appears likely that overall GAG charge distribution and steric structure, rather than particular disaccharide sequences, determine specificity for protein binding (Dreyfuss et al., 2009).

3. Integrin Interactions In parallel with their influence over growth factor signaling, proteoglycans also affect multiple intracellular signaling networks via the integrins. Named for their ability to integrate outside signals toward the cytoplasm, integrins represent a dichotomous family of alpha and beta glycoprotein subunits that sense and bind to specific extracellular matrix components. At least 18 alpha and 8 beta subunits have been identified to date, and integrins are found as a noncovalent pairing of one alpha and one beta subunit (van der Flier and Sonnenberg, 2001). Each paired combination, of which 24 have been identified, is able to bind with high affinity to a specific matrix protein, such as fibronectin, laminin, von Willebrand factor, and collagen, although there is some degree of overlap among the pairing specificities (Hood and Cheresh, 2002). Integrins are capable of relaying extracellular signals toward intracellular compartments, and are conversely able to transform intracellular signaling into alterations of extracellular attachment (Hynes, 2002). Similarly, proteoglycans and integrins often bind identical extracellular matrix components, and influence the same signaling networks. Such cross talk between ligand binding and signal transduction has been reported for years, although only recently have the notions of mutual dependence and synergy been ascribed to the signaling properties of these two protein families. Assembling focal contacts between cells and the extracellular matrix, establishing cell polarity, and the processes of wound healing and angiogenesis, for example, are all dependent upon the cooperative activity of both integrins and proteoglycans in various cell types (Morgan et al., 2007).

4. Autonomous Signaling Beyond the coreceptor functions served by proteoglycans in growth factor and integrin signaling are numerous circumstances under which proteoglycans also participate in signal transduction independently (Kirn-

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Safran et al., 2009; Tkachenko et al., 2005). Because proteoglycans frequently initiate signaling events within multimeric complexes comprised of other receptors, the isolation of independent proteoglycan capabilities represents a nontrivial experimental challenge. One method to overcome this issue is to induce proteoglycan activation by way of ligands that do not concurrently initiate signaling via other receptors. In the study of syndecan 4, a heparan sulfate proteoglycan known to initiate intracellular signaling cascades, this has been accomplished by addition of a particular fibronectin fragment (Bass et al., 2007) or antibodies against syndecan 4 (Tkachenko et al., 2006). Such antibodies (directed against the extracellular proteoglycan domain) do not induce simultaneous activation of other receptors, and their multivalency facilitates proteoglycan oligomerization. The extracellular epitopes of numerous proteoglycans, however, have proven notoriously difficult to immunologically target with great efficacy. This is likely due to the variable steric effects of their GAG chains, and has perhaps resulted in the underestimation of proteoglycan signaling potency when induced solely by means of antibody clustering. For this reason, models of chimeric proteoglycan activation have been developed for the study of their downstream signaling effects (Tkachenko and Simons, 2002). These chimeras contain native proteoglycan transmembrane and intracellular domains, thus preserving their intracellular signaling capabilities, and have a substituted GAG-free extracellular receptor domain. In the study of syndecans, the extracellular domain has been successfully replaced with the human Fc receptor (FcR), a molecule exclusively found in cells of the immune system. By introducing an exogenous, immunogenic target to proteoglycan core domains, it is possible to study their interactions, localization, and signaling capabilities with a high level of specificity. As detailed in Section 8, this model of proteoglycan oligomerization first involves expression of the chimeric construct and confirmation of its appropriate localization at the cell membrane. Nonimmune IgG is used to occupy the chimeric FcR receptor sites, and oligomerization is subsequently induced by multivalent anti-IgG antibodies or antibody fragments, such as F(ab)2 subunits. The use of fluorescently labeled antibodies in such experiments also enables the visualization of both spatial distributions and kinetics of oligomerization at the cell surface. In the case of syndecan 4, clustering of a chimeric receptor construct as described above leads to an enhanced rate of cell migration (Tkachenko et al., 2006). This is due to the unique signaling capabilities afforded by this proteoglycan’s cytoplasmic domains, involving the downstream activation of protein kinase Ca (PKCa) and two Rho family GTPases: RhoG and Rac1 (Elfenbein et al., 2009; Tkachenko et al., 2005). The active form of Rac1 is typically found at the leading edge of migrating cells, where it coordinates the actin polymerization machinery required for membrane protrusion and directional migration (Vicente-Manzanares et al., 2005).

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Syndecan 4 assembles multimeric protein complexes that suppress the activity of such GTPases until activation during cell migration; inactive pools of Rho GTPases are thus sequestered until syndecan 4 both releases and activates them at specifically localized regions cell regions (Elfenbein et al., 2009). Chimeric syndecan 4 constructs have been used to demonstrate that protein core mutations lead to an inability to form GTPase-inhibitory complexes, resulting in GTPase dysregulation and a cell migration defect (Elfenbein et al., 2009). In this way, the autonomous signaling potential of proteoglycan receptors has been shown to supersede that of simple liganddependent activation, and additionally encompasses the ability to orchestrate multimeric, localized signaling complexes (Naccache et al., 2006; Zimmermann et al., 2005). Assembly of these multiprotein complexes at proteoglycan cytoplasm domains, in concert with integrin activation, likely underlie the mechanism by which GTPases are precisely activated and regulated in response to extracellular signaling cues (Morgan et al., 2007). Despite effectively excluding other receptors from antibody-mediated clustering, the proteoglycan chimera model of signal transduction bears several limitations. First, the stereochemistry of antibody-mediated chimera oligomerization represents an approximation of physiological activation by ligands such as growth factors or extracellular matrix components. Whether this corresponds to comparable molecular proximities and conformational interactions with native ligand binding is unknown; both factors are critical for effective activation and deactivation in other receptor systems, and are likely to impact proteoglycan signaling similarly. Second, the spatial distribution of proteoglycan clustering is not controlled by antibody treatment in the same way as it commonly is, for example, at the leading edge of a migrating cell. Instead, the chimera becomes clustered on all apical and lateral surfaces, partially undermining the proteoglycan function of spatial guidance and the establishment of cell polarity for directional migration (Elfenbein et al., 2009; Pankov et al., 2005). Third, it is unclear whether the immunological isolation of proteoglycans during oligomerization also functionally excludes the activation of other receptors; this is potentially a confounding issue that occurs by mass effect of multiprotein oligomerization, thus diminishing the specificity of antibody-directed proteoglycan clustering. Finally, as with other model systems involving receptor overexpression, chimeric proteoglycan oligomerization is fraught with issues of the stoichiometric imbalance between the receptor and downstream signaling molecules. This stoichiometric inconsistency may further be complicated by a high degree of intercellular variability, depending on whether cells used for assay exhibit stable and similar levels of chimeric construct expression. For these reasons, while chimeric oligomerization models provide insight into the signaling potential of cell surface proteoglycans, characterization of their respective signaling networks is best achieved when supported by complementary methodologies. In the case of syndecans, the generation of

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syndecan 4 genetic knockout models have validated the role of this proteoglycan and its associated signaling proteins in cell migration, and demonstrated their importance in physiological processes such as wound healing, neurodevelopment, and maintenance of vascular integrity (Chittenden et al., 2006; Gorovoy et al., 2007; Matthews et al., 2008; Partovian et al., 2008). In such animal models of targeted genetic deletion, the redundancy of other expressed proteoglycan receptors often serves to compensate for defects generated by a particular receptor’s absence. It follows that alternative approaches may be used in vitro to better define proteoglyans’ signaling roles. These include RNA interference (RNAi)-mediated genetic knockdown of the studied proteoglycan(s), the addition of antibodies that preclude ligand binding, introduction of dominant-negative constructs, and the use of specific proteases and/or pharmacological inhibitors. Collectively, such approaches enhance the ability to identify the requirement and define the specific involvement of proteoglycans within signaling networks.

5. Proteoglycan Downregulation: Endocytosis and Ectodomain Shedding Following membrane-bound receptor activation, resultant signaling complexes are generally subjected to endocytosis in order to downregulate cell surface expression. Once internalized, these protein complexes may be recycled back to the membrane, directed toward lysosomal vesicles for degradation, or sorted toward numerous other vesicle types and cell compartments. In recent years, the role of proteoglycans as key regulators of endocytic processes has become more apparent (Kobialka et al., 2009; Tkachenko et al., 2004; Valdembri et al., 2009). Endocytosis is studied by cell surface labeling of the protein or membrane region of interest with a fluorescent dye, fluorophore, or immunologic tag. Because the internalization of labeled protein increases over time, endocytosis assays should be performed within consistently and strictly timed intervals. Synchronization for the start of endocytosis is therefore performed by bringing cells to a temperature of 0
C, at which point all endocytic machinery is arrested. After incubation with the labeling agent, cells are returned to 37
C to permit endocytosis to occur for a set period of time. Several techniques exist for microscopic observation of endocytic processes or the quantification of internalized protein (Fig. 1.2), and are detailed in Section 8. Microscopic approaches are taken when the goal is to dissect the particular endocytic pathway taken by internalized proteoglycans, or to ascertain whether certain other labeled proteins are found within close proximity to labeled endocytic vesicles. More quantitative approaches rely on measurements of rate and amount of internalized receptor.

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Cell surface receptors, including proteoglycans, may be internalized by several endocytic pathways (Mosesson et al., 2008). The kinetics, accompanying proteins, impact on signal duration and eventual receptor fate differ significantly from pathway to pathway. It is for this reason that a receptor’s preference for one route of internalization will often dictate its signaling properties. Proteoglycans primarily influence endocytosis of other cell surface receptors through the recruitment of proteins involved with directing vesicular traffic. Perhaps the best characterized of these proteins are the Rabs, GTPases that coordinate the translocation of nascent vesicles to specific cellular compartments. A reported connection between Rab5 and syndecan 1 ectodomain cleavage also implies the presence of an additional mechanism by which a proteoglycan receptor’s recruitment of endocytic machinery results in subsequent receptor downregulation by nonendocytic means (Hayashida et al., 2008). Ectodomain shedding in transmembrane proteoglycans has been previously described as a constitutive process that is accelerated during conditions such as tissue inflammation (Alexopoulou et al., 2007). Cleavage of extracellular domains, which is catalyzed by a variety of metalloproteases and other proteases, results in the extracellular accumulation of GAG chains that retain their protein-binding properties. The physiological significance of ectodomain shedding ranges from the formation of growth factor gradients to the attenuation of escalating inflammatory responses (Alexopoulou et al., 2007). In contrast to proteoglycan endocytosis, methods to study extracellular domain cleavage generally rely on biochemical techniques of quantifying both GAG production and the enzymatic activation of relevant proteases. Among the remaining elusive issues in this field is how proteoglycan removal from the membrane is regulated (via either endocytosis or ectodomain shedding) and how balanced proteoglycan turnover is consistently maintained.

6. Cell Adhesion Matrix attachment and subsequent migration are among the cellular processes largely mediated by proteoglycan signaling. Because of the significant overlap in signaling mediators and temporal characteristics of these discrete processes, they represent a functional continuum that is often experimentally challenging to dissect. Migration and matrix attachment take place simultaneously when cells are exposed to novel surfaces, thus temporally obscuring the distinction between both events. Likewise, the characterization of cells with a decreased ability to migrate is frequently confounded by the presence of a concurrent deficit in matrix adhesion. The differentiation between adhesion and migration is even further encumbered

Auxiliary and Autonomous Proteoglycan Signaling Networks

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by a large degree of overlap between the growth factors, receptors, and activation pathways that mediate each event. Three predominant mechanisms underlie the proteoglycan contribution to cell adhesion, and each is exemplified by the syndecans. First, these proteoglycans are able to directly bind extracellular matrix components, as demonstrated in the cases of fibronectin, laminin, and vitronectin engagement (Couchman, 2003). Second, the syndecans function synergistically with integrins during cell adhesion; this cooperation involves downstream signaling cross talk, the formation of vinculin-rich focal adhesions, and the activation of Rac1 (Morgan et al., 2007). In some systems, the function of syndecans and integrins is even more directly related, as evidenced by the requirement for syndecan 1 in avb3 integrin activation (Beauvais et al., 2004). Finally, growth factor signaling in the context of adhesion is partially mediated by syndecans (Gopal et al., 2010), particularly as it applies to the described Rho GTPases and their maintenance of balance between adhesion and motility. The above mechanisms afford a wide range of experimental approaches for studying cellular adhesion to a novel matrix. Among them are the assay of various adhesion substrates, incubation with proteoglycan-binding growth factors, treatment with pharmacologic modulators of proteoglycan signaling, and antibodies that either oligomerize or functionally block the matrix-binding properties of proteoglycans and/or integrins. Cell adhesion studies in the context of proteoglycan signaling typically involve timed measurements of attachment strength. In such experiments, cells in suspension are plated above a substrate coated with a selected matrix. At specified time points, nonattached cells are washed away with an isotonic medium, leaving the adherent cells for quantification. Although the fixation, staining, and counting of individual cells is a commonly employed approach to measure the percentage of remaining adherent cells, fluorescent membrane dyes may also be used to provide a more sensitive readout of this value; these dyes are generally used as part of a preincubation solution prior to the time of assay. Other variations of this technique involve fluorogenic enzymatic reactions that correlate linearly with adherent cell number (Tolosa and Shaw, 1996). On account of the inherent variability in the force of washing and between plated samples, statistically meaningful results may only be realized upon assay of multiple duplicated experimental and control conditions. Related to the described whole-cell assay are several microscopic approaches to studying cell–matrix adhesion quantitatively. One such method serves to characterize the extent of cell spreading (total area of interface contact) upon a two-dimensional matrix such as fibronectincoated glass. The degree and morphology of spreading on a particular matrix differs significantly among cell types, thus necessitating appropriate cell type-matched controls. Experiments involve fixation of cells after a time period during which matrix adhesion occurs, with subsequent microscopic imaging and quantification of the two-dimensional region of contact

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between the cell membrane and matrix. It is important to note that although increased adhesive surface areas imply a greater degree of surface interaction between the cell and extracellular matrix, such values are strictly morphometric and do not necessarily reflect adhesion strength. Live-cell microscopic imaging approaches may also be used to study cell attachment, and these techniques yield particular insight into the morphologic and temporal characteristics of adhesion. The use of total internal reflection fluorescence microscopy (TIRFM) represents one frequently employed method for studying cell signaling events, morphological changes, and cell adhesion processes occurring exclusively at the membrane–matrix interface. TIRFM imaging relies on light refraction properties to illuminate a distance above a reflecting glass surface, typically less than 200 nm in depth (Axelrod, 2003). This is accomplished by controlling a laser beam’s angle of incidence, which is related to light refraction according to Snell’s law. When directed at angles of total internal reflection, an ‘‘evanescent wave’’ is formed, which illuminates basal membrane surfaces. Evanescent wave intensity decays exponentially with distance above the reflective surface, thus enabling the effective exclusion of all structures not within immediate proximity to the cell’s basal membrane. For TIRFM studies, cells must be plated upon thin surfaces with amenable indices of refraction, typically glass coverslips. TIRFM also requires objective lenses with wide numerical apertures (NAs) that permit the manipulation of laser light toward oblique angles of incidence. When coupled with fluorescent probes for proteoglycans that localize to adhesion sites and mediate adhesion signaling events, TIRFM represents a valuable tool for studying the intersection between signaling and localized generation of adhesive force. Perhaps the best-studied nexus between these processes is the focal adhesion, a structure that serves to anchor cells within their surrounding matrices. Focal adhesions are dynamic multiprotein complexes that establish points of structural contact between the cytoskeleton and the extracellular matrix, and are required for the generation of tensile strength required for cell movement. These multimeric complexes also represent sites of numerous localized signaling processes, and generally contain enriched pools of proteins such as vinculin, talin, paxillin, integrins, in addition to proteoglycans, which may be used experimentally as focal adhesion markers (Albiges-Rizo et al., 2009). In this way, early studies of proteoglycan involvement revealed that chondroitin sulfate and hyaluronic acid are not associated with focal adhesions, while heparan sulfate is intimately associated with these cell structures (Turley, 1984). Heparan sulfate proteoglycans are now appreciated as requisite components of focal adhesion assembly, where their action is mutually dependent upon (and synergistic with) the activity of integrins (Couchman, 2003). Syndecan 4 overexpression causes increased focal adhesion formation with a subsequent decrease in cell motility (Longley et al., 1999); this proteoglycan

Auxiliary and Autonomous Proteoglycan Signaling Networks

13

is also hypothesized to directly interface between the extracellular matrix and the actin cytoskeleton at focal adhesions on account of its ability to bind both fibronectin and the actin bundling protein, a-actinin (Greene et al., 2003). The quantification of focal adhesion formation after cell fixation and focal adhesion immunostaining has been used to characterize cell–matrix adherence (Partridge and Marcantonio, 2006). TIRFM’s applicability to live-cell imaging holds great potential to expand upon these quantitative studies by enabling real-time study of proteoglycan-mediated fluxes of focal adhesion generation and disassembly. The forces exerted by regions of cells during adhesion may also be discretely measured in studies of cell–matrix interactions to precisely quantify the vectors of tensile strength. This is accomplished either by measurement of the ensuing matrix deformity (passive) or by the direct probing of cellular resistive forces (active). The former principle generally entails the creation of an observable and malleable extracellular array, such as sheets constructed from silicone or similarly pliable materials, or beads embedded within a matrix. Microdeformities or shifts in bead arrays resulting from cellular adhesion are subsequently interpolated to their respective forces and mapped to their corresponding cellular sites (Harris et al., 1980; Munevar et al., 2001). Direct probing of cell resistance and adhesive strength, on the other hand, involves a direct challenges to the integrity of a cell’s adherence, and is exemplified by the techniques of shear flow generation, atomic force microscopy, magnetic tweezers, and optical tweezers. Such techniques have permitted the localization and quantification of cellular forces to piconewton levels of resolution, and have been extensively reviewed elsewhere (Addae-Mensah and Wikswo, 2008). Although the technical considerations of subcellular adhesion force measurement is beyond the scope of this chapter, the application of force mapping to proteoglycan signaling is significant for two reasons. First, as a collectively predominant component of the extracellular matrix, proteoglycans are largely responsible for both the cell-to-matrix and intercellular adherent forces that sustain tissue growth. The structural integrity of most tissues is therefore strongly influenced by proteoglycan support and signaling, the mechanisms of which are likely to be more clearly delineated by future studies of subcellular force generation. Second, emerging evidence in recent years has led to the implication of proteoglycans in mechanotransduction within various signaling roles. In endothelial cells, for example, the detection of shear flow is also likely to be partly accomplished by heparan sulfate proteoglycans (Moon et al., 2005). It has furthermore been postulated that the bulky mass of these proteoglycans’ heparan chains serve as sail-like sensors for shear stress, leading to flow-dependent oligomerization. Such mechanisms currently remain without definitive demonstration, and will likely rely upon the intersection of live-cell imaging modalities and force reactivity assays for complete characterization (Florian et al., 2003; Mochizuki et al., 2003).

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7. Migration Cell adhesion is a requisite stage of migration, although the former represents only one element of the coordinated process needed to propel a cell through a given matrix. In order for effective and directional migration to occur, a cell must first establish opposing leading and retracting edges. As described above, this cell polarity is largely mediated by proteoglycans and their effects on Rho family GTPase signaling (Burridge and Wennerberg, 2004). Studies of cell polarization may be performed by either morphologic characterization (as with directionality of cell protrusions or migration) (Pankov et al., 2005) or on a molecular level by spatial mapping of enzymatic activity over time. In recent years, the mechanistic insight yielded by the latter approach has been significantly expanded by the development of fluorescent biosensors; such engineered constructs allow investigators to observe spatial and temporal features of signaling downstream of proteoglycans within living, migrating cells. Among these are probes that exploit the potential transfer of energy that occurs when two molecules come within close proximity to each other (Nakamura et al., 2006). This physical property, known as Fo¨rster resonance energy transfer (FRET), has permitted the engineering of constructs containing both a donor and acceptor fluorophore, with the spatial proximity of both dictated by construct conformation. Intramolecular FRET-based probes are designed to shift conformation in a reversible manner that is dependent upon ligand binding or enzymatic activity. Cells expressing such probes are subjected to light wavelengths that exclusively excite the donor fluorophores, and the resultant emission from both the donor and acceptors are subsequently measured. Regions of acceptor emission indicate that energy transfer has occurred. Thus, this technique allows the mapping of donor–acceptor proximity throughout the living cell (directly correlating with probe conformation), and so far have successfully been used to characterize cell polarity with respect to phosphoinositide concentrations, Rho family GTPase activity, kinase activity, cell surface receptor activation, among others (Aoki and Matsuda, 2009). For effective migration to occur, cells must also form new points of matrix adhesion for leverage, dismantle existing adhesion points at the trailing edge, disengage the cell–cell connections that maintain tissue structural integrity, and perform these events in synchrony with neighboring cells. The mechanisms underlying the mobilization and orchestration of all involved signaling networks remain incompletely understood, although the contributions made by proteoglycans are indispensable. In cells lacking syndecan 4, for example, Rac1 activity is not appropriately suppressed at regions of the cell uninvolved with leading edge formation, resulting in noncoordinated membrane protrusion and a subsequent defect in cell migration ability (Bass et al., 2007; Elfenbein et al., 2009; Matthews et al., 2008; Pankov et al., 2005).

Auxiliary and Autonomous Proteoglycan Signaling Networks

15

Several approaches may be employed for the characterization and quantification of cell migration. Perhaps the most basic characterization of cell migration involves the tracking of single-cell movement in two dimensions by low-magnification live-cell microscopy over time. Cellular displacement over time is recorded by sequential image acquisition, with a readily identifiable cell structure such as the nucleus generally used for tracking motion. From these images, characteristics of migration such as velocity, distance, and directionality may be calculated for comparison among various experimental conditions. This technique requires the plating of living cells on surfaces such as glass, or plastics that are engineered to exhibit similar refractive indices. Because migration occurs over the course of hours, microscope configurations amenable to migration studies necessitate thermal control and the maintenance of a stable pH in the cell culture medium. Thermal regulation is best achieved with an insulated enclosure and thermostat-heater circuitry, while control of pH may be accomplished by several methods. Culturing chambers may be hermetically sealed at the initiation of assay to prevent changes in CO2 partial pressure within the medium over time. Alternatively, a layer of mineral oil may be added above the culturing medium, providing a barrier to CO2-dependent acidification. Cells used for assay may also be grown in media containing buffers such as 100 mM HEPES, which render the cultures more resistant to pH fluxes than most conventional culturing medium formulations. The latter approach requires controls to exclude the possibility that buffering agents alone influence the cell signaling events under investigation. Finally, environmental O2 and CO2 partial pressures may be kept constant by control of their supply by direct gas perfusion. When combined with a programmable motorized stage, the migration of multiple adherent cells in different microscopic fields may be tracked over time, providing a well-controlled and statistically meaningful comparison of cells’ migration behaviors. Tracking the migration of individual cells enables the study of proteoglycan roles in whole-cell displacement over time. It largely in this manner that the ability of proteoglycans to direct cell migration via Rho family GTPases has been characterized (Morgan et al., 2007; Pankov et al., 2005; Tkachenko et al., 2006). Despite the wide range of experimental flexibility and described future potential offered by single-cell migration assays, however, several inherent limitations also exist with these techniques. The first is related to characterizing a cell’s direction of migration: migration is a purposeful and energy-intensive process that typically occurs in response to a spatially directed signal, often established as a protein gradient. Although single-cell tracking techniques using conventional cell culture chambers (dishes and/or wells) often serve to identify migration deficits or enhanced proclivities, such experimental setups do not facilitate the formation of gradients that guide migration under physiological conditions.

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To that end, a variety of cell culture devices have been designed for the purpose of establishing a linear gradient of chemoattractant or migrationinhibiting factors (Boyden, 1962; Zicha et al., 1991; Zigmond, 1988). In the study of proteoglycan signaling, these gradients may involve extracellular matrix components, growth factors, and/or antibodies that induce oligomerization at the cell membrane. Commonly used devices for this purpose are Boyden and Dunn chambers, and their respectively modified variants. These methods of culturing cells involve two interfacing chambers, one in which cultured cells are placed and the one for the chemoattractant(s) being assayed. In Dunn chambers, cells are adherent to the chamber surface at the time of assay and migrate horizontally in two dimensions toward the established gradient. In contrast, the Boyden chamber model of migration has suspended cells in an upper chamber, with a porous filter separating it from a lower chamber in which chemoattractant is typically placed. Cellular migration through the porous filter is generally quantified in this assay by counting cells that traverse the membrane (see Section 8). Alternatively, cells may be preincubated with a fluorescent membrane dye, with subsequent lower chamber fluorescence quantification of trans-filter migration. It is important to note that in measuring migration, the Boyden model actually serves to quantify the entire continuum of adhesion and migration, as cells must first successfully attach to the interchamber filter before migrating to the lower chamber. For this reason, such experiments do not clearly discriminate between cellular adhesion and migration. Given this constraint of interpretation, the ability to modify which extracellular signaling ligands are (1) immobilized upon the filter and (2) soluble within the lower chamber medium nevertheless confers enormous experimental versatility to this method. The second limitation of single-cell migration assays relates to the fundamental behavioral difference between cells that migrate in isolation and those that move as part of a multicellular front. The migration of numerous cells is by far more commonly observed within physiologically intact tissues than is lone cell migration, and the former is exquisitely sensitive to factors such as intercellular contact inhibition and paracrine signaling. Multicellular migration may be studied by quantifying en masse movement of cells in confluent two-dimensional monolayers. In such experiments, an acellular discontinuity, or ‘‘wound’’ is created within a monolayer, and the rate of cellular reinfiltration of the void is subsequently quantified. This is most simply accomplished by subjecting the monolayer to a uniform linear disruption or scratch, and imaging the resultant migration into this region at defined time points. Results of multicellular migration studies are particularly sensitive to artifacts of proliferation; a multiplication of total cell number during the course of an assay can result in falsely accelerated level of migration. It is for this reason that cell cycles should be synchronized by adequate serum-starvation prior to

Auxiliary and Autonomous Proteoglycan Signaling Networks

17

the time of assay. Additionally, the degree of cellular confluence profoundly impacts migration rate; cultured cells that have been permitted to overgrow (subsequent divisions upon reaching complete confluence) exhibit severe migration defects, even after several subsequent cycles of cell passaging. It is for this reason that cells to be used for multicellular migration studies are recommended to invariably undergo passaging as they approach 75–80% confluence, thus minimizing such intercellular contact inhibition artifacts. These and other considerations for tissue culture of monolayer cells are addressed in Section 8. An alternative approach to the study of cell migration involves the creation of a three-dimensional matrix for the quantification of cell movement. Such techniques were historically used to assay tumor cell invasive potential, and initially involved basement membrane preparations from the amnion (Liotta et al., 1980). Development of reconstituted extracellular matrices, such as Matrigel, and refinement of three-dimensional imaging techniques have since facilitated the ability to readily characterize the velocity, directionality, and morphology of cells as they negotiate defined extracellular matrices in three dimensions. Migration assays involving Matrigel and related reconstituted matrices permit the selection of numerous experimental conditions, including growth factors, structural proteins, concentration gradients, and matrix viscosity. These create an environment that ideally mimics physiological migration better than two-dimensional cultures. These three-dimensional matrices may be used in conjunction with techniques such as Boyden chamber assays, providing a three-dimensional medium above the filter that separates upper from lower chambers (Albini et al., 2004). Reconstituted matrices may furthermore be used as implants in animal models of migration, particularly as they apply to nerve regeneration or angiogenesis (Malinda, 2009). Finally, such three-dimensional models of migration are indispensable for the assay of morphological features not observed in twodimensional cultures, such as endothelial tube, which is also partially mediated by proteoglycan signaling (Ferrari do Outeiro-Bernstein et al., 2002; Ponce, 2009).

8. Experimental Procedures 8.1. Immunoblotting for heparan sulfate proteoglycans On account of their variable GAG chain modifications, proteoglycans typically exhibit patters of poorly defined streaks when subjected to gel electrophoresis. This prevents their effective separation and visualization by Western blot analysis, and necessitates several modifications to classical immunoblot techniques.

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GAG chain cleavage is typically performed prior to electrophoresis in order to resolve molecular weights and quantities of proteoglycan naked protein cores. Samples to be analyzed by immunoblotting are incubated with heparitinase (otherwise known as heparanase III) or chondroitinase ABC, depending on the proteoglycan under investigation. The former cleaves heparan sulfate chains, leaving dermatan, keratan, and chondroitin sulfate modifications intact. On the other hand, incubation with chondroitinases generally results in the exclusive cleavage of dermatan and chondroitin sulfates. (1) Cell lysates are incubated in one of two buffers, depending upon whether the enzyme of choice is a heparitinase or chondroitinase. In the case of heparitinase, the final buffer concentration is: 0.1 mM calcium acetate and 0.1 M sodium acetate with pH adjusted to 7.0. Depending upon whether heparitinase is isolated from a microbial source, the addition of a protease inhibitor is frequently recommended (Couchman and Tapanadechopone, 2001). If chondroitinase is to be used, the following final buffer concentrations are preferred: 50 mM Tris–HCl, 20 mM ethylenediaminetetraacetic acid (EDTA), 30 mM sodium acetate, 10 mM N-ethylmaleimide (NEM), and 0.2 mM phenylmethylsulfonyl fluoride (PMSF). Concentrations of enzyme should be adjusted in accordance with estimated proteoglycan concentration. Heparitinase is added in approximate quantities of 1 mU for up to 1–5 mg of proteoglycan, whereas chondroitinase ABC is added at of 1 mU for every 2–10 mg of proteoglycan. An incubation time of 3 h at 37
C is recommended for complete digestion. Experimental conditions of optimal enzymatic reaction times and concentrations may be conducted with known quantities of purified proteoglycan. (2) Samples are added to 2 SDS-PAGE sample buffer in a 1:1 ratio (final concentrations: 10% (v/v) glycerol, 62.5 mM Tris–HCl (pH ¼ 6.8), 2% sodium dodecyl sulfate (SDS), 0.01 mg/ml bromophenol blue, 5% (v/v) b-mercaptoethanol (BME)). All samples are heated to 95
C for 5 min, and either frozen at 80
C for future electrophoresis or run as follows. (3) Samples are loaded onto a poly-acrylamide gel (concentration should be optimized for the proteoglycan of interest), subjected to electrophoresis and then transfer onto a polyvinylidene fluoride (PVDF) membrane. (4) Nonspecific membrane binding sites are blocked by either 5% nonfat milk in Tris-buffered saline (TBS: 50 mM Tris/HCl (pH ¼ 7.4), 150 mM NaCl) or 5% bovine serum albumin (BSA), also dissolved in TBS. Primary antibodies directed against proteoglycan epitopes are added at dilutions according to their specifications (in either 1% nonfat milk/TBS or 1% BSA/TBS), and incubated for 1 h at room temperature or overnight at 4
C. (5) Following three sequential, 10-min washes with TBS containing 0.1% Tween-20 (TBST), secondary antibodies (conjugated with either

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Auxiliary and Autonomous Proteoglycan Signaling Networks

horseradish peroxidase or a specified fluorescent dye) are added in either 1% nonfat milk/TBS or 1% BSA/TBS at concentrations determined by their specifications. (6) The membranes are washed three times for 10 min with TBST, and once for 10 min with TBS. They are then imaged by standard luminance-based or fluorescence-based methods, as determined by the secondary antibody conjugates.

8.2. Proteoglycan chimera oligomerization In order to isolate the signaling effects of proteoglycan clustering, a chimeric proteoglycan model of antibody-induced oligomerization may be employed (Fig. 1.1). To begin, DNA expression vectors should be designed to conserve both transmembrane and intracellular domains, and the proteoglycan extracellular domain should be substituted with a nonendogenously expressed antibody receptor. For stoichiometric consistency among cells undergoing antibody clustering and between experiments, we recommend the generation of clonal A

B Non-immune human lgG

C

D

F(ab)2

F(ab) Domains:

HS

Extracellular

FcR

Transmembrane Cytoplasmic Endogenous syndecan 4

Syndecan 4FcR chimera

Rho GTPase activation / migration

(oligomerization)

Endocytosis PKCa activation

Figure 1.1 Clustering of proteoglycan chimeras. Endogenous syndecan 4 is membrane bound, and contains heparan sulfate chain modifications on its extracellular domain (A). The syndecan 4-FcR chimera consists of the human Fc receptor extracellular domains substituted for the native (heparan sulfate (HS)-bearing) extracellular domain of syndecan 4 (B). Clustering is initiated by the addition of nonimmune human IgG. Excess antibody is removed by washing, and F(ab)2 fragments are added to induce oligomerization (C). This leads to multiple downstream signaling events, as described in the figure. As a negative control, univalent F(ab) fragments may be added, which do not induce proteoglycan clustering (D).

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populations of cells exhibiting stable expression of the chimera construct. Alternatively, pooled cell populations with comparable stable chimera expression, generated by fluorescence-assisted cell sorting (FACS), may be used for these studies. The following experimental procedures apply to a model chimera receptor system: rat fat pad endothelial cells (RFPECs) with stable expression of a syndecan 4/human Fc receptor chimera (Tkachenko and Simons, 2002). (1) Prior to assay, cells are plated after sequentially passaging, with confluence not exceeding 75–80%. Because of the described cross talk between proteoglycans and integrins, an extracellular matrix component, such as fibronectin, should be chosen to control for integrin activation. Plates may be coated with fibronectin by covering plating surfaces with a solution of 10 mg/ml fibronectin dissolved in sterile phosphate-buffered saline (PBS) for 30 min at 37
C. In our experience, failure to select an extracellular matrix (and thus, reliance upon ionically treated plastic substrates for adhesion) results in unacceptable levels of interexperimental variability on account of uncontrolled integrin engagement. (2) Cells are serum-starved in a culturing medium, such as Dulbecco’s modified Eagle medium (DMEM) containing 0.5% fetal bovine serum (FBS) and unchanged antibiotic concentrations from those of typical culture conditions (100 U/ml penicillin, 100 mg/ml streptomycin). (3) Human nonimmune IgG ( Jackson ImmunoResearch Laboratories) is added to cells at a final concentration of 1 mg/ml. Cells are incubated with these antibodies for 10 min at 37
C. (4) Excess IgG is washed away three times with serum-free culture medium that has been prewarmed to 37
C to avoid thermal disruption. (5) The cells are left to incubate in serum-free medium for 30 min at 37
C to allow shear-mediated signaling to return to baseline. In our experience, this does not result in significant downregulation of chimerabound IgG at the cell membrane, and is critical for establishing an unstimulated baseline for various signaling pathways. (6) Clustering is initiated by addition of F(ab)2 fragments at a final concentration of 2–5 mg/ml while keeping cells at 37
C. As a negative control, monovalent F(ab) fragments may be added at 4–10 mg/ml in place of an F(ab)2 incubation. (7) At determined time points, cells are placed on ice and lysed with lysis buffers appropriate for subsequent assay(s). For assay of Rho family GTPase activity (as described below), a time course of 15–20 min after clustering is recommended to detect the primary wave of activation (Elfenbein et al., 2009). Upon lysis, samples may be flash frozen in liquid nitrogen and stored at 80
C for future assay, or run immediately.

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21

8.3. Qualitative and quantitative endocytosis assays Studies of proteoglycan internalization involve characterizing both the rate of endocytosis and pathway involved in transport. Several common principles apply to studies of both, and the following experimental methods detail such qualitative and quantitative approaches. RFPECs with stable expression of the described syndecan 4 Fc receptor chimera will again be used as a model system for these studies (Fig. 1.2). (1) For purposes of microscopy, cells are plated on glass-bottom dishes or glass coverslips (in either case precoated with an extracellular matrix component, such as fibronectin (10 mg/ml in PBS, coated at 37
C for 30 min)). For cells to be analyzed by flow cytometry, matrix-coated 12well or 24-well dishes may be used for plating. A confluence of 75–80% should not be exceeded, and cells are serum-starved in 0.5% FBS/ DMEM with antibiotics (100 U/ml penicillin, 100 mg/ml streptomycin) for 24 h prior to assay. (2) Chimera clustering is performed as described above, with two modifications. First, the 10 min incubation with primary antibody (IgG) and subsequent washes must be performed on ice in order to halt all endocytic processes. Addition of F(ab)2 fragments (chimera oligomerization and initiation of endocytosis) is performed at 37
C. Second, either the primary antibody (IgG) or the clustering antibody fragment F(ab)2 must be conjugated to a fluorescent dye compatible with available microscope filters. (3) Endocytosis is allowed to proceed after chimera oligomerization for set time points, with optimal experiments containing several samples in a time course. Typical endocytic time points might span anywhere from 30 s to 30 min, depending upon the route of internalization. Following the completion of a time point, the sample is again placed on ice to arrest endocytosis. (4) In order to remove noninternalized antibody from the cell surface, a solution of ice-cold PBS titrated with HCl to a pH of 2.5 is added to the cells as a first wash. The cells are subsequently washed three times more with ice-cold standard PBS (pH ¼ 7.4). (5) For samples to be used in microscopy analyses, a solution of 4% paraformaldehyde diluted in PBS is added to cells for fixation. The cells are then brought to room temperature and permitted to incubate for 10 min. At that time, the paraformaldehyde solution is replaced by PBS. Cells may then be mounted for future analysis, or visualized immediately by fluorescent microscopy. Endocytic vesicles may be quantified by imaging sampled cells, and their subcellular localization is most effectively determined by this technique. Vesicular characterization (with designations such as early endosome, recycling endosome,

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F(ab)2 - Cy5

A

H+ B H+ H+

lgG

H+ H+

H+

H+

H+

H+

Acid wash

Endocytosis

Early endosome

GFP (transfected cell) Fixation, with analysis by microscopy

Trypsin treatment, with analysis by flow cytometry D 1000

C

SSC-H

800 600 400 200

64

0 0

200 400 600 800 1000 FSC-H

GFP

Cell selection by morphology

F(ab)2 - Cy5 104 0.18

104 4.8

0.15

3

FL1-H

FL1-H

102 101

Merge

DIC

99.5 100 0 10 101

37.3

103

10

102 101

0.19 102

103

104

FL4-H

Negative control

100 20.8 0 101 10

37.1 102

103

104

FL4-H

Transfected cells with endocytosis

Figure 1.2 Quantification of proteoglycan endocytosis. Internalization of proteoglycans following oligomerization is quantified by first inducing receptor oligomerization, as described in Fig. 1.1. For these experiments, fluorescently labeled antibodies (either IgG or F(ab)2 fragements) are used to label endocytosed proteoglycans. In this figure, the fluorescent dye Cy5 is conjugated to F(ab)2 fragments, and cells used for assay express both the S4-FcR chimera and green fluorescent protein (GFP) (A). Incubations are performed on ice, and endocytosis is permitted to proceed at 37
C for specified periods of time. At the conclusion of an endocytosis time point, cells are brought to ice again to arrest all endocytic processes, and washed with PBS that has been titrated with HCl to pH ¼ 2.5 (B). This disrupts antibody–antigen binding, and permits the effective washing of remaining surface-bound fluorescent antibodies. One approach for

Auxiliary and Autonomous Proteoglycan Signaling Networks

23

macropinosomes, etc.) may be accomplished with costaining using additional fluorescent dyes or conjugated antibodies. See notes below for preparation of samples for staining with additional markers. For samples to be analyzed by flow cytometry, cells are removed from their dishes by addition of trypsin–EDTA (0.25%) in a volume of approximately 20–25 ml. Trypsin activity is slowed by transfer of cells to ice and addition of 150 ml of 1% BSA. The activity of trypsin may also optionally be quenched by the addition of trypsin soybean inhibitor. Cells are then either fixed with 4% paraformaldehyde/PBS for 10 min, followed by centrifugation, resuspension in PBS, and storage at 4
C until assay, or immediately analyzed for internalized fluorescence by flow cytometry. Appropriate negative controls for autofluorescence, including chimera clustering in the absence of fluorescent antibodies, is critical to establish baseline fluorescence. Notes (1) Because of cellular sensitivity to excessive periods of exposure to 4
C ambient temperatures and endocytic differences at various temperature ranges, experimental timing of each step should be strictly observed for the generation of reliably reproducible data. characterizing endocytosed vesicles is fixation and analysis by microscopy (C). These panels show two cells, both of which express S4-FcR, and have undergone clusteringmediated endocytosis for 15 min (scale bar ¼ 10 mm). Vesicles are apparent throughout the cytosol, and are not found in nuclear compartments. One of the two cells also coexpresses GFP, and this technique of construct coexpression permits the investigation of other endocytosis-specific plasmids, such as clathrin, dynamin or Rab mutants, or other endocytic markers. All the visualized fluorescent vesicles are known to originate from the cell surface in this technique, as the source of vesicular fluorescence is derived from the dye-conjugated clustering antibodies added to the cells prior to fixation. Note that the cell membrane contains minimal fluorescent signal, on account of the acid wash step described above; this acid wash effectively excludes signals generated by endocytic vesicles from that of the cell membrane. A second approach involves treatment of the cells by trypsin to remove them from their culture dishes, with subsequent analysis performed by flow cytometry (D). The first panel demonstrates morphologic selection of cells for analysis using side-scattered (SSC) and forward-scattered (FSC) light. The region selected represents  64% of the total cell population in this example, excluding debris and cells that have been inadvertently lysed. Of this subpopulation, fluorescence is measured in the bottom two panels with GFP represented on the y-axis (FL1-H) and Cy5 on the x-axis (FL4-H). The left panel shows a negative control condition of untransfected cells that were not exposed to fluorescent antibodies, used to establish a baseline of autofluorescence for gating of experimental samples. The right panel demonstrates a cell population that was transfected with a GFP construct, and underwent clustering-mediated endocytosis of the S4-FcR chimera for 15 min. Variability in GFP expression and fluorescent antibody internalization is readily apparent, and median or mean values are typically used to characterize fluorescence values in each condition. GFP in this experiment may also be replaced with endocytosis-related fluorescence constructs to specifically quantify the endocytic rates of cells expressing such constructs.

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(2) In microscopic analysis and flow cytometry experiments, sample fixation with paraformaldehyde may in some cases diminish the fluorescence of dyes and fluorophores used for vesicular identification. (3) In the case of endogenous proteoglycan endocytosis, antibodies generated against extracellular epitopes may be used as above to visualize and/or characterize their internalization. (4) In order to stain cytoplasmic protein markers of endosomal stage or function, cells must first be permeabilized to allow diffusion of antibodies across the membranes toward their targets. This is performed after fixation with 4% paraformaldehyde, and after the subsequent PBS washes. Triton X-100 (0.1%) dissolved in PBS is added to samples for 10 min at room temperature, after which three successive washes with PBS are performed. Nonspecific sites are blocked with 5% BSA in PBS for 30 min, after which three more washes with PBS are again performed. Incubation with primary antibody ensues, with antibody dilution being dependent upon affinity specifications. This is performed in 1% BSA/PBS for 1 h, after which the primary antibody solution is washed three times with PBS. Secondary antibody incubation follows (also in 1% BSA/PBS), with three washes of PBS concluding the 1-h incubation. The cells are then visualized by microscopy. (5) Live-cell microscopy may be performed as above in a temperaturecontrolled stage environment to visualize proteoglycan endocytosis with greater temporal resolution than that provided with fixed samples. In these experiments, the addition of acidic PBS (pH ¼ 2.5) is not advised. Instead, endocytic vesicles are differentiated from residual membrane-associated antibody fluorescence by morphologic features rather than by acidic removal of the latter.

8.4. Rac1 pull-down assay Rho family GTPases function as molecular switches, alternating between GDP-bound inactivity and the active, GTP-bound form. Determination of Rac1 activity will be considered here to exemplify the GTPase pull-down technique. Rac1 activity is typically quantified as a ratio of the cellular fraction of active Rac1 to total expressed Rac1. Active Rac1 is isolated by exploiting the enhanced affinity it has for its effector, p21 activated kinase (PAK). Immobilized, PAK-binding domain (PBD) peptides are therefore used as bait to bind active (GTP-bound) Rac1, and to separate this protein pool from all other cellular proteins. The immobilized Rac1 PBD is purified as a GST-fusion construct (GST-PBD), and agarose glutathione beads are subsequently used for pull-down assay (del Pozo et al., 2000).

Auxiliary and Autonomous Proteoglycan Signaling Networks

25

(1) As described in the experimental procedures for syndecan 4 FcR chimera oligomerization, cells are grown to approximately 75–80% confluence. For the purpose of Rac1 activation assays, these are plated on 10 cm or larger plastic dishes coated with a given extracellular matrix component, such as fibronectin. (2) The cells are serum-starved in 0.5% FBS/DMEM with unchanged antibiotic concentrations for a minimum of 24 h. Although longer serum-starvation periods (and those of lower serum concentrations) result in a furthermore diminished baseline of Rac1 activity, the ability to tolerate such conditions is cell-type dependent and should be determined prior to assay. After stimulation (as with chimera clustering or growth factor addition), lysis is performed on ice using cell lifters/scrapers and the following lysis buffer: 50 mM Tris (pH ¼ 7.4), 150 mM NaCl, 1 mM EDTA, 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). A substitution of 0.5–1% NP-40 for CHAPS may be made, although stronger detergents such as SDS are not recommended, as they are more likely to disrupt GTPase–bait binding interactions. The lysis buffer is also supplemented with a protease inhibitor cocktail, or addition of 1 mg/ml aprotinin, 1 mg/ml leupeptin, and 1 mM PMSF. The lysis buffer may also be supplemented with 1 U/ml benzonase nuclease (Novagen) to prevent DNA agglutination. A relatively large lysis volume is recommended (750–1000 ml) for each sample. Ten microliters of aliquots are then taken for protein concentration measurement prior to flash freezing of the sample for future assay. Protein concentration measurement may be performed by standard methods, such as Bradford, bicinchoninic acid (BCA), or Lowry protein assays. (3) Agarose glutathione beads with conjugated GST-PAK are prepared prior to thawing samples for assay. The frozen samples are thawed on ice, with corrections for concentration variabilities made with lysis buffer. Once the samples have thawed, they may be precleared with addition of agarose glutathione beads alone (50 ml slurry) and centrifugation for 2 min at 16,000 rpm in a microcentrifuge that is precooled to 4
C. The supernatant is transferred to new sample tubes, also at 4
C. At this point, a small, equal volume of each supernatant is removed for immunoblotting of total Rac1 expression (20 ml is sufficient for most cell types). (4) 50 ml of glutathione agarose bead slurry and 50 mg of recombinant GST-PAK is added to each sample, and the tubes are subjected to gentle agitation or rotation for 20–30 min at 4
C. (5) The beads are then centrifuged using a precooled (4
C) microcentrifuge at 16,000 rpm for 2 min, and washed with ice-cold lysis buffer. This sequence of washing and centrifugation is performed sequentially three times. An equal volume of 2 sample buffer (final concentrations: 10% (v/v) glycerol, 62.5 mM Tris–HCl (pH ¼ 6.8), 2% SDS, 0.01 mg/ml bromophenol blue, 5% (v/v) BME) is added to the beads and to the total

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protein samples taken in step 3. All samples are heated to 95
C for 5 min, and either frozen at 80
C for future electrophoresis or run on an acrylamide gel that is optimized for resolution of this 24 kDa protein (e.g., 12% acrylamide). Immunoblotting for Rac1 is performed as described in the above Western blotting protocol, with anti-Rac1 primary antibodies and horseradish peroxidase- or fluorescent dye-conjugated secondary antibodies used for detection of active and total Rac1 bands. (6) The ratios between active and total Rac1 for each sample are quantified using densitometry. Notes (1) The intrinsic GTPase activity of Rac1 renders this assay particularly sensitive to time and temperature; upon thawing flash-frozen samples, all steps until the addition of sample buffer should be performed at 4
C or on ice, and without excessive delays. (2) Alternative protocols based upon the enzyme-linked immunosorbent assay (ELISA) method have been developed, and may similarly be used to quantify the activity of Rho family GTPases (Elfenbein et al., 2009; Zhou et al., 2007). These protocols also rely upon bait peptides to isolate pools of active (GTP-bound) enzyme within each sample, and have a lower requirement for total protein input. (3) Because pull-down methods measure total cellular pools of active GTPases, they are unable to aid in characterizing the spatial localization of these signaling events. For example, the activation of Rac1 exclusively at the leading edge of migrating cells, although comprising a small proportion of total Rac1 activity, is critical for effective directional migration. It is therefore advisable to conduct experiments complementary to pulldowns in the study of Rho GTPase signaling, including those involving live-cell imaging with GTPase-specific biosensors. The development, technical considerations, and applications of such biosensors have been extensively reviewed elsewhere (Ibraheem and Campbell, 2010).

8.5. Multicellular migration assay This technique measures the distance that a migrating front of cells in a monolayer traverse in two dimensions over time. (1) Cells are plated in 12-well format and grown until reaching 50% confluence, at which time they are serum-starved for a minimum of 24 h with 0.5% FBS in DMEM, supplemented with penicillin and 100 mg/ml streptomycin. Because high-power microscopy is not required for this technique, plastic dishes are acceptable for this assay.

Auxiliary and Autonomous Proteoglycan Signaling Networks

(2)

(3)

(4)

(5)

(6)

27

Dishes should be premarked with a grid or pattern of demarcated lines on the microscope objective side; this enables the accurate imaging of identical regions over time. Proteoglycan and integrin engagement of extracellular matrix components is a critical factor in migrations. For this reason, dishes should invariably be precoated with a known matrix substrate, such as fibronectin, collagen, or vitronectin. Assay is optimally begun as cells approach 90–95% confluence, as overconfluent cells frequently exhibit diminished migrational capabilities. At the time of assay, a uniform linear disruption is created in the monolayer. This may be accomplished by using a sterile plastic pipette tip and scratching the dish surface in a linear fashion. Upon formation of the monolayer discontinuity, the dish is imaged at each site marked by the underside grid. Attempts should be made to maximize the number of imaged fields, as this enhances the statistical robustness of migration measurements. Cell stimulation or inhibition is performed, as with the addition of growth factor, pharmacologic agent, proteoglycan-oligomerizing antibody, or proteoglycan-blocking antibody. Cells are then returned to 37
C. Reagents targeting integrins may also be used for these experiments in combination with those affecting proteoglycan function. At determined time points, identical fields marked by the dish markings are imaged under low power. These regions should ideally register perfectly with those imaged in step 3. Optimal migration time points are cell type dependent. Ranges of 12–36 h typically yield reproducible results. The distance migrated is quantified by either determining the linear distance traversed (difference between division edges) or the approximately columnar area invaded by migrating cell fronts. Either method is appropriate if identical regions are measured consistently, with a statistically meaningful number of data points.

8.6. Two-chambered migration assay (adhesion and migration) In contrast to multicellular migration assays, the use of a two-chambered assay enables the measurement of single-cell adhesion and invasion through a given matrix. These devices, such as the Boyden chamber and its variants, contain an upper chamber into which cells are placed, and a lower chamber that often contains a chemoattractant or stimulatory molecule such as a clustering antibody. Between the two chambers is a porous membrane that permits cell migration, or a three-dimensional matrix such as Matrigel. The ability of cells to adhere to the membrane/matrix, or migrate from the top chamber to the bottom is quantified in this assay.

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(1) Cells are grown to approximately 75–80% confluence on plastic dishes precoated with specified matrix components, removed from their plates by treatment with trypsin–EDTA (0.25%), and their concentration is then determined by a cell counter. (2) An equal number of cells is added to the top chamber of the device described above, and cells are stimulated by addition of growth factor, pharmacological agent, or receptor-clustering antibody to the lower chamber. The effect of function-blocking antibodies (preventing ligand–proteoglycan interactions) may also be assayed as an experimental condition. (3) For adhesion studies, the supernatant is aspirated after a specified period of time, and the top chambers are washed three times sequentially with equal force, using PBS or culture medium. Cells adherent to the porous filter or matrix separating both chambers are then fixed by treatment with 4% paraformaldehyde in PBS for 10 min. They are stained with a dye such as crystal violet and quantified by low-power microscopy. Typically, a time course of 20–60 min is used for adhesion studies, although this is highly dependent upon cell type and matrix composition. For studies of cell migration, cells are transferred to the top well and stimulated as described above. At the specified migration endpoint, the upper chamber is aspirated and cells adhering to the upper surface of the membrane/matrix are removed with a cotton swab. Cells in the lower chamber are quantified by a cell counter. Alternatively, if migration has not occurred for long enough for complete invasion into the lower chamber, cells that have migrated through the filter/matrix and remain adherent to the lower aspect of this barrier may be stained with a dye (as above) and quantified by low-power microscopy. (4) Quantification is typically expressed as percent adherent or migrated, as compared to total cell number per sample. Note An alternative method of quantification involves the preincubation of cells with a fluorescent membrane dye. Upon conclusion of the migration assay, the fluorescence of the bottom chamber is measured with a fluorimeter. This value likewise correlates linearly with number of successfully migrated cells.

ACKNOWLEDGMENTS This work was supported by the National Heart, Lung, and Blood Institute (grant HL62289 to M. Simons). The authors thank the members of the Simons laboratory and the Matsuda laboratory (Kyoto University, Japan) for their experimental guidance and expertise.

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Dual Roles of Drosophila Glypican Dally-Like in Wingless/Wnt Signaling and Distribution Yihui Wu,*,† Tatyana Y. Belenkaya,‡ and Xinhua Lin*,†,‡ Contents 1. Introduction 2. Generation of Dally and Dlp Null Alleles 3. Examination of Dally and Dlp in Wg Signaling and Gradient Formation in the Wing Disc 3.1. Loss-of-function experiments show that dally and dlp are required for the proper distribution of extracellular Wg 3.2. The effects of overexpressing Dally and Dlp on extracellular Wg distribution 4. Dlp Core Protein Determines Its Biphasic Activity in Wg Morphogen Signaling 4.1. Generation of Dlp core protein transgenic line 4.2. Expression of Dlp core protein in the wing disc 4.3. Immunofluorescent staining of imaginal discs 5. Dlp Core Protein Can Interact with Wg Independent of Its GAG Chains 5.1. Generation of constructs 5.2. Generation of stable cell lines 5.3. Wg conditioned medium preparation 5.4. Cell-binding experiment 5.5. Immunostaining of cultured S2 cells 5.6. Co-immunoprecipitaton of Dlp or Dlp core protein with Wg-GFP 6. The Ratio of Dlp/Fz2 Determines the Biphasic Activity of Dlp or Dlp Core Protein in Wg Signaling 6.1. Luciferase reporter assay 6.2. Co-immunoprecipitation

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* State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Key Laboratory of Stem Cell, Institute of Zoology, Chinese Academy of Sciences, Beijing, China { Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA {

Methods in Enzymology, Volume 480 ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)80002-3

#

2010 Elsevier Inc. All rights reserved.

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7. Conclusion Acknowledgments References

48 48 49

Abstract Heparan sulfate proteoglycans (HSPGs) are cell-surface and extracellular matrix (ECM) macromolecules that comprise a core protein to which heparan sulfate (HS) glycosaminoglycan (GAG) chains are attached. Glypican is a major family of HSPGs that is linked to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Over the past decade, fruit fly Drosophila has been used as a powerful model system to examine the functions of HSPGs in cell signaling and development. There are two members of Drosophila glypicans named division abnormally delayed (Dally) and Dally-like (Dlp). To study the functions of these two glypicans in development, we have generated the null mutants of dally and dlp. Here, we describe the methods employed to analyze their functions in development with a focus on Dlp in the context of Wingless signaling. Our data suggest that Dlp shows biphasic activity in Wingless/Wnt signaling and distribution.

1. Introduction Heparan sulfate proteoglycans (HSPGs) are cell-surface and extracellular matrix (ECM) macromolecules. They are composed of heparan sulfate (HS) glycosaminoglycan (GAG) chains attached to specific sites on a core protein (Lin, 2004). Glypican is a major family of HSPGs that is linked to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor (Lin, 2004). There are six members of glypicans in mice. Due to the overlapping functions of individual glypican members, it has been difficult to determine the functions of glypicans in development using mouse model. Fruit fly Drosophila shows its advantage in examining the activities of glypicans in cell signaling and development. There are two kinds of glypicans in Drosophila: division abnormally delayed (dally) (Nakato et al., 1995) and dally-like (dlp or dly) (Khare and Baumgartner, 2000). Over the past decade, we have focused our studies on the roles of Dally and Dlp in development by examining their functions in various developmental signaling pathways including Wingless (Wg), Hedgehog (Hh), Decapentaplegic (Dpp; a Drosophila BMP member), and FGF (Lin, 2004). Our analyses demonstrated the essential roles of Dally and Dlp in Wg, Hh, Dpp, and FGF signaling pathways (Lin, 2004). Here, we will describe the methods employed to analyze Dally and Dlp functions in development with a focus on Dally and Dlp in the context of Wg signaling and gradient formation. Our results demonstrated that Dally and Dlp play cooperative and distinct roles in modulating Wg gradient and signaling in development. Importantly, our recent analyses of Dlp provide

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Mechanisms of Dlp in Wg Signaling Distribution

strong evidence that Dlp core protein shows its biphasic activity in Wg signaling and distribution.

2. Generation of Dally and Dlp Null Alleles As a first step to characterize the roles of Dally and Dlp in development, we have generated null alleles of dally and dlp (Fig. 2.1). dally80 is a null mutant generated by P-element-mediated mutagenesis using dallyP1 (Nakato et al., 1995). dally80 contains a deletion from
724 to þ415 (the ATG start codon is designated as 1) that covers the ATG followed by 54 amino acids including the signal peptide as well as part of the first intron (Han et al., 2004). P

ATG

dally80 Deletion –724

54 AAs

+415

Coded by first exon

ATG

dipA187

Deletion of 26 bp

ORF shift from AA205

Signal peptide

5⬘UTR

GPI anchoring signal

3⬘UTR

CDS P-element

GAG Cysteine residue

Figure 2.1 Diagram of dally and dlp null allele mutations. Dally and Dlp proteins both contain a signal peptide, an N-terminal cysteine-rich domain, a GAG attachment domain and a GPI-anchoring signal. dally80 is generated by P-element-mediated excision. dlpA187 contains a reading frame shift from AA 205. dlpA187 disrupts part of cysteine-rich region, all the GAG attachment sites and a GPI-anchor signal, therefore is a null allele.

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dlpA187 is a null allele generated by EMS mutagenesis (Han et al., 2004). dlp contains 26 nucleotides deletion causing open reading frame shift from amino acid 205. dlpA187disrupts part of cysteine-rich region, all the GAG attachment sites and GPI-anchor signal as shown in Fig. 2.1 (Han et al., 2004). dally80–dlpA187 double mutant allele was generated by recombination of dally80 and dlpA187 (Han et al., 2004). A187

3. Examination of Dally and Dlp in Wg Signaling and Gradient Formation in the Wing Disc 3.1. Loss-of-function experiments show that dally and dlp are required for the proper distribution of extracellular Wg Drosophila wing disc is a great system to examine Wg signaling and distribution. In the wing disc, Wg is secreted from the D/V border and acts as a morphogen to form a concentration gradient along D/V axis (Neumann and Cohen, 1997; Nolo et al., 2000; Zecca et al., 1996). This concentration gradient can be detected by an extracellular Wg staining protocol (Baeg et al., 2001; Strigini and Cohen, 2000). Wg can induce the expression of Wg-target genes in a concentration-dependent manner to activate sens expression at a short range, and to activate dll at a long range (Neumann and Cohen, 1997; Nolo et al., 2000; Zecca et al., 1996). To determine whether Dally and Dlp can affect Wg gradient formation, we analyzed the extracellular Wg distribution in wing discs bearing clones mutant for dally and dlp (Fig. 2.2; Han et al., 2005). Large mutant clones were generated by the FLP–FRT method in combination with Minute technique (Xu and Rubin, 1993). Then extracellular Wg staining was performed to determine the roles of Dally and Dlp on Wg distribution. The experimental procedures are described below: (1) Generation of the following genotypes of flies by crossing dally80, dlpA187, or dally80–dlpA187 flies with flies of genotype y w hsp70-flp; hsp70-Myc-GFP M(3)i55 FRT2A y w hsp70-flp/þ or Y; hsp70-Myc-GFP M(3)i55 FRT2A/dally80 FRT2A y w hsp70-flp/þ or Y; hsp70-Myc-GFP M(3)i55 FRT2A/dlpA187 FRT2A y w hsp70-flp/þ or Y; hsp70-Myc-GFP M(3)i55 FRT2A/dally80-dlpA187 FRT2A (2) Induction of the clones of mutant cells and extracellular Wg staining in the wing disc: The mutant clones were induced by exposing first- or second-instar larvae to a heat-shock at 37  C for 1 h. Several days after heat-shock, third-instar larvae were dissected in ice-cold PBS and put into ice-cold M3 medium (Sigma) in flat-bottom tube. Discs were just immerged in pre-cold

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dally A

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E

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Figure 2.2 Dally and Dlp are required for the proper distribution of extracellular Wg. (A–C0 ) Extracellular Wg staining in discs bearing large clones mutant for dally. The yellow arrows indicate the wild-type cells that have reduced extracellular Wg level. (D–E0 ) Extracellular Wg staining in discs bearing large clones mutant for dlp. Within the clones, extracellular Wg is reduced outside of a zone that is 7–10 cell diameters wide and centered at the D/V boundary. (F–G0 ) Extracellular Wg staining in discs bearing large clones mutant for both dally and dlp. All wing discs are oriented dorsal topright, anterior top-left. All mutant clones are marked by the absence of GFP and outlined by broken lines. Red, extracellular Wg.

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primary Wg antibody (mouse anti-Wg 4D4 at 1:3; Developmental Studies Hybridoma Bank, DSHB), and incubate in ice water. 1 h later, discs were washed three times quickly with ice-cold PBS (phosphate-buffered saline, pH 7.4) and fixed with 3.7% formaldehyde/PBS for 17–20 min at room temperature while shaking. Then, 15 min incubation was performed for four times in PBST (PBS with 0.1% Triton X-100). After blocking for 15 min in block solution PBST/5% NHS (PBST with 5% normal horse serum (NHS)), the discs were incubated with secondary antibody (rabbit anti-GFP Alexa Fluor 488 at 1:1000 Molecular Probes and Goat anti-mouse Cy3 at 1:400) in PBST/5% NHS for 1.5 h. After being washed with PBST, the discs were mounted in DABCO solution (2.2% DABCO (Sigma) in 70.0% glycerol) for observation. The data is analyzed by using Zeiss LSM510 confocal laser scanning microscope. Results indicate that loss of dally or dlp activity in the wing disc both lead to reduction of extracellular Wg levels (Fig. 2.2A–E0 ), However, the Wg gradient defects associated with dally and dlp mutant clones are noticeably different. In dally clones (Fig. 2.2A–C0 ), the defect is rather cell nonautonomous because extracellular Wg levels reduced gradually close to the D/V boundary between wild-type cells and dally mutant cells. In dlp mutant clones (Fig. 2.2D–E0 ), the reduction of extracellular Wg level is only obvious when the mutant cells are far away from the D/V boundary. Moreover, the extracellular Wg levels in double mutant clone dally80–dlpA187 have a stronger reduction of the extracellular Wg than in dally or dlp clones (Fig. 2.2F–G0 ). On the basis of these data, we suggest that Dally and Dlp have both distinct and overlapping roles in shaping the Wg gradient (Han et al., 2005).

3.2. The effects of overexpressing Dally and Dlp on extracellular Wg distribution (1) Expression of dally and dlp in P compartment by enGal4 or hhGal4. WT control: enGal4-UASGFP dlp: tub1a-Gal80ts/UAS-dlp; hhGal4/þ dally: hhGal4/UAS-dally Previous studies and our aforementioned loss-of-function data suggest that Dally and Dlp may have differential intrinsic abilities to influence Wg distribution. To examine further this possibility, we expressed Dally and Dlp in the posterior (P) compartment of the wing disc by using enGal4 or hhGal4 drivers, and then compared the extracellular Wg distribution in the anterior (A) and P compartments. To eliminate the problem of deleterious effect of early induction of UAS-dlp, we used a temperature-sensitive allele of Gal80 (Gal80ts) to keep Gal4 inactive until the late stage of larval development. Gal80ts functions as a repressor of Gal4 at the permissive temperature (19  C) but allows Gal4 to be active at the nonpermissive temperature (30  C)

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(McGuire et al., 2003). Extracellular Wg staining is the same as mentioned above. The genotypes of flies generated for dally and dlp overexpressing analyses are: (2) Image processing and evaluation of Wg levels in cells expressing dally and dlp To measure the level of the extracellular Wg, the plot values were measured from selected regions in Image J [http://rsb.info.nih.gov/ij/], and then used to generate plot profiles in Microsoft Excel. For each experiment, plot profiles were generated from at least three discs and similar results were obtained (Han et al., 2005). Overexpression of dally or dlp results in dramatic difference on extracellular Wg gradient distribution. In the wild-type disc, the patterns of the extracellular Wg gradient are virtually identical in the A and P compartments (Fig. 2.3A–A00 ). When UAS-dlp is induced by hhGal4 for 24 h at the nonpermissive temperature (30  C) in the presence of Gal80ts, extracellular Wg is significantly increased on the surface of dlp overexpressing cells and the Wg gradient extends to the whole wing pouch along the A/P axis (Fig. 2.3B, B0 ). The plot profile suggests that the Wg gradient becomes broader in the P compartment (Fig. 2.3B00 ). However, consistent with previous results (Strigini and Cohen, 2000), overexpression of dally by enGal4 causes only mild alteration of extracellular Wg levels (Fig. 2.3C, C0 ). In summary, our loss-of-function studies and overexpression experiments demonstrated that Dally and Dlp play cooperative but distinct roles in modulating Wg gradient and signaling (Han et al., 2005). As dally mutant exhibits wing margin defects and shows genetic interactions with Wg signaling components (Lin and Perrimon, 1999), our analyses as well as studies from other argue that Dally plays a positive role in Wg signaling (Franch-Marro et al., 2005; Fujise et al., 2001; Han et al., 2005; Lin and Perrimon, 1999). However, the data suggested that Dlp plays dual roles in Wg signaling and distribution. Dlp suppressed high levels of Wg signaling, but enhanced low levels (Baeg et al., 2001, 2004; Franch-Marro et al., 2005; Han et al., 2005; Kirkpatrick et al., 2004; Kreuger et al., 2004; Lin and Perrimon, 1999).

4. Dlp Core Protein Determines Its Biphasic Activity in Wg Morphogen Signaling We further investigated the mechanisms of dual functions of Dlp in Wg gradient formation. We hypothesized that Dlp core protein may be involved in its dual activities in Wg signaling and distribution. In the following section,

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A

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Anterior compartment

Posterior compartment

hhGal4 Gal80ts UAS-DIp 24 h HS

WT

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enGal4 UAS-dally

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enGal4 UAS-dally

Figure 2.3 The effects of overexpressing Dally and Dlp on extracellular Wg distribution. Extracellular Wg staining in a wild-type wing disc (A, A0 ), and discs overexpressing Dlp (B, B0 ) and Dally (C, C0 ) in the posterior compartment. The posterior compartment is marked by either GFP (green in A0 and C0 ) or the absence of Ci (green in B0 ). Signal profiles of extracellular Wg from selected areas in the anterior and posterior compartments of each wing disc are plotted and compared (A00 , B00 , C00 ). In the inset in each plot profile, the areas used for analysis are shown in box. The wing discs are oriented dorsal top-right, anterior top-left. The left–right axis of the plot profiles corresponds to the dorsoventral axis of the boxed regions.

we will describe the methods employed to examine the activities of Dlp core protein in Wg signaling and distribution (Yan et al., 2009).

4.1. Generation of Dlp core protein transgenic line To examine the roles of Dlp core protein in Wg signaling and distribution, we established Dlp core protein ((dlp(-HS)) transgenic line. The construct pUASTdlp(-HS) was generated using Invitrogen’s gene-tailor mutagenesis kit.

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All five potential GAG attachment sites, Ser625, Ser629, Ser631, Ser643, and Ser686 were converted to Ala. Therefore, Dlp(-HS) will not contain any GAG chains (Yan et al., 2009).

4.2. Expression of Dlp core protein in the wing disc To examine the activity of Dlp core protein in Wg signaling and gradient formation, we expressed UAS-dlp and UAS-dlp(-HS) in the P compartment of wing disc using enGal4. Both wild-type and dlpA187 wing discs are used as controls.

4.3. Immunofluorescent staining of imaginal discs To determine Wg levels in cells expressing Dlp core proteins, third-instar larvae were dissected in ice-cold PBS in the 60 mm Petri dish lid. The dissected discs were fixed in 4% formaldehyde for 20 min. After 15 min incubation for three times in PBST (PBS with 0.1% Triton X-100) with shaking, the discs were pre-blocked for 15 min in PBST/5% NHS (PBST with 5% NHS). Then, discs were immerged in 50 ml primary antibody diluted in PBST/5% NHS. After shaking for 1.5–2 h at room temperature, 15 min incubation in PBST with shaking was performed for three times. Then, the discs were preblocked and the secondary antibody was added and incubated for 1.5 h. After another washing in PBST, discs were mounted in a drop of DABCO solution on the glass slide for observation. The primary antibodies were used at the following dilutions: mouse antiDll 1:50 (Duncan et al., 1998), guinea pig anti-Sens 1:50 (Nolo et al., 2000). The results of above experiments are shown in Fig. 2.4. In dlp homozygous mutant discs, the region of sens expression is broadened, while the range of dll expression is significantly reduced (Fig. 2.4D–D00 ). Overexpression of dlp and dlp core protein in the P compartment of the disc eliminates sens expression, but expands the dll expression range (Fig. 2.4E–E00 ). Although the dll expression range is enhanced, the dll expression level is reduced in areas close to the D/V boundary (Fig. 2.4E0 ). These results suggest that Dlp acts as a positive cofactor to enhance Wg signaling activity in areas distant from the Wg source, while it acts as a negative cofactor to suppress Wg signaling in areas close to the Wg source (Franch-Marro et al., 2005; Hufnagel et al., 2006; Kirkpatrick et al., 2004; Kreuger et al., 2004). As the Dlp core protein (Dlp(-HS)) generates virtually identical effects on Wg signaling and gradient formation, we concluded that Dlp protein core is essential for its biphasic activity in Wg signaling (Yan et al., 2009).

A

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enGal4, UAS-dlp

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Figure 2.4 Dlp core protein has biphasic activity in Wg signaling. (A) Major Dlp constructs used in this study. (B) (C–C00 ) sens (C) and dll (C0 ) expression were analyzed by antibody staining in wild-type wing discs. (D–D00 ) sens (D) and dll (D0 ) expression in dlp homozygous mutant discs. The domain of sens expression is broadened and the domain of dll expression is significantly narrowed. Wing imaginal discs in all the figures are oriented anterior to the up and dorsal to the left except in Figures 2.2 and 2.3. (E– F00 ) Expression of Dlp (E–E00 ) or Dlp(-HS) (F–F00 ) in the P compartment (bottom part of the broken line) by enGal4 diminishes sens expression (E, F) and expands dll expression domain (E0 , F0 ). Modified, reproduced with permission from Yan et al. (2009).

5. Dlp Core Protein Can Interact with Wg Independent of Its GAG Chains Since Dlp core protein exhibits its biphasic activities in Wg signaling, we hypothesized that Dlp core protein might interact with Wg to regulate

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Wg signaling and distribution. To test this, we examined the interaction between Wg and Dlp (or Dlp core protein) by performing the cell-binding and co-immunoprecipitation experiments (Yan et al., 2009).

5.1. Generation of constructs pUAST-dlp(-HS)-GFP was generated by inserting the PCR amplified GFP fragment from pEGFP-N1 (Clontech) at a unique NdeI site in dlp(-HS). This insertion site is the same as in Dlp-GFP (Baeg et al., 2004). GFP–GPI contains EGFP sequence followed by the GPI signal from Dlp (amino acids 695–765).

5.2. Generation of stable cell lines Drosophila S2 cells were maintained at 25  C in HyQ SFX-INSECT cell culture medium (Hyclone SH30278.01). Drosophila S2-Act-Wg-puro cell line was established by transfected pAC-wg and pBS-puro in ratio of 1:10 in Drosophila S2 cell and selected with 10 mg/ml Puromycin. For about 1 month, the cells grow healthily and the cell line was established. This cell line is maintained at 25  C in HyQ SFX-INSECT medium (Hyclone) with 10 mg/ml Puromycin.

5.3. Wg conditioned medium preparation To collect adequate conditioned medium, Drosophila S2-Act-Wg-puro cells were amplified in 10 mm dishes with HyQ SFX-INSECT medium supplied with 10 mg/ml Puromycin. When cells reached the exponential phase of growth, the medium was removed completely and the fresh drug-free medium was added. After 24–30 h, the medium was collected and centrifuged at 5000g for 10 min. Then the supernatant was 10 concentrated with Amicon Ultra-15 Centrifugal Filter (10 kDa).

5.4. Cell-binding experiment Firstly, Drosophila S2 cells were seeded in 3.5 mm dishes at 70% confluency. 16 h later, cells were transfected with dlp-GFP, dlp(-HS)-GFP, and GFP– GPI expression vectors using Effectene (Qiagen). Twenty-four hours after transfection, the cells were reseeded onto the 0.5% poly-D-lysine pretreated cover slips. After another 24 h-growth, the cells were incubated in pre-cold Wg conditioned medium for 3 h on ice. Then, the cells were fixed and stained with anti-Wg and anti-GFP as indicated in the Fig. 2.5 (Bhanot et al., 1996; Franch-Marro et al., 2005).

A

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IP: GFP IB: Wg

Figure 2.5 Dlp core protein can interact with Wg in vitro and in vivo. (A–C0 ) Transfection of dlp-GFP, dlp(-HS)-GFP or GFP–GPI in S2 cells causes accumulation of exogenous Wg at the cell surface. Notice that dlp-GFP expression cells accumulate more Wg than dlp (-HS)-GFP expression cells (A0 , B0 ).The control cells transfected with GFP–GPI plasmid do not cause Wg accumulation at the cell surface. (C) Transfected cells are recognized with anti-GFP (A–C) anti-Wg(A0 –C0 ), respectively. (D) Wg can co-immunoprecipitate with Dlp core protein. Top and middle panels: S2 cells were transfected with indicated expression vectors, and cell lysates were immunoprecipitated (IP) and analyzed by Western blotting with the antibodies indicated. Dlp forms a smear typical of heparan sulfate proteoglycans, while Dlp(-HS) displays a sharp band in protein gel indicating it is a nonglycanated form. Note that more Wg-GFP is coprecipitated with Dlp than Dlp(-HS)(arrow). Modified, reproduced with permission from Yan et al. (2009).

5.5. Immunostaining of cultured S2 cells Cells grown on cover slips were rinsed two times with PBS and then fixed in 3.7% formaldehyde in PBS for 10 min. Then, the cells were incubated in PBS for 5 min. The cells were permeabilized in PBST (PBS þ 0.1% Triton X-100) for 7 min at room temperature. After being rinsed and washed with

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PT (PBS þ 0.1% Tween-20), cells were blocked in block solution (PT þ 5% Normal Serum) at room temperature for 15 min. Then the cells were incubated with 100–200 ml primary antibody (mouse anti-Wg 4D4 1:3; DSHB) diluted in block solution 1 h at room temperature. In order to avoid drying out, the staining is carried out in a humidified chamber. The cover slips are incubated in antibody solution by facing down on a piece of parafilm. After being rinsed two times and 10-min incubation for three times in PT, cells were incubated with secondary antibody (Donkey anti-mouse Cy3 1:400, Jackson Immuno Research Laboratories, Inc.; rabbit anti-GFP Alexa Fluor 488 1:800 Molecular Probes) diluted in block solution for 1 h at room temperature. Then cells were washed two times rinsed in PT and washed three times for 10 min. After that, the cover slips with cells were mounted facing down on a drop of mounting medium for observation.

5.6. Co-immunoprecipitaton of Dlp or Dlp core protein with Wg-GFP S2 cells were transfected in 100 mm dishes with 4 mg total DNA at 80% confluency, including pUAST-dlp (or other dlp construct), pAc-wg-GFP, and pArmadilloGal4. Cells were harvested 48 h later and lysed in 900 ml lysis buffer containing 150 mM NaCl, 20 mM Tris–HCl (pH 7.5), 1% Triton X100, 1 mM EDTA, 1% bovine serum albumin (BSA), and 10 ml/proteinase inhibitor tablet (Roche Molecular Biochemicals) on ice for 40 min with occasional stirring. After preclearance with protein G Sepharose 4 Fast Flow (Amersham) beads for 1 h at 4  C, each lysate sample was divided into two parts and incubated with primary antibodies for 4 h at 4  C and for an additional 2 h in the presence of 25 ml of beads. Then, the beads were washed once with 1 ml of lysis buffer and four times with 1 ml of 150 mM NaCl, 20 mM Tris–HCl (pH 7.5), 0.2% Triton X-100, 1 mM EDTA for 10 min at 4  C. Material bound to the beads was eluted by boiling in sample loading buffer, and resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 7.5% gels under reducing conditions, and then transferred onto PVDF membrane (BioRad) and blocked with 5% nonfat dry milk, and then analyzed by Western blotting using the ECL detection system (Pierce). The antibodies for precipitation are guinea pig anti-GFP (made in our lab) and guinea pig anti-Dlp (made in our lab). The primary antibodies for protein blot are mouse anti-Wg 4D4 (DSHB) and guinea pig (GP) anti-Dlp (made in our lab). The secondary antibodies are HRP-conjugated goat anti-GP immunoglobulin G (IgG) and HRP-conjugated goat anti-mouse IgG, light chain ( Jackson Immuno Research Laboratories, Inc.) (Belenkaya et al., 2002). The experiments mentioned above demonstrated that Dlp’s core protein determines its major and specific activity in Wg signaling. Dlp core protein

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shows biphasic activity for both short- and long-range signaling similar to that of wild-type Dlp. Dlp core protein can also interact with Wg in both co-IP experiment and cell-binding assay. However, our experiments suggest that the attached HS GAG chains on Dlp can increase the binding affinities between Dlp and Wg (Fig. 2.5) (Yan et al., 2009).

6. The Ratio of Dlp/Fz2 Determines the Biphasic Activity of Dlp or Dlp Core Protein in Wg Signaling We further examined the mechanisms by which the biphasic activities of Dlp and Dlp core protein in Wg signaling and distribution. We hypothesized that the Dlp/Fz2 ratio is essential for Dlp’s biphasic activity. We performed the co-IP experiment and Luciferase reporter assay to test our hypothesis.

6.1. Luciferase reporter assay 5  104 S2 cells were transfected in 24-well plates by using Effectene transfection reagent (Qiagen). In each well, 450 ng of total DNA was added, including 12 dTOP luciferase reporter, PolIII-RL normalization vector (DasGupta et al., 2005), pUAST-fz2-V5, and pUAST-dlp or pUASTdlp(-HS). The amounts of fz2-V5 and dlp plasmids are indicated in the figures. After 48 h, concentrated Wg conditioned medium was applied on cells for additional 20 h. Cells were then lysed and luciferase activities were measured using Dual-Luciferase Assay Kits (Promega).

6.2. Co-immunoprecipitation S2 cells were transfected in 100 mm dishes with 6 mg total DNA by using Effectene transfection reagent (Qiagen), including pUAST-fz2-V5, pUAST-dlp/pUAST-dlp(-HS), pAc-wg-GFP, and pArmadilloGal4. The ratio of dlp/dlp(-HS) to fz2-V5 are indicated in the figures. Cells were harvested 60 h later and lysed in 900 ml of 150 mM NaCl, 20 mM Tris–HCl (pH 7.5), 2% Triton X-100, 1 mM EDTA plus proteinase inhibitors (Roche) on ice for 1 h. The subsequent steps are the same as in methods described before. The primary antibodies used for IP and Western blot were guinea pig antiDlp (made in our lab), rabbit anti-V5 (Sigma), guinea pig anti-GFP (made in our lab), mouse anti-V5 (Invitrogen), mouse anti-Wg (4D4, DSHB). Our results are shown in Fig. 2.6. The biphasic activity of Dlp and Dlp core protein depends on the Dlp/Fz2 ratio, with a low level of Dlp increasing Wg signaling reporter activity and a high level of Dlp reducing

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Figure 2.6 Fz2/Dlp ratio determines Dlp’s biphasic activity in Wg signaling. (A, B) S2 cells were transfected with the 12 dTOP-Luciferase reporter, the Renilla normalization vector, 20 ng Fz2 expression plasmids, variable amount of Dlp, or Dlp(-HS) expression plasmids and then incubated with Wg conditioned medium. (A) The columns represent luciferase activities in the absence of Dlp or in the presence of Dlp with the dlp/fz2 DNA ratio of 1, 2, 4, and 8 as indicated. (B) Luciferase activities in the absence of Dlp(-HS) or in the presence of Dlp(-HS) with the dlp(-HS)/fz2 DNA ratio of 2, 4, 8, and 16 as indicated. While a low amount of Dlp or Dlp(-HS) enhances Wg signaling, a high amount of Dlp/Dlp(-HS) inhibits Wg signaling. The error bars represent standard deviations. (C, D) Fixed amount of Wg-GFP, Fz2-V5, and variable amount of Dlp or Dlp(-HS) expression vectors were transfected individually or together into S2 cells. Top three panels: cell lysates were immunoprecipitated and analyzed by Western blotting with the antibodies indicated. Bottom panel: The amount of Wg-GFP in 5% of cell lysates input was assessed by Western blot. A low level of Dlp or Dlp(-HS) helps Fz2 pull down more Wg, but a high level of Dlp or Dlp(-HS) reduces Wg coprecipitated by Fz2 (arrows). Note that the total amount of Wg in the lysates increases as more Dlp/Dlp(-HS) was added, probably reflecting its ability to stabilize Wg. Also Dlp was not found coprecipitated with Fz2, suggesting that Dlp does not form a stable complex with Fz2 as a coreceptor. (E, F) Dlp’s biphasic curve changes in different Wg and Fz2 concentrations. (E) S2 cells were transfected with the

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its activity. Co-IP experiments show that a low amount of Dlp provides Wg for Fz2 receptor, while a high amount of Dlp sequesters the Wg ligand. Moreover, we found that for a constant amount of Dlp, it is more likely to repress Wg signaling in high Wg concentration, but to promote signaling in low Wg concentration. On the contrary, Dlp is more likely to promote Wg signaling in high Fz2 concentration, but to repress signaling in low Fz2 concentration.

7. Conclusion Drosophila Dally and Dlp show overlapping and distinct activities in shaping the extracellular Wg gradient. While Dally plays a positive role in Wg signaling likely acting as a coreceptor, Dlp exhibits biphasic activities in Wg signaling and distribution. Our detailed analyses demonstrated that the core protein of Dlp plays a major roles the biphasic activity of Dlp in Wg signaling and distribution. The ratio of Dlp/Fz2 determines its biphasic activity in cell culture and in the wing disc. While a low ratio of Dlp/Fz2 can help Fz2 obtain more Wg, a high ratio of Dlp/Fz2 prevents Fz2 from capturing Wg. On the basis of our data, we argue that the main activity of Dlp in Wg signaling is to retain Wg on the cell membrane and mediate the exchange of Wg between receptors and itself. The net flow of the ligand depends on the ratios of the ligand, receptor, and Dlp.

ACKNOWLEDGMENTS We thank Guolun Wang for comments on the manuscript. We thank G. Baeg, R. Cohen, R. Nusse, J. Vincent, and the Bloomington Stock Center for Drosophila stocks; H. Bellen, S. Cumberledge, and the Iowa Developmental Studies Hybridoma Bank (DSHB) for antibodies. This work was supported partially by a NIH grant (2R01 GM063891), American

Luciferase reporter, the normalization vector, 20 ng Fz2 expression plasmids, variable amount of Dlp expression plasmids as indicated, and then incubated with two different concentrations of Wg conditioned medium. High Wg conditioned medium is 10 times concentrated than low Wg medium. In low Wg condition, data are plotted on the right secondary axis. In high Wg condition, the biphasic point shifts to the left. (F) S2 cells were transfected with the Luciferase reporter, the normalization vector, 10 or 60 ng Fz2 expression plasmids, variable amount of Dlp expression plasmids as indicated, and then incubated with Wg conditioned medium. In high Fz2 condition, the biphasic point shifts to the right. The error bars represent standard deviations. Reproduced with permission from Yan et al. (2009).

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Cancer Society (RSG-07-051), and the Knowledge Innovation Program of the Chinese Academy of Sciences KSCX2-YW-R-263 and KSCX2-YW-R-240.

REFERENCES Baeg, G. H., et al. (2001). Heparan sulfate proteoglycans are critical for the organization of the extracellular distribution of Wingless. Development 128, 87–94. Baeg, G. H., et al. (2004). The Wingless morphogen gradient is established by the cooperative action of Frizzled and Heparan Sulfate Proteoglycan receptors. Dev. Biol. 276, 89–100. Belenkaya, T. Y., et al. (2002). pygopus Encodes a nuclear protein essential for wingless/Wnt signaling. Development 129, 4089–4101. Bhanot, P., et al. (1996). A new member of the frizzled family from Drosophila functions as a Wingless receptor. Nature 382, 225–230. DasGupta, R., et al. (2005). Functional genomic analysis of the Wnt-wingless signaling pathway. Science 308, 826–833. Duncan, D. M., et al. (1998). Control of distal antennal identity and tarsal development in Drosophila by spineless-aristapedia, a homolog of the mammalian dioxin receptor. Genes Dev. 12, 1290–1303. Franch-Marro, X., et al. (2005). Glypicans shunt the Wingless signal between local signaling and further transport. Development 132, 659–666. Fujise, M., et al. (2001). Regulation of dally, an integral membrane proteoglycan, and its function during adult sensory organ formation of Drosophila. Dev. Biol. 235, 433–448. Han, C., et al. (2004). Drosophila glypicans control the cell-to-cell movement of Hedgehog by a dynamin-independent process. Development 131, 601–611. Han, C., et al. (2005). Drosophila glypicans Dally and Dally-like shape the extracellular Wingless morphogen gradient in the wing disc. Development 132, 667–679. Hufnagel, L., et al. (2006). On the role of glypicans in the process of morphogen gradient formation. Dev. Biol. 300, 512–522. Khare, N., and Baumgartner, S. (2000). Dally-like protein, a new Drosophila glypican with expression overlapping with wingless. Mech. Dev. 99, 199–202. Kirkpatrick, C. A., et al. (2004). Spatial regulation of Wingless morphogen distribution and signaling by Dally-like protein. Dev. Cell 7, 513–523. Kreuger, J., et al. (2004). Opposing activities of Dally-like glypican at high and low levels of Wingless morphogen activity. Dev. Cell 7, 503–512. Lin, X. (2004). Functions of heparan sulfate proteoglycans in cell signaling during development. Development 131, 6009–6021. Lin, X., and Perrimon, N. (1999). Dally cooperates with Drosophila Frizzled 2 to transduce Wingless signalling. Nature 400, 281–284. McGuire, S. E., et al. (2003). Spatiotemporal rescue of memory dysfunction in Drosophila. Science 302, 1765–1768. Nakato, H., et al. (1995). The division abnormally delayed (dally) gene: A putative integral membrane proteoglycan required for cell division patterning during postembryonic development of the nervous system in Drosophila. Development 121, 3687–3702. Neumann, C. J., and Cohen, S. M. (1997). Long-range action of Wingless organizes the dorsal-ventral axis of the Drosophila wing. Development 124, 871–880. Nolo, R., et al. (2000). Senseless, a Zn finger transcription factor, is necessary and sufficient for sensory organ development in Drosophila. Cell 102, 349–362. Strigini, M., and Cohen, S. M. (2000). Wingless gradient formation in the Drosophila wing. Curr. Biol. 10, 293–300.

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Xu, T., and Rubin, G. M. (1993). Analysis of genetic mosaics in developing and adult Drosophila tissues. Development 117, 1223–1237. Yan, D., et al. (2009). The core protein of glypican Dally-like determines its biphasic activity in wingless morphogen signaling. Dev. Cell 17, 470–481. Zecca, M., et al. (1996). Direct and long-range action of a wingless morphogen gradient. Cell 87, 833–844.

C H A P T E R

T H R E E

Use of a Phage Display Antibody to Measure the Enzymatic Activity of the Sulfs Kenji Uchimura,* Hassan Lemjabbar-Alaoui,† Toin H. van Kuppevelt,‡ and Steven D. Rosen† Contents 1. Overview 2. ELISA for Sulf Activity Against the RB4CD12 Epitope in Immobilized Heparin/HS 2.1. Materials 2.2. Method 3. Flow Cytometry-Based Sulf Assay Against the Cell Surface RB4CD12 Epitope 3.1. Materials 3.2. Method 4. Ex Vivo Sulf Assay Against the RB4CD12 Epitope in Cryostat-Cut Sections 4.1. Materials 4.2. Method Acknowledgments References

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Abstract Sulf-1 and Sulf-2 are extracellular endoglucosamine 6-sulfatases, which selectively liberate the 6-O-sulfate groups on glucosamines present in N, 6-O, and 2-O trisulfated disaccharides of intact heparan sulfate (HS)/heparin chains. The Sulfs are known to regulate signaling of heparin/HS-binding protein ligands, such as morphogens and growth factors, presumably through their ability to decrease the association between the ligands and HS proteoglycans.

* Section of Pathophysiology and Neurobiology, National Center for Geriatrics and Gerontology, Aichi, Japan Department of Anatomy, University of California, San Francisco, California, USA { Department of Biochemistry 280, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands {

Methods in Enzymology, Volume 480 ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)80003-5

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These enzymes serve important roles in development and are dysregulated in many cancers. We previously described arylsulfatase and endoglucosamine 6sulfatase assays for the Sulfs. RB4CD12 is a phage display anti-HS antibody. N-sulfation, 2-O-sulfation, and 6-O-sulfation are involved in its binding. In this chapter, we describe the application of RB4CD12 in ELISA, flow cytometry, and immunohistochemistry assays to measure the enzymatic activity of the Sulfs. These newly established methods should facilitate further investigation of the Sulfs in vitro and in vivo.

1. Overview Sulfatases are enzymes that catalyze the hydrolysis of sulfate esters of complex macromolecules such as glycosaminoglycans and sulfatides (DiezRoux and Ballabio, 2005). The Sulfs are the most recently identified members of this family. The field began with the discovery of QSulf-1 in quail (Dhoot et al., 2001) and its rodent and human orthologs (MorimotoTomita et al., 2002; Ohto et al., 2002). After the identification of QSulf-1, the closely related Sulf-2 was cloned and characterized (Ai et al., 2006; Morimoto-Tomita et al., 2002; Nagamine et al., 2005). Although the best understood of the eukaryotic sulfatases are localized in lysosomes where they participate in catabolism, the Sulfs are secreted into the extracellular space or anchored on the cell surface. Each Sulf contains a signal peptide and two sulfatase-related domains which are separated by a large hydrophilic domain (Ai et al., 2006; Frese et al., 2009; Morimoto-Tomita et al., 2002). Posttranslational modification with formylglycine (Cosma et al., 2003; Dierks et al., 2003) and N-linked glycans (Ambasta et al., 2007; Morimoto-Tomita et al., 2002) are required for Sulf activity. Human Sulf-1 and Sulf-2 are processed by furin-like endoproteases and form disulfide-bond-linked heterodimers of 75 and 50 kDa polypeptides (Morimoto-Tomita et al., 2002; Tang and Rosen, 2009). This processing may modulate the Sulfs’ localization in specialized membrane microdomains (Tang and Rosen, 2009). Both Sulf-1 and Sulf-2 remove sulfate groups on C-6 positions of glucosamines in [–IdoA(2-OSO3)GlcNSO3(6-OSO3)–], trisulfated disaccharides of heparin/heparan sulfate (HS) glycosaminoglycan chains (Ai et al., 2003; Morimoto-Tomita et al., 2002; Saad et al., 2005; Viviano et al., 2004) as well as having arylsulfatase activity against a pseudosubstrate (Morimoto-Tomita et al., 2002). The Sulfs are thus endosulfatases, which remove sulfate esters from glucosamine within the appropriate contexts of heparin and HS chains (Ai et al., 2003; Dai et al., 2005; Morimoto-Tomita et al., 2002). Lysosomal sulfatases are, in contrast, exosulfatases, which remove sulfate esters from the nonreducing termini of sugars. In further contrast to lysosomal sulfatases which function

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at acidic pH’s, the Sulfs demonstrate optimal enzymatic activity in the neutral pH range (Morimoto-Tomita et al., 2002). HS proteoglycans (HSPGs) consist of a restricted set of core proteins that are covalently modified by HS chains. HSPGs are critical in many biological processes through their ability to interact with a multiplicity of protein ligands, including growth factors, morphogens, cytokines, proteases, apolipoproteins, and matrix proteins (Bishop et al., 2007). These interactions, which regulate the localization and biological activities of the ligands, are mediated mainly via internal subdomains of the HS chains (Bernfield et al., 1999; Gallagher, 2001). HS sequences are highly variable, but are controlled in a tissue- and developmental stage-specific manner (Nakato and Kimata, 2002). With their ability to edit the glucosamine 6-O-sulfate status of intact chains, the Sulfs provide a novel postsynthetic mechanism to dynamically regulate HS sequences and thereby regulate signaling of heparin/HS-binding protein ligands. Thus far, Sulf effects have been documented for signaling of Wnts (Dhoot et al., 2001; Lemjabbar-Alaoui et al., 2010; Nawroth et al., 2007; Tang and Rosen, 2009), BMP (Viviano et al., 2004), FGF-2 (Lai et al., 2003; Li et al., 2005; Narita et al., 2006; Wang et al., 2004), HGF (Lai et al., 2004a,b), TGF-b (Yue et al., 2008), and GDNF (Ai et al., 2007). It has been reported that HSulf-1 possesses tumor suppressor function (Lai et al., 2008a), and that HSulf-2 is pro-oncogenic in several cases (Lai et al., 2008b; Lemjabbar-Alaoui et al., 2010; Nawroth et al., 2007). Sulf-2 has also been shown to be proangiogenic, presumably through its ability to reverse the association between angiogenic factors and heparin/HSPGs (Morimoto-Tomita et al., 2005; Uchimura et al., 2006a). Furthermore, studies of quail, Xenopus, and sea urchin embryos (Dhoot et al., 2001; Freeman et al., 2008; Fujita et al., 2009) and of Sulf-deficient mice have demonstrated developmental roles for the Sulfs (Ai et al., 2007; Holst et al., 2007; Kalus et al., 2008; Lamanna et al., 2006; Lum et al., 2007; Ratzka et al., 2008). The defects in double-null mice are most severe than in single knockouts, consistent with overlapping and redundant functions for the enzymes in development (Ai et al., 2007; Holst et al., 2007; Lamanna et al., 2006). Antibodies against HS have been established as useful tools to evaluate the expression and localization of HS in cultures and tissues (David et al., 1992; van den Born et al., 2005). The HS epitopes of recently developed phage display antibodies have been defined using derivatives of HS and heparins (van Kuppevelt et al., 1998). RB4CD12, one of the phage display antibodies, recognizes N- and O-sulfated saccharides of HS/heparin (Dennissen et al., 2002; Jenniskens et al., 2000). N-sulfation, 2-O-sulfation, and 6-O-sulfation are involved in its binding (Jenniskens et al., 2002). Establishing the direct detection of Sulf activity by utilizing antibodies specific for HS sequences has been greatly desirable to facilitate investigation of the Sulfs (Hossain et al., 2010). Here, we describe RB4CD12-based

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assays which measure the enzymatic action of the Sulfs: on immobilized heparin/HS in ELISAs; on the cell surface of transfected cells; on cancer cells with or without RNAi knockdown of Sulf-2; and on the extracellular matrix of murine brain vessels.

2. ELISA for Sulf Activity Against the RB4CD12 Epitope in Immobilized Heparin/HS 2.1. Materials Recombinant HSul-1 and HSulf-2 are expressed and prepared as described (Hossain et al., 2010; Tang and Rosen, 2009; Uchimura et al., 2006a). A native form of HSulf-2 can be obtained from MCF-7 cells and HS66T cells. Albumin from bovine serum (BSA, further purified, pH 7, suitable for diluent in ELISA applications) and heparin–BSA are from Sigma. HS conjugated with BSA was prepared as described previously (Uchimura et al., 2006b). Heparin–BSA or HS–BSA is dissolved at 1 mg/ml in PBSþ (PBS with Ca2þ and Mg2þ), 0.01% NaN3 and stored at 4  C. Blocking reagent: 3% BSA, 0.01% NaN3 in PBS (PBS without Ca2þ and Mg2þ) filtered by a 0.45-mm filter and stored at 4  C. Wash buffer: 0.1% Tween-20 in PBS (PBS-T). A 96-well polystyrene ELISA plate (Immulon 2HB, DYNEX Technologies, Chantilly, VA). Phage display-derived RB4CD12 anti-HS antibody (also known as HS3A8) and MPB49 non-anti-HS antibody were produced in a VSVtag version and purified as described previously (Dennissen et al., 2002). Polyclonal rabbit anti-VSV-G antibody was from Bethyl Laboratories (Montgomery, TX). Alkaline phosphatase-conjugated polyclonal goat antirabbit IgG (H þ L) is from Jackson Immuno Research Laboratories (West Grove, PA). ImmunoPure PNPP tables (p-nitrophenyl phosphate, Pierce, Rockford, IL) are dissolved in an ice-cold solution of diethanolamine buffer (Pierce). Tablets of PNPP are freshly dissolved in each assay and kept on ice until use. A microplate reader (Model 680) from Bio-Rad Laboratories (Hercules, CA).

2.2. Method 1. In order to immobilize heparin or HS, 100 ng/ml of heparin–BSA or 1 mg/ml of HS–BSA in PBS is added to the wells (100 ml/well) of a 96well ELISA plate (Immulon 2HB). The plate is placed at 4  C overnight.

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2. The wells are washed three times with PBS-T and then blocked with the blocking reagent at room temperature (RT) for 2 h. To detect the Sulf activity on the immobilized heparin or HS, the wells are washed as above and then incubated with bead-bound or purified FLAG/Histagged Sulfs or concentrated conditioned medium of HSulf2-expressing cells in 100 ml of a reaction mixture containing 50 mM of HEPES, pH 7.5, 10 mM of MgCl2 at 37  C. 3. The wells are washed three times with PBS-T and then incubated with 100 ml/well of RB4CD12 (1:750 diluted by 0.1% BSA in PBS) at RT for 1 h. 4. The wells are washed three times with PBS-T and then incubated with 100 ml/well of polyclonal rabbit anti-VSV-G antibody (1 mg/ml in 0.1% BSA in PBS) at RT for 45 min. 5. The wells are washed as above and then incubated with 100 ml/well of alkaline phosphatase-conjugated polyclonal goat antirabbit IgG (H þ L) (0.3 mg/ml in 0.1% BSA in PBS) at RT for 45 min. The wells are washed three times. 6. Finally, the wells are incubated with PNPP in diethanolamine buffer (100 ml/well) at RT for 5–10 min. OD 405 nm is read on a microplate reader (Fig. 3.1).

3. Flow Cytometry-Based Sulf Assay Against the Cell Surface RB4CD12 Epitope 3.1. Materials Phage display-derived RB4CD12 anti-HS antibody and MPB49 non-antiHS antibody were produced in a VSV-tag version (Dennissen et al., 2002). Cy3-conjugated monoclonal mouse anti-VSV-G antibody is from Sigma. pcDNA3.1 Myc/His-HSulf1, -HSulf2, -HSulf1DCC, or -HSulf2DCC expression plasmid (Morimoto-Tomita et al., 2002). Dulbecco’s modified Eagle medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented 10% fetal bovine serum for human embryonic kidney (HEK) 293 cells and HeLa cells. OptiMEM I reduced serum medium (Invitrogen). PBS supplemented with 0.02% ethylenediamine tetraacetic acid (EDTA; Sigma). FuGENE6 transfection reagent (Roche, Indianapolis, IN). 1 FACS buffer: 1% BSA, 0.1% NaN3 in PBS filtered by a 0.22-mm filter and stored at 4  C. 2% paraformaldehyde in PBS.

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Figure 3.1 ELISA for Sulf activity against the RB4CD12 epitope in immobilized heparin and heparan sulfate. (A) RB4CD12 was tested for binding to varying amounts of immobilized heparin–BSA or heparan sulfate–BSA by ELISA. (B) The purified Sulfs were tested for their effects on immobilized heparin–BSA in the RB4CD12 epitope ELISA. The Sulfs were prepared from 0.5 ml of CM collected from the transfected HKE293 cells. Different letters among treatments indicate significant differences (P < 0.05). (C and D) The activity of MCF-7 CM on the RB4CD12 epitope as a

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5 ml polystyrene round-bottom tube capped with a cell-strainer cap (BD, Franklin Lakes, NJ). FACSCalibur flow cytometer and CellQuest software (BD). FlowJo software (Tree Star, Ashland, OR).

3.2. Method 1. HEK293 and HeLa cells are cultured on 75 cm2 flasks and passaged until they reach 50% confluence. The cells are rinsed with warm PBS and OptiMEM I and then transiently transfected with a Myc–His-tagged HSulf expression plasmid using FuGENE6 transfection reagent according to the manufacturer’s instructions. An empty vector is used as a control (‘‘mock’’ control). The cells are incubated for 48 h with OptiMEM I (HEK293) or DMEM 10% fetal bovine serum (HeLa). Nonsmall cell lung cancer (NSCLC) cell lines (H292, H460, and Calu-6) are stably transduced with Sulf-2 specific shRNA (PLV-1413) via lentivirus or with a control irrelevant shRNA (PLV-Ctr) as described (LemjabbarAlaoui et al., 2010; Nawroth et al., 2007). 2. Adherent cells are detached from culture flasks by incubating in 0.02% EDTA/PBS at 37  C for 10 min and then dissociated into monodispersed cells. 3. Cells (1  105) are collected and suspended in 100 ml 1 FACS buffer in a 1.5 ml tube. RB4CD12 (1:50 dilution) or MPB49 (1:50 dilution) is added to the suspension. 4. After incubation at 4  C for 30 min, 400 ml 1 FACS buffer is added to the tube. Cells are pelleted and suspended in 400 ml 1 FACS buffer. Then, cells are pelleted and suspended in 100 ml 1 FACS buffer containing Cy3-conjugated anti-VSV-G (4 mg/ml). 5. After incubation at 4  C for 30 min, cells are washed as above, suspended in 100 ml 2% paraformaldehyde/PBS, and then transferred into a 5 ml polystyrene round-bottom tube. Antibody-bound cells are then analyzed by FACS using a FACSCalibur flow cytometer. The mean fluorescence intensity (MFI) of FL2 is analyzed by CellQuest and FlowJo softwares. Figure 3.2 shows the MFI in Sulf-transfected cell samples compared to that in mock-transfected controls. Figure 3.3 shows flow cytometry histograms for NSCLC cells transduced with Sulf-2 shRNA and control shRNA. function of CM volume (1-h incubation) and reaction time (1 ml of CM). (E) Inhibition of the activity in MCF-7 CM by H2.3 anti-HSulf2 antibody. Two microgram of H2.3 antibody or rabbit IgG was incubated with MCF-7 CM (1 ml, 4-h incubation). MPB49, a non-HS-binding scFv antibody, was used as a control for RB4CD12. A mixture of bacterial heparinases I–III was used as a positive control to produce epitope degradation (heparinases). **P < 0.001. Figure taken from Hossain et al. (2010) with permission.

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Figure 3.2 Cell surface expression of the RB4CD12 epitope diminished in HSulf-transfected cells. HEK293 (A) and HeLa (B) cells transiently transfected with pcDNA3.1 empty vector alone (Mock), cDNA of HSulf-1 (HSulf-1), HSulf-1DCC (HSulf-1DCC), HSulf2 (HSulf-2), or HSulf-2DCC (HSulf-2DCC) were analyzed by flow cytometry with RB4CD12. These data are representative of at least three independent experiments. Mean fluorescence intensity (MFI) relative to ‘‘Mock’’ of three independent experiments are shown. MPB49, a non-HS-binding scFv antibody, was used as ‘‘control.’’ **P < 0.001; *P < 0.01; #P < 0.05 as determined by one-way ANOVA with Tukey’s post hoc test. Figure adapted from Hossain et al. (2010) with permission.

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Figure 3.3 Cell surface expression of the RB4CD12 epitope increased in NSCLC cells with Sulf-2 knockdown. H292, H460, and Calu-6 cells, each of which expresses Sulf-2 endogeneously, were transduced with Sulf-1 shRNA or control shRNA and analyzed by flow cytometry with RB4CD12. Flow cytometry profiles are shown with ‘‘2’’ denoting cells transduced with control shRNA (PLV-Ctr) and ‘‘3’’ cells transduced with Sulf-2 specific shRNA (PLV-1413). ‘‘1’’ denotes cells stained with the control scFV antibody, MPB49. Figure taken from Lemjabbar-Alaoui et al. (2010).

4. Ex Vivo Sulf Assay Against the RB4CD12 Epitope in Cryostat-Cut Sections 4.1. Materials Adult (>12-week-old) C57BL/6 mice. Phage display-derived RB4CD12 anti-HS antibody. Cy3-conjugated monoclonal mouse anti-VSV-G antibody (Sigma). Rabbit anti-laminin antibody (Sigma). Cy2-conjugated polyclonal goat antirabbit IgG (Jackson Immuno Research Laboratories). Heparinase I (2 mU/ml) (EC 4.2.2.7), heparinase II (0.5 mU/ml), and heparinase III (0.2 mU/ml) (EC 4.2.2.8) from Sigma are stored in small aliquots at 20  C. Fresh brains from 12-week-old C57BL/6 mice were embedded in the O.C.T. compound (Sakura Finetek, Torrance, CA) and frozen in liquid nitrogen. Solutions, 0.5 M HEPES, pH 7.5, and 0.5 M MgCl2, are stored at RT. Ice-cold acetone. O.C.T. compound and plastic molds are from Sakura Finetek (Torrance, CA). Blocking reagent: 3% BSA, 0.01% NaN3 in PBS filtered by a 0.45-mm filter and stored at 4  C. FluorSaverTM Reagent (EMD Chemicals, Gibbstown, NJ). MAS-coated glass slide with three 15 mm wells (Matsunami Glass, Osaka, Japan). Cryostat (model CM1950, Leica Microsystems).

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Figure 3.4 Ex vivo Sulf activity against the RB4CD12 epitope in the basement membrane of mouse brain microvessels. (A) Cryostat-cut sections of mouse brains were incubated overnight with recombinant HSulf-1 (0.4 mg) and HSulf-2 (0.4 mg) prepared from CM of transfected HEK293 cells (HSulf-1, HSulf-2), buffer only (no enzyme) or CM of MCF-7 human breast cancer cells (MCF-7 CM). The Ni–NTA resin-bound materials that were prepared from HEK293 cells transfected with the empty vector were eluted and used (Mock). A mix of bacterial heparinases (heparinases) served as a positive control. RB4CD12 binding was visualized by a Cy3-conjugated anti-VSV tag antibody with a confocal laser scanning microscopy (red). Basement membranes of vessels were stained by an anti-laminin antibody in conjunction with a Cy2-conjugated secondary antibody (green). The signal intensities along the line markers (blue lines) indicated by arrows in the images were measured for RB4CD12

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Laser scanning confocal microscopy (model LSM 510, Carl Zeiss). Image-Pro Plus software (Media Cybernetics, Bethesda, MD).

4.2. Method 1. Fresh brains of adult C57BL/6 mice are dissected out, embedded in the O.C.T. compound-containing plastic molds and then frozen on dry ice. 2. Sections of the embedded brain block are cut by cryostat in 10 mm thick and prepared onto MAS-coated glass slides. After air dry for 30–60 min, sections are fixed in ice-cold acetone for 15 min. 3. Sections are incubated with blocking reagent for 15 min at RT after air drying for 30 min. Sections are then pretreated with bead-bound or purified FLAG/His-tagged Sulfs (0.4 mg of HSulf-1 or HSulf-2) or 100 concentrated conditioned medium of HSulf2-expressing MCF-7 cells (10 ml) in 100 ml of a reaction mixture containing 50 mM of HEPES, pH 7.5, 10 mM of MgCl2 at 37  C overnight. A mix of heparinase I (2 mU), heparinase II (0.5 mU), and heparinase III (0.2 mU) is served as a positive control. 4. Sections are washed twice with PBS and then incubated with a mixture of RB4CD12 (1:100 dilution) and a rabbit anti-laminin antibody (1:100 dilution) for 1 h at RT. 5. After washing twice with PBS, primary antibodies are detected with Cy3-conjugated monoclonal anti-VSV-G (4 mg/ml) and Cy2-conjugated polyclonal goat antirabbit IgG (3 mg/ml). Sections are mounted in FluorSaverTM Reagent. 6. Digital images are captured by a laser scanning confocal microscopy at the same setting for all images. Magnification used is 630. Basement membranes of brain vessels are costained by both RB4CD12 and antilaminin antibodies. 7. The fluorescence signal intensities along the line (100 pixels in length) within vessels in the images are determined semiquantitatively for RB4CD12 and anti-laminin staining by Image Pro Plus software. The relative intensity of RB4CD12 to laminin is measured in 10 randomly selected vessels and plotted on to a graph. The values are analyzed by one-way ANOVA with Dunnet’s post hoc test (Fig. 3.4).

and laminin staining. Representative images and measurements of 10 randomly selected vessels are shown. (B) The relative intensity of RB4CD12 to laminin is shown. n ¼ 10 vessels per treatment. Each symbol represents one vessel image; small horizontal lines indicate the mean. Magnification used 630. **P < 0.001. Figure taken from Hossain et al. (2010) with permission.

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ACKNOWLEDGMENTS Supported by the Japanese Health and Labour Sciences Research Grant (H19-001 to K. U.); a Tobacco-Related Disease Research Program Grant (17RT-0117 and the National Institutes of Health Grant P01 AI053194 to S. D. R.); and in parts by the Uehara Memorial Foundation and the Takeda Science Foundation (to K. U.).

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Fujita, K., Takechi, E., Sakamoto, N., Sumiyoshi, N., Izumi, S., Miyamoto, T., Matsuura, S., Tsurugaya, T., Akasaka, K., and Yamamoto, T. (2009). HpSulf, a heparan sulfate 6-O-endosulfatase, is involved in the regulation of VEGF signaling during sea urchin development. Mech. Dev. 127, 235–245. Gallagher, J. T. (2001). Heparan sulfate: Growth control with a restricted sequence menu. J. Clin. Invest. 108, 357–361. Holst, C. R., Bou-Reslan, H., Gore, B. B., Wong, K., Grant, D., Chalasani, S., Carano, R. A., Frantz, G. D., Tessier-Lavigne, M., Bolon, B., French, D. M., and Ashkenazi, A. (2007). Secreted sulfatases Sulf1 and Sulf2 have overlapping yet essential roles in mouse neonatal survival. PLoS ONE 2, e575. Hossain, M. M., Hosono-Fukao, T., Tang, R., Sugaya, N., van Kuppevelt, T. H., Jenniskens, G. J., Kimata, K., Rosen, S. D., and Uchimura, K. (2010). Direct detection of HSulf-1 and HSulf-2 activities on extracellular heparan sulfate and their inhibition by PI-88. Glycobiology 20, 175–186. Jenniskens, G. J., Oosterhof, A., Brandwijk, R., Veerkamp, J. H., and van Kuppevelt, T. H. (2000). Heparan sulfate heterogeneity in skeletal muscle basal lamina: Demonstration by phage display-derived antibodies. J. Neurosci. 20, 4099–4111. Jenniskens, G. J., Hafmans, T., Veerkamp, J. H., and van Kuppevelt, T. H. (2002). Spatiotemporal distribution of heparan sulfate epitopes during myogenesis and synaptogenesis: A study in developing mouse intercostal muscle. Dev. Dyn. 225, 70–79. Kalus, I., Salmen, B., Viebahn, C., von Figura, K., Schmitz, D., D’Hooge, R., and Dierks, T. (2008). Differential involvement of the extracellular 6-O-endosulfatases Sulf1 and Sulf2 in brain development and neuronal and behavioral plasticity. J. Cell. Mol. Med. 13, 4505–4521. Lai, J., Chien, J., Staub, J., Avula, R., Greene, E. L., Matthews, T. A., Smith, D. I., Kaufmann, S. H., Roberts, L. R., and Shridhar, V. (2003). Loss of HSulf-1 up-regulates heparin-binding growth factor signaling in cancer. J. Biol. Chem. 278, 23107–23117. Lai, J. P., Chien, J., Strome, S. E., Staub, J., Montoya, D. P., Greene, E. L., Smith, D. I., Roberts, L. R., and Shridhar, V. (2004a). HSulf-1 modulates HGF-mediated tumor cell invasion and signaling in head and neck squamous carcinoma. Oncogene 23, 1439–1447. Lai, J. P., Chien, J. R., Moser, D. R., Staub, J. K., Aderca, I., Montoya, D. P., Matthews, T. A., Nagorney, D. M., Cunningham, J. M., Smith, D. I., Greene, E. L., Shridhar, V., et al. (2004b). hSulf1 Sulfatase promotes apoptosis of hepatocellular cancer cells by decreasing heparin-binding growth factor signaling. Gastroenterology 126, 231–248. Lai, J. P., Sandhu, D. S., Shire, A. M., and Roberts, L. R. (2008a). The tumor suppressor function of human sulfatase 1 (SULF1) in carcinogenesis. J. Gastrointest. Cancer 39, 149–158. Lai, J. P., Sandhu, D. S., Yu, C., Han, T., Moser, C. D., Jackson, K. K., Guerrero, R. B., Aderca, I., Isomoto, H., Garrity-Park, M. M., Zou, H., Shire, A. M., et al. (2008b). Sulfatase 2 up-regulates glypican 3, promotes fibroblast growth factor signaling, and decreases survival in hepatocellular carcinoma. Hepatology 47, 1211–1222. Lamanna, W. C., Baldwin, R. J., Padva, M., Kalus, I., Ten Dam, G., van Kuppevelt, T. H., Gallagher, J. T., von Figura, K., Dierks, T., and Merry, C. L. (2006). Heparan sulfate 6-Oendosulfatases: Discrete in vivo activities and functional co-operativity. Biochem. J. 400, 63–73. Lemjabbar-Alaoui, H., van Zante, A., Singer, M. S., Xue, Q., Wang, Y. Q., Tsay, D., He, B., Jablons, D. M., and Rosen, S. D. (2010). Sulf-2, a heparan sulfate endosulfatase, promotes human lung carcinogenesis. Oncogene 29, 635–646. Li, J., Kleeff, J., Abiatari, I., Kayed, H., Giese, N. A., Felix, K., Giese, T., Buchler, M. W., and Friess, H. (2005). Enhanced levels of Hsulf-1 interfere with heparin-binding growth factor signaling in pancreatic cancer. Mol. Cancer 4, 14. Lum, D. H., Tan, J., Rosen, S. D., and Werb, Z. (2007). Gene trap disruption of the mouse heparan sulfate 6-O-endosulfatase gene, Sulf2. Mol. Cell. Biol. 27, 678–688.

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Morimoto-Tomita, M., Uchimura, K., Werb, Z., Hemmerich, S., and Rosen, S. D. (2002). Cloning and characterization of two extracellular heparin-degrading endosulfatases in mice and humans. J. Biol. Chem. 277, 49175–49185. Morimoto-Tomita, M., Uchimura, K., Bistrup, A., Lum, D. H., Egeblad, M., Boudreau, N., Werb, Z., and Rosen, S. D. (2005). Sulf-2, a proangiogenic heparan sulfate endosulfatase, is upregulated in breast cancer. Neoplasia 7, 1001–1010. Nagamine, S., Koike, S., Keino-Masu, K., and Masu, M. (2005). Expression of a heparan sulfate remodeling enzyme, heparan sulfate 6-O-endosulfatase sulfatase FP2, in the rat nervous system. Brain Res. Dev. Brain Res. 159, 135–143. Nakato, H., and Kimata, K. (2002). Heparan sulfate fine structure and specificity of proteoglycan functions. Biochim. Biophys. Acta 1573, 312–318. Narita, K., Staub, J., Chien, J., Meyer, K., Bauer, M., Friedl, A., Ramakrishnan, S., and Shridhar, V. (2006). HSulf-1 inhibits angiogenesis and tumorigenesis in vivo. Cancer Res. 66, 6025–6032. Nawroth, R., van Zante, A., Cervantes, S., McManus, M., Hebrok, M., and Rosen, S. D. (2007). Extracellular sulfatases, elements of the Wnt signaling pathway, positively regulate growth and tumorigenicity of human pancreatic cancer cells. PLoS ONE 2, e392. Ohto, T., Uchida, H., Yamazaki, H., Keino-Masu, K., Matsui, A., and Masu, M. (2002). Identification of a novel nonlysosomal sulphatase expressed in the floor plate, choroid plexus and cartilage. Genes Cells 7, 173–185. Ratzka, A., Kalus, I., Moser, M., Dierks, T., Mundlos, S., and Vortkamp, A. (2008). Redundant function of the heparan sulfate 6-O-endosulfatases Sulf1 and Sulf2 during skeletal development. Dev. Dyn. 237, 339–353. Saad, O. M., Ebel, H., Uchimura, K., Rosen, S. D., Bertozzi, C. R., and Leary, J. A. (2005). Compositional profiling of heparin/heparan sulfate using mass spectrometry: Assay for specificity of a novel extracellular human endosulfatase. Glycobiology 15, 818–826. Tang, R., and Rosen, S. D. (2009). Functional consequences of the subdomain organization of the sulfs. J. Biol. Chem. 284, 21505–21514. Uchimura, K., Morimoto-Tomita, M., Bistrup, A., Li, J., Lyon, M., Gallagher, J., Werb, Z., and Rosen, S. D. (2006a). HSulf-2, an extracellular endoglucosamine-6-sulfatase, selectively mobilizes heparin-bound growth factors and chemokines: Effects on VEGF, FGF1, and SDF-1. BMC Biochem. 7, 2. Uchimura, K., Morimoto-Tomita, M., and Rosen, S. D. (2006b). Measuring the activities of the Sulfs: Two novel heparin/heparan sulfate endosulfatases. Methods Enzymol. 416, 243–253. van den Born, J., Salmivirta, K., Henttinen, T., Ostman, N., Ishimaru, T., Miyaura, S., Yoshida, K., and Salmivirta, M. (2005). Novel heparan sulfate structures revealed by monoclonal antibodies. J. Biol. Chem. 280, 20516–20523. van Kuppevelt, T. H., Dennissen, M. A., van Venrooij, W. J., Hoet, R. M., and Veerkamp, J. H. (1998). Generation and application of type-specific anti-heparan sulfate antibodies using phage display technology. Further evidence for heparan sulfate heterogeneity in the kidney. J. Biol. Chem. 273, 12960–12966. Viviano, B. L., Paine-Saunders, S., Gasiunas, N., Gallagher, J., and Saunders, S. (2004). Domain-specific modification of heparan sulfate by Qsulf1 modulates the binding of the bone morphogenetic protein antagonist Noggin. J. Biol. Chem. 279, 5604–5611. Wang, S., Ai, X., Freeman, S. D., Pownall, M. E., Lu, Q., Kessler, D. S., and Emerson, C. P., Jr. (2004). QSulf1, a heparan sulfate 6-O-endosulfatase, inhibits fibroblast growth factor signaling in mesoderm induction and angiogenesis. Proc. Natl. Acad. Sci. USA 101, 4833–4838. Yue, X., Li, X., Nguyen, H. T., Chin, D. R., Sullivan, D. E., and Lasky, J. A. (2008). Transforming growth factor-beta1 induces heparan sulfate 6-O-endosulfatase 1 expression in vitro and in vivo. J. Biol. Chem. 283, 20397–20407.

C H A P T E R

F O U R

Glycomics Profiling of Heparan Sulfate Structure and Activity Jeremy E. Turnbull, Rebecca L. Miller, Yassir Ahmed, Tania M. Puvirajesinghe, and Scott E. Guimond Contents 1. Overview 1.1. HS structure and biosynthesis 1.2. Heparan sulfates: A family of multifunctional cell regulators 1.3. Decoding HS structure–function: Toward glycomics approaches 2. Experimental 2.1. Rapid purification of HS 2.2. Profiling disaccharide composition 2.3. Profiling HS oligosaccharides 2.4. Profiling HS oligosaccharide function 3. Conclusions and Future Perspectives Acknowledgments References

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Abstract The heparan sulfate (HS) family of glycosaminoglycans are highly complex and structurally diverse polysaccharides with information encoded within the chains that imparts the ability to bind selectively to a wide range of proteins—the ‘‘HS interactome’’—and to regulate their biological activities. However, there are two key questions which need to be addressed; first, the extent of structural variation of expressed HS structures—the ‘‘heparanome’’—in specific biological contexts and second, the degree of functional selectivity exerted by these structures in regulating biological processes. There is a clear need to develop more systematic and high throughput approaches in order to address these questions. Here, we describe a cohort of protocols for profiling different aspects of HS structure and activity, focusing particularly on disaccharide building blocks and larger oligosaccharide domains, the latter representing the functional units of HS chains. A range of other complementary methods in the literature are also discussed. Together these provide a new and more Centre for Glycobiology, School of Biological Sciences, University of Liverpool, Liverpool, United Kingdom Methods in Enzymology, Volume 480 ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)80004-7

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2010 Elsevier Inc. All rights reserved.

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comprehensive toolkit to investigate HS structure and activity in a higher throughput manner in selected biological systems. The implementation of such a glycomics strategy will enable development of a systems biology view of HS structure–function relationships and help to resolve the significant puzzle of the extensive interactome of HS, which remains a key question in the glycobiology field. We anticipate that the next decade will see major advances in our understanding of the complex biology of HS.

1. Overview 1.1. HS structure and biosynthesis Heparan sulfate (HS) is a member of the glycosaminoglycan (GAG) family of linear polysaccharides made up of repeating disaccharide unit backbones onto which are superimposed specific modification patterns, most notably addition of sulfate groups. Within this family, HS has the most highly varied structure, with polymorphic sulfated sequences expressed found in its chains which are responsible for the many protein binding and regulatory properties of HS (Turnbull et al., 2001). HS chains are normally attached to core proteins in the form of HS proteoglycans (HSPGs), which are located at the cell surface and in the extracellular matrix. HS biosynthesis involves a complex set of Golgi enzymes which initially produce a nonsulfated polysaccharide chain precursor, heparosan. A sequential series of modifications then follows which superimpose complex patterns of sulfate group addition and uronate epimerization at selective positions. Most important, the system is not template-driven and the reactions only proceed to a partial extent, resulting in a high degree of structural diversity of HS sugar sequences. There is an ordered structure to HS chains in which domains with differing types and density of modifications are spaced apart along the molecule (Turnbull and Gallagher, 1991). Domains where the heparosan has undergone relatively few modifications consist mainly of GlcA-GlcNAc repeats, and are called N-acetylated (NA) domains. These act to space apart the highly modified, sulfated domains (S-domains; typically 3–8 disaccharide units in length) in which extensive modifications occur, especially N- and O-sulfate group additions and epimerization of glucuronic acid (GlcA) to iduronic acid (IdoA). Flanking domains that have alternating N-acetylated and N-sulfated disaccharides (NA/NS domains) are also present (Lindahl et al., 1998; Turnbull et al., 2001). Variant HS chain modifications provide structural diversity that underpins its many protein binding and regulatory properties. The potential variations in even short saccharide sequences are very high, making HS a very information dense molecule (Nugent, 2000), though the theoretical

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variations are restricted by biosynthetic constraints. Reproducible differences in HS structure have been observed at both the tissue (Brickman et al., 1998; Ford-Perriss et al., 2002; Maccarana et al., 1996) and cell level (Kato et al., 1994; Sanderson et al., 1994), and upon cell transformation (Jayson et al., 1998). Identical core proteins can display different HS structures when expressed by different cell types (Kato et al., 1994; Sanderson et al., 1994). Furthermore, it is increasingly evident that HS structures are biosynthesized in a dynamic manner: HSPGs have a rapid turnover (half-life 3–4 h) in cultured cells (Yanagishita and Hascall, 1984); cells can alter the HS structures they make, for example, in response to extracellular signals such as growth factors (Schmidt et al., 1995); and changes in HS structure have been shown to occur over short time spans in developing tissues (Ford-Perriss et al., 2002). HS is thus not as a single molecule but is in fact a highly diverse family of related molecules with dynamic and specific functional roles (Turnbull et al., 2001).

1.2. Heparan sulfates: A family of multifunctional cell regulators The HS family is now viewed as multifunctional players with vital roles in cell regulation (Turnbull et al., 2001). Their strategic location at the cell surface and in the extracellular matrix, positions them ideally for selective regulatory interactions with many proteins (Ori et al. 2008; Powell et al., 2004). These interactions can result in functional regulation of protein activities and are critical for a range of physiological processes (Bishop et al., 2007). Genetic studies in model organisms have clearly demonstrated that HS is essential for many aspects of development and normal physiological functions (Lander and Selleck, 2000). HS interacts with an extraordinary range of proteins (the HS ‘‘interactome’’) including growth factors, enzymes, extracellular matrix proteins, and proteins found on the surface of pathogens (Ori et al. 2008). In many examples of HS–protein interactions there is evidence that these interactions are selective to varying degrees and serve regulatory roles. A principal role of HS is to act as a ‘‘catalyst of molecular encounters’’ (Lander et al., 1999). The mechanisms of action of HS are an active area of investigation which has revealed a number of distinct modes of action in mediating protein–protein interactions, including altering protein conformations (e.g., antithrombin III), increasing protein stability and restricting protein mobility, acting as a cell surface receptor, and acting as a template for controlled assembly of protein complexes (Turnbull et al., 2001). Probably one of the best studied examples of the latter is the action of HS as a coreceptor for the fibroblast growth factor (FGF) family, where it modulates

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the interaction of FGF with its receptor tyrosine kinase (Ornitz et al., 1996; Rapraeger et al., 1991; Yayon et al., 1991). HS activates signaling by participating in the formation of a ternary complex, mediated by specific interactions of the HS with both the growth factor and its receptor, and the presence or absence of specific sulfate groups within HS can dictate which members of the FGF family can signal through which receptors (Guimond and Turnbull, 1999; Pye et al., 1998).

1.3. Decoding HS structure–function: Toward glycomics approaches Two key questions in the field are now the extent of natural diversity of HS structures—the ‘‘heparanome’’—in specific biological systems (Turnbull et al., 2001) and the level of sequence specificity or selectivity in terms of regulation of biological responses (Lindahl and Ping, 2009). It has become increasingly clear that HS does not influence protein functions in a ‘‘digital’’ on/off manner via highly specific single sequences with single functions, but rather via ‘‘analog tuning’’ in which ensembles of restricted motifs are able to exert a range of modulatory functions. Further work is now clearly required to decode HS structural diversity and how this relates to functional properties in a systematic manner. Glycomics technologies have recently been expanding in the wider glycobiology field (Turnbull and Field, 2007), and strategies appropriate for HS can now be envisaged which integrate a variety of methods in order to develop a systems biology view of the structural selectivity underlying the diverse functions of HSPGs. The first step for this type of strategy is isolation of HS from the appropriate cells or tissues under study. Second, the ‘‘building blocks’’ present can be determined by quantitative disaccharide compositional analysis, permitting a first (basic) level of comparison of tissue and cell type specificity of HS structures. This is typically achieved using HPLC methods employing UV or fluorescence detection, or by mass spectrometry, using authentic standards. Third, and increasingly important, the higher order organization of disaccharide building blocks into oligosaccharide level domains can be profiled (or ‘‘mapped’’) using a variety of methods including gel electrophoresis, gel filtration, HPLC, and mass spectroscopy. Finally, it is also possible to profile the bioactivities of particular saccharides, allowing screening and determination of the functional specificity of particular saccharides. Here, we present protocols for a number of methods in this type of work flow and further information on additional alternative methods.

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2. Experimental 2.1. Rapid purification of HS In order to study the variant spatial and temporal regulation of HS biosynthesis in cells and tissues, efficient methods for extraction of HS are required to facilitate subsequent structural analysis. Isolation of HS and other GAGs from tissues can be achieved through several means. The most common involves the dissolution of tissues using proteases, detergents, and/or chaotropic salts. Alkaline treatment of tissues can also be used to release GAG chains from their protein cores. The exact method used is chosen based on the amount and nature of the starting material and the availability of equipment and reagents. These methods for the isolation of HS are more suited to use on larger amounts of tissues or have multiple, lengthy steps. Previous methods could also suffer loss of material during transfer steps. Use of the recently developed RIP (Rapid Isolation of Proteoglycans) method to isolate HS from small cell or tissue samples overcomes these problems (Guimond et al., 2009). 2.1.1. RIP method for isolation of proteoglycans 1. Tissues are ground in a tissue homogenizer with 1 ml of Trizol reagent for every 100 mg of wet weight of starting tissue. The samples are left for 5 min at room temperature (RT), and then 0.2 ml of chloroform is added per 1 ml of Trizol originally used. Samples are shaken vigorously for 15 s and then left for 3 min at RT. Centrifuge samples at no more than 12,000
g for 15 min at 4  C. 2. Remove the upper, aqueous layer containing the proteoglycans as well as RNA. Dilute 1:10 in water and apply to DEAE-Sephacel (100 ml beads/100 mg starting material; GE Life Sciences) or a comparable weak anion exchange matrix. This can be done as a batch absorption or as a column. Wash with 10 column volumes of phosphate buffered saline (PBS) followed by 10 column volumes of PBS (0.25 M NaCl) and elution with 10 column volumes of PBS (2 M NaCl). Desalt samples and freeze dry. Samples can be stored at 20  C. 3. After the first weak anion exchange step, the samples should be reconstituted in 100 ml of HPLC-grade water and passed through a 5000 MWCO spin filter to remove any impurities that were not removed in the previous steps that may affect subsequent enzyme and labeling steps. 4. Digestion and removal of contaminating molecules is achieved by the sequential addition of enzymes, followed by weak anion exchange chromatography and desalting. Common choices are DNase, RNase, chondroitin ABC lyase, neuraminidase, keratanase, hyaluronidase, and proteases, depending on the molecule and level of purity required.

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The RIP method is much faster than previous methods, with samples ready for initial weak anion exchange chromatography in as little as 30 min and ready for many structural analyses in 2 days (Guimond et al., 2009). It also requires only one transfer before the first weak anion exchange step, thus minimizing sample loss. The weak anion exchange step and subsequent enzyme steps are common to all methods of purification. This method can also be used to isolate other GAGs by altering the enzymes used in the additional purification step. It can also be used on cultured cells. The main disadvantage is the nature of the phenol/chloroform limits the method to small amounts of tissue (up to 1 g) to avoid the handling and accumulation of large amounts of chemical waste. Figure 4.1 shows an example of analysis of HS derived from mouse tissues by the RIP method.

2.2. Profiling disaccharide composition The most common method of profiling HS from different sources is the analysis of disaccharides. This type of analysis is an essential first step for characterization of HS. Although common disaccharides are present in different tissues, the proportion of each disaccharide may vary quantitatively, resulting in structural differences between tissues. Disaccharide analysis is most commonly achieved using a mixture of heparinases I, II, and III, derived from Flavobacterium, which are able to cleave HS chains to disaccharide products via an elimination reaction (Hovingh and Linker, 1970; Linhardt et al., 1990). This introduces a C4–C5 unsaturated double bond in the uronic acid residue at the nonreducing end, making direct absorbance measurements possible at 232 nm (molar extinction coefficient of 5500 M 1 cm 1; Linhardt et al., 1988). Analysis of the resulting disaccharide structures is most commonly undertaken using HPLC separation techniques with UV detection. However, extraction of HS from small amounts of cells or tissues often produces only small quantities of purified HS sample, making more sensitive analysis techniques essential. High sensitivity detection can be achieved by labeling the products of heparinase digestion, with newly created reducing ends, with fluorescent tags having amine or hydrazide functional groups. Fluorophores which have been coupled to HS disaccharide structures include 2-cyanoacetamide (Toyoda et al., 1997), 2-aminoacridone (AMAC; Deakin and Lyon, 2008; Hitchcock et al., 2008a,b; Kitagawa et al., 1995), 2-aminobenzamide (Kinoshita and Sugahara, 1999; Yamada et al., 1999), and 2-aminobenzoic acid (Sato et al., 2005; Turnbull et al., 1999; Volpi et al., 2009). Reported sensitivities are in the picomol range, with enhanced sensitivity possible using laser-induced fluorescence, often coupled to capillary electrophoresis (Hitchcock et al., 2008a; Karamanos et al., 1996; Lamari et al., 1999; Militsopoulou et al., 2002).

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Figure 4.1 Disaccharide compositional profiling of HS from different mouse tissues using RIP extraction and fluorescence detection. HS is purified from 100 mg of each tissue by RIP, digested with heparitinases, labeled with BODIPY hydrazide, and separated by HPLC. Representative data are shown for the tissues: (A) spleen and (B) liver. Disaccharide structures are identified by comparison of retention time with authentic standards (Dextra Ltd). Disaccharide standards are (1) UA-GlcNAc, (2) UA-GlcNAc(6 S), (3) UA-GlcNS, (4) UA-GlcNS(6 S), (5) UA(2 S)-GlcNS, (6) UA (2 S)-GlcNS(6 S), (7) UA(2 S)-GlcNAc, and (8) UA(2 S)-GlcNAc(6 S).

A more recently developed fluorescent tag is 4,4-difluoro-5,7dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY) hydrazide (Atrih et al., 2009; Skidmore et al., 2006, 2010). This label has several advantages including a detection limit of 100 fmol for an HS disaccharide mixture, which provides a >1000-fold increase in sensitivity over the use of UV absorbance and 10- to 100-fold increase in

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sensitivity over previous fluorescent labels. The method for labeling HS disaccharides with BODIPY fluorescent tag is described below. Another recent technology is quantification of disaccharides using offline and online mass spectrometry. The advantages of mass spectrometry are accurate molecular mass confirming the compound, as contaminants from extracts can elute at the same position as the known standard on an HPLC system and the other reason being the sensitivity. Even though six of the disaccharides have isomeric structures, by use of collision-induced dissociation (CID), it is possible to differentiate between them. Quantification by off-line mass spectrometry on biological samples has been achieved using tandem MS, since the ratios of CID products giving a complete quantification analysis without the use of standards. Off-line disaccharide analysis takes minutes and requires no chromatography cleanup (Desaire and Leary, 1999, 2000; Saad and Leary, 2003). Online mass spectrometry has advantages over off-line due to the chromatographic separation; graphitized carbon columns and hydrophilic interaction liquid chromatography (HILIC) may not give full disaccharide separation (but can provide analysis with reference to standards), whereas reverse-phase ion pairing (RPIP) is able to separate all eight common disaccharides, though the ion pairing reagents can cause a small decrease in sensitivity and quality of the spectrum (Zhang et al., 2009). LC–MS using RPIP has also been used for quantitation of disaccharides reductively labeled with aniline-containing stable isotopes (Lawrence et al., 2008). 2.2.1. Fluorescence HPLC analysis of HS disaccharide composition using BODIPY label 1. Tissue and cell samples are prepared using the RIP protocol, as described above (Guimond et al., 2009). 2. A preclearing step is carried out to remove low MW contaminants and improve labeling efficiency. Samples are aliquoted into Ultrafree 0.5 spin filters (NMWL 5 kDa; Millipore) and centrifuged for 5 min at 12,000 rpm. 3. HS oligosaccharides are digested into disaccharides using a mixture of heparinases I, II, and III (Ibex Ltd.). Complete digestion with heparinase enzymes is achieved by incubating at 37  C using initial digestion with heparinase I for 2 h, followed by heparinase III for 2 h and finally addition of heparinase II, with an overnight incubation. Heparinase enzymes were used at 2.5 mU each (in 10 ml of 5
heparinase buffer, 500 mM sodium acetate, 0.5 mM calcium acetate, pH 7.0). Total volume used is typically 50 ml, and these conditions are suitable for digestion of up to 1 mg of sample. 4. For reducing-end labeling with the BODIPY dye, samples are initially lyophilized and centrifuged in a 500-ml Eppendorf tube. For the labeling

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steps, 10 ml of 5 mg/ml BODIPY hydrazide in methanol is added to the lyophilized sample. Methanol is evaporated using a vacuum centrifuge set at 36  C for 30 min. The labeling reaction is carried out in dimethyl sulfoxide (DMSO): glacial acetic acid (17:3, v/v) and incubated at RT for 4 h. The imine (Schiff’s base) structures are then reduced to stable derivatives by incubating with 5 ml of 1 M sodium borohydride solution (SigmaAldrich) for 30 min at RT. Samples are then flash frozen using liquid nitrogen and lyophilized. Before separation and analysis, samples are reconstituted with DMSO: water solution (1:1, v/v) and resuspended in sample loading solution (150 mM NaOH). BODIPY-labeled unsaturated HS disaccharides are separated and analyzed using a linear sodium chloride gradient over an isocratic sodium hydroxide solution mobile phase, using a Propac PA1 column (Dionex) on an HPLC system. The column is first equilibrated in sample loading solution (150 mM NaOH). The fluorescently labeled disaccharides are then loaded using 150 mM NaOH eluent, with a 10- to 20-min wash step during which the free label elutes and baseline fluorescence levels are restored. The disaccharides are then eluted using a linear gradient of 0–1 M sodium chloride (in 150 mM NaOH), over 30 min at a flow rate of 2 ml/min. Fluorescence measurements are recorded using in-line fluorescent detection at an excitation wavelength of 488 nm and an emission wavelength of 520 nm. The column is washed using 2 M NaCl in 300 mM NaOH.

This approach has been used to rapidly profile the disaccharide composition of tissue HS and small amounts of HS from cell culture (Guimond et al., 2009). An example of disaccharide compositional profiling of HS from different mouse tissues using RIP extraction, BODIPY labeling, and strong anion exchange (SAX)-HPLC with fluorescence detection is shown in Fig. 4.1. Previously calculated correction factors are applied to quantitate the observed disaccharides, since there are differences in efficiency of labeling (Skidmore et al., 2006).

2.3. Profiling HS oligosaccharides Following disaccharide compositional analysis, the next stage is to examine the structural profiles of larger oligosaccharides obtained by partial cleavage of HS chains, in order to provide insights into the higher order domain structure. Previous work has exploited methods including gel electrophoresis (Linhardt et al., 1988; Turnbull and Gallagher 1988), gel filtration (Turnbull and Gallagher, 1990, 1991), and HPLC methods including SAX-HPLC (Guimond and Turnbull, 1999; Powell et al., 2010) and

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reverse-phase HPLC (Thanawiroon and Linhardt, 2003). More recently, strategies to profile differences in oligosaccharide domains have been developed employing online mass spectrometry techniques. These strategies look at the total ion abundances from online separations, which yields information on the structures present, including number and types of monosaccharide units, the presence of unsaturated and saturated uronate residues, number of sulfates, number of acetates, and also presence of linker region structures. However, quantitative comparisons by total ion abundance are not possible, since each structure can give different intensities due to differential ionization properties, and standards are simply not available for all structures. However, comparisons of tissue oligosaccharides with the additional data from tandem MS are quantifiable, by comparison between the B-, C-, and Y- type ions based on the nomenclature of Domon and Costello (1988). This strategy has recently been used to compare differences in total ion abundance oligosaccharide patterns between normal bovine cartilage and diseased adult human cartilage, and agreed with similar findings that the sulfation of CS changes with age (Estrella et al., 2007; Hitchcock et al., 2008b). It has also been used to study differences between HS chains between organs (Staples et al., 2009). Thus, this developing methodology is providing structural insights into the differences between HS from different tissues using microgram amounts of sample. 2.3.1. Liquid chromatography electrospray mass spectrometry Oligosaccharide masses were analyzed using liquid chromatography electrospray mass spectrometry (LC-ESI MS) using graphitized carbon columns. 1. SAX-HPLC-purified HS saccharides were desalted and separated using a porous graphitized carbon column (100 mm
0.32 mm, 5 mm; Thermo Hypersil, Runcorn, UK). Eluent A was 10 mM ammonium bicarbonate in HPLC-grade water, and eluent B was 80% acetonitrile/ 10 mM ammonium bicarbonate. 2. The samples of varying concentrations were injected and a linear gradient of 100% eluent A to 40% eluent B employed over 40 min at a flow rate of 4 ml/min. 3. Elution into the electrospray source of the mass spectrometer (LCQXPþ Thermo Finnigan) was at a flow rate of 4 ml/h, via a 1-mm tip heated to 270  C. 4. The negative ion mass spectrum of the sample was recorded using a spray voltage of 3.5 kV. Measuring the total ion count allows a profile of the separated oligosaccharides to be obtained, for example, allowing comparison of different size-fractionated pools of HS oligosaccharides (Fig. 4.2). The masses of saccharides can be examined in each peak to determine a profile of the

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200 cm, bead size 34 mm). Fractions corresponding to particular size-defined peaks were collected, freeze dried,

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oligosaccharide constituents. This type of analysis can provide accurate profiling information on the types of structures found in a particular HS chain and permits comparisons to be made between samples (Staples et al., 2009). 2.3.2. Software tools for HS oligosaccharide characterization The analysis of GAGs is most often undertaken using HPLC and mass spectrometry techniques. HPLC techniques alone provide constructive data via quantitative compositional analysis, and also comparative profiling, and the latter can be supported by mathematical methods for chemogenic comparisons (Puvirajesinghe et al., 2009). More recently, a leading technique for oligosaccharide profiling, compositional analysis, and identification of rare structures is mass spectrometry. Both ESI and MALDI (Laremore et al., 2007; Tissot et al., 2007) have been employed, ESI providing soft ionization and LC–MS options for accurate analysis and MALDI providing rapid analysis, particularly for backbone structures. Now that mass spectrometry in this field has developed, a stream of oligosaccharide data are beginning to emerge, following strategies that proteomics have adopted. Consequently, bioinformatics programs for the analysis of mass spectrometry spectra are becoming a valuable asset for data analysis. These programs include HOST (Saad and Leary, 2005) and a modified version of Glycoworkbench adapted for GAG analysis (Tissot et al., 2008), based on the Domon and Costello (1988) nomenclature. HOST is a simple Excel software program which uses a combination of clues from MS, MSMS, and enzymatic digestion. For example, heparinase I and III digestion provide clues to the oligosaccharide sequence because of their specificities, assisting MS and MSMS data collection from the same oligosaccharide products. Both data from MS and tandem MS give clues to the sequence, MS revealing the number of sulfates, acetates, and disaccharide units by the [M  H]  1 ions and MSMS producing fragmentation patterns based on known comparisons (Saad and Leary, 2005). Combined with compositional analysis, HOST will give a score between 1 and 0, with 1 being the correct sequence, and 0 being an incorrect sequence. The only other data interpretation software available to date is Glycoworkbench (Ceroni et al., 2008). This software analyses spectra by assigning mass ions for both MALDI and ESI data sets. Analysis of the MSMS spectra is also possible, and gives a graphical representation of all and subjected to LC–MS analysis using a graphitized carbon column (100 mm
0.32 mm, 5 mm; Thermo Hypersil, Runcorn, UK) and an LCQ-XPþ Thermo Finnigan instrument as described in Section 3. Each fraction (A)–(C) was run using the same conditions 0–40% acetonitrile and 10 mM ammonium bicarbonate. (A) LC–MS of a dp4 fraction, (B) LC–MS of a dp6 fraction, and (C) LC–MS of a dp10 fraction.

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possible cleavage products, in which the user can pick the number of cleavage events. These data sets have all been verified using known heparin and chondroitin oligosaccharides. It can also be used independently to build structures in silico from building blocks, and examine their theoretical fragmentation patterns. With mass spectrometry becoming an established tool for the analysis of GAGs, it may be expected that data production will increase exponentially, with consequent requirements for rapid and automatic interpretation of data.

2.4. Profiling HS oligosaccharide function Natural heparan sulfate (HS) oligosaccharide libraries are an important source for exploring HS structure–activity relationships (SAR) and now permit the development of functional glycomics studies. Libraries of HS oligosaccharides can be produced by partial degradation of tissue-derived HS polysaccharide chains and chromatographic fractionation of the resulting oligosaccharide mixtures (Powell et al., 2010). The initial step in HS oligosaccharides production is partial digestion, for example, with heparinase III (to isolate predominantly the sulfated domains), followed by fractionation based on hydrodynamic volume, using size exclusion chromatography (SEC). Subsequent to SEC fractionation, selected size-defined oligosaccharide pools can be subjected to SAX-HPLC, and the resulting products desalted and quantified. These HS oligosaccharide fractions have been shown to contain purified or partially purified oligosaccharides. A brief protocol is provided below, and a comprehensive protocol has been described by Powell et al. (2010). 2.4.1. Method for HS oligosaccharide library production 1. Partial enzyme digestion is performed on a purified HS sample after resuspension in heparinase buffer [100 mM sodium acetate, 0.1 mM calcium acetate (pH 7.0)]. Trial digestion is conducted first in order to determine a suitable amount of heparinase III to use and the time required for digestion to the required extent. Bulk digestion to completion of 400 mg HS was conducted by heparinase III at 0.1 mU/mg (5 mU/ml) for 2 h, then 10 mU heparinase III was added and incubated for at 37  C for 22 h, and then further 10 mU was added for 2 h. The reaction was stopped by boiling for 2 min; partially digested saccharides were lyophilized and stored at  20  C. 2. SEC was carried out using a SuperdexTM 30 column (self-poured SuperdexTM 30, 16 mm I.D.
200 cm length) connected to an AKTA purifier 10 system (GE Healthcare). The flow rate was set at 0.5 ml/ min with 0.5 M ammonium hydrogen carbonate and elution was monitored at labs ¼ 232 nm. Fractions of 1 ml were collected (typically

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sample elution occurs between 150 and 350 ml of column volume for this column). Fractions were pooled individually according to the selected partially resolved SEC peaks. The pooled fractions were then concentrated by serial cycles of lyophilization and stored at 20  C. 3. SAX-HPLC was performed on Shimadzu SCL-10AVP system equipped with a Propac PA1 column (9 mm
250 mm, Dionex). An analytical trial run (50 mg) was performed in order to establish where fraction collection was required, followed by preparative fractionation. About 5 ml of 0.1 M NH4HCO3 was added to each tube, this amount being sufficient to neutralize 0.25 ml of elution solvent (pH 3.5). Sample (maximum 5 mg) was made up to a final volume of 0.5 ml in Milli Q H2O and injected into the prewashed 1 ml injection loop using a Hamilton syringe. The column was prewashed for 10 min with 2 M NaCl in water, pH 3.5 (solvent B) at 1 ml/min flow rate and then equilibrated with Milli Q H2O, pH 3.5 (solvent A) for 10 min at the same flow rate. The flow was isocratic in Milli Q H2O (pH 3.5) for 1 min at flow rate 1 ml/min for sample loading onto the column. Samples were then eluted with a linear gradient of 0–50% solvent B (0–1 M NaCl) over 180 min. The column temperature was set at 40  C using column oven. Detection was performed online using a UV detector set at labs ¼ 232 nm. Fractions were collected at 15-s intervals (0.25 ml volume) using a fraction collector calibrated at 1 ml/ml flow rate, and fraction collector racks were changed at suitable time points where peak elution was absent. Fractions corresponding to the resolved or partially resolved SAX peaks were pooled. The pooled fractions were concentrated by lyophilization and stored at 20  C (Guimond and Turnbull, 1999; Powell et al., 2010). 4. Desalting of fractions was performed on HiPrepTM 26/10 desalting column (packed with Sephadex G-25 fine) equilibrated with water on an AKTA purifier 10 system. Elution was monitored at labs ¼ 232 and 215 nm. The fractions corresponding to the oligosaccharide peak (which elutes first) were pooled together, after ensuring that the salt peak (which elutes later) did not overlap with the saccharide peak. 5. Oligosaccharide concentrations were calculated by measuring the absorbance at labs ¼ 232 nm and using the extinction coefficient for the double bond of 5500 mol 1 cm 1 (Linhardt et al., 1988). Following library production, it is possible to screen the binding of saccharides using a variety of methods including affinity selection, ELISA assays and optical biosensors (Popplewell et al., 2009; Powell et al., 2004) and more recent technologies such as microarrays (Powell et al., 2009; Zhi et al., 2006, 2008). Saccharides can also be used for studying their effects on protein complex formation, for example, ligand–receptor interactions (Hussain et al., 2006). Perhaps most important, the activity of the saccharides

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can be screened in bioassays, to obtain a bioactivity profile which provides insights into actual SARs. Here, we describe an example of screening modulation of FGF signaling. Cell signaling by FGFs through FGF receptors (FGFRs) depend on HS saccharides (Rapraeger et al., 1991; Yayon et al., 1991). In bioassays, tissue-derived HS oligosaccharides showed diverse selectivity for both activation and inhibition of FGF signaling (Guimond and Turnbull, 1999), using cell proliferation assays with a BaF3 lymphoblastoid cell line which lacks both endogenous HS and FGFRs. These cells can be transfected with specific FGFRs to study FGF signaling (Ornitz et al., 1996) and have been exploited to investigate HS saccharides function (Guimond and Turnbull, 1999; Pye et al., 1998). Established transfectants which express specific FGFR isoforms can be used to screen the abilities of HS saccharides to regulate signaling; in the absence of IL-3 the cells will proliferate in response to appropriate FGFs only in the presence of exogenous activating HS oligosaccharides. 2.4.2. FGF signaling assay screening using BaF3 cells expressing FGFR1c 1. BaF3 cells transfected with FGFR1c (Ornitz et al., 1996) were maintained in RPMI 1640 growth medium supplemented with 10% fetal calf serum, L-glutamine, penicillin G, streptomycin, and 1 ng/ml rmIL-3 (R&D systems). 2. FGF-1 or FGF-2 (1 nM, R&D), and selected HS saccharides are prepared in RPMI growth medium. The cells were added to the wells at final density of 104–105 cells/ml in the medium without IL-3, and incubated at 37  C in 5% CO2 for 72 h. 3. Cell viability is determined using 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl-tetrazolium bromide (MTT). MTT was added for 4 h in the above conditions before solubilization by the addition of 10% SDS/ 0.01 M HCl for 16 h. Absorbance at labs 570 nm is measured using a microtiter plate reader. An example of the SAX chromatogram profile of an 8-mer fraction of porcine mucosal HS is shown in Fig. 4.3A, demonstrating a complex set of peaks from this tissue. Fractions from the start, middle, and the end of the salt gradient were selected for screening in FGF signaling assays. The selected fractions displayed variant capabilities to activate signaling by FGFR1c in response to FGF-1 (Fig. 4.3B). In addition, the BaF3 bioassay on these fractions was also performed for FGF-2 with same FGFR1c isoform, since it has been documented that FGFR1c has the same proliferative response for both FGF-1 and FGF-2 (Ornitz et al., 1996). The 8-mer SAX subfractions also displayed variant activities with FGF-2, but the profile differed from that for FGF-1 (Fig. 4.3C). In particular, a number of fractions were more active than a heparin 8-mer for activation of FGF-2,

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Figure 4.3 Functional profiling of HS saccharides that regulate FGF-1 and FGF-2 through FGFR1c signaling. (A) Porcine mucosal heparan sulfate 8-mer pools obtained by heparinase III digestion followed by SEC were further resolved by high resolution SAX-HPLC, performed on Propac PA1 columns (9 mm
250 mm) equilibrated in Milli Q H2O, pH 3.5 at flow rate of 1 ml/min. Samples were eluted with a linear gradient of sodium chloride pH 3.5 (0–1 M over 180 min; detection by absorbance at l ¼ 232 nm). The figure shows a typical preparative run. Multiple separations of 5 mg per run were performed, and identical peak fractions corresponding to selected peaks were pooled and desalted on HiPrepTM desalting columns, freeze dried, and quantified. BaF3 cells were grown in the presence of (B) 1 nM FGF-1 or (C) 1 nM FGF-2. Briefly, BaF3 cells were grown in the presence of FGFs and HS 8-mer SAX-HPLC fractions at 3 mg/ml (A, G, I, L, M, N, P, Q, S, U, V, and Z) or an 8-mer heparin saccharide; FGF with no saccharide was the control. Cell proliferation was measured at absorbance at l ¼ 570 nm using a 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT) colorimetric assay. Values are the means of triplicate samples. Error bars represent the standard deviation, and the data are representative of two separate experiments.

whereas only fraction 8P was more active for FGF-1. These results confirmed previous report that HS saccharides differentially activate FGF signaling (Guimond and Turnbull, 1999), but indicate that the FGF-FGFR ligand activation specificities previously described in the literature (Ornitz et al., 1996) will need to be revised. Finally, it is also possible to select saccharides with defined activities for sequencing (Guimond and Turnbull, 1999; Turnbull et al., 1999), thus permitting detailed SARs to be explored. This is an area in which significant advances can be expected over the next few years.

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3. Conclusions and Future Perspectives The cohort of protocols described above provide one set of approaches for profiling different aspects of HS structure and activity, focusing particularly on disaccharide building blocks and larger oligosaccharide domains, the latter representing the functional units of HS chains. A range of other complementary methods in the literature have also been discussed. Taken together, these provide a new and more comprehensive toolkit to investigate HS structure and activity in a higher throughput manner in selected biological systems. They allow investigation of key questions about the actual structural variation of the heparanome in specific biological contexts and the degree of functional selectivity exerted by these structures in regulating biological processes. The extensive interactome of HS (Ori et al., 2008) provides a significant glycobiology puzzle; its solution clearly requires a move toward systems biology approaches. Further advances in the application of mass spectroscopy to sequencing of HS saccharides can be expected to make a major contribution, since more complete structure–activity surveys are essential. In addition, development of databases, bioinformatics and computational biology approaches are required to automate, catalog, integrate, and model the ensuing flow of data. We anticipate that the applications of glycomics strategies over the next decade will see major advances in our understanding of the complex biology of HS.

ACKNOWLEDGMENTS The authors were funded by grants from the Biotechnology and Biological Sciences Research Council (to J. E. T., Y. A., and S. E. G.), the Engineering and Physical Sciences Research Council (Basic Technology Grant GR/S79268/01 and Basic Technology Translation Grant EP/G037604 to J. E. T.), the Medical Research Council Senior Research Fellowship (to J. E. T.), and the Wellcome Trust (to J. E. T. and S. E. G.). We also thank Dr Andrew Powell, Dr Mark Skidmore, Dr Ed Yates, and Prof. Dave Fernig for stimulating discussions.

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Brickman, Y., Nurcombe, V., Gallagher, J., Ford, M., and Turnbull, J. E. (1998). Structural modification of FGF-binding HS at a determinative stage of neuroepithelial development. J. Biol. Chem. 273, 4350–4359. Ceroni, A., Maass, K., Geyer, H., Geyer, R., Dell, A., and Haslam, S. M. (2008). Glycoworkbench: A tool for the computer-assisted annotation of mass spectra of glycans. J. Proteome Res. 7, 1650–1659. Deakin, J. A., and Lyon, M. (2008). A simplified and sensitive fluorescent method for disaccharide analysis of both heparan sulfate and chondroitin/dermatan sulfates from biological samples. Glycobiology 18, 483–491. Desaire, H., and Leary, J. (1999). Multicomponent quantification of diasteromeric hexosamine monosaccharides using ion trap tandem mass spectrometry. Anal. Chem. 71, 4142–4147. Desaire, H., and Leary, J. (2000). Detection and quantification of the sulfated disaccharides in chrondroitin sulfate by electrospray tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 11, 916–920. Domon, B., and Costello, C. E. (1988). A systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS spectra of glycoconjugates. Glycoconj. J. 5, 397–409. Estrella, R. P., Whitelock, J. M., Packer, N. H., and Karlsson, N. G. (2007). Graphitized carbon LC–MS characterisation of the chrondroitin sulfate oligosaccharides of aggrecan. Anal. Chem. 79, 3597–3606. Ford-Perriss, M., Guimond, S., Greferath, U., Kita, M., Grobe, K., Habuchi, H., Kimata, K., Esko, J. D., Murphy, M., and Turnbull, J. E. (2002). Variant heparan sulfates synthesised in the developing mouse brain differentially regulate FGF signalling. Glycobiology 12, 721–727. Guimond, S. E., and Turnbull, J. E. (1999). Fibroblast growth factor receptor signalling is dictated by specific heparan sulphate saccharides. Curr. Biol. 9, 1343–1346. Guimond, S. E., Puvirajesinghe, T. M., Skidmore, M. A., Kalus, I., Dierks, T., Yates, E. A., and Turnbull, J. E. (2009). Rapid purification and high sensitivity analysis of heparan sulfate from cells and tissues: Toward glycomics profiling. J. Biol. Chem. 284, 25714–25722. Hitchcock, A. M., Bowman, M. J., Staples, G. O., and Zaia, J. (2008a). Improved workup for glycosaminoglycans disaccharide analysis using CE with LIF detection. Electrophoresis 29, 4538–4548. Hitchcock, A. M., Yates, K. E., Costello, C. E., and Zaia, J. (2008b). Comparative glycomics of connective tissue glycosaminoglycans. Proteomics 8, 1384–1397. Hovingh, P., and Linker, A. (1970). The enzymatic degradation of heparin and heparitin sulfate. 3. Purification of a heparitinase and a heparinase from flavobacteria. J. Biol. Chem. 245, 6170–6175. Hussain, S., Piper, M., Fukuhara, N., Strochlic, L., Cho, G., Howitt, J. A., Ahmed, Y., Powell, A. K., Turnbull, J. E., Holt, C. E., and Hohenester, E. (2006). A molecular mechanism for the heparan sulfate dependence of slit-robo signaling. J. Biol. Chem. 281, 39693–39698. Jayson, G., Lyon, M., Paraskeva, C., Turnbull, J. E., Deakin, J. A., and Gallagher, J. T. (1998). Heparan sulfate undergoes specific structural changes during the progression from adenoma to carcinoma, in vitro. J. Biol. Chem. 273, 51–57. Karamanos, N. K., Vanky, P., Tzanakakis, G. N., and Hjerpe, A. (1996). High performance capillary electrophoresis method to characterize heparin and heparan sulfate disaccharides. Electrophoresis 391–395. Kato, M., Wang, H.-M., Bernfield, M., Gallagher, J. T., and Turnbull, J. E. (1994). Cell surface syndecan-1 on distinct cell types differs in fine structure and ligand binding of its HS chains. J. Biol. Chem. 269, 18881–18890.

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C H A P T E R

F I V E

Microbe-Associated Molecular Patterns in Innate Immunity: Extraction and Chemical Analysis of Gram-Negative Bacterial Lipopolysaccharides Cristina De Castro,* Michelangelo Parrilli,* Otto Holst,† and Antonio Molinaro* Contents 1. Overview 2. LPS and LOS Extraction Procedures 2.1. PCP extraction of LOS 2.2. Methods 2.3. Phenol/TEA/EDTA extraction 2.4. Methods 2.5. Hot phenol/water extraction 3. Purification of the Crude Extracts 3.1. Enzymatic hydrolysis 3.2. Method 4. SDS-PAGE 4.1. Kittelberg and Hilbink protocol 4.2. Methods 4.3. Tsai and Frasch (1982) protocol 4.4. Method 4.5. Detection of acidic polysaccharides 4.6. Method 5. Carbohydrate Analysis: Monosaccharide Determination, Absolute Configuration, and Definition of Branching Points 5.1. Monosaccharide determination: Acetylated methyl glycosides 5.2. Method

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* Universita` di Napoli Federico II, Dipartimento di Chimica Organica e Biochimica, Complesso Universitario Monte Santangelo, Via Cynthia, Napoli, Italy Division of Structural Biochemistry, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Borstel, Germany

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Methods in Enzymology, Volume 480 ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)80005-9

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2010 Elsevier Inc. All rights reserved.

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5.3. Monosaccharide absolute configuration 5.4. Methods 5.5. Determination of the absolute configuration of carbohydrates in a sample 5.6. Determination of monosaccharides branching points (methylation analysis): protocol for neutral and uronic acid containing polysaccharides 6. Fatty Acids Compositional Analysis (GC-MS) 6.1. Total fatty acid composition by methanolysis 6.2. Methods 6.3. O-Linked fatty acid 6.4. Methods 6.5. Absolute configuration determination of hydroxyl fatty acids 6.6. Methods References

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Abstract Bacterial lipopolysaccharides (LPSs) are the major component of the outer membrane of Gram-negative bacteria. They have a structural role since they contribute to the cellular rigidity by increasing the strength of cell wall and mediating contacts with the external environment that can induce structural changes to allow life in different conditions. Furthermore, the low permeability of the outer membrane acts as a barrier to protect bacteria from host-derived antimicrobial compounds. They also have a very important role in the elicitation of the animal and plant host innate immunity since they are microbe-associated molecular patterns, namely, they are glycoconjugates produced only by Gramnegative bacteria and are recognized as a molecular hallmark of invading microbes. LPSs are amphiphilic macromolecules generally comprising three defined regions distinguished by their genetics, structures, and function: the lipid A, the core oligosaccharide and a polysaccharide portion, the O-chain. In some Gram-negative bacteria, LPS can terminate with the core portion to form rough-type LPS (R-LPS, LOS). In this chapter, we will describe the isolation of both kinds of LPSs and their full chemical analysis, pivotal operations in the complete description of the primary structure of such important glycoconjugates.

Abbreviation list Kdo DMSO

3-deoxy-D-manno-oct-2-ulosonic acid dimethylsulfoxide

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e.i. EDTA GC-MS Glc LOS LPS ML M AAPM PCP PAGE SDS TEA

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electronic impact ethylendiamine tetracetate gas chromatograph equipped with mass spectrometer glucose lipooligosaccharide lipopolysaccharide McLafferty molecular ion partially methylated and acetylated alditol phenol/chloroform/light petroleum extraction polyacrylamide gel electrophoresis sodium dodecyl sulfate triethylamine

1. Overview Gram-negative bacterial lipopolysaccharides (LPSs) are powerful virulence factors and among the most important elicitors of eukaryotic innate immune response both in animals and plants (Alexander and Rietschel, 2001; Raetz and Whitfield, 2002, Silipo et al., 2010). They are heat-stable complex amphiphilic macromolecules of the Gram-negative cell envelope, and indispensable for bacterial growth, viability, and for the correct assembly of the outer membrane. They represent a defensive barrier which helps bacteria to resist to antimicrobial compounds and environmental stresses and are involved in many aspects of host–bacterium interactions as recognition, adhesion, colonization, and, in the case of extremophile bacteria, in the survival under harsh conditions. LPS are also called endotoxins because they are cell-bound and, once released from dead bacteria, play a key role in the pathogenesis of Gram-negative infections, in mechanisms as virulence, tolerance for commensal bacteria, and symbiosis. In mammalian hosts, they trigger the activation of both the innate and the adaptative immune system whereas in plants they activate the innate immunity defense system, also called basal defense. LPSs are biosynthesized according to their common structural architecture. They are composed of a hydrophilic heteropolysaccharide (formed by core oligosaccharide and a polysaccharide moiety) covalently linked to a lipophilic domain termed lipid A, which is embedded in the outer leaflet of the outer membrane and anchors these macromolecules there. The LPS leaflet is stabilized by electrostatic interaction of the negatively charged

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groups present in the lipid A and in the core region (phosphate groups, uronic acids) with divalent cations (Mg2þ, Ca2þ) which contribute to link the LPS molecules to each other. While a typical glycerophospholipid bilayer is a flexible and fluid system, the LPS layer is a semirigid structure with a highly ordered structure and low fluidity. Such a highly structured barrier is obtained by the rigidity of the lipid A-inner core saccharide backbone, the tight packing of the fatty acid residues together with the presence of such strong electrostatic interactions. The low permeability of the Gram-negative outer membrane explains their lower susceptibility to hydrophobic molecules and/or negatively charged antibiotic than Grampositive microorganisms. The three LPS domains are genetically, biosynthetically, biologically, and chemically distinct. When present, the polysaccharide moiety confers a smooth appearance to the colony on agar plates and in this case, LPS are called smooth (S)-form LPS. The polysaccharide can be the O-specific polysaccharide (OPS, O-antigen), the enterobacterial common antigen (only in Enterobacteriaceae), or a capsular polysaccharide (CPS) as, for example, colanic acid. The absence of the polysaccharide gives the colony a rough aspect and, thus, the LPS is named rough (R) form or, chemically more correct, lipooligosaccharide (LOS). R-Form LPS may occur in both wild and laboratory strains possessing mutations in the genes encoding the polysaccharide biosynthesis or transfer. It was demonstrated that laboratory deep-rough mutants are able to survive in vitro, and, thus, it has been thought for a long time that a short core region linked to lipid A represents the minimal structure with which bacteria are still viable. However, recently viable mutants were generated which contained only a precursor of LPS biosynthesis (the lipid IVA) in the outer membrane, proving that under certain conditions only a lipid A partial structure is required to guarantee outer membrane function and bacterial survival (Meredith et al., 2006). However, in tissues or body fluid, many pathogenic bacteria can only survive expressing the polysaccharide, which protects them from the host environment. Nevertheless, a good number of highly pathogenic Gramnegative bacteria possess an R-form LPS: Neisseria meningitidis, N. gonorrhoeae, Haemophilus influenzae, Bordetella pertussis, Campylobacter jejuni, and several other opportunistic pathogens as Pseudomonas and Burkholderia. In these species, the outer core oligosaccharide is often branched, contributes to the viability and to the membrane function, and its relative variability (similar to that of the polysaccharide in S-form LPS) leads to serological specificity. In this chapter, we describe the protocols for the extraction of both LPS forms and their chemical characterization, that is, analyses of the carbohydrate and fatty acid content, their absolute configuration, and their attachment sites. Spectroscopical approaches are out of the aim of this chapter.

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Readers should note that the protocols described in this chapter represent commonly used original referenced protocols, however, there are changes originating from state-of-art revisions created in our laboratories.

2. LPS and LOS Extraction Procedures In the course of the analysis of microbial carbohydrate, the first important step represents the extraction procedure in order to isolate the material of interest from the biomass in rather pure form. In particular, the first two protocols, phenol/chloroform/light petroleum (PCP) and phenol/triethylamine (TEA)/EDTA operate without breaking the cells which instead happens with the third one, the hot phenol/water extraction, which gives good LPS yields together with nucleic acid or protein contaminants. Consequently, the first two methods have the advantage that low amounts of nucleic acid and/or proteins are coextracted with the LPS, even though the LPS extraction may be incomplete. Then, in the case of a new bacterial strain, it is not possible to know in advance the partition behavior of its LPS molecules, and therefore applying only the PCP or hot phenol/water extraction may result in no LPS. In addition, it is advisable to operate two or more extraction protocols in tandem, with the hot phenol/ water method placed at the end of the sequence (Fig. 5.1). The three methodologies proposed yield five extraction phases (Fig. 5.1), which must be carefully checked for carbohydrate material before being discarded. Finally, LPS yields range from 0.1% to 6% (wLPS/wdry_cells), therefore higher yields might be related to higher amounts of contaminating substances.

Dry cells

PCP

Solid + Supern

PhOH / TEA / EDTA

• LOS precipitation with water • Wash the precipitate with acetone, suspend it in water and freeze dry it • Dialysis of the supernatant and freeze-drying

Solid + Supern

H2O / PhOH / 65–70 ºC

• Dialysis • NaCl • Water • Freeze-drying

• Dialysis Water phase • Freeze-drying + Phenol phase (+ solid)

• Dialysis • Removal of the solid (centrifugation) • Freeze-drying

Figure 5.1 Tandem application of the three extraction protocols to a sample. PCP and water/phenol are the most widely used, but phenol/EDTA/TEA can be inserted among the other two. Fractions resulting from the freeze-drying are those which might contain sample.

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2.1. PCP extraction of LOS (Galanos et al., 1969) This protocol is suitable for LOS isolation and it is advisable to start with dry cells which are obtained from wet bacteria by successive extraction/washing with absolute ethanol, then with acetone (twice) and diethylether (all at 20–22  C, RT), and then drying under a fume hood. Reagents and equipment 2.1.1 PCP solution (work under a fume hood): mix 90% aqueous phenol, chloroform, and light petroleum in the proportion 2:5:8 (v/v/v). If the solution appears opalescent, gradually add pieces of solid phenol until the solution is clear. 2.1.2 Stirrer 2.1.3 Centrifuge 2.1.4 Rotary evaporator 2.1.5 Freeze-drier

2.2. Methods 2.2.1 Suspend the dry biomass in the PCP solution ( 2.5%, w/v) and stir for 30 min at RT. A blender or an ultra-turrax disperser may be used, but pay attention and avoid the excessive warming of the solution. 2.2.2 Centrifuge the slurry, collect the supernatant in the evaporation flask, and treat twice the pellet as in step 1, collecting the supernatants together. 2.2.3 Save the pellet for later extraction(s). 2.2.4 Remove the low boiling solvents (chloroform and light petroleum) from the supernatant by rotary evaporation. Phenol and the water traces will remain in the evaporation flask. 2.2.5 Transfer the phenol solution in a tube suitable for centrifugation. 2.2.6 Add water dropwise to the phenol solution, slowly and mixing every time, until the LOS has precipitated. Let the sample stand every now and then for some time. Be careful and avoid phase separation, otherwise any precipitated LOS cannot be sedimented by centrifugation since it is present at the phenol/water phase border. In such cases, the whole sample has to be evaporated again and the procedure of dropwise adding water has to be repeated. If no LOS precipitates and there is the danger of phase separation, let the sample stand overnight, a precipitate may be present next morning. 2.2.7 Centrifuge the precipitate, wash it with 85% aqueous phenol, then with acetone (two to three times), let it dry, suspend it in water, and freeze-dry it. 2.2.8 With regard to the phenol supernatant, dilute it 10 times with water, dialyze it until the phenol smell has disappeared, and freeze-dry it.

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2.3. Phenol/TEA/EDTA extraction (Ridley et al., 2000) This procedure works well either on dry or on wet cells. Reagents and equipment 2.3.1 Extracting solution: 0.25 M EDTA, 5% phenol, pH 6.9, corrected with TEA 2.3.2 Dialysis tube, cutoff 12,000–14,000 2.3.3 Oven 2.3.4 Stirrer 2.3.5 Centrifuge 2.3.6 Freeze-drier

2.4. Methods 2.4.1 Equilibrate the oven with the stirrer inside and the extracting solution at 37  C. 2.4.2 Suspend the biomass in the extraction buffer (dry cell 2.5%, wet cells 20%) and stir the slurry at 37  C, 30 min. 2.4.3 Centrifuge, collect the supernatant, and extract the pellet a second time. 2.4.4 Save the pellet for further extractions. 2.4.5 Combine the two supernatants and dialyze them against 100 mM NaCl, changing the solution every 3–4 h, at least for six times (see Notes and tips). 2.4.6 Dialyze against water and recover the sample via freeze-drying. Notes and tips Point 2.4.5: EDTA is slowly removed during the dialysis process and its presence may cause the turgidity (and the rupture) of the dialysis bag. The use of ionic strength (at least 100 mM) facilitates EDTA removal and avoids the above-mentioned problems.

2.5. Hot phenol/water extraction (Westphal and Jann, 1965) This method works equally well on dry or wet cells and it is not selective for LPS or LOS. Reagents and equipment 2.5.1 2.5.2 2.5.3 2.5.4

90% phenol Dialysis tubes, cutoff 12,000–14,000 Oven Stirrer

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2.5.5 Refrigerated centrifuge 2.5.6 Freeze-drier Methods 2.5.7 Equilibrate the oven with the stirrer inside and the solutions (water and 90% phenol) at 65–70  C. 2.5.8 Suspend the biomass in the warm water and add an equal volume of phenol (final concentration: dry cell 2.5%, wet cells 20%). 2.5.9 Stir at 65–70  C for 30 min. Let it cool to RT then. 2.5.10 Centrifuge at 4  C: cell debris will sediment on the bottom and the solution will divide in two to three areas: the phenol layer (bottom), eventually a milky interphase, and the top (water) layer. 2.5.11 Separate the water layer from the rest, and add an equal volume of prewarmed water to the remaining material (milky interphase, the phenol phase, and the cell debris). 2.5.12 Repeat the extraction twice, pooling the water supernatants. 2.5.13 Dialyze (separate flasks) the water and the phenol phases until the phenol smell has disappeared and freeze-dry. 2.5.14 The exhaust pellet can be saved or discarded.

3. Purification of the Crude Extracts Mixtures of LPS, LOS, and CPS (if present) are difficult to purify. There is a general protocol to remove nucleic acids and CPS by repeated ultracentrifugation (see below); however, separation of LPS and CPS may not be obtained. In such cases, it is recommended to remove the (usually negatively charged) CPS and the nucleic acids by precipitation with Cetavlon ( Jones, 1953; Westphal and Jann 1965). However, proteins and nucleic acids may be removed enzymatically, as described in the following. The LPS or LOS obtained possess a good degree of purity and are ready for chemical characterization (scheme presented in Fig. 5.2).

3.1. Enzymatic hydrolysis Reagents and equipment 3.1.1 Digestion buffer: 100 mM Tris, 50 mM NaCl, 10 mM MgCl2, buffer at pH 7.5 with 1 M HCl 3.1.2 RNAse solution: 2 mg/ml water 3.1.3 DNAse solution 2 mg/ml water 3.1.4 Proteinase K solution: 5 mg/ml water

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Absolute configuration

GC–MS

n-Hexane (Top layer)

GC–MS Lipids and monosaccharides identification

Fatty acid methyl esters

(1) Methanolysis

LPS (2) n-Hexane extraction Methanol

Methylglycosides

(Bottom layer)

Acetylation

Acetylated methylglycosides

(1) Octanolysis (2) Acetylation

Methylation analysis (Fig.5.5)

GC–MS (Absolute configuration)

Figure 5.2 Convenient strategy for GC-MS chemical analysis of LPS (or LOS) purified samples.

3.1.5 3.1.6 3.1.7 3.1.8

Oven Dialysis tube, cutoff 12,000–14,000 Freeze-drier Centrifuge and ultracentrifuge (optional)

3.2. Method 3.2.1 3.2.2 3.2.3 3.2.4 3.2.5 3.2.6 3.2.7 3.2.8 3.2.9

Dissolve the crude extract (50 mg) in the digestion buffer (9 ml). Add RNAse and DNAse solution, 0.5 ml each. Incubate at 37  C overnight with no stirring. Add 0.1 ml of Proteinase K solution and incubate at 55  C for 4–5 h. If necessary, add a second aliquot of Proteinase K solution and prolong the incubation overnight. Dialyze and freeze-dry the sample. For separation of CPS, dissolve the sample in water at a concentration 2.5 mg/ml and centrifuge at 10,000g, 4  C, 30 min (see Notes and tips). Remove the precipitate (if present) and ultracentrifuge the supernatant at 300,000–500,000g, 4  C, overnight. Collect the supernatant, suspend the solid in the same amount of water originally used, and repeat the ultracentrifuge treatment for another two times. Do not combine the three supernatants; analyze them and the precipitate with the methodologies illustrated in the following sections.

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Notes and tips Point 3.2.6: Such ultracentrifugation treatment may lead to separation of CPS from LPS or LOS molecules. It is recommended in those cases in which the occurrence of CPS is suspected.

4. SDS-PAGE Detailed protocols for the preparation and run of an electrophoresis gel are available from the instrument manufacturer. This section will cover the two main staining procedures used for LPS detection and their adaptation to anionic polysaccharides. Performing a typical 12% SDS-PAGE followed by silver staining, LOS molecules, by virtue of their low molecular mass, migrate faster and are found almost at the bottom of the gel. The LPS banding pattern is polydisperse, due to the occurrence of molecules with a different number of repeating units in the O-antigen. As a result, LPS bands start (from the top) in the upper 30% of the gel and end with the LOS band, giving rise to the so-called ladder-like pattern. Differently from LPS or LOS, capsular anionic polysaccharides can be seen only if prestained (or fixed) with alcian blue, and usually are located above the LPS bands, although they can ‘‘invade’’ the LPS area (due to diffusion).

4.1. Kittelberg and Hilbink protocol (Kittelberg and Hilbink, 1993) Reagents and equipment 4.1.1 Rotatory shaker 4.1.2 Fixing solution: 40% EtOH, 5% AcOH 4.1.3 Oxidizing solution: 0.7% sodium metaperiodate (NaIO4) in the fixing solution (4.1.2.), 100 ml 4.1.4 0.1% Silver nitrate (AgNO3) in water, 100 ml 4.1.5 Developing solution: formaldehyde (20 ml) in 100 ml of 3% Na2CO3 4.1.6 Stop solution: AcOH 1% or 7% 4.1.7 Farmer solution: 0.3% sodium thiosulfate, 0.15% potassium ferricyanide, 0.05% Na2CO3

4.2. Methods At any step, even if not explicitly mentioned, the gel is gently shaken on a rotatory shaker.

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4.2.1 Fix the gel with the fixing solution (4.1.2) for at least 1 h. 4.2.2 Treat the gel with the oxidizing solution (4.1.3) for 10 min. 4.2.3 Wash the gel with water: 3  100 ml, 30 min each. If NaIO4 is not perfectly removed, the gel background may overstain. 4.2.4 Treat the gel with silver solution (4.1.4) for 30 min. 4.2.5 Wash the gel with water for few seconds. 4.2.6 Color development: treat the gel with the formaldehyde solution (4.1.5). The color should start to appear within a few minutes and should become more intense as time passes. The gel background might turn to gray or dark gray. Do not prolong the development for more than 30 min. 4.2.7 Stop the stain development with 1% AcOH (10 min) if the gel has to be treated with the Farmer solution afterwards. Otherwise, use 7% AcOH (10 min). 4.2.8 Wash the gel with water: 3  100 ml, 10 min each. The gel can now be stored, dried, scanned, or treated with Farmer solution. 4.2.9 To optimize staining contrast, treat the gel with Farmer solution (4.1.7) for a few seconds (see Notes and tips). 4.2.10 Stop decoloration with 1% or 7% AcOH if you wish to enhance the silver staining with a second staining passage. 4.2.11 Wash the gel with water: 3  100 ml, 10 min. 4.2.12 Store the gel, or restart from point 4.2.4. Notes and tips Point 4.2.9: Farmer solution: this solution allows to attenuate silver overstaining, especially that appearing as background. It is important to remember that the background may destain faster depending on the sample; however, excessive time exposure of the gel to the solution might eliminate everything. The destaining is reversible and the gel can be recovered starting from point 4.2.4.

4.3. Tsai and Frasch (1982) protocol Reagents and equipment 4.3.1 Oxidizing solution: 0.07% aqueous same as 4.1.2) 4.3.2 Silver nitrate solution (150 ml) 142 ml H2O, 0.7 ml 4 M NaOH, 20% AgNO3 4.3.3 Developing solution: formaldehyde 150 ml H2O

NaIO4 as fixative (150 ml, the freshly prepared as follows: 2 ml 25% aqueous NH3, 5 ml (80 ml), 8 mg sodium citrate in

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4.4. Method At any step, even if not explicitly mentioned, the gel must be gently shaken on a rotatory shaker. 4.4.1 Fix the gel with the fixing solution (4.1.2) for at least 1 h. 4.4.2 Treat the gel with the oxidizing solution (4.3.1) for 5 min. 4.4.3 Wash the gel with water: 8  100 ml, 10 min each. If NaIO4 is not perfectly removed, the gel background may overstain. 4.4.4 Treat the gel with the silver nitrate solution (4.3.2): 10 min. 4.4.5 Wash the gel with water: 4  100 ml, 10 min each. 4.4.6 Color development: treat the gel with the formaldehyde solution (4.3.3). The color should start to appear within a few minutes and should become more intense as time passes. Stop the staining with 7% AcOH (10 min) when it reaches the desired intensity. 4.4.7 Wash the gel with water, 3  100 ml, 10 min each, and store it as appropriate.

4.5. Detection of acidic polysaccharides (Min and Cowman, 1986) Reagent 4.5.1 0.05% (w/v) alcian blue in fixing solution (4.1.2)

4.6. Method 4.6.1 Wash the gel with 100 ml fixing solution for 1 h (see Notes and tips). 4.6.2 Leave the gel in the alcian blue solution (4.5.1) overnight. 4.6.3 Wash the gel with the fixing solution until the background is decolored (see Notes and tips). 4.6.4 Proceed with the preferred silver staining procedure. Notes and tips Point 4.6.1: Removal of SDS from the gel is important or precipitation of alcian may occur. Point 4.6.3: Alcian blue complexes and fixes anionic polysaccharides that will appear azure after overnight incubation. The presence of alcian blue does not affect the silver staining but may confer to this material unexpected colors (usually green).

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5. Carbohydrate Analysis: Monosaccharide Determination, Absolute Configuration, and Definition of Branching Points 5.1. Monosaccharide determination: Acetylated methyl glycosides The amounts of sample indicated refer to samples extracted from the biomass without further purification. With the analysis of acetylated methyl glycosides it is possible to detect different types of monosaccharides, that is, deoxyhexoses, hexoses, uronic acids, aminosugars, 3-deoxy-D-manno-oct2-ulosonic acid (Kdo), neuraminic acid; however, sugars, as fructose and 2-deoxy ribose, are completely destroyed. Each class of monosaccharides (hexoses, deoxyhexoses, etc.) is eluted in a particular range of the chromatogram (see Fig. 5.3), and displays the same electronic impact (e.i.) fragmentation pattern (fragmentation of different types of monosaccharides are reported in Fig. 5.4A–F). For a given mass spectrum, the peaks most relevant for the recognition of the type of residue are those at high mass values. It must be noted that the molecular ion is never observed because it is unstable and prone to fragmentation. The most diagnostic ion usually observed, even if with low intensity, is the oxonium ion descending from the molecular ion after loss of the anomeric carbon substituent, together with the secondary fragments arising from the loss of acetic acid (60 u), acetic anhydride (120 u), or ketene (42 u) (Lo¨nngren and Svensson, 1974).

220 210 200 190 180 170 160 150

ºC Hexosamine Deoxyhexosamine Dideoxyhexose

Pentose

Uronic acid

Deoxyhexose 5

10

15

Hexosaminuronic acid

Hexose 20

Time (min) 25

Figure 5.3 Elution order of the monosaccharide as acetylated methyl glycoside derivatives in the GC-MS chromatogram with the temperature program: 150  C 3 min, 3  C/min up to 280  C, on a SPB-5 column (0.25 mm  30 m, He as carrier gas). The dotted gray line indicates the temperature reached at the specific time point of the chromatographic run. Heptoses are found around 27 min, Kdo around 28 min, legionaminic acid-type residues gives at least a couple of peaks between 32 and 35 min, sialic acid is detected at 38 min.

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B

A 43

43

69

57 40

C

60

86 102

115128 139 157170187

40

D

43

157 184 213 74 87 102115 129 142 171 201

57

217 259 80 100 120 140 160 180 200 220 240 260

60

273

80 100 120 140 160 180 200 220 240 260

43

101

61 40

81 98115 145 169183 215 200 229 243 129 289 331 80 120 160 200 240 280 320

59 40

F

E 43

40

80

120

80

114 143 127 156 181199 228242 120 160 200 240

302 288 330 280 320

43

153 115 59 8199 131

84

101 143 181 213 273 197 231255 300316333

160

200

240

280

320

375 360

403 400

61 83 40

242 284 325 362 124 165 199224 266 302 343 386 414 446

80 120 160 200 240 280 300 320 360 400 440

Figure 5.4 Fragmentation pattern of acetylated methyl glycosides prepared from different types of monosaccharides: (A) pentose, (B) deoxyhexose, (C) hexose, (D) 2-aminohexose, (E) Kdo, (F) sialic acid.

Reagents and equipment 5.1.1 Screw cap pyrex tubes with caps lined with inert material (Teflon). Alternatively, pyrex tubes can be sealed by fusing 5.1.2 Anhydrous hydrochloric methanol, 1–1.25 M, n-hexane, dry pyridine, and acetic anhydride 5.1.3 Heating block 5.1.4 GC-MS equipped with Supelco SPB-5, or SPB-1 column (0.25 mm  30 m) or equivalent columns from other manufacturers

5.2. Method 5.2.1 Dry the sample (0.5–1.0 mg of crude product or 0.2 mg of purified sample) over a drying agent, under continuous vacuum, for a couple of hours (see Notes and tips). 5.2.2 Add 1 ml of methanolic hydrochloric acid and close the tube tightly. 5.2.3 Incubate the sample at 80  C overnight (see Notes and tips). 5.2.4 Cool the sample and add n-hexane until the two layers are almost equal.

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5.2.5 Collect the hexane phase (top) separately and repeat the extraction twice, combining all top layers. Follow point 6.2.1. 5.2.6 Dry the methanolic phase in a stream of air, eventually warming it gently (40  C). 5.2.7 Acetylate the dried methanolic extract with pyridine (200 ml) and Ac2O (100 ml), 80  C, 30 min. 5.2.8 Dry the products in a stream of air. Last traces of pyridine can be removed by adding few drops of toluene and evaporation. 5.2.9 Dissolve the product in acetone (100 ml) and analyze (1–3 ml) by GCMS with the temperature program 150  C 3 min, 3  C/min up to 280  C, 280  C 10 min. Notes and tips Point 5.2.1: Due to the kind of chemical cleavage, each monosaccharide will give rise to different O-methyl glycosides (a- and b-, furanose and pyranose ring) and more than one peak will be observed in the chromatogram. Point 5.2.3: Overnight incubation is necessary for samples of unknown composition. If occurrence of 2-amino-2-deoxy-sugars can be ruled out, methanolysis can proceed for 2 h.

5.3. Monosaccharide absolute configuration (octyl glycosides, see Notes and tips: Leontein et al., 1978) The identification of the absolute configuration of a monosaccharide is possible by the preparation of the appropriate octyl glycoside standard, as shown in Fig. 5.5. This approach avoids the use of rare (if available) monosaccharides and minimizes the use of the (cost-effective) enantiomeric pure alcohol. The rationale behind the use of racemic 2-octanol is to produce two diastereoisomers starting from an enantiopure monosaccharide (D or L). For instance (see Fig. 5.5), starting from D-Glc, the diastereoisomers D-Glc-(þ)-oct. and D-Glc-()-oct. are synthesized, and these two products have the same chromatographic behavior of their enantiomers, L-Glc-()-oct. and L-Glc-(þ)-oct., respectively. As a result, analysis and comparison of the chromatograms obtained after the reaction of D-Glc with the racemic and the enantiopure 2-octanol yield to the determination of the retention time of D-Glc-(þ)-oct. and L-Glc-(þ)-oct. Reagents and equipments 5.3.1 Screw cap pyrex tubes with caps lined with inert material (Teflon). Alternatively, pyrex tubes can be fused by heat 5.3.2 Acetyl chloride, pure 2-(þ)-octanol (or the enantiomer), racemic 2-()-octanol, dry pyridine, and acetic anhydride 5.3.3 Heating block

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2-(+)-octanol, H+

D-Glc-(+)-oct.

Acetyl

L-Glc-(–)-oct.

GC–MS D-Glc-(+)-oct.

2-(±)-octanol, H+ D-Glc

D-Glc-(+)-oct. D-Glc-(–)-oct.

Acetyl

L-Glc-(–)-oct. L-Glc-(+)-oct.

GC–MS

L-Glc-(+)-oct.

Figure 5.5 Strategy used for the construction of the appropriate octyl glycoside standard. D-glucose is used as an example and L-Glc is not necessary for the construction of the standard. Reaction of 2-()-octanol with D-Glc produces a mixture of diastereoisomers, D-Glc-(þ)-oct., and D-Glc-()-oct.; the retention time of these compounds is the same as the corresponding enantiomers, L-Glc-()-oct., and L-Glc-(þ)-oct., respectively (gray tone in the scheme), making the identification of the L-Glc-(þ)oct. possible without the use of this rare monosaccharide.

5.3.4 GC-MS equipped with Supelco SPB-5, or SPB-1 column (0.25 mm  30 m) or analog columns from other manufacturers

5.4. Methods Construction of an octyl glycoside standard necessary for the determination of the absolute configuration. 5.4.1 Prepare two vials, each with the same amount of the reference compound (0.2 mg). 5.4.2 Dry the samples over a drying agent, under continuous vacuum, for a couple of hours. 5.4.3 Add to one tube 100 ml 2-(þ)-octanol, and to the other tube the same amount of the racemic alcohol. 5.4.4 Add to each tube 15 ml of acetyl chloride: pipette the liquid dipping the tip in the octanol solution. 5.4.5 Close the vials and incubate overnight at 60  C 5.4.6 Remove the solvent in a stream of air warming at 40  C. 5.4.7 Acetylate with pyridine (200 ml) and Ac2O (100 ml), 80  C, 30 min. 5.4.8 Dry the products in a stream of air. Last traces of pyridine can be removed by evaporation after adding a few drops of toluene. 5.4.9 Dissolve the product in acetone (200 ml) and analyze it (1 ml) via GC-MS with the temperature program: 150  C 3 min, 3  C/min up to 280  C, 280  C 10 min.

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5.5. Determination of the absolute configuration of carbohydrates in a sample 5.5.1 In this case it is necessary to have only one vial containing the sample. It is possible to start from polysaccharide material, or better, from the acetylated methyl glycoside. 5.5.2 When starting with the pure polysaccharide, the sample is treated as from point 5.4.2. When starting from the acetylated methyl glycosides, then this step is skipped. 5.5.3 Prepare the 2-(þ)-octanol derivative as described in 5.4.3. 5.5.4 Dissolve the sample in 100 ml chloroform and inject 1 ml via GC-MS. Use the reference compound for the appropriate assignment. Notes and tips Point 5.3 The diastereoisomers obtained with glucosamine and 2-()octanol are hardly separated by GC-MS, whereas if 2-butanol is used instead, it works (Gerwig et al., 1978).

5.6. Determination of monosaccharides branching points (methylation analysis): protocol for neutral (Ciucanu and Kerek, 1984) and uronic acid containing polysaccharides The determination of the substitution pattern of one monosaccharide residue in a polysaccharide is possible by analyzing partially methylated alditol acetates (AAPM) that result after a series of reactions (Fig. 5.6). Basically, the methylation reaction transforms the available free hydroxyl functions of the polysaccharide into methyl ethers (Fig. 5.6, methylation step), N-acyl aminosugars are N-methylated as well. Uronic acids are esterified and the methyl ester function needs to be reduced before the hydrolysis step, otherwise the residue cannot be detected. The reduction reaction with NaBD4 transforms the methyl ester function in a hydroxymethyl group with two deuterium atoms (Fig. 5.6, ester reduction step). The hydrolysis step cleaves all the glycosidic linkages and partially methylated monosaccharides are released, the free hydroxyl groups that now appear are those originally involved in glycosidic linkages (Fig. 5.6, hydrolysis step). Successively, the anomeric position is marked with one deuterium atom using NaBD4 reduction, and two new hydroxyl functions appear originating from ring opening (Fig. 5.6, reduction). Acetylation of these compounds yields to the so-called AAPM. The molecular ion is never detected in the spectrum, and interpretation of GC-MS fragmentations follows few rules (given below), leading to the localization of the methyl and acetyl groups on the alditol backbone (examples in Fig. 5.7).

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6 HOH2C O 4 HO 3

5

6 HOOC

O

O 4 HO

2 OH 1

3

HOD2C H3COH2C HO H3CO

HO H3CO

O

5

Methylation

O

OCH3

O

O H3CO

O OCH3

Ester reduction (NaBD4)

O OH OCH3

OH

H3COOC

O

O H3CO

O

2 OH 1

H3COH2C

Hydrolysis

H3COH2C

OCH3

HOD2C

O

O H3CO

OCH3

O

O H3CO

O OCH3

Reduction (NaBD4) AAPM CHDOH H H3CO H H

OCH3

CHDOH H

H OH

H3CO

OH

H

H

CH2OCH3

OCH3

1

OCH3

2

H OAc

3 H3CO 4 H

OAc

5

H Acetylation

H OH

CHDOAc

H3CO H

OH

H

CD2OH

CH2OCH3

CHDOAc OCH3

H

H OAc

H

6

OAc CD2OAc

Figure 5.6 Derivatization protocol used to determine the substitution pattern of the monosaccharide residues of a polysaccharide. Carbon atoms of the monosaccharide are numbered and the same numbers are used for the corresponding hydroxyl functions, protons are omitted for clarity. At the end of the reaction sequence, the monosaccharide derivative detected is a partially methylated and acetylated alditol (AAPM). O-Methyl groups (at C-2, C-3, and C-6) indicate free hydroxyl function of the original polysaccharide; O-acetyl groups position is related to the substitution point (C-4) and on the type of the ring closure (pyranosidic, C-1 and C-5) of the monosaccharide. C-6 of a uronic residue is doubly deuterated and acetoxylated.

A

B

C

CHDOAc 277

H

233 H CO 3

OCH3 H

CHDOAc

CHDOAc 307 118 263 162

H H3CO

OCH3 H

318 118 162

H

N

233 H3CO

H

CH3 Ac

H

OAc

H

OAc

H

OAc

H

OAc

H

OAc

H

OAc

CH2OCH3

CD2OAc

159 203

CH2OCH3

Figure 5.7 Ions expected from the primary fragmentation of the AAPM descending from: (A) 4-substituted glucose, (B) 4-substituted glucuronic acid, (C) 4-substitued N-acetyl glucosamine

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– Primary fragments are originating from the carbon–carbon bond cleavage of the alditol backbone. – Intensities of the primary fragments decrease with increasing molecular mass. – The (positive) charge is placed on the methoxylated (or on the nitrogen bearing) carbon. – Fission between two methoxylated carbons is favored with respect to an acetoxylated and a methoxylated carbon, in turn favored with respect to that between two acetoxylated carbons. – For neutral and uronic descending alditols, primary fragments containing C-1 are even numbers, whereas those containing C-6 are odd numbers, the reverse is true for amino sugars when the fragment contains the nitrogen atom. – Secondary fragments are formed from the primary ones by single or consecutive eliminations of formaldehyde (30 u), methanol (32 u), ketene (42 u), acetic acid (60 u), and methyl acetate (74 u). – Elimination of methanol is observed when methoxyl group is situated at the b-position to the carbon having the formal charge, the same happens for acetoxyl group elimination. Reagents and equipment 5.6.1 Screw cap pyrex tubes with caps lined with inert material (Teflon) 5.6.2 Dry DMSO, NaOH pellets, pure CH3I, CHCl3, 2 M trifluoroacetic acid, NaBD4, pyridine, Ac2O, EtOH, MeOH, i-PrOH, glacial AcOH, 1 M HCl 5.6.3 Stirrer and stirring bars appropriate for the pyrex tube 5.6.4 Ultrasound bath 5.6.5 Centrifuge 5.6.6 Heating block 5.6.7 GC-MS equipped with Supelco SPB-5, or SPB-1 column (0.25 mm  30 m) or analog columns from other manufacturers 5.7. Methods 5.7.1 Dry the sample (0.5–1.0 mg of a purified product) together with the stirring bar over a drying agent, if possible in a thermoregulated desiccator, overnight. 5.7.2 From now on, work under a fume hood. Dissolve the sample in  1 ml of dry DMSO (see Notes and tips). 5.7.3 Pulverize in a pestle 2–3 NaOH pellets and add the content of the spun of a small spatula ( 100 mg) to the solution (see Notes and tips).

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5.7.4 Seal the sample and stir the solution for 2 h (see Notes and tips). At the end of this time perform one last sonication cycle (5 min). 5.7.5 Freeze the solution in an ice bath and add 200 ml of CH3I (see Notes and tips). 5.7.6 Leave the solution stirring for at least 2 h (if necessary, overnight) after DMSO has melted. 5.7.7 Apply a gentle stream of air for 1 h to remove CH3I. 5.7.8 Extract the solution with water (10–15 ml) and CHCl3 (1–2 ml), five times, replacing the top layer with water, each time. Phase separation is better achieved if the solution is spun in a centrifuge. 5.7.9 Dry the organic phase (see Notes and tips). 5.7.10 If the sample contains uronic acids, follow the next steps, otherwise go to point 5.7.15. 5.7.11 Dissolve the sample in 50% MeOH solution in water (1 ml) and add the tip of a small spun of NaBD4 (5 mg). Stir at 4  C, overnight. 5.7.12 Destroy the NaBD4 excess with one to two drops of 1 M HCl and dry the solution in a stream of air, eventually warming at 40  C. 5.7.13 Add 300 ml of MeOH and one drop of acetic acid and dry by warming at 40  C. Repeat three times. 5.7.14 Add 300 ml of MeOH and dry by warming at 40  C. Repeat three times. 5.7.15 Hydrolyze the sample with 2 M trifluoroacetic acid (200 ml) at 120  C, 1 h (see Notes and tips). 5.7.16 Dry the sample in a stream of air, adding few drops of i-PrOH. Do not warm: partially methylated monosaccharides produced after hydrolysis are volatile. 5.7.17 Once the sample is dried, repeat the evaporation process with i-PrOH only for three times. This treatment is necessary to remove trifluoroacetic acid traces. 5.7.18 For reduction, add to the sample the tip of a small spun of NaBD4 (5 mg) and 200 ml of EtOH. Keep the sample capped, at RT for at least 1 h. 5.7.19 Destroy the NaBD4 excess with one to two drops of glacial AcOH, and dry the solution in a stream of air. 5.7.20 Add 300 ml of MeOH and one drop of acetic acid and dry in a stream of air, without warming. 5.7.21 Add 300 ml of MeOH and dry without warming. Repeat three times. 5.7.22 Keep the sample in a desiccator, without vacuum, for 1 h. 5.7.23 Acetylate with Pyr (200 ml) and Ac2O (100 ml), at 80  C for 30 min. 5.7.24 Dry in a stream of air and add with water (4 ml) and CHCl3 (1–2 ml). 5.7.25 Extract three times with CHCl3, replacing each time the water phase. Use of a centrifuge to separate the two phases is recommended.

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5.7.26 Dry the organic phase, dissolve it in 50–100 ml of acetone and analyze 1–3 ml by GC-MS under the conditions indicated for the analysis of acetylated methyl glycosides. Please note that AAPM are volatile, do not exceed in drying the sample more than necessary. Notes and tips Point 5.7.2: Polysaccharides are not very soluble in DMSO, therefore sample dispersion is important for the outcome of the whole procedure. Alternate sonication and stirring to reach the best dispersal of the sample. Point 5.7.3: NaOH is hygroscopic and adsorbs both humidity and carbonic anhydride. When pulverizing it, work quickly and avoid to weigh the powder which would spoil your reactive. NaOH in DMSO is partly soluble, for better results stir and sonicate the solution after addition of the solid. A good result is obtained when the solution is opalescent. If a strong excess of NaOH is used, then a precipitate is observed, even though this usually does not affect the reaction outcome. In this methodology, NaOH in DMSO acts as both a dryer (for the water traces) and as a base (to deprotonate the monosaccharide hydroxyl functions). Point 5.7.4: The time contact between the sample and the NaOH is somehow dependent on the solubility of the sample in DMSO. If it is soluble, or if a good dispersion of the polysaccharide is reached easily, then stirring the solution for a couple of hours is usually enough; otherwise, it is a good practice to prolong the contact time between the reactives. Point 5.7.5: CH3I and NaOH react together and the reaction is rather exothermic. It seems that a good result is reached when the reaction proceeds slowly, so that the CH3I contact with the DMSO solution is gradual and dictated from the melting speed of the frozen solution. Point 5.7.9: If after drying, the organic phase appears deliquescent, it means that it contains still some traces of DMSO; extract it again. Point 5.7.15: Hydrolysis time may need some optimization. This condition is rather general, but amino hexoses may be underestimated. Alternatively, prolong hydrolysis time to 2 h or use 4 M trifluoroacetic acid at 100  C for 4 h. In this last case hydrolysis is rather complete and monosaccharides degradation is low.

6. Fatty Acids Compositional Analysis (GC-MS) 6.1. Total fatty acid composition by methanolysis Fatty acids occurring in LPS can be divided in essentially three different classes: (1) simple and saturated fatty acids, as 14:0; (2) C-3 hydroxylated fatty acids, as 14:0(3-OH); and (3) C-2 hydroxylated fatty acid. According

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to the formalism adopted, ‘‘14’’ identifies a 14-carbon atom fatty acid, ‘‘:0’’ indicates that the residue is saturated (without double bonds), ‘‘2-OH’’ (or 3-OH) indicates that a hydroxyl function occurs at carbon 2 (or 3) of the molecule. The assignment of different fatty acid methyl esters is possible by comparison of their retention time with those of commercially available standards (Fig. 5.8) and by analysis of their fragmentation pattern. In general, e.i. spectra of the methylester derivatives are rather informative and the molecular ion (M) is usually visible for type 1 and 2 fatty acids, although its intensity is rather low. For type 1 fatty acids, the spectrum (Fig. 5.9A) is dominated from the fragment at 74 u originated from McLafferty (ML) rearrangement, which is produced from the fission of the Cb–Cg linkage, together with the extraction of a g-proton, the protonation of the carbonyl oxygen, and the formation of a radical cation. Most of the other ions differ for 14 u and descend from carbon–carbon linkage ruptures. The series containing the methylester group is predominant. With regard to type 2 lipids (Fig. 5.9B), identification of the substitution pattern is possible observing the ML ion at 90 u, which indicates the hydroxylation at C-2. In addition, when the molecular ion is not detected, the rather intense ion at M-59 u (where 59 originates from the carboxymethyl function) indicates the length of the hydrophobic tail of the compound. The other fragmentations observed for this type of lipid are less indicative to define its structure.

35000

1

5

2

67

8

13 11 12

14 15 16

30000

19 20 21

22 23

24

18

25000 20000

3

15000

4

9 10 17

10000 5000 8.00

10.00

12.00

14.00

16.00

18.00

20.00

22.00

24.00

26.00

28.00

Figure 5.8 GC-MS chromatogram of fatty acid methyl ester derivatives commercially available. GC-MS operative conditions are those listed for acetylated methylglycosides. Compound list: (1) C12:0, (2) C13:0, (3) C12:0(2-OH), (4) C12:0(3-OH), (5) C14:0, (6) i-C15:0, (7) a-C15:0, (8) C15:0, (9) C14:0(2-OH), (10) C14:0(3-OH), (11) i-C16:0, (12) 16:19, (13) C16:0, (14) i-C17:0, (15) 17:0D, (16) C17:0, (17) C16:0(2-OH), (18) C18:29,12, (19) C18:19 cis, (20) C18:19 trans þ C18:111 (21) C18:0, (22) C19:0D, (23) C19:0, (24) C20:0. i- and a- indicate the iso- or anteiso-terminal type of the aliphatic chain of the fatty acid, respectively; D indicates the presence of a ciclopropane ring; ‘‘:19’’ means that the fatty acid possesses one unsaturation at C-9.

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A

8000

ML 74

7000 6000

87

5000 4000

M-31

3000 2000

43

55

143

1000

115 129

97

0 60

40 B 900 800 700 600 500 400 300 200 100 0

100

80

120

157 171 140

160

M-59 199 211 185

180

200

M 242

220

240

55 69 83 97

43

M-59 199

ML 90 111

125 145 40

60

80

100 120

156

140 160

M 258

213 180

200

220

240

260

C 2400 2200 2000 1800 1600 1400 1200 1000 43 800 600 400 200 0 40

103

ML 74

55 67 60

83 80

96 100

111 123 120

141 140

M-92 166 183 160

180

M-50 208 200

Figure 5.9 Electronic impact MS spectra of three fatty acid methyl esters representative of the different types of lipids occurring in LPS (or LOS): (A) C14:0, (B) C14:0(2-OH), (C) C14:0(3-OH). M ¼ molecular ion, ML ¼ fragment from McLafferty rearrangement.

Differently from type 1 and 2, type 3 lipid fragmentation pattern (Fig. 5.9C) is dominated by the ion at 103 u: this ion is originating from the Cb–Cg fission and it contains the first three carbon atoms of the fatty acid and the positive charge is stabilized from the b-hydroxyl function. The molecular ion is usually not visible, but an indication of the length of the tail

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is obtained observing the ions at M-50 (loss of water and methanol) and M-92 (loss of water, methanol, and ketene). The other fragmentations observed for this type of lipid are less indicative to define its structure. Please note that hydroxylated fatty acid methyl esters might tail on the GC column, up to be lost in baseline. This behavior is observed when the column is not performing correctly, for example, it is an aged one. Reagents and equipment 6.1.1 Screw cap pyrex tubes with caps lined with inert material (Teflon). Alternatively, pyrex tubes can be fused by heat 6.1.2 Anhydrous hydrochloric methanol (1–1.25 M), n-hexane 6.1.3 Heating block 6.1.4 GC-MS equipped with Supelco SPB-5, or SPB-1 column (0.25 mm  30 m) or analog columns from other manufacturers

6.2. Methods 6.2.1 Follow the protocol from point 5.2.1 to 5.2.5. 6.2.2 Dry the combined n-hexane phases in a stream of air. Please note that lipid methylesters are volatile, do not exceed in drying the sample more than the necessary. 6.2.3 Dissolve the product in n-hexane (100 ml) and analyze it (1-3 ml) via GC-MS with the temperature program: 150  C 3 min, 3  C/min up to 280  C, 280  C 10 min.

6.3. O-Linked fatty acid Reagents and equipment 6.3.1 Screw cap pyrex tubes with caps lined with inert material (Teflon). Alternatively, pyrex tubes can be fused by heat 6.3.2 0.5 M NaOH, 1–0.5 M HCl, chloroform, diazomethane 6.3.3 Heating block 6.3.4 Centrifuge 6.3.5 GC-MS equipped with Supelco SPB-5, or SPB-1 column (0.25 mm  30 m) or analog columns from other manufacturers

6.4. Methods 6.4.1 Dissolve the sample (2 mg) in 0.5 M NaOH (1 ml). 6.4.2 Incubate the sample at 37  C for 2 h.

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6.4.3 Add HCl to the solution to reach a pH  3. 6.4.4 Extract twice the solution with chloroform, pool the organic phases, and dry them in a stream of air. 6.4.5 Treat the dry organic phase with diazomethane, 0.3–0.4 ml for 15 min or until the solution destains. If destaining is too fast, dry the solution and repeat the treatment. 6.4.6 Dry the solution in a stream of air, dissolve the product in hexane (100 ml) and analyze it (1–3 ml) via GC-MS with the temperature program 150  C 3 min, 3  C/min up to 280  C, 280  C 10 min.

6.5. Absolute configuration determination of hydroxyl fatty acids (Rietschel, 1976) Reagents and equipment 6.5.1 Screw cap pyrex tubes with caps lined with inert material (Teflon). Alternatively, pyrex tubes can be fused by heat 6.5.2 4 M HCl, 8 M HCl, 1 M NaOH, 5 M NaOH, dry NaOH, CHCl3, methyl iodide, DMSO, thionylchloride, D- and L-phenylethylamine, pyridine 6.5.3 Heating block 6.5.4 GC-MS

6.6. Methods Treat the sample [0.5–1.0 mg of LPS or 0.1 mg of 14:0(3-OH)] as follows, or use the methylesters from methanolysis starting from point 6.6.6. 6.6.1 4 M HCl (200 ml), 2 h, 100  C. 6.6.2 5 M NaOH (200 ml), 30 min, 100  C. 6.6.3 Acidify with 8 M HCl (200 ml). 6.6.4 Extract three times with 0.5 ml of CHCl3 and combine all the three extracts in a vial, dry with air. 6.6.5 Dissolve in CHCl3 and treat the sample twice with diazomethane (under a fume hood), dry the sample. 6.6.6 Dissolve the methyl esters with in 1 ml of DMSO. 6.6.7 Add a spatula tip of pulverized NaOH. 6.6.8 Add 0.5 ml methyl iodide and stir for 30–60 min. 6.6.9 Cautiously add water, 5–10 ml. 6.6.10 Extract three times with 1 ml of CHCl3, combine the extracts. 6.6.11 Wash the CHCl3 phase one time with 50 ml of water. 6.6.12 Remove the CHCl3 in a stream of air.

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6.6.13 Add 100 ml of 1 M NaOH (in MeOH/H2O 1/1, v/v), and heat for 1 h at 85  C. 6.6.14 Add 50 ml of 4 M HCl and 400 ml of H2O. 6.6.15 Extract three times with CHCl3 and dry with a stream of air. 6.6.16 Add 50 ml of thionylchloride and incubate 10 min at 85  C. 6.6.17 Dry in a stream of air. 6.6.18 Add D- or L-phenylethylamine (0.5 ml/100 ml pyridine, without NaOH). 6.6.19 Add 50 ml of this mixture to the sample, incubate 20 min at 85  C and dry in a stream of air. 6.6.20 Add 600 ml of 1 M HCl and extract twice with 600 ml of hexane. 6.6.21 Separate and dry the organic phase, dry, dissolve it in CHCl3, analyze via GC-MS 0.2/500 ml of sample in the conditions mentioned for fatty acid methyl esters. Note that R-configured fatty acids elute first.

REFERENCES Alexander, C., and Rietschel, E. T. (2001). Bacterial lipopolysaccharides and innate immunity. J. Endotoxin Res. 7, 167–202. Ciucanu, I., and Kerek, F. (1984). A simple and rapid method for the permethylation of carbohydrates. Carbohydr. Res. 131, 209–217. Galanos, C., Lu¨deritz, O., and Westphal, O. (1969). A new method for the extraction of R lipopolysaccharides. Eur. J. Biochem. 9, 245–249. Gerwig, G. J., Kamerling, J. P., and Vliegenthart, J. F. G. (1978). Determination of the D and L configuration of neutral monosaccharides by high-resolution capillary g.l.c. Carbohydr. Res. 62, 349–357. Jones, A. S. (1953). The isolation of bacterial nucleic acids using cetyltrimethylammonium bromide (Cetavlon). Biochem. Biophys. Acta 10, 607–612. Kittelberg, R., and Hilbink, F. J. (1993). Sensitive silver-staining detection of bacterial lipopolysaccharides in polyacrylamide gels. Biochem. Biophys. Methods 26, 81–86. Leontein, K., Lindberg, B., and Lo¨nngren, J. (1978). Assignment of absolute configuration of sugars by g.l.c. of their acetylated glycosides formed from chiral alcohols. Carbohydr. Res. 62, 359–362. Lo¨nngren, J., and Svensson, S. (1974). Mass spectrometry in structural analysis of natural carbohydrates. Adv. Carbohydr. Chem. Biochem. 29, 41–106. Meredith, T. C., Aggarwal, P., Mamat, U., Lindner, B., and Woodard, R. W. (2006). Redefining the requisite lipopolysaccharides structure in Escherichia coli. ACS Chem. Biol. 1, 33–42. Min, H., and Cowman, M. K. (1986). Combined alcian blue and silver staining of glycosaminoglycans in polyacrylaminde gels: Application to electrophoretic analysis of molecular weight distribution. Anal. Biochem. 155, 275–285. Raetz, C. R., and Whitfield, C. (2002). Lipopolysaccharide endotoxins. Annu. Rev. Biochem. 71, 635–700. Ridley, B. L., Jeyaretnam, B. S., and Carlson, R. W. (2000). Sensitive silver-staining detection of bacterial lipopolysaccharides in polyacrylamide gels. Glycobiology 10, 1013–1023. Rietschel, E. T. (1976). Absolute configuration of 3-Hydroxy fatty acid present in lipopolysaccharides from various bacterial groups. Eur. J. Biochem. 64, 423–428.

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Silipo, A., Erbs, G., Shinya, T., Dow, J. M., Parrilli, M., Lanzetta, R., Shibuya, N., Newman, M.-A., and Molinaro, A. (2010). Glyco-conjugates as elicitors or suppressors of plant innate immunity. Glycobiology 20, 406–419. Tsai, C. M., and Frasch, C. E. (1982). A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal. Biochem. 119, 115–119. Westphal, O., and Jann, K. (1965). Bacterial lipopolysaccharides extraction with phenolwater and further applications of the procedure. Methods Carbohydr. Chem. 5, 83–91.

C H A P T E R

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Structural and Functional Analysis of Glycosphingolipids of Schistosoma mansoni Irma van Die,* Caroline M. W. van Stijn,* Hildegard Geyer,† and Rudolf Geyer† Contents 1. Overview 2. Isolation and Purification of S. mansoni Glycosphingolipids 3. Structural Characterization of S. mansoni Glycosphingolipids 3.1. High-performance thin-layer chromatography 3.2. Mass spectrometric analysis of glycosphingolipids 3.3. Analysis of the ceramide moiety 3.4. Survey of S. mansoni glycosphingolipid structures 4. Immunochemical Characterization of Glycan Antigens 4.1. Overlay technique for glycosphingolipids and immunostaining 4.2. Analysis of glycan antigens of glycosphingolipids by ELISA 5. Interaction of S. mansoni Glycosphingolipids with Dendritic Cell Receptors 5.1. Dendritic cells in the immune system 5.2. Preparation of human monocyte-derived immature dendritic cells 5.3. Adhesion of immature dendritic cells and K562 cells, expressing recombinant CLRs, to glycosphingolipids 5.4. Modulation of dendritic cell function by S. mansoni glycosphingolipids 6. Conclusions References

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* Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University Giessen, Giessen, Germany

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Methods in Enzymology, Volume 480 ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)80006-0

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2010 Elsevier Inc. All rights reserved.

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Abstract Glycosphingolipids are ubiquitous membrane components that play important roles in signal transduction events thereby affecting many cellular functions, including modulation of the immune response. Whereas many studies focus on the functional roles of glycosphingolipids in mammals, relatively little is known about the structures of glycosphingolipids of pathogenic organisms, and how such pathogen-derived glycosphingolipids influence immune functions of their hosts. Many different glycosphingolipids of the human parasitic helminth Schistosoma mansoni have been structurally characterized. Recent evidence indicates that glycosphingolipids isolated from different life-cycle stages of the parasite have the potential to modulate the function of human dendritic cells, a cell population that is crucial to regulate adaptive immunity in the host. A remarkable finding in this context is that glycosphingolipids derived from adult worms induce maturation of dendritic cells, in contrast to glycosphingolipids of eggs or cercariae. The glycosphingolipid-induced dendritic cell activation requires intact fucose residues on the glycolipids, and is induced via a mechanism that involves both the dendritic cell receptors TLR4 and DC-SIGN. In this chapter, we describe methods to extract glycosphingolipids from the different life-cycle stages of the parasite, techniques to separate them by thin-layer chromatography or high-performance liquid chromatography as well as strategies to structurally characterize the glycan and ceramide moieties of the glycosphingolipids. Moreover, an overview is provided of the structural diversity in the glycosphingolipid-derived glycan moieties found in this helminth. Finally, we discuss methods used to isolate monocyte-derived dendritic cells from human blood and to study the modulation of dendritic cell function by these molecules.

1. Overview Glycosphingolipids, also called glycolipids, are abundantly present in cell membranes of animals and plants. They often occur in defined microdomains in the membrane (lipid rafts, glycosynapses) and are involved in signal transduction events that affect cellular phenotypes (Hakomori, 2008; Lopez and Schnaar, 2009; Todeschini and Hakomori, 2008). Whereas the functional roles of glycosphingolipids in mammalian membranes are being unraveled rapidly, relatively little is known about the structures and the biological roles of glycosphingolipids in pathogens. In this chapter, we will focus on the structures of glycosphingolipids of the human parasitic helminth Schistosoma mansoni and their effects on the host’s immune system. Infections with parasitic helminths (worms) are a major cause for human suffering and death. Despite the pathology observed, most chronic helminth infections are relatively asymptomatic, which can be ascribed to the capacity

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of the worms to regulate the degree of inflammatory responses in their hosts. S. mansoni is a trematode (liver fluke) and the causative agent of schistosomiasis, which is the second most prevalent human parasitic disease worldwide after malaria (Chitsulo et al., 2004; Engels et al., 2002). S. mansoni infection of the mammalian host is acquired by penetration of the skin by cercariae, which are released from an intermediate snail host in water. Inside the host the cercariae transform via schistosomula into adult worms that produce hundreds of eggs daily over a life span of 5–30 years. Many eggs end up in host tissues thereby causing pathology, and a substantial amount of the eggs is secreted/excreted by the host into the environment thereby facilitating new infections. In the water, miracidia are released from the eggs that infect the intermediate snail host (Pearce and MacDonald, 2002). During infection, the immune system is continuously triggered by an array of molecules derived from different life-cycle stages of S. mansoni. Many of these molecules are glycoconjugates, proteins, and lipids carrying covalently linked glycan molecules, which are playing important roles in host–parasite interplay and induce both humoral and cellular immune responses in the host (Cummings and Nyame, 1999; Harn et al., 2009; Hokke and Yazdanbakhsh, 2005; van Die and Cummings, 2006, 2010). Mammalian glycosphingolipids are implicated in basic cellular functions but also in the pathogenesis of various diseases. Recent literature shows that S. mansoni presents unique glycolipids that strongly differ from mammalian glycosphingolipids. Such glycosphingolipids may have important roles for the development of the different helminth stages, establishment of the infection, and/or modulation of the host’s immune response. Therefore, one focus of this chapter is to summarize methods suitable for characterization of these compounds and to provide a survey of S. mansoni glycosphingolipids structures described so far. The second focus is laid on the biological activities of schistosomal glycosphingolipids as we have shown that schistosome glycosphingolipids have the potential to modulate the function of dendritic cells (DCs) via interaction with C-type lectin receptors, indicating a potential role in host– pathogen interactions.

2. Isolation and Purification of S. mansoni Glycosphingolipids Glycosphingolipids are composed of a carbohydrate moiety linked to a ceramide part, thus leading to a high variability and complexity of structures. The glycan portion may vary considerably in length, ranging from one (ceramide monohexoside, CMH) up to more than 20 monosaccharide units (Lochnit et al., 2001; Schnaar, 1994). In the same manner, the lipid

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entity usually comprises hydroxylated and/or nonhydroxylated fatty acids of varying chain lengths as well as different types of sphingoid bases (Karlsson, 1970a,b; Makita and Taniguchi, 1985). A variety of methods has been reported in the literature for the isolation and purification of glycosphingolipids (Dennis et al., 1998; Schnaar, 1994; van Echten-Deckert, 2000). S. mansoni glycosphingolipids are usually isolated from freeze-dried material obtained from the different parasitic stages, such as eggs, cercariae, and adult worms, by consecutive extraction with different organic solvents, bulk separation from lipid and nonlipid contaminants, and chromatographic fractionation. Extraction of Glycosphingolipids: In each case, 100 ml of organic solvent per gram dry weight of parasite material are used. Extraction is performed twice with chloroform:methanol:water (CMW) 10:10:1 (v/v/v) with sonication (Branson sonifier B15; Branson, Danbury, CT) of the suspension for 30 min and incubation at 50
C for 30 min, once with chloroform: methanol:0.8 M aqueous sodium acetate 30:60:8 (v/v/v) employing a 30 min sonication step and overnight incubation at 4
C, and again twice with 2-propanol:n-hexane:water 50:20:25 (v/v/v), followed by a 30 min sonication step and incubation at 50
C. After each extraction step, the sample is centrifuged at 10,000g for 10 min. The obtained sediment is subjected to the next extraction cycle, and the resultant supernatants are combined and dried by rotary evaporation (Kantelhardt et al., 2002; Meyer et al., 2005; Wuhrer et al., 2000c). Saponification of Lipid Contaminants: Contaminating phospholipid- and/ or triglyceridesters can be cleaved by mild alkali treatment leaving S. mansoni glycosphingolipids intact. To this end, obtained raw extracts are saponified using methanolic 0.1 M sodium hydroxide (100 ml per 1 g dry weight) for 2 h at 37
C. The reaction is terminated by the addition of acetic acid until the pH reaches 5–7. Desalting of Glycosphingolipids by Reversed-Phase Chromatography: Salt and hydrophilic contaminants are removed by reversed-phase chromatography (Chromabond C18ec, Macherey and Nagel, Du¨ren, Germany). For small amounts of glycosphingolipids, C18ec-cartridges (100 or 500 mg) can be used, whereas higher amounts of lipids (>0.5 g) are preferentially purified on a reversed-phase column. Cartridges or columns are washed prior to use with 10 column volumes of methanol, chloroform:methanol (CM) 2:1 (v/v), and methanol. Samples are suspended in 2 column volumes of CMW 3:98:74 (v/v/v), sonicated for 10 min, and centrifuged for 10 min at 2500g. The sediment is taken up again in CMW 3:98:74, sonicated, and centrifuged. Combined supernatants are applied to the reversed-phase material which has been equilibrated with 5 volumes of CMW 3:98:74 before use. The cartridge/column is washed with 10 volumes of water and glycosphingolipids are eluted by 5 volumes of methanol and 10 volumes of CM 2:1 (v/v).

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Purification of Glycosphingolipids by Anion-Exchange Chromatography: To remove charged impurities, glycosphingolipids are dissolved in CMW 30:60:8 (v/v/v) and applied to a QAE-SephadexA-25 column (10  80 mm, acetate form; GE Healthcare, Uppsala, Sweden) equilibrated with CMW 30:60:8 (v/v/v). The neutral glycosphingolipid fraction is collected as the flowthrough with 50 ml of CMW 30:60:8 (v/v/v) and 10 ml methanol (Wuhrer et al., 2000c). Fractionation of Glycosphingolipids by Silica Gel Chromatography: The neutral fraction of S. mansoni glycosphingolipids can be subfractionated chromatographically. To this end, samples are dissolved in CM 98:2 (v/v), sonicated, and applied on a silica gel cartridge (1 or 5 ml, depending on the amount of sample; Waters, Eschborn, Germany), equilibrated with chloroform. Stepwise elution is achieved with CM and CMW of increasing polarity, for instance with CM 90:10 (v/v), 60:40 (v/v), 50:50 (v/v), 40:60 (v/v), CMW 65:25:4 (v/v/v), and CMW 10:70:20 (v/v/v) (Meyer et al., 2005). If further fractionation is desired, additional elution steps can be employed using eluents of intermediate polarity (van Stijn et al., 2010). Alternatively, or when the fractionation on silica gel cartridges is insufficient, a porous silica gel column (Iatrobeads 6RS-8010, 10 mm, 4.6  500 mm; Macherey and Nagel) can be used at a flow rate of 1 ml/ min for separation of glycosphingolipids according to their carbohydrate and ceramide compositions. Different conditions have to be used for small neutral species or complex S. mansoni glycosphingolipids: (a) Small neutral species (ceramide mono-/dihexosides; CMH/CDH) are dissolved in CM 9:1 (v/v), and the column is equilibrated with 2% methanol in chloroform (v/v). After injection, the column is run isocratically for 15 min, and then the methanol content of the eluting solvent is increased to 38% (v/v) within a further 60 min period. Finally, the column is washed with methanol. (b) Complex glycosphingolipids (CDH) are separated using a binary linear gradient from 100% solvent A (CMW; 83:16:1, v/v/v) in 60 min to 60% solvent B (CMW 10:70:20 (v/v/v)) followed by a 20-min elution step with 100% solvent B (Meyer et al., 2005; Wuhrer et al., 2004). Resulting fractions (2 ml) are analyzed by high-performance thin-layer chromatography (HPTLC) and orcinol/H2SO4 staining (see below).

3. Structural Characterization of S. mansoni Glycosphingolipids Comprehensive structural characterization, including analysis of both carbohydrate and ceramide moieties of glycosphingolipids, is still a challenging task and requires application of different analytical strategies and

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techniques (see, e.g., Levery, 2005; Mu¨thing, 2000; Mu¨thing and Distler, 2010). Therefore, it is not intended to review comprehensively all methods available for this purpose, as this would clearly exceed the scope of this chapter. Instead, this chapter is focused mainly on the use of HPTLC and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for characterization of intact glycosphingolipids as well as MALDI-TOF-MS and electrospray ionization mass spectrometry (ESI-MS) for the analysis of free oligosaccharides derived thereof. In addition, analysis of fatty acids and sphingoid bases by gas chromatography/mass spectrometry (GC/MS) is briefly described.

3.1. High-performance thin-layer chromatography HPTLC is a robust and highly valuable tool for separation of glycosphingolipids in both analytical and preparative scale (Schnaar and Needham, 1994; van Echten-Deckert, 2000). A particular advantage of this technique is that it can be combined with mass spectrometric approaches, thus allowing a detailed structural characterization of carbohydrate and ceramide moieties (see Levery, 2005; Mu¨thing and Distler, 2010, and references therein). Furthermore, HPTLC can be easily used in the context of socalled overlay techniques (Ishikawa and Taki, 2000; Magnani et al., 1980, 1987; Mu¨thing, 1996, 1998) for studying specific binding of ligands such as antibodies, lectins, or bacterial toxins. Recent improvements of this technology even allow a sequential immunochemical detection of individual glycosphingolipids on a single HPTLC plate which are then further characterized by mass spectrometry (Souady et al., 2009). Usually different solvent systems are employed for separation of neutral glycosphingolipids and gangliosides. Complex neutral glycosphingolipids of S. mansoni can be resolved on silica gel 60 plates (Merck, Darmstadt, Germany) with chloroform:methanol:0.25% KCl 50:40:10 (v/v/v) as the developing solvent in an automatic HPTLC-developing tank (DC-MAT; Baron, Reichenau, Germany). After HPTLC separation, glycosphingolipids are routinely visualized chemically by orcinol/H2SO4 staining and iodine vapor or, alternatively, by immunostaining. Orcinol Staining: The dried HPTLC plate is sprayed moderately with a solution of orcinol (Sigma) in 2 M aqueous sulfuric acid (0.2 g orcinol/ 100 ml 2 M H2SO4) and incubated in an oven at 110
C for about 5–15 min. Glycosphingolipids are detected as purple spots with a sensitivity of approximately 200 ng total carbohydrate. Iodine Staining: The dried HPTLC plate is placed at room temperature for 30 min to 16 h in a closed chamber saturated with iodine vapor. Iodine vapor reversibly detects lipid components as yellow or brown spots with a sensitivity of about 1 mg which disappear in a warm stream of air.

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3.2. Mass spectrometric analysis of glycosphingolipids For MALDI-TOF-MS experiments, 6-aza-2-thiothymine (ATT; Sigma) is used as matrix. Matrix solution (0.5 ml; 5 mg/ml in water) is spotted onto the stainless-steel target. Glycosphingolipids are dissolved in CMW 10:10:1 (v/v/v), and 1 ml is added to a dry matrix spot under a stream of warm air. MALDI-TOF-MS experiments are performed in the positive-ion reflectron mode throughout. Typically, about 100–500 individual spectra are summarized in each case (Kantelhardt et al., 2002; Meyer et al., 2005, 2007; Wuhrer et al., 2000b,c, 2002). Due to the vast structural heterogeneity of the glycosphingolipids, usually highly complex spectra are thus obtained. Preparation of Free Oligosaccharides: Carbohydrate moieties are liberated from glycosphingolipids by treatment with recombinant endoglycoceramidase II (from Rhodococcus spp.; Takara Shuzu Co., Ltd., Otsu, Shiga, Japan). Aliquots of the complex, neutral glycosphingolipid fraction are dissolved in 200 ml 50 mM sodium acetate buffer (pH 5.0; 0.1% sodium taurodeoxycholate) and sonicated for 5 min at 50
C. After addition of the recombinant enzyme (20 ml, 40 mU), the sample is incubated at 37
C for 72 h and 20 mU of fresh enzyme are added each day. Samples are applied to a reversed-phase cartridge (see above), which is washed with 10 ml of water to obtain the released oligosaccharides. Uncleaved glycosphingolipids and free ceramides are subsequently eluted with 10 ml methanol and 20 ml CM 2:1 (v/v). Aqueous and organic phases are lyophilized or rotary evaporated to dryness, respectively. Amounts of released oligosaccharides as well as uncleaved glycosphingolipids are quantified by carbohydrate composition analysis as outlined elsewhere (Meyer et al., 2005; Wuhrer et al., 2000c). Mass Spectrometric Analysis of Oligosaccharides: For analysis of free or fluorescently tagged (Wuhrer et al., 2000c) glycans, again ATT (5 mg/ml in water) is used as matrix. Oligosaccharides are dissolved in water. One microliter of this solution is added to a fresh 1 ml droplet of matrix solution and allowed to cocrystallize with matrix in a gentle stream of cold air. MALDI-TOF-MS experiments are performed in the positive-ion reflectron mode employing the experimental conditions and instrumentation detailed elsewhere (Geyer et al., 2005; Kantelhardt et al., 2002; Lehr et al., 2007; Meyer et al., 2005, 2007; Wuhrer et al., 2000c, 2002). An example for oligosaccharide profiling by MALDI-TOF-MS is given in Fig. 6.1. For further structural characterization, individual compositional species can be analyzed by tandem mass spectrometry (MS/MS) in the laser-induced dissociation (LID) or collision-induced dissociation (CID) mode as described elsewhere (Geyer et al., 2005; Lehr et al., 2007; Meyer et al., 2005, 2007). Alternatively, released glycosphingolipid-glycans or fluorescently tagged derivatives obtained thereof may be analyzed by tandem mass spectrometry employing an ESI-ion trap-MS instrument and multiple cycles of ion

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Figure 6.1 Mass spectrometric analysis of glycosphingolipid-derived glycans. Oligosaccharides were released from glycosphingolipids of S. mansoni cercariae by endoglycoceramidase treatment and analyzed by MALDI-TOF-MS. Monoisotopic masses of pseudomolecular ions ([MþNa]þ) and deduced monosaccharide compositions are assigned using the software tools Glyco-peakfinder (Maass et al., 2007) and GlycoWorkbench (Ceroni et al., 2008). Glycan species containing a LeX-epitope (m/z 917.3, 1120.3, and 1323.3) or a pseudo-LeY-unit (m/z 1063.6, 1266.3, and 1469.3) are marked. Dark square, GlcNAc; open circle, Gal; triangle, Fuc. H, hexose; N, N-acetylhexosamine; F, fucose.

isolation and fragmentation as described previously (Wuhrer et al., 2002). An advantage of the latter experimental setup is that even oligofucosylated fragments can be easily detected as shown in Fig. 6.2.

3.3. Analysis of the ceramide moiety For fatty acid analysis, glycosphingolipids are treated with 500 ml of 1 M HCl and 10 M H2O in methanol for 16 h at 100
C according to Gaver and Sweeley (1965). Fatty acids, released as their methyl esters, are recovered by a threefold phase partition using n-hexane and dried down under a stream of nitrogen. An aliquot of the obtained fatty acid methyl esters is then

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Figure 6.2 Mass spectrometric analysis of a glycosphingolipid-derived, pyridylaminated glycan. The oligosaccharide was released from glycosphingolipids of S. mansoni eggs by endoglycoceramidase treatment, isolated after fluorescent tagging by HPLC on an amino-phase column and analyzed by ESI-MS/MS. The doubly charged pseudomolecular ion ([MþNaþH]2þ) at m/z 839.8 was subjected to further fragmentation. Obtained fragments are illustrated and assigned according to Domon and Costello (1988) using the GlycoWorkbench software tool (Ceroni et al., 2008). The diagnostically relevant Y4ßB4 fragment at m/z 664.9 is marked in bold type. PA, pyridylamine; dark square, GlcNAc; open square, GalNAc; dark circle, Glc; triangle, Fuc; þ, sodium adduct; *, proton adduct.

acetylated by incubation with 250 ml acetic anhydride overnight at room temperature in the dark, dried down under N2, purified by a threefold phase partition using n-hexane and water, and dried down again under N2. Native and O-acetylated fatty acid derivatives are analyzed by GC/MS in the positive-ion mode after either chemical ionization with ammonia or electron impact ionization using the instrumentation described elsewhere (Wuhrer et al., 2000b, 2001). For sphingoid base analysis, 2 ml of water and 500 ml of 25% NH3 are added to the lower methanol phase, and the sphingoid bases are purified by a threefold phase partition using 2 ml aliquots of chloroform. The chloroform fractions are dried down under a stream of nitrogen. After addition of 200 ml acetonitrile and

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25 ml pentafluoropropionic anhydride (Supelco, Deisenhofen, Germany), the sample is heated for 30 min at 150
C, dried, and taken up in acetonitrile. Samples are analyzed by GC using flame-ionization detection or GC/MS in the negative-ion mode after chemical ionization with ammonia or in the positive-ion mode after electron-impact ionization as described previously (Wuhrer et al., 2000b, 2001). Alternatively, free native sphingoid bases may be analyzed by MALDI-TOF-MS (Wuhrer et al., 2000b).

3.4. Survey of S. mansoni glycosphingolipid structures The glycan structures of schistosomes have been extensively reviewed elsewhere (Cummings and Nyame, 1996, 1999; Dennis et al., 2007; Hokke and Deelder, 2001; Jang-Lee et al., 2007; Khoo, 2001; Nyame et al., 2004; Wuhrer and Geyer, 2006). Therefore, only a brief summary of published glycosphingolipid structures is given here. While elongated mammalian glycosphingolipids are based on lactosylceramide (Gal(b1-4)Glc (b1-1)ceramide), S. mansoni expresses a unique GalNAc(b1-4)Glc(b1-1) disaccharide attached to ceramide which has been termed the ‘‘schisto’’ core (Makaaru et al., 1992). Besides glucosylceramide and galactosylceramide, this schisto-specific glycosphingolipid GalNAc(b1-4)Glc(b1-1)ceramide seems to be expressed in all life-cycle stages, although considerable variation in the ceramide structures is exhibited (Wuhrer et al., 2000b). For more complex S. mansoni glycosphingolipids, epitope typing with monoclonal antibodies (mAbs) and infection sera revealed a marked antigenicity and stage-specific expression of distinct glycolipid species (Weiss et al., 1986; Wuhrer et al., 1999). Elongated cercarial glycosphingolipids, for example, were shown to be based on the GalNAc(b1-4)Glc(b1-1)ceramide ‘‘schisto’’ core and contained mainly terminal Gal(b1-4)[Fuc(a1-3)] GlcNAc (Lewis X, LeX) trisaccharide units (Wuhrer et al., 2000c, Fig. 6.3). Some of these glycosphingolipids carried an additional fucose (a1-3)-linked to galactose, that is, a structural unit which has been termed a pseudo-Lewis Y motif in analogy to the Lewis Y blood group determinant (Fig. 6.3). Both Lewis Y and pseudo-Lewis Y are based on the Lewis X trisaccharide unit and differ only in the (a1-2)- or (a1-3)-linkage of a second fucose to galactose, respectively. While the majority of S. mansoni cercarial glycosphingolipids carries Lewis X or pseudo-Lewis Y units, another group of cercarial glycosphingolipids is highly antigenic and shares antigenic motifs with the large glycosphingolipids found in eggs (Weiss et al., 1986; Wuhrer et al., 1999). The precise carbohydrate structures of these glycosphingolipids, however, have not been characterized yet. Detailed structural analysis of S. mansoni egg glycosphingolipids (Khoo et al., 1997; Wuhrer et al., 2002) revealed a backbone of N-acetylhexosamine residues decorated, at least in part, with oligofucosyl side chains. In contrast to cercarial glycosphingolipids, S. mansoni egg glycosphingolipids

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Structure Gal(b1-1)Cer Glc(b1-1)Cer GalNAc(b1-4)Glc(b1-1)Cer GlcNAc(b1-3)GalNAc(b1-4)Glc(b1-1)Cer Gal(b1-4)GlcNAc(b1-3)GalNAc(b1-4)Glc(b1-1)Cer Gal(b1-4)GlcNAc(b1-3)GalNAc(b1-4)Glc(b1-1)Cer

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Fuc(a1-2)

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±Fuc(a1-2)

Figure 6.3 Carbohydrate structures and terminal epitopes of glycosphingolipids expressed by S. mansoni in different life-cycle stages. Data are compiled as described in the literature (Khoo et al., 1997; Makaaru et al., 1992; Wuhrer et al., 2000b,c, 2002). Cer, ceramide; dark square, GlcNAc; open square, GalNAc; open circle, Gal; triangle, Fuc.

are dominated by the presence of terminal Fuc(a1-3)GalNAc(b1-4)GlcNAc (F-LDN), GalNAc(b1-4)[Fuc(a1-3)]GlcNAc (LDN-F), Fuc(a1-3)GalNAc (b1-4)[Fuc(a1-3)]GlcNAc (F-LDN-F), GalNAc(b1-4)[Fuc(a1-2)Fuc(a13)]GlcNAc (LDN-DF), Fuc(a1-3)GalNAc(b1-4)[Fuc(a1-2)Fuc(a1-3)] GlcNAc (F-LDN-DF), and Fuc(a1-2)Fuc(a1-3)GalNAc(b1-4)[Fuc(a1-2)

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Fuc(a1-3)]GlcNAc (DF-LDN-DF) motifs (Fig. 6.3). A major antigenic determinant of these glycosphingolipids is recognized by the mAb M2D3H (Wuhrer et al., 1999; Wuhrer et al., 2000a), and has been identified as Fuc (a1-3)GalNAc(b1)-epitope (Kantelhardt et al., 2002). In contrast to cercarial and egg glycosphingolipids, glycolipids from adult worms are still poorly defined. So far, only respective CMH and CDH have been described, the latter of which comprise the ‘‘schisto’’ core (Makaaru et al., 1992; Wuhrer et al., 2000b). Likewise, information on the composition of the ceramide moieties of schistosomal glycosphingolipids is limited. Whilst ceramide units of S. mansoni and S. japonicum egg glycosphingolipids have been reported to be mainly composed of t18:0 or t20:0 sphingoid derivatives and C14:0, C16:0, C16h:0, and C18h:0 fatty acids (Khoo et al., 1997; Levery et al., 1992; Wuhrer et al., 2000b), CMH units of cercariae and adult worms are characterized by a greater structural heterogeneity comprising, for example, d18:0 and/or d20:0, d18:1 and t19:0 sphingoid bases in addition to t18:0 or t20:0, as well as C14h:0, C17h:0, C18:0, C24:0, and C24h:0 in addition to the fatty acid species mentioned above. Intriguingly, extended cercarial glycosphingolipids were characterized by increased amounts of long-chained fatty acids with 24–27 carbon atoms (Wuhrer et al., 2000b).

4. Immunochemical Characterization of Glycan Antigens 4.1. Overlay technique for glycosphingolipids and immunostaining Following HPTLC separation, glycosphingolipids can be visualized with high sensitivity and specificity by the overlay technique using epitope-specific antiglycan antibodies. Many mAbs have been described that recognize glycan antigens of S. mansoni (Bickle and Andrews, 1988; Nyame et al., 1999, 2000; van Remoortere et al., 2000; van Roon et al., 2005). In the case of schistosomal glycosphingolipids mAbs recognizing distinct carbohydrate motifs, such as GalNAc(b1-4)GlcNAc (LDN), Fuc(a1-3)GalNAc(b1-4)GlcNAc (F-LDN), GalNAc(b1-4)[Fuc(a1-3)]GlcNAc (LDN-F), Fuc(a1-3)GalNAc(b1-4)[Fuc (a1-3)]GlcNAc (F-LDN-F), GalNAc(b1-4)[Fuc(a1-2)Fuc(a1-3)]GlcNAc (LDN-DF), and Gal(b1-4)[Fuc(a1-3)]GlcNAc (Lewis X; LeX), or polyclonal antibodies directed, for example, against soluble egg antigens (SEA) of S. mansoni are frequently employed. To enable the necessary multiple incubations of the plate in aqueous solutions without flaking of the silica gel sorbent, the plates have to be fixed by incubation in a hydrophobic polymer solution. To this end, the plate is dried thoroughly after HPTLC separation in a stream of warm air and dipped carefully for 60 s in a solution of 0.5%

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polyisobutylmethacrylat (Plexigum P28; Aldrich) in n-hexane:chloroform 9:1 (Baumeister et al., 1994; Ishikawa and Taki, 2000; Mu¨thing, 1998). After drying in a stream of warm air for at least 2 min the immersion is repeated once. The plate is blocked for 1 h at room temperature with PBS (100 mM sodium phosphate buffer, pH 7.2, 150 mM NaCl) containing 0.5 % Tween 20 (Sigma) and 2 % bovine serum albumin, and incubated with the primary antibody for at least 2 h at room temperature (Wuhrer et al., 1999). Suitable primary antibody concentrations have to be individually optimized in order to achieve sensitive staining without overloading the plate. Subsequently, horseradish peroxidase-coupled, anti-mouse immunoglobulins (Ig) or anti-rabbit Ig (Dako Diagnostics, Hamburg, Germany) can be used as secondary antibodies (Wuhrer et al., 1999, 2000c). Following secondary antibody incubation, the plate is washed twice with PBS and equilibrated once for 5 min with 100 mM sodium citrate buffer, pH 6.0. For staining, 240 ml of a substrate stock solution (97.5 mg of 4-chloro-1-naphthol and 60 mg diethylphenylenediamine (both from Sigma) in a mixture of 9 ml acetonitrile and 1 ml methanol, stored at 20
C) and 8 ml of 30% H2O2 (Merck) are added to 10 ml of sodium citrate buffer. The plate is overlaid with this substrate solution and bound secondary antibody is visualized by a blue precipitate (Conyers and Kidwell, 1991). Alternatively, alkaline phosphatase-coupled secondary antibodies can be applied, and binding is visualized by use of 10 mg of 5-bromo-4-chloro-3indolyl phosphate (Biomol, Hamburg, Germany) and 5 mg nitro-blue tetrazolium chloride (Sigma) as substrates in 10 ml of 100 mM glycine buffer, pH 10.4, containing 1 mM ZnCl2 and 1 mM MgCl2 (Wuhrer et al., 1999, 2000c, 2001, 2004). An example of this type of analysis is shown in Fig. 6.4 for glycosphingolipids obtained from S. mansoni cercariae, eggs, and adult worms utilizing mAbs with different carbohydrate-binding specificity.

4.2. Analysis of glycan antigens of glycosphingolipids by ELISA ELISA represents a fast method to identify the presence of known glycan antigens within more complex glycans on glycosphingolipid stages of S. mansoni, using similar antiglycan antibodies as described above, or (commercial) peroxidase-conjugated/biotinylated lectins. Glycosphingolipids are diluted in ethanol, sonicated, and coated (1–10 ng/well) on a NUNC maxisorb plate (Roskilde, Denmark) for 1 h at 37
C, or at room temperature until dryness is reached. After coating, a standard ELISA method can be used. Plates are blocked with 1% ELISA grade BSA (Fraction V, fatty acid free; CalBiochem, San Diego, CA) in TSM (20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2) for 30 min at 37
C. The use of fat-free milk as blocking agent and diluent should be avoided, since the presence of milk-oligosaccharides may inhibit binding of antiglycan antibodies. Subsequently, the glycosphingolipids are incubated for 1 h at 37
C

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3 a

b 4

F-LDN

b 4

3 a

Lex

Eggs Eggs Adults Adults Cercariae Cercariae

Figure 6.4 HPTLC-immunostaining of complex S. mansoni glycosphingolipids. HPTLC-resolved glycosphingolipids from S. mansoni cercariae, adult worms, and eggs are visualized by immunostaining using monoclonal antibodies directed against Fuc(a1-3)GalNAc(ß1-4)GlcNAc (F-LDN) or Gal(ß1-4)[Fuc(a1-3)]GlcNAc (LeX) epitopes. Dark square, GlcNAc; open square, GalNAc; open circle, Gal; triangle, Fuc.

with antiglycan mAbs, followed by incubation with peroxidase-coupled goat-anti-mouse Ig. After extensive washing with TSM/0.1% Tween-20, the binding is detected by adding 100 ml/well tetramethylbenzidine (TMB) substrate (10 ml 0.1 M sodium acetate/citric acid buffer, pH 4.0 þ 100 ml TMB [10 mg/ml in DMSO] þ 1 ml H2O2 [30%]). The color reaction is stopped after 5–10 min by adding 50 ml/well 0.8 M sulfuric acid, and optical density is measured by a spectrophotometer at 450 nm. Similar approaches can be followed using commercial peroxidase-labeled lectins, or recombinant chimaeric lectins, such as DC-SIGN-Fc, as described (Geijtenbeek et al., 2002; van Die et al., 2003).

5. Interaction of S. mansoni Glycosphingolipids with Dendritic Cell Receptors 5.1. Dendritic cells in the immune system DCs are crucial for the initiation of innate and adaptive immune responses, and control the immune balance between inflammation and tolerance (Kapsenberg, 2003). DCs capture and process putative pathogenic and

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other ‘‘foreign’’ antigens in peripheral tissues throughout the body. This interaction modulates the DC to provide naı¨ve T cells with a polarization signal, thereby instructing the T cell to develop into particular T cell subsets such as T helper (Th) and T regulatory (T-reg) cells. Immature DCs (iDCs) survey peripheral tissues to detect pathogens and after recognition of pathogens they migrate to T cell-rich areas in the lymph nodes to activate naı¨ve T cells. During the migration process they ‘‘mature’’ by downregulation of surface molecules (such as DC-SIGN (DC-specific ICAM3-grabbing nonintegrin)) involved in antigen uptake, and upregulation of molecules (such as CD86) that are important to activate T cells. In general, maturation signals are provided by interaction of a specific class of receptors on DCs, the Toll-like receptors (TLRs), with ‘‘foreign’’ antigens (danger signals) (Medzhitov and Janeway, 2000; Trinchieri and Sher, 2007). In addition, DCs use a family of calcium-dependent carbohydrate (glycan)binding proteins called C-type lectins (CLRs) as receptors for glycosylated antigens (Weis et al., 1998). Triggering of CLRs plays an important role in determining the phenotype of the DCs by cross talk with TLRs which is critical for the outcome of the immune response (Gringhuis et al., 2007; van Kooyk, 2008; van Vliet et al., 2008). We have studied the interaction of CLRs of DCs with S. mansoni glycosphingolipids and showed that S. mansoni glycosphingolipids differentially modulate the function of DCs.

5.2. Preparation of human monocyte-derived immature dendritic cells To analyze the ability of schistosome glycosphingolipids to interact with human DCs, monocytes are isolated from buffycoats of healthy donors. The packed cells (50 ml) of one buffycoat (Sanquin, Amsterdam, the Netherlands) are resuspended in PBS containing 1% citric acid monohydrate, to a final volume of 150 ml. The cell suspension is carefully layered onto a layer of Lymphoprep (Axis-Shield, Oslo, Norway), in 50 ml tubes (25 ml cells/ 10 ml of Lymphoprep), and the tubes are centrifuged at 700g for 30 min without brake. The layer of peripheral blood mononuclear cells (PBMCs) on top of the Lymphoprep gradient is collected and washed several times in PBS with 1% citrate. To isolate the monocytes from the PBMCs, the PBMCs are taken up in PBS containing 2 mM EDTA and 1% fetal calf serum (FCS) (40 ml/107 cells), and anti-CD14 magnetic microbeads (5 ml beads/107 cells MACS, Miltenybiotec, Bergisch Gladbach, Germany) are added. After an incubation of 30 min at 4
C, the cells are washed and passed over a MACS column standing in a magnet. The MACS column is subsequently taken out of the magnet and the CD14 positive cells are collected. The obtained monocytes are cultured for 5 days in the presence of IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (500 and 800 U/ml, respectively; Miltenybiotec) in RPMI 1640 (Gibco Life

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Figure 6.5 Dendritic cell characterization. Human monocytes were incubated with IL-4 and GM-CSF for 5 days and the expression of DCs markers was measured to assess the phenotypical change from monocytes to iDCs. Monocytes and iDCs (cultured for 5 days) were characterized by FACS analysis for the expression of the CLRs DC-SIGN and MGL, using anti-DC-SIGN and anti-hMGL mAbs. The gray histogram shows iDCs stained as indicated, and the open figures represent unstained cells. From each time point 105 cells are washed with PBS containing 0.05% BSA and incubated for 30 min at 4
C with AZN-D1 (anti-DC-SIGN mAb, Geijtenbeek et al., 2000b) or 18E4 (antiMGL mAb, van Vliet et al., 2006a). After washing, the cells were incubated for 30 min at 4
C with rabbit-anti-mouse IgG1-PE. The unbound antibodies were washed away and cells were analyzed by flow cytometry by FACSScan. During the differentiation of monocytes to DCs, both MGL and DC-SIGN are upregulated (van Vliet et al., 2006b).

Technologies, Gibco-BRL, Eggenstein, Germany) containing 10% FCS, 10,000 U/ml penicillin, 10,000 U/ml streptomycin (BioWhittaker Inc. Walkersville, MD, USA), and 10,000 U/ml glutamine (Invitrogen, Carlsbad, CA, USA), to obtain iDCs. During differentiation from monocytes to iDCs, the surface expression of the CLRs DC-SIGN and MGL (macrophage galactose-type C-type lectin) is strongly upregulated (Geijtenbeek et al., 2000b; van Vliet et al., 2006b). To assess the phenotype of the cells, the expression of these CLRs is routinely measured at day 0 (monocytes) and day 5 (iDCs) (Fig. 6.5).

5.3. Adhesion of immature dendritic cells and K562 cells, expressing recombinant CLRs, to glycosphingolipids To assess the physical interaction between DCs and S. mansoni glycosphingolipids derived from eggs, cercariae, and adult worms, the glycosphingolipids are diluted in ethanol, sonicated, and coated (3 ng/well) on a NUNC

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maxisorb 96-well plate for 1 h at 37
C until the ethanol is completely evaporated. The coated plates are washed and blocked with 1% ELISA graded BSA in TSM (20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2) for 30 min at 37
C. To visualize the binding to the glycosphingolipids, DCs are labeled with a fluorescence dye, Calceine-AM (25 ml/7  106 cells; Molecular Probes, Eugene, OR), for 15 min at 37
C and incubated with the coated glycosphingolipids for 1.5 h at 37
C. The nonadherent cells are gently washed away and subsequently the adherent cells are lysed (50 mM Tris–HCl, pH 7.4, 0.1% SDS). The fluorescence released from the DCs is quantified on a Fluostar spectrofluorimeter (BMG Labtech, Offenburg, Germany) at 485/520 nm. The fluorescence of total added DCs is set at 100%. The degree of DC binding to the glycosphingolipids is expressed as a percentage of the fluorescence of total added DCs. An example of a DC-binding experiment is shown in Fig. 6.6, demonstrating that iDCs bind to glycosphingolipids of adult worms and cercariae, but not to egg glycosphingolipids (Meyer et al., 2005; van Stijn et al., 2010). To define the contribution of CLRs expressed by the iDCs in the binding to the glycosphingolipids, the binding assay is performed in the presence of EDTA (to assess the Ca2þ dependence of the binding), or by performing the binding experiment in the presence of specific antibodies that inhibit the binding, for example, AZN-D1 (an anti-DC-SIGN mAb). The data in Fig. 6.6 show that DC-SIGN is involved in the binding of iDCs to

Adhesion iDCs (%)

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Figure 6.6 Dendritic cell adhesion to S. mansoni glycosphingolipids. Binding of immature DC to the glycosphingolipids of different life-cycle stages of S. mansoni was determined by a solid-phase adhesion assay. iDCs did not bind to the glycosphingolipids of S. mansoni eggs. However, iDCs strongly bound to glycosphingolipids from adult worms and cercariae. The binding to these glycosphingolipids was completely blocked by preincubation of the iDCs with the DC-SIGN-specific mAb AZN-D1 (20 mg/ml), indicating that DC-SIGN plays a prominent role in recognition of these glycosphingolipids by iDCs.

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glycosphingolipids of both cercariae and adult worms (Meyer et al., 2005; van Stijn et al., 2010). In subsequent studies, we showed that DC-SIGN recognizes both Lewis X and pseudo-Lewis Y antigens of cercarial glycosphingolipids (Meyer et al., 2005). The structural characterization of the DC-SIGN-binding glycosphingolipids of adult worms is underway. A similar cell adhesion ELISA can be performed with cell lines expressing recombinant CLRs, such as K562 cells expressing recombinant L-SIGN or DC-SIGN (Geijtenbeek et al., 2000a). L-SIGN is a CLR that shows a high sequence identity with DC-SIGN, which is not expressed on DCs but on human liver sinusoidal endothelial cells. We have demonstrated that K562 cells expressing DC-SIGN, similar to iDCs, do not bind to egg glycosphingolipids. However, K562 cells expressing L-SIGN bind highly fucosylated egg glycosphingolipids of S. mansoni (Meyer et al., 2007). These data indicate that DC-SIGN and L-SIGN show a different glycan specificity, which has also been observed in other studies (Guo et al., 2004; Van Liempt et al., 2004).

5.4. Modulation of dendritic cell function by S. mansoni glycosphingolipids The cell adhesion ELISA demonstrates which cellular surface receptors may enable recognition of S. mansoni glycosphingolipids. However, these findings cannot predict whether these interactions have functional consequences. To assess the potential of S. mansoni glycosphingolipids to modulate the function of iDCs, the cells are incubated with the glycosphingolipids, in the presence or absence of TLR agonists. In these assays, iDCs are plated in a round-bottom 96-well plate (105 cells/ml, 100 ml/well; NUNC) and left to settle for 30 min at 37
C. S. mansoni glycosphingolipids (diluted in ethanol) are subsequently sonicated for 30 s and immediately added to the wells (concentrations ranging from 0.1 to 1.5 mg/ml), and incubated at 37
C. As a control for maturation of the iDCs, cells that are stimulated only with LPS from E. coli 0111:B4 (10 ng/ml; Sigma-Aldrich, St. Louis, USA) are included. After 24 h incubation, the DCs are tested for their expression of maturation markers such as CD80, CD86, and MHC class II. The cells are washed in PBS and incubated with anti-CD80-PE, anti-CD86-PE, or anti-HLA-DR-FITC antibodies (BD biosciences Pharmingen, San Jose, CA, USA) for 30 min at 4
C. Unbound antibodies are washed away and cells are analyzed for binding of the labeled antibodies by flow cytometry (FACSScan). In addition to the upregulation of surface molecules, DCs produce cytokines upon activation. The production of cytokines by DCs is a sensitive readout to assess modulation of DC function. The induction of specific cytokines can be assessed by PCR to detect upregulation of mRNA expression, but it is more reliable to evaluate the levels of secreted protein. Cytokines are measured in the culture

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medium after 24 h culture of iDCs with pathogen-derived molecules using ELISA, or a multiplex bead immunoassay (Invitrogen). In the latter method, a whole array of cytokines is measured simultaneously. A mixture of antibody-conjugated beads (each specific for one cytokine) is added in one well of a 96-well filter plate and the beads are washed. Cytokinecontaining medium, or the manufacturers standards (Human TwentyFive-Plex), are added to the antibody-conjugated beads and specific biotinylated detection antibodies. The plate is incubated for 3 h at room temperature (orbital shaker at 500 rpm shielded from light). After the incubation the unbound medium components and antibodies are washed away. The beads are subsequently incubated with R-Phycoerythrin conjugate for 30 min at room temperature (orbital shaker at 500 rpm shielded from light). The amount of cytokines present in the medium is measured by Luminex100 (Bio-Rad, Hercules, CA, USA) and compared with the included standards. Using these procedures, we showed that glycosphingolipids of adult S. mansoni worms activate DCs as deduced from the upregulation of the maturation markers CD80, CD83, or CD86 (Fig. 6.7) and the production of cytokines (van Stijn et al., 2010). Glycosphingolipids from S. mansoni adult worms trigger the secretion of a broad panel of cytokines including IL-12 p40, IL-10, IL-1b, IL-6, IL-8, and TNF-a (van Stijn et al., 2010). By contrast, egg and cercarial glycosphingolipids are not able to induce DC activation using similar conditions (Fig. 6.7). To assess the role of CLR or TLR surface receptors in DC maturation and cytokine production, blocking antibodies against these surface receptors can be used by adding them to the iDCs 30 min prior to incubation with the glycosphingolipids. This blocks the interaction of the glycosphingolipids

80 60 40 20 0

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Figure 6.7 Activation of dendritic cells by S. mansoni glycosphingolipids. Human immature DCs were incubated with different concentrations of S. mansoni glycosphingolipids (GL) of different life-cycle stages of S. mansoni. After 24 h the expression of CD86 on the DCs was determined by FACS analysis, and shown as a percentage of the expression of CD86 induced by 10 ng/ml LPS (100%). A representative experiment is shown, out of at least three experiments, using DCs derived from monocytes isolated from different donors.

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with the respective surface receptors. The results of such experiments contributed to our conclusion that both TLR4 and DC-SIGN are essential receptors by which worm glycosphingolipids induce DC maturation and cytokine production (van Stijn et al., 2010).

6. Conclusions S. mansoni glycosphingolipids are characterized by both high structural variability and multiple biological activities. In order to correlate observed immunomodulatory properties with defined structural features, more detailed information is required with regard to a potentially life-cycle stage-dependent expression of distinct carbohydrate and ceramide entities. Using this information, targeted synthetic chemical and/or chemoenzymatic approaches could lead to better defined schisto-type glycosphingolipids or derivatives thereof which would allow more in-depth studies on structure/function relationships of this class of molecules. In a long term, this knowledge could be eventually used to interfere with the highly successful evasion and survival strategies of this parasite, thus enabling a more efficient treatment of schistosomiasis.

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Biotoxicity Assays for Fruiting Body Lectins and Other Cytoplasmic Proteins ¨nzler,* Silvia Bleuler-Martinez,* Alex Butschi,† Markus Ku ¨thy,* Michael O. Hengartner,† and Mattia Garbani,* Peter Lu Markus Aebi* Contents 142 143 145 147 147 148 148 148

1. Overview 2. Expression of Fruiting Body Lectins in E. coli 3. Toxicity Test Toward the Insect A. aegypti 4. Toxicity Test Toward the Nematode C. elegans 5. Toxicity Test Toward the Amoeba A. castellanii 6. Statistics Acknowledgments References

Abstract Recent studies suggest that a specific class of fungal lectins, commonly referred to as fruiting body lectins, play a role as effector molecules in the defense of fungi against predators and parasites. Hallmarks of these fungal lectins are their specific expression in reproductive structures, fruiting bodies, and/or sclerotia and their synthesis on free ribosomes in the cytoplasm. Fruiting body lectins are released upon damage of the fungal cell and bind to specific carbohydrate structures of predators and parasites, which leads to deterrence, inhibition of growth, and development or even killing of these organisms. Here, we describe assays to assess the toxicity of such lectins and other cytoplasmic proteins toward three different model organisms: the insect Aedes aegypti, the nematode Caenorhabditis elegans, and the amoeba Acanthamoeba castellanii. All three assays are based on heterologous expression of the examined proteins in the cytoplasm of Escherichia coli and feeding of these recombinant bacteria to omnivorous and bacterivorous organisms. * Institute of Microbiology, Department of Biology, ETH Zu¨rich, Zu¨rich, Switzerland Institute of Molecular Life Sciences, University of Zu¨rich, Zu¨rich, Switzerland

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Methods in Enzymology, Volume 480 ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)80007-2

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2010 Elsevier Inc. All rights reserved.

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1. Overview The recognition of non-self-carbohydrate structures by endogenous lectins is being recognized as a basic and probably very ancient strategy in the defense of organisms against competitors, predators, parasites, and pathogens. Many plant lectins have been shown to exhibit insecticidal or nematicidal activity and thus to act as direct effector molecules in the defense of plants against herbivorous predators and parasites (Michiels et al., 2009; Peumans and Van Damme, 1995). Expression of these lectins is often spatially and temporally regulated in that they are synthesized in specific organs (bark, leaves, seeds, etc.) of the plant and/or are induced, often systemically, by herbivore attack (Oka et al., 1997; Vandenborre et al., 2009). At subcellular level, most of these lectins are localized in the vacuole of the plant cell but there are also recent reports about cytoplasmic plant lectins (Lannoo and Van Damme, 2010). Lectins have also been implicated in innate immunity of animals (Dann and Eckmann, 2007; Kurata et al., 2006; Pace and Baum, 2004; Schulenburg et al., 2008; Vasta, 2009; Wilson et al., 1999; Zhu et al., 2006). These lectins are, similar to plant lectins, often induced upon parasite or pathogen attack but usually rely on additional molecules or cells to exert their role in defense (Ao et al., 2007; French et al., 2008; Ideo et al., 2009; Kim et al., 2006; Tsuji et al., 2001, 2007). However, recent demonstrations of the antimicrobial activity and pathogeninduced expression of human mucosal lectins (Cash et al., 2006; French et al., 2008; Ideo et al., 2009; Kohatsu et al., 2006; Stowell et al., 2010; Tsuji et al., 2001) suggest that the function of lectins as direct effectors in defense is also conserved in animals and may be especially important in cases where adaptive immunity fails (Stowell et al., 2010). Recent studies now demonstrate that lectin-mediated defense is also conserved in fungi (Hamshou et al., 2009; Trigueros et al., 2003; Wang et al., 2002). Since plants and fungi are (1) nonmotile organisms and therefore at the mercy of predators and parasites and (2) do not have additional defense strategies such as circulating immune cells or adaptive immunity, these organisms have elaborated this defense strategy and thus are a rich source of lectins. Most fungal lectins, identified so far, belong to the class of fruiting body lectins which are characterized by their spatial and temporal expression pattern and their subcellular localization: these lectins accumulate in fungal reproductive organs, that is, the fruiting bodies or the sclerotia, and are synthesized on free ribosomes in the cytoplasm (Goldstein and Winter, 2007). Preliminary data suggests that their expression can be induced by fungivory (S. Bleuler-Martinez, M. Ku¨nzler and M. Aebi, unpublished data). Fungal predators and parasites comprise parasitic bacteria and fungi, amoeba, nematodes, insects, mites, springtails, slugs, and mammals. In order to assess the toxicity of fruiting body lectins and other cytoplasmic proteins toward such organisms, we have developed biotoxicity assays toward three different model organisms: the insect Aedes

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Strategy

Fungus

E. coli

Bacterial predator

Aedes aegypti (Insect)

Caenorhabditis elegans (Nematode)

Acanthamoeba castellanii (Amoeba)

Feeding setup

Bacterial suspension

Bacterial lawn

Bacterial lawn

Larval survival

Larval development

Area of clearing zone

Score

Figure 7.1 Concept of assaying the biotoxicity of fruiting body lectins toward model organisms. The fungal lectins (indicated by the four-circle symbol) were expressed in the cytoplasm of E. coli strain BL21(DE3) and the recombinant bacteria were fed to the indicated omnivorous and bacterivorous model organisms. Bacteria were applied either as suspension in water (A. aegypti) or as lawn on agar-solidified medium (C. elegans and A. castellanii). Lectin-mediated toxicity was assessed by scoring larval survival (A. aegypti), larval development (C. elegans), or the area of the clearing zone in the bacterial lawn (A. castellanii). The surface electron micrograph of E. coli was provided by E. Fischer (NIH/NIAID), the light micrograph of A. castellanii by D. Patterson (MBL), and the light micrograph of the A. aegypti imago by J. Gathany (US CDC).

aegypti, the nematode Caenorhabditis elegans, and the amoeba Acanthamoeba castellanii. All three assays are based on the feeding of omnivorous and bacterivorous organisms with recombinant Escherichia coli bacterial cells expressing the heterologous protein in their cytoplasm (see Fig. 7.1). The C. elegans biotoxicity assay can be combined with C. elegans genetics to identify the in vivo target of nematotoxic lectins and toxins (Barrows et al., 2006; Butschi et al., 2010; Griffitts et al., 2005; Titz et al., 2009).

2. Expression of Fruiting Body Lectins in E. coli Examined fruiting body lectins were either previously characterized or newly isolated based on carbohydrate-binding (by carbohydrate-affinity chromatography) or abundance (by comparative 2D-gelelectrophoresis)

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from soluble protein (SP) extracts of fruiting bodies or sclerotia of fungi of which an annotated genome sequence was available. Isolated proteins were identified by separation over SDS-PAGE, tryptic in-gel-digestion of the protein, extraction and m/z determination of the peptides by LC-ESI–MS, and matching of the peptide m/z values with the predicted proteome of the respective fungus. Double-stranded cDNA coding for the identified proteins was synthesized from mRNA using the OneStep RTPCR Kit (Qiagen) with specific primers designed on the basis of the available genome sequence and cloned using the pGEMÒ-T Easy Vector System (Promega) and the E. coli cloning strain DH5a according to the manufacturers’ protocols. The sequence of the cloned cDNA was verified by DNA sequencing. The verified cDNAs were amplified using specific primers carrying suitable restriction sites (and eventually sequences encoding an N- or C-terminal His8-tag for affinity purification of the protein) and recloned into the pET24 E. coli expression vector (Novagen). The expression plasmids were verified by DNA sequencing and transformed into the E. coli expression strain BL21(DE3; Studier et al., 1990). Protein expression was typically achieved by cultivating the E. coli transformants to OD600 nm of 1 in Luria-Bertani (LB) broth containing 50 mg/l Kanamycin at 37
C, adding 1 mM IPTG and further incubating the cultures, depending on the protein expressed, for a period of 4–14 h at a temperature of 23–37
C. Protein expression and solubility were assessed by harvesting the equivalent of 20 ml of cells with an OD600 nm of 1 and resuspending the cells in 2 ml of phosphate-buffered saline (PBS) containing 1 mM of phenylmethanesulfonylfluoride (PMSF). A whole-cell extract (WCE) was prepared by mixing 100 ml of this cell suspension with an equal volume of La¨mmli sample buffer (Laemmli, 1970), boiling the cell suspension for 5 min at 95
C and sonicating the lysate for 5–10 s at 15 amplitude microns. SPs were isolated by mixing of 1 ml of the cell suspension with 1 g of glass beads (0.1 mm diameter, BioSpec), lysing the cells by spinning the mixture for 35 s at level 6 in a FastPrep device (Savant), spinning the cell lysate for 5 min at 5000  g and collecting the supernatant. This low-speed supernatant was spun for 30 min at 16,000  g. Hundred microliters of the high-speed supernatant was mixed with an equal volume of La¨mmli sample buffer and boiled for 5 min at 95
C. Twenty microliters of each WCE and SP was run out on an SDS-PAGE and the proteins were stained with Coomassie Brilliant Blue. Expression of the recombinant protein was indicated by the presence of a strong band at the expected molecular weight in the WCE sample compared to a WCE sample derived from a BL21(DE3) control transformant with empty pET24. Solubility of the recombinant protein was judged by comparing the relative band intensities of the recombinant protein and the endogenous bacterial proteins between and within the SP samples. These control experiments are crucial, since the toxicity of the

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recombinant protein strictly correlates with its expression and solubility in the bacterial host. This was exemplified by the analysis of the fruiting body lectins XCL and CGL2 from the mushrooms Xerocomus chrysenteron and Coprinopsis cinerea (Butschi et al., 2010; Trigueros et al., 2003), respectively, at two different induction conditions (see Fig. 7.2). In contrast to CGL2, whose expression, solubility, and toxicity were hardly affected by the induction conditions, XCL was well expressed, soluble, and very toxic upon induction for 14 h at 23
C but only poorly expressed, hardly soluble, and absolutely nontoxic upon induction for 5 h at 37
C.

3. Toxicity Test Toward the Insect A. aegypti Eggs of A. aegypti isolate Rockefeller were obtained from the Swiss Tropical and Public Health Institute (Basel, Switzerland). The eggs were hatched by preincubation of 30 mg grinded (by mortar and pestle) fish food (Vitakraft) in 200 ml deionized water in a suitable, open glass container, for example, a glass petri dish, for 20 h at 28
C before addition of another 30 mg of grinded fish food and approximately 600–800 eggs and incubation for another 18 h at 28
C. To avoid stress, eggs and larvae were shielded from direct light exposure during incubations. Hatched larvae were transferred to 700–800 ml of fresh deionized water by pipetting. After adding 50 mg grinded fish food, the larvae were incubated for another 10 h at 28
C. This schedule yielded a nearly synchronous population of L2 larvae. For each data point, 10 L2 larvae were transferred to 100 ml of fresh deionized water in suitable, open glass containers, for example, 100 ml Schott flasks, and starved by incubating for another 6 h at 28
C without the addition of any food. For statistical evaluation of the results, five replicates of each data point were performed. To set up the assay, 100 ml of BL21(DE3) cells expressing the recombinant proteins were prepared as described above, washed once with sterile deionized water to remove the bacterial growth medium, and adjusted to OD600 nm of 20. Protein expression was controlled in each experiment by preparing and analyzing a WCE sample of the bacterial suspension as described above. One milliliter of bacterial suspension (OD600 nm ¼ 20) was added to 100 ml of water containing the larvae and the flasks were incubated for 96 h at 28
C in the dark. After this period, the number of the living larvae was evaluated. In some cases of toxicity, the number of larvae was reduced due to cannibalism among the larvae. In addition, the final OD600 nm of the culture was determined as a means for uptake inhibition.

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4. Toxicity Test Toward the Nematode C. elegans The C. elegans Bristol N2 isolate and pmk-1(km25) strain were obtained from the Caenorhabditis Genetics Center (CGC) at the University of Minnesota (USA), maintained on nematode growth media (NGM) and fed with E. coli strain OP50 as described (Stiernagle, 2006). Eggs from gravid hermaphrodites were prepared as described (Stiernagle, 2006), transferred to an unseeded NGM plate, and incubated overnight at 23
C for hatching. This procedure yielded a nearly synchronous population of L1 larvae. The L1 larvae were washed from the plate using sterile deionized water, collected in a sterile 15-ml screw cap tube (Falcon), and counted. The number of larvae was adjusted to 10–30 per 10 ml. In parallel, 5 ml of a stationary culture of BL21(DE3) transformants harboring the expression plasmid for the recombinant protein of interest was prepared. Three to five NGM plates containing 1 mM IPTG and 50 mg/l of Kanamycin were seeded with 300 ml of each culture and incubated overnight at 23
C. Expression of the recombinant protein was controlled by scraping the bacterial lawn off a separate plate. A WCE sample of these cells was prepared and analyzed as described above. For the assay, 50–100 L1 larvae (the exact number was determined for each plate) were placed onto each plate seeded with BL21(DE3) cells expressing the recombinant protein. After 72 h at 20
C, the fraction of animals that reached the L4 stage was determined by visual inspection under the stereo microscope.

5. Toxicity Test Toward the Amoeba A. castellanii A. castellanii (ATCC 30234) was obtained from the American Type Culture Collection (Manassas, USA) and maintained in axenic static culture in 75 cm3 cell culture flasks in peptone-yeast extract-glucose (PYG) medium at 30
C as described (Neumeister, 2004). For the assay, trophozoites in exponential growth phase (2 days after splitting), were collected in a sterile 15-ml screw cap tube (Falcon), pelleted by spinning for 5 min at 750  g, and resuspended in 5 ml of sterile deionized water. The concentration of the amoeba in the suspension was determined in a counting chamber (Thoma, 0.1 mm) and adjusted to 200 amoeba/ml. In parallel, a 50-ml induced culture of BL21(DE3) expressing the recombinant protein of interest was prepared as described above, pelleted by spinning for 5 min at 4000  g, washed with 10 ml of sterile deionized water and adjusted to OD600 nm of 50. Bacterial expression of the

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recombinant protein was controlled by preparing and analyzing a WCE sample as described above. For each data point, 400 ml of induced bacterial cell suspension was spread as uniformly as possible on a water agar plate (1.5% agar) and let dry for 30 min. Five plates were prepared per protein of interest. For the assay, sterile susceptibility disks (Oxoid) were placed in the middle of the seeded plates and 5 ml of amoeba suspension was applied on top of the disk. The inoculated plates were sealed with parafilm and incubated for 5–6 days at 30
C. The area of the clearing zone was determined by transferring the border of the clearing zone to a transparency, scanning of the transparency, and calculating the area using an image analysis software.

6. Statistics The statistical significance of the toxicity assays was evaluated by pairwise comparisons, using the nonparametric Kolmogorov-Smirnov and Mann–Whitney U tests for A. aegypti and C. elegans and the parametric T-student test for A. castellanii.

ACKNOWLEDGMENTS We acknowledge Sonja Ka¨ser, and Yannick Duport for excellent technical assistance, Laurent Paquereau for providing a plasmid containing the XCL-encoding cDNA and Pie Mu¨ller for the constant supply of A. aegypti eggs. We thank Elizabeth Fischer (NIH/NIAID Rocky Mountain Laboratories, Hamilton, MT, USA) for allowing us to use her scanning electron micrograph of E. coli as well as David Patterson (CoPI Life Sciences, Marine Biological Laboratory, Woods Hole, MA, USA) and James Gathany (US CDC) for granting free use of their light micrographs of A. castellanii and the A. aegypti imago, respectively. This work was financially supported by the Swiss National Science Foundation (grant no. 31003A-116827 to M. K., M. O. H. and M. A.) and ETH Zu¨rich.

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Cash, H. L., Whitham, C. V., Behrendt, C. L., and Hooper, L. V. (2006). Symbiotic bacteria direct expression of an intestinal bactericidal lectin. Science 313, 1126–1130. Dann, S. M., and Eckmann, L. (2007). Innate immune defenses in the intestinal tract. Curr. Opin. Gastroenterol. 23, 115–120. French, A. T., Knight, P. A., Smith, W. D., Brown, J. K., Craig, N. M., Pate, J. A., Miller, H. R., and Pemberton, A. D. (2008). Up-regulation of intelectin in sheep after infection with Teladorsagia circumcincta. Int. J. Parasitol. 38, 467–475. Goldstein, I. J., and Winter, H. C. (2007). Mushroom lectins. In ‘‘Comprehensive Glycoscience: From Chemistry to Systems biology,’’ ( J. P. Kamerling, ed.), Vol. 3. Elsevier Ltd., Amsterdam. Griffitts, J. S., Haslam, S. M., Yang, T., Garczynski, S. F., Mulloy, B., Morris, H., Cremer, P. S., Dell, A., Adang, M. J., and Aroian, R. V. (2005). Glycolipids as receptors for Bacillus thuringiensis crystal toxin. Science 307, 922–925. Hamshou, M., Van Damme, E. J., and Smagghe, G. (2010). Entomotoxic effects of fungal lectin from Rhizoctonia solani towards Spodoptera littoralis. Fungal Biol. 114, 34–40. Ideo, H., Fukushima, K., Gengyo-Ando, K., Mitani, S., Dejima, K., Nomura, K., and Yamashita, K. (2009). A Caenorhabditis elegans glycolipid-binding galectin functions in host defense against bacterial infection. J. Biol. Chem. 284, 26493–26501. Kim, Y. M., Park, K. I., Choi, K. S., Alvarez, R. A., Cummings, R. D., and Cho, M. (2006). Lectin from the Manila clam Ruditapes philippinarum is induced upon infection with the protozoan parasite Perkinsus olseni. J. Biol. Chem. 281, 26854–26864. Kohatsu, L., Hsu, D. K., Jegalian, A. G., Liu, F. T., and Baum, L. G. (2006). Galectin-3 induces death of Candida species expressing specific beta-1, 2-linked mannans. J. Immunol. 177, 4718–4726. Kurata, S., Ariki, S., and Kawabata, S. (2006). Recognition of pathogens and activation of immune responses in Drosophila and horseshoe crab innate immunity. Immunobiology 211, 237–249. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lannoo, N., and Van Damme, E. J. (2010). Nucleocytoplasmic plant lectins. Biochim. Biophys. Acta 1800, 190–201. Michiels, K., Van Damme, E. J., and Smagghe, G. (2009). Plant–insect interactions: What can we learn from plant lectins? Arch. Insect Biochem. Physiol. 73, 193–212. Neumeister, B. (2004). Intracellular multiplication of Legionella species and the influence of amoebae on their intracellular growth in human monocytes: Mono mac 6 cells and Acanthamoeba castellanii as suitable in vitro models. Methods Mol. Biol. 268, 141–151. Oka, Y., Chet, I., and Spiegel, Y. (1997). An immunoreactive protein to wheat-germ agglutinin antibody is induced in oat roots following invasion of the cereal cyst nematode Heterodera avenae, and by jasmonate. Mol. Plant Microbe Interact. 10, 961–969. Pace, K. E., and Baum, L. G. (2004). Insect galectins: Roles in immunity and development. Glycoconj. J. 19, 607–614. Peumans, W. J., and Van Damme, E. J. (1995). Lectins as plant defense proteins. Plant Physiol. 109, 347–352. Schulenburg, H., Hoeppner, M. P., Weiner, J., 3rd, and Bornberg-Bauer, E. (2008). Specificity of the innate immune system and diversity of C-type lectin domain (CTLD) proteins in the nematode Caenorhabditis elegans. Immunobiology 213, 237–250. Stiernagle, T. (2006). Maintenance of C. elegans. WormBook 1–11. Stowell, S. R., Arthur, C. M., Dias-Baruffi, M., Rodrigues, L. C., Gourdine, J. P., Heimburg-Molinaro, J., Ju, T., Molinaro, R. J., Rivera-Marrero, C., Xia, B., Smith, D. F., and Cummings, R. D. (2010). Innate immune lectins kill bacteria expressing blood group antigen. Nat. Med. 16, 295–301.

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Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990). Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185, 60–89. Titz, A., Butschi, A., Henrissat, B., Fan, Y. Y., Hennet, T., Razzazi-Fazeli, E., Hengartner, M. O., Wilson, I. B., Kuenzler, M., and Aebi, M. (2009). Molecular basis for galactosylation of core fucose residues in invertebrates: Identification of Caenorhabditis elegans N-glycan core alpha1, 6-fucoside beta1, 4-galactosyltransferase GALT-1 as a member of a novel glycosyltransferase family. J. Biol. Chem. 284, 36223–36233. Trigueros, V., Lougarre, A., Ali-Ahmed, D., Rahbe, Y., Guillot, J., Chavant, L., Fournier, D., and Paquereau, L. (2003). Xerocomus chrysenteron lectin: Identification of a new pesticidal protein. Biochim. Biophys. Acta 1621, 292–298. Tsuji, S., Uehori, J., Matsumoto, M., Suzuki, Y., Matsuhisa, A., Toyoshima, K., and Seya, T. (2001). Human intelectin is a novel soluble lectin that recognizes galactofuranose in carbohydrate chains of bacterial cell wall. J. Biol. Chem. 276, 23456–23463. Tsutsui, S., Iwamoto, K., Nakamura, O., and Watanabe, T. (2007). Yeast-binding C-type lectin with opsonic activity from conger eel (Conger myriaster) skin mucus. Mol. Immunol. 44, 691–702. Vandenborre, G., Miersch, O., Hause, B., Smagghe, G., Wasternack, C., and Van Damme, E. J. (2009). Spodoptera littoralis-induced lectin expression in tobacco. Plant Cell Physiol. 50, 1142–1155. Vasta, G. R. (2009). Roles of galectins in infection. Nat. Rev. Microbiol. 7, 424–438. Wang, M., Trigueros, V., Paquereau, L., Chavant, L., and Fournier, D. (2002). Proteins as active compounds involved in insecticidal activity of mushroom fruitbodies. J. Econ. Entomol. 95, 603–607. Wilson, R., Chen, C., and Ratcliffe, N. A. (1999). Innate immunity in insects: The role of multiple, endogenous serum lectins in the recognition of foreign invaders in the cockroach, Blaberus discoidalis. J. Immunol. 162, 1590–1596. Zhu, Y., Ng, P. M., Wang, L., Ho, B., and Ding, J. L. (2006). Diversity in lectins enables immune recognition and differentiation of wide spectrum of pathogens. Int. Immunol. 18, 1671–1680.

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Carbohydrate Signaling by C-Type Lectin DC-SIGN Affects NF-kB Activity Sonja I. Gringhuis*,† and Teunis B. H. Geijtenbeek*,† Contents 1. Overview 2. Dendritic Cell Stimulation with LPS and ManLAM 3. RNA Interference in Dendritic Cells 4. NF-kB Activation in DCs 5. Phosphorylation and Acetylation of NF-kB by DC-SIGN Signaling 6. Activity of Acetyltransferases in DC 7. Transcription Regulation by Acetylation of p65 8. Concluding Remarks Acknowledgments References

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Abstract Pathogen recognition is central to the induction of adaptive immunity. Dendritic cells (DCs) express different pattern recognition receptors (PRRs), such as Tolllike receptors and C-type lectins, that sense invading pathogens. Pathogens trigger a specific set of PRRs, leading to activation of intracellular signaling processes that shapes the adaptive immunity. It is becoming clear that cross talk between these signaling routes is crucial for pathogen-tailored immune responses. The C-type lectin DC-SIGN interacts with different mannose-expressing pathogens such as Mycobacterium tuberculosis and HIV-1. Notably, DC-SIGN triggering by these pathogens results in a specific Raf-1-dependent signaling pathway that modulates TLR-induced NF-kB activation. Here, we will discuss the various methods that we have used to identify the innate signaling by the C-type lectin DC-SIGN, and how to analyze the consequences on NF-kB activation. * Center of Infection and Immunity Amsterdam, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

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1. Overview Dendritic cells (DCs) are crucial to the induction of adaptive immune responses to bacteria, viruses, and fungi (Ueno et al., 2007). DCs initiate and dictate T helper (TH) cell differentiation, which is required for pathogenspecific adaptive immunity (Steinman and Banchereau, 2007). Upon pathogen interaction, DCs produce specific cytokines and express surface molecules that provide important cues to modulate the effector functions of responding TH cells (Medzhitov, 2007). DCs express a diverse array of pattern recognition receptors (PRRs) that sense the presence of invading pathogens and trigger signaling pathways that lead to DC activation. These PRRs include the Toll-like receptors (TLRs), C-type lectins, nucleotidebinding oligomerization domain (NOD) proteins, and caspase-recruiting domain (CARD) helicases, and are present on the cell surface, in endosomes, in phagosomes, and in the cytosol, where they capture highly conserved structures expressed on microorganisms, the so-called pathogen-associated molecular patters (PAMPs) (Geijtenbeek and Gringhuis, 2009; Kanneganti et al., 2007; O’Neill and Bowie, 2007). Each PRR triggers a distinct innate signaling pathway which contributes to the overall adaptive immune response mounted against the invading pathogen. TLRs elicit innate signaling pathways via either MyD88 or TRIF adaptor proteins, leading to activation of NF-kB and other transcription factors (Ouaaz et al., 2002). Activation of NF-kB is pivotal for adaptive immunity (Geijtenbeek and Gringhuis, 2009). C-type lectins constitute a major class of non-TLR PRRs that recognize carbohydrates present on pathogens, and either by themselves induce immune responses or modulate TLR signaling to tailor immune responses (Gringhuis et al., 2007). We have recently elucidated the molecular basis for the cross talk between TLRs and the C-type lectin DC-SIGN, and here we will describe the methods that have been used to elucidate the signaling by DC-SIGN that modulates TLR signaling at the level of NF-kB p65 phosphorylation and acetylation (Geijtenbeek and Gringhuis, 2009). DC-SIGN is a key player in pathogen recognition and pathogen-tailored adaptive immunity due to its broad carbohydrate specificity (Geijtenbeek and Gringhuis, 2009). A plethora of pathogens interacts with DC-SIGN through both high mannose structures and fucose-containing glycans, which are differentially expressed on bacteria, viruses, parasites, and fungi. Remarkably, the immunological outcome of DC-SIGN triggering depends on the pathogen involved. DC-SIGN binding by distinct pathogens can lead to either inhibition or promotion of TH1 polarization, TH2 responses as well as the induction of regulatory T cell differentiation (Gringhuis et al., 2007). Carbohydrate-specific signaling by DC-SIGN might play an important role

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in these distinct T cell responses (Gringhuis et al., 2009a). The mechanisms behind these pathogen-specific immune responses of DC-SIGN are unclear. DC-SIGN binding by the mycobacterial cell-wall component ManLAM increases the TLR4-induced cytokines such as IL-10, IL-6, and IL-12p70 (Gringhuis et al., 2009a). We have previously shown that triggering of DC-SIGN by ManLAM induces a distinct innate signaling pathway that leads to the activation of the serine–threonine kinase Raf-1, which is central to modulation of TLR-specific immune responses by DC-SIGN in response to several mannose-carrying pathogens, including mycobacteria, HIV-1, and measles virus (Gringhuis et al., 2007). Here, we will describe the methods for elucidation of this signaling pathway by DC-SIGN.

2. Dendritic Cell Stimulation with LPS and ManLAM Immature DCs are cultured as described before (Gringhuis et al., 2009b). In short, human blood monocytes are isolated from buffy coats by use of a Ficoll gradient and a subsequent CD14 selection step using a MACS system (Miltenyi Biotec). Purified monocytes are differentiated into immature DCs in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colonystimulating factor (500 and 800 U/ml, respectively; Schering-Plough). DCs are used for experiments at days 6 and 7. To investigate DC-SIGN cross talk with TLR4, DCs are incubated with the DC-SIGN ligand ManLAM and the TLR4 ligand LPS. 100,000 DCs are plated per well (96-well plate) in 100 ml medium (RPMI/10% FCS). In order to investigate the role of DC-SIGN, DCs are preincubated with the blocking DC-SIGN antibody AZN-D2; 1 ml AZN-D2 (2 mg/ml) is added to the wells and the cells are incubated for 2 h at 37
C. Next, DCs are stimulated by addition of 1 ml LPS (stock 1 mg/ml LPS from Salmonella typhosa (Sigma)) and/or 1 ml ManLAM (stock 1 mg/ml ManLAM from Mycobacterium tuberculosis; a kind gift from J. Belisle, Colorado State University, NIH, NIAID Contract No. HHSN266200400091C). After 6 h incubation, cells are lysed by addition of equal volume (100 ml) of cell lysis buffer from mRNA capture kit (Roche). Lysates can be stored frozen or used directly for mRNA isolation. mRNA is isolated by capturing of polyA RNA in streptavidin-coated tubes with the mRNA capture kit (Roche) following the manufacturer’s guidelines. cDNA is synthesized with the reverse transcriptase kit (Promega) as recommended by the manufacturer. For real-time PCR analysis, PCR amplification is performed with the Fast SYBR green method in an ABI 7900HT sequence detection system (Applied Biosystems). The reactions are set by mixing 4 ml of Fast SYBR green master mix (Applied Biosystems) with 2 ml primer mix (containing 10 mM of both primers), and 2 ml cDNA. Specific primers for IL-10 and

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GAPDH have been designed using Primer Express 2.0 (Applied Biosystems): IL-10/F 50 -GAGGCTACGGCGCTGTCAT-30 , IL-10/R 50 -CCACGGCCTTGCTCTTGTT-30 ; GAPDH/F 50 -CCATGTTCGTCATGGGTGTG-30 , GAPDH/R 50 -GGTGCTAAGCAGTTGGTGGTG-30 . The Ct value is defined as the number of PCR cycles where the fluorescence signal exceeds the detection threshold value, fixed at 0.2 relative fluorescence units. This threshold is set constant throughout the study and corresponds to the log linear range of the amplification curve. For each sample, the normalized amount of target mRNA is calculated from the obtained Ct values for both target and GAPDH mRNA with Nt ¼ 2Ct(GAPDH)Ct(target). The relative mRNA expression is obtained by setting Nt in LPS-stimulated samples at 1 within one experiment and for each donor. Real-time PCR analyses demonstrate that ManLAM alone does not induce IL-10 but that costimulation of DCs with ManLAM and LPS increases IL-10 expression several fold compared to LPS alone (Fig. 8.1A). Notably, preincubation of DCs with anti-DC-SIGN abrogates the

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Figure 8.1 DC-SIGN signaling modulates TLR4-dependent IL-10 induction. (A) ManLAM induces IL-10 after TLR4 triggering. Human DCs were treated with TLR4 ligand LPS, ManLAM, or combinations. Specificity for DC-SIGN was determined by preincubating the cells for 2 h with the blocking DC-SIGN antibody AZND2. After 6 h of stimulation, relative IL-10 mRNA expression was determined by quantitative real-time PCR analysis, using GAPDH as an internal control. IL-10 mRNA expression in LPS-stimulated cells was set at 1. Results are presented as the means  s.d. from at least three independent experiments. (B) Raf-1 was specifically silenced by RNA interference. DCs were transfected with 50 nM Raf-1 SMARTpool siRNA, individual Raf-1 siRNAs 1–3 or control nontargeting siRNA. At 72 h after transfection, Raf-1 protein was measured by flow cytometry. A representative experiment from three donors is shown. (C) Raf-1 is essential to ManLAM-induced IL-10 upregulation. Raf-1 or control siRNA knockdown DCs were incubated with LPS alone or in combination with ManLAM, and IL-10 mRNA was determined as described in (A). Results are presented as the means  s.d. from at least three independent experiments.

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increased expression of IL-10 demonstrating that DC-SIGN binding to ManLAM enhances LPS-induced IL-10 expression (Gringhuis et al., 2007). Thus, DC-SIGN increases TLR4-induced IL-10 expression.

3. RNA Interference in Dendritic Cells Next, we investigate the role of Raf-1 kinase in ManLAM-induced IL-10 expression by silencing Raf-1 in DCs using RNA interference. Different transfection reagents (DF1–DF4) are supplied by the manufacturer for transecting cells (Dharmacon). DF4 is optimal for primary monocyte-derived DCs (Hayden et al., 2006). DCs are transfected with 50 nM siRNA using transfection reagent DF4 (Dharmacon). The siRNAs used are Raf-1 SMARTpool (M-003601-00), Raf-1 siRNA-1 (D-003601-01), Raf-1 siRNA-2 (D-003601-02), Raf-1 siRNA-3 (D-003601-04), and nontargeting siRNA pool (D-001206-13) as a control (Dharmacon). This protocol results in nearly 100% transfection efficiency as determined by flow cytometry of cells transfected with siGLO-RISC free-siRNA (D-001600-01). DCs are used at day 4 of differentiation. At day 4, DCs are counted and resuspended at a concentration of 1  106 cells/ml in medium (RPMI/10% FCS) but without antibiotics. Antibiotic-free medium is essential to preserve cell viability after transfection. Next, a 10 mM siRNA stock is prepared by resuspending siRNA in 1 siRNA buffer (Dharmacon). Also a DF4 mix is prepared per condition by mixing 0.5 ml DF4 (Dharmacon) with 12 ml Hank’s balanced salt solution (HBSS) and leave at RT for 5 min. Next, dilute siRNA for every condition by diluting 0.3125 ml siRNA stock to 6.25 ml with 1 siRNA buffer. Next, add 6.25 ml HBSS medium to diluted siRNA and add 12.5 ml diluted DF4 to siRNA/HBSS mixture (total volume of 25 ml). Leave this siRNA/DF4 mixes at RT for 30 min. After the incubation, transfer 25 ml of siRNA/DF4 mixture to 96-well round-bottom plate and add 100 ml (100,000 DCs) per well. Culture these cells for 48 h at 37
C (this time point will largely depend on the gene expression you try to silence; in general, change medium 24 h before stimulation of cells). After 48 h, wash the cells to remove any residual DF4. Resuspend cells and transfer to 96-well Vbottom plate and centrifuge for 2 min, 1500 rpm and discard supernatant. Next, resuspend cells in 100 ml medium (RPMI/10% FCS) without antibiotics and transfer to the original 96-well round-bottom plate. Culture the cells for another 24 h at 37
C. DCs are used 72 h after transfection. Silenced expression of Raf-1 is confirmed at the mRNA level by quantitative real-time PCR and at the protein level by flow cytometry (Fig. 8.1B). RNA interference completely silences Raf-1 expression in primary DCs, both by using individual Raf-1

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siRNAs 1–3 as well as a SMARTpool consisting of different individual Raf-1 siRNAs (Fig. 8.1B). Knockdown of Raf-1 abrogates ManLAMmediated upregulation of LPS-induced IL-10 mRNA (Fig. 8.1C). These data demonstrate that Raf-1 signaling by DC-SIGN modulates TLRinduced IL-10 expression (Gringhuis et al., 2007).

4. NF-kB Activation in DCs TLR4 triggering by LPS leads to NF-kB activation, and we investigate here whether DC-SIGN triggering by ManLAM modulates TLRinduced NF-kB activity. We first examine the composition of the active NF-kB dimers, as the components that make up the transcription factor (p50, p52, p65, RelB, c-Rel) have different binding affinities and transactivation features (Gringhuis et al., 2007). Nuclear extracts are prepared from 5  106 DCs, unstimulated or stimulated with either ManLAM, LPS, or the combination using the NucBuster protein extraction kit (Novagen). Resuspend DCs (from day 6 or 7) at a concentration of 1  106 cells/ml in RPMI/10% FCS. Make sure to use at least 5  106 cells per condition for an optimal isolation. Plate DCs in a 6-well plate (5 ml per well). Add stimuli LPS (10 ng/ml final concentration) and/or ManLAM (10 mg/ml final concentration) and incubate for 2 h at 37
C. Stop the incubation by adding 5 ml ice-cold PBS. Transfer the cells to a 50 ml tube and add ice-cold PBS to 50 ml. Centrifuge for 5 min at 1200 rpm (4
C) and discard the supernatant. Resuspend the cells in 300 ml ice-cold PBS and transfer to a 1.5 ml tube. Centrifuge 10 s (14,000 rpm) and discard supernatant. Resuspend the cell pellet in x ml NucBuster Reagent 1 (x ml ¼ three times the volume of the packed cells). Vortex for 15 s, leave on ice for 5 min, and vortex again for 15 s. Isolate the nuclei by centrifugation for 5 min (14,000 rpm at 4
C) and discard the supernatant. Lyse the nuclei by resuspending the pellet in y ml (y ¼ ½x) NucBuster Reagent 2 supplemented with protease inhibitor cocktail and 100 mM DTT (both 1 ml per 75 ml NucBuster Reagent 2). Vortex for 15 s, leave on ice for 5 min, and centrifuge for 5 min (14,000 rpm at 4
C). Transfer the supernatant (¼ nuclear extract) to a clean 1.5 ml tube. Store at 80
C until performing protein measurement and transcription factor binding assay. To determine the composition of DNA-binding NF-kB complexes, we used the TransAM NF-kB family kit (Active Motif). Dilute 5–10 mg of nuclear extract to 20 ml with complete lysis buffer (supplied in the kit; complete lysis buffer: 5 ml 1 M DTT and 10 ml of protease inhibitor cocktail per ml of lysis buffer AM2). Add 30 ml complete binding buffer (complete binding buffer: 2 ml 1 M DTT, 10 ml 1 mg/ml Herring sperm DNA per ml of binding buffer AM3) to TransAM wells and add subsequently 20 ml diluted

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lysate to the wells. Incubate for 1 h at RT (mild shaking). Wash the wells carefully three times with 200 ml wash buffer. To detect the different NF-kB subunits that have bound to the precoated nucleotides containing the consensus NF-kB binding sequence, 100 ml primary anti-NF-kB subunit antibody (1:1000 diluted in 1 Antibody binding buffer (supplied in the kit); anti-p65, c-Rel, RelB, p50, and p52) are added to the wells and incubated for 1 h at RT. Wells are washed three times with 200 ml wash buffer and after 100 ml secondary HRP-conjugated antibody (1:1000 diluted in 1 Antibody binding buffer) is added, the plate is incubated for 1 h at RT. Next, wells are washed (three times, 200 ml wash buffer) and the reaction is developed by adding 100 ml TMB substrate solution and incubating for 2–10 min at RT. The reaction is stopped by addition of 100 ml stop solution. Absorbance is measured at 450 nm. As expected, LPS strongly induces the nuclear translocation and DNA binding of p65 and p50, whereas we detect only a modest increase in binding of c-Rel, and a negligible change in p52 and RelB binding in nuclear extracts from LPS-stimulated DCs (Fig. 8.2A). Stimulation of DCs with ManLAM alone or in combination with LPS does not induce the nuclear translocation and binding of any of the NF-kB components (Fig. 8.2A). These data demonstrate that DC-SIGN signaling does not induce translocation of NF-kB into the nucleus.

5. Phosphorylation and Acetylation of NF-kB by DC-SIGN Signaling NF-kB activity can be regulated by covalent modifications such as phosphorylation and acetylation (Hayden et al., 2006). Here, we investigate the phosphorylation status of the key serine residues Ser276 and Ser536 of p65, since these have been shown to be induced by Raf-1 and TLR signaling, respectively (Yang et al., 2003). It has been shown that phosphorylation at Ser276 of p65 induces acetylation of p65 on several lysines, since phosphorylation of Ser276 allows binding of the histone acetyltransferases (HATs) CREB-binding protein (CBP) and p300 (Gringhuis et al., 2007). Therefore, we also determined the acetylation state of p65 using antibodies that recognize acetylated lysine residues (Gringhuis et al., 2007). 2.5  106 DCs are plated in wells (6-well plate) in 2.5 ml medium (RMPI/10% FCS) and incubated with Raf inhibitor GW5074 (1 mM final concentration) for 2 h at 37
C. Next cells are stimulated with ManLAM and LPS as described above for 30 min at 37
C. Stop the incubation by adding 5 ml ice-cold PBS. Transfer the cells to a 50 ml tube and add ice-cold PBS to 50 ml. Centrifuge for 5 min, 1200 rpm, 4
C and discard the supernatant. Use the residual fluid to resuspend the cells and transfer the

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Figure 8.2 DC-SIGN-mediated Raf-1-dependent signaling induces p65 phosphorylation on Ser276 and acetylation, after concurrent TLR signaling. (A) DC-SIGN signaling does not influence nuclear translocation of different NF-kB subunits. DNAbinding ELISA of the NF-kB subunits p65, RelB, c-Rel, p50, and p52 in nuclear extracts of human DCs treated as indicated for 1 h. NF-kB was allowed to bind to oligonucleotides containing the NF-kB-binding consensus sequence; specific antibodies were used to detect the different subunits within the bound complexes. Results are presented as the means  s.d. from two independent experiments. (B,C) DC-SIGN signaling induces p65 phosphorylation on Ser276 through Raf-1. ELISA of phosphoSer536-p65 (B) or phospho-Ser276-p65 (C) in cell lysates of human DCs treated as indicated for 30 min; cells were preincubated for 2 h with Raf inhibitor GW5074 as indicated. Results are presented as the means  s.d. from three independent experiments. (D) DC-SIGN induces acetylation of p65. ELISA with acetyl-lysine antibodies on captured p65 from cell lysates of human DCs treated as indicated for 30 min; cells were preincubated with Raf inhibitor GW5074 as in (C). Results are presented as the means  s.d. from three independent experiments. (E) Acetylation of p65 is essential to the ManLAM-induced IL-10 upregulation. Relative IL-10 mRNA production by human DCs treated with different ligands for 6 h as indicated; cells were preincubated for 2 h with anacardic acid (AA), an inhibitor of the histone acetyltransferases p300 and CBP. IL-10 mRNA was determined as described in Fig. 8.1A. Results are presented as the means  s.d. from three independent experiments.

cells to a clean 1.5 ml tube. Pellet the cells by centrifuging 10 s at 14,000 rpm. Discard the supernatant and lyse the cells by adding per 1  106 cells 100 ml ice-cold cell lysis buffer (20 mM Tris–HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 2.5 mM Na pyrophosphate, 1 mM Na b-glycerophosphate, 1% Triton X-100; Cell Signaling Technology) supplemented with protease inhibitors (10 mg/ml leupeptin, 10 mg/ml pepstatin A, 0.4 mM PMSF). Incubate the mixture for

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5 min on ice and sonicate 3  10 s, with a 10 s pause between each pulse, in a waterbath sonicator (at medium settings). Leave on ice for 45 min and centrifuge for 15 min (14,000 rpm) at 4
C. The cell lysate can be stored at 80
C until the PathScan ELISA (Cell Signaling) to determine p65 phosphorylation or acetylation is performed. For the PathScan ELISA, dilute per detection 10 mg cell lysate with sample diluent (blue solution, PathScan kit) to 120 ml end volume. Transfer 100 ml diluted cell lysate to wells of anti-p65-coated strips and incubate for 2 h at 37
C or overnight at 4
C. Wash wells four times with 200 ml wash buffer (supplied with the kit) and add 100 ml/well detection antibody (either anti-S536P-p65 rabbit mAb (S536P-p65 PathScan kit, Cell Signaling) undiluted (green); anti-S276Pp65 rabbit pAb (Cell Signaling) 1:50 (diluted in 3% BSA in TBS); anti-p65 rabbit mAb (Total p65 PathScan kit, Cell Signaling) undiluted (green); or anti-Ac-lysine rabbit pAb (HAT kit, Upstate Biotechnology) 1:100 (diluted in 3% BSA in TBS)). Incubate for 1 h at 37
C and wash wells four times with 200 ml wash buffer. Add 100 ml/well undiluted HRP-conjugated antirabbit IgG (supplied with the kit) and incubate for 30 min at 37
C. Wash wells four times with 200 ml wash buffer and add 100 ml/well TMB substrate solution. Let the reaction continue until the background starts to develop. Terminate the coloring reaction by adding 100 ml/well stop solution and measure the absorbance at 450 nm. Phosphorylation of p65 at Ser536 is detected upon stimulation of DCs with LPS (Fig. 8.2B), which supports previous reports that phosphorylation of p65 at Ser536 is required for LPS-dependent NF-kB activation (Gringhuis et al., 2007). Stimulation of DCs with LPS and ManLAM together did not further enhance phosphorylation of p65 at Ser 536 (Fig. 8.2B). Notably, combined triggering of TLR4 and DC-SIGN signaling results in phosphorylation of Ser276 (Fig. 8.2C), whereas the stimuli alone do not induce phosphorylation (Fig. 8.2C). These data indicate that LPS-mediated activation of NF-kB is required in order to allow for ManLAM-induced phosphorylation of p65 at Ser276. Phosphorylation at Ser276 of p65 is a downstream target of Raf-1 in the DC-SIGN signaling pathway, since Ser276 phosphorylation after LPS/ManLAM costimulation is abrogated by Raf-1 inhibition (Fig. 8.2B). Thus, only when TLR signaling has activated NF-kB can DC-SIGN-mediated signaling result in phosphorylation of p65 at Ser276, supporting our data that ManLAM binding to DC-SIGN only induces IL-10 after TLR activation. Phosphorylation at Ser276 of p65 has been linked to the acetylation of p65 on several lysines, induced by the binding of the HATs CBP and p300 to the phosphorylated Ser276 residue (Gringhuis et al., 2007). Indeed, DC-SIGN and TLR4 signaling cooperated in inducing the acetylation of p65 (Fig. 8.2D). We could not detect any p65 acetylation after stimulation with either LPS or ManLAM alone (Fig. 8.2D). Similarly to the phosphorylation of Ser276, the acetylation of p65 could be blocked by the Raf

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inhibitor (Fig. 8.2D). Furthermore, acetylation of p65 increases IL-10 expression, since inhibition of the HATs by anacardic acid (AA) abrogates DC-SIGN-induced IL-10 expression (Fig. 8.2D). These findings show that the Raf-1-dependent signaling pathway induced by DC-SIGN leads first to the phosphorylation of p65 on Ser276 and subsequently to its acetylation.

6. Activity of Acetyltransferases in DC To investigate whether DC-SIGN signaling regulates acetylation of p65 only at the level of p65 phosphorylation, we investigated the activity of the p300/CBP acetyltransferases using the HAT assay kit (Upstate Biotechnology). DCs are used at day 6 or 7 of differentiation. Count the cells and resuspend them at a concentration of 1  106 cells/ml in medium (RPMI/ 10% FCS). Use a minimum of 5  106 cells per condition. Plate DCs in a 6-well plate (5 ml/well). Stimulate the cells with ManLAM and LPS alone or in combination as described above for 30 min. Transfer the cells to 50 ml tubes and add ice-cold PBS to 50 ml, centrifuge for 4 min at 1500 rpm by 4
C. Resuspend the cells in 500 ml HAT lysis buffer (supplied in the kit) and leave for 45 min on ice. Centrifuge for 10 min, 1200 rpm, at 4
C and resuspend the nuclei pellet in 20 ml glycerol buffer (glycerol buffer: 50 mM Tris–HCl [pH 7.5], 5 mM MgCl2, 0.1 mM EDTA, 40% glycerol), and transfer supernatant to 1.5 ml tubes. Add 2 ml anti-p300/CBP antibodies and incubate overnight at 4
C (rotating). Next, immunoprecipitate p300/ CBP by adding 30 ml prot A/G Plus Agarose (Santa Cruz Biotechnology) and incubate for 2 h at 4
C (rotating), followed by centrifugation for 1 min at 14,000 rpm (4
C). Discard the supernatant, resuspend pellet in 400 ml HAT lysis buffer, and centrifuge for 1 min at 14,000 rpm (4
C). Repeat this wash step once with HAT lysis buffer and once with 100 ml HAT assay buffer. Next, the HAT activity is measured by resuspending the pellet in 50 ml HAT assay cocktail (10 ml 5 HAT assay buffer, 10 ml 500 mM acetyl CoA, 2 ml (0.1 mg) biotin-histone H4 peptide, and 28 ml water). Incubate 30 min at 30
C while shaking. Next, isolate the supernatant by centrifugation (2 min at 14,000 rpm) and add supernatant to streptavidin-coated wells. Incubate 30 min at RT and wash the wells five times with 200 ml TBS buffer (supplied in the kit). Block the wells by incubation with 200 ml 3% BSA/ TBS for 30 min at 30
C. Wash wells with 200 ml TBS and add 100 ml antiAc-lysine antibody (1:250 dilution in 3% BSA/TBS). Incubate 90 min at RT, wash five times with 200 ml TBS and incubate with 100 ml HRPconjugated anti-goat-rabbit antibody (1:1000 dilution in 3% BSA/TBS) for 30 min at RT. Wash five times with TBS and add 100 ml TMB substrate mixture (supplied in the kit) and after development stop the reaction with 100 ml stop solution and measure absorbance at 450 nm.

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Figure 8.3 DC-SIGN-induced acetylation enhances the IL-10 transcription rate. (A) Constitutive acetyltransferase activity in DCs. DCs were stimulated with ManLAM, LPS, and the combination for 30 min and the activities of the histone acetyltransferases p300/CBP were measured by the histone acetyltransferase (HAT) assay. (B) DC-SIGN signaling enhances the Il10 transcription rate. Il10 transcription rate in nuclei isolated from human DCs treated with LPS, ManLAM, or the combination; cells were preincubated for 2 h with the Raf inhibitor GW5074 or CBP/p300 HAT inhibitor anacardic acid (AA) as indicated. Transcription by isolated nuclei was initiated in the presence of 16-biotin-UTP; after 20 min of transcription, nuclei were lysed, RNA with incorporated biotin was isolated, and quantitative real-time PCR analysis was performed to determine the nascent IL-10 RNA production. The amount of RNA produced in 20 min is representative of the transcription rate. The IL-10 RNA production by LPS-stimulated cells was set at 1. Results are presented as the means  s.d. from two independent experiments.

Notably, our data show that unstimulated DCs already have high p300/ CBP acetyltransferase activities, whereas DC-SIGN triggering does not further affect activity (Fig. 8.3A). These data suggest that the acetyltransferases are poised to respond immediately upon TLR-dependent nuclear translocation of NF-kB and DC-SIGN/Raf-1-dependent phosphorylation of p65.

7. Transcription Regulation by Acetylation of p65 Acetylation of p65 results in enhanced transcriptional activity of NF-kB (Chen and Greene, 2004). Our data have shown that ManLAMinduced phosphorylation of p65 at Ser276 prolongs nuclear activity of p65 at least twofold compared to p65 activated by LPS alone (Gringhuis et al., 2007). However, transcriptional activity of p65 might also be affected by phosphorylation of Ser276 and subsequent p65 acetylation (Chen and Greene, 2004; Gringhuis et al., 2007). Therefore, we determine the effect of DC-SIGN signaling on the transcription rate from the Il10 gene using the nuclear run-on assay. DCs are used at day 6 or 7 of differentiation. Count the cells and resuspend them at a concentration of 1  106 cells/ml in medium

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(RPMI/10% FCS). Use a minimum of 5  106 cells per condition. Plate DCs in a 6-well plate (5 ml/well). Stimulate the cells with ManLAM and LPS alone or in combination as described above for 3 h. Transfer the cells to 50 ml tubes and add ice-cold PBS to 50 ml, centrifuge for 4 min, 1500 rpm, 4
C. Resuspend the cells in 5 ml ice-cold NRO lysis buffer (10 mM Tris– HCl [pH 7.5], 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40) and leave for 5 min on ice. Centrifuge for 10 min, 1200 rpm, at 4
C and resuspend the nuclei pellet in 20 ml glycerol buffer (glycerol buffer: 50 mM Tris–HCl [pH 7.5], 5 mM MgCl2, 0.1 mM EDTA, 40% glycerol) and transfer to 1.5 ml tubes. Add 20 ml transcription buffer (12.5 mM Tris–HCl [pH 7.5], 16 mM KCl, 2 mM DTT, 1 mM ATP, 1 mM GTP, 1 mM CTP, 1 mM 16biotin-UTP (Roche), 0.5 U/ml RNasin). Mix gently and incubate at 26
C for 20 min. Add 40 ml cell lysis buffer (mRNA capture kit; Roche) and leave at RT for 3 min. Newly synthesized biotin-containing RNA are captured in streptavidin-coated tubes provided with the mRNA capture kit: transfer transcription mix to streptavidin-coated 0.5 ml 8-tube strips (do this in two steps with each 40 m1) and incubate at 37
C for 5 min. Wash twice with 100 ml washing buffer (mRNA capture kit) and add 40 ml of cDNA synthesis enzyme-mixture/well (Reverse transcription system, Promega; 5 mM MgCl2, 1 reverse transcription buffer, 0.33 mM dNTPs, 0.25 U RNasin ribonuclease inhibitor, 0.2 U AMV reverse transcriptase, 0.01 mg random hexamer primers). Incubate at RT for 10 min, followed by incubation at 42
C for 90 and 5 min at 95
C. Transfer cDNA to clean 96 well of 96-well plate. To obtain residual cDNA, add 40 ml water to streptavidincoated tubes, and incubate again at 95
C for 2 min: pool cDNA with first 40 ml of cDNA. cDNA synthesis and real-time PCR analyses for IL-10 and GAPDH are performed as described above. Stimulation of DCs with ManLAM enhances the LPS-induced Il10 transcription rate approximately threefold (Fig. 8.3B). This ManLAMinduced increase is completely blocked by the addition of the Raf and HAT inhibitors, GW5074, and AA, respectively (Fig. 8.3B). Thus, the increase in IL-10 by DCs after ManLAM and LPS stimulation is regulated at the level of NF-kB by prolonging NF-kB activity and enhancing the transcription rate from the Il10 gene, both processes triggered by DC-SIGN through the Raf-1-acetylation-dependent pathway.

8. Concluding Remarks Here, we have described the methods that led to the identification of a novel molecular mechanism by which mycobacteria, fungi, and viruses target DC-SIGN to modulate TLR signaling in human DCs (Gringhuis et al., 2007). Binding of mycobacterial ManLAM to DC-SIGN impairs

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LPS-induced maturation of DCs and increases the production of the immunosuppressive cytokine IL-10 (Gringhuis et al., 2007). These data strongly suggested that DC-SIGN modulates the TLR4-induced gene expression program. The cross talk of DC-SIGN is not limited to TLR4, but also other TLR-induced immune responses are modulated, such as TLR5 and intracellular TLR3 (Gringhuis et al., 2007). Our data show that DC-SIGN induces intracellular signaling that converges with the TLR signaling pathway at the level of NF-kB activation (Gringhuis et al., 2007). DC-SIGN triggering by ManLAM activates the serine/threonine kinase Raf-1, which induces phosphorylation of the NF-kB subunit p65 at serine 276 (Ser276) (Gringhuis et al., 2007). This phosphorylation subsequently induces binding of the HATs CBP and p300 to p65, which leads to acetylation of p65 on several lysines. Notably, DC-SIGN-mediated acetylation leads to both increased and prolonged transcriptional activation from the Il10 promoter. In contrast to the activation of Raf-1, the effect of DCSIGN signaling on p65 only occurs after simultaneous activation by TLRs (Gringhuis et al., 2007). Raf-1 by itself cannot induce NF-kB activation, but enhances its transcriptional activity only after other signaling routes have induced NF-kB activation, indicating that the DC-SIGN signaling route functions as a modulatory pathway. The Raf-1-induced acetylation of p65 is induced by DC-SIGN in response to several mannose-carrying pathogens, including M. tuberculosis, M. bovis BCG, and M. leprae; DC-SIGN-signaling is induced by several other classes of pathogens (Gringhuis et al., 2007, 2009a). Fungi (Candida albicans and Saccharomyces cerevisiae) and viruses (HIV-1 and measles virus) also induce the Raf-1 pathway through DC-SIGN to modulate TLR responses (Gringhuis et al., 2007). This underscores that DC-SIGN has an important role in the regulation of DC-mediated immune responses to numerous pathogens. Acetylation of p65 does not only increase transcription of IL-10 but also transcription of proinflammatory cytokines IL-12p35, IL-12p40, and IL-6 is upregulated by DC-SIGN-induced acetylation of p65 (Gringhuis et al., 2009a). These data suggest that the interactions of mannose-expressing pathogens with DC-SIGN do not necessarily suppress immune responses by induction of anti-inflammatory cytokines but the complete cytokine expression profile needs to be investigated.

ACKNOWLEDGMENTS We are grateful to the members of the Host Defense group for their valuable input. This work was supported by the Netherlands Organisation for Scientific Research (NWO 91746-367 and NWO 912-04-025 to T. B. H. G.) and the Dutch Asthma Foundation (3.2.03.39 to S. I. G.).

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REFERENCES Chen, L.-F., and Greene, W. C. (2004). Shaping the nuclear action of NF-[kappa]B. Nat. Rev. Mol. Cell Biol. 5, 392–401. Geijtenbeek, T. B., and Gringhuis, S. I. (2009). Signalling through C-type lectin receptors: Shaping immune responses. Nat. Rev. Immunol. 9, 465–479. Gringhuis, S. I., den Dunnen, J., Litjens, M., van het Hof, B., van Kooyk, Y., and Geijtenbeek, T. B. H. (2007). C-type lectin DC-SIGN modulates toll-like receptor signaling via Raf-1 kinase-dependent acetylation of transcription factor NF-kappa B. Immunity 26, 605–616. Gringhuis, S. I., den Dunnen, J., Litjens, M., van der Vlist, M., and Geijtenbeek, T. B. H. (2009a). Carbohydrate-specific signaling through the DC-SIGN signalosome tailors immunity to Mycobacterium tuberculosis, HIV-1 and Helicobacter pylori. Nat. Immunol. 10, 1081–1088. Gringhuis, S. I., den Dunnen, J., Litjens, M., van der Vlist, M., Wevers, B., Bruijns, S. C. M., and Geijtenbeek, T. B. H. (2009b). Dectin-1 directs T helper cell differentiation by controlling noncanonical NF-[kappa]B activation through Raf-1 and Syk. Nat. Immunol. 10, 203–213. Hayden, M. S., West, A. P., and Ghosh, S. (2006). NF-[kappa]B and the immune response. Oncogene 25, 6758–6780. Kanneganti, T. D., Lamkanfi, M., and Nu´n˜ez, G. (2007). Intracellular NOD-like receptors in host defense and disease. Immunity 27, 549–559. Medzhitov, R. (2007). Recognition of microorganisms and activation of the immune response. Nature 449, 819–826. O’Neill, L. A. J., and Bowie, A. G. (2007). The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling. Nat. Rev. Immunol. 7, 353–364. Ouaaz, F., Arron, J., Zheng, Y., Choi, Y., and Beg, A. A. (2002). Dendritic cell development and survival require distinct NF-kappaB subunits. Immunity 16, 257–270. Steinman, R. M., and Banchereau, J. (2007). Taking dendritic cells into medicine. Nature 449, 419–426. Ueno, H., Klechevsky, E., Morita, R., Aspord, C., Cao, T., Matsui, T., Di, P. T., Connolly, J., Fay, J. W., Pascual, V., Palucka, A. K., and Banchereau, J. (2007). Dendritic cell subsets in health and disease. Immunol. Rev. 219, 118–142. Yang, F., Tang, E., Guan, K., and Wang, C.-Y. (2003). IKK{beta} plays an essential role in the phosphorylation of RelA/p65 on serine 536 induced by lipopolysaccharide. J. Immunol. 170, 5630–5635.

C H A P T E R

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Engineered CarbohydrateRecognition Domains for Glycoproteomic Analysis of Cell Surface Glycosylation and Ligands for Glycan-Binding Receptors Alex S. Powlesland, Adria´n Quintero-Martinez, Paik Gee Lim, Zoi Pipirou, Maureen E. Taylor, and Kurt Drickamer Contents 1. Overview 2. Engineering Glycan-Binding Specificity 2.1. Protocol 1: Characterization of engineered CRDs using glycan arrays 2.2. Characterization of engineered CRDs using solid-phase binding assays 3. CRDs as Probes for Detection of Glycans on Blots 3.1. Protocol 2: Labeling and detection of ligands 4. CRDs as Affinity Tools for Probing of Cell Surface Glycosylation 4.1. Protocol 3: Generation of affinity columns 4.2. Protocol 4: Cell fractionation on affinity columns 4.3. Protocol 5: Gel analysis of isolated glycoproteins and identification using in-gel trypsin digestion followed by mass spectrometry 4.4. Protocol 6: Analysis of purified ligands by in-solution trypsin digestion followed by LC-MS/MS 4.5. Protocol 7: Mass spectrometry of tryptic peptides 4.6. Protocol 8: Informatics 4.7. Glycomics Acknowledgments References

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Division of Molecular Biosciences, Department of Life Sciences, Imperial College, Biochemistry Building, London, United Kingdom Methods in Enzymology, Volume 480 ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)80009-6

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2010 Elsevier Inc. All rights reserved.

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Abstract Modular calcium-dependent carbohydrate-recognition domains (CRDs) of mammalian glycan-binding receptors (C-type lectins), engineered to have novel glycan-binding selectivity, have been developed as tools for the study of glycans on cell surfaces. Structure-based specificity swapping between domains can be complemented by empirical characterization of ligand-binding specificity using glycan arrays. Both natural and modified CRDs can be used as probes for detecting and isolating glycoproteins that bear specific glycan epitopes and that act as target ligands for glycan-binding receptors. CRD-based affinity chromatography facilitates proteomic and glycomic analysis of such ligands.

1. Overview Calcium-dependent carbohydrate-recognition domains (C-type CRDs) form the sugar-binding portions of a large and diverse family of mammalian glycan-binding receptors (C-type lectins) (Drickamer, 1999). Although these protein modules share conserved framework amino acids, the sugar-binding sites are diverse. These sites fall broadly into two classes, with primary specificity either for mannose and GlcNAc or for galactose and GalNAc, based on interaction of the 3- and 4-hydroxyl groups with a conserved calcium ion (Drickamer, 1999; Weis and Drickamer, 1996). Additional interactions in some of the binding sites lead to more selective binding to specific types of oligosaccharides. Binding of C-type CRDs to sugars on glycoproteins and cell surfaces forms a basis for cell adhesion, serum glycoprotein turnover, and recognition of pathogens in the innate immune system, which reflect the roles of additional domains in the receptors (Garcı´a-Vallejo and van Kooyk, 2009; Taylor and Drickamer, 2007; van Kooyk and Rabinovich, 2008). For example, when CRDs in serum mannose-binding protein interact with sugars on the surfaces of bacterial and fungal pathogens, proteases bound to collagen-like stalks adjacent to the CRDs are able to activate complement (Wallis, 2007). Several features of the C-type CRDs make them attractive as potential tools for characterizing glycosylation of cells and proteins. The CRDs are modular and many can be readily expressed in bacterial expression systems. The presence of several disulfide bonds makes the protein stable to immobilization and labeling chemistries. In addition, the calcium dependence of the ligand-binding activity means that bound ligands can be efficiently released with chelating agent, such as EDTA, following which binding activity is readily restored by readdition of calcium ions. In addition to these features of naturally occurring C-type CRDs, protein engineering experiments have demonstrated the feasibility of modifying the sugar-binding characteristics of the CRDs with relatively minor changes in their amino acid

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sequences. For example, changes in just 10 residues are sufficient to convert the CRD of mannose-binding protein from mannose/GlcNAc specificity to galactose/GalNAc specificity (Iobst and Drickamer, 1994).

2. Engineering Glycan-Binding Specificity Attempts to modify sugar-binding specificity of C-type CRDs have been informed by knowledge of the structures of multiple CRD–ligand complexes. Combination of sequence comparisons with structural and mutagenesis studies has led to an appreciation for the role of acidic and amide side chains that ligate a calcium ion and also form part of the sugar-binding site (Weis and Drickamer, 1996). In addition, in galactose-binding sites, an aromatic residue, often a tryptophan, usually plays a key role in packing against the apolar B face of the sugar (Iobst and Drickamer, 1994; Kolatkar and Weis, 1996). These features are encoded in a contiguous stretch of 9–14 amino acid residues that forms one side of the sugar-binding region (Fig. 9.1). Changes of the sequences in this region reliably lead to predictable swaps in specificity between mannose-type and galactose-type binding sites. Additional specificity is conferred by interactions with protuberances in the surface of the CRD on the other side of the binding site, but in a manner that is less readily predictable from sequence comparisons (Feinberg et al., 2000, 2007; Somers et al., 2000). Thus, defining the specificity of engineered carbohydrate-binding domains depends on the ability to screen for interesting sugar-binding selectivity. Glycan arrays have recently emerged as excellent tools for the broad characterization of binding specificity, providing a powerful empirical complement to the structure-based design approach (Blixt et al., 2004; Feizi et al., 2003; Taylor and Drickamer, 2009). The importance of glycan array screening is illustrated in Fig. 9.2, in which galactose-binding activity has been introduced into three different CRDs that naturally bind to mannose-type sugars. Although all three modified proteins contain the same galactose-specificity sequence and bind to galactose-Sepharose affinity columns, they show very different interactions with oligosaccharide ligands. All three proteins are oligomeric, so they have the potential to make multivalent interactions that provide the avidity necessary to withstand the washing steps used in processing of the arrays. The galactose-binding version of mannose-binding protein (Gal-MBP) binds multiple classes of galactose-terminated glycans (Powlesland et al., 2009). Amongst the better ligands are oligosaccharides that bear terminal fucosylated epitopes, although some nonfucosylated ligands also rank high amongst the ligands. By comparison, the galactose-binding version of langerin (Gal-langerin) binds exclusively to terminal Lewisa and Lewisx groups, and the galactose-binding version of DC-SIGN (Gal-DC-SIGN) (Guo et al., 2006) does

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Figure 9.1 Strategy for introduction of galactose-binding activity into CRDs. (A) Alignment shows sequences of the galactose-binding major subunit of the human asialoglycoprotein receptor (human hepatic lectin 1, HHL1), rat serum mannosebinding protein, human langerin, and human DC-SIGN. Conserved framework residues are highlighted in green and residues that ligate Ca2þ and sugar are highlighted in yellow. The blue region has been transferred from the galactose-binding CRD to the three mannose-binding CRDs. (B) The corresponding region of the crystal structure of Gal-MBP is shown in cyan. Figure was created with PyMol using coordinates from Protein DataBank entry 1afb.

not show strong binding to any ligands on the array. Interestingly, none of these patterns matches the specificities seen for natural galactose-binding proteins from which the inserted sequence was derived. These results reflect the effects of the surface residues on the unmodified side of the binding site, and they emphasize the importance of experimental characterization of the glycanbinding activity of engineered binding proteins.

2.1. Protocol 1: Characterization of engineered CRDs using glycan arrays For oligomeric proteins, direct labeling with fluorescein isothiocyanate gives good signals on the array (Guo et al., 2004). Proteins need to be transferred out of Tris-containing buffers, either by dialysis or by repurification on a

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Figure 9.2 Glycan array analysis of CRDs modified to bind galactose. The arrays were screened with approximately 200 mg/ml of each of the fluorescein-labeled proteins. Gal-MBP and Gal-langerin are trimers, while Gal-DC-SIGN is a tetramer. Consortium for Functional Glycomics array version 2.1 was screened with Gal-MBP (A) while the other proteins were screened on array version 4.1 (B,C). Glycans terminating in Lewisa and Lewisx epitopes are highlighted in red, while other glycans containing galactose residues with free 3- and 4-hydroxyl groups are shaded green, and those with GalNAc residues with free 3- and 4-hydroxyl groups are shaded cyan.

sugar-Sepharose column. Buffers containing 150–500 mM NaCl and either 25 mM Na-HEPES, pH 7.8, or 25 mM bicine-Cl, pH 9.0, can be used. For a 1-ml column, after application of the protein, the column is rinsed with 5 ml

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of buffer containing 25 mM CaCl2 and eluted with 5 aliquots of 0.5 ml of buffer containing 2.5 mM EDTA. Aliquots of approximately 2 ml containing 200–400 mg of protein are mixed with 5 aliquots of 10 ml of fluorescein isothiocyanate dissolved at 1 mg/ml in dimethyl sulfoxide and left to react overnight at 4  C. The reaction is quenched by addition of buffer containing 25 mM Tris–Cl and 25 mM CaCl2 and the protein is separated from unreacted reagent by column repurification as described above, but with buffers containing 25 mM Tris–Cl, pH 7.8. For expression of biotin-tagged protein, synthetic oligonucleotides are used to append the biotinylation sequence GlyLeuAsnAspIlePheGluAlaGlnLysIleGluTrpHisGlu at the C-termini of the extracellular domain fragments (Schatz, 1993). Proteins are expressed in Escherichia coli strains containing plasmid pACYC-184 with the birA gene (Avidity, Denver CO) and grown in the presence of 20 mg/ml chloramphenicol and 12.5 mg/ml biotin. The system works both for proteins expressed in a T5T system in strains, such as BL21(DE3), in which proteins are expressed as inclusion bodies followed by refolding (Graham et al., 2009), or in an ompA system in strains, such as JA221, in which the protein is directed to the periplasm and folds directly (Powlesland et al., 2008). Following purification, the tagged protein is adjusted to 25 mM CaCl2 and Alexa 488-labeled streptavidin (Invitrogen) is added so that the ratio of CRD to streptavidin monomers is roughly 2:1. After incubation overnight at 4  C, complexes are isolated by affinity chromatography on 1-ml monosaccharide affinity columns as described above. Fractions containing labeled protein are used to probe the glycan array developed by the Consortium for Functional Glycomics (www.functionalglycomics.org) following their standard protocol. The presence of 2 mM CaCl2, in the binding and wash buffers, is sufficient to support binding. Glycan arrays provide a broad overview of the types of ligands bound by receptors with engineered CRDs, but there is need for caution in making quantitative interpretations of the results. For this purpose, solid-phase binding assays can often be employed. Because of the weak intrinsic affinity of C-type CRDs for individual oligosaccharides, these assays are usually best carried out in a competition format using a highly valent neoglycoprotein reporter ligand (Coombs et al., 2010). Provided that the ligand is used at levels well below saturation, the observed inhibition constants provide a close approximation to the binding affinity constants (Levitzki, 1997). An example is shown in Fig. 9.3, in which a segment of LSECtin, a receptor with a mannose-type binding site, has been transferred into the CRD of the major subunit of the human asialoglycoprotein receptor, which would naturally bind galactose-type sugars (Spiess, 1990). In this case, the competition illustrates that modified receptor binds mannose, but the very high affinity of LSECtin for the disaccharide GlcNAcb1-2Man compared to mannose (Powlesland et al., 2008) is not conferred by the region that has been transferred.

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HHL1 WKWV DG T DY E T G F KNWR P E Q P DDWYGHG L GGG E D C AH F T DDGRWNDD V C Q R P – Y RWV C E LSECtin Y QWV DG V S – – L S F S HWNQG E P NDA – – – – – WGR E N C VMML H T G LWNDA P C D S E KDGW I C E

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Figure 9.3 Binding competition assays to characterize CRDs modified to bind mannose. (A) Alignment shows sequences of the galactose-binding major subunit of the human asialoglycoprotein receptor (human hepatic lectin 1, HHL1) and the receptor LSECtin, which binds selectively to the disaccharide GlcNAcb1-2Man. Conserved framework residues are highlighted in green and residues that ligate Ca2þ and sugar are highlighted in yellow. The blue region has been transferred from the LSECtin CRD to the HHL1 CRD. (B,C) Wild-type LSECtin and Man-HHL1 were immobilized on wells and probed with 125I-Man33-BSA in the presence of varying concentrations of inhibitors as described in (Powlesland et al., 2008).

2.2. Characterization of engineered CRDs using solid-phase binding assays Details of solid-phase binding assays have been described in this series (Taylor and Drickamer, 2003).

3. CRDs as Probes for Detection of Glycans on Blots Radiolabeling of natural and engineered CRDs that have a range of specificities toward different carbohydrate structures creates a panel of molecular probes that can be used to detect the presence of distinct glycan structures on glycoproteins. These labeled probes are particularly useful for detecting glycoprotein ligands on blots of SDS–polyacrylamide gels of cell extracts (Coombs et al., 2005; Powlesland et al., 2008).

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3.1. Protocol 2: Labeling and detection of ligands Protein for radiolabeling should ideally be at concentrations of at least 0.5–1 mg/ ml and needs to be transferred into buffer containing 100 mM bicine-Cl, pH 8.5, by dialysis or affinity chromatography as described in Protocol 1. Labeling of 1-ml aliquots of protein is performed with 250 mCi of 125I Bolton–Hunter reagent (Bolton and Hunter, 1973) dried from 20 ml of benzene under a stream of nitrogen immediately prior to use. After incubation at room temperature for 30 min, the labeled protein is separated from unreacted reagent as described in Protocol 1. Labeling efficiency can be checked by counting 1 ml of each fraction in a gamma counter. Fractions containing the highest levels of radioactivity are diluted into 25 ml of binding buffer (150–500 mM NaCl, 25 mM Tris–Cl, pH 7.8, 25 mM CaCl2) containing 2% hemoglobin. For detection of receptor-binding, glycoproteins resolved on SDS– polyacrylamide gels are electroblotted onto nitrocellulose membranes (Burnette, 1981). After blocking in 2% hemoglobin in loading buffer overnight at 4  C, the membrane is incubated with radiolabeled receptor for 120 min at room temperature with gentle shaking and washed four times with 100 ml of binding buffer, with 5 min shaking for each wash. Radioactivity is detected using a phosphorimager.

4. CRDs as Affinity Tools for Probing of Cell Surface Glycosylation Immobilized CRDs provide a convenient tool for enrichment of glycoproteins that are target ligands for glycan-binding receptors (Fig. 9.4). Proteomic and glycomic approaches can be used to confirm the presence of distinct glycan structures and to identify the major protein carriers of these structures within the enriched pool. While natural CRDs from receptors can sometimes be used in such studies, it is often difficult to produce sufficient quantities of oligomeric forms of these proteins for construction of affinity columns. In such cases, it is often more practical to use CRDs that can be readily produced in bacterial expression systems but which have been engineered to share ligandbinding properties with the receptor of interest. Figure 9.5 illustrates how this strategy has been used to identifying a highly restricted population of glycoproteins responsible for presenting Lewisx and other fucosylated glycan structures in the breast cancer cell line MCF7 (Powlesland et al., 2009).

4.1. Protocol 3: Generation of affinity columns Protein for preparing affinity columns should be at high concentration, typically at least 1–2 mg/ml, as the efficiency of the coupling reaction is considerably lower for more dilute samples. The protein is dialyzed

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Protocol 2 Blotting with Gel Direct trypsin lectins and antibodies digestion electrophoresis

Protocol 5

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In-gel trypsin Protocol 6 digestion

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Protocol 7 Protocol 8

Figure 9.4 Flow diagram for use of CRD affinity columns in proteomic and glycomic analysis. Relationships between the steps described in individual protocols are shown.

overnight against coupling buffer (150 mM NaCl, 100 mM HEPES, pH 8.0, 50 mM CaCl2). Most CRDs couple efficiently to Affigel 10 agarose-N-hydroxysuccinimide conjugate (BioRad Laboratories), which is rinsed with 10 ml of cold H2O in a 10-ml Polyprep chromatography column (BioRad Laboratories) before 2 ml of the dialyzed protein is added. Both ends of the column are sealed and the suspension is mixed vigorously followed by end-over-end mixing at 4  C for 4 h. The column is drained and rinsed with 10 ml of blocking buffer (150 mM NaCl, 25 mM Tris–Cl, pH 7.8, 25 mM CaCl2). Capping of remaining N-hydroxysuccinimide groups is ensured by storage in blocking buffer overnight before use.

4.2. Protocol 4: Cell fractionation on affinity columns Membrane glycoproteins are typically purified from 0.5–1  108 cells. Adherent cells are washed twice with 10 ml of phosphate-buffered saline (PBS) and harvested by scraping into 10 ml of PBS followed by centrifugation at 450g for 2 min. Suspension cells are harvested by centrifugation at

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Column 2

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Figure 9.5 Example of purification of glycoproteins ligands from MCF7 cells. Glycoproteins on metastatic breast cancer cells that are capped with Lewisx and related sugar structures may bind to the scavenger receptor C-type lectin on endothelial cells, leading to extravasation at sites of secondary tumor formation (Elola et al., 2007). As the trimeric extracellular domain of this protein is not readily produced in large amounts, the engineered galactose-binding CRD from mannose-binding protein (Gal-MBP) was used for affinity purification of the potential ligands from the surface of MCF-7 cells. After two rounds of affinity chromatography, a single major glycoprotein ligand, CD98, and its associated light chain, LAT1, were identified by proteomic analysis (Powlesland et al., 2009).

450g for 2 min and washed twice by resuspension in 10 ml PBS. Pellets resuspended in 10 ml of cell lysis buffer (150 mM NaCl, 25 mM Tris–Cl, pH 7.8, 2 mM CaCl2, 0.1% Triton X-100) supplemented with Triton to 1% and containing protease inhibitors (cocktail set I from CalBiochem), are sonicated for 10 s using a small probe at low power, as only gentle sonication is required. After incubation on ice for 30 min, the lysate is precleared by centrifugation at 100,000g for 15 min at 4  C and passed over the affinity column. The column is washed with 5 ml of cell lysis buffer and glycoproteins are eluted in five 1-ml fractions of elution buffer (150 mM NaCl, 25 mM Tris–Cl, pH 7.8, 2.5 mM EDTA, 0.1% Triton X-100). The column can be regenerated by washing with loading buffer so that the purity of glycoproteins that bind to the affinity resin can be increased by a second round of purification. Pooled elution fractions are adjusted to 25 mM CaCl2 and passed back over the affinity column. The column is washed with 5 ml of high salt loading buffer (1.25 M NaCl, 25 mM Tris–Cl, pH 7.8, 25 mM CaCl2, 0.1% Triton X-100) and 5 ml of low salt loading buffer (150 mM NaCl, 25 mM Tris–Cl, pH 7.8, 25 mM CaCl2, 0.1% Triton X-100) and eluted with five 1-ml fractions of elution buffer. Purified

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glycoproteins can be monitored on SDS–polyacrylamide gels followed by staining with Coomassie blue. Two alternative methods of protein identification, both based on the analysis of tryptic peptides by mass spectrometry (MS), can be employed in the proteomic strategy. For purified glycoproteins that are present in high concentration and are well resolved on SDS–polyacrylamide gels, excision of the protein from the gel and subsequent identification provides a useful means of identifying the major species present. However, glycoproteins are often present at concentrations not suitable for such in-gel analysis. Also, highly glycosylated proteins often do not form well-resolved bands for excision. In these cases, it is possible to analyze the total population of glycoproteins purified by in-solution digestion and purification of the resulting peptides by liquid chromatography followed by sequencing using tandem mass spectrometry (LC-MS/MS).

4.3. Protocol 5: Gel analysis of isolated glycoproteins and identification using in-gel trypsin digestion followed by mass spectrometry Proteins in the elution fractions are precipitated by addition of trichloroacetic acid to a final concentration of 10% and incubation on ice for 10 min. Typically, 20% of the elution fraction is precipitated, but this fraction can be adjusted to ensure that any major populations of glycoproteins are visible by Coomassie blue staining. Samples are centrifuged at 16,000g for 5 min, and the pellets are washed twice with 1 ml of ethanol:ether (1:1), and dried for 2 min under vacuum. Precipitated fractions are analyzed on SDS– polyacrylamide gels followed by staining with Coomassie blue. After the gel is destained and soaked in water, major protein bands are excised in sections of approximately 1 mm and cut into 1 mm  1 mm pieces. Coomassie blue stain is removed from the gel pieces by incubation in a mixture of 200 ml of 50 mM ammonium bicarbonate, pH 8.4, and 200 ml of acetonitrile for 5 min at room temperature, and the gel pieces are dried in a vacuum centrifuge. Incubation at 56  C for 30 min in 200 ml of 10 mM dithiothreitol dissolved in 50 mM ammonium bicarbonate, pH 8.4, is followed by washing in 200 ml of acetonitrile and drying in a vacuum centrifuge. Alkylation is performed on the dried gel pieces in the dark at room temperature for 30 min with 200 ml of 55 mM iodoacetic acid dissolved in 50 mM ammonium bicarbonate, pH 8.4. The iodoacetic acid solution is removed and the gel pieces are washed in 500 ml of 50 mM ammonium bicarbonate, pH 8.4, for 15 min at room temperature. The volume of the gel pieces is reduced by adding 200 ml of acetonitrile for 5 min and drying in a vacuum centrifuge. The dried gel pieces are rehydrated in 20 ml of 25 ng/ml sequencing grade modified trypsin (E.C.3.4.21.4, Promega) dissolved in 50 mM ammonium bicarbonate,

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pH 8.4, and incubated for 15 min at room temperature. After addition of a further 20 ml of 50 mM ammonium bicarbonate, pH 8.4, to the gel pieces, the digestion is allowed to proceed overnight at 37  C. The supernatant from the gel pieces is transferred to a new tube. Peptides are extracted from the gel pieces by incubation in 50 ml of 0.1% trifluoroacetic acid in H2O for 10 min at 37  C followed by the addition of 100 ml of acetonitrile and incubation for a further 15 min at 37  C. The supernatant is combined with the previous supernatant and the elution process is repeated. The volume of the final pooled supernatants is reduced to approximately 30 ml using a vacuum centrifuge.

4.4. Protocol 6: Analysis of purified ligands by in-solution trypsin digestion followed by LC-MS/MS Peak elution fractions are precipitated by the addition of trichloroacetic acid to a final concentration of 10% and incubation on ice for 10 min. Samples are centrifuged at 16,000g for 5 min, washed twice with 1 ml of ethanol: ether (1:1), and dried for 2 min under vacuum. Precipitated fractions are dissolved in 25 ml 8 M urea containing 10 mM HEPES, pH 8.0, diluted with 25 ml 50 mM ammonium bicarbonate, pH 8.4, and reduced with 2 ml 0.5 mg/ml dithiothreitol at room temperature for 30 min. Alkylation is performed with 10 ml of 0.5 mg/ml iodoacetic acid at room temperature for 20 min. After dilution with 325 ml of 50 mM ammonium bicarbonate and digestion overnight at 37  C with 2 mg of sequencing grade modified trypsin (E.C.3.4.21.4, Promega), samples are dried on a vacuum centrifuge and resuspended in 80 ml of 0.1% trifluoroacetic acid. Peptides are desalted and purified by nanoliquid chromatography on a 75 mm  15 cm C18 nanocapillary column (Pepmap analytical column from LC Packings). Peptides are loaded in buffer A (2% acetonitrile containing 0.1% trifluoroacetic acid) and eluted with a gradient of 8–45% buffer B (90% acetonitrile containing 0.1% trifluoroacetic acid) over 60 min at a flow rate of 0.3 ml/min. Peptides with retention times of between 12 and 60 min are collected in 30-s intervals by spotting directly onto a stainless steel target plate using a Probot (LC Packings) with a-cyano-4 hydroxycinnamic acid used as matrix.

4.5. Protocol 7: Mass spectrometry of tryptic peptides For in-gel trypsin digestion, 0.5 ml of sample is spotted onto a stainless steel target plate and 0.5 ml of 10 mg/ml a-cyano-4 hydroxycinnamic acid dissolved in acetonitrile:0.1% trifluoroacetic acid (1:1) is spotted on top of the sample, and allowed to air dry before analysis. Matrix-assisted laserdesorption mass spectrometry (MALDI-MS) profiling is complemented with tandem mass spectrometry (MS/MS) of the 10 most abundant ions in each sample. A typical instrument for this analysis is an Applied

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Biosystems 4800 MALDI time-of-flight tandem mass spectrometer, and the Applied Biosystems GPS Explorer software can be used for analysis of peptide mass fingerprint data and peak picking. A signal-to-noise threshold of 10 is employed and sequazyme peptide mass standards can be used as external standards for calibration. For peptides purified by liquid chromatography, prespotted samples are analyzed by MALDI-MS profiling, and major peaks are selected for collision-induced dissociation and sequencing by MS/MS. The Applied Biosystems GPS Explorer software can be used to select a maximum of 10 peaks from each sample for sequencing. Peaks with identical masses present in multiple spots should only be sequenced once.

4.6. Protocol 8: Informatics The MS and MS/MS data for the tryptic peptides eluted from gels is used to search the SWISSPROT protein database with the Mascot database search algorithm (http://www.matrixscience.com) using the following parameters: peptide masses are stated as monoisotopic, methionine residues are assumed to be partially oxidized, carboxymethylation of cysteine residues is considered; the mass tolerance is kept at 75 ppm, fragment ion tolerance is kept at 0.1 Da, and tryptic digests are assumed to have no missed cleavages. To increase the speed of searching, the taxonomy group searched should be fixed based on the origin of the sample. Peptides positively identified from MS and MS/MS data can be combined to give a probability-based Mowse protein score (Perkins et al., 1999), equal to 10 log(P), where P is the probability that the observed match is a random event. Protein identifications with a probability score of greater than 95% are considered significant. Additional searches can subsequently be conducted with varied parameters. Due to the complexity of the sample, only MS/MS data from LCpurified tryptic peptides is used to search the SwissProt database with the Mascot database search algorithm for peptide sequences consistent with the ion fragments observed. Probability-based Mowse scores are generated for each peptide sequence identified and only values of greater than 95% significance should be considered (Perkins et al., 1999). The Mowse scores of peptides from an individual protein can be combined to give a total ion score for that protein.

4.7. Glycomics N-linked and O-linked glycans on glycoproteins that have been purified by affinity chromatography can be either enzymatically or chemically cleaved from protein carriers using well-established procedures (Sutton-Smith and Dell, 2005). Information about the structural composition of these glycans can be obtained by a variety of mass spectrometric approaches. In particular,

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MALDI-MS profiling coupled with fragmentation data from MS/MS analysis of selected ions can be combined with knowledge of biosynthetic pathways to predict the major glycan structures present (North et al., 2009).

ACKNOWLEDGMENTS This work was supported by grant 075565 from the Wellcome Trust and grant GM62116 from the National Institute of General Medical Sciences to the Consortium for Functional Glycomics. We thank David Smith and Jamie Heimburg-Molinaro of the Consortium for Functional Glycomics for performing the array screening experiments and Paul Hitchen and Anne Dell, Imperial College London, for information on the MS protocols.

REFERENCES Blixt, O., Head, S., Mondala, T., Scanlan, C., Huflejt, M. E., Alvarez, R., Bryan, M. C., Fazio, F., Calarese, D., Stevens, J., Razi, N., Stevens, D. J., et al. (2004). Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. Proc. Natl. Acad. Sci. USA 101, 17033–17038. Bolton, A. E., and Hunter, W. M. (1973). The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent: Applications to the radioimmunoassay. Biochem. J. 133, 529–539. Burnette, W. N. (1981). ’’Western blotting’’: Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112, 195–203. Coombs, P. J., Graham, S. A., Drickamer, K., and Taylor, M. E. (2005). Selective binding of the scavenger receptor C-type lectin to Lewisx trisaccharide and related glycan ligands. J. Biol. Chem. 280, 22993–22999. Coombs, P. J., Harrison, R., Pemberton, S., Quintero-Martinez, A., Parry, S., Haslam, S. M., Dell, A., Taylor, M. E., and Drickamer, K. (2010). Identification of novel contributions to high-affinity glycoprotein–receptor interactions using engineered ligands. J. Mol. Biol. 396, 685–696. Drickamer, K. (1999). C-Type lectin-like domains. Cur. Opin. Struct. Biol. 9, 585–590. Elola, M. T., Capurro, M. I., Barrio, M. M., Coombs, P. J., Taylor, M. E., Drickamer, K., and Mordoh, J. (2007). Lewis x antigen mediates adhesion of human breast carcinoma cells to activated endothelium: Possible involvement of the endothelial scavenger receptor C-type lectin. Breast Cancer Res. Treat. 101, 161–174. Feinberg, H., Torgersen, D., Drickamer, K., and Weis, W. I. (2000). Mechanism of pHdependent N-acetylgalactosamine binding to a function mimic of the hepatic asialoglycoprotein receptor. J. Biol. Chem. 275, 35176–35184. Feinberg, H., Taylor, M. E., and Weis, W. I. (2007). Scavenger receptor C-type lectin binds to the leukocyte cell surface glycan Lewis x by a novel mechanism. J. Biol. Chem. 282, 17250–17258. Feizi, T., Fazio, F., Chai, W., and Wong, C. H. (2003). Carbohydrate microarrays—A new set of technologies at the frontiers of glycomics. Curr. Opin. Struct. Biol. 13, 637–645. Garcı´a-Vallejo, J. J., and van Kooyk, Y. (2009). Endogenous ligands for C-type lectin receptors: The true regulators of immune homeostasis. Immunol. Rev. 230, 22–37.

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Graham, S. A., Je´gouzo, S. A. F., Yan, S., Powlesland, A. S., Brady, J. P., Taylor, M. E., and Drickamer, K. (2009). Prolectin: A glycan-binding receptor on dividing B cells in germinal centers. J. Biol. Chem. 284, 18537–18544. Guo, Y., Feinberg, H., Conroy, E., Mitchell, D. A., Alvarez, R., Blixt, O., Taylor, M. E., Weis, W. I., and Drickamer, K. (2004). Structural basis for distinct ligand-binding and targeting properties of the receptors DC-SIGN and DC-SIGNR. Nat. Struct. Mol. Biol. 11, 591–598. Guo, Y., Atkinson, C. E., Taylor, M. E., and Drickamer, K. (2006). All but the shortest polymorphic forms of the viral receptor DC-SIGNR assemble into stable homo- and hetero-tetramers. J. Biol. Chem. 281, 16794–16798. Iobst, S. T., and Drickamer, K. (1994). Binding of sugar ligands to Ca2þ-dependent animal lectins: II Generation of high affinity galactose binding by site-directed mutagenesis. J. Biol. Chem. 269, 15512–15519. Kolatkar, A., and Weis, W. I. (1996). Structural basis of galactose recognition by C-type animal lectins. J. Biol. Chem. 271, 6679–6685. Levitzki, A. (1997). Ligand binding. In ‘‘Protein Function: A Practical Approach, Vol. 101– 129,’’ (T. E. Creighton, ed.).Oxford University Press, Oxford. North, S. J., Hitchen, P. G., Haslam, S. M., and Dell, A. (2009). Mass spectrometry in the analysis of N-linked and O-linked glycans. Curr. Opin. Struct. Biol. 19, 498–506. Perkins, D. N., Pappin, D. J., Creasy, D. M., and Cottrell, J. S. (1999). Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551–3567. Powlesland, A. S., Fisch, T., Taylor, M. E., Smith, D. F., Tissot, B., Dell, A., Po¨hlmann, S., and Drickamer, K. (2008). A novel mechanism for LSECtin binding to Ebola virus surface glycoprotein through truncated glycans. J. Biol. Chem. 283, 593–602. Powlesland, A. S., Hitchen, P. G., Parry, S., Graham, S. A., Barrio, M. M., Elola, M. T., Mordoh, J., Dell, A., Drickamer, K., and Taylor, M. E. (2009). Targeted glycoproteomic identification of cancer cell glycosylation. Glycobiology 19, 899–909. Schatz, P. J. (1993). Use of peptide libraries to map the substrate specificity of a peptidemodifying enzyme: A 13 residue consensus peptide specifies biotinylation in Escherichia coli. Biotechnology 11, 1138–1143. Somers, W. S., Tang, J., Shaw, G. D., and Camphausent, R. T. (2000). Insights into the molecular basis of leukocyte tethering and rolling revealed by structures of P- and Eselectin bound to SLeX and PSGL-1. Cell 103, 467–479. Spiess, M. (1990). The asialoglycoprotein receptor: A model for endocytic transport receptors. Biochemistry 29, 10008–10019. Sutton-Smith, M., and Dell, A. (2005). Analysis of carbohydrates/glycoproteins by mass spectrometry. (J. E. Celis, ed.)Vol. 4, pp. 415–425. Academic Press, San Diego. Taylor, M. E., and Drickamer, K. (2003). Structure-function analysis of C-type lectins. Methods Enzymol. 363, 3–16. Taylor, M. E., and Drickamer, K. (2007). Paradigms for glycan-binding receptors in cell adhesion. Curr. Opin. Cell Biol. 19, 572–577. Taylor, M. E., and Drickamer, K. (2009). Structural insights into what glycan arrays tell us about how glycan-binding proteins interact with their ligands. Glycobiology 19, 1155–1162. van Kooyk, Y., and Rabinovich, G. A. (2008). Protein-glycan interactions in the control of innate and adaptive immune responses. Nat. Immunol. 9, 593–601. Wallis, R. (2007). Interactions between mannose-binding lectin and MASPs during complement activation by the lectin pathway. Immunobiology 212, 289–299. Weis, W. I., and Drickamer, K. (1996). Structural basis of lectin-carbohydrate interaction. Annu. Rev. Biochem. 65, 441–473.

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Mannose 6-Phosphate Receptor Homology Domain-Containing Lectins in Mammalian Endoplasmic Reticulum-Associated Degradation Nobuko Hosokawa,* Koichi Kato,†,‡,§ and Yukiko Kamiya†,‡ Contents 1. 2. 3. 4.

Overview Quality Control of Newly Synthesized Glycoproteins Primary Structures of Yos9p, OS-9, and XTP3-B OS-9 and XTP3-B form a Complex with Membrane-Embedded Ubiquitin Ligase 4.1. Method for immunoprecipitation followed by Western blotting 5. Sugar Recognition Specificity of OS-9 and XTP3-B 5.1. Method for FAC analysis 6. The Effect of OS-9 and XTP3-B on ERAD in Mammals 6.1. Method for pulse-chase analysis 7. Concluding Remarks Acknowledgments References

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Abstract Quality control of glycoproteins synthesized in the endoplasmic reticulum (ER) is mediated by lectins and molecular chaperones. N-linked Glc3Man9GlcNAc2 oligosaccharides attached to the nascent polypeptides are processed and recognized by lectins in the ER. OS-9 and XTP3-B/Erlectin, mannose 6-phosphate receptor homology (MRH) domain-containing lectins in mammals, were recently identified as ER luminal glycoproteins that participate in ER-associated

* Department of Molecular and Cellular Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan Institute for Molecular Science and Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Japan { Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan } The Glycoscience Institute, Ochanomizu University, Tokyo, Japan {

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degradation (ERAD) of misfolded proteins. Frontal affinity chromatography (FAC) and cell-surface expressed lectin assay revealed that both OS-9 and XTP3-B recognize high-mannose type N-glycans that lack the terminal mannose on the C branch. Furthermore, these lectins associate with the HRD1-SEL1L ubiquitin ligase complex on the ER membrane. In this chapter, we describe the FAC methods used to analyze the carbohydrate-recognition specificity of OS-9 and methods to examine the interaction and the effect on ERAD of these proteins in vivo. We also discuss the structure and function of OS-9 and XTP3-B, and the effect of these lectins on ERAD.

1. Overview Modification of mannose 6-phosphate (M6P) is a well-characterized sorting signal for lysosomal enzymes (Kornfeld, 1986; von Figura and Hasilik, 1986). M6P is recognized by the M6P receptor (MPR) (Ghosh et al., 2003; Kornfeld and Mellman, 1989), and the binding of cationdependent MPR (CD-MPR) with M6P was confirmed by cocrystallization (Roberts et al., 1998). Subsequently, protein sequences similar to the carbohydrate-binding domain of CD-MPR are named MRH domains (Munro, 2001). Yos9p, an MRH domain-containing protein in Saccharomyces cerevisiae, was identified as a lectin important for endoplasmic reticulumassociated degradation (ERAD) (Bhamidipati et al., 2005; Buschhorn et al., 2004; Kim et al., 2005; Szathmary et al., 2005). In the ER membrane, there are several ubiquitin ligases (E3) which target misfolded proteins for ERAD (Hirsch et al., 2009). Hrd1p/Der3p in S. cerevisiae is a RING-finger ubiquitin ligase with six membrane-spanning regions (Bordallo et al., 1998; Hampton et al., 1996). Hrd1p forms a stoichiometric complex with Hrd3p, a type I transmembrane protein in the ER (Gardner et al., 2000). Yos9p then binds to Hrd3p at the luminal domain, forming a large membrane complex for the recognition and degradation of misfolded glycoproteins (Carvalho et al., 2006; Denic et al., 2006; Gauss et al., 2006). Furthermore, recent studies have identified the a1,6-mannose of the N-glycan as the residue that is recognized by Yos9p, and shown that Man7A, an isomer lacking the terminal mannoses on both the B and C branches, serves as the glycan-tag for ERAD in yeast (Quan et al., 2008). Recently, OS-9 and XTP3-B/Erlectin, two mammalian MRH domain-containing lectins, were reported to function as homologs of yeast Yos9p (Christianson et al., 2008; Hosokawa et al., 2008). Both OS-9 and XTP3-B were shown to localize to the ER, and contribute to the regulation of ERAD by associating with the HRD1-SEL1L ubiquitin ligase complex on the ER membrane.

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2. Quality Control of Newly Synthesized Glycoproteins Many proteins synthesized in the ER are glycoproteins, which are covalently modified with N-linked oligosaccharides on the consensus peptide sequence Asn-Xaa-Ser/Thr when they enter the ER through the translocon. Because of the large number of secretory and membrane proteins that are synthesized and folded in the ER, it is equipped with resident proteins that support the folding of nascent polypeptides (Anelli and Sitia, 2008; van Anken and Braakman, 2005). Only correctly folded proteins are allowed to exit the ER to their final destinations, and polypeptides that have failed to obtain the correct conformation are retained in the ER, a process called ER quality control (ERQC) (Ellgaard and Helenius, 2003; Hurtley and Helenius, 1989). The folding status of glycoproteins is monitored by chaperone proteins that recognize unfolded polypeptide regions and lectins that bind N-linked oligosaccharide moieties. To discriminate nascent polypeptides in the process of folding from terminally misfolded proteins, the conformation of N-linked oligosaccharides serves as a ‘‘tag’’ to identify these polypeptides (Hebert et al., 2005; Helenius and Aebi, 2004). The processing of N-glycans on newly synthesized proteins as well as the enzymes and lectins that regulate glycoprotein ERQC are summarized schematically in Fig. 10.1. Trimming of glucose residues in the ER begins immediately after N-linked oligosaccharides are attached to nascent polypeptides, and the removal of mannose from the middle branch of N-glycans destines misfolded glycoproteins for degradation (Cabral et al., 2001; Helenius and Aebi, 2004; Jakob et al., 1998). a1,2-Linked mannoses are then further trimmed while the misfolded proteins are retained in the ER (Lederkremer and Glickman, 2005). Proteins recognized as terminally misfolded are subsequently retrotranslocated out of the ER, ubiquitylated, and then degraded by cytoplasmic proteasomes (McCracken and Brodsky, 2003; Raasi and Wolf, 2007; Tsai et al., 2002). This mechanism is called ERAD (Kostova and Wolf, 2003; Vembar and Brodsky, 2008). Meanwhile, correctly folded glycoproteins are recognized by mannose-binding L-type lectins ERGIC-53, VIP36 and VIPL, and allowed to exit the ER (Appenzeller-Herzog and Hauri, 2006; Kamiya et al., 2005, 2008). Recent studies have revealed that Yos9p, the MRH domain-containing lectin in S. cerevisiae, recognizes Man7A glycan for ERAD (Quan et al., 2008), and that Htm1p/Mnl1p is the processing a1,2-mannosidase that removes the terminal mannose on the C branch (Clerc et al., 2009). In mammals, OS-9 and XTP3-B have been experimentally identified as homologs of yeast Yos9p, and the N-glycan species that yeast Yos9p and mammalian OS-9 recognize are essentially the same, suggesting that these

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ERGIC-53

Productive folding

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Figure 10.1 Processing and recognition of N-glycans in the mammalian ER. N-linked oligosaccharides attached to polypeptides are processed by enzymes in the ER. Each glycan is recognized by specific lectins.

proteins are functionally homologous. A number of recent reviews focus on N-glycan structures and the role of lectins in glycoprotein quality control (Aebi et al., 2010; Hosokawa et al., 2010a; Kamiya et al., 2009; Lederkremer, 2009; Yoshida and Tanaka, 2010).

3. Primary Structures of Yos9p, OS-9, and XTP3-B Yos9p and OS-9 have one MRH domain and XTP3-B has two MRH domains. The primary structures and splice variants of OS-9, XTP3-B, and yeast Yos9p are shown in Fig. 10.2A. Proteins predicted to be homologs of OS-9 or Yos9p are identified in various eukaryotes listed in the database, including protists, plants, fungi, sea anemones, nematodes, and vertebrates, except insects. The overall lengths of the proteins are variable and contain no significant homologous sequences other than the MRH domain. On the other hand, proteins with two MRH domains that are predicted to be homologs of XTP3-B are found in sea anemones, nematodes, insects, and vertebrates. A putative homolog of XTP3-B was also identified in choanoflagellate Monosiga brevicollis (King et al., 2008), a unicellular organism that is one of the closest living relatives of metazoans (Fig. 10.2B). All these XTP3-B

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Figure 10.2 (Continued)

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C XTP3-B_MRH-1 XTP3-B_MRH-2 OS-9_human Yos9p Consensus XTP3-B_MRH-1 XTP3-B_MRH-2 OS-9_human Yos9p Consensus XTP3-B_MRH-1 XTP3-B_MRH-2 OS-9_human Yos9p Consensus

. . SSCSYRIESYWTYEVCHGKHIRQYH-E-EKE-TGQKINI--HEYY-LGNMLAKNLL SYCFRGGVGWWKYEFCYGKHVHQYH-E-DKD-SGK------TSVV-VGTWNQEEHI APCLLKTKDWWTYEFCYGRHIQQYHME-DSEIKGE-------VLY-LGYYQSAF-ERCIFYQAGFWIYEYCPGIEFVQFHGRVNTK-TGEIVNRDESLVYRLGKPKANVEE C W YE C G Q H G G . --FEKEREAEEKEKSNEIPTKNIEGQMTPYYPVGMGNGTPCSLKQNRPRSSTVMYI -EWAKKNTARAYHLQDDGT------QTVRMVSHFYGNGDICDIT-DKPRQVTVKLK -DWDD-ETAKASK-QHR---------LKRYHSQTYGNGSKCDLN-GRPREAEVRFL REFELLYDD-----------------VGYYISEIIGSGDICDVT-GAERMVEIQYV G G C R . . CH--PE-SK-HEI-LSVAEVTTCEYEVVILTPLLCSHPK CKE-SD-SP-HAVTVYMLEPHSCQYILGVESPVICKILD CDEGAGISG-DYI-DRVDEPLSCSYVLTIRTPRLCPHPL CGG-SN-SGPSTI-QWVRETKICVYEAQVTIPELCNLEL C S E C Y P C

D XTP3-B MRH2 XTP3-B MRH 2 human XTP3-B MRH 2 Daniorerio XTP3-B MRH 2 D.melanogaster XTP3-B MRH 2 C.elegans XTP3-B MRH 2 M.brevicollis XTP3-B MRH 1 human XTP3-B MRH 1 Daniorerio XTP3-B MRH 1 C.elegans XTP3-B MRH 1 D.melanogaster XTP3-B MRH 1 M.brevicollis XTP3-B MRH1

Figure 10.2 Primary structures of Yos9p, OS-9, and XTP3-B. (A) Domain organization of Yos9p, four splice variants of hOS-9, and three splice variants of hXTP3-B. The MRH domain is shown in gray, and the signal sequence is in light gray. The ER retrieval signal in Yos9p is shown in dark gray. Numerals indicate the number of the amino acid residues. (B) Molecular phylogenic tree of full-length XTP3-B. This tree was constructed by the neighbor-joining (NJ) method (Saitou and Nei, 1987) based on the alignment of human XTP3-B (NP_056516.2) and predicted XTP3-B proteins of Zebrafish (Danio rerio, NP_955464.1), nematode (Caenorhabditis elegans, NP_740930.2), fruit fly (Drosophila melanogaster, NP_609537.1), and choanoflagellate (Monosiga brevicollis, XP_001747310.1). The open circle indicates the choanoflagellate-animal split. (C) Alignment of human XTP3-B MRH domain 1, MRH domain 2, human OS-9 MRH domain, and yeast Yos9p MRH domain (CLASTALW). Cysteines conserved among the MRH domains are indicated in light gray, and identical amino acid residues

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homologs exhibit high sequence homology throughout. Aligning the MRH domains of OS-9 and XTP3-B revealed that six cysteine residues are well conserved, as well as several amino acids among these MRH domains in this group (Fig. 10.2C). However, only two of the six amino acids that are shown to interact with mannose in CD-MPR (Roberts et al., 1998) are conserved (Fig. 10.2C). Both the first and second MRH domains (MRH1 and MRH2) of XTP3-B from metazoans and M. brevicollis form separate clusters on the molecular phylogenetic tree (Fig. 10.2D), suggesting that XTP3-B emerged by duplication of the MRH domain before the separation of choanoflagellates and metazoans. In human, four splice variants of OS-9 (isoforms 1–4) and three splice variants of XTP3-B (isoforms 1–3) are listed in the database, although their functional differences remain to be clarified (see below for OS-9). Association of the HRD1-SEL1L ubiquitin ligase complex has been reported only with XTP3-B iso1 (long form) but not iso3 (short form) (Hosokawa et al., 2008). Both XTP3-B isoforms 2 and 3 lack part of the MRH domain 2, which recognizes N-glycans (Cruciat et al., 2006; Yamaguchi et al., 2010). Thus, the function of these lectins might be regulated by alternative splicing of the proteins.

4. OS-9 and XTP3-B form a Complex with Membrane-Embedded Ubiquitin Ligase HRD1/synoviolin, a homolog of yeast Hrd1p, is a ubiquitin ligase embedded in the ER membrane with six transmembrane regions and a RING ubiquitin ligase domain in the cytoplasm (Amano et al., 2003; Kikkert et al., 2004). It forms a stoichiometric complex with SEL1L, the mammalian homolog of yeast Hrd3p (Lilley and Ploegh, 2005; Mueller et al., 2006). Mass spectrometric analysis using epitope-tagged OS-9 (Christianson et al., 2008), XTP3-B (Christianson et al., 2008; Hosokawa et al., 2008), or SEL1L (Mueller et al., 2008) revealed that OS-9 and XTP3-B associate with SEL1L. Combined with the results of coimmunoprecipitation experiments, it was revealed that these lectins form a large complex with the HRD1-SEL1L ubiquitin ligase complex on the ER membrane. This large complex also contains a ubiquitin-conjugating among the MRH domains of OS-9 and XTP3-B are shown in dark gray. Amino acid residues that interact with mannose in CD-MPR are marked by dots. (D) Molecular phylogenic tree of the two MRH domains of XTP3-B from various organisms as listed in (B). This tree was also created using the NJ method. The open circle indicates the choanoflagellate-animal split, and the filled rhombus indicates duplication of the MRH domain before the choanoflagellate-animal split.

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enzyme (E2) and other ER membrane proteins necessary for ubiquitylation (Mueller et al., 2008). AAA ATPase p97, which extracts proteins from the ER to the cytosol (Ye et al., 2004), and Derlins, which comprise the putative retrotranslocation channel, also interact with this E3 complex (Lilley and Ploegh, 2005; Schulze et al., 2005; Ye et al., 2005). To analyze complex formation, we performed coimmunoprecipitation in metabolically labeled cells, immunoprecipitation analysis followed by western blotting, and immunoprecipitation in fractions separated by sucrose density gradient ultracentrifugation. In order to detect complex formation with transmembrane proteins or weak protein–protein interactions, digitonin was used to solubilize the cells.

4.1. Method for immunoprecipitation followed by Western blotting 1. Cells are lysed in a buffer (150 mM NaCl, 50 mM Tris–HCl, pH 7.5) containing the appropriate detergent (3% digitonin or 1% NP-40) supplemented with protease inhibitors. The supernatant is then collected by centrifugation at 4
C. 2. Antibodies are added to the lysate and incubated at 4
C for several hours to overnight. 3. Immune complexes are collected with Protein A- or Protein G-Sepharose beads, and separated by SDS-PAGE. 4. Proteins are transferred to a nitrocellulose or nylon membrane. 5. After blocking the membrane, proteins are probed with specific antibodies, and the signal is detected by enhanced chemiluminescence. It is also possible that the interaction of OS-9 with the HRD1-SEL1L complex at the ER membrane is dynamic. Based on the observation that larger amounts of OS-9 associate with GRP94 when SEL1L or HRD1 is silenced by shRNA-mediated knockdown, Christianson and coworkers suggested a model whereby the OS9-GRP94 complex transports ERAD substrates to the SEL1L-containing complex at the membrane (Christianson et al., 2008). More recently, the ERAD substrate BACE476D was shown to bind to OS-9 when SEL1L or HRD1 is silenced (Bernasconi et al., 2010). Studies in our laboratory showed that endogenous OS-9 distributes in two peaks of lower and higher molecular weights when fractionated by sucrose density gradient ultracentrifugation (Hosokawa et al., 2008). In the high molecular weight fractions, endogenous SEL1L and HRD1 are also detected (Fig. 10.3A and manuscript in preparation). Therefore, a portion of OS-9 may preferentially associate with the HRD1–SEL1L complex at the ER membrane, while OS-9 recovered in the lower molecular weight fractions is possibly involved in carrying the ERAD substrates to the ER membrane (Fig. 10.3B). On the other hand, yeast Yos9p associates with the ER membrane (Buschhorn et al.,

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Figure 10.3 Association of ERAD components with the ER membrane. (A) Cofractionation of OS-9 and XTP3-B with the HRD1-SEL1L ubiquitin ligase complex. Cell lysates were fractionated by sucrose density gradient ultracentrifugation (10–40%). An aliquot of each fraction was separated by SDS-PAGE, and endogenous SEL1L, OS-9 and FLAG-tagged XTP3-B long form (isoform 1) were detected by specific antibodies. The proteins highlighted by the square were detected in the fractions close to the bottom. The positions of the molecular weight standards are indicated on the left. Two variants of OS-9 are shown by arrowheads. Asterisks indicate nonspecific bands detected by the anti-SEL1L antibody. (B) ERAD components localized to the ER membrane. In the ER lumen, OS-9, XTP3-B and BiP bind to SEL1L, and the HRD1-SEL1L complex associates with other transmembrane proteins, including the putative retrotranslocation channel Derlins. It remains unknown whether both OS-9 and XTP3-B are incorporated in the same complex.

2004) and proofreads the oligosaccharide structures on the misfolded proteins that are bound to the luminal domain of SEL1L to ensure their degradation (Denic et al., 2006; Gauss et al., 2006). If there are two populations of mammalian OS-9, one that remains at the membrane complex and another

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that cycles between the lumen and the membrane, then the mechanism that regulates these two populations remains to be clarified.

5. Sugar Recognition Specificity of OS-9 and XTP3-B Frontal affinity chromatography (FAC) analysis revealed that the N-glycan structures that yeast Yos9p (Quan et al., 2008) and mammalian OS-9 (Hosokawa et al., 2009; Mikami et al., 2010) recognize are highmannose N-glycans lacking the terminal mannose from the C branch (Fig. 10.1). To analyze the interaction of the human OS-9 (hOS-9) MRH domain with oligosaccharides, recombinant proteins were expressed in E. coli and purified. Because a recombinant hOS-9 MRH domain was primarily incorporated into the inclusion body, proteins were solubilized and denatured in guanidinium chloride, and refolded. An N-terminally hexahistidine-tagged hOS-9 MRH domain was conjugated to Ni-NTA Sepharose beads and fixed in a small column for FAC analysis (see Chapter 10 of Vol. 478).

5.1. Method for FAC analysis FAC was established by Dr. Kasai (Hirabayashi et al., 2003; Kasai et al., 1986) for analyses of low affinity lectin–oligosaccharide interactions. Using HPLC, oligosaccharides are applied individually to a column of immobilized lectin. The oligosaccharides that specifically interact with the lectin are eluted later than the analyte with no lectin interaction. A dissociation constant (Kd) of lectin for oligosaccharides was determined with Eq. (10.1): ½A0 ðVf  V0 Þ ¼ Bt  Kd ðVf  V0 Þ;

ð10:1Þ

where [A]0, V0, and Bt are the initial concentrations of the oligosaccharide, the elution volume of the control sugar, and the total amount of immobilized lectin in the column, respectively. If [A]0 is negligibly small compared to Kd, then Eq. (10.1) can be simplified as follows: Vf  V0 ¼ Bt =Kd

ð10:2Þ

We analyzed the interaction between pyridilaminated (PA-) oligosaccharides and a recombinant hOS-9 MRH domain using the following technique:

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1. The recombinant hOS-9 MRH domain is immobilized on Ni2þ-Sepharose (GE Healthcare) via its N-terminal histidine-tag. After immobilization, the sepharose beads are packed into a high-performance stainless steel column (4.0  10 mm, GL Sciences). 2. Each PA-oligosaccharide is dissolved in 10 mM HEPES (pH 7.4), 150 mM NaCl, and 1 mM CaCl2, and subsequently applied to the column at a flow rate of 0.25 ml/min at 20
C. 3. To determine the relative affinity of each oligosaccharide using Eq. (10.2), Vf values of PA-oligosaccharides dissolved at a concentration of 10 nM are measured. Retardation of the oligosaccharide compared to the control oligosaccharide (PA-GD1b-hexasaccharide) is estimated based on the difference in elution volume, Vf. The elution profiles are monitored by fluorescence intensity at 400 nm (excitation at 320 nm). 4. Kd values for Glc1Man7GlcNAc2-PA are determined using Eq. (10.1), which has a relatively high affinity for the hOS-9 MRH domain. VfV0 values, depending on the ligand concentration, are measured in the range of 2–24 mM. Fifty micromolar of p-nitrophenyl-b-D-galactopyranoside is used as a control sugar for determination of the V0 value of the lectin-immobilized column. The elution profile is monitored by UV absorption at 300 nm to avoid possible quenching caused by the relatively high concentration of the PA-oligosaccharide. Kd values of other PA-oligosaccharides are estimated based on their relative affinities with respect to that of Glc1Man7GlcNAc2-PA. Recently, the sugar-binding specificity of the XTP3-B MRH domain was analyzed using cell-surface expressed lectin assay (see Chapter 11 of Vol. 478), revealing that the repertoire of N-glycans that the XTP3-B MRH domain recognizes is similar to that of OS-9 and Yos9p (Yamaguchi et al., 2010). Interestingly, the authors report that only the MRH domain 2 of XTP3B has lectin activity. This result is consistent with the analysis of Erlectin in Xenopus laevis, which indicated that MRH domain 2, but not MRH domain 1, associates with glycoproteins within the cells (Cruciat et al., 2006).

6. The Effect of OS-9 and XTP3-B on ERAD in Mammals In yeast, the degradation of ERAD luminal (ERAD-L) substrates with misfolded regions in the ER lumen is delayed in Dyos9 cells (Carvalho et al., 2006). On the other hand, the effect of either knockdown or overexpression of OS-9 and XTP3-B on ERAD in mammalian cells is not as simple. Knockdown of OS-9 delayed degradation of the misfolded glycoprotein NHK

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(Bernasconi et al., 2008; Christianson et al., 2008; Hosokawa et al., 2008), but did not affect the degradation kinetics of other ERAD substrates, such as TCRa and RI332 (Christianson et al., 2008). Furthermore, overexpression of OS-9 was reported to delay the degradation of RI332 or NHK (Bernasconi et al., 2008; Mueller et al., 2008), or retain the ERAD substrate NHK in the ER (Bernasconi et al., 2008). Recently, it was suggested that degradation of soluble ERAD luminal (ERAD-LS) substrates, defined as misfolded soluble proteins in the ER (such as BACE476D and CD3-dD), depends on both OS-9 and XTP3-B (Bernasconi et al., 2010). The authors showed that simultaneous silencing of both OS-9 and XTP3-B inhibited the degradation of ERAD-LS substrates, although knockdown of either of them alone did not affect degradation. While this is an interesting finding, further analysis is needed to determine its general implications. Although the XTP3-B MRH domain recognizes carbohydrates, XTP3-B has also been reported to bind to (Christianson et al., 2008; Hosokawa et al., 2008) and delay ERAD of misfolded nonglycosylated proteins (Hosokawa et al., 2008) when overexpressed. Two reports so far have suggested functional differences among the OS-9 splice variants. First, BACE476D binds specifically to OS-9v1, but not to v2, when SEL1L is silenced by RNAi (Bernasconi et al., 2010). Secondly, although silencing only OS-9v1 does not affect ERAD of NHK, silencing both OS-9v1 and v2 delays ERAD (Christianson et al., 2008), suggesting a complementary or alternatively, different function between the two variants. In addition, we have also observed that OS-9v2 preferentially binds to SEL1L (manuscript in preparation). Although OS-9 and XTP3-B have lectin activity, the carbohydraterecognition domains that recognize N-glycans on misfolded glycoproteins or those on SEL1L are controversial at present. Further analysis is required to clarify the molecular mechanisms and binding activity of these lectins. To analyze the function of OS-9 and XTP3-B, we overexpressed or knocked down these lectins in human embryonic kidney (HEK) 293 cells. To manipulate the N-glycan structures on the ERAD substrate NHK, we overexpressed processing a-mannosidases at the same time. To analyze ERAD of glycoproteins in mammalian cells, pulse-chase experiments are preferred over cycloheximide chase methods because the addition of cycloheximide decreases the amount of ER a-mannosidase I (Wu et al., 2007), resulting in stabilization of the misfolded glycoproteins.

6.1. Method for pulse-chase analysis 1. Cells are plated approximately 16–24 h prior to transfection (day 0). 2. siRNA is mixed with transfection reagent (Lipofectamine RNAi TM MAX ) and added to the medium, according to the protocol provided by the manufacturer (day 1).

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3. Plasmids encoding ERAD substrates are transfected (FuGENE6 , LipoTM TM fectamine 2000 , Effectene ), according to the protocol provided by the manufacturer (day 2). 4. When plasmids expressing shRNA are used to silence gene expression, pSUPER and plasmids encoding the ERAD substrates are transfected simultaneously on day 1. 5. Cells are preincubated with medium lacking methionine and cysteine supplemented with dialyzed fetal bovine serum for 20–30 min prior to metabolic labeling (day 3). 6. Cells are metabolically labeled with [35S]methionine/cysteine ([35S]protein-labeling mixture) in medium lacking methionine and cysteine for 15 min, and then chased in normal growth medium. 7. Cell lysates are mixed with antibodies and incubated at 4
C for several hours to overnight. 8. Immune complexes are collected by Protein A- or Protein G-Sepharose beads, and separated by SDS-PAGE. 9. Gels are dried and exposed to phosphor-imaging plates, and the radioactivity is measured for analysis.

7. Concluding Remarks Characterization of mammalian OS-9 and XTP3-B revealed that these lectins recognize specific N-glycans and target misfolded glycoproteins for ERAD. Questions that remain to be answered are discussed in recent reviews (Aebi et al., 2010; Hosokawa et al., 2010a). Here, we briefly discussed the functional interaction of these lectins with ER-degradation enhancing a-mannosidase-like proteins (EDEMs), a subgroup of the glycosylhydrolase family 47 in mammals. The yeast ortholog Htm1p/Mnl1p was shown recently to process the mannose from the C branch of N-glycans, which is subsequently recognized by Yos9p (Clerc et al., 2009). Mammalian EDEMs have three homologs (EDEM1, EDEM2, and EDEM3), and the mannose-processing activities of these proteins in vivo appear to be quite different (for review, see Aebi et al., 2010; Moremen and Molinari, 2006). Recently, we have suggested that EDEM1 is able to process a1,2-linked mannose on the C branch, although the mannosidase activity is dispensable for the enhancement of glycoprotein ERAD (Hosokawa et al., 2010b). Moreover, the interaction of EDEM1 and the SEL1L–HRD1 complex was reported (Cormier et al., 2009), although we were unable to detect a significant interaction between these proteins (Hosokawa et al., 2008). Thus, functional analysis of MRH domain-containing lectins and proteins in glycosylhydrolase family 47 would improve our understanding of how N-glycans regulate the glycoprotein ERAD.

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ACKNOWLEDGMENTS We thank Dr. N. Iwabe (Kyoto University) for the phylogenetic analysis of OS-9 and XTP3-B. This work was supported by the Hayashi Memorial Foundation for Female Natural Scientists (N.H.) and by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (K. K. and Y. K.).

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Multiple Functional Targets of the Immunoregulatory Activity of Galectin-1: Control of Immune Cell Trafficking, Dendritic Cell Physiology, and T-Cell Fate Dianne Cooper,*,1 Juan M. Ilarregui,†,1 Susana A. Pesoa,†,1 Diego O. Croci,† Mauro Perretti,*,2 and Gabriel A. Rabinovich†,‡,2 Contents 1. General Introduction 2. Regulation of Immune Cell Trafficking, Recruitment, and Chemotaxis 2.1. Conceptual framework 2.2. In vitro approaches to study galectins in leukocyte chemotaxis, trafficking, and recruitment 2.3. In vivo methods to study the role of galectin-1 in leukocyte trafficking and recruitment 3. Galectin–Glycan Lattices in the Control of DC Physiology 3.1. Conceptual framework 3.2. In vitro strategies to study the role of galectins in DC physiology 3.3. In vivo strategies to study the role of galectins in DC physiology 4. Galectin–Glycan Lattices in the Control of T Helper Cell Fate 4.1. Conceptual framework 4.2. In vitro studies to study the role of galectins in Thelper cell survival

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* The William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London, London, United Kingdom { Laboratorio de Inmunopatologı´a, Instituto de Biologı´a y Medicina Experimental, Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas, Buenos Aires, Argentina { Departamento de Quı´mica Biolo´gica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina 1 Contributed equally to this work 2 Senior authors Methods in Enzymology, Volume 480 ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)80011-4

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4.3. In vivo assays to study the role of galectin-1 in T helper cell fate 5. Final Remarks and Future Directions Acknowledgments References

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Abstract In the postgenomic era, the study of the glycome—the whole repertoire of saccharides in cells and tissues—has enabled the association of unique glycan structures with specific physiological and pathological processes. The responsibility for deciphering this biological information belongs to endogenous glycan-binding proteins or lectins. Galectin-1, a prototypic member of a family of structurally related proteins, has demonstrated selective antiinflammatory and immunoregulatory effects either by controlling immune cell trafficking, ‘‘finetuning’’ dendritic cell physiology and regulating T-cell fate. These regulatory functions mediated by an endogenous glycan-binding protein may contribute to fulfill the needs for immune cell homeostasis, including preservation of fetomaternal tolerance and prevention of collateral damage as a result of microbial invasion or autoimmune pathology. We will discuss here the conceptual framework which led to the study of galectin–glycan lattices as a novel paradigm of immune cell communication in physiological and pathological processes and will highlight selected methods and experimental strategies which have contributed to the study of the immunoregulatory activities of this multifaceted glycan-binding protein both in in vitro and in vivo biological settings.

1. General Introduction The immune system has evolved as a highly effective and dynamic cellular network which signals the presence of invading pathogens and growing tumors and initiates a protective response that is specific for danger signals; yet maintaining tolerance to self. Active investigation performed over the last decade has disclosed multiple regulatory pathways composed of several checkpoints and fail–safe processes preventing self-reactivity and limiting aberrant or unfaithful immune responses. Important developments include the identification of a number of gene products responsible for central and peripheral T-cell deletion, T-cell anergy, and cytokine deviation, as well as the dissection of the molecular mechanisms underlying the differentiation and function of T regulatory (Treg) cells and tolerogenic dendritic cells (DCs; Bluestone et al., 2007; Tarner and Fathman, 2006). These regulatory processes involve a number of receptors, cytokines, and inhibitory signaling pathways, which may act in concert during the lifespan of immune cells to achieve homeostasis (Fife and Bluestone, 2008; Maynard and Weaver, 2008; Peggs et al., 2008).

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In spite of the well-established mechanisms governing cognate ligand– receptor interactions during cytokine signaling, chemotaxis, and cell adhesion paradigms, the immunological relevance of supramolecular lattices established by lectin–saccharide interactions and their role in immune cell tolerance and homeostasis is just emerging (Toscano et al., 2007a; van Kooyk and Rabinovich, 2008). Galectins, a family of soluble lectins widely distributed in the animal kingdom, have emerged as pleiotropic regulators of innate and adaptive immune responses (Rabinovich et al., 2007a,b; Yang et al., 2008). To date, 15 galectins have been identified in mammals, most of them with wide tissue distribution; yet expression of some galectins is confined to a restricted set of tissues. Galectins share a common structural fold and contain a conserved carbohydrate-recognition domain (CRD) of about 130 amino acids that mediates carbohydrate binding. A traditional classification based on structural similarities includes: (a) ‘‘proto-type’’ galectins (galectin-1, -2, -5, -7, -10, -11, -13, 14, and -15) which have one CRD and may exist as monomers or dimers; (b) ‘‘tandem-repeat type’’ galectins (galectin-4, -6, -8, -9, and -12) which contain two different CRDs separated by a linker of up to 70 amino acids, and (c) the unique ‘‘chimera-type’’ galectin-3 which contains a CRD connected to a nonlectin N-terminal region. Most galectins are either bivalent or multivalent with regard to their carbohydrate-binding activities, which enable recognition of multiple binding partners and activation of distinct signaling pathways; one-CRD galectins can dimerize, two-CRD galectins are at least bivalent; and galectin-3 can form oligomers upon binding to multivalent glycoproteins (Cummings and Liu, 2008; Rabinovich et al., 2007a,b). Although galectins do not contain a classical signal sequence, they are frequently found in the extracellular compartment and are released through an unusual route which requires intact b-galactoside-specific activity of the secreted protein (Cummings and Liu, 2008). Once outside the cell, galectins can bind multiple glycosylatedbinding partners, form distinct types of multivalent lectin–glycoprotein lattices and convey glycan-containing information into immune cell activation, differentiation, trafficking, and signaling programs (Brewer et al., 2002; Rabinovich et al., 2007b). In this way, galectins are capable of eliciting autocrine or paracrine regulatory effects in acute and chronic inflammatory microenvironments. Galectin-1, a prototypical member of the galectin family, was discovered more than 20 years ago as a b-galactoside-binding lectin of 14.5 kDa with typical hemagglutinating activity (Levi and Teichberg, 1981). Since then, several studies have identified and characterized galectin-1 in many different tissues of several species, demonstrating its widespread distribution in multiple cells and tissues. However, it was only in the last decade that this endogenous lectin appeared in the center of the scene as a fine-tuner of innate and adaptive immune responses (Rabinovich et al., 2002a). Within the immune system, galectin-1 is synthesized and secreted by activated but not resting T and B cells (Blaser et al., 1998; Fuertes et al., 2004; Rabinovich et al., 2002b; Zun˜iga et al., 2001a) and it is significantly upregulated in activated endothelial cells, activated

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macrophages, CD4þCD25þ Treg cells and decidual natural killer (NK) cells (Garı´n et al., 2007; Kopcow et al., 2008; Rabinovich et al., 1996, 1998; Sugimoto et al., 2006). This regulated secretion and preferential localization suggested a potential function for galectin-1 in negative regulation of leukocyte trafficking and inhibition of effector T-cell responses. Relevantly to the latter, expression of galectin-1 is abundant in immune privileged sites such as placenta (Blois et al., 2007; Iglesias et al., 1998; Kopcow et al., 2008), testis (Dettin et al., 2003; Wollina et al., 1999), and retina (Ishida et al., 2003; Romero et al., 2006) and is significantly altered (up- or downregulated) during several pathological conditions, including cancer, infections, and autoimmunity (Harjacek et al., 2001; Rabinovich, 2005; Rabinovich and Gruppi, 2005; Zun˜iga et al., 2001b). Research over the past years using experimental models of autoimmunity, acute and chronic inflammation, fetomaternal tolerance and cancer, has provided proof-of-concept of the pivotal role of galectin-1 and its specific saccharide ligands in immune tolerance and homeostasis, highlighting multiple functional targets of its immunoregulatory activity. While galectin-1based gene and protein therapy strategies suppress chronic inflammation and autoimmunity in experimental models of arthritis, diabetes, inflammatory bowel disease, multiple sclerosis, and uveitis (reviewed in Rabinovich and Toscano, 2009), targeted disruption of galectin-1 gene expression results in heightened T-cell-mediated tumor rejection in B16 melanoma (Rubinstein et al., 2004) and classical Hodgkin lymphoma (Juszczynski et al., 2007), highlighting novel therapeutic opportunities for immune-intervention based on the selective manipulation of galectin-1-glycan interactions. Here, we will discuss the most important methods and experimental strategies used to investigate the role of galectin-1 in acute and chronic inflammatory microenvironments. Particularly, we will focus on three well-established functions exerted by this glycan-binding protein, namely modulation of immune cell trafficking and recruitment, control of DC physiology and selective regulation of T-cell fate, which have led to the notion of galectin1 as a selective and ‘‘nonredundant’’ regulator of immune cell homeostasis.

2. Regulation of Immune Cell Trafficking, Recruitment, and Chemotaxis 2.1. Conceptual framework The trafficking and selective recruitment of leukocytes throughout the vasculature toward inflamed tissues is a life-saving process. Three decades of intense research have allowed the development of a model whereby different molecules, spanning from proteins, lipids, glycans, and autacoids, may act in concert to orchestrate the process of leukocyte migration and extravasation.

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Concomitantly, a wealth of knowledge on the structural features and functional properties of adhesion molecules, chemoattractants, and extracellular matrix proteins has been acquired. The spatiotemporal regulation of this response is accomplished through the coordinated action of pro- and antiinflammatory mediators that appear in the inflammation scene in a tightly controlled sequential fashion. In this way, the complex series of events ‘‘switched-on’’ by proinflammatory signals are slowly ‘‘switched-off’’ by the action of the latter until tissue homeostasis is reestablished. Interestingly, galectin-1 has been shown to downmodulate innate immune responses by inhibiting leukocyte trafficking and extravasation. Illustrating this concept, in a model of noninfective and resolving acute inflammation, high levels of galectin-1 were detected in peritoneal macrophages as well as in inflammatory exudates only at later stages, when the inflammatory reaction is being extinguished, but not at earlier time periods (Gil et al., 2006). Initially, studies on the biological properties of galectin-1 on T cells have revealed important modulatory functions of this endogenous lectin in the context of adaptive immunity (see Sections 3 and 4 for more details); in contrast, the study of galectin-1’s effects on specific innate immune components has lagged behind. In a model of rat paw oedema, administration of recombinant galectin-1 resulted in a robust antiinflammatory effect, evidenced both in terms of decreased paw swelling and diminished cellular infiltration (Rabinovich et al., 2000). Histological analysis of galectin-1treated paws showed significantly reduced numbers of infiltrated polymorphonuclear neutrophils (PMN) and degranulated mast cells when compared to controls. Furthermore, galectin-1 was also found to inhibit arachidonic acid release and prostaglandin E2 production by lipopolisaccharide (LPS)-stimulated macrophages (Rabinovich et al., 2000), suggesting a potential mechanism for the beneficial effects observed in this model. The effects of galectin-1 in the process of leukocyte migration and extravasation have been further studied in detail in a mouse model (La et al., 2003). Here, administration of low doses of recombinant galectin-1 (0.3–1 mg per mouse equivalent to 20–66 pmol) induced potent inhibition of PMN migration elicited by interleukin (IL)-1b (see below for technical details on the peritonitis protocol). Studies in vivo were complemented by doseresponse analyses using an established in vitro system to study leukocyte chemotaxis. These assays demonstrated that addition of low concentrations of galectin-1 (ranging from 0.04 to 4 mg/ml) induced downregulation of IL-8-induced human PMN locomotion (La et al., 2003). These inhibitory properties were also evident when human PMN transmigration across endothelial cell monolayers was assessed under static conditions. In these studies, addition of low concentrations of galectin-1 (0.04 mg/ml corresponding to 3 pmol/ml) induced a sustained inhibitory effect. Thus, galectin-1-induced inhibition of PMN chemotaxis and

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transmigration followed a bell shape response, being pronounced at very low and high concentrations (La et al., 2003). The same was observed when cell–cell interactions were studied under flow (Cooper et al., 2008). Cummings and colleagues obtained provocative results when studying the effects of galectin-1 on human PMN physiology. In contrast to the proapoptotic effects observed on T cells (Perillo et al., 1997), exposure of activated human PMN resulted in sustained phosphatidylserine (PS) exposure, an effect which might reflect changes in scramblase and/or flipase activation in the absence of overt apoptosis (Dias-Baruffi et al., 2003). Although PMN remained viable over time, binding to annexin V was significantly enhanced in a time-dependent fashion. Moreover, induction of PS exposure was found to be reversible upon removal of galectin-1 suggesting that this lectin does not initiate irreversible cell death in human PMN, but rather prepares PMN for phagocytosis (Dias-Baruffi et al., 2003; Stowell et al., 2009). Remarkably, binding of galectin-1 to PMN was markedly increased when PMN were activated, suggesting exposure of lectin-binding sites during the activation process, an effect which might reflect changes in the cell surface ‘‘glycome’’ of these cells (Dahlgren et al., 2000; Dias-Baruffi et al., 2003; La et al., 2003; Sengelv et al., 1995). Collectively, these data identify PMN as unequivocal targets of galectin-1 activity, but do not shed light as to its modes of action. In order to address this question, protocols of intravital microscopy (IVM), which allow direct visualization of the inflamed microcirculation, were performed. Following administration of an antiinflammatory dose of galectin-1, inhibition of all three processes typical of an inflamed microcirculation, namely cell rolling, adhesion, and migration was observed (La et al., 2003). These results were confirmed for human PMN by monitoring interactions of these cells with monolayers of human umbilical vein endothelial cells (HUVECs) under flow (using the flow chamber system; protocol detailed below). This study showed that preincubation of PMN with recombinant galectin-1 significantly decreased the extent of capture, rolling, and adhesion of these cells on activated endothelial cell monolayers (Cooper et al., 2008). Of note, these assays were done with increasing concentrations of galectin-1 (0.04, 0.4, and 4 mg/ml), showing that the lowest concentrations affected both PMN rolling and adhesion, while higher concentrations only abolished rolling. The fact that distinct cellular processes are affected by galectin-1 in a concentration-dependent fashion suggests that unique receptors could mediate different biological processes during neutrophil activation. Following this reasoning, PMN rolling on endothelial cell monolayers might induce cell surface exposure of a second and distinct glyco-receptor which might be subsequently activated by a different concentration of this lectin, leading to inhibition of PMN adhesion and transmigration. Additional work is needed in order to define and characterize specific receptors involved in these effects and their selective glycosylation pattern during the lifespan of PMN. In addition, galectin-1-binding sites have also been described on platelets

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which are involved in the aggregation and activation of these cells (Pacienza et al., 2008). Future studies are warranted to determine whether galectin-1 affects platelet adhesion to the endothelium and to define whether galectin-1mediated platelet/leukocyte interactions are critical to delineate the fate of systemic inflammatory responses. The role of endogenous galectin-1 in immune cell trafficking may be assessed experimentally using two different approaches: (a) siRNAmediated silencing of galectin-1 in vitro and in vivo; (b) gene deleted mice lacking the galectin-1 gene (Lgals1/). As determined by flow chamber assays, downregulation of galectin-1 in endothelial cells using siRNA strategies favored their interaction with leukocytes resulting in increased number of captured cells (Cooper et al., 2008). Collectively, these findings indicate that galectin-1 functions to limit PMN recruitment onto a tumor necrosis factor (TNF)-treated endothelium, a property that may underline its inhibitory effects during acute inflammation. To verify the effects of galectin-1 in an in vivo system, IVM of Lgals1/ mice was performed in comparison with their wild-type counterpart (Poirier and Robertson, 1993). Consistent with an inhibitory function of galectin-1 in acute inflammation, leukocyte trafficking, adhesion, and migration were significantly increased in the cremasteric circulation of galectin-1 null mice (Cooper et al., 2008). Of interest, these inhibitory properties of endothelial galectin-1 were not restricted to the PMN compartment, but were also evident on flowing lymphocytes (Norling et al., 2008). In addition to regulating cell trafficking, endothelial cell-derived galectin-1 has been shown to play an important role in angiogenesis (Thijssen et al., 2006). Hence, targeting endothelial galectin-1 may represent a novel strategy with potential anticancer applications (Thijssen et al., 2007). Furthermore, studies performed in galectin-1-deficient mice showed that endothelial-derived galectin-1 activates inhibitory signals to reduce lymphocyte trafficking during the early phases of an inflammatory response (Norling et al., 2008). The same ‘‘buffering’’ effect was seen, as mentioned above, when the cremaster microcirculation was exposed to IL-1b (Cooper et al., 2008), indicating again the potent inhibitory effects of galectin-1 on cell migration and endothelial–leukocyte interactions. Thus, mimicking endothelial galectin1 during the development of inflammatory responses, would represent a novel strategy to control aberrant cell trafficking. Figure 11.1 illustrates the working model that prompted us to investigate whether exogenous and endogenous galectin-1 acts as a tonic inhibitory mediator of leukocyte recruitment. This effect could also underlie the antiinflammatory effects of this glycan-binding protein or its genetic delivery in experimental models of autoimmune disease, including collagen-induced arthritis, diabetes, uveitis, concanavalin A-induced hepatitis, hapten-induced colitis, and experimental autoimmune encephalomyelitis (EAE; reviewed in Rabinovich and Ilarregui, 2009; Rabinovich and Toscano, 2009).

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CD43 / 45 Putative receptor

Leukocyte

Exogenous galectin-1

Leukocyte

More interactions (rolling and adhesion)

Endothelial galectin-1 Gal1 siRNA

Figure 11.1 Galectin-1 acts as a tonic inhibitor of leukocyte interaction with endothelial cells. Endothelial cells express high amounts of galectin-1, a good proportion of which is exposed on the luminal face. Exposure to exogenous galectin-1 inhibits leukocyte rolling and adhesion. In contrast, reducing galectin-1 expression by using silencing RNA approaches (or genetically modified animals, or cells thereof) would augment the extent of white blood cell interaction with the endothelial monolayer.

2.2. In vitro approaches to study galectins in leukocyte chemotaxis, trafficking, and recruitment A number of experimental approaches in vitro have allowed the investigation of the mechanisms underlying leukocyte chemoattraction and migration toward inflamed tissues. Among them, the chemotaxis assay can assess the migration of a single population of leukocytes through a filter in response to a chemotactic stimulus whereas the flow chamber assay allows examination of the interactions between endothelial cells and leukocytes under flow. Details of these assays are described below. 2.2.1. Human blood leukocyte isolation The isolation of pure leukocyte populations is required for chemotaxis, as well as for flow chamber assays. We typically collect blood from healthy volunteers with a 21-gauge needle and transfer to a 50-ml centrifuge tube containing 1/10 volume of 3.2% (w/v) sodium citrate. This is further diluted 1:1 with warm RPMI 1640 medium. A double density gradient is formed by layering an equal volume (3 ml) of Histopaque 1077 over Histopaque 1119. This must be prepared immediately before use to avoid any diffusion occurring between the two layers. Using a sterile Pasteur pipette prediluted blood should be layered onto the Histopaque and centrifuged at room temperature for 30 min at 400g. Two distinct layers of leukocytes can be seen after centrifugation, with red blood cells (RBCs) pelleted to the bottom of the centrifuge tube. PMN are found above the RBCs and peripheral blood mononuclear cells (PBMCs) are found at the plasma/Histopaque interface. 2.2.2. Lymphocyte isolation For lymphocyte isolation, the PBMC layer should be removed first with a Pasteur pipette to avoid crosscontamination with PMN. Aliquots of 25 ml should be placed in 50 ml centrifuge tubes and an equal volume of RPMI 1640

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should be added. Following centrifugation at 300g for 15 min the supernatant should be discarded. PBMCs are resuspended in 50 ml RPMI 1640 and centrifuged at 300g for 10 min. This step is repeated until the supernatant is clear so that contaminating platelets are removed. The cell pellet is then resuspended in 2 ml RPMI medium supplemented with 10% (v/v) heatinactivated fetal calf serum (FCS), 2 mM L-glutamine, 100 U penicillin and 100 mg/ml streptomycin (complete media) to count (see below). To separate monocytes and lymphocytes, PBMC are plated into 6-well culture plates (4  106 cells/well) and placed at 37  C for 1 h to allow monocytes to adhere. Lymphocytes can then be removed for use in chemotaxis or flow assays. 2.2.3. PMN isolation Following collection of the PMN layer with a Pasteur pipette (6 ml PMN/ 15 ml falcon tube) an equal volume (6 ml) of RPMI 1640 is added. Cells are then centrifuged at 300g for 15 min, the supernatants are discarded and the pellet is resuspended by flicking tubes gently. If the pellet is not completely resuspended red cell lysis is not achieved. Next, 7.5 ml of ice-cold water is added, the tube is inverted three times and 2.5 ml of 3.6% (w/v) sodium chloride solution is subsequently added. The ice-cold water is employed to induce lysis of contaminating erythrocytes, whilst sodium chloride is added to preserve PMN. Cells are finally centrifuged at 300g for 10 min and resuspended in 2 ml RPMI medium supplemented with 0.1% (w/v) BSA to count. 2.2.4. Cell counting For cell counting, we remove a 10-ml aliquot of cells and add to 990 ml Turk’s [0.01% (w/v) crystal violet in 3% (v/v) acetic acid]. This allows a differential cell count (distinguishing between PMN, monocytes, and lymphocytes based on nuclear morphology) to be performed. 2.2.5. Chemotaxis Assay Chemotaxis assays can be performed using any purified leukocyte subset and chemoattractant of choice. IL-8 (30 ng/ml) is commonly used for testing potential inhibitors of PMN chemotaxis, whilst SDF-1a (10 ng/ml) or MIP1a (10 ng/ml) is typically used for T cells or monocytes respectively. Chemotaxis plates are commercially available (e.g., the Neuroprobe ChemoTxplateTM). The plate is based on a 96-well microplate format with an upper filter and lid. The filters are available with different pore sizes that can be used for studying different cell populations (3 mm for PMN, 5 mm for T lymphocytes and 8 mm for monocytes and macrophages). PMN are isolated as described above and resuspended in RPMI containing 0.1% (w/v) BSA at a concentration of 4  106 ml 1. Twenty-seven microliters of the chemoattractant or medium alone (negative control) is pipetted into the bottom well of the chamber. Care must be taken to avoid development of any air bubbles. The upper filter is then carefully placed on top of the plate

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and fixed in place at each corner. The filter should be checked visibly to ensure that the membrane is in contact with the fluid in the bottom compartment of every well and that there are no air bubbles between the filter and the media in the bottom well as this will cause false negative results. Twenty-five microliters of cell suspension is then carefully pipetted onto the top wells and the lid put on the plate. The plate is then placed in a humidified chamber in 5% CO2 at 37  C for the desired time, which in the case of neutrophil transmigration is 1.5 h. At the end of this experimental period, the remaining cells/media are removed from the top of the membrane using a cotton bud. Each well is then washed with 25 ml RPMI to remove any remaining cells. The plate and membrane is centrifuged for 1 min at 312g for 5 min to pellet cells in the bottom well, the filter is then removed and the cell pellet resuspended. Leukocytes that have migrated to the bottom chamber are quantified by diluting in Turk’s dye and cells are counted as described above. To test the effect of galectin-1 in this model, leukocytes are preincubated with recombinant galectin-1 for 10 min at 37  C prior to addition to the filter. We have found that PMN chemotaxis is inhibited when PMN are preincubated with 0.04–4 mg/ml galectin-1 (La et al., 2003). To determine whether galectin-1 itself has chemotactic potential, a range of galectin-1 concentrations may be placed in the lower well and chemotaxis may be compared to that induced by a known chemoattractant such as IL-8 for PMN. As galectins are known to induce agglutination of some cell types this should be assessed prior to performing chemotaxis assays as false negative results could be obtained if leukocytes agglutinate in the top well of the chamber. 2.2.6. Flow chamber assay: Studying the effects of galectin-1 on human PMN and lymphocyte interactions with human endothelial cells under flow The major advantage of the in vitro flow chamber over static assays such as the chemotaxis assay described above is that it is performed under flow. This enables a more physiologically relevant examination of the effects of single agents and compounds on the various steps of the leukocyte recruitment cascade. Flow assays are required to investigate mechanisms by which adhesion molecules such as selectins capture leukocytes so that they initiate rolling across the endothelium. If the correct stimulations are applied, all aspects of leukocyte recruitment, such as capture, rolling, adhesion, and transmigration, can be monitored. A detailed protocol for carrying out these assays is detailed below. 2.2.7. Isolation and culture of primary HUVECs Primary HUVECs are isolated from umbilical cords. Cords are collected in cord buffer [PBS containing penicillin (100 U), streptomycin (100 mg/ml), and fungizone (2.5 mg/ml)] and stored at 4  C until endothelium isolation.

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Endothelial cells are purified from umbilical cords by collagenase digestion of the interior umbilical vein. The veins are perfused with approximately 50 ml of cord buffer using a sterile syringe to wash out the residual blood. The other end of the cord is then clamped and approximately 20 ml of 0.1% (w/v) collagenase type II in serum-free medium 199 containing penicillin (100 U), streptomycin (100 mg/ml), fungizone (2.5 mg/ml), and L-glutamine (2 mM) is added. Another clamp is then placed at the top end of the cord and the vein is incubated in a humidified chamber in 5% CO2 at 37  C for 15 min. Following incubation, the collagenase solution is collected into a 50-ml centrifuge tube and the vein flushed with 30 ml of cord buffer to remove endothelial cells. Cells are then centrifuged at 560g for 5 min, supernatants are removed and the pellet is resuspended in 15 ml of complete medium (M199 containing 20% human serum, 100 U penicillin, 100 mg/ ml streptomycin, 2.5 mg/ml fungizone, and 2 mM L-glutamine) and transferred to a T75 flask. Typically the yield of this procedure is in the range of 0.5–1.5  106 cells per cord. Cells are seeded into T75 flasks or 35 mm plates (for flow assays) precoated in 0.5% (w/v) bovine gelatin prior to use. Briefly, 2% (w/v) gelatin is diluted 1:4 with PBS and tissue culture plastics are coated with the 0.5% solution for 20 min at room temperature. Following this coating procedure, gelatin is aspirated before seeding. Then, cells are incubated in a humidified chamber in 5% CO2 at 37  C, medium is replaced after 24 h to remove residual erythrocytes and changed every 48 h thereafter. When cells cultured in the flask reach approximately 80% confluency they are rinsed once with PBS and subcultured using 0.025% (w/v) trypsin/0.01% (v/v) EDTA solution (2 ml for a 75-cm2 flask). When 90% of the cells have rounded and started to detach, the flask is then tapped firmly on the side to release the cells and an appropriate volume of complete media is added. For flow chamber assays, HUVEC should ideally be used at passage 1–2 and not beyond passage 3; as high passage HUVECs begin to lose their responsiveness and expression levels of adhesion molecules. 2.2.8. In vitro flow chamber assay HUVECs are seeded in 35 mm2 gelatin-coated dishes at a density of 3  105 cells in complete medium and used 24 h after plating at confluence. Confluent monolayers can be stimulated with TNF (10 ng/ml) for 4 h to upregulate adhesion molecules such as E-Selectin, ICAM-1, and VCAM-1 (Cooper et al., 2008). Human PMN, isolated as outlined above, are resuspended to 1  107 cells/ml in Dulbecco phosphate buffered saline (DPBS; without calcium and magnesium) containing 0.1% (w/v) BSA and kept on ice before use. Immediately prior to flow, 5  106 (0.5 ml) PMN are diluted to 5 ml in DPBS supplemented with Ca2þ and Mg2þ, yielding a cell suspension of 1  106 cells/ml. PMN are then incubated for 10 min

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Step 1

Step 3

HUVEC monolayer in 35 mm2 dish

Gasket

Parallel plate flow chamber Step 2

Vacuum pump Neutrophil inlet

PBS Syringe pump

Figure 11.2 Assembly of the parallel plate flow chamber. The chamber is first covered in vacuum grease to hold the gasket in place, and residual grease is removed using an alcohol wipe. PBS is then added to the window in the gasket to cover both the inlet and outlet, ensuring no air bubbles are present. Finally, the HUVEC monolayer is added to the vacuum-sealed chamber, inverted, and placed under an inverted microscope.

at 37  C following which the endothelium is rinsed with PBS prior to attachment to the parallel plate laminar flow chamber (e.g., the one from GlycoTech), as shown in Figure 11.2, and the cells are flowed for 8 min prior to recording. A shear stress of 1 dyne/cm2 is generated using an automated syringe pump (Harvard Apparatus, South Natick, MA). This is calculated according to an adaptation of Poiseuille’s law that states: Wall shear stressðdyne=cm2 Þ ¼ Mean flow velocityðmm=sÞ  ½8=tube diameterðmmÞ  viscosityðPoiseÞ This approximation takes into account the flow rate through a cylindrical vessel; the equation relating wall shear stress to volumetric flow rate through the chamber is given by: Tw ¼ 6mQ=a2b where tw, wall shear stress (dynes/cm2); m, coefficient of viscosity (P); Q, volumetric flow rate (ml/s); a, channel height (i.e., gasket thickness, cm); b, channel width (i.e., gasket width, cm). Experiments are usually performed at a constant volumetric flow rate of 0.00707 ml/s. The coefficient of viscosity is determined by the fluid in which the PMN are resuspended and the temperature of the solution. For PBS at a constant temperature of 37  C, the viscosity is 0.0076 Poise.

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• HUVEC stimulated with 10 ng / ml of hr-TNF for 4h • 5 × 106 PMN / ml flowed over a HUVEC mono-layer 8-min • Six 10s clips recorded for offline analysis

Video

Vacuum

Syringe pump

Flow chamber

Figure 11.3 Equipment required for the flow chamber assay. An example of a full set up used to run the flow chamber assay with human PMN and HUVEC and used, for instance, to determine the effect of exogenously applied or endogenous (endothelial) galectin-1.

The flow channel has a set size determined by the dimensions of the gasket; the channel height relates to the thickness of the gasket, which is 0.0254 cm with a channel width of 0.5 cm. These conditions create a wall shear stress of precisely 0.9994172 dyne/cm2. The entire flow chamber is placed under an inverted microscope fitted with 10 and 20 phase contrast objectives (Nikon, Melville, NY). Figure 11.3 shows a complete flow chamber setup. PMN are perfused over HUVEC monolayers for a period of 8 min, and six, 10-s frames are chosen from random fields of view using a Q-imaging Retiga EXi digital video camera (Q-imaging) and recorded in Streampix capture software (Norpix) ready for off-line analysis. Sequences are loaded into ImageProPlus software (Media Cybernetics, Wokingham), PMN can be manually tagged and their migration monitored. Three measurements are usually made in the analysis: the total number of interacting cells are quantified as number of cells initially captured during the 10-s frame, which is further

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classified as either rolling or firmly adherent (those which remain stationary for the 10-s observation period); the total number of interacting cells for each category (capture, rolling, and adhesion) is expressed as cells per field, counting six fields per treatment. To determine the effect of galectin-1 on leukocyte recruitment either the leukocyte population to be studied or the endothelial cells can be preincubated with the recombinant lectin. We have found that preincubation of PMN with low concentrations of galectin-1 (0.04 mg/ml) significantly reduced the number of captured and rolling PMN (Cooper et al., 2008), whereas lymphocyte interactions are inhibited by preincubating either lymphocytes or endothelial cells with recombinant galectin-1 (Norling et al., 2008).

2.3. In vivo methods to study the role of galectin-1 in leukocyte trafficking and recruitment All animal studies are conducted following local Ethics Committee’s approval and in accordance with national regulations (in the United Kingdom, following the Home Office Guidance on the Operation of Animals Scientific Procedures Act, 1986). 2.3.1. Experimental model of mouse peritonitis: Studying the effect of galectin-1 on PMN trafficking Peritonitis is a clinical feature observed in patients undergoing long-term peritoneal dialysis or in complications following peritoneal surgery. Experimental peritonitis can help to understand the mechanisms of inflammation in that particular cavity; in some cases, the local inflammatory reaction in the peritoneum can rapidly disseminate and lead to a systemic syndrome associated with high morbidity. Experimental peritonitis is widely used as an inflammatory model for drug screening and testing. Depending on the triggering agent used, specific inflammatory mediators, enzymes, or receptors can contribute to the leukocyte recruitment process, facilitating assessment of the effects of compounds or drugs of potential interest. More recently, this self-resolving model has been used to study the mechanisms and molecules that contribute to the resolution of inflammation. Rodents are usually the species of choice for peritonitis induction, although the use of larger animals has also been reported. The peritoneal cavity is a suitable model for injection of different biological or chemical agents capable of mediating the recruitment of specific leukocyte subsets. For example, nonspecific inflamogens such as zymosan or carrageenan (Ajuebor et al., 1998a) attract multiple cell types, whereas more specific inflammatory mediators, such as IL-1b or chemokines (CCL2, CXCL1, and so forth; Ajuebor et al., 1998a,b; La et al., 2003) induce selective recruitment of specific subsets. IL-1b-induced PMN recruitment represents an in vivo model where chemotaxis occurs in the absence of many features that characterize acute

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inflammation, for example, exudation. In fact, PMN recruitment observed in response to application of IL-1b is not strictly direct; that is, it does not result from a direct activation of the circulating PMN, at variance from TNF (Young et al., 2002) and it may require activation of intermediate cells, including endothelial cells and mesothelial cells. 2.3.2. Induction of peritonits IL-1b injection induces peritonitis in both mice and rats; commonly doses of 0.5–50 ng per mouse cavity and 10–100 ng per rat cavity are given. On the day of the experiment we remove an aliquot of IL-1b from the freezer and resuspend to a concentration suitable for injecting a volume of 0.5–1 ml per mouse cavity or 1–5 ml per rat cavity. We inject the lower part of the abdominal cavity, being careful not to damage the liver in order to minimize a potential hemorrhage that will interfere with the evaluation of leukocyte recruitment. 2.3.3. Harvesting of recruited cells After a given time period, animals are sacrificed and the skin is separated from the abdomen wall with help of forceps via a careful incision; ensure the abdominal wall is not perforated. Next, the abdomen is exposed and up to 2–3 or 6–10 ml of ice-cold PBS per mouse or rat respectively is injected using a syringe and 21-gauge needle. The abdomen is gently massaged to enable complete washing of the cavity and recovery of recruited leukocytes. The use of heparin, EDTA (or both) in the wash buffer minimizes leukocyte aggregation (25 U/ml heparin, 2 mM EDTA in sterile PBS). The recovery of the wash fluid can be performed via an incision and by the use of a plastic Pasteur pipette. If sterile conditions are required, lavage fluid can be recovered by using a syringe without opening the cavity. For best practice, access to a cell culture hood will ensure the most likely sterile conditions. Migrated cells can be determined by light microscopy after staining in Turk’s dye, which allows a differential cell count (distinguishing PMN from mononuclear cells based on nuclear morphology) to be performed. For more detailed identification of migrating cell populations, peritoneal cells harvested from the cavities may be stained with specific monoclonal antibodies (mAb) and analyzed by flow cytometry. For instance Gr-1þ cells can be determined with a Ly6C/Ly6G mAb (Clone RB6-8C5; BD Pharmingen). However, since this mAb can also react with a monocyte population with intermediate level of Gr-1 expression, it is advisable to quantify Gr-1bright cells as PMN. Alternatively, the Ly6G mAb (Clone 1A8; BD Pharmingen), which only stains mouse PMN may be used. 2.3.4. Summary of the peritonitis model in the mouse 1. Inject IL-1b (5–10 ng/cavity) in a final volume of 500 ml pyrogen-free PBS, with control animals receiving an identical volume of PBS.

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2. At the selected timepoint, use a humane procedure to sacrifice the animals according to the most rigorous ethical guidelines. 3. Expose the abdominal cavity via an incision in the skin, without opening the cavity. 4. Detach the skin from the abdominal wall with the help of forceps. 5. Inject 3 ml of ice-cold wash buffer with a syringe equipped with a 21gauge needle. 6. Perform a gentle massage of the abdomen (for 10–20 s). 7. Make a small incision and carefully insert a Pasteur pipette for lavage fluid collection. 8. Transfer the collected fluid to a 15-ml tube on ice. 9. Repeat the collection step until the fluid has been completely harvested. Note that usually the volume recovered is 2.5 ml. 10. Take a 100-ml aliquot and dilute in 900 ml of Turk’s solution for total leukocyte count in a Neubauer chamber. 11. The remaining wash fluid can be centrifuged for collection of the cellfree lavage fluid and subsequent determination of soluble mediators. The remaining cells can be pelleted and used for cellular analysis by flow cytometry, Western blot and real time RT-PCR. 2.3.5. IVM: Studying leukocyte trafficking in vivo IVM is used to directly visualize the microcirculation of animals in vivo, thus allowing assessment of leukocyte–endothelial cell interactions. Since many immunological disease mechanisms are reflected by primary interactions at the microcirculation level, IVM can be used to study the underlying mechanisms, their contributions to leukocyte trafficking and implications for tissue damage and immunopathology. 2.3.6. Microvascular beds Early IVM studies are typically carried out in an array of tissue beds and animals with the bat wing, hamster cheek, rabbit ear, and cat mesentery all being used. The advent of genetically modified animals has led to rodents now being the preferred choice with the cremaster (the muscle surrounding the testicle) and mesentery being the most studied tissues due to their thin and transparent nature. However, the surgical preparation required to analyze these microvascular beds may result in activation of the tissue causing rapid and pronounced upregulation of rolling through partial degranulation of perivascular mast cells and endothelial expression of P-selectin; yet this effect has been exploited for studies investigating selectin-dependent recruitment. A further limitation of IVM has been the inability to differentiate granulocyte responses from those of mononuclear cells. However, this problem has been overcome recently with the use of leukocyte subtype-specific antibodies.

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2.3.7. Intravital setup The basic setup for IVM requires a microscope suitable for epi- or transillumination, a camera, a video, or DVD recorder for recording images to be analyzed off-line and a Doppler velocimeter for measuring blood flow within vessels. 2.3.8. Mesentery preparation Mice are anesthetized using xylazine (7.5 mg/kg) and ketamine (150 mg/kg) i.p. Once anesthetized the mouse is placed on a specialized plexiglas board and the right jugular vein is cannulated for administration of saline, pharmacological agents or antibodies. A midline laparotomy is then performed and the small bowel is gently exteriorized using moist cotton buds. Care should be taken to avoid pulling on the bowel as this may activate the tissue and reduce blood flow within the microcirculation. A loop of bowel is placed over the viewing platform and the mouse is placed on the microscope. The exposed tissue is constantly perfused with 37  C bicarbonate buffered saline (BBS: 132 nM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 20 mM NaHCO3) and the microcirculation is observed. Common methods for inducing inflammation in the murine mesentery include i.p. injection of cytokines such as IL-1b (5 ng) or TNF (500 ng) or proinflammatory agents such as zymosan (1 mg in 0.5 ml sterile saline) or LPS (0.5 mg/kg derived from Escherichia Coli serotype 0111:B4 in a volume of 0.5 ml sterile PBS) for 2–4 h prior to observation. The mesentery is also a good option for studying ischemia reperfusion injury as ischemia can be easily established through clamping the superior mesenteric artery (45 min ischemia is typical with reperfusion times ranging from 30 min to 4 h). The major limitations of the mesentery are that young mice weighing no more than 12 g should be used, as beyond this age the postcapillary venules become obscured by fat limiting visibility of transmigrated cells. Movement of the bowel due to peristalsis can also prove challenging although this can be limited to some degree by overnight fasting of mice prior to IVM. 2.3.9. Cremaster preparation Mice are anesthetized as described above and the skin over the ventral aspect of the right scrotum is removed. The fascia surrounding the cremasteric sack is removed with care being taken to minimize manipulation of the cremaster itself. At this point the animal is positioned using a gel heating pack in a supine position with the cremaster sack resting on the central glass region of the stage. Throughout the isolation procedure the preparation is kept moist with 37  C BBS. A suture is placed through the distal end of the cremaster and secured to the Plexiglas board with tape. A longitudinal line down the center and a line from left to right along the top edge of the sack is then scored in the cremaster using a cauterizer so that it can be opened flat against the viewing pedestal. The muscle is held in place using four hooks made from 6-gauge needles.

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The vessel connecting the testicle to the cremaster is cauterized and the remaining connective tissue is cut so that the testicle can be moved aside leaving a clear view of the cremasteric microcirculation. Larger mice can be used for studies in the cremaster as fat deposits are not an issue in this preparation. It is good practice to let the cremaster stabilize for 30 min once surgery is finished. In comparison to the mesentery the cremaster preparation is stable and can be observed for a number of hours. This is advantageous for studies involving superfusion of inflamogens such as PAF (100 nM superfused in BBS for up to 2 h). Superfusion protocols allow recordings to be made prior to induction of inflammation and the inflammatory response is then monitored over time usually in one vessel section. End-point experiments are performed by inducing inflammation through injection of proinflammatory agents, such as TNF (300 ng in 400 ml sterile saline) or IL-1b (30 ng in 400 ml sterile saline) intrascrotally 2–6 h prior to observation. In this case numerous vessels and vessel sections can be analyzed and compared to mice injected with saline alone. 2.3.10. Parameters analyzed by IVM Various parameters can be analyzed by IVM. These include leukocyte rolling, velocity, adhesion, and transmigration (vascular permeability can also be quantified although fluorescence is required to measure this parameter). Figure 11.4 illustrates how these parameters are quantified. The shear rate within postcapillary venules can also be calculated using a Doppler velocimeter to measure mean RBC velocity (shear-dependent leukocyte recruitment has been shown to occur at rates lower than 500 s 1 (Bienvenu and Granger, 1993). Mean RBC velocity is calculated from the formula Vmean ¼ centerline velocity/1.6. Wall shear rate (WSR) is then derived from the Newtonian formula: WSR ¼ 8000(Vmean/diameter). With the availability of galectin-1-deficient mice, these experiments allow both the endogenous and exogenous proteins to be studied. Figure 11.5 shows data from IVM experiments performed in galectin-1deficient mice with significantly increased leukocyte transmigration apparent in the absence of this endogenous lectin.

3. Galectin–Glycan Lattices in the Control of DC Physiology 3.1. Conceptual framework During the past years great progress has been made in our understanding of the cellular and molecular pathways that regulate immune cell tolerance and homeostasis (Rabinovich et al., 2007c). Active immunosuppression can be achieved through the secretion of antiinflammatory cytokines or through

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Transmigrated leukocytes 50 mm

Post-capillary venule

100 mm Vessel diameter 20–40 mm

Adherent leukocyte

Rolling leukocyte

50 mm Transmigrated leukocytes

Figure 11.4 Parameters analyzed by IVM. Postcapillary venules with diameters between 20 and 40 mm are selected for analysis by IVM. Leukocyte flux is quantified by counting the number of cells that role past a fixed point over 1 min and is expressed as number of cells/min. Rolling velocity is calculated from the time taken for a given leukocyte to travel a fixed distance and is expressed as mm/s. Adherent cells are classified as those being stationary for a 30-s period. The number of adherent cells within a 100-mm vessel section are quantified over a period of 1 min. Transmigrated cells are quantified at both sides of the selected 100 mm vessel section in an area of 100  50 mm2.

specialized suppressor cells, including CD4þCD25þFoxP3þ Treg cells, IL10-producing type-1 FoxP3 Treg cells, myeloid-derived suppressor cells, ‘‘alternatively activated’’ macrophages, and tolerogenic DCs (Rabinovich et al., 2007c). DCs are the central players in all immune responses, both innate and adaptive (Steinman et al., 2003). Conventional DC subsets described in humans include myeloid DCs and plasmacytoid DCs. Through antigen recognition, processing, and presentation, DCs can orchestrate adaptive immune responses; yet these cells can also attenuate inflammatory reactions irrespective of their maturation status by promoting T-cell anergy or by favoring the expansion and/or differentiation of Treg cells (Steinman

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Gal-1 null mice show increased leukocyte recruitment in response to PAF superfusion Baseline WT Gal1 KO

18 16

* 28 mm

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14 12 10

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8 6 4 2 0 0

15

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45 60 75 Time (min)

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Figure 11.5 Data obtained by IVM of the cremasteric microcirculation. Cremasters were exteriorized and baseline parameters in one vessel section were quantified. PAF (100 nM) was then superfused onto the tissue for 2 h and the vessel was recorded and analyzed every 15 min. Significantly increased numbers of leukocytes were observed to transmigrate in galectin-1 knockout mice compared to their wild-type counterparts. Images of vessels in galectin-1 knockout mice at the 0 and 2 h timepoint post-PAF are also shown.

et al., 2003). Research over the past decade has identified a number of upstream and downstream signaling events on DCs which can be manipulated in a selective manner to amplify either an immunogenic or a tolerogenic response (Rabinovich et al., 2007c). Several stimuli may influence the decision of DCs to become tolerogenic, including transforming growth factor-b (TGF-b), IL-10, vasoactive intestinal peptide (VIP), and 1,25dihydroxyvitamin D3 (Steinman et al., 2003). In spite of early observations assigning a predominant immunogenic function to mature DCs (which express high levels of major histocompatibility complex (MHC) II and costimulatory molecules CD80/CD86), recent evidence challenged this paradigm showing that DC maturation itself is neither an immunogenic nor a tolerogenic hallmark of DCs (Reis e Sousa, 2006). Supporting this concept, fully mature DCs are abundant throughout the peak and resolution phase of autoimmune inflammation and are capable of promoting the expansion of Treg cells instead of inciting a primary or memory T-cell response (Reis e Sousa, 2006). Hence, it is the flexibility of specialized DCs to respond to selective environmental signals, which may determine the amplification or silencing of adaptive immunity, which may in turn shape the course of chronic inflammation. The mechanisms and pathways underlying these regulatory processes are the subject of intensive research with still more questions than answers. While DCs producing high amounts

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of IL-12 favor the differentiation of Th1 effector cells, IL-23-secreting DCs favor a Th17 pathogenic phenotype, and those producing high amounts of IL-27 determine the differentiation of IL-10-producing FoxP3 Treg (Tr1) cells (Ilarregui and Rabinovich, 2010; Steinman et al., 2003). Hence, promotion of Treg cell expansion and induction of tolerogenic DCs have emerged as rational therapeutic strategies aimed at tempering autoimmune inflammation and protecting from immune-mediated pathology. To understand the cellular and molecular mechanisms underlying the broad immunosuppressive activity of galectin-1 in autoimmunity and tumor settings, we investigated the impact of this glycan-binding protein on human and mouse DC physiology using a series of in vivo models (Ilarregui et al., 2009). Notably, DCs differentiated or matured in a galectin-1-enriched microenvironment acquired a distinctive ‘‘regulatory signature’’ characterized by high expression of the cell surface marker CD45RB, phosphorylation of the transcription factor STAT3 and abundant secretion of IL-27 and IL-10. More importantly, when transferred in vivo, these DCs promoted T-cell tolerance in antigen-specific and neoplastic settings, blunted Th1 and Th17 responses and halted autoimmune neuroinflammation through mechanisms involving DC-derived IL-27 and T-cell-derived IL-10. Thus, using IL-27 receptor-deficient (Il27ra/) and IL-10-deficient (Il10/) mice, we have identified an immunoregulatory circuit linking galectin-1 signaling, IL-27producing tolerogenic DCs and IL-10 secreting Tr1 cells (Fig. 11.6; Ilarregui

Gal1

STAT-3

IL-27

IL-10 Tr1 cell

Tolerogenic DC

Figure 11.6 Identification of an immunoregulatory circuit linking galectin-1 signaling, IL-27-producing DCs and IL-10-secreting Tr1 cells. During the peak and resolution stages of autoimmune inflammation, galectin-1 expression augments and promotes the differentiation of CD11clo, CD45RBþ tolerogenic DCs which express high levels of phosphorylated STAT3 (pSTAT3) and IL-10. These DCs also secrete high amounts of IL-27, a heterodimeric cytokine composed of the p28 and the EBI3 subunits, which interacts with its specific receptor (gp130/WSX1) and promotes the expansion of IL10-producing FoxP3 regulatory type 1 T cells (Tr1 cells). Delivery of these tolerogenic signals from DCs to T cells blunt Th1 and Th17 responses and promotes the resolution of autoimmune inflammation.

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et al., 2009). Other studies have found that DCs engineered to overexpress galectin-1 can induce contrasting effects on naı¨ve and stimulated T cells similar to direct exposure of T cells to soluble recombinant galectin-1 (Perone et al., 2006), suggesting that these cells could also be used as vehicles of immunomodulatory target genes. Moreover, exposure to galectin-1 also promoted the migration of DCs through mechanisms involving Syk and PKC signaling (Fulcher et al., 2009), suggesting that DCs exposed to galectin-1 may acquire a distinctive immunomodulatory program characterized by a ‘‘mature’’ or ‘‘semimature’’ cell surface phenotype, increased migration profile, and enhanced tolerogenic potential. Given its tolerogenic effects, we also investigated the relevance of endogenous galectin-1 during the evolution of central nervous system (CNS) autoimmune inflammation. Galectin-1 expression augmented during the peak and recovery phases of EAE (see below) and was dramatically upregulated by tolerogenic stimuli including VIP, vitamin D3, and IL-10, but significantly downmodulated by proinflammatory agents such as TNF, IFN-g, and most Toll-like receptor (TLR) agonists (Ilarregui et al., 2009). Moreover, DCs lacking Lgals1 gene had lower expression of IL-27, higher production of IL23 and reduced STAT3 phosphorylation and were not capable of promoting T-cell tolerance during ongoing EAE. In contrast, galectin-1-expressing DCs restored T-cell tolerance and contributed to the resolution of autoimmune neuroinflammation, suggesting a crucial role of endogenous galectin-1 in ‘‘fine-tuning’’ the immunogenicity of DCs. Thus, galectin-1-differentiated DCs producing IL-27 can be harnessed to silence Th1- and Th17-mediated responses and promote the differentiation of IL-10-producing Treg type 1 (Tr1) cells, suggesting a hierarchy of tolerogenic signals which may represent potential targets in T-cell-mediated immunopathology.

3.2. In vitro strategies to study the role of galectins in DC physiology 3.2.1. Preparation of recombinant galectin-1 For all the studies described in this chapter, including those detailed above, recombinant galectin-1 is purified as described (Barrionuevo et al., 2007; Pace et al., 2003). Briefly, soluble fractions are obtained for subsequent purification of the recombinant protein by affinity chromatography on a lactosyl-Sepharose column (Sigma). LPS should be carefully removed using Detoxi-GelTM (Pierce) and LPS content should be tested using Gel Clot Limulus Test (Cape Code) until levels are lower than 0.5 IU/mg. Removal of LPS is particularly important in the case of DCs as low traces of endotoxin contamination may promote DC maturation and mask galectin-1 inhibitory effects. Endotoxin-free recombinant galectin-1 is lyophilized for long-term storage, and when needed reconstituted at 1 mg/ml either in PBS alone or in PBS containing 1 mM 2-mercaptoethanol to avoid intramolecular

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disulfide bonding between unpaired cysteines present in the carbohydratebinding site and consequent reduction of biological activity. Remarkably, in our assays both preparations showed a comparable capacity to modulate DC physiology. 3.2.2. Differentiation and maturation of human DCs PBMCs are isolated from healthy volunteers by standard density gradient centrifugation on Ficoll-PaqueTM Plus (GE Healthcare). Monocytes are purified by centrifugation on a discontinuous PercollTM Plus (GE Healthcare) gradient. In brief, PBMCs are suspended in Ca2þ/Mg2þ-free tyrode solution supplemented with 0.2% (w/v) EDTA and incubated for 30 min at 37  C. Three different PercollTM fractions are layered in polypropylene tubes: 50% at the bottom, followed by 46% and 40%. PBMCs (5  106 ml 1) are layered at the top and centrifuged at 400g for 20 min at 4  C. Monocytes are recovered at the 50/46% interface. The purity can be checked by flow cytometry using an anti-CD14 mAb (>85% is appropriate). To differentiate immature DCs, monocytes are plated onto 6-well culture plates or Petri dishes at 1  106 cells/ml in complete medium [RPMI 1640 supplemented with 10% (v/v) heat-inactivated FCS, 40 mg/ml gentamicin, 50 mM 2-mercaptoethanol and 2 mM L-glutamine (all from Gibco)] containing 5 ng/ml IL-4 (Sigma) and 35 ng/ml recombinant human granulocyte macrophage-colony stimulating factor (GM-CSF; Sigma) in the absence or presence of galectin-1 at concentrations ranging from 0.3 to 3 mM. Cells are fed on day 3 by adding 50% fresh medium, restoring the same concentration of IL-4, GM-CSF, and galectin-1. To obtain human mature DCs, immature DCs differentiated in the absence of galectin-1 are harvested at day 6, washed with PBS, resuspended in complete medium and counted. Cells are then plated at a concentration of 1  106 cells/ml and exposed to 1 mg/ml LPS (0111:B4 E. coli strain; Sigma) in the absence (DC) or presence (DCGal1) of galectin-1 at concentrations ranging from 0.3 to 3 mM. To analyze the role of the JAK2-STAT3 signaling pathway in the immunoregulatory activity of galectin-1, immature DCs are cocultured with LPS, recombinant galectin-1, and different concentrations (0.25, 2.5, and 25 mM) of the pharmacological inhibitor AG490 (Calbiochem). 3.2.3. Differentiation and maturation of mouse DCs Six- to eight-week-old female or male C57BL/6 or BALB/c mice are used for these assays. The protocol is performed as described by Inaba et al. (1992) with minor modifications. After removing all muscle tissues with gauze from the femurs and tibias, the bones are transferred into a fresh dish with Dulbecco’s modified Eagle’s medium (DMEM). Both ends of the bones are cut with scissors and then the marrow is flushed out using 1 ml syringe and 25-gauge needle filled with DMEM. Bone marrow cells are then passed

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through a 21-gauge needle to obtain a single cell suspension. After washing, RBCs are lysed in 5 ml of ACK buffer (NH4Cl 0.15 M; KHCO3 10 mM; Na2EDTA 0.1 mM) for 5 min, washed with PBS and resuspended in DMEM complete medium [DMEM supplemented with 10% FCS, 40 mg/ml gentamicin, 50 mM 2-mercaptoethanol, and 2 mM L-glutamine (all from Gibco)] containing 20 ng/ml recombinant mouse GM-CSF (BD Biosciences) or 10% conditioned medium from the GM-CSF-producing J588L cell line. Cells are counted and plated at 2  106 cells/ml into 6-well culture plates or Petri dishes in the absence or presence of recombinant galectin-1 (0.3–3 mM). On day 2 and 5, floating cells are gently removed and resuspended in fresh medium restoring the same GM-CSF and galectin1 concentrations. On day 7 or 8 of cell culture, nonadherent cells and loosely adherent DC aggregates are harvested for analysis or stimulation (approximately 80% of nonadherent cells should express CD11c). In certain experiments, to obtain highly purified populations for subsequent analysis, DCs are isolated using bead-conjugated anti-CD11c mAb (Miltenyi Biotec) followed by positive selection through paramagnetic columns (LS columns; Miltenyi Biotec). The purity of the selected cell fraction is typically >95%. For maturation, immature DCs (1  106 cells/ml) are exposed for 48 h to 1 mg/ml LPS in DMEM complete medium. To study the regulated expression of endogenous galectin-1, we selected a panel of tolerogenic stimuli such as VIP (10 8 M; Calbiochem), 1,25dihydroxyvitamin D3 (10 8 M; Sigma), IL-10 (50 ng/ml; R&D), or proinflammatory signals such as IFN-g (50 ng/ml; R&D), TNF (20 ng/ml; Sigma), CD40-specific agonistic antibody (10 mg/ml HM40-3;BD Biosciences), and TLR agonists including synthetic bacterial lipoproteins Pam2CSK4 (100 ng/ml; Invivogen) and Pam3CSK4 (1 mg/ml; Invivogen), Bacillus subtilis PGN (10 mg/ml; Invivogen), poly(I:C) (10 mg/ml; Invivogen), zymosan (10 mg/ml; Invivogen), heat-killed Proprionibacterium acnes (20 mg/ml; van Kampen Group), B. subtilis flagellin (200 ng/ml; Invivogen), endotoxin-free Schistosome egg antigen (SEA; 50 mg/ml), or apoptotic splenocytes (irradiated with 12,000 rad) in a 5:1 ratio. To study the intracellular mechanisms underlying these processes, immature DCs are exposed to pharmacological inhibitors of JAK2-STAT3 (2.5 mM AG490; Calbiochem), JNK-SAP (20 mM SP600125; Calbiochem), ERK1/2 (5 mM U0126; Sigma), NF-kB (1 mM BAY11-7082; Sigma), PI3K-AKT (2 mM Ly294002; Sigma), and p38 (10 mM SB202190; Calbiochem) signaling pathways. For phenotypic and functional analyses of in vivo-expanded DCs, spleens are typically placed in 60 mm Petri dishes and injected with 1 mg/ml collagenase IV (Invitrogen) in DMEM (500 ml per spleen), minced into small fragments, transferred into 15 ml polypropylene tubes and incubated for 30 min at 37  C with 5 ml DMEM plus 1 mg/ml collagenase IV. The reaction is stopped with 500 ml FCS and 50 U/ml DNase I (Roche). Splenic DCs are purified with magnetic beads (MACS) as described above,

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resuspended in DMEM complete medium and cell viability is assessed using 0.2% (w/v) Trypan blue dye exclusion. 3.2.4. Galectin-1-binding assays To use in binding assays, galectin-1 is biotinylated with the EZ-Link SulfoNHS-LC-Biotinylation kit (Pierce; cat #21435) according to manufacturer’s instructions. DCs (5  105) are washed with PBS in 1.5 ml tubes and resuspended in 50 ml PBS plus 50 mM 2-mercaptoethanol. Biotinylated galectin-1 is added to cells at concentrations ranging from 0.3 to 3 mM and incubated for 1 h at 4  C in the absence or presence of 30 mM lactose or 30 mM sucrose as specific or nonspecific disaccharides respectively. Cells are then washed with 1 ml PBS, incubated for 30 min at 4  C with fluorescently labeled streptavidin, washed, and analyzed in a FACSAriaTM cytometer (BD Biosciences). Nonspecific binding is assessed with conjugated streptavidin alone. 3.2.5. Cell surface phenotypic analysis To analyze cell surface markers, DCs (1  105) are normally washed with PBS and resuspended in 20 ml PBS plus 1% (v/v) FCS and 0.05% (w/v) sodium azide. Then, cells are incubated for 30 min at 4  C with fluorochrome-labeled mAb (all from BD Biosciences) at 10 mg/ml. Human cells are typically stained with fluorochrome (phycoerythrin, PE, or fluorescein isothiocyanate, FICT)-conjugated anti-CD1a (HI149), anti-CD14 (M5E2), anti-CD86 (2331-FUN-1), anti-HLA-DR (G46-6), and anti-CD83 (HB15e) mAb. Mouse cells are typically stained with fluorochrome (PE or FITC)-labeled anti-CD11c (HL3), anti-CD40 (HM40-3), anti-I-Ab (AF6-120.1), anti-H-2Kb (AF6-88.5), anti-CD80 (16-10A1), anti-CD86 (GL1), and antiCD45RB (16A) mAb. Nonspecific binding is determined using appropriate fluorochrome-conjugated, isotype-matched irrelevant mAb. Cells are acquired on a FACSAriaTM cytometer (BD Biosciences) and analyzed by FACSDiva software (BD Biosciences). 3.2.6. Endocytosis assays Human immature DCs (1  106) are incubated in RPMI 1640 in the presence or absence of 300 mg/ml FITC-conjugated ovalbumin (OVA) for different time periods either at 37 or 4  C as a negative control. After incubation, cells are washed twice with cold PBS and resuspended in 200 ml of PBS. Endocytosis is analyzed by flow cytometry. 3.2.7. Receptor segregation To analyze receptor segregation, DCs (2  106) are washed with PBS in 1.5 ml tubes, resuspended in 50 ml PBS containing 50 mM 2-mercaptoethanol, treated with optimal doses of recombinant galectin-1 or buffer control for 1 h and fixed for 30 min at 4  C with 2% (w/v) paraformaldehyde. Then, cells are washed with 1 ml PBS, resuspended in 50 ml PBS containing 1% (v/v) FCS and

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incubated for 1 h with anti-CD43 (8.4 mg/ml; DF-T1, Dako) or anti-CD45 (14.5 mg/ml; 2B11, Dako) human mAb at 4  C. Next, cells are washed with 1 ml PBS containing 1% (v/v) FCS and incubated with FITC-conjugated antimouse IgG (F0479; Dako) for 30 min at 4  C. Cells are then washed and resuspended in 20 ml ProLong Gold antifade Reagent (Invitrogen, Cat. No. P36930) with 1 mg/ml propidium iodide and 5 ml of these preparations are mounted on each slide. Receptor segregation is analyzed on a Nikon laser confocal microscope (Eclipse E800). 3.2.8. Real-time quantitative RT-PCR Total RNA is prepared using Trizol (Invitrogen) following manufacturer’s instructions. Real-time quantitative PCR is performed using the SYBR Green PCR Master Mix (Applied Biosystem) in an ABI PRISM 7500 Sequence Detection Software (Applied Biosystem) by means of the absolute quantification protocol. Primers and conditions used are as follows: mouse galectin-1 forward: 50 -TGAACCTGGGAAAAGACAGC-30 ; mouse galectin-1 reverse: 50 -TCAGCCTGGTCAAAGGTGAT-30 , Tm ¼ 62  C. Mouse GAPDH forward: 50 -CCAGAACATCATCCCTGCAT-30 ; mouse GAPDH reverse: 50 -GTTCAGCTCTGGGATGACCTT-30 , Tm ¼ 62  C. 3.2.9. Western blot analysis Cells (1106) are extensively washed and lysed in 30 ml lysis buffer (5 mM EDTA, 1% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 0.15 M NaCl, 50 mM Tris–HCl) in the presence of a mixture of protease inhibitors (Sigma). For detection of protein phosphorylation, 1 mM Na3VO4, 50 mM NaF and 10 mM b-glycerophosphate is added to the lysis buffer. Protein concentration is measured using the MicroBCATM Protein Assay Reagent Kit (Pierce) as described by the manufacturer. Equal amounts of protein are resolved by SDS-PAGE, blotted onto nitrocellulose membranes (GE Healthcare) and probed with a series of commercially available antibodies or a rabbit anti-galectin-1 IgG (1.5 mg/ml) generated and used as described (Juszczynski et al., 2007; Rubinstein et al., 2004; Toscano et al., 2007a) in PBS containing 1% BSA (w/v). Bound antibodies are detected using peroxidase-labeled anti-IgG (BioRad) and immunoreactivity is developed using ECL Plus Western blotting detection system (GE Healthcare) following the manufacturer’s guidelines. Films are analyzed using Scion Image Analysis software (Scion Corp.). The intensity of each band is recorded and expressed as relative expression (RE) to the expression of actin or the unphosphorylated forms of signaling molecules when appropriate. 3.2.10. Human allogeneic mixed leukocyte reaction Human CD4 T cells are purified from PBMCs of healthy donors by negative selection using the CD4 T Cell Isolation kit (RosetteSepTM; StemCell Technologies) as specified by the manufacturer. Human control DC (DC)

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or DC differentiated in the presence of galectin-1 (DCGal1) are extensively washed with PBS and resuspended in RPMI complete medium, irradiated (3000 rad, 137Cs source) and cocultured with allogeneic CD4 T cells (1105) in RPMI complete medium at various DC:T ratios (1:1, 1:5, 1:10, 1:50) in U-bottom 96-well culture plates in duplicates. At day 3, supernatants are harvested and used for cytokine assessment. After 4 days of culture, 1 mCi/ well [3H]-thymidine (specific activity 5 Ci/mM; PerkinElmer NENÒ) is added during the last 18 h of culture, cells are then harvested and thymidine incorporation is monitored in a liquid scintillation counter. To determine whether DCGal1 have regulatory function, human allogeneic CD4 T cells (1  105) isolated as above are cocultured in RPMI complete medium for 5 days with LPS-matured DCs (1  104) in the absence or presence of variable numbers of DCGal1 (1  103, 1  104, and 3  104). Cells are then plated in U-bottom 96-well culture plates in a final volume of 200 ml/well in duplicates. At day 3, supernatants are harvested for cytokine determination. After 4 days of culture, 1 mCi/well [3H]-thymidine (specific activity 5 Ci/mM) is added during the last 18 h of culture and thymidine incorporation is monitored in a liquid scintillation counter. To examine whether allogeneic T cells are rendered regulatory following exposure to DCGal1, CD4 T cells are purified from mixed leukocyte reaction (MLR) cultures and their suppressive capacity is analyzed. Human CD4 T cells primed with allogeneic DC or DCGal1 for 5 days (as stated above) are further purified by cell sorting. For this, 107 cells from MLR are washed with PBS and resuspended in 100 ml of PBS plus 1% (v/v) FCS. Then, cells are incubated for 30 min at 4  C with fluorochrome-conjugated anti-human CD4 mAb at a concentration of 10 mg/ml. Next, cells are washed with 2 ml PBS plus 1% (v/v) FCS, resuspended in 1 ml of RPMI complete medium, sorted in a FACSAriaTM (BD Biosciences) and collected in 1 ml FCS. CD4 T cells are finally resuspended in RPMI complete medium and their viability and recovery is assessed by 0.2% (w/v) Trypan blue staining. To evaluate the regulatory capacity of allogeneic T cells on subsequent MLR cultures, CD4 T cells primed with DC or DCGal1 (104 or 105) are cocultured in a secondary MLR involving 105 fresh CD4 T cells (isolated as stated above) and 2  104 DCs. In some experiments, anti-human cytokine neutralizing Abs can be added to the MLR in order to evaluate the requirements of endogenous cytokines for the observed inhibitory effects. 3.2.11. Mouse allogeneic MLR Mouse naı¨ve CD4 T cells (CD62LþCD44lo) are isolated from spleens of 6–8-week-old BALB/c or C57BL/6 wild-type mice using the MagCellect Isolation kit (R&D) as described by the manufacturer and resuspended in DMEM complete medium. For this, spleens are removed, transferred to a well of a 6-well culture plate with 1 ml DMEM and cut in small pieces. Then, tissue is placed on a mesh and gently pressed; the obtained suspension

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(splenocytes) is washed with DMEM (200  g, 10 min at room temperature) and treated with the MagCellect Isolation kit. DCs differentiated in the absence or presence of galectin-1 or obtained from Lgals1/ mice are extensively washed with PBS and resuspended in DMEM complete medium, irradiated (3000 rad, 137Cs source) and cocultured for 5 days with naı¨ve CD4 splenocytes (2  105) in DMEM complete medium at various DC:T cell ratios (1:5, 1:10, 1:20, 1:50), in U-bottom 96-well culture plates in duplicates. At day 3, supernatants are collected for cytokine determination. After 4 days of culture, 1 mCi/well [3H]-thymidine (specific activity 5 Ci/mM) is added during the last 18 h of culture, cells are harvested and thymidine incorporation is analyzed in a liquid scintillation counter. In selected experiments, anti-IL-27p28 (AF1834, R&D), anti-TGF-b1 (1D11, R&D), or anti-IL-10 receptor (CD210; 1B1.3a, BD Biosciences) neutralizing mAbs (10 mg/ml) are incorporated at the beginning of MLR to determine potential cytokines and mediators responsible for the tolerogenic activity of galectin-1-differentiated DCs.

3.3. In vivo strategies to study the role of galectins in DC physiology All animal work should be approved by the Institutional Ethics Committees in agreement with Institutional Animal House and NIH guidelines. 3.3.1. Adoptive transfer experiments For adoptive transfer, DCs are washed with PBS, resuspended in DMEM complete medium at 1  106 cells/ml and pulsed with OVA (200 mg/ml; Sigma) overnight. Thereafter, cells are extensively washed, resuspended in PBS and injected (3  105 in 200 ml per mouse) i.p. with a 23-gauge needle into recipient mice. After 7 days, mice are challenged subcutaneously with OVA (100 mg) in CFA (IFA plus 2 mg/ml of Mycobacterium tuberculosis, H37Ra; Difco). Typically 7 days later, splenocytes are isolated as stated above and 1  105 cells are plated in U-bottom 96-well culture plates in DMEM complete medium in the absence or presence of 75 mg/ml OVA (final volume 200 ml/well). After 2 days of culture, 1 mCi/well [3H]thymidine (specific activity 5 Ci/mM) is added during the last 18 h of culture, cells are harvested and thymidine incorporation is analyzed with a liquid scintillation counter. Additionally, at day 3 supernatants are harvested and either used immediately to measure cytokine production or frozen at 80  C until further use. 3.3.2. Tumor protection assays The mouse B16/F0 or F1 melanoma cell line (C57BL/6 background) obtained from ATCC is used for these experiments. Cells are typically cultured in DMEM complete medium and subcultured every 2 days after

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treatment with TrypLETM Express solution (Invitrogen). For induction of B16 lysates (i.e., necrotic cells), cells are collected using TrypLETM Express solution, washed twice in PBS and resuspended in PBS at 107 cells/ml in 2 ml cryotubes. Then, B16 melanoma cells are subjected to four cycles of rapid freeze (liquid nitrogen) and thaw (42  C water bath), spun down at 200  g at 4  C for 10 min to remove cellular debris and resuspended at 1  107 cells/ml. Tumor cells are then examined for the degree of necrosis using FITC-annexin-V and propidium iodide (typically necrotic cells are annexin-Vþ, propidium iodideþ). For immunization C57BL/6 control DC or DCGal1 are washed with PBS, resuspended in DMEM complete medium and cocultured in the presence or absence of necrotic cells at a 1:3 DC:B16 cell ratio in roundbottom 12 ml polypropylene tubes overnight. Then, DCs are extensively washed, resuspended with PBS at 2.5  106 cells/ml and injected (200 ml, 5  105 DCs) in C57BL/6 mice twice subcutaneously at 7-days intervals. After 14 days of the last immunization, mice are challenged subcutaneously with 2  105 viable B16 cells in 200 ml with a 25-gauge needle. B16 melanoma cells are collected using TrypLETM Express solution, washed twice in PBS and resuspended in PBS at 106 cells/ml prior to injection. Tumor development is monitored every second day by measuring tumor perpendicular diameters with a digital caliper. Tumor volume is estimated as (d2  D  0.5), where d and D are the minor and major diameters, respectively. For ethical reasons, animals should be sacrificed when tumors reach a volume greater than 2 cm3. Mice with tumor volumes of less than 0.5 cm3 are normally considered as tumor free. Some animals from each group are sacrificed at day 12 following challenge to analyze immunological parameters. Briefly, tumor-draining lymph nodes are isolated, transferred to a 24-well culture plate in 200 ml DMEM and cut in small pieces. Then, tissue is placed into a mesh and gently pressed, the suspension obtained (lymph node cells) is washed with DMEM (200  g, 10 min). Lymph node cells (2  105) are cocultured with 1  104 irradiated (5000 rad, 137Cs source) B16 melanoma cells for 72 h in F-bottom 96-well culture plates in DMEM complete medium. After 2 days of culture, 1 mCi/well [3H]-thymidine (specific activity 5 Ci/mM) is added during the last 18 h of culture, cells are then harvested and thymidine incorporation is monitored in a liquid scintillation counter. At day 3, supernatants are harvested and either used immediately or frozen at 80  C until further use. 3.3.3. Induction and assessment of EAE Multiple sclerosis is a major inflammatory and demyelinating disease of the CNS characterized by a relapsing-remitting stage followed by a secondary progressive phase (Ilarregui and Rabinovich, 2010; Sospedra and Martin, 2005). Animal models of experimental EAE recapitulate the clinical and immunological features of the disease and have been of crucial importance

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for the validation of many therapeutic targets (Sospedra and Martin, 2005). From an immunological standpoint, the activity of the disease is controlled through a delicate balance of Th1, Th2, Th17, and Treg cells (Lopez-Diego and Weiner, 2008). There are several models of EAE which may reflect the relapsing-remitting, primary progressive or secondary progressive disease and may be induced in Lewis rats, SJL mice, 129/Sv mice, C57BL/6 mice, or guinea pigs. In addition, for EAE induction a number of different immunogens may be used including a peptide (35–55) of myelin oligodendrocyte glycoprotein (MOG), proteolypid peptide (PLP), and myelin basic protein (MBP; Sospedra and Martin, 2005). MOG-induced EAE is typically induced in 6–8-week-old female mice. While C57BL/6 mice are highly susceptible to disease development, 129/Sv are quite resistance to disease induction. Therefore different strains should be used based on the requirements of each particular experiment. For example, to evaluate whether endogenous galectin-1 is critical for limiting autoimmune brain inflammation, we used 129/Sv Lgals1/ and wild-type mice as this mouse strain provides a window of opportunity to visualize an aggravation of the disease. In contrast, we used mice of the C57BL/6 background to assess the ameliorating and therapeutic activity of recombinant galectin-1 (i.p. injections from days 3–9) or DCGal1 (a single injection at the day of disease onset) as it allows clear visualization of an antiinflammatory effect (Ilarregui et al., 2009; Toscano et al., 2007a). MOG-induced EAE is induced by s.c. immunization with 200 mg MOG35–55 (MEVGWYRSPFSRVVHLYRNGK) in CFA 4 mg/ml of M. tuberculosis (H37Ra; Difco) using a 23gauge needle. On days 0 and 2, mice receive 200 ng of pertussis toxin (List Biological Labs) in 100 ml PBS i.p. using a 27-gauge needle. Mice are examined daily for signs of EAE and assigned scores as follows: 1, limp tail; 2, hindlimb weakness; 3, hindlimb paralysis; 4, hindlimb and forelimb paralysis; and 5, moribund. Mice with established EAE (clinical score 1) are injected with syngeneic MOG35–55-pulsed or unpulsed DC or DCGal1 (Ilarregui et al., 2009). For this, DC or DCGal1 are washed with PBS, resuspended in DMEM complete medium and cocultured with 75 mg/ml MOG35–55 in round bottom 12 ml polypropylene tubes overnight. Then, DCs are extensively washed, resuspended with PBS at 1  106 cells/ml and injected (200 ml, 2  105 DCs) i.p. with a 25-gauge needle. At day 25 after immunization, lymph nodes draining sites of immunization are isolated (as stated above) and 1  105 cells are plated in U-bottom 96-well culture plates in DMEM complete medium in the absence or presence of 50 mg/ml MOG35–55 (final volume 200 ml/well). After 2 days of culture, 1 mCi/well [3H]-thymidine (specific activity 5 Ci/mM) is added for the last 18 h of culture, cells are harvested and thymidine incorporation is monitored in a liquid scintillation counter. Following 3 days of ex vivo restimulation, supernatants from lymph node cells are harvested and either used immediately or frozen at 80  C until further use for cytokine

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determination. For histopathologic analysis, spinal cords are removed on days 25–30 after the first immunization and immediately fixed with neutral 10% (v/v) formalin. Six micrometer paraffin-embedded sections are examined histopathologically following staining with hematoxilin & eosin (H&E) to evaluate inflammatory infiltrates and Luxol Fast blue to assess demyelination.

4. Galectin–Glycan Lattices in the Control of T Helper Cell Fate 4.1. Conceptual framework In addition to their tolerogenic effects on DC biology, galectin–glycan interactions can also modulate the T-cell compartment. In fact, a number of regulatory checkpoints may be targeted by galectin-1 during the lifespan of T cells including T-cell maturation, activation, cytokine secretion, and survival (Rabinovich and Toscano, 2009). Apoptotic mechanisms are critical to regulate the development and shaping of the T-cell repertoire in the thymus (Strasser and Bouillet, 2003). Moreover, they are also crucial to peripheral tolerance by dampening self-reactive T cells, restoring T-cell number following execution of effector functions and/or preventing immune-mediated pathology (Bide`re et al., 2006). Compelling evidence has been accumulated regarding the role of galectin-1 in the control of T-cell viability from developing thymocytes to activated and fully differentiated effector T cells (Kopcow et al., 2008; Perillo et al., 1997; Rabinovich and Ilarregui, 2009; Rabinovich et al., 1997, 1998, 2002b; Toscano et al., 2007a,b). T-cell susceptibility to galectin-1 may be regulated at least at three distinct levels. First, galectin-1 sensitivity may be influenced by the presence of specific glycoprotein receptors. Interestingly, while many cell surface glycoproteins contain substantial amounts of LacNAc glycans, galectin-1 binds to a restricted set of Tcell surface glycoproteins (e.g., CD45, CD43, CD2, CD3, CD7; Pace et al., 1999; Stillman et al., 2006; Walzel et al., 2000) and glycolipids such as GM1 (Wang et al., 2009). Second, galectin-1 binding is limited to those cells that are able to generate specific saccharide ligands by expressing a particular repertoire of glycosyltransferases responsible for creating or modifying cell surface glycoconjugates (Fig. 11.7). In this regard, cell death triggered by galectin-1 involves the expression and activity of the core 2 b-1,6-Nacetylglucosaminyltransferase (GCNT1), an enzyme responsible for creating the core 2 branch on O-glycans, thus allowing the exposure of poly-Nacetyl-lactosamine sequences, which are the preferred saccharide ligands of galectin-1. In this regard, lymphoma T cells lacking core 2-O-glycans are resistant to galectin-1-induced cell death (Cabrera et al., 2006). Moreover,

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N-glycans

O-glycans a3 b4

a2

a2 a2

a2

a3

a3 b4 b4

ST6Gal1 a6

a3

a6

a3

b4

b4

b4

b4

a6

b2

a6

a3 b4

a6

b4

Asn

a6

b2

b2

a6

a3 b4 b4

Asn

High mannose

b2

a3

b3

ST3Gal1

a6 Ser / Thr

b3

b6

Core 1

Tri-antennary

b3

Ser / Thr

Ser / Thr

FucTVII

b3 b4

a3 GCNT1

b3

a6

b3 b4

Ser / Thr

b6 a6 GnT5

Asn

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Figure 11.7 Schematic representation of N- and O-glycan biosynthesis. This scheme includes relevant glycosyltransferases, such as GCNT1, GnT5, ST3Gal1, and ST6Gal1, the coordinated actions of which lead to the generation or masking of common glycosylated ligands for galectin-1 (LacNAc). Galectin-1 binding to specific glycoepitopes is indicated in orange and lectins used as tools for glycophenotyping are depicted in green.

T-cell susceptibility to galectin-1-induced death may also be regulated by the controlled expression of the a2,6-sialyltransferase 1 (ST6Gal1), which is responsible for the addition of sialic acid in a2,6 position of terminal galactose. Increased ST6Gal1 activity results in masked galactose residues on T-cell surface glycoproteins which are no longer able to bind galectin-1 (Amano et al., 2003; Earl et al., 2010). However, a given glycosylation profile is not always permissive or restrictive for galectin-1 as CD45þ T cells lacking GCNT1, which are not able to generate core 2-O-glycans, are resistant to galectin-1-induced cell death, while galectin-1 binds to CD43 modified with either unbranched core 1 or branched core 2-O-glycans (Hernandez et al., 2006). We have provided proof-of-concept of the critical role of endogenous galectin-1 in the control of T helper cells in antigen specific and inflammatory settings. Using in vitro and in vivo experiments, we found a link between differential glycosylation of T helper cells, susceptibility to galectin-1-

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induced cell death and termination of the inflammatory response (Toscano et al., 2007a). While Th1- and Th17-differentiated cells express the repertoire of cell surface glycans that are critical for galectin-1 binding and cell death, Th2 cells are protected from galectin-1 through differential a2,6-sialylation of cell surface glycoproteins (Toscano et al., 2007a), demonstrating the critical role of endogenous galectin-1 in controlling T-cell homeostasis. Remarkably, in vivo-differentiated antigen-specific T helper cells (i.e., Th1 cells generated in vivo by adoptive transfer of DCs pulsed with the bacteria P. acnes and Th2 cells driven by DCs pulsed with SEA) exhibited comparable glycophenotypes and susceptibility to galectin-1 as in vitro human polarized T helper cells. Accordingly, in the EAE model, galectin-1-deficient mice showed greater Th1 and Th17 responses and enhanced susceptibility to autoimmune brain inflammation than their wild-type counterpart (Toscano et al., 2007a). Collectively, these data indicate that differential glycosylation of cell surface glycoproteins can selectively control the survival of T helper cells by modulating their susceptibility to galectin-1 (Fig. 11.8). In line with this evidence, Motran et al. (2008) showed that Th2 cells can promote Th1 cell apoptosis through secretion of galectin-1, suggesting a lectin-dependent mechanism of cross-regulation between distinct T helper cell subsets. Consistent with the ability of galectin-1 to dampen Th1 responses, silencing of Lgals1 gene expression resulted in increased IFN-g and IL-2 production at sites of tumor growth, an effect which was associated with heightened tumor rejection (Juszczynski et al., 2007; Rubinstein et al., 2004). Finally, the dramatic immunosuppressive effects of galectin-1 in vivo in experimental models of autoimmunity and cancer (Rabinovich et al., 2007b), prompted us to investigate the ability of this glycan-binding protein to modulate the Treg cell compartment. Remarkably, administration of galectin-1 in experimental models of autoimmune ocular inflammation (Toscano et al., 2006) and stress-induced pregnancy failure (Blois et al., 2007) restored T-cell tolerance and resulted in considerable expansion of

Th1

a2-6 sialic acid

Apoptosis

Th2

Apoptosis Th17

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Figure 11.8 Differential glycosylation of T helper cell subsets and susceptibility to cell death. While Th1 and Th17 cells share the repertoire of cell surface glycans that are critical for galectin-1 binding and cell death, Th2 cells are protected from galectin-1 through differential a2–6 sialylation of cell surface glycoproteins.

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IL-10-producing CD4þCD25high Treg cells. Interestingly, these cells showed no significant variations in the levels of Foxp3 expression; yet they displayed considerable immunosuppressive activity in vivo. However, using in vitro differentiation systems, exposure of T cells to galectin-1 resulted in significant expansion of a population of CD4þCD25high Treg cells with high expression of FoxP3 (Juszczynski et al., 2007). Whether galectin-1 stimulates the differentiation and/or expansion of both Foxp3þ and Foxp3 Treg cells still remains unclear. Interestingly, analysis of gene expression profiles of regulatory versus effector T cells revealed a substantial increase in Lgals1 mRNA in naturally occurring Treg cells (Garı´n et al., 2007; Sugimoto et al., 2006). Notably, Lgals1 overexpression was found to be Foxp3-independent similar to other upregulated genes such as granzyme B and Helios (Sugimoto et al., 2006). Remarkably, Ab-mediated blockade of galectin-1 significantly reduced the suppressive effects of human and mouse Treg cells indicating that endogenous galectin-1 is required for maximal Treg cell function (Garı´n et al., 2007). This effect appeared to be mediated by galectin-1 cross-linking of the GM1 ganglioside and activation of the TRPC5 ion channel on effector T cells (Wang et al., 2009). These results suggest that protein–glycan systems can also operate within the Treg cell compartment to modulate their expansion and immunosuppressive activity. As discussed here, like many cytokines and growth factors, galectins and their specific glycan partners are critical regulators of innate and adaptive immune cells (particularly DCs and T CD4þ cell subsets; Toscano et al., 2006), which makes them attractive therapeutic targets for limiting autoimmune inflammation, preventing allograft rejection, and potentiating antitumor responses.

4.2. In vitro studies to study the role of galectins in Thelper cell survival 4.2.1. Polarization of Th1, Th2, and Th17 cells When naı¨ve CD4 T cells are primed with appropriate antigens, they undergo a process of activation, proliferation (clonal expansion) and differentiation. In response to IL-12 and the adequate activation stimuli (anti-CD3 mAb, antiCD28 mAb, and IL-2), naı¨ve T cells differentiate into a Th1-type phenotype which typically produce IFN-g, IL-2, and TNF and express high levels of the T-bet and STAT1 transcription factors, while in the presence of IL-4, T cells differentiate to a Th2 subset which produces considerable amounts of IL-4, IL-13, and IL-5 and expresses high levels of GATA-3 and c-Maf. While Th1 cells are involved in the defense against intracellular pathogens and tumors and play a pathogenic role in the development of autoimmune reactions, Th2 responses are critical in fighting helminthic infections and play a pathogenic role in allergic disorders (Rabinovich and Toscano, 2009). In addition, other T helper subsets have been incorporated in recent years to the portfolio of T helper cells that regulate adaptive immunity; these include Th17 cells that are

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generated in the presence of IL-6 and TGF-b1, produce IL-17 and IL-6, express considerable amounts of the RORgt and STAT3 transcription factors and are involved in autoimmune reactions and defense against certain types of bacteria and fungi. More recently other cell subsets have been identified including Th9 and Th22 cells, the pathophysiologic relevance of which still remains to be unveiled. Although a variety of factors such as antigen dose, costimulatory molecules and genetic polymorphism may play a role in dictating differentiation of T helper cells, the cytokine microenvironment encountered by the naı¨ve CD4 T cells during activation, plays a dominant role in the subsequent T helper profile. In this section we will focus on current methods used to polarize human and mouse Th1, Th2, and Th17 cells in vitro. 4.2.2. Polarization of human T helper cells Human CD4 T cells are purified from PBMCs of healthy donors by negative selection with magnetic beads (Dynabeads CD4 T Cell Isolation kit Invitrogen) according to the manufacturer’s protocol. After purification, CD4 T cells are cultured at a concentration of 1  106 cells/ml for 5 days with phytohemagglutinin (PHA; 1 mg/ml) and IL-2 (8 ng/ml; BD Biosciences) in neutral or polarizing conditions essentially as described (Hannier et al., 2002; Fitch et al., 2008). In order to polarize cells toward a Th1 cytokine profile, RPMI 1640 complete medium supplemented with PHA (2 mg/ml), IL-2 (16 ng/ml; R&D), IL-12 (4 ng/ml; BD Biosciences), plus IL-4-specific neutralizing mAb (200 ng/ml; clone MP4-25D2; BD Biosciences) must be used. Five hundred microliters of this Th1 supplemented medium is added to the purified CD4 T-cell suspension cultured in 24-well plates. To obtain Th2-polarized cells, RPMI 1640 complete medium supplemented with PHA (2 mg/ml), IL-2 (16 ng/ml; R&D), IL-4 (10 ng/ml; Sigma) plus anti-IL-12 mAb (4 mg/ml; clone C8.6; BD Biosciences) is prepared. As above, 500 ml of the Th2 supplemented medium is added to the same volume of a purified CD4þ T-cell suspension in a 24-well plate. In either case, cells are cultured at 37  C for 4–5 days. CD4 T cells will develop blast morphology after 1–2 days. In cases where cells have overgrown and consume medium, additional fresh supplemented medium without PHA should be added. Of note, the presence of IL-4 in the Th2 culture medium for the first 72 h is critical to sustain IL-4 production by Th2 cells upon subsequent reactivation. After 4 or 5 days of culture, cells are harvested in 15–50 ml tubes in PBS and centrifuged. The cell pellet is resuspended, washed, and counted and the viability is assessed using the Trypan blue dye exclusion method. Polarization toward a Th1 cytokine profile is checked by the expression levels of IFN-g (ELISA) or the T-bet transcription factor (Western blot), while Th2 differentiation is characterized by the expression of IL-5 and GATA-3 (Toscano et al., 2007a). For evaluation of the function of sialylation, cells were pretreated for 1 h at 37  C with Clostridium perfringens a2–6/a2–3 neuraminidase (500 mU/ml),

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Salmonella typhimurium a2–3 neuraminidase (500 mU/ml; New England Biolab), or buffer control (50 mM sodium citrate, pH 6.0) before lectinbinding and cell death assays. 4.2.3. Polarization of mouse T helper cells Naı¨ve CD4þ T cells (CD62LþCD44lo) are isolated from mouse spleens using the MagCellect Isolation kit (R&D Systems) following the manufacturer’s recommended protocol. Purified cells are resuspended at a concentration of 2  106 cells/ml and stimulated with plate-bound anti-CD3 mAb (5 mg/ml; clone 145-2C11; BD Biosciences) and soluble anti-CD28 mAb (1 mg/ml; clone 37.51; BD Biosciences) in neutral or polarizing conditions as described (Morgan et al., 2004). Briefly, 150 ml (5 mg/ml in PBS) of anti-CD3 mAb is added to each well of 24-well plates and incubated for 2 h at 37  C. Subsequently, plates are washed three times with PBS. Care should be taken to avoid plates getting dry and touching the bottom of the wells during the washing step. Then, 500 ml of cell suspensions and 500 ml of Th1, Th2, or Th17 polarizing media (see below) are added to each well and cultures are placed in the incubator for 5 days. Polarized cells are then harvested and their number and viability are assessed as described above. – Culture medium for Th1 cell polarization contains RPMI 1640, IL-12 (10 ng/ml; BD Biosciences), anti-IL-4 Ab (2 mg/ml; clone 11B11; BD Biosciences), and IL-2 (100 U/ml; R&D). – Culture medium for Th2 cell polarization contains RPMI 1640, IL-4 (20 ng/ml; BD Biosciences), anti-IL-12 Ab (2 mg/ml; clone C 17.8; BD Biosciences), anti-IFN-g Ab (20 mg/ml; clone XMG1.2; BD Biosciences), and IL-2 (100 U/ml; R&D). – Culture medium for Th17 cell polarization contains RPMI 1640, TGF-b1 (6 ng/ml; R&D), IL-6 (40 ng/ml; R&D), IL-23 (40 ng/ml; R&D), anti-IL-4 mAb (20 mg/ml; clone 11B11), and anti-IFN-g neutralizing mAb (20 mg/ml; clone XMG1.2). Cultures were supplemented with IL2 (50 U/ml) on days 2 and 4 of the polarization protocol as described (Bettelli et al., 2006; Mangan et al., 2006). 4.2.4. Glycophenotype analysis The binding activity of endogenous lectins is limited to those cells that are able to generate specific saccharide ligands by expressing a set of particular glycosyltransferases (Fig. 11.7). The cell surface glycophenotype can be assessed by flow cytometry using a panel of plant lectins (conjugated with either biotin or different fluorescent probes) with specificity for particular saccharide structures (Fig. 11.7). Also, some mAb which recognize specific carbohydrate structures decorating particular glycoproteins may be used for glycophenotyping; this is the case of the 1D4 mAb which recognizes the core 2-O-glycan epitope and the 1B11 mAb which recognizes the same saccharide

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decorating CD43 on mouse T cells (Toscano et al., 2007a,b). Although these approaches are highly reliable and offer a broad overview of the cell surface glycans expressed in a given cell population, the systematic study of glycans in cells and tissues (glycomic analysis) relies on effective analytical techniques for correlation of glycan structure with function (Hsu and Mahal, 2009). Hence, chromatographic analysis, mass spectrometry, and nuclear magnetic resonance have been traditionally used for glycan profiling; yet lectin-based flow cytometry and lectin microarrays have emerged as simple, rapid, and reliable methods for deciphering the complex nature of glycan-mediated recognition. While lectin cytometry detects a restricted number of cell surface glycans in an individual fashion and provides a broad idea of the ‘‘glycosylation signature’’ of different cell types, it is recommended to combine this technique with other high-throughput profiling methods of glycan detection including lectin-based microarrays, glyco-gene arrays and MALDI-MS analysis (Tateno et al., 2007). For lectin cytometry, polarized Th1, Th2, and Th17 cells (5  105) are incubated with a panel of biotinylated or fluorescent-conjugated plant lectins and processed for flow cytometry as described (Toscano et al., 2007a). The specificity of these lectins is illustrated in Fig. 11.7. In brief, 5  105 polarized CD4 T cells are suspended in 50 ml of PBS containing 1% (w/v) BSA and biotinylated lectins (20 mg/ml) including Sambucus nigra agglutinin (SNA; E-Y Labs), Peanut agglutinin (PNA; Sigma), Maackia amurensis agglutinin (MAL II; Vector), Helix pomatia agglutinin (HPA; Vector), Lycopersicon esculentum agglutinin (LEA; Vector), and L-phytohemagglutinin (L-PHA; Sigma) for 1 h at room temperature. After incubation, cells are washed with PBS containing 1% (w/v) BSA and further incubated with FITC-conjugated streptavidin (10 mg/ml in PBS-1% (w/ v) BSA) for 30 min in the dark. After washing twice, cells are analyzed in a FACScalibur flow cytometer (BD Bioscience). Nonspecific binding is determined with conjugated streptavidin alone. Galectin-1 binding to T cells is evaluated essentially as described in Section 3 for DCs. 4.2.5. Cell death assays Polarized CD4 T cells (1  106 cells/ml) are incubated for various time periods with galectin-1 (at concentrations of ranging from 5 to 10 mM). These assays are traditionally performed in 1 mM dithiothreitol in RPMI medium in order to keep galectin-1’s CRD in reducing conditions. However, it is our experience that, when cells are polarized toward Th1 or Th17 profiles, such concentrations of a reducing agent may not be required to induce cell death. Apoptotic cells can be identified by double staining using annexin V and propidium iodide (BD Biosciences) or by using the TUNEL assay (ApopTag kit; Chemicon) according to manufacturers’ recommended protocols. At least three types of assays including morphological, cytofluorometric, and biochemical analyses are recommended for the study of

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a given cell death pathway as recently indicated (Galluzzi et al., 2009). Using the annexin V-propidium iodide kit for analysis of early and late apoptotic cells, polarized T cells (1  105) are exposed to recombinant galectin-1, washed twice with cold PBS and resuspended in 100 ml of 1 binding buffer containing with 2.5 ml FITC-Annexin V according to the manufacturer’s recommended protocol (BD Biosciences). Cell death is determined as the percent annexin V-positive polarized cells with stimulus minus the percent annexin V-positive polarized cells without stimulus. These assays are often complemented by assessment of cell viability using the Trypan blue dye exclusion test described above.

4.3. In vivo assays to study the role of galectin-1 in T helper cell fate In order to validate the in vitro methods of T-helper cell polarization, different approaches can be used to differentiate T helper cells in vivo and further evaluate their glycophenotype and susceptibility to cell death. These approaches include experimental models of autoimmunity such as EAE (see Section 3.3) and adoptive transfer experiments of antigen-pulsed DCs. Although experimental models of autoimmunity offer the possibility of analyzing T helper cell differentiation in a complete organism in a setting of pathophysiologic relevance, adoptive transfer experiments, although more artificial, can raise cleaner and unequivocal results based on the administration of DCs pulsed with Th1 or Th2-polarizing antigens. For these experiments, we take advantage of the ability of DCs to tailor T helper cell responses. Hence, bone marrow-derived DCs pulsed with the bacteria P. acnes (Pa) selectively promote Th1 responses when adoptively transferred into naı¨ve mice, whereas DCs pulsed with Schistosoma mansoni egg antigen (SEA) selectively induce Th2-polarized responses (Cervi et al., 2004). The experimental strategy is illustrated in Fig. 11.9. Briefly, bone marrowderived DCs (C57BL/6 mice) are differentiated in the presence of GM-CSF as described in Section 3.2. On day 8, when 80% or more of nonadherent cells express CD11c, immature DCs are exposed to 20 mg/ml heat-killed P. acnes (Van Kampen Group) or 50 mg/ml of endotoxin-free SEA (supplied by Dr. M. Doenhoff or Dr. E. Pearce for our experiments) for 18 h. As control, DCs are exposed to medium alone. The activated phenotype of DCs is controlled by expression of CD11c, MHC II, and costimulatory molecules using flow cytometry as described (see Section 3.2). To generate in vivo Th1 and Th2 responses, syngeneic naı¨ve mice are injected i.p. with 5  105 DCs that had been pulsed with Pa, SEA, or neither antigen as described above. Seven days later, mice are sacrificed and spleens are removed. Splenocytes are then restimulated with Pa (25 mg/ml) or SEA (50 mg/ml) for 48 h and polarization toward Th1 and Th2 profiles are verified by measuring in culture supernatants the levels of IFN-g and IL-5

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i.p.

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Pa GM-CSF Bone marrow myeloid precursors

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Spleen

Figure 11.9 Schematic representation of adoptive transfer experiments used to analyze the glycophenotype and function of T helper cells in vivo. Bone marrow-derived DCs pulsed with P. acnes (Pa) promote selective Th1 responses when adoptively transferred into naı¨ve mice, whereas DCs pulsed with Schistosoma mansoni egg antigen (SEA) induce selective Th2 responses. Mice transferred with DCs pulsed with medium alone are used as controls. After 7 days, CD4 T cells are purified and analyzed for glycophenotype, susceptibility to cell death and antigen-specific cytokine production to check the in vivo generated Th1 and Th2 responses.

by capture ELISA. CD4 T cells are further purified and processed for glycophenotypic analysis and cell death assays as described in Section 4.2.

5. Final Remarks and Future Directions In the present review, we summarize emerging evidence on the immunoregulatory activity of galectin-1, its most important cellular targets within the innate and adaptive immune cell compartments and a number of experimental strategies used to determine its biological activities both in in vitro and in vivo settings. Galectin-1 signaling has been consistently associated with T helper cell death, generation of tolerogenic DCs, leukocyte mobility, cytokine production, and Treg cell function. Similar to many cytokines, autacoids and growth factors, it is not surprising that galectin-1 exhibits a ‘‘double-edge sword’’ effect with opposing biological outcomes depending on different intrinsic factors such as the physicochemical properties of the protein (monomer/dimer equilibrium), stability of the protein in oxidative versus reducing microenvironments, as well as extrinsic factors such as the target cell type and its activation and/or differentiation status. Thus, an integrated study of galectin-1 functions should comprise: (a) generation of endotoxin-free recombinant protein (and evaluation of its biochemical and biophysical properties); (b) in vitro assays including reliable readouts such as PMN chemotaxis and transmigration, DC differentiation, T helper cell apoptosis, cytokine production (particularly IL-10 which is a very consistent readout), and endothelial cell morphogenesis (angiogenesis);

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1

Production of endotoxin-free recombinant galectin

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In vitro assays T cell apoptosis tolerogenic DCs

DNA fragments to be cloned

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In vivo assays Experimental models: Multiple sclerosis Arthritis Uveitis Diabetes Inflammatory bowel disease Graft vs host disease Cancer

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Pre-clinical assesments Patient samples binding to -T cell -DCs

Figure 11.10 Multidisciplinary approach to study the immunoregulatory activity of galectin-1. In general, the extracellular functions of this lectin are studied using endotoxin-free recombinant galectin-1 while the biological effects of the endogenous protein are typically studied following antisense- or siRNA-mediated silencing of cells expressing high levels of galectin-1 or overexpression of the Lgals1 gene in cells which do not express or express low levels of this protein. A variety of in vitro readouts and in vivo experimental models are illustrated and mentioned in detail in the text.

(c) in vivo assays in different experimental mouse models; and (d) correlation of the glycophenotype of immune cells (DCs and T cells) of patients with different pathological conditions with galectin-1-binding and signaling capacity (Fig. 11.10). In light of the broad spectrum of immunoregulatory effects, challenges for the future will embrace a rational manipulation of galectin-1-glycan interactions toward attenuating immune responses in autoimmune diseases, graft rejection and recurrent fetal loss. Important proof-of-concept data must be generated using galectin-1-deficient mice and the application of predictive disease models, as emerged from initial analyses of the inflammatory response. Moreover, with the diverse range of glycosyltransferase knockout mice that

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are available it will now be feasible to determine the impact of glycosylation in galectin-1-mediated effects. However, before galectin-1-based therapeutic agents can be extrapolated to clinical settings, a more thorough understanding of the mechanisms involved in galectin-1 functions is essential. In this regard, it will be critical to evaluate the results of side-by-side studies of the antiinflammatory activities of different members of the galectin family, dissect the biological activity of different galectin-1 variants, evaluate the influence of proinflammatory and tolerogenic microenvironments, and establish the most adequate routes of administration as well as the underlying toxicity of this glycan-binding protein in vivo. As a reverse side of the same coin, interrupting galectin-1-glycan interactions may contribute to overcome T-cell tolerance. Hence, galectin-1 inhibitors/antagonists may serve as adjuvants in preventive or therapeutic vaccines against chronic infections and cancer (Liu and Rabinovich, 2005). In order to validate this concept, the design of specific antagonists as well as a comparative study of already established inhibitors is essential. Given the complexity of galectin-1-glycan interactions and the multiple parameters influencing these molecular contacts, further work is required, involving multidisciplinary efforts from different laboratories, to achieve a global comprehensive view of the role of endogenous galectin-1 and its specific carbohydrate ligands in immunoregulation. It is our hope that this conceptual and methodological review will contribute to this goal.

ACKNOWLEDGMENTS Work in authors’ laboratories is supported by grants from The Argentinean Agency for Promotion of Science and Technology (PICT 2006-603; Argentina), Sales Foundation for Cancer Research (Argentina), The Cancer Research Institute (USA), and The Prostate Cancer Research Foundation (UK) to G. A. R., Arthritis Research UK fellowship to D. C. (18103) and the William Harvey Research Foundation to D. C. and M. P. We thank all members of our laboratories, especially M. Toscano for critical reading of the manuscript.

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C H A P T E R

T W E LV E

Manipulating Cell Surface Glycoproteins by Targeting N-Glycan–Galectin Interactions Ani Grigorian*,‡ and Michael Demetriou*,†,‡ Contents 1. Overview 2. Galectins and Their N-Glycan Ligands 3. Regulation of Glycoprotein Concentration at the Cell Surface by the Galectin–Glycoprotein Lattice 4. T Cells and the Galectin–Glycoprotein Lattice 5. Genetic and Metabolic Regulation of the Galectin–Glycoprotein Lattice 6. Overview of Methods to Measure and Modulate the Galectin–Glycoprotein Lattice 6.1. Methods to measure N-glycan branching and galectin– glycoprotein interactions at the cell surface 6.2. Methods to modulate galectin–glycoprotein interactions at the cell surface 6.3. Methods to examine regulation of T cell receptor signaling and cell growth by the galectin–glycoprotein lattice References

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Abstract The interaction of cell surface receptors and transporters with cognate ligands depends on their concentration, distribution, and organization at the cellular surface. The majority of cell surface receptors and transporters are co- and/or post-translationally modified with asparagine (N)-linked oligosaccharides (glycans). N-Glycan number and structure combine to control the concentration of glycoproteins at the cell surface through interactions with endogenous lectins such as galectins. ER/Golgi enzyme activity and hexosamine pathway supply of Golgi metabolites co-dependently regulate N-glycan biosynthesis and * Department of Neurology, University of California, Irvine, California, USA Department of Microbiology and Molecular Genetics, University of California, Irvine, California, USA Institute for Immunology, University of California, Irvine, California, USA

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Methods in Enzymology, Volume 480 ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)80012-6

#

2010 Elsevier Inc. All rights reserved.

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combine to provide adaptive control over cell growth and differentiation. Studies in mice and humans have revealed metabolic and genetic dysregulation of N-glycosylation in T-cell-mediated autoimmunity. In this chapter, we describe methods used to analyze N-glycan–galectin interactions in controlling the distribution and organization of cell surface receptors and transporters.

1. Overview The great abundance, size, and complexity of glycan structures that dominate the cell surface topology provide for information encoding distinct from the genome. The extracellular domains of most cell surface glycoproteins display N- and/or O-linked glycan structures produced by a sequential series of glycohydrolases and glycosyltransferases in the ER and Golgi (Fig. 12.1; Kornfeld and Kornfeld, 1985; Schachter, 1991). The ensuing heterogeneity of N-glycans following Golgi modification culminates in the same protein existing as multiple glycoforms that differ in binding avidity for carbohydrate-binding proteins (lectins) such as the galectins, siglecs, and selectins (Brewer et al., 2002; Crocker et al., 2007; Dennis et al., 2009a; Grigorian et al., 2009; Marth and Grewal, 2008). In this chapter, we focus on N-glycan–galectin interactions and their role in controlling the distribution and concentration of cell surface receptors and transporters to affect cell growth and differentiation, with a focus on T cells.

2. Galectins and Their N-Glycan Ligands Galectins are a 15-member family of lectins that possess a conserved carbohydrate-recognition domain (CRD) specific for N-acetyllactosamine (galactose b1,4 N-acetylglucosamine ¼ Galb1,4GlcNAc ¼ LacNAc; Hirabayashi et al., 2002). Golgi N-glycosylation of proteins cooperates with gene-encoded differences in N-glycan number per protein (i.e., occupied N-X-S/T sites) to differentially regulate glycoprotein concentration and distribution at the cell surface based on differences in avidity for the galectins (Chen et al., 2007; Lau et al., 2007). Galectins are classified into three groups based on whether they possess a single CRD and frequently dimerize (galectin-1, -2, -5, -7, -10, -11, -13, -14, and -15), two CRDs in tandem repeat connected by a short linker region that can also dimerize (galectin-4, -6, -8, -9, and -12) or a single CRD connected to an N-terminal region that can form pentamers (galectin-3; Ahmad et al., 2004; Hirabayashi and Kasai, 1993; Stowell et al., 2008; Yang et al., 2008). Binding of galectins to individual glycoproteins increases in proportion to the number of LacNAc units per N-glycan and the number of

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Branched N-glycan biosynthesis Hexosamine pathway Glucose-6phosphate NH4 Glucosamine N-Acetylglucosamine (Gln) (GlcNAc) AcetylGlutamine Glucosamine- CoA Fructose-6N-Acetylglucosamine-6-phosphate 6-phosphate phosphate

Glucose

Glycolysis

N-Acetylglucosamine-1-phosphate UTP Uridine UDP-GlcNAc UMP Cytosol ER

Cytosol Golgi

UDP-GlcNAc transporter

N-Glycan pathway UMP

UDP-GlcNAc (~1.5 mM)

OT

GI

GII

MGAT1

MI

MII

Km~0.04 mM

DMN

Mannose Glucose

Galactose N-Acetylglucosamine (GlcNAc) N-Acetyllactosamine

GalT3

MGAT 4a/b

MGAT2

MGAT5

Km~0.9 mM Km~5 mM Km~11 mM

SW

Mono

GalT3

Bi

GalT3

Tri

GalT3 IGnT

Tetra

Galectin binding L-PHA binding

Galectin avidity Endocytosis

Figure 12.1 Regulation of GlcNAc-branched N-glycan biosynthesis by the hexosamine and N-glycan pathways. UDP-GlcNAc is required by the N-acetylglucosaminyltransferases Mgat1, 2, 4, and 5 and iGnT. Size of the arrows for Mgat1, 2, 4, and 5 depicts relative affinity for UDP-GlcNAc, with Km below arrows. Cytosolic UDP-GlcNAc enters the Golgi via antiporter exchange with Golgi UMP, a reaction product of the N-acetylglucosaminyltransferases. Galectins bind N-acetyllactosamine, with avidity increasing in proportion to the number of N-acetyllactosamine units (i.e., branching). b-1,6GlcNAc-branching by Mgat5 promotes poly-N-acetyllactosamine production, further enhancing avidity for galectins. DMN, deoxymannojirimycin; SW, swainsonine; GI, glucosidase I; GII, glucosidase II; MI, mannosidase I; MIIx, mannosidase IIx; GalT3, galactosyltransferase3. This research was originally published by Grigorian et al. (2007). # The American Society for Biochemistry and Molecular Biology.

N-glycans per protein (Lau et al., 2007). Although this suggests redundancy among galectins, the presence of fucose and/or terminal a2,6-sialic acid alter binding of some galectins (Hirabayashi et al., 2002; Stillman et al., 2006; Stowell et al., 2008; Toscano et al., 2007). CRD orientation and valency as well as N-glycan topology (i.e., the orientation of N-glycans attached to individual glycoproteins) also influence selectivity of galectins for glycoprotein partners (Bi et al., 2008; Brewer et al., 2002; Stillman et al., 2006).

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3. Regulation of Glycoprotein Concentration at the Cell Surface by the Galectin–Glycoprotein Lattice In the presence of multivalent glycan ligands, galectins form higher order lattices (Ahmad et al., 2004; Brewer et al., 2002; Demetriou et al., 2001; Hirabayashi et al., 2002; Nieminen et al., 2007). At the cell surface, these interactions regulate the concentration of receptors and transporters by controlling membrane localization, clustering, and endocytosis rates (Cha et al., 2008; Chen et al., 2007; Demetriou et al., 2001; Demotte et al., 2008; Kuball et al., 2009; Lajoie et al., 2007; Lau et al., 2007; Ohtsubo et al., 2005; Pace et al., 1999; Partridge et al., 2004). Here we define the galectin– glycoprotein lattice as a macromolecular structure that encompasses all galectins and glycoproteins interacting at the cell surface. In this model, any transmembrane glycoprotein with appropriate N-glycan ligands may interact with one or more galectins at the cell surface and thereby be regulated by the galectin–glycoprotein lattice. Experimentally defining all endogenous galectin–glycoprotein interactions at the cell surface is a daunting task. However, computational modeling of the Golgi branching pathway, when coupled with gene-encoded differences in N-glycan number, is highly predictive of cell surface glycoprotein regulation by the galectin– glycoprotein lattice (Lau et al., 2007). The ability to computationally predict regulation of individual glycoproteins based on Golgi activity and N-glycan number provides a powerful tool to probe the function of the galectin– glycoprotein lattice. In addition to Golgi enzyme activity, biosynthesis of N-glycans in the Golgi is sensitive to uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) supply and hexosamine pathway metabolism, allowing adaptive regulation of glycoproteins at the cell surface (Chen et al., 2007; Demetriou et al., 2001; Demotte et al., 2008; Grigorian et al., 2007; Lau et al., 2007; Lee et al., 2007; Morgan et al., 2004; Sasai et al., 2002; Togayachi et al., 2007). In this manner, metabolism and Golgi N-glycosylation combine to adaptively regulate cellular responsiveness to extracellular signals. Convergent studies by multiple groups provide two primary molecular mechanisms of cell surface glycoprotein regulation by the galectin–glycoprotein lattice: (1) regulation of membrane localization, lateral mobility, and clustering and (2) inhibition of endocytosis (Cha et al., 2008; Chen et al., 2007; Demetriou et al., 2001; Demotte et al., 2008; Kuball et al., 2009; Lajoie et al., 2007; Lau et al., 2007; Ohtsubo et al., 2005; Pace et al., 1999; Partridge et al., 2004). Cytoplasmic domains of transmembrane glycoproteins bind various adaptor proteins that are components of the cytoskeletal network, the endocytic machinery, and/or lipid microdomains. The same receptor or transporter can also interact with galectins on the cell surface via their glycosylated extracellular domains, directly opposing or enhancing cytoskeletal-mediated movement in the plane of the membrane

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and/or endocytosis (Chen et al., 2007; Demetriou et al., 2001; Lajoie et al., 2007; Lau et al., 2007; Partridge et al., 2004). As exemplified for the T cell receptor (TCR), galectin–glycoprotein interactions function by physically keeping receptors apart, thereby preventing spontaneous ligand-independent receptor clustering and signaling (Chen et al., 2007).

4. T Cells and the Galectin–Glycoprotein Lattice Accumulating evidence over the past decade provides strong evidence that the galectin–glycoprotein lattice coordinates basal, activation, and arrest/ differentiation signaling to control T cell homeostasis and self-tolerance (Chen et al., 2007, 2009; Demetriou et al., 2001; Demotte et al., 2008; Grigorian et al., 2007; Lau et al., 2007; Lee et al., 2007; Morgan et al., 2004; Rabinovich and Toscano, 2009; Togayachi et al., 2007). T cells play a central role in the antigen-specific adaptive immune response and when dysregulated can lead to loss of self-tolerance and autoimmune pathogenesis. The association of the TCR with the galectin–glycoprotein lattice prevents ligand-independent basal TCR signaling via Lck by inhibiting spontaneous TCR clustering, CD4-Lck recruitment to TCR, and consequent filamentous (F)-actin-mediated transfer of the TCR complex to GM1-enriched membrane microdomains (GEM; Chen et al., 2007). The galectin–glycoprotein lattice also negatively regulates basal TCR signaling through Lck by promoting partition of the tyrosine phosphatase CD45 to GEMs (Chen et al., 2007). In the presence of TCR ligand, the galectin–glycoprotein lattice raises T cell activation thresholds by inhibiting ligand-dependent TCR clustering and promoting CD45 retention at the immune synapse (Chen et al., 2007; Chung et al., 2000; Demetriou et al., 2001; Kuball et al., 2009). Once activated, T cells undergo several rounds of cell division followed by growth arrest. The galectin–glycoprotein lattice promotes arrest signaling by inhibiting endocytosis of the growth inhibitor CTLA-4 (Lau et al., 2007). The critical interplay between activating and inhibitory receptors defines the nature and outcome of the immune response, highlighting the importance of a global regulatory network such as the galectin–glycoprotein lattice in controlling cell growth and activity.

5. Genetic and Metabolic Regulation of the Galectin–Glycoprotein Lattice Glycoprotein–galectin interactions are highly influenced by the extent of N-glycan branching produced in the Golgi as well as the number of N-glycans specific to each glycoprotein (i.e., occupied N-X-S/T sites; Dennis et al., 2009b; Lau et al., 2007). The number of N-glycans per glycoprotein is an encoded

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feature of protein sequence and varies widely among glycoproteins. Growth promoting receptors, such as the TCR and receptor tyrosine kinases, usually have large numbers of N-glycans (i.e., five or more), while growth inhibitory receptors, such as CTLA-4 and transforming growth factor-b receptors I and II (TbR), usually have few N-glycans per protein molecule (i.e., four or less; Lau et al., 2007). When Golgi branching activity is limited, growth promoting receptors are predicted to predominate at the cell surface, positively regulating cell growth. However, with increasing N-glycan branching by the Golgi, growth inhibitory receptors become incorporated into the galectin– glycoprotein lattice enhancing cell surface retention to induce growth arrest. This paradigm has been demonstrated for TCR/CTLA-4 in T cells and receptor tyrosine kinases/TbR in epithelial cells (Lau et al., 2007). N-Glycan branching is regulated by multiple Golgi enzymes, including a-mannosidases I and II and N-acetylglucosaminyltransferases Mgat1, 2, 4, and 5. The latter catalyze the addition of N-acetylglucosamine (GlcNAc) from the sugar-nucleotide substrate, UDP-GlcNAc, to N-glycan precursors transiting the Golgi (Fig. 12.1). Additional Golgi processing generates mono-, bi-, tri-, and tetra-antennary N-acetyllactosamine-branched N-glycans which can be modified further with sialic acid, fucose, and/or sulfate to alter galectin-binding avidity (Fig. 12.1). The b1,6GlcNAc-branched N-glycans catalyzed by Mgat5 are preferentially extended by poly-N-acetyllactosamine, maximizing avidity for galectins (Fig. 12.1; Cummings and Kornfeld, 1984; Hirabayashi et al., 2002; Stowell et al., 2008; Ujita et al., 1999a,b). The activities of the late Golgi branching enzymes (Mgat4 and Mgat5) are highly sensitive to metabolic production of UDP-GlcNAc by the hexosamine pathway (Fig. 12.1; Grigorian et al., 2007; Hirschberg et al., 1998; Lau et al., 2007; Sasai et al., 2002). Biosynthesis of UDP-GlcNAc requires key metabolites of carbohydrate, amino acid, lipid, and nucleotide metabolism, positioning the hexosamine pathway as a critical sensor of cellular nutrient availability. Several groups have demonstrated that supplementing various cell types with one or more of these metabolites enhance N-glycan branching by the Golgi (Grigorian et al., 2007; Lau et al., 2007; Sasai et al., 2002). For example, hexosamine pathway metabolite supplementation (i.e., GlcNAc) and consequent increases in N-glycan branching suppress T cell activity, including inhibiting TCR signaling, CD69 expression, T cell proliferation, TH1 differentiation, and CTLA-4 endocytosis (Grigorian et al., 2007). Therefore, metabolic regulation of Golgi N-glycosylation provides an adaptive mechanism to modulate glycoproteins at the cell surface.

6. Overview of Methods to Measure and Modulate the Galectin–Glycoprotein Lattice In addition to the cell surface and extracellular matrix, galectins are also expressed in the cytoplasm, where they appear to have intracellular activities distinct from cell surface interactions with glycoproteins (Liu et al., 2002). This complicates interpretation of studies utilizing galectin-deficient

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mice. In contrast, altering production of galectin ligands (i.e., N-acetyllactosamine) allows a more specific evaluation of glycoprotein–galectin interactions at the cell surface. In this section, we describe methods used to measure and modulate cell surface galectin–glycoprotein interactions and subsequent functional effects on receptor signaling and cell growth. First, we describe methods to assess changes in N-glycan branching and galectin–glycoprotein interactions at the cell surface: (Section 6.1.1) co-immunoprecipitation of galectins and glycoproteins, (Section 6.1.2) flow cytometry to measure N-glycan branching and glycoprotein surface levels, (Section 6.1.3) immunofluorescence microscopy to examine glycoprotein clustering and mobility, (Section 6.1.4) mass spectroscopy of N-glycans, (Section 6.1.5) Golgi enzyme assays, and (Section 6.1.6) quantitative real-time PCR and mRNA half-life measurements of Golgi enzymes. Next, we describe methods to modulate N-glycan branching and galectin–glycoprotein interactions: (Section 6.2.1) Disrupting galectin–glycoprotein interactions by (Section 6.2.1.1) gene-targeted deletions of Golgi enzymes, (Section 6.2.1.2) N-X-S/T site mutation, (Section 6.2.1.3) small molecule inhibitors of N-glycan branching, and (Section 6.2.1.4) competitive inhibition of galectin binding and (Section 6.2.2) Strengthening galectin–glycoprotein interactions by (Section 6.2.2.1) overexpression of Golgi branching enzymes and (Section 6.2.2.2) metabolic supplementation of the hexosamine pathway. Finally, we describe methods to examine downstream receptor signaling and cell growth/differentiation regulated by the galectin–glycoprotein lattice: (Section 6.3.1) TCR signaling, (Section 6.3.2) T cell activation and proliferation, and (Section 6.1.2) flow cytometry to measure receptor surface levels and cell growth markers.

6.1. Methods to measure N-glycan branching and galectin– glycoprotein interactions at the cell surface 6.1.1. Co-immunoprecipitation of galectins with glycoprotein partners Co-immunoprecipitation is a powerful tool for protein and glycan interaction discovery and can be used to identify unknown glycoproteins that comprise the galectin–glycoprotein lattice by targeting specific galectins. Since galectins are non-covalently associated with surface glycoproteins and may be washed away during in vitro manipulation of cells, intact cells are first chemically cross-linked to stabilize complexes at the cell surface with the homobifunctional crosslinker dithio-bis-sulphosuccinimydylpropionate (DTSSP; Pierce) at 0.1 mg/ ml with 1  106 cells/ml in phosphate-buffered saline (PBS), pH 8.0, for 10 min at room temperature. After cross-linking cell surface proteins, the reaction is quenched by adding Tris–HCl, pH 7.2, to a final concentration of 50 mM and incubating for 15 min. Cells are then lysed in ice-cold lysing buffer (50 mM Tris–HCl, pH 7.2, 300 mM NaCl, 0.5% Triton X-100, 2 mM sodium orthovanadate) containing a proteinase inhibitor cocktail (Roche Applied Science). One milliliter of the cell lysates (100–500 mg total protein) are incubated with 0.2–2 mg anti-galectin primary antibody (Ab; optimal Ab

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concentration should be determined by a titration) for 2 h at 4  C followed by incubation with 20 ml Protein G PLUS-Agarose beads (Santa Cruz Biotechnology) at 4  C overnight on a rotating device. After six washes with PBS, samples are boiled for 2 min and eluted protein concentrations are measured using a BCA protein assay kit (Pierce). Equivalent amounts of protein are separated on SDS-polyacrylamide gel electrophoresis gels under reducing conditions, transferred electrophoretically to polyvinylidene difluoride membranes (Millipore), and blocked with 10% dry milk in Tris-buffered saline (TBS; pH 7.2) containing 0.1% Tween-20. These membranes are then immunoblotted with antibodies to the glycoprotein of interest to determine if it interacts with one or more galectins at the cell surface. Alternatively, the glycoprotein of interest can be immunoprecipitated and then blotted for specific galectins (Demetriou et al., 2001). 6.1.2. Flow cytometry to measure N-glycan branching and glycoprotein surface levels Flow cytometry is a rapid and quantitative method to analyze expression levels of cell surface proteins. A major advantage is the ability to analyze multiple parameters of specific cell types within a heterogeneous population, including viability, proliferation, cell cycle progression, apoptosis, and cellular processes such as calcium flux and phagocytosis. The various participants in the galectin–glycoprotein lattice can be quantified and characterized under specific conditions at the cell surface. The extent of N-glycan branching can also be directly quantified using various lectins specific for particular carbohydrate structures. Lectins isolated from a wide variety of plants have highly specific carbohydrate-binding properties and are valuable in analyzing cell surface N-glycosylation status. L-PHA is a plant lectin from Phaseolus vulgaris (Leucoagglutinin) that specifically recognizes b1,6GlcNAc-branched N-glycans produced by Mgat5 (Cummings and Kornfeld, 1982; Demetriou et al., 2001). LEA (tomato lectin) is a plant lectin from Lycopersicon esculentum that exhibits specificity for elongated polylactosamine chains. Cells are washed with FACS buffer (PBS, 1% bovine serum albumin (BSA), 0.1% sodium azide) prior to staining. 1  106 cells in 100 ml FACS buffer are incubated with antibodies or lectins directly conjugated to various fluorescent dyes (e.g., anti-CD4-FITC and anti-CD3-PE at 0.5 mg per sample, L-PHA-FITC at 2–20 mg/ml) for 30 min at 4  C (optimal Ab/ lectin concentration and time should be determined by a titration). Cells are washed twice with FACS buffer and can be analyzed right away or fixed with 1% paraformaldehyde for analysis at a later time. Data are analyzed with a FACScan flow cytometer using the CellQuest program (BD Biosciences) or WinMDI (The Scripps Research Institute; Chen et al., 2009; Grigorian et al., 2007; Lau et al., 2007; Lee et al., 2007). Detecting galectins at the cell surface with antibodies is complicated by the non-covalent attachment of galectins to the cell surface. Repeated

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washing of cells in buffer removes cell surface galectins and can result in the false conclusion that galectins are not present. This can be mitigated by minimizing washes and the volume of buffer for the washes. Chemical cross-linking of the cell surface with DTSSP (see Section 6.1.1 above) prior to staining with anti-galectin Ab can also be used to avoid this issue. 6.1.3. Immunofluorescence microscopy to examine glycoprotein clustering and co-localization with membrane microdomains Compartmentalization of cell surface glycoproteins into functional microdomains influences cell signaling. Immunofluorescence microscopy can be used to study the effect of the galectin–glycoprotein lattice on receptor clustering, mobility, as well as co-localization of galectins, glycoproteins, and membrane microdomains. To examine ligand-induced receptor clustering, receptor ligand is coated onto polystyrene beads (Polysciences) in PBS for 1 h, followed by blocking with 200 mg/ml BSA. Cells are then mixed with beads in media at 37  C for 10 min, placed on poly-L-lysine-coated cover slips, fixed with 10% formalin, stained with fluorescently labeled antibodies to the receptor of interest and then visualized by immunofluorescence microscopy (either by confocal or deconvolution). To costain for actin microfilaments, the nucleus and/or intracellular proteins, the membrane is permeabilized with 0.2% Triton X-100 and labeled with rhodamine-phalloidin, Hoechst, and/or relevant Ab prior to microscopy. Co-localization of glycoproteins within GM1-enriched microdomains (GEM) can be assessed using patching techniques. The ability of the B subunit of cholera toxin (CTB) to bind GM1 can be utilized to visualize membrane microdomains enriched in cholesterol and sphingolipids by immunofluorescence microscopy. As individual GEMs are too small for direct visualization, they are first coalesced/patched via incubation with CTB-TRITC at 10 mg/ml in 0.1% BSA/TBS for 45 min on ice, washed, and then cross-linked with antiCTB Ab (1/250 dilution in 0.1% BSA/TBS, Calbiochem) for 30 min on ice and an additional 20 min at 37  C. Patched cells are placed on poly-L-lysinecoated slides, fixed with 10% formalin for 30 min at room temperature, and blocked with 0.5% BSA/TBS for 1 h. Cells can then be incubated with one or more fluorescently labeled antibodies to the glycoproteins or galectins of interest. Slides are mounted with Vectashield (Vector Laboratories) and visualized by confocal and/or deconvolution immunofluorescence microscopy (Chen et al., 2007). 6.1.4. Mass spectroscopy of N-glycans MALDI-TOF mass spectroscopy is a useful technique to validate changes in N-glycan branching patterns observed using plant lectins and flow cytometry described in Section 6.1.2 above. This technique requires significant expertise and for this purpose, we have utilized the service provided by the Glycotechnology Core Resource at the Glycobiology Research and

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Training Center, University of California, San Diego. N-glycans are isolated by lysis with 1% SDS in 100 mM Tris, pH 7.4, dialyzed to remove SDS, digested with trypsin to generate glycopeptides, and treated with PNGase F to release the N-glycans. To focus on N-glycan branching, Nglycans are desialylated by mild acid treatment prior to permethylation and analysis by MALDI-TOF mass spectroscopy. Monoisotopic peaks in the spectra are identified using GlycanMass and GlycoMod online software, with specific targeting of the N-glycan intermediates occurring during Golgi processing (Grigorian et al., 2007; Lau et al., 2007; Lee et al., 2007). 6.1.5. Golgi enzyme assays Effective antibodies to the Golgi branching enzymes are lacking and thus Western blotting/immunoprecipitation to assess protein expression is not available. However, examination of enzyme activity provides a more quantitative alternative. The activity of the Golgi N-acetylglucosaminyltransferases I, II, and V (i.e., Mgat1, Mgat2, and Mgat5) are measured using highly specific synthetic glycan acceptors and radiolabeled UDP-GlcNAc, with the enzymes transferring radiolabeled GlcNAc to the synthetic acceptor. Cell lysates (10 ml) are added to 1 mM acceptor, 1 mM [6-3H] UDPGlcNAc (Amersham Biosciences) in 50 mM MES, pH 6.5, 0.1 mM GlcNAc, and 25 mM AMP for a total reaction volume of 20 ml. The acceptor for Mgat5 (GnTV) is bGlcNAc(1,2)aMan(1,6)bGlc-O (CH2)7CH3, Mgat2 (GnTII) is bGlcNAc(1,2)aMan(1,3)[aMan(1,6)]b Man-O(CH2)7CH3, and Mgat1 (GnTI) is aMan(1,3)bMan-O(CH2)7CH3 (Toronto Research Chemicals). Mgat2 and Mgat1 reactions also contain 5 mM MnCl2 and are incubated for 1 h. Mgat5 reactions are incubated for 3 h at 37  C. All reactions are stopped with 1 ml of ice-cold water. Enzyme products are separated from radioactive substrates by binding to 50 mg C18 cartridges (Alltech) preconditioned with methanol rinsing and water washing. Reactions are loaded and the columns are washed five times with 1 ml of water. Radiolabeled products are eluted directly into scintillation vials with two 0.5 ml aliquots of methanol. Radioactivity is determined by liquid scintillation counting (Lau et al., 2007; Lee et al., 2007). 6.1.6. Quantitative real-time PCR and mRNA half-life measurements of Golgi enzymes Real-time PCR provides quantitative measurements of gene transcription and can be used to profile changes in gene expression of the various N-glycan branching enzymes. Since N-glycan remodeling is dependent upon the sequential action of a series of enzymes, it is important to assess changes in multiple Golgi genes in the N-glycan branching pathway. For these assays, RNA is isolated using the RNeasyÒ Mini Kit (Qiagen). Reverse transcription is performed with the RETROscriptÒ Kit (Ambion) according to the manufacturer’s instructions. Taqman probes and primers are purchased from

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Applied Biosystems. To measure mRNA half-life/stability, cDNA is derived from cells treated with the transcriptional inhibitor actinomycin D (5 mg/ml, Sigma). mRNA half-life/stability is determined by comparing Ct values from actinomycin D-treated with mock-treated cells using a standard curve generated by two-fold serial dilutions for each primer (Chen et al., 2009).

6.2. Methods to modulate galectin–glycoprotein interactions at the cell surface 6.2.1. Disrupting galectin–glycoprotein interactions 6.2.1.1. Gene-targeted deletions of Golgi enzymes The structural diversity of N-glycans produced by the Golgi enzymes dictates the affinity and specificity of glycoproteins for the galectin–glycoprotein lattice. Genetargeted deletion of Golgi enzymes can limit N-glycan processing at specific stages and thereby disrupt galectin–glycoprotein interactions. Targeted deletion of genes is a well-established technique described elsewhere. As glycoprotein affinities for galectins increase in proportion to N-glycan branching, absence of Mgat5, 4, 2, and 1 enzymatic activity is expected to result in increasing deficiencies in galectin ligands. T cells intrinsically express little N-glycan branching relative to other cell types, and therefore, the most dramatic phenotypes observed in Mgat5-deficient mice appear to be related to T cell biology. Mgat5 deficiency enhances TCR clustering and signaling, lowers T cell activation thresholds, promotes TH1 differentiation, reduces CTLA-4 surface levels, and results in spontaneous autoimmunity (Demetriou et al., 2001; Lau et al., 2007). Similarly, mice deficient in b1,3-N-acetylglucosaminyltransferase-2 (b3GnT2), an enzyme that extends branched N-glycans with poly-N-acetyllactosamine, display lower T cell activation thresholds (Togayachi et al., 2007). Mgat4 has two isoenzymes, Mgat4a and Mgat4b, which produce b1,4GlcNAc-branching present in triand tetra-antennary N-glycans. Mgat4a is predominantly expressed in pancreatic b cells and targeted deletion disrupts glucose transporter 2 (Glut2) interaction with galectins, thereby enhancing loss to endocytosis and markedly reducing Glut2 surface levels in b cells (Ohtsubo et al., 2005). This reduces the ability of b cells to sense glucose and results in Type II diabetes. Mgat4b is more ubiquitously expressed, however, Mgat4b deficiency and Mgat4a/b double deficiency results in compensatory upregulation of Mgat4a, Mgat5, and/or b3GnT1/T2, thereby largely maintaining overall N-acetyllactosamine content in N-glycans and associated avidity for galectins (Takamatsu et al., 2010). Thus, the galectin-glycoprotein lattice appears largely undisturbed in Mgat4a/b double deficient mice and indeed overt phenotypes other than those induced by Mgat4a deficiency are not observed. Mannosidase II activity is required to produce N-glycan acceptors utilized by Mgat4 and Mgat5, but is encoded by two isoenzymes termed amannosidase II (MII) and a-mannosidase II (MIIx) that have overlapping

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expression patterns (Akama et al., 2006). Targeted deletion of either isoenzyme has minimal effects on N-glycan branching in most tissues, although specific phenotypes are observed in red blood cells and the kidney in MII deficient mice and in sperm in MIIx deficient mice (Akama et al., 2002; Chui et al., 1997, 2001). MII/MIIx double knockout mice as well as deficiencies in Mgat1 and Mgat2 markedly reduce N-glycan branching and result in severe phenotypes, suggesting that a minimal galectin–glycoprotein lattice is required for embryonic development and postnatal viability. Mgat1-deficiency eliminates N-glycan ligands for galectins and embryos die by E9.5 (Ioffe and Stanley, 1994; Metzler et al., 1994). Mgat2 deficiency and MII/ MIIx double deficiency result in mono-antennary N-glycans with minimal binding avidity for galectins and severe developmental and postnatal defects in multiple organs that result in pre- and peri-natal death (Akama et al., 2006; Wang et al., 2001). To avoid these developmental defects and examine adult tissue and phenotypes, the cre/loxP system can be utilized. Jamey Marth and colleagues have produced mice containing floxed alleles of many Golgi enzymes (Lowe and Marth, 2003). For example, targeted deletion of Mgat1 in neuronal cells results in normal neuronal development but accelerated apoptosis in adult brains and associated neurological phenotypes (Ye and Marth, 2004). Another important consideration is variation in N-glycan branching among inbred laboratory mouse strains. Indeed, certain inbred strains are intrinsically hypomorphic for N-glycan branching, displaying partial deficiencies of multiple Golgi enzymes and consequent phenotypes (Lee et al., 2007). For example, the PL/J strain is known to be highly susceptible to the multiple sclerosis model EAE; has partial deficiencies of Mgat-1, -2, and -5 enzyme activities; and displays the lowest levels of N-glycan branching observed among multiple inbred strains (Lee et al., 2007). This leads to T cell hyperactivity and development of a spontaneous multiple sclerosislike disease that is further enhanced by Mgat5 deficiency. 6.2.1.2. N-X-S/T site mutation Genetic alterations in the number of N-glycans per glycoprotein (i.e., occupied N-X-S/T sites) can influence the specificity of individual glycoproteins for the galectin–glycoprotein lattice. Genetic manipulation that removes individual N-X-S/T sites can be achieved by site-directed mutagenesis as widely described elsewhere. For example, selective removal of the single N-X-S/T site in GLUT4 eliminated the ability of the Golgi branching pathway to enhance GLUT4 surface expression (Lau et al., 2007). Similarly, removal of specific N-X-S/T sites from TCR enhanced diffusion, clustering, and activation signaling (Kuball et al., 2009), phenocopying the effects of N-glycan branching deficiency on TCR. Changes in N-X-S/T sites and/or usage are also observed in many human disease-associated mutations and polymorphisms (Anjos et al., 2002; Kavvoura and Ioannidis, 2005; Vogt et al., 2005). For example, a common

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polymorphism in the signal peptide of human CTLA-4 (Thr17Ala) reduces average N-glycan occupancy at the two N-X-S/T sites by 50% (i.e., from two to one) and is associated with decreased CTLA-4 cell surface retention, T cell hyperactivation, and development of Type I diabetes (Anjos et al., 2002; Kavvoura and Ioannidis, 2005; Maurer et al., 2002). 6.2.1.3. Inhibitors of N-glycan branching Supplementation of cell cultures with small molecule inhibitors of Golgi processing enzymes can be utilized to downregulate N-glycan branching and weaken the galectin– glycoprotein lattice. Swainsonine (used at a range of 50–200 nM) inhibits a-mannosidase II, blocking N-glycan branching at the mono-antennary stage, and leads to enhanced T cell proliferation and TH1 differentiation (Elbein, 1987; Morgan et al., 2004; Wall et al., 1988). At higher concentrations, swainsonine may also inhibit lysosomal a-mannosidases. Blocking N-glycan branching prior to Mgat1 (i.e., absence of GlcNAc branching) can be achieved with deoxymannojirimycin (used at a range of 0.1–2 mM), an inhibitor of a-mannosidase I (Elbein, 1987; Wall et al., 1988). As the expression and activity of these enzymes may vary significantly between different cells, optimal concentrations need to be determined by titration. It is also important to note that these inhibitors only partially reduce N-glycan branching. Moreover, positive feedback to restore N-acetyllactosamine content in N-glycans may also occur. This effect may counteract reductions in N-glycan branching to restore or even enhance galectin avidity and thereby, paradoxically strengthen the lattice and associated phenotypes. For example, we have recently observed that in T cell blasts, but not resting T cells, swainsonine increases poly-N-acetyllactosamine content at the cell surface 10-fold, as determined by flow cytometry with LEA (tomato lectin; Grigorian et al., unpublished data). It is unclear why this was observed in blasting but not resting T cells. This type of feedback parallels that observed in Mgat4a and Mgat4a/b double knockout mice described above (Takamatsu et al., 2010) and implies homeostatic mechanisms to maintain N-acetyllactosamine content in N-glycans within a narrow range. Thus, the use of small molecule inhibitors to disrupt the galectin lattice needs to be used with caution and potential positive feedback that strengthens the lattice needs to be carefully assessed. 6.2.1.4. Competitive inhibition of galectin binding The galectin– glycoprotein lattice can also be disrupted by direct competition of galectin– N-glycan interactions at the cell surface. The galectins possess a conserved CRD with a minimal binding specificity for N-acetyllactosamine (LacNAc ¼ Galb1,4GlcNAc; Hirabayashi et al., 2002). Addition of lactose (Galb1,4Glc) or LacNAc disaccharides to cultured cells competes with cell surface N-glycans for binding to galectins, effectively disrupting galectin–glycoproteins at the cell surface (Demetriou et al., 2001). LacNAc is more effective than lactose, while

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NS Mgat5 Lactose Cross WB: TCRa WB: CD3e

IP:Gal-3 –/– +/+ – – – + – + + – + + + +

Figure 12.2 Association of galectin-3 with TCR complex proteins. Cell lysates were immunoprecipitated with anti-galectin-3 Ab (e.g., H-160, Santa Cruz Biotechnology) and membranes were blotted with anti-TCRa Ab (H-142, Santa Cruz Biotechnology) and anti-CD3e Ab followed by incubation with peroxidase-conjugated Ab. Signals are detected using an ECL kit (Pierce). Shown is galectin-3 association with CD3e and TCRa chain, and its disruption by Mgat5 deficiency and lactose. This research was originally published by Demetriou et al. (2001). # Nature Publishing Group.

increasing the number of LacNAc repeats further enhances competitive binding to galectins with tri-LacNAc (Galb1,4GlcNAc)3 > bi-LacNAc (Galb1,4GlcNAc)2 (Ohtsubo et al., 2005). For mouse or human T cells, cells are incubated with lactose or LacNAc at concentration ranges of 2–50 mM for 20 min at 37  C in media and subsequently maintained in the lactose/LacNAc containing media at all times for the duration of the assay. This includes all wash steps up to the time the cells are fixed or lysed. Control disaccharides, such as sucrose, are used at the same concentrations. Concentrations of lactose or LacNAc required to disrupt galectin–N-glycan binding vary with different cell types as a function of their intrinsic lattice strength. As such, careful titrations are an obligatory step to determine effective inhibitory concentrations. Figure 12.2 shows the co-immunoprecipitation of galectin-3 with TCR complex proteins in the absence but not presence of lactose. Figure 12.3 shows enhanced ligandinduced TCR clustering by lactose but not sucrose (Chen et al., 2007; Demetriou et al., 2001). 6.2.2. Strengthening galectin–glycoprotein interactions 6.2.2.1. Overexpression of Golgi branching enzymes Transient or stable transfection of epithelial cells with Mgat5 cDNA markedly increases b1,6GlcNAc branching, leading to increased cell motility, reduced substratum adhesion, and epithelial to mesenchymal transition (Demetriou et al., 1995). This represents a straightforward approach to strengthen the galectin–glycoprotein lattice and is widely applicable to most cell types. However, T and B cells are small cells with little cytoplasm, making them very difficult to transfect by traditional methods (e.g., electroporation, liposomes). Lentiviral vectors can be utilized; however, this approach requires dividing cells and therefore cannot be used on resting T cells.

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Mgat5

Bead

+/+

a -CD3

Bright field

GM1

CD45

Lck-pY394

Merge

14 of 20*

–/–

a-CD3 16 of 20*

Figure 12.3 Immunofluorescence microscopy images of Mgat5þ/þ and Mgat5/ T cells. Mgat5þ/þ and Mgat5/ T cells were conjugated as described above with microbeads coated with 0.5 mg/ml anti-CD3e Ab (2C11 for mouse, OKT3 for Jurkat cells, eBioscience), and co-spun for 15 s, incubated for 3 min at 37  C and fixed. Slides were stained with anti-CD45-FITC (30-F11 for mouse and H130 for Jurkat, eBioscience) and CTB-TRITC (non-patched cells, List Biology Laboratory), permeabilized with 0.2% Triton X-100 in blocking solution, and then intracellularly stained with antiphospho-Src family Tyr416 Ab (Cell Signaling Technology) which cross-reacts with phospho-Lck-Tyr394 followed by aminomethylcoumarin acetic acid-conjugated secondary Ab ( Jackson ImmunoResearch). Slides were mounted with Vectashield (Vector Laboratories). Images were collected on a Nikon TE-2000-U microscope with a 60 objective. Deconvolution and co-localization was performed on images collected at 0.2 mm Z-intervals with MetaMorph and quantification of the co-localization coefficient was accomplished by WCIF ImageJ software. Twenty T cell-bead conjugates were scored for presence or absence of CD45 in GM1-enriched microdomain clusters at the contact site. A deficiency in b1,6 N-acetylglucosaminyltransferase V (Mgat5) reduces CD45 clustering at the early immune synapse. This research was originally published by Chen et al. (2007). # The American Society for Biochemistry and Molecular Biology.

6.2.2.2. Metabolic supplementation of the hexosamine pathway Activity of the distal Golgi branching enzymes, Mgat4 and Mgat5, are highly dependent on UDP-GlcNAc concentrations in the Golgi. As such, N-glycan branching is influenced by the nutrient environment and metabolite supply to the hexosamine pathway for UDP-GlcNAc biosynthesis (Grigorian et al., 2007; Lau et al., 2007; Sasai et al., 2002). Supplementing the hexosamine pathway with glucose, glutamine, acetoacetate (metabolized to acetyl-CoA), and uridine (metabolized to UTP) increases de novo UDP-GlcNAc biosynthesis by the hexosamine pathway (Fig. 12.1). In T cells, daily supplementation at a range of 1–80 mM concentrations for at least 2–3 days is required to see enhancement of N-glycan branching at the cell surface. The addition of N-acetylglucosamine (GlcNAc; 40 mM) to culture media also supplements UDP-GlcNAc pools after uptake by bulk endocytosis, 6-phosphorylation, and conversion to UDP-GlcNAc (Fig. 12.1). Uptake of GlcNAc and associated increases in N-glycan branching depends on bulk endocytosis

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rates/membrane turnover, which in turn often reflects cellular growth rates. Thus, nondividing cells may be relatively resistant to GlcNAc uptake, which may be resolved by daily supplementation. Glucosamine is also salvaged into the hexosamine pathway for UDP-GlcNAc biosynthesis, but unlike GlcNAc, it may also be metabolized to fructose-6-phosphate to enter glycolysis (Fig. 12.1). Consistent with this, we have observed that increasing concentrations of glucosamine initially enhance N-glycan branching to a small degree, but then decrease N-glycan branching (Grigorian et al., 2007). Cosupplementation with various metabolites can be more effective than higher concentrations with a single metabolite. For example, co-supplementing resting Jurkat and primary mouse T cells with GlcNAc and uridine is more effective than doubling the GlcNAc concentration (Grigorian et al., 2007). The strength of the T cell galectin–glycoprotein lattice can also be enhanced in live mice by oral supplementation with GlcNAc (Grigorian et al., 2007). Age- and sex-matched mice are treated with GlcNAc orally by adding GlcNAc to their drinking water at various concentrations (62.5– 1000 mg/ml; 10–160 mg/kg dose daily). Fresh GlcNAc is given daily with the amount consumed approximated by determining the volume remaining after 24 h. In vivo and in vitro changes in UDP-GlcNAc/UDP-HexNAc levels can be measured by HPLC and/or MS/MS mass spectroscopy (Grigorian et al., 2007; Lau et al., 2007; Sasai et al., 2002), while changes in surface levels of b1,6GlcNAc-branched N-glycans and poly-N-acetyllactosamine are assessed by flow cytometry analysis with L-PHA and LEA, respectively. In summary, metabolic manipulation of the N-glycosylation pathway provides a simple tool to increase glycan ligands for the galectins, strengthening the galectin–glycoprotein lattice, and adaptively regulating surface retention of receptors and transporters.

6.3. Methods to examine regulation of T cell receptor signaling and cell growth by the galectin–glycoprotein lattice 6.3.1. TCR signaling The effects of the galectin–glycoprotein lattice on TCR signaling under specific conditions (e.g., glycosyltransferase gene knockout cells) can be assessed by measuring sensitivity to TCR agonist. For these assays, naı¨ve T cells are purified from spleens of mice at 8–12 weeks of age by negative selection using CD3þ T cell purification columns (R&D Systems). 5  106 polystyrene beads (6 mm, Polysciences) are coated with 0.5 mg/ml antiCD3e Ab (2C11 for mouse, OKT3 for Jurkat or human T cells, eBioscience) at 4  C overnight. These beads are then mixed with 1  106 T cells, pelleted at 5000 rpm for 15 s, incubated at 37  C for various times, and solubilized with ice-cold lysing buffer (50 mM Tris–HCl, pH 7.2,

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300 mM NaCl, 0.5% Triton X-100, 2 mM sodium orthovanadate) containing a proteinase inhibitor cocktail (Roche Applied Science). Protein concentrations are measured using a BCA protein assay kit (Pierce). Equivalent amounts of protein are separated on SDS-polyacrylamide gel electrophoresis gels under reducing conditions, transferred electrophoretically to polyvinylidene difluoride membranes (Millipore), and blocked with 10% dry milk in TBS (pH 7.2) containing 0.1% Tween-20. TCR signaling can be determined by tyrosine phosphorylation of multiple proteins including enhanced phosphorylation of Zap70, LAT, and Lck-Tyr394. Membranes are immunoblotted with anti-phospho-Zap70 Ab (Cell Signaling Technology), anti-phospho-LAT Ab (Upstate), and anti-phospho-Src family Tyr416 Ab (Cell Signaling Technology), which cross-reacts with phospho-Lck Tyr394, as a measure of signal transduction. Signals are detected using an ECL kit (Fig. 12.4; Pierce; Chen et al., 2007). 6.3.2. T cell activation and proliferation To assess activation status following TCR stimulation, 1  106 purified T cells (see above) are plated on anti-CD3e Ab-coated 24-well plates (2C11 for mouse, OKT3 for Jurkat cells, eBioscience; concentration range of 62.5–1000 ng/ml) for 24–72 h in RPMI-1640 medium with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin, and 50 mM b-mercaptoethanol. Cells are then harvested and analyzed by flow cytometry (see Section 6.1.2) for cell surface activation markers such as CD69 (at 24 h) and CD25 (at 48–72 h). To directly examine cell division, the fluorescent dye 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probes) is utilized to label cells. Each cell division results in half of the fluorescence being distributed to each daughter cell, which can be tracked by flow cytometry. 10  106 cells/ml purified T cells are labeled with 1 mM CFSE in PBS for 20 min at 4  C and cultured in anti-CD3e Ab-coated 24-well plates as above. After 3–5 days of Mgat5+/+ Time (min)

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culture, the cells are analyzed by flow cytometry. The percentage of cells that have proliferated can be quantified, as well as the number of cells within each cell division (Fig. 12.5; Grigorian et al., 2007). Flow cytometry, as described in Section 6.1.2, can also be utilized to measure changes in receptor surface levels (e.g., CTLA-4) in T cell blasts as a function of changes in the galectin–glycoprotein lattice. For example, Mgat5 deficiency, swainsonine, and lactose treatment reduce surface levels of CTLA-4 in activated T cells due to disruption of the galectin– glycoprotein lattice. TCR signaling upregulates Mgat5 gene expression and N-acetyllactosamine levels in N-glycans 3–5 days after T cell activation, promoting CTLA-4 incorporation into the galectin–glycoprotein lattice and retention at the cell surface to signal growth arrest (Lau et al., 2007).

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C H A P T E R

T H I R T E E N

Galectin-1 and HIV-1 Infection Christian St-Pierre,* Michel Ouellet,† Michel J. Tremblay,† and Sachiko Sato* Contents 1. Overview 1.1. Human immunodeficiency virus 1.2. Galectins 2. Experimental 2.1. General precautions for HIV-1-related studies (biosafety level for HIV-1 research; Byers et al., 2004) 2.2. HIV-1 production 2.3. Cell lines and primary cells isolation for HIV-1 infection 2.4. Purification of galectin-1 and -3 2.5. Quality control of galectins 2.6. S-Carboxyamidomethylation of galectin-1 2.7. Inhibition of lectin activity of galectin in physiological condition 2.8. Virus attachment assays 2.9. HIV-1 infection assay References

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Abstract Initial binding of human immunodeficiency virus-1 (HIV-1) to its susceptible CD4þ cells is the limiting step for the establishment of infection as the avidity of viral envelope gp120 for CD4 is not high and the number of viral envelope spikes on the surface is found to be low compared to highly infectious viruses. Several host factors, such as C-type lectins, are listed as being able to enforce or facilitate the crucial interaction of HIV-1 to the susceptible cell. Recent works suggest that a host soluble b-galactoside-binding lectin, galectin-1, also facilitates both virion binding and the infection of target cells in a manner dependent on lactose but not mannose, suggesting that this soluble galectin can be

* Glycobiology and Bioimaging Laboratory, Research Centre for Infectious Diseases, Faculty of Medicine, Laval University, Quebec, Canada Laboratory of Human Immuno-Retrovirology, Research Centre for Infectious Diseases, Faculty of Medicine, Laval University, Quebec, Canada

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considered as a host factor that influences HIV-1 pathogenesis. In this chapter, we describe methods used to investigate the potential role of the galectin family in HIV-1-mediated disease progression.

1. Overview 1.1. Human immunodeficiency virus Human immunodeficiency virus-1 (HIV-1) is a lentivirus that belongs to the retrovirus family and causes acquired immunodeficiency syndrome (AIDS). Over 100 million people have been infected with this retrovirus and more than 25 million people have already died of AIDS. The impact of HIV-1 infections has been particularly devastating in the developing world. In some countries, as high as 25% of the adult population is reported to be infected with HIV-1, eliminating a large proportion of the working force that could support the development of financial and intellectual infrastructures in the near future (Greene, 2007). Since HIV-1 mainly infects cells of the immune system, namely CD4þ T lymphocytes and macrophages, and cripples the adaptive immune system, the impact of HIV-1 infection on emergence or spread of other infectious diseases is enormous (Weiss, 2001). First, HIV-1 infected individuals in poor settings, where access to medication is difficult, rapidly develop AIDS and thus become especially sensitive to infection by pathogens, opportunistic or not, present in the environment. Secondly, the ensuing immunocompromised status leads to high loads of coinfecting pathogens, including tuberculosis, a phenomenon increasing the chances of transmission to immunocompetent hosts. Thirdly, immunosuppression of millions of people in a population can also reduce the effectiveness of immunization campaigns and even make hazardous the use of live ‘‘attenuated’’ vaccines. When imagining the worst possible scenario, one can hypothesize that bacteria, fungi, and protozoa in the environment or zoonotic pathogens now have 30 million immunocompromised people in which to learn and adapt as human pathogens. Thus, the control of the HIV/AIDS pandemics is urgent and a serious matter not only for the most afflicted countries, but also for the industrialized countries to reduce the risk of pandemics by other emerging infectious agents. Despite the relentless progression toward AIDS in HIV-1 infected individuals, it is also known that HIV-1 is a relatively inefficient virus in regards to its potential of transmission and its overall infectivity. Epidemiological studies suggest that the possibility of transmission from a sexual intercourse ranges from 1 in 2000 to 1 in 200 depending on the type of sexual intercourse and the viral load of the infected individual. Simple preventative measures such as condom usage further reduce drastically the risk of HIV-1 transmission but cultural reasons hinder its acceptance in

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many countries. Education about HIV/AIDS prevention and use of efficient physical or chemical barriers to sexual transmission of HIV-1 are certainly the most practical approaches to control the spread of the epidemic and to address the cost and availability issues associated with antiretroviral therapy in infected individuals. Very recent works indicate that at the port of entry in mucosal membranes, a very small proportion of HIV-1 reaches the lamina propria where resident dendritic cells can interact with the virus and transmit it in cis or in trans to a small number of ‘‘resting yet previously activated’’ effector memory CD4þ T lymphocytes, establishing a founder HIV-1þ cell population (Haase, 2005; Hel et al., 2006; Mehandru et al., 2004). Once this founder population initiates viral replication, a significant number of CD4þ memory T lymphocytes found in the gut-associated lymphoid tissue are destroyed within 4–10 days. By 3 weeks postinfection, close to 80% of CD4þ T lymphocytes are depleted and the virus has already spread to the whole organism while entering the chronic stage of the infection (Brenchley et al., 2004; Li et al., 2005b; Mattapallil et al., 2005; Mehandru et al., 2004). Since viral spread and memory CD4þ T lymphocytes depletion arise from infection of such a limited number of susceptible cells initially, this port of entry also represents a major vulnerability for HIV-1. Thus, information related to the very early stage of HIV-1 infection could provide a possible means of prevention at this bottleneck of viral transmission. To infect CD4þ cells, HIV-1 must first stably attach to its target cells. This is mediated by the interaction between the viral envelope gp120 and the host integral membrane protein CD4, leading to a conformational change of gp120, which allows its interaction with a chemokine receptor, most notably CCR5 or CXCR4. This complex formation is crucial for viral entry through membrane fusion, which is initiated by insertion of the viral transmembrane glycoprotein gp41 into the target cell membrane (Chan and Kim, 1998; Gallo et al., 2003). Importantly, however, the avidity of oligomeric gp120 for CD4 appears to be much lower than originally expected (Fouts et al., 1997; Moore and Sweet, 1993; Moore et al., 1992; Sattentau and Moore, 1995). It is suggested that the equilibrium binding at 37  C is only achieved after 1–2 h (Moore and Sweet, 1993). The very low avidity and the slow binding kinetics displayed by oligomeric gp120 suggest that the gp120– CD4 interaction alone could not be sufficient to initiate fusion, especially in cells expressing low surface levels of CD4 (Ugolini et al., 1999). In addition, HIV-1 carries less than 30 envelope spikes on its surface (Chan and Kim, 1998; Gallo et al., 2003), which is in contrasts to the highly infectious influenza virus, which surface contains about 350 viral spikes (Karlsson Hedestam et al., 2008). Indeed, even under optimal in vitro condition, less than 0.5% of HIV-1 associates with target cells, suggesting that the initial interaction occurs under suboptimal conditions (Bobardt et al., 2003; Cantin et al., 2005; Mondor et al., 1998; Tremblay et al., 1998; Ugolini et al., 1999).

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Thus, viral attachment represents a rate-limiting step for virus entry and requires additional reinforcement especially at the beginning of HIV-1 pathogenesis (Mondor et al., 1998; Ugolini et al., 1999). Thus, attention has been drawn to identify other virus–cell interactions that could prompt efficient attachment and entry in vivo since disruption of such host factormediated interactions in the early stage of infection could greatly reduce the initial infection of susceptible cells and further limit the expansion of HIV-1 at the mucosal site (Bobardt et al., 2003; Cantin et al., 2005; Mondor et al., 1998; Tremblay et al., 1998; Ugolini et al., 1999). To date, information concerning those host factors has been relatively limited. At least two types of host membrane proteins, that is, b-integrins and C-type lectins, have been shown to facilitate viral attachment (Bobardt et al., 2003; Cantin et al., 2005; Mondor et al., 1998; Saphire et al., 2001; Tremblay et al., 1998; Ugolini et al., 1999). For example, it has been shown that CD4þ T lymphocytes express a b-integrin, LFA-1, that promotes HIV-1 attachment by binding to ICAM-1, a host molecule acquired by HIV-1 into its envelope during the budding process (Cantin et al., 1997; Fortin et al., 1997; Hildreth and Orentas, 1989; Tremblay et al., 1998). Dendritic cells, which can transmit virus to permissive cells, express C-type lectins such as DC-SIGN, dendritic cell immunoreceptor (DCIR), CD207, and CD206. Those membrane C-type lectins capture many pathogens, including HIV-1, by binding to surface protein oligomannose glycans (Feinberg et al., 2001; Geijtenbeek et al., 2000; Kwon et al., 2002; Lambert et al., 2008; Lin et al., 2003; McDonald et al., 2003; Turville et al., 2003). In the case of HIV-1, this can result in dissemination of the virus to permissive cells (Pope and Haase, 2003). In addition to these molecules, we recently found that at least one member (galectin-1) of another type of lectin family, galectin, which has affinity for b-galactoside, can also contribute to HIV-1 binding to CD4þ T cells and macrophages.

1.2. Galectins 1.2.1. Structures of galectins Galectin-1 belongs to the galectin family, which is defined by conserved peptide sequence elements in the carbohydrate recognition domain (CRD), consisting of 130 amino acid (Barondes et al., 1994a). Up to 14 galectins (galectin-1–14) have been found in mammals so far, as well as in many other phyla including birds, amphibians, fish, nematodes, drosophila, sponges, and fungi (Leffler, 2002). While all galectins share a core sequence in their CRD, galectins exhibit interesting structural differences in the presentation of their CRD (Hirabayashi and Kasai, 1993). Some galectins contain one CRD (prototype), and exist as monomers (galectin-5, -7, -10) or dimers (galectin-1, -2, -11, -13, -14) while other galectins, such as galectin-4, -6, 8, -9, -12 contain two CRD connected by a short linker region (tandem

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repeat) (Hirabayashi and Kasai, 1993). In contrast, galectin-3 uniquely occurs as a chimeric protein with one CRD and an additional non-CRD domain, which is involved in its oligomerization. Upon binding to its glycan ligands at the cell surface, the conformation of galectin-3 appears to be altered, and galectin-3 oligomerizes by self-assembly of its N-terminal regulatory domain. This oligomerization results in the formation of galectin-3 molecules with multivalent CRDs (Ahmad et al., 2004; Nieminen et al., 2007). 1.2.2. Glycan-binding specificity The minimum binding unit recognized by galectins is the galactose residue (Gal), linked to an adjacent saccharide in the b configuration (called b-galactoside), such as lactosamine residues (Galb1-4N-acetlyglucosamine, Galb1-4GlcNAc; Barondes et al., 1994b; Rabinovich et al., 2002; Sato, 2002; Sato and Nieminen, 2004; Sparrow et al., 1987). While this type of glycans (i.e., b-galactoside-containing glycans) is often found in N-linked ‘‘complex-type’’ glycans attached to proteins (glycoproteins), each galectin binds to a relatively limited set of ligands. 1.2.3. Expression of galectin-1 Galectin-1 is expressed in thymus and lymphoid parenchymal epithelial cells, lamina muscularis mucosae, endothelial cells, trophoblasts, activated T lymphocytes, macrophages, activated B cells, and follicular DCs (Baum et al., 1995; Blaser et al., 1998; Dettin et al., 2003; Jeschke et al., 2004; Rabinovich et al., 1996; Stillman et al., 2006; Zuniga et al., 2001). Similar to human tonsil lymphoid tissue, gut-associated lymphoid tissues could contain as high as 20 mM of galectin-1 (Ouellet et al., 2005). 1.2.4. Release/secretion of galectins With the unique exception of galectins, all the other mammalian lectins are synthesized in the lumen of the endoplasmic reticulum and Golgi apparutus and delivered to the extracellular space through the classical secretory pathway. In contrast, all galectins are synthesized as cytosolic proteins, even though their glycan ligands are found in the extracellular space (Hughes, 1999). The biological significance of this localization remains speculative (Sato, 2002; Sato and Nieminen, 2004; Sato et al., 2009; Vasta, 2009). It has, however, been clearly established that extracellular release of galectins is a highly regulated process that involves active secretion through an ‘‘alternative’’ secretory pathway, which is also used by fibroblast growth factors and IL-1 (Bianchi, 2007; Cooper et al., 1991; Hughes, 1999; Nickel, 2003, 2005; Oppenheim et al., 2007; Sato and Hughes, 1994; Sato et al., 1993). Although the alternative secretory pathway is still poorly understood, an original report on the observation that differentiated myoblasts can secrete massive amounts of galectin-1 suggests that (rather than direct transport through a hypothetical transporter) galectin-1 is first

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accumulated below the plasma membrane, and is then exported by a mechanism involving ectocytosis (Cooper and Barondes, 1990). This has been confirmed by other reports on other galectins (Hughes, 1999; Nickel, 2003; Sato et al., 1993). The exported galectins in vesicles are then passively released to the extracellular space (Hughes, 1999; Mehul and Hughes, 1997). Galectin-1 is secreted by activated B cells, activated (but not quiescent) T lymphocytes, activated macrophages, and certain epithelial cells (Baum et al., 1995; Blaser et al., 1998; Rabinovich et al., 1998; Zuniga et al., 2001). In addition, galectin-1 can be passively released from necrotic cells (Sato, 2002; Sato and Nieminen, 2004; Sato et al., 2009).

2. Experimental Our previous reports suggest that galectin-1, but not galectin-3, drastically increases the kinetics of HIV-1 binding to CD4þ cells (T lymphocytes and macrophages; Mercier et al., 2008; Ouellet et al., 2005). This enhancement in binding thus greatly facilitates infection of susceptible cells and leads to a more robust viral replication. In the following sections, the various methods that were used to investigate the role of galectins in HIV-1 infection and new set of data related to the activity of galectin-1 in HIV-1 infection, will be described.

2.1. General precautions for HIV-1-related studies (biosafety level for HIV-1 research; Byers et al., 2004) Selection of an appropriate biosafety level for work with a particular pathogen depends on a number of factors, such as the virulence, the pathogenicity, the route of spread, manipulations involving the agent and the availability of effective therapeutic measures. In the case of HIV-1, routine diagnostic work with clinical specimens to detect the presence of antibodies against HIV-1 can be performed in a biosafety level 2 (BSL2) facility using practices and procedures relevant to this biosafety level (Jackson and Balfour, 1988). However, the majority of experimental procedures involved in fundamental research of HIV-1, including infection of susceptible cells, replication and harvest of the virus, or the use of concentrated viral preparations must be carried out using BSL3 practices and procedures. Further, medium- to largescale viral production and concentration of viral preparation by ultracentrifugation also require a BSL3 facility and use of BSL3 practices and procedures (Delenda et al., 2002). It is strongly recommended to strictly follow the laboratory safety and biosecurity measures recommended by both your country and institution. Some information can be found on web sites such as http://www.cdc.gov and http://www.phac-aspc.gc.ca.

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2.2. HIV-1 production Human embryonic kidney (HEK) 293T cells are normally used to obtain highly concentrated and infectious HIV-1 preparations, as this cell line consistently produces higher virus yields compared to methods using primary CD4þ cells. However, when compared to primary target cells such as CD4þ lymphocytes and macrophages (Brockhausen, 1999; Garcia et al., 1991), it is possible that established cancerous cell lines introduce different glycosylation patterns on both the viral envelope glycoproteins and the host glycoproteins that are being acquired by the virus. Thus, it is recommended to verify that any effects observed by a host lectin on HIV-1 infection are consistently found regardless of producing cells, or at least by the most physiological cellular targets. In the case of galectin-1 and -3, our previous works confirmed that galectin-1, but not galectin-3, enhances both HIV-1 attachment and infection, regardless of the virus producer cells (Mercier et al., 2008; Ouellet et al., 2005). The method to purify or maintain cells will be described in Section 2.3. 2.2.1. HIV-1 production in HEK 293 T cells HEK cells are seeded at 2  106 cells in a ventilated 75 cm2 cell culture flask (T-75) for 16 h prior to a transient transfection with infectious HIV-1 molecular clones, such as pNL4-3 (X4-tropic; Adachi et al., 1986) or pNL4-3 Bal env (R5-tropic; Cantin et al., 1996; Dornadula et al., 1999). For the preparation of this calcium phosphate transfection, all solutions should be at room temperature. The plasmid DNA (30 mg) is suspended in 500 ml of 0.25 M calcium chloride in sterile distilled water. This DNA solution is slowly added dropwise into 500 ml of 2 HEPES-buffered saline (280 mM NaCl supplement with 10 mM KCl, 1.5 mM Na2HPO4, 12 mM dextrose, 50 mM HEPES (pH 7.05–7.12)) under continuous mild agitation. Once these reagents are mixed, they should be incubated for 20 min at RT (milky cloudiness should appear during the incubation). The culture medium of HEK cells in the flask is replaced with 9 ml of fresh DMEM supplemented with 10% FBS, and the premixed transfection reagent is slowly added to cells. The flask is moved gently back and forth to distribute the transfection solution evenly. Rotation movements should be avoided, as this would increase the possibility of accumulation of the DNA–calcium phosphate precipitate in the center of the flask, which would affect the yield of virus production. After 4–16 h at 37  C, the medium is removed and cells are washed gently with PBS and then 10 ml of fresh DMEM–10% FBS is added. Two days posttransfection, virus-containing cell-free medium is collected, filtered through a 0.22-mm sterile syringe filter, and aliquoted to prepare frozen stocks at 80  C. If necessary, this viral preparation can be further concentrated and/or purified (see the sections

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below). Quality control of prepared virus is routinely carried by measuring both p24 capsid protein and infectivity (see below for detail). 2.2.2. HIV-1 production in primary cells such as peripheral blood mononuclear cells, CD4þ T lymphocytes and monocytederived macrophages In most cases, it is important to confirm observations made using cell linederived viruses with a viral preparation produced by human primary target cells. In the case of HIV-1, however, it is important to mention that the expected yield in primary CD4þ cells such as CD4þ T lymphocytes and macrophages is often several orders of magnitude less than with HEK cells. For CD4þ T lymphocytes, cells (1–2  106 cells/ml) are first activated with 1 mg/ml of phytohemagglutinin-L (PHA-L, Sigma-Aldrich, Cat. # L4144) and 50 U/ml of recombinant human IL-2 (NIH AIDS Research & Reference Reagent Program) for 3 days in RPMI1640 supplemented with 10% FBS. Cells are then infected with HIV-1 (NL4-3, X4-tropic virus) at a concentration of 10 ng of p24 equivalent per 1  105 cells for 4–10 days. Virus-containing cell-free medium is then collected at 4, 7, and 10 days postinfection, and replaced by fresh culture medium to continue viral harvest. Supernatants can be concentrated by ultracentrifugation and further purified by OptiprepTM gradient (see below for the methods). For macrophages (monocyte-derived macrophages, MDMs), cells are plated at a density of 5–10  106 cells per T-75 flask and infected with HIV-1 (NL4-3 Bal env) at a dose of 20 ng of p24 per 1  105 cells for 2–4 h at 37  C. After this short exposure, MDMs are washed two times with PBS, followed by a brief wash with RPMI1640–5% FBS. Then, MDMs are incubated with RPMI1640–5% FBS for the production of virus. Half of the cell-free medium is collected at day 7, 14, 21, and 28 postinfection and replaced with fresh medium. Peak of virus production (based on p24 levels in the medium) is between 10 and 21 days postinfection. From this cell-free supernatant, virus is further purified or concentrated as described below. 2.2.3. p24 ELISA Our previous observations indicate that the majority of the p24 capsid protein (>90%) is associated with HIV-1 particles (Fortin et al., 1997). Thus, virus stocks are normalized using p24 levels as estimated by an inhouse p24 test. In brief, an ELISA 96-well plate (high binding, Immuno Plate MaxiSorp, NUNC) is first coated with a monoclonal anti-p24 antibody (183-H12-5C, 2.5 mg/ml, 100 ml/well, NIH AIDS Research & Reference Reagent Program) either overnight at 4  C or for 1 h at 37  C. After three washes with 300 ml PBST (PBS with 0.05% Tween20), free surface sites are blocked by incubation with 200 ml of 1% bovine serum albumin (BSA) in PBST at RT for 30 min. Following another round of three washes, viral preparations are added to the wells (100 ml/well) at

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various dilutions in 1% BSA-PBST. Recombinant purified p25gag/SF2 (31.25–2000 pg/ml, NIH AIDS Research & Reference Reagent Program) is used as a standard. To lyse viral particles, 25 ml/well of disruption buffer (2.5% Triton X-100, 0.05% Tween-20, thimerosal 0.02% in PBS) is added. After incubation for 1 h at RT followed by three washes, 100 ml/well of 0.5 mg/ml biotinylated anti-p24 antibody (clone 31-90-25, ATCC #HB9725) is added and incubation is carried out for 1 h at RT. After removing unbound antibody by three washes, streptavidin-conjugated horseradish peroxidase (100 ml/well, 66 ng/ml, HRP40-streptavidin, Fitzgerald industries international) is added and incubated at RT for 20 min. Following the last series of wash, the wells are incubated for 10 min with a peroxidase substrate, TMB-S (100 ml/well, Fitzgerald industries international), and the reaction is terminated by adding 50 ml of 1 M H3PO4. The absorbance is measured using a microplate reader at 450 nm with a reference at 630 nm and p24 values are estimated based on regression analysis of p24 standards over a linear range (Bounou et al., 2002). 2.2.4. Concentration of HIV-1 preparation by ultracentrifuge The cell-free supernatant containing HIV-1 can be further concentrated to obtain higher titer viral preparations by ultracentrifugation at 58,000g (28,000 rpm with Beckman 70Ti rotor) for 45 min at 4  C without brake. As the virus pellet is not visible at the end of the run, it is recommended to mark the expected pellet position. Discard supernatant by pouring it off in one continuous motion, being careful not to shake or blot off any drop that may remain hanging. The virus pellet is then thoroughly resuspended with 1 ml of PBS (the virus pellet can be located both at the bottom and on the side of the tube). The concentration of virus is then reevaluated with a p24 ELISA. 2.2.5. Purification of HIV-1 with OptiprepTM For highly purified HIV-1, an OptiprepTM gradient is used to separate virus particles from cell-released exosomes. OptiprepTM is a 60% (w/v) iodixanol solution in water (Axis-shield Cat. # 1030061) with a density of 1.32 g/ml. The gradient is composed of 11 fractions (900 ml each) of iodixanol in PBS with concentrations starting from 6% to 18% with 1.2% increments, that is, 6.0%, 7.2%, 8.4%, 9.6%, 10.8%, 12.0%, 13.2%, 14.4%, 15.6%, 16.8%, 18.0%. Under a laminar flow hood, stock solutions of these fractions are prepared and can be kept at 4  C. Using a serological 1 ml pipette, 18% iodixanol–PBS is placed at the bottom of an OptisealTM tube (Beckman Cat. # 362181). Then, the tube is tilted at 45 and 900 ml of the 16.8% solution is slowly but continuously deposited onto the 18% iodixanol layer. The end of the pipette should be maintained in a position so it barely touches the 18% layer. After the addition of a new layer, the interface of the layers becomes visible for a short period of time. The position of each

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layer should be marked to facilitate the collection of a desired fraction after centrifugation. Nine more layers are placed in a similar manner to obtain a 6– 18% gradient of iodixanol. Four hundred microliters of virus suspension, which was concentrated by ultracentrifugation, is slowly applied to the top of the iodixanol gradient (6%). Aluminum caps are inserted and firmly closed on OptisealTM tubes to seal them in the NVT65 rotor and caps are then screwed over the filled positions of the rotor by applying pressure between 80 and 100 psi using the provided tool (Allen key with a dynamometer). The gradient is ultracentrifuged for 1 h 15 min at 219,000g (52,000 rpm) at 4  C with slow acceleration and deceleration. With a 1-ml pipette, each gradient fraction is gently removed. HIV-1 is normally found between fractions 14.4% and 16.8%. Pooled fractions (3 ml) are diluted 10-fold with PBS followed by ultracentrifugation at 58,000g as above to obtain purified HIV-1. Quality control of prepared virus should be carried out following each purification using both the p24 ELISA described above and infectivity assays with reporter cell lines described below. 2.2.6. AT-2 inactivation of HIV-1 For some studies that do not require infectious HIV-1 virus particles, such as virion structure studies, a treatment with 2-aldrithiol (2,20 -dithiodipyridine or AT-2, Sigma-Aldrich, Cat. # D5767) can be used to inactivate HIV-1, thereby giving the possibility to perform experiments in a BSL2 facility using BSL2 practices and procedures. AT-2 covalently modifies the essential zinc fingers in the viral nucleocapsid proteins, reverse transcriptase and integrase, thereby inactivating HIV-1 while preserving the integrity of viral envelope proteins (Chertova et al., 2003; Rossio et al., 1998). AT2 powder should be kept at 4  C and protected from moisture and the stock solution (100 mM in methanol) should be prepared under a chemical hood (this solution can be stored at 20  C for 1 month). After filtration of the viral solution using a 0.22-mm syringe filter, the AT-2 stock solution (10 ml/ 1 ml of virus solution, final concentration 1 mM) is added and incubated overnight at 4  C and protected from light. Virus particles are then purified by ultracentrifugation at 58,000g (Beckman rotor type 70Ti at 28,000 rpm) at 4  C for 45 min without brake. After carefully removing the supernatant, the virus pellet is resuspended in PBS and the virus concentration is estimated by monitoring the p24 content. It is important to remove the AT-2-containing supernatant as much as possible since concentrations of more than 8 mM have been observed to induce apoptosis of peripheral blood mononuclear cells (PBMCs) after 24 h of treatment. Lack of infectivity of each viral preparation treated with AT-2 has to be confirmed if the experiments with AT-2-treated HIV-1 are to be performed outside a BSL3 facility using a sensitive luciferase-based HIV-1 infection assay involving reporter cell lines, such as LuSIV or TZMBL cells. Typically, concentrations of HIV-1 between 1 and 100 ng of p24

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per 1  105 cells are used to evaluate inactivation. Luciferase activity is checked after 24–48 h to be sure that viral particles do not carry any residual infectivity. It is only after this verification that the viral preparations can be used outside a BSL3 facility.

2.3. Cell lines and primary cells isolation for HIV-1 infection 2.3.1. LuSIV The LuSIV cell line (NIH AIDS Research & Reference Reagent Program) is derived from the CEMx174 cell line, which stably expresses a firefly luciferase reporter gene driven by the SIVmac 239 long terminal repeat (LTR) region. This cell line is highly sensitive to infection by primary and laboratory T-cell-tropic strains of HIV-1 and SIV, resulting in Tatmediated expression of luciferase correlating with viral infectivity. The LuSIV system is a powerful tool to analyze HIV/SIV infectivity, providing a unique assay system that can detect virus replication as soon as 24 h, due to the initial presence and early expression of Tat in infected cells (Wu, 2004). Since primate lentiviruses, such as HIV-1 and SIV, require an average of 24 h to complete one cycle of replication, LuSIV enables measurements after a single round of infection (Roos et al., 2000). Cells are cultured in RPMI1640 supplemented with 10% FBS, 300 mg/ml of hygromycin B at a cellular concentration between 2.5  105 and 2  106 cells/ml. Hygromycin B is used to maintain cells containing the episomal reporter plasmid (Yates et al., 1985). 2.3.2. TZM-bl The TZM-bl cell line (NIH AIDS Research & Reference Reagent Program), also called JC53BL-13, is a CXCR4 positive HeLa cell clone that is engineered to express both CD4 and CCR5 (Platt et al., 1998). These cells have been further modified to contain integrated reporter genes for firefly luciferase and b-galactosidase under the control of the HIV LTR sequence (Wei et al., 2002). TZM-bl cells are permissive to infection by a wide variety of HIV-1, SIV, and SHIV strains, including primary HIV isolates and molecularly cloned Env-pseudotyped viruses. These adherent cells are grown in DMEM supplemented with 10% FBS. For the passage of this cell line, a 0.25% trypsin solution is used (Li et al., 2005a). 2.3.3. Peripheral blood mononuclear cells PBMCs are purified from blood of healthy donors by Ficoll–Hypaque centrifugation. Fresh blood is collected and treated with anticoagulants, heparin or citrate depending on the system used to collect blood. Fresh blood (25 ml) is carefully applied over 20 ml of Ficoll–Hypaque (GE Healthcare Cat. # 17-1440-02) in a 50-ml conical tube and then centrifuged at 400g for 30 min at RT without brake. After centrifugation,

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the upper layer, containing diluted autologous plasma, is removed by aspiration to leave the interphase layer, rich in mononuclear cells, undisturbed. The cells at the interphase are then carefully transferred to a new conical tube, which is filled with Hank’s balanced salt solution (HBSS), and cells are centrifuged at 300g for 10 min at RT. Supernatant is removed and two additional washing steps are carried out by resuspending cells with 50 ml of HBSS and centrifugation at 200g for 10 min at RT. Cells are resuspended at 1  106 cells/ml in RPMI1640 with 10% FBS. PBMCs can be used immediately or activated (see below for conditions). 2.3.4. CD4þ T lymphocytes CD4þ T lymphocytes are purified from PBMCs using the human CD4þ T lymphocytes enrichment kit (Stemcell Technologies Inc., EasySep CD4þ T cell enrichment kit Cat. # 19052). Following the manufacturer’s protocol, PBMCs are resuspended at a concentration of 5  107 cells/ml in PBS supplemented with 2% FBS and placed in a 5-ml polystyrene tube. The enrichment cocktail (50 ml/ml) is added, mixed, and incubated at RT for 10 min. EasySep D magnetic particles are vortexed for at least 30 s to prevent aggregation, and then the particles (100 ml/ml) are added to resuspended PBMCs, mixed and incubated at RT for 5 min. Then, PBS supplemented with 2% FBS is added to the cell suspension to obtain a total volume of 2.5 ml. The entire suspension is mixed gently and placed in a magnet stand, without cap, for 5 min. The cell suspension is then poured into a new tube by inversion of the magnet stand using a continuous motion. The CD4þ enriched cells can be used immediately or activated. 2.3.5. Monocyte-derived macrophages PBMCs (1.25  108 cells per T-75 flask) are plated and incubated at 37  C for 2 h. Nonadherent cells are then removed and adherent monocytes are washed twice with RPMI 1640–5% FBS and further cultured for a week in medium supplemented with 100 ng/ml macrophage colony-stimulating factor (M-CSF; GenScript Corporation) for differentiation into MDMs. MDMs are then removed from the flask by briefly incubating with AccutaseÒ (Sigma-Aldrich, Cat. # A6964) followed by gentle scraping with a cell scraper. Cells are replated in a 48-well plate at 5  104 cells/ well for infection studies (Mercier et al., 2008). 2.3.6. Cell activation It is known that resting CD4þ T lymphocytes are only weakly permissive to productive HIV-1 infection due to host factors affecting different steps of the virus life cycle (Tremblay et al., 1998). Thus, to maximize infection, CD4þ T lymphocytes are activated with 1 mg/ml of PHA-L and 50 U/ml of recombinant human IL-2 (Scott and Nahm, 1984). Alternatively, PBMCs or CD4þ T lymphocytes can be activated through the T cell

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receptor with the antibody OKT3 (1 mg/ml, ATCC) and a costimulation signal with an anti-CD28 antibody (clone 9.3 at 1 mg/ml) provided by J.A. Ledbetter (Bristol-Myers Squibb Pharmaceutical Research Institute), followed by cross-linking with goat anti-mouse IgG 5 mg/ml (Tardif and Tremblay, 2005).

2.4. Purification of galectin-1 and -3 To get consistent data obtained from experiments with galectins, it is essential to be fully aware that galectins are not necessarily stable (Hirabayashi and Kasai, 1991; Hsu et al., 1992) (unpublished observations in the laboratory of SS). Thus, constant monitoring of the quality of purified galectins is extremely important. 2.4.1. Purification Human recombinant galectins are produced using BL21 Escherichia coli transformed with an expression plasmid containing the galectin gene as previously published (Mercier et al., 2008; Nieminen et al., 2005; Ouellet et al., 2005; Pelletier and Sato, 2002; Pelletier et al., 2003; Sato et al., 2002). E. coli are grown at 37  C in LB culture medium with 100 mg/ml of ampicillin until optical density reaches 0.7 at 600 nm. Once in their exponential growth phase, bacteria are incubated with IPTG (1 mM) for 3 h at 37  C. Then, bacteria are collected by centrifugation at 4  C. The following steps are done on ice or at 4  C and all buffers used for the preparation are required to be ice-cold. The bacteria pellet is resuspended with lysis buffer (22 mM Tris–HCl (pH 7.5)) supplemented with 5 mM EDTA, 1 mM DTT and a protease inhibitor cocktail (Sigma-Aldrich Cat. # P8465). We generally use at least 15 ml of lysis buffer to suspend bacteria derived from a 1 l culture to ensure sufficient disruption of membranes. The suspension is subjected to sonication in an ice-bath at 120 W for 30 s every 2 min eight times to disrupt bacterial membranes. Cell lysates are then centrifuged at 84,000g (Beckman rotor 70.1Ti, 35,000 rpm) for 30 min at 4  C to obtain a soluble fraction. Since galectins have high affinity for bgalactoside, lactosyl-agarose, or asialofetuin-agarose is used for purification of galectins. The soluble fraction is applied onto 2 ml of lactosyl-agarose column (Sigma-Aldrich Cat. # L7634), which is equilibrated with 50 mM Tris–HCl (pH 7.2) supplemented with 105 mM NaCl (TBS). A 25 column volumes wash with TBS is then carried out to remove unbound materials as well as EDTA and DTT in the lysis buffer. Since any trace of reducing agents such as DTT (as low as 1 mM) inhibits HIV-1 infection assays (unpublished data, CSP, SS) (Koken et al., 1994), it is important to completely remove DTT during purification. Active galectins are eluted from lactosyl-agarose with TBS supplemented with 150 mM lactose. The galectin-containing fractions (1 ml per fraction) are monitored by the

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presence of protein with the Bradford reaction. Typically, the first 5–7 fractions (5–7 ml) contain galectin. Eluted galectin fractions are pooled and either ultrafiltration or gel filtration is used to remove lactose from the galectin preparation and to replace TBS with PBS. For galectin-3, we typically use gel filtration with a Hiprep 26/10 desalting column (53 ml column volume, GE Healthcare), which is equilibrated with PBS. Galectincontaining fractions (maximum volume to apply for this column is 15 ml) are applied and the fractions (1 ml/fraction) are collected. The presence of galectin is tracked by Bradford. Typically, the 15th to 30th fractions ( 15 ml) contain galectin. To avoid dilution of purified lectin, only the peak fractions (5–7 ml, corresponding to 80% of applied galectin) are pooled. For galectin-1, ultrafiltration is employed because it gives better yield and higher quality galectin than the gel filtration method. Galectin-1containing fractions are placed in a dialysis tube (Spectrum, MWCO 68000) and extensively dialyzed against PBS at 4  C for at least four rounds (1:500/dialysis, which theoretically reduces the lactose concentration from 150 mM to less than 2 pM). All purified galectins are then passed through Acticlean ETOX endotoxin-removing gels following manufacturer recommendations (Sterogene, Carlsbad, CA). Briefly, 1 ml of Acticlean ETOX is placed in a poly-prep chromatography column (Bio-Rad, #731-1550). Any other type of column can be used as long as the flow rate is well controlled to enable maximal interaction of samples with Acticlean. Endotoxin-binding capacity of this matrix is 20,000 EU/ml. Prior to use, Acticlean column must first be resuspended with 5 ml of 1 M NaOH and incubated overnight at 4  C. The column is washed extensively with endotoxin-free sterile water until a neutral pH is reached in the eluate. After extensive washes with PBS, galectin solution is applied slowly, and the flowthrough fraction is collected. When necessary, this step is repeated. Galectin preparations are then sterilized by filtration through a 0.22 mm filter and kept a 4  C until use. For both galectin-1 and -3, we found that freeze-thaw cycles significantly reduce the quality (specific lectin activity per protein, see the section on quality control for details). Mock preparations are also prepared when necessary using E. coli that does not express galectins by using the same purification protocol (Mercier et al., 2008; Nieminen et al., 2005). In our laboratory, these purification protocols yield 2–5 mg of galectin from every 3 l of bacteria culture medium.

2.5. Quality control of galectins 2.5.1. SDS-PAGE Several procedures are used to ensure the quality of purified galectins. Purified galectins are subject to SDS-PAGE with a 12% or 15% reducing gel to confirm that galectin-1 and -3 migrate at their respective position of 14 and 30 kDa, without any additional protein band.

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2.5.2. Endotoxin The endotoxin level is measured using Limulus amebocyte lysate (LAL) assay kit (Associates of Cape Cod Incorporated). Protein preparations that exceed 10 EU/mg of protein are rejected or passed through the Acticlean ETOX endotoxin-removing gel. 2.5.3. Lectin activity (specific activity per weight of protein) Specific lectin activity per protein concentration is then evaluated using a hemagglutination assay. To prepare the suspension of red blood cells (RBCs) for hemagglutination assay, heparine-treated human peripheral blood (10 ml) is first centrifuged at 2000g for 5 min. Buffy coat is removed as much as possible and RBCs are washed three times with 50 ml PBS. Then RBCs are diluted with PBS to obtain 100 ml of an 8% cell suspension. RBC suspension is treated with glutaraldehyde (at a final concentration of 3%) under rotation for 1 h at RT, followed by washing with 0.0025% NaN3 in PBS. Fixed RBCs are resuspended at 3–4% in PBS–NaN3. Calibration of RBCs is required to obtain the appropriate concentration for lectinmediated hemagglutination. Series of RBCs dilutions (2–20-fold) are distributed into a U-shape 96-well plate (100 ml/well) and incubated at 37  C for 30 min. When RBCs are aggregated (hemagglutination), they are spread out like a sheet covering the entire surface of the well. In contrast, RBCs form very tight button-like precipitation at the bottom of the well when there is no aggregation. For lectin hemagglutination, we then use the minimum concentration of RBCs that results in this very tight button-like precipitation. RBC suspension can be kept at 4  C for more than 3 months, although a routine check of the quality of RBCs is necessary. Serial dilutions of galectins (from 10 to 0 mM) are mixed with the appropriate quantity of RBCs in the wells of a U-shape 96-well plate and incubated at 37  C for 30 min. Galectin-1 and -3 induce hemagglutination at concentrations of around 15 and 10 mg/ml, respectively (Butler, 1963; Giguere et al., 2006).

2.6. S-Carboxyamidomethylation of galectin-1 Among members of the galectin family, galectin-1 is a unique lectin that is sensitive to oxidation due to a cysteine residue proximal to its dimerization site (Cho and Cummings, 1996). Oxidation leads to its inactivation as a lectin and therefore loss of its hemagglutinin activity. Thus, some groups have been using relatively high concentrations of thiol-reducing agents such as DTT to prevent its oxidative inactivation and to maintain carbohydratebinding activity upon purification and storage. However, treatment of live cells with DTT could seriously hamper cell viability and make interpretations about the effects of galectin-1 difficult as previously pointed out by the

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group of Cummings (Stowell et al., 2007). Indeed, as little as 5 mM of DTT was sufficient to halt virtually all natural migration and movement of T lymphocytes (time-lapse live cell imaging, unpublished observations by CSP and SS). Moreover, the presence of 1 mM of DTT inhibited HIV-1 infection (unpublished observations by CSP and SS). Thus, it is highly recommended to avoid any reducing agents in assays related to galectins. It also raises some concerns over data obtained using commercially available galectins, as some of those galectins (galectin-1 as well as galectin-3) contain high levels of 2-mercaptoethanol (a reducing agent) as preservative. Other commercial sources provide preservative-free lyophilized form of galectin-1, although the state of oxidation of these reagents remains unknown. As recent works suggest, the oxidated form of galectin-1 also possesses some biological functions but they are independent of its lectin-binding activity (Horie et al., 2004; Scott et al., 2009). Thus, cautions must be applied for the analysis of data obtained with commercially available galectin-1. Recently, Stowell and colleagues found that S-carboxyamidomethylation of galectin-1 by mild treatment with iodoacetamide makes galectin-1 resistant to atmospheric oxygen in absence of disulfide-reducing reagents and yet preserves some of its biological functions (Stowell et al., 2009). Our data (Fig. 13.1) also suggest that Scarboxyamidomethylated galectin-1 facilitates HIV-1 infection to similar levels than freshly purified galectin-1. Thus, in some cases, we have begun to use this method to stabilize galectin-1, although it is highly recommended that each laboratory verifies whether this treatment has an impact on their own assays. Carboxyamidomethylation of galectin-1 is achieved using the method published by Stowell et al. (2009). First, galectin-1 is resuspended in a 100 mM lactose–PBS solution to protect the CRD of the protein. Then, galectin-1 (2–5 mg/ml) is incubated overnight at 4  C with iodoacetamide at a final

RLU (luciferase activity)

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Figure 13.1 Carboxyamidoacetylated galectin-1 facilitates HIV-1 infection. LuSIV cells were infected with HIV-1 (NL4-3) for 24 h in different concentrations of either galectin-1 (Gal-1) or carboxyamidoacetylated galectin-1 (Gal-1IODO). Levels of infection were estimated by luciferase activity.

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concentration of 100 mM. Free iodoacetamide and lactose are extensively removed by series of dialysis (ultrafiltration) against PBS, followed by sterilization as described above. The quality of galectin-1 is tested by hemagglutination assay. The treated protein remains stable over prolonged period at 4  C (Stowell et al., 2008b; Whitney et al., 1986).

2.7. Inhibition of lectin activity of galectin in physiological condition 2.7.1. Sugar antagonists to inhibit galectin’s lectin activity Being a b-galactoside-binding protein, the lectin activity of galectins can be readily inhibited by relatively small b-galactoside-containing sugars, such as lactose (Galb1-4glucose) or other appropriate b-galactoside-containing oligosaccharides. While lactose is one of the most used inhibitors for galectins, affinity of galectins for lactose is often low and thereby high concentrations of lactose are necessary to inhibit their lectin activity. In our laboratory, 50–150 mM lactose is routinely used to ensure inhibition. The isotonicity of the medium has to be taken into account when such high concentrations of saccharide are used for cell assays as cells exposed to hypertonic solutions would become fragile to any successive treatment. To overcome this problem, the concentration of sodium chloride in PBS is reduced for the accommodation of high doses of lactose so that the lactose-containing PBS has the same osmolarity as saline (i.e., 317 mOsml/l). Recently, Krishnamoorthy and coworkers reanalyzed the NIH consortium glycan array data (http://www.functionalglycomics.org), which the group of Cummings originally produced (Stowell et al., 2008a), in regard to the affinities of galectin-1 for oligomannose glycans (Krishnamoorthy et al., 2009), suggesting that galectin-1 is potentially a mannose-binding lectin. This suggestion is not consistent with previously published data concerning the specificity of galectin-1. In addition, our published data and Fig. 13.2 also indicate that galectin-1 promotes HIV-1 binding to the target cells in a b-galactoside-dependent manner, and cannot be inhibited by mannose (Fig. 13.2). Since the previous reports consistently suggest that binding of galectin-1 requires an intact nonreducing pyranose ring of GlcNAc residue that is linked to Gal residue, the same set of glycan array data are reanalyzed based on the number of those nonreducing lactosamine residues in the glycans used for this array (Fig. 13.3). Indeed, galectin-1 shows a weak affinity specific for only one mannose-containing glycan (#197; Man6) among five different oligomannose type glycans (#192, 193, 194, 197, 198; Man5, 6, 7, 8, and 9). However, galectin-1 binding to glycans containing one or two nonreducing lactosamine residues exhibits a 6.2-fold increase compared to those without, suggesting the superior binding preference of galectin-1 for b-galactoside over a-mannoside. A modification of lactosamine with a2–6 sialylation completely abolishes its binding,

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6.0

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Figure 13.2 Galectin-1 promotes HIV-1 infection in b-galactoside-dependent manner. PBMC cells were infected with HIV-1 (NL4-3) for 3 days in the presence or absence of galectin-1 together with 50 mM lactose (galectin-1 antagonist) or with 50 mM mannose. HIV-1 infection was estimated by p24 levels in the cell-free medium.

consistent with the previous report of the group of Rabinovich (Toscano et al., 2007). This analysis, together with previously published data, confirms that galectin-1 is an almost exclusive b-galactoside-binding protein. 2.7.2. Dominant-negative form of galectins The possibility to use the dominant-negative form of galectin molecules, which binds to similar ligands but lack cross-linking activity, has been put forward. For galectin-1, a modified protein unable to dimerize could be created by performing a point mutation of the valine residue at the amino position 5 (galectin-1-V5D) which is crucial for dimerization (Cho and Cummings, 1996). However, our unpublished observations suggest that this monomeric galectin-1 binds to lactosyl-agarose at 4  C but not asialofetuin-agarose, which also contains b-galactoside (unpublished observation by J. Nieminen and SS). Furthermore, the monomeric form of galectin-1 could not inhibit dimeric galectin-1-induced hemagglutination (unpublished observation by JN and SS), suggesting that lack of avidity for b-galactoside prevents the dominant-negative galectin-1 from persistently binding to its ligand and from inhibiting binding by native galectin-1. For galectin-3, a potential dominant negative could be a truncated lectin that lacks the N-terminal repeating domain involved in its oligomerization. Truncated galectin-3 is easily prepared using collagenase VII (C0773; Sigma-Aldrich) (Nieminen et al., 2005, 2007). This truncated galectin-3 binds stably to the cell surface at 4  C. However, immediately after increasing the temperature of the cell culture medium to 37  C, the majority of truncated galectin-3 is released from the endothelial cell layer (Nieminen et al., 2007), suggesting that this form of truncated galectin-3 does not bind stably to the cell membrane at physiological temperatures. Indeed, the

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#143 Siaa2-3 biantennary complex type (2 lactosamine residues) #147 Galb1-4 GlcNAcb1-3Galb1-4GlcNAc-sp0 (1 lactosamine residues) #147 Galb1-4GlcNAc-sp0 (1 lactosamine residues) #153 Galb1-4GlcNAc-sp8 (0 lactosamine residues) #53 Siaa2-6 biantennary complex type (2 lactosamine residues) #192 Oligomannose type-Man7 (0 lactosamine residues) #193 Oligomannose type-Man8 (0 lactosamine residues) #194 Oligomannose type-Man9 (0 lactosamine residues) #197 Oligomannose type-Man6 (0 lactosamine residues) #198 Oligomannose type-Man5 (0 lactosamine residues)

Figure 13.3 Analysis of galectin-1 binding to glycan microarrays from the Consortium for Functional Glycomics (Stowell et al., 2008a). Levels of galectin-1 binding estimated by fluorescence intensity are plotted against the concentrations of galectin-1 applied to the microarray. The light gray area indicates the maximum level of galectin-1 binding to either with oligomannose glycans (a-mannoside) or glycans that lacks the nonreducing ring of pyranose of GlcNAc residue. The glycan-containing a2–6 sialylated lactosamine (#53) also exhibits very low affinity as previously published. Only the glycans containing nonreducing lactosamine residues (#143 and #147) were preferentially bound by galectin-1, confirming that galectin-1 is principally b-galactoside-binding protein.

truncated form of galectin-3 could not compete with full-length galectin-3 binding, also suggesting that this form may not be considered as a dominant negative in certain conditions when stable binding of galectin-3 is necessary for its function.

2.8. Virus attachment assays In physiological settings, binding kinetics of virion-associated gp120 to cellular CD4, the first step for virus infection, is known to be often slow. Further, in the early stages of HIV-1 infection, the majority of permissive

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cells express low levels of CD4 (Ugolini et al., 1999). Thus, it has been proposed that a number of distinct interactions with cellular proteins may influence and facilitate virus attachment (Arthur et al., 1992). Indeed, even in vitro, binding of HIV-1 particles to CD4þ cells is found to be <1% after 60 min of binding at 37  C. Our previous reports suggest that galectin-1 facilitates HIV-1 infection by increasing the kinetics of HIV-1 binding to its target cell (Mercier et al., 2008; Ouellet et al., 2005). To study whether a lectin could increase HIV-1 binding to target cells or not, HIV-1 permissive cells, such as LuSIV cells, primary PBMC, or primary CD4þ cells are incubated with HIV-1 virions in the presence or absence of a lectin. Several approaches can be used to estimate the levels of HIV-1 attached to the target cells; for LuSIV, luciferase activity is evaluated while direct measurement of cell-associated p24 can be performed when primary cells are used. 2.8.1. Indirect evaluation of HIV-1 binding (luciferase activity) Cells are first pretreated with various concentrations of galectins (0–4 mM) for 1 h at 4  C. In some cases, a galectin antagonist, lactose (50 mM), is included to verify specificity. Then HIV-1 (virus suspension containing 10 ng of p24) is added to the well with 1  105 target cells and binding assays are performed by incubating for various times (0–2 h) at 4  C. In some cases, higher temperatures such as RT or 37  C can be used, especially when the dynamics or the tertial structures of membrane proteins on the viral surfaces are a concern, although those temperatures also induce both viral– host membrane fusions and endocytosis, which may make the analysis of the impact of a lectin on binding difficult. Fusion inhibitors, such as enfuvirtide (T-20) (NIH AIDS Research & Reference Reagent Program, 100 ng/ml) can be included in such assays. After the incubations, cells are washed three times with PBS to remove both unbound lectin and virus. Cells are then resuspended in RPMI1640–10% FBS (100 ml/well) and cultured at 37  C for 24 h. The initial binding of HIV-1 to LuSIV followed by incubation at 37  C leads to membrane fusion, reverse transcription, integration, and production of the viral Tat protein. This viral transactivator binds to the TAR region of the integrated LTR located upstream of the luciferase gene, thereby increasing by 1000-fold the biosynthesis of the luciferase protein within 24 h. Since LuSIV cells are highly permissive to HIV-1 infection, the level of HIV-1 binding is directly correlated with the level of infection and therefore to the level of luciferase activity. To measure the activity of luciferase, cells in 100 ml/well are directly lysed by adding 25 ml of 5  luciferase lysis buffer (Tris–HCl, pH 7.8 supplemented with 10 mM DTT, 5% Triton X-100, and 50% glycerol) and incubated for 30 min under agitation. The cell lysate (20 ml/well) is added to a 96 wells luminometer plate and the plate is placed in a Dynex MLX microplate luminometer. The luminometer injects 100 ml/well of luciferase assay buffer (20 mM tricine

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supplemented with 1.07 mM (MgCO3)4 Mg(OH)2, 2.67 mM MgSO4, 0.1 mM EDTA, 220 mM coenzyme A, 4.7 mM D-luciferin potassium salt, 530 mM ATP, and 33.3 mM DTT). After a 2-s delay, the luciferase activity is monitored for 20 s per well (Ouellet et al., 1999). 2.8.2. Direct evaluation of HIV-1 binding (p24) When natural cellular reservoirs, PBMCs, CD4þ T lymphocytes, or MDMs as well as LuSIV cells, are used, HIV-1 binding to target cells is estimated by measuring cell-associated p24 with a p24 ELISA. Thus, a similar type of viral binding at 4  C is carried out as described above and then, after extensive washing to remove unbound virus particles, the cells lysate is prepared for p24 ELISA. For example, MDMs at 5  104 cells/well are plated in a 48-well plate and incubated with R5-tropic virus (the viral input is usually 10 ng of p24) with or without galectin-1 for 1 h. After having been washed, cells are lysed with 25 ml/well of disruption buffer (see Section 2.2.3) and p24 levels are measured (Ouellet et al., 2005).

2.9. HIV-1 infection assay For the function of a lectin in HIV-1 infection, it is recommended to initiate the study with a sensitive method like the LuSIV reporter cell system. This assay allows the quantitative evaluation of single-cycle infection events through activation of integrated LTR sequences driving the luciferase reporter gene following the production of the viral protein Tat by de novo viral infection. Once the lectin has been observed to influence HIV1 infection in this reporter cell system, it is recommended to verify whether these effects can be observed in other HIV-1 susceptible cells, as different glycosylation patterns expressed by different cells may have an influence on the role of the lectin. For galectin-1, we have observed that galectin-1, but not galectin-3, facilitates HIV-1 infection in LuSIV cells, PBMC, both activated and naı¨ve CD4þ T lymphocytes and macrophages (Mercier et al., 2008; Ouellet et al., 2005 and manuscript in preparation by CSP, MO, MJT, SS). We have also verified that this effect could be reproduced using HIV-1 produced in various cellular settings such as HEK cells, PBMCs, or macrophages. Thus, at least for galectin-1 activity in the context of HIV-1 infection, the involved glycan ligands appear to be similarly glycosylated, regardless of the cell type used for virus production and/or infectivity assays (manuscript in preparation by CSP, MO, MJT, SS). This further suggests that, in the case of the galectin-1 ligands involved in HIV-1 infection, the dominant glycosylation pattern of surface proteins could be mainly determined by their structure, rather than by the type of cells that produced the proteins.

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2.9.1. LuSIV cells Infectivity assays are performed in a similar manner to the HIV-1-binding assay described above with some modifications. LuSIV cells (1  105 cells/ well) in RPMI1640–10% FBS (100 ml/well) are infected with HIV-1 (virus suspension containing 10 ng of p24) with various concentrations of galectins (0–4 mM) at 37  C for 24 h to initiate a single round of viral replication. No washes are thus performed for infectivity assays. Lactose (50 mM) can be added to the cell culture medium (see 2.7.1 for some caution related to osmolarity) as a specificity control. To evaluate HIV-1 replication, cells are directly lysed with 20 ml of 5 luciferase lysis buffer, as above, and luciferase activity is monitored (Ouellet et al., 1999). 2.9.2. Primary cells For infection in primary cells, PBMCs, CD4þ T lymphocytes, and MDMs, the amount of HIV-1 capsid protein p24 in the culture medium is utilized to estimate viral replication as papers previously published by others and we have shown that the amount of p24 in the culture medium is closely correlated with the amount of HIV-1 produced by infected cells. Typically, for PBMCs or CD4þ T lymphocytes, 1  105 cells/well are exposed to HIV-1. The levels of p24 in the culture medium collected at day 3, 6, and 9 posttreatment with HIV-1 are measured by p24 ELISA as described above. This multiround of infection assay allows us to study the role of a lectin in a more physiological context, since it provides an opportunity to address not only the role in a single viral replication (LuSIV cell system) but also to examine whether a lectin is involved in later steps of viral replication or in further rounds of infection.

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C H A P T E R

F O U R T E E N

The Glycomics of Glycan Glucuronylation in Drosophila melanogaster Kazuhiro Aoki* and Michael Tiemeyer*,† Contents 1. Introduction 2. Experimental Procedures and Results 2.1. Extraction of proteins and lipids 2.2. Preparation of glycopeptides and release of N-linked glycans 2.3. Permethylation 2.4. Glycan analysis by NSI-MSn and automated data acquisition 2.5. Release of O-linked oligosaccharides by b-elimination 2.6. Isolation and fractionation of glycosphingolipids 2.7. Iatrobead chromatography for purification of total GSL 2.8. QAE Sephadex chromatography for neutral GSL 2.9. DEAE Sephadex chromatography for fractionation of acidic and zwitterionic GSLs 2.10. Analysis of intact GSLs by NSI-MSn data-dependent acquisition 3. Discussion Acknowledgments References

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Abstract As glycan characterization methods increase in sensitivity, new opportunities arise to undertake glycomic analyses on limiting amounts of material. Developing systems present special challenges since the amount of available tissue can restrict deep glycan characterization. We have optimized mass spectrometric methods with the goal of obtaining full glycan profiles from small amounts of tissue derived from organisms of particular interest. A major target of our efforts has been the Drosophila embryo, allowing us to leverage the tools already developed in this organism to meld glycomics, genomics, and molecular * Complex Carbohydrate Research Center, The University of Georgia, Athens, Georgia, USA Department of Biochemistry and Molecular Biology, The University of Georgia, Athens, Georgia, USA

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genetics. Our analysis of the N-linked, O-linked (non-GAG), and glycosphingolipid (GSL) glycans of the Drosophila embryo have identified expected and unexpected glycan structures. We have verified previous findings regarding the predominance of high-Man and pauci-Man N-linked glycans, but have also detected minor families of sialylated and glucuronylated N-linked structures. Glucuronic acid (GlcA) also presents itself as an abundant modification of O-linked and GSL glycans. We describe critical advancements in our methodology and present the broad range of contexts in which GlcA is found in the Drosophila embryo.

1. Introduction Advances in somatic cell genetics have yielded extremely important insights into glycan biosynthesis and function in cultured vertebrate cells (Esko, 1991, 1992; Patnaik and Stanley, 2006). These efforts have highlighted the value of random mutagenesis and cDNA expression screens for identifying unknown gene functions and have benefited from the great depth of knowledge regarding the glycan structures present in vertebrate systems (Fukuda et al., 1996; Mitoma and Fukuda, 2006; Varki, 1993). Similarly, random mutagenesis in whole organisms offers intriguing opportunities to identify genes that regulate glycan function in multicellular contexts. Drosophila melanogaster, with its prodigious genetic armamentarium, is an ideal organism for advancing whole organism glycobiology. Unfortunately, characterization of invertebrate glycan diversity has generally lagged behind that of vertebrate systems. In large part, this reflects the difficulty associated with performing in-depth glycan characterization on limited amounts of material. To alleviate this limitation, we have undertaken the optimization of methods for characterizing glycan structural diversity in small amounts of material, such that are obtainable from developing tissues of invertebrate embryos (Aoki et al., 2007, 2008). Our goal has been to elaborate a sufficient body of knowledge regarding glycan diversity in the Drosophila embryo so that we might leverage the genetic advantages of the system to understand glycan function in the context of embryonic development. Our methods utilize mass spectrometry (MS and multidimensional MS, i.e., MSn) and other orthogonal approaches to profile complex glycan mixtures released from embryonic tissues. We begin with efficient sample delipidation, which enhances subsequent glycoprotein processing and simultaneously yields glycolipids for analysis (Fig. 14.1). Characterization of N-linked, O-linked, and glycosphingolipid (GSL) glycans extracted from Drosophila embryos has revealed the presence of many structures also found in vertebrate tissues, but has also identified sets of glycans unique to

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Homogenization and organic extraction Starting material Precipitate proteins by centrifugation

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Figure 14.1 Glycomic strategy for characterizing N-linked, O-linked, and glycosphingolipid glycan diversity in the Drosophila embryo. Embryo collections are homogenized and delipidated by organic extraction. Centrifugation causes precipitation of glycoproteins, simultaneously producing a total lipid extract as supernatant. The pellet is dried to give a protein powder, which yields N-linked glycans following protease digestion and glycan release by PNGaseF (or A). Alternately, O-linked glycans are released from the protein powder by reductive or nonreductive b-elimination. Liberated N-linked and O-linked glycans are permethylated for subsequent analysis by NSI-MSn. The total lipid extract contains glycerophospholipids, sterols, and glycosphingolipid (GSL), in addition to other hydrophobic components. Saponification and Iatrobead clean-up produces a total GSL preparation, which contains neutral, zwitterionic, and acidic GSLs. The total GSL preparation is fractionated by sequential QAE and DEAE chromatography to give separate pools of neutral, zwitterionic, and acidic GSLs. Intact GSLs are analyzed by NSI-MSn either as underivatized molecules or following permethylation.

invertebrates (Aoki et al., 2007, 2008; Itonori and Sugita, 2005; Seppo et al., 2000). Many of these unique glycans carry glucuronic acid (GlcA) in novel contexts, sometimes reminiscent of the use of sialic acid in vertebrates (Breloy et al., 2008; Roth et al., 1992; Schauer, 2001; Wiegandt, 1992).

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However, GlcA is not strictly a substitute for sialic acid since Drosophila possesses its own capacity to sialylate glycans and because GlcA in Drosophila appears in contexts not previously ascribed to sialic acid in any other organism (Aoki et al., 2008; Koles et al., 2004; Repnikova et al., 2010; Seppo et al., 2000). In this report, we describe essential enhancements of our glycomic methods and present an overview of the varied uses of GlcA in the Drosophila embryo. The vast structural heterogeneity of the GlcA-containing glycosaminoglycan family has been reported elsewhere by other investigators with more appropriate expertise and is, therefore, not discussed here (Toyoda et al., 2000).

2. Experimental Procedures and Results 2.1. Extraction of proteins and lipids Drosophila embryo homogenates are treated with organic solvents to extract lipids and precipitate proteins as described previously (Aoki et al., 2007; Fredman and Svennerholm, 1980). The method is broadly applicable to other types of cells, whole tissues, or biological fluids. Drosophila embryos of desired stage (approximately 50–100 ml volume, equivalent to approximately 50–100 mg wet weight) are homogenized in a minimum of 5 volumes of ice-cold 50% methanol with a glass Dounce homogenizer. Lipids are extracted by adjusting the solvent composition to 4:8:3::chloroform:methanol:water (C:M:W, v:v:v). After adjusting the solvent composition, the suspension is further disrupted by Dounce homogenization to maximize lipid extraction. The suspension is transferred to a Teflon-lined screw-top glass tube and agitated for 3 h at room temperature, if the extraction is performed on fresh material. If lyophilized or previously dried material is extracted, the agitation is continued overnight. Following agitation, the suspension is clarified by centrifugation at 4
C (3000 rpm, 2000g). For small-scale sample preparation, conical bottom tubes are useful. The resulting pellet is reextracted twice with fresh 4:8:3 solvent and all of the resulting lipid extracts are combined and dried under a stream of nitrogen for further work-up (see below). For long-term storage, the lipid extract is dried completely by adding absolute ethanol and redrying to remove residual water. Every biological sample that we have encountered is contaminated with a hexose polymer of unknown origin or composition. In some cases, this polymer is of such high prevalence that it can be the major component detected by mass spectrometric analysis. In addition, plastic polymers enter into samples during homogenization and can contribute to background noise, especially at low m/z values. Therefore, we investigated methods for removing these contaminants without sacrificing recovery of glycans of

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interest. Optimal removal of contaminants was achieved by washing the protein pellet with 4:1::acetone:water (A:W, v:v; Fig. 14.2). The pellet is overlaid with 4:1::A:W at 4
C and vigorously vortexed. Proteins are reprecipitated by centrifugation and the resulting pellet is reextracted with fresh 4:1. After recentrifugation, the pellet is extracted once more with pure acetone. The final pellet is gently dried under a nitrogen stream at 40
C and the resulting protein powder is stored at 20
C for subsequent preparation of N- and O-linked glycans.

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2000

m/z

Figure 14.2 Removal of hexose polymer contaminant by acetone wash. The profile of N-linked glycans released from Drosophila embryo protein powder is dominated by a ladder of hexose polymers. These ions, indicated by asterisks in the full MS scans shown in (A) and (B), are characteristically found at m/z ¼ 477.2, 681.3, 885.4, 1089.5, 1293.6, etc. (A) Without removal, hexose polymers overwhelm the permethylated N-linked glycans. (B) Following wash of the protein pellet with cold 4:1::acetone: water, signals associated with N-linked glycan are equal to or greater than residual hexose polymer. Note difference in the full-scale relative intensity values for MS scans of unwashed and washed protein powders. Glycan representations are consistent with the recommendations of the Consortium for Functional Glycomics. Unshaded circles indicate Hex of unassigned identity.

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2.2. Preparation of glycopeptides and release of N-linked glycans Protein powder, generally 1–3 mg (equivalent to 50–200 mg of crude protein by BCA assay), is resuspended in 200 ml of trypsin buffer (0.1 M Tris–HCl, pH 8.2, containing 1 mM CaCl2) by sonication and boiling for 5 min. After cooling to room temperature, 25 ml of trypsin solution (2 mg/ml in trypsin buffer) and 25 ml of chymotrypsin solution (2 mg/ml in trypsin buffer) are added and incubated for 18 h at 37
C. For glycoproteomic analysis, trypsin in a standard volatile ammonium bicarbonate buffer can be used without chymotrypsin. The glycan profile is similar with dual or single enzyme digestion, but the total yield of glycans is enhanced by trypsin/chymotrypsin. Protease digestions are terminated by boiling for 5 min. Insoluble material is removed by centrifugation and the supernatant is collected and dried by vacuum centrifugation. To remove residual peptide and reagent contaminants, the dried material is resuspended in 250 ml of 5% acetic acid (v/v) and purified on a C18 cartridge column (Baker C18 column, 100 mg size) as described previously (Aoki et al., 2007; Dell et al., 1993). Prior to use, the C18 cartridge is washed with at least 3 ml of 100% acetonitrile and preequilibrated with at least 3 ml of 5% acetic acid. The resuspended peptide/glycopeptide mixture is loaded onto the prepared C18 cartridge column and the column is washed with 10 ml of 5% acetic acid. Glycopeptides are eluted stepwise, first with 2 ml of 20% isopropanol in 5% acetic acid, and then with 2 ml of 40% isopropanol in 5% acetic acid. The 20% and 40% isopropanol steps are pooled and evaporated to dryness. Dried glycopeptides are resuspended in 50 ml of 20 mM sodium phosphate buffer, pH 7.5, for digestion with PNGaseF (Prozyme), or in 50 ml of 0.2 M citrate phosphate buffer, pH 5.0, for digestion with PNGaseA (Calbiochem), or in 200 ml of anhydrous hydrazine. Following PNGase digestion for 18 h at 37
C, released oligosaccharides are separated from peptide and enzyme by passage through a C18 cartridge column. The digestion mixture is adjusted to 5% acetic acid and loaded onto the cartridge column. The column runthrough and an additional wash with 2 ml of 5% acetic acid, containing released oligosaccharides, are collected together and evaporated to dryness. Glycan release by hydrazinolysis is performed in small, crimp-top glass tubes with Teflon-lined lid inserts. Re-N-acetylation and desalting over AG50-X8 are performed as previously described (Aoki et al., 2007; Hanneman et al., 2006; Patel et al., 1993). Protein content is determined by the BCA method on dilutions of solubilized protein powder or on a portion of the peptides following trypsin/chymotrypsin digestion (Pierce Chemical).

2.3. Permethylation To facilitate analysis by mass spectrometry, released glycans are permethylated according to the method of Anumula and Taylor (1992). The NaOH/DMSO reagent is freshly prepared for a single day’s use and unused reagent is discarded.

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2.4. Glycan analysis by NSI-MSn and automated data acquisition Following permethylation, released glycans are analyzed by nanospray ionization mass spectrometry (NSI-MSn) using a linear ion-trap mass spectrometer (LTQ, Thermo-Fisher) or an LTQ-Orbitrap FT instrument (LTQ-Orbi, Thermo-Fisher). The permethylated glycan sample is reconstituted in 50 ml of 1 mM sodium acetate in 50% aqueous methanol and infused into the mass spectrometer at a syringe flow rate of 0.40 ml/min (Aoki et al., 2007, 2008; Ashline et al., 2005). The capillary temperature is set to 210
C and MS analysis is performed in positive ion mode. In addition to routine instrument tuning and maintenance, a standard mixture of permethylated glycans is infused before performing analyses of unknowns in order to monitor for day-to-day variation of signal intensity that might mandate instrument optimization. Normalized collision energy (30–50%) is used for fragmentation by collision-induced dissociation (CID) in MS/MS and MSn modes. Detection and relative quantification of the prevalence of glycans are accomplished using the total ion mapping (TIM) functionality of the Xcalibur software package version 2.07 (Thermo-Fisher) as previously described (Aoki et al., 2007, 2008). For TIM analysis, the m/z range from 200 to 2000 is automatically scanned in successive 2.8 mass unit windows with a window-to-window overlap of 0.8 mass units, allowing the naturally occurring isotopes of glycans to be summed into a single response thereby increasing detection sensitivity. Furthermore, the 2-mass unit overlap facilitates capture of glycans that might be present at the edges of each scan window, ensuring representative and unbiased sampling of all glycans in complex mixtures. The TIM scans also automatically acquire MS/MS spectra for each overlapping m/z window, providing fragmentation in support of structural assignments and a means to identify isobaric mixtures. Subsequent manual MSn analyses are focused on m/z values of interest, highlighted in the automated TIM scan, to obtain additional fragmentation as needed in order to assign structural topologies or resolve isobars. Glycans are identified as singly, doubly, triply, or quadruply charged ions and the peak intensities for all charge states of each individual glycan are summed for quantification. The predictive fragmentation tools available in the Glycoworkbench suite (EurocarbDB) are extremely useful for calculating monoisotopic masses, and for interpreting and annotating spectra (Ceroni et al., 2008). The profile of N-linked glycans released from Drosophila embryo proteins by PNGaseF or PNGaseA are grossly comparable, indicating that both enzymes are able to access and act on similar glycopeptide populations (Fig. 14.3). However, PNGaseF fails to liberate structures that carry Fuc in a3-linkage to the innermost GlcNAc of the chitobiose core. These glycans are predominantly difucosylated, since they also possess an

304

Relative intensity (%)

A

Kazuhiro Aoki and Michael Tiemeyer

PNGaseF release 100 90 80 70 60 50 40 30 20 10 0

2+ 2+

2+

2+ 2+

2x

2+ 2+ 2+

2+ 2+

400

500

600

700

800

900

2+

2+

1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z

B

Relative intensity (%)

PNGaseA release 100 90 80 70 60 50 40 30 20 10 0

2+

400

500

600

2x

2+ 2+

2+ 2+ 2+

2+

2+

700

800

900

1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z

Figure 14.3 PNGaseF and PNGaseA generate similar profiles of the dominant Nlinked glycans in the Drosophila embryo. Embryo protein powder was digested with trypsin/chymotrypsin and then treated with PNGaseF (A) or PNGaseA (B) to release glycans. Following permethylation, full MS profiles were obtained. The major glycans of the Drosophila embryo are detected in similar relative proportions by both enzymes. Arrows in (A) and (B) (2) indicate a twofold zoom of the plot scale for m/z values greater than 1500.

a6-linked Fuc residue on the same GlcNAc and can be released by PNGaseA (Tarentino et al., 1985; Tretter et al., 1991). Recognized by antibodies raised against the plant glycoprotein horseradish peroxidase (HRP), a3fucosylated glycans are known as HRP-epitopes and exhibit tissue-specific expression, characterized by enrichment in the nervous system during all stages of the Drosophila life cycle (Jan and Jan, 1982; Snow et al., 1987). They are, therefore, of significant interest as developmental markers, albeit of currently incompletely defined function (Desai et al., 1994; Seppo et al., 2003; Sun and Salvaterra, 1995; Wang et al., 1994; Whitlock, 1993). Glycan profiles resulting from hydrazinolysis failed to reveal the presence of any glycans not previously detected by PNGaseF or PNGaseA release, indicating that elaboration of branching Fuc residues described in other organisms does not pertain in Drosophila (Hanneman et al., 2006; Schachter, 2004). Altogether, the complete set of HRP-epitopes accounts for less than 1% of the total glycan profile in the embryo (Aoki et al., 2007). Accordingly, the

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signal intensities associated with HRP-epitopes are extremely low in full MS profiles of total embryonic N-linked glycans. TIM profiling reports the presence of extremely minor glycans without requiring prior knowledge of the structures expected to be found within a complex sample. This advantage derives from the increased sensitivity inherent in MS/MS detection compared to full MS, allowing attention to be focused on minor parent ions that generate fragmentation profiles predictive of glycans. Complete TIM scans generate in excess of 700 MS/MS spectra. Systematic manual inspection of these spectra, although tedious, has resulted in the identification of hybrid and complex glycans in Drosophila embryos, including extremely minor structures bearing a6-linked terminal sialic acid (Aoki et al., 2007). Recently, we have detected and characterized additional, low prevalence sialylated glycans as well as complex and hybrid N-linked glycans capped by LacdiNAc (HexNAc–HexNAc) or GlcA, rather than sialic acid, including four additional HRP-epitopes (Sasaki et al., 2007; Table 14.1 and Fig. 14.4). Although the linkage position and anomericity of the GlcA is yet to be determined, the terminal arrangement of the uronic acids is evocative of sialic acid addition and suggests that Drosophila may deploy capping residues of differing acidity for specific, but yet-to-be-determined functions.

2.5. Release of O-linked oligosaccharides by b-elimination Protein powder, generally 1–3 mg, is resuspended in 500 ml of 100 mM NaOH containing 1 M NaBH4 and incubated for 18 h at 45
C in a glass tube sealed with a Teflon-lined screw-top cap (Aoki et al., 2008; Greis et al., 1996). The base borohydride solution is prepared by diluting 50% aqueous NaOH (Fisher Scientific) to 100 mM with HPLC grade water and the resulting base solution is then used to prepare a 1 M NaBH4 reagent. Incubation times were empirically determined to maximize release of glycan and minimize side-reactions, such as peeling and release of N-linked glycans (Aoki et al., 2008). Following incubation, the pH of the reaction mixture is reduced by dropwise addition of 10% acetic acid on ice until foaming is no longer observed. The material is desalted by passage over an AG50W-X8 column, 1 ml bed volume (Hþ form, BioRad). Free oligosaccharide alditols are collected in the column run-through and combined with a wash of 3 bed volumes of 5% acetic acid. Following lyophilization, complexed borate is removed from the glycans as an azeotrope with methanol by adding 0.5 ml of 10% acetic acid in methanol, drying under a nitrogen stream at 37
C, and repeating four additional times. Surprisingly, excessive drying from methanol/acetic acid results in significantly reduced recovery of a subset of O-linked glycans. Therefore, careful attention is required so that the drying time is limited to the minimum necessary to

Table 14.1 Terminal modifications of N-linked glycans in the Drosophila embryo, including sialylation, GalNAc extension (LacdiNAc formation), and Glucuronylation

a

m/z + (m+Na)

Structure

% Total Profileb

Structure

m/z + (m+Na)

% Total Profile

Glucuronylated N-linked Glycans:

Sialylated N-linked Glycans: c

GlcA-GalNM3N2

1838.9

<0.1

SA-GalNM3N2

1982.0

<0.1

SA-GalN2M3N2

2227.1

<0.1

GlcA-GalN2M3N2

2084.0

<0.1

SA-Gal2N2M3N2

2431.2

<0.1

GlcA-Gal2N2M3N2

2288.1

<0.1

SA2-Gal2N2M3N2

2792.4

<0.1

GlcA2-Gal2N2M3N2

2506.2

<0.1

2013.0

0.1

2258.1

<0.1

2462.2

<0.1

2680.3

<0.1

2187.7

<0.1

2432.2

<0.1

6

SA-GalNM3N2F

2156.1

6

SA-GalN2M3N2F

2402.0

GlcA-GalNM3N2F

<0.1

6

GlcA-GalN2M3N2F

<0.1

6

6

SA-Gal2N2M3N2F

2605.3

6

2966.5

SA2-Gal2N2M3N2F

GlcA-Gal2N2M3N2F

<0.1

6

GlcA2-Gal2N2M3N2F

<0.1

GlcA-GalNM3N2F

LacdiNAc N-linked Glycans: GalNAcNM3N2 GalNAcN2M3N2 6

GalNAcNM3N2F

6

GalNAcN2M3N2F

3, 6

GalNAcNM3N2F

3, 6

GalNAcN2M3N2F

6

1661.8

GlcA-GalN2M3N2F

<0.1

1907.0

<0.1

1835.9

<0.1

2081.1

<0.1

2010.0

<0.1

2255.0

<0.1

a

3, 6

3, 6

m / z values are given for the permethylated, singly charged, monoisotopic, sodiated ion of each intact glycan. for glycans with m / z>2000, the doubly charged ions were characterized. b Glycan prevalence is described as “% Total profile” and is calculated by normalizing the signal intensity of an individual glycan to the summed intensities for all detected glycans. n c Glycans detected at <0.1 % Total profile generate strong MS fragmentation signals but are present at levels that give MS intensities below the threshold for quantification (>3-fold above background). Only one of the minor glycans presented in this table reached the quantification threshold, GlcA-GalNM3N2F6.

A

MS2 at m / z = 1053

1052.0

2+

(= 2081.0, singly charged) 9222+

922

100 90 Relative intensity

80 2+

70

793

60

1053.52+ (= 2081.0, singly charged)

50

2+

915

40 30

704

20

923 1403

793 9152+

10 704 1403

0

B

500

1000

1500

m/z

2000 1053

704

3

MS at m / z = 704

477

100 90 Relative intensity

472

704

80 477

70

O

MeO COOMe O

50 MeO

40 472

30 20

MeO

MeO

60

OMe

MeO

O

NMeAc

630

459

OMe

MeO

630

O

O

477 704

459

10 0 200

300

400

500 m/z

600

700

800

704

C 4

MS at m / z = 477 100

477

329

90

245

477

Relative intensity

80 MeO

445

70

273

60 50

MeO

373

40

477

MeO

COOMe O

20

O OMe

O

329 373

273 301

10

O

OMe

245

30

MeO

359

301 359

0 200

300 m/z

400

500

Figure 14.4 MSn fragmentation of a terminally glucuronylated, biantennary N-linked glycan. (A) MS2 fragmentation observed at m/z ¼ 1053  1.1. This collection window is wide enough to include fragments for the indicated triantennary core-fucosylated glycan (m/z ¼ 1052.0, doubly charged) and the monoglucuronylated biantennary glycan (m/z ¼ 1053.5, doubly charged). The MS2 fragment ion at 704 is signature for the composition HexA-Hex-HexNAc (singly charged). (B) MS3 fragmentation of the m/z ¼ 704 ion dissects the trisaccharide into its component monosaccharides and defines the sequence as a linear, terminally glucuronylated LacNAc. (C) MS4 fragmentation of the m/z ¼ 477 ion generates a wealth of cross-ring cleavages that place the HexA at the 3-position of the subterminal Hex residue.

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remove solvent for each wash. Released oligosaccharide alditols are purified on a C18 cartridge column as described previously for N-linked glycans. We compared the recovery of O-linked glycans prepared by b-elimination from protein powder or from tryptic or tryptic/chymotryptic peptides derived from protein powder. We routinely achieve greater yield and reproducibility using undigested protein powder (Aoki et al., 2008). In contrast, O-linked glycan recovery from standard glycoproteins (fetuin/ ovalbumin) is equivalent whether b-elimination is performed on intact protein or on tryptic peptides. We suspect that the heterogeneity of the peptide/glycopeptide mixtures produced from tryptic digests of complex samples yields an excess of free amino termini that may interfere with the efficacy of the b-elimination reaction. Alternatively, protease digestions of complex samples may produce a significant proportion of O-linked glycopeptides possessing undersized peptides. Small peptides with small glycans may not be efficiently captured during the C18 cartridge column clean-up after proteolysis. Therefore, we recommend that O-linked glycans in biological matrices be harvested from intact proteins or from peptides generated by rare-cutting proteases to ensure representative sampling. Nonreductive b-elimination is extremely useful for interrogating linkage positions and defining specific structural topologies at the reducing terminal of O-linked glycans. For release of O-linked glycans with an intact reducing terminal, 1–3 mg of protein powder in a screw-top plastic tube is resuspended with 500 ml of 28% aqueous ammonium hydroxide saturated with ammonium carbonate (Huang et al., 2002). An additional 100 mg of solid ammonium carbonate is added and the release reaction is incubated for 40 h at 60
C. At the end of the incubation, volatile reagent and salts are removed by vacuum centrifugation and the dried material is washed 5 times, or until residual salt is not evident, by resuspension in 500 ml of water followed by redrying. Released oligosaccharides are purified on a C18 cartridge column as described for N-linked glycans. After combining and drying the C18 run-through and wash fractions, glycosylamines are converted to free reducing termini by incubation in 50 ml of 0.5 M boric acid at 37
C for 1 h. The reaction mixture is dried and borate is removed by adding 0.5 ml of 10% acetic acid in methanol, drying under a nitrogen stream at 37
C, and repeating four additional times. Again, as described for reductive release of O-linked glycans, overdrying during the wash steps should be avoided to maximize glycan recovery. MS analysis of O-linked glycans released by reductive or nonreductive b-elimination revealed the expression of expected and novel structures in the Drosophila embryo. Sialylated O-linked glycans were not detected, but glucuronylation emerges as a major modification of standard and unexpected O-linked core structures (Table 14.2). GlcA extension on a standard Core 1 disaccharide (GlcAb3Galb3GalNAc-O-S/T) has been previously described in Drosophila cell lines and is clearly detected in embryo extracts

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Table 14.2 Glucuronylated O-linked glycans in the Drosophila embryo are found on multiple core types as internal and terminal residues m / za (m+Na)+

% Total profile b

752.4

9.7

752.4

1.3

997.5

0.1

997.5

0.1

1201.6

0.1

722.4

11.3

1215.6

<0.1

or

793.4

0.9

or

997.5

0.1

or

1215.6

<0.1

Structure Core 1 Type:

O-Fuc Type:

Core 2 Type:

Other Type:

c

am / z

values are given for the permethylated, singly charged, monoisotopic, sodiated ion of each intact glycan. bGlycan prevalence is described as “% Total Profile” and is calculated by normalizing the signal intensity of an individual glycan to the summed intensities for all detected glycans. cUnshaded square indicates a HexNAc of unassigned identity, either GlcNAc or GalNAc.

(Aoki et al., 2008; Breloy et al., 2008). In addition, however, the resolving power of TIM scans and subsequent MSn analysis demonstrates the presence of an isobaric, branched structure that carries GlcA in b4-linkage to the reducing terminal GalNAc of the Core 1 disaccharide (Galb3(GlcAb4) GalNAc-O-S/T). This branched trisaccharide is approximately sevenfold less abundant than its linear isobar, but still ranks among the moderately prevalent O-linked glycans in the embryo (Aoki et al., 2008). Other O-linked glycans with branching and/or terminal GlcA residues are built

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from other standard cores and from O-Fuc initiated structures. A glucuronylated O-Fuc trisaccharide is the only detectable O-Fuc structure in the embryo and exhibits spatially distributed expression in larval wing discs consistent with the activity of the Fringe GlcNAc transferase; homozygous null Fringe embryos are substantially reduced, but not devoid, of the O-Fuc trisaccharide (Aoki et al., 2008). The linkage anomericity of the major glucuronylated O-linked glycans in the Drosophila embryo was verified by digestion with b-glucuronidase from Helix pomatia (Aoki et al., 2008). A few minor O-linked glycans present terminal and internal HexNAc-HexA disaccharides evocative of glycosaminoglycan repeat units. Whether these proto-glycosaminoglycan structures, which have also been described in Caenorhabditis elegans, are substrates for sulfation or are capable of mediating the developmental functions of extended glycosaminoglycan chains remains to be determined (Guerardel et al., 2001).

2.6. Isolation and fractionation of glycosphingolipids Embryo lipid extracts are freed of contaminating glycerophospholipids by saponification as previously described (Schnaar, 1994; Seppo et al., 2000). Generally, enough base is used to ensure a 20-fold molar excess to phospholipid in the extract. When dried from 4:8:3 without ethanol wash, the lipid extract retains water, forming a moist residue. Therefore, 500 ml of 0.5 M methanolic KOH or NaOH is added for each 10 mg of moist lipid residue, based on the assumption that 90% of the residual mass is glycerophospholipid. Following incubation at 37
C for a minimum of 6 h to overnight, the reaction mixture is desalted by dialysis or by passing through a C18 cartridge column as previously described (Schnaar, 1994).

2.7. Iatrobead chromatography for purification of total GSL The alkali-stable lipid preparation resulting from saponification contains sterols and free fatty acids released from glycerophospholipids, in addition to GSLs. Purification of the total GSL away from these contaminants enhances MS analysis and is achieved by Iatrobeads (Iatron Laboratories, Tokyo, 6RA8010) chromatography. The saponified, desalted lipid mixture is dried, resuspended in the minimal necessary volume of 2:1::C:M, and loaded onto an Iatrobead column washed and equilibrated in chloroform (1.5 ml bed volume packed in a 2-ml glass pipette). Fatty acids and sterols are eluted with 3 bed volumes of chloroform, followed by 3 bed volumes of 95:5::C:M. GSLs are then eluted by stepwise increases in solvent polarity, using 3 column volumes each of 9:1::C:M, 8:2::C:M, 1:1::C:M, and finally 30:60:8::C:M:W. Fractions equivalent to 1 column volume are collected

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for each solvent step. The 9:1 and 8:2 fractions may contain some residual sterol and free fatty acids in addition to neutral GSLs with short sugar chains, such as mono- and diglycosylceramide. Therefore, fractions should be monitored by thin layer chromatography (TLC). Solvent systems useful for TLC include 60:35:8::C:M:W, 60:40:10::C:M:W, and 75:25:5::n-propanol/water/ammonium hydroxide. Orcinol-H2SO4, Dittmer-Lester, and ninhydrin reagents are used for the detection of sugar, phosphate, and amino group, respectively. Iatrobead fractions containing GSLs are combined as desired to produce the total GSL fraction, which is suitable for glycosphingolipidomic analysis by MS.

2.8. QAE Sephadex chromatography for neutral GSL The total GSL fraction contains neutral, acidic, and zwitterionic components that are resolved by ion-exchange chromatography. A strong anion exchange media is used to separate neutral GSLs from the other species. A 10-fold excess of QAE Sephadex A-25 (GE Healthcare), relative to total GSL mass, is required (e.g., 100 mg resin for 10 mg of moist lipid residue). Before use, the QAE ion-exchange resin is activated with 1 M NaOH at 60
C for 30 min. The activated resin is washed with deionized water until the pH becomes neutral, then with methanol before storage in 30:60:8::C:M:W. The desired amount of the total GSL fraction is dried, resuspended in 30:60:8, and mixed with one-third of the required volume of activated QAE resin. GSL are absorbed to the resin for 30 min at room temperature. The remaining twothirds of the resin is packed into a column and the suspension of absorbed GSL is then loaded onto the bed. Neutral GSLs are collected in the runthrough, which is combined with the eluates resulting from washing the column with 5 volumes of 30:60:8 and 1 volume of methanol. The combined run-through and washes are dried as the neutral GSL fraction. Acidic and zwitterionic GSLs are eluted stepwise with 3 bed volumes each of 0.05, 0.15, and 0.45 M ammonium acetate in methanol. The 0.05 M fraction contains most of the acidic and zwitterionic GSLs, although GSLs with stronger charge may be eluted in the 0.15 and 0.45 M fractions. Following dialysis, the mixture of acidic and zwitterionic GSLs are lyophilized.

2.9. DEAE Sephadex chromatography for fractionation of acidic and zwitterionic GSLs The GSLs that are bound and then eluted from QAE are further resolved by weak anion exchange using DEAE Sephadex A-25 (GE Healthcare). A 10-fold excess of resin to GSL is required as described for the QAE column. The resin is activated in 30:60:8::C:M:0.8 M sodium acetate and then washed and equilibrated with 30:60:8::C:M:W. The dried GSLs obtained by stepwise elution from the QAE column are resuspended in

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Kazuhiro Aoki and Michael Tiemeyer

30:60:8::C:M:W and mixed with one-third of the required DEAE resin. Following absorption, the suspension is loaded onto a column prepared from the remaining two-thirds of the DEAE resin. Zwitterionic GSLs are eluted with 3 bed volumes of 30:60:8::C:M:W and 1 bed volume of methanol, which are combined before drying under a stream of nitrogen. Acidic GSLs are then eluted with 3 bed volumes each of 0.05, 0.15, and 0.45 M ammonium acetate in methanol. Most of acidic GSLs will be obtained from the 0.05 M fraction. The acidic fraction(s) are dialyzed and then lyophilized for storage.

2.10. Analysis of intact GSLs by NSI-MSn data-dependent acquisition The well-characterized endoglycosylceramidase from leech (Macrobella decora) can release glycans en bloc from Drosophila GSLs (Zhou et al., 1989). However, the enzyme’s activity is not uniform across all GSL structures, thereby skewing GSL glycomic profiles derived from released glycans (Levery, 2005; Li and Li, 1994). To avoid this bias, we analyze GSLs by MS either as intact, underivatized molecules or as intact, permethylated structures. Previous work by several groups has described the complexity of Drosophila GSLs and their relation to the GSLs of other arthropods, resulting in the definition of the ‘‘arthro-series’’ structural conservation (Chen et al., 2007; Rietveld et al., 1999; Seppo et al., 2000; Wiegandt, 1992). The neutral arthro-series core is built from a Manb4Glc disaccharide linked to ceramide. This so-called mactosylceramide acceptor (named to parallel the lactosylceramide core of vertebrate GSLs, Galb4GlcCer) is extended by GlcNAc, GalNAc, and Gal residues, ultimately generating the longest identified arthro-series GSL, At9Cer: Galb3GalNAcb4GlcNAcb3Galb3GalNAca4GalNAcb4GlcNAcb3Manb4GlcbCer (Sugita et al., 1990). Variations on the neutral arthro-series core include the addition of phosphoethanolamine (PEtn) at the 6-position of one or both GlcNAc residues and/or terminal extension by GlcA (Helling et al., 1991; Seppo and Tiemeyer, 2000; Seppo et al., 2000; Weske et al., 1990; Wiegandt, 1992). Addition of PEtn imparts zwitterionic character to the neutral core, while GlcA addition generates an acidic GSL. Relatively little is understood regarding the enzymatic machinery that adds PEtn to Drosophila GSLs, although progress has been made in identifying and characterizing relevant genes in other organisms, most notably C. elegans and other nematodes (Cipollo et al., 2005; Grabitzki et al., 2008; Lochnit et al., 2006; Poltl et al., 2007). Unlike the sialic acid of vertebrate GSLs, which exists both as a terminal modification and as a branch from an internal Gal, the GlcA of the arthro-series has only been found attached to a terminal Gal residue

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The Glycomics of Glycan Glucuronylation in Drosophila melanogaster

Table 14.3 Glucuronylated O-linked glycosphingolipids in the Drosophila embryo belong to the arthro-series

Structure

Molecular weighta

% Total profileb

1457.1

<1

1863.5

<1

1986.5

4

2431.8

<1

2554.8

6

2677.8

21

GlcAGal-At3Cer Cer GlcA-At6Cer Cer GlcA(PEtn)-At6Cer

c Cer

GlcA-At9Cer Cer GlcA(PEtn)-At9Cer Cer GlcA(PEtn)2-At9Cer Cer aMolecular

weights are presented for the indicated GSLs as intact structres without permethylation and assuming the most prevalent ceramide form (C20:0 arachidic acid on a C14:1 tetradecasphingenine). bGSL prevalence is described as “% total profile” and is calculated by normalizing the signal intensity of an individual GSL to the summed intensities for all detected GSLs. cOrange oval represents phosphoethanolamine (PEtn) addition to the 6-position of the indicated GlcNAc residues.

(Table 14.3). In comparison to other insects, the acidic GSL profile of the Drosophila embryo is skewed toward longer species that possess At6 and At9 cores decorated with one or two PEtn moieties (Itonori and Sugita, 2005; Seppo et al., 2000; Sugita et al., 1989; Weske et al., 1990; Wiegandt, 1992). We have detected very little GlcA-extended At5Cer in Drosophila embryos, although it is a major acidic lipid in the adult form of another dipteran, Calliphora vicina (Seppo et al., 2000; Weske et al., 1990). It remains to be determined whether the enrichment of longer chain acidic structures is a result of developmental regulation or of species differences.

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The characterization of GSLs as intact molecules preserves information regarding ceramide heterogeneity. We find that ceramide distributions are generally consistent when compared across Drosophila GSLs. In the embryo, the predominant lipid is a C20:0 arachidic acid linked to a C14:1 tetradecasphingenine (m/z ¼ 548, permethylated), consistent with the ceramides detected in other dipterans (Seppo et al., 2000). The dominant fragment ion derived from MS/MS of all GSLs corresponds to loss of ceramide, leaving an intact oligosaccharide. Thus, NSI-MS efficiently performs the same service as endoglycosylceramidase digestion without imparting structural bias. We have harnessed the reproducible production of this fragment to trigger data-dependent acquisition of MSn fragmentation profiles when analyzing complex GSL mixtures. Since ion-trap instruments provide significantly greater sensitivity in MS/MS than in full MS, this workflow enhances the identification of minor species and generates deeper fragmentation for structural assignment. If data-dependent acquisition fails to capture sufficient information for determining structure, it still identifies specific m/z values associated with GSLs for subsequent manual fragmentation experiments. Using such an approach, we have identified previously undetected GSLs in the Drosophila embryo, including a single example of a neutral core glycan that deviates from the arthro-series consensus. This GSL, GlcAGal-At3Cer, possesses a Gal residue, rather than a GalNAc, as the fourth monosaccharide away from the Cer (Table 14.3). In addition, prior characterization of the glucuronylated GSLs of the Drosophila embryo indicated that all acidic GSLs possess at least one PEtn (Seppo et al., 2000). However, with more sensitive instrumentation and the ability to identify fragment ions of interest based on loss of ceramide, we now detect acidic forms of At6- and At9-Cer that lack PEtn (Table 14.3 and Fig. 14.5). If these minor, undecorated acidic lipids represent biosynthetic precursors, captured before PEtn addition, then the enzymatic machinery must be able to act on fully extended structures and we may have reached saturation in characterizing Drosophila GSLs. Alternatively, these minor modifications on structural themes may represent important regulatory molecules in specific developmental contexts and the diversity of Drosophila GSLs may still be incompletely defined, requiring additional systematic studies using methods of enhanced sensitivity.

3. Discussion It has been almost 25 years since the publication of preliminary data under the title ‘‘Insects: animals without gangliosides,’’ in which some of the first parallels were drawn between the acidic GSLs of dipterans and the sialylated GSLs of vertebrates (Dennis et al., 1986; Wiegandt, 1992). In the

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Figure 14.5 MSn fragmentation of the longest acidic glycosphingolipid found to lack phosphoethanolamine (PEtn) in the Drosophila embryo. (A) MS2 fragmentation at m/z ¼ 1443 detects a major ion at m/z ¼ 1169 (doubly charged), corresponding to an intact glycosphingolipid (GSL) glycan lacking ceramide. Loss of ceramide produces the dominant MS2 fragment for all GSLs, providing a signature ion for data-dependent acquisition of MSn data. This ceramide loss reports the predominant form in the embryo (C20:0 arachidic acid on a C14:1 tetradecasphingenine). (B) MS3 fragmentation of the resulting free glycan ion (m/z ¼ 1169 doubly charged) provides fragments that place the HexA at the nonreducing terminal, give the linear sequence for the three subterminal residues, and verify the mactosyl disaccharide at the reducing terminal. The mass of the parent ion and the observed fragment ions eliminate the possibility of PEtn addition to the expected At9-Cer structure.

intervening years, the characterized insect glycome has grown to include a reasonable diversity of N-linked, O-linked, and GSL glycans. In the Drosophila embryo, we now possess an understanding of relative glycan

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prevalences sufficient to appreciate that sialylated glycans exist as N-linked structures, but only in very limited amounts. Similarly, glucuronylated N-linked glycans are also present in small amounts. On the other hand, glucuronylation is a highly prevalent elaboration of O-linked and GSL glycans. The restricted expression of acidic N-linked glycans likely reflects cell-specific distributions of the glycosyltransferases needed for their production. Restricted enzyme expression has been clearly demonstrated for the single sialyltransferase identified in Drosophila (Koles et al., 2004; North et al., 2006), but similar characterization of relevant glucuronyltransferases that act on N-linked, O-linked, or GSL cores is not yet available. The functions of glucuronylated glycans are essentially unexplored in Drosophila, although the GlcA residues of glycosaminoglycan chains have received considerable attention since the demonstration that these glycoconjugates modulate growth factor signaling (Bornemann et al., 2004; Esko and Selleck, 2002; Lin, 2004; Lin and Perrimon, 2002). In this context, the significant phenotypes associated with mutations that affect GlcA biosynthesis have been attributed exclusively to altered GAG function, ignoring the likelihood that loss of GlcA from other classes of glycans may also impinge on development (Hacker et al., 1997; Toyoda et al., 2000). The total mass of GAG (as heparan and chondroitin sulfates) in the Drosophila embryo is approximately 12-fold higher than the total mass of the major glucuronylated Core 1 glycan, but approximately fourfold lower than the total mass of glucuronylated GSL (assuming average chain length equivalent to a hepatosylceramide); 121, 10, and 480 ng glycoconjugate per mg dry weight for GAG, O-linked, and GSL, respectively (Aoki et al., 2008; Seppo et al., 2000; Toyoda et al., 2000). On a molar basis, the abundance of GSL and the Core 1 structure far exceeds that of the GAG chains, further emphasizing that any evaluation of the biological relevance of glycan glucuronylation must consider the presentation of this acidic moiety on multiple glycoconjugate classes. The glucuronylated Core 1 O-linked glycan of the Drosophila embryo is structurally similar to a developmentally regulated family of glycoconjugates in vertebrate tissues known as HNK-1 antigens (GlcAb3Galb3GlcNAc, with and without sulfate on the GlcA residue; Chou et al., 1991; Kruse et al., 1984). This epitope is characteristically expressed on several cell adhesion molecules and also on glycolipids in the nervous system of a wide range of species, where it has been suggested to mediate cell–cell and cell–matrix adhesion (Terayama et al., 1997). A nonsulfated form of the HNK-1 antigen (L2) is present in the mouse kidney and in insects, including Drosophila (Dennis et al., 1988, 1991; Tagawa et al., 2005). Two glucuronyltransferases capable of synthesizing the HNK-1 epitope have been described in mammals: GlcAT-P and GlcAT-S, both of which were initially thought to be specific for glycoprotein oligosaccharides but were subsequently shown to act on glycolipids as well. GlcAT-P prefers

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N-acetyllactosamine (Galb4GlcNAc) acceptor substrates, and GlcAT-S prefers to act on N-acetylneolactosamine (lacto-N-biose, Galb3GlcNAc). Another glucuronyltransferase, GlcAT-I, initiates GAG synthesis, and Drosophila homologues for all three of these enzymes have been identified (Kakuda et al., 2005; Kim et al., 2003). Targeted deletion of the GlcAT-P gene in mice reduced HNK-1 antigen expression and induced neurological deficits (Yamamoto et al., 2002), but the biosynthetic pathway and consequences of altered HNK-1 production in Drosophila remain unknown. Likewise, the enzymes responsible for the novel addition of GlcA to O-Fuc or to the antennae of N-linked glycans are yet to be identified. With the appropriate genetic and biochemical tools available to us, we will be able to assess the extent to which novel Drosophila glucuronylated glycans contribute to cell adhesion, cell signaling, and other cellular functions that drive tissue development.

ACKNOWLEDGMENTS This work was supported by NIH grants 1-R01-GM072839 from NIGMS (to M. T.), a Toyobo Biotechnology Foundation Long-Term Research Grant (to K. A.), and a Mizutani Foundation for Glycoscience Award (100065 to K. A.). The authors are indebted to all members of the Tiemeyer laboratory, past and present, as well as Lance Wells (CCRC, University of Georgia), Shoko Nishihara (Soka University), Vlad Panin (Texas A&M), and Kelly Ten Hagan (NIH/NIDCR) for critical input and encouragement.

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C H A P T E R

F I F T E E N

Glycosyltransferases and Transporters that Contribute to Proteoglycan Synthesis in Drosophila: Identification and Functional Analyses Using the Heritable and Inducible RNAi System Shoko Nishihara Contents 1. Overview of Glycosaminoglycan Biosynthesis in Drosophila 1.1. Glycosaminoglycan structures 1.2. Biosynthesis of Drosophila GAGs: Glycosyltransferases, sulfotransferases, and sugar-nucleotide transporters 1.3. Core proteins of proteoglycans 1.4. Function of proteoglycans 2. Identification of Drosophila Glycosyltransferases and SugarNucleotide Transporters that Contribute to Proteoglycan Synthesis 2.1. Cloning and expression of Drosophila glycosyltransferases 2.2. Assay of glycosyltransferase activity 2.3. Cloning and expression of Drosophila sugar-nucleotide transporters including PAPS transporters 2.4. Preparation of subcellular fractions of yeast and assay of sugar-nucleotide transport activity 3. Establishment and Functional Analysis of RNAi Flies for the Glycosyltransferases and Sugar-Nucleotide Transporters that Contribute to Proteoglycan Synthesis 3.1. Establishment of RNAi flies 3.2. Functional analyses of RNAi flies References

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Abstract Drosophila melanogaster is an important model organism that can be used as a powerful genetic tool to analyze the physiological functions of various molecules. Recently, many successful analyses of the function of glycans in vivo have been performed using Drosophila. The glycan portion of proteoglycans, namely glycosaminoglycans (GAGs), which include heparan sulfate (HS) and chondroitin sulfate (CS), is conserved structurally between Drosophila and mammals, including humans. The analysis of mutant and RNAi flies has demonstrated that HS proteoglycans play key roles in the regulation of various basic developmental signaling pathways, including those of fibroblast growth factor (FGF), Wingless (Wg)/Wnt, Hedgehog (Hh), and Decapentaplegic (Dpp, a BMP-type ligand that belongs to the TGFb family). In this chapter, I give an overview of glycosaminoglycan biosynthesis in Drosophila and then describe the methods that can be used to identify and perform functional analyses of the molecules involved in this process, namely glycosyltransferases, sulfotransferases, sugar-nucleotide transporters including PAPS transporters, and core proteins, using the heritable and inducible RNAi system.

1. Overview of Glycosaminoglycan Biosynthesis in Drosophila Proteoglycans are composed of negatively charged glycosaminoglycans (GAGs) and core proteins. Various growth factors, morphogens and cytokines interact with GAGs, which regulate the signaling pathways of these factors. First, I provide an overview of GAG structures, the proteins that contribute to GAG synthesis (glycosyltransferases, sulfotransferases, and sugar-nucleotide transporters), core proteins, and the physiological functions of these molecules.

1.1. Glycosaminoglycan structures Drosophila GAGs comprise two major types, heparan sulfate (HS) and chondroitin sulfate (CS) (Fig. 15.1). Each of these types contains unique disaccharide repeats, (GlcNAca1-4GlcAb1-4)n in HS and (GalNAcb1-4GlcAb1-3)n in CS, but they share a so-called linkage region, which is a common tetrasaccharide structure, GlcAb1-3Galb1-3Galb1-4Xylb1-O-Ser. Structural analyses of GAGs from Drosophila melanogaster have been performed (Lawrence et al., 2008; Toyoda et al., 2000a,b; Yamada et al., 2002). Drosophila HS contains N-, 2-O-, and 6-O-sulfated disaccharide structures, and sulfation can give rise to mono-, di-, and tri-sulfated forms (Fig. 15.1). The relative amount of each type of sulfation is regulated developmentally, probably by the expression of sulfotransferases (Toyoda et al., 2000b). Drosophila CS contains nonsulfated and 4-O-sulfated disaccharide structures. The nonsulfated

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Heparan sulfate GlcAb1-(4GlcNAca1-4GlcAb1-)n4GlcNAca1-4GlcAb1-3Galb1-3Galb1-3Galb1-4Xylb1-O-Ser ΔUA-GlcNH2 **

ΔUAGlcNAc

ΔUA-GlcNAc6S ΔUA2S-GlcNS6S ΔUAGlcNS

(2P)*

ΔUA- ΔUA GlcNS 2S6S GlcNS

Repeating disaccharide region

Linkage region

GlcAb1-(3GalNAcb1-4GlcAb1-)n3GalNAcb 1-4GlcAb1-3Galb1-3Galb1-4Xylb1-O-Ser **

ΔUA-GalNAc Chondroitin sulfate

2P ΔUA-GalNAc4S

Figure 15.1 Pattern diagrams of Drosophila glycosaminoglycan structures. Heparan sulfate (HS) and chondroitin sulfate (CS) share a common tetrasaccharide structure (the so-called linkage region), GlcAb1-3Galb1-3Galb1-4Xylb1-O-Ser, but they also contain unique disaccharide repeats, (GlcNAca1-4GlcAb1-4)n and (GalNAcb14GlcAb1-3)n in HS and CS, respectively. This pattern diagram is based on published information. *, only 30% of the Xyl residues of HS are phosphorylated at the C2position in adult flies. **, the relative amount of each disaccharide structure in the unique disaccharide repeats of HS and CS. The abbreviations indicate the following disaccharide structures: DUA-GlcNAc, DHexUAa1-4GlcNAc; DUA-GlcNS, DHexUAa1-4GlcN(2-N-sulfate); DUA-GlcNAc6S, DHexUAa1-4GlcNAc(6-O-sulfate); DUA-GlcNS6S, DHexUAa1-4GlcN(2-N-,6-O-disulfate); DUA2S-GlcNS, DHexUA(2-O-sulfate)a1-4GlcN(2-N-sulfate); DUA2S-GlcN6S, DHexUA(2-O-sulfate) a1-4GlcN(2-N-,6-O-disulfate); DUA-GalNAc, DHexUAb1-3GalNAc; DUA-GalNAc4S, DHexUAb1-3GalNAc(4-O-sulfate); 2P, 2-O-phosphate.

structure is the more common type and corresponds to approximately 80–90% of CS in adult flies (Fig. 15.1). However, the ratio of the two types is also regulated developmentally (Toyoda et al., 2000b). In addition to the sulfation described above, the xylosyl (Xyl) residue in the linkage region can be phosphorylated in both HS and CS. In adult flies, approximately 70% of Xyl residues in HS are unphosphorylated, whereas 100% of Xyl residues in CS are phosphorylated at the C2-position (Yamada et al., 2002).

1.2. Biosynthesis of Drosophila GAGs: Glycosyltransferases, sulfotransferases, and sugar-nucleotide transporters The first step of GAG biosynthesis is the formation of the shared linkage tetrasaccharide structure, GlcAb1-3Galb1-3Galb1-4Xylb1-O-Ser (Fig. 15.2). Initially, a Xyl residue is transferred to a Ser residue on the core protein by peptide O-xylosyltransferase (O-XylT). Then, two galactosyl (Gal) residues

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Repeating disaccharide region

Linkageregion

Heparan sulfate Heparan sulfate 3-O-sulfotransferase Heparan sulfate 6-O-sulfotransferase Heparan sulfate 2-O-sulfotransferase C5 epimerase

(4) Proteoglycan glucuronyltransferase I (3) Proteoglycan b1-3galactosyltransferase II (2) Proteoglycan b1-4galactosyl(7) Heparan N-deacetylase / N-sulfotransferase transferase I (6) DEXT1(Ttv), DEXT2(Sotv) (1) Peptide GlcAb1-(4GlcNAca1-4GlcAb1-)n4GlcNAca1O-xylosyltransferase SO3– SO3– (5) DEXTL3 (Botv) (11) (10) (9) (8)

4GlcAb1-3Galb1-3Galb1-4Xylb1-O-Ser SO3–

(5) Chondroitin N-acetylgalactosaminyltransferase

GlcAb1-(3GalNAcb1-4GlcAb1-)n3GalNAcb1(6) Chondroitin synthase, Chondroitin polymerization factor

Core protein

(7) Chondroitin 4-O-sulfotransferase Chondroitin sulfate

Figure 15.2 Biosynthetic pathway of Drosophila glycosaminoglycans. The biosynthesis of glycosaminoglycan (GAG) is initiated by the synthesis of the linkage tetrasaccharide structure, GlcAb1-3Galb1-3Galb1-4Xylb1-O-Ser, which is common to heparan sulfate (HS) and chondroitin sulfate (CS). The numbers in parentheses indicate the order in which the sugar residues are transferred. Steps (1)–(4) illustrate the synthesis of the linkage tetrasaccharide structure. At step (5), the GAG biosynthetic pathway divides to allow synthesis of the sulfated disaccharide repeats of either HS or CS.

are added sequentially to the growing chain with b1-4 and b1-3 linkages by two different galactosyltransferases, namely, proteoglycan b1-4galactosyltransferase I (b4GalTI) and proteoglycan b1-3galactosyltransferase II (b3GalTII). Subsequently, glucuronic acid (GlcA) is added with a b1-3 linkage by proteoglycan glucuronyltransferase I (GlcATI). These steps are common to both CS and HS. In Drosophila, all the glycosyltransferase genes involved in the synthesis of the linkage tetrasaccharide structure have been cloned and the in vitro activities of the encoded proteins have been identified. The activities correspond to O-XylT (Wilson, 2002), b4GalTI (Nakamura et al., 2002; Takemae et al., 2003; Vadaie et al., 2002), b3GalTII (Ueyama et al., 2008), and three glucuronosyltransferases (GlcAT) (Kim et al., 2003) (Table 15.1). It is not yet clear which of the three Drosophila GlcATs transfers GlcA to Galb1-3Galb1-4Xylb1-O-Ser in vivo because all three enzymes have this GlcATI activity in vitro.

Table 15.1

Glycosyltransferases, sulfotransferases, and sugar-nucleotide transporters contributing to glycosaminoglycan biosynthetic pathway

Transferases or transporters

Glycosyltransferases Peptide O-xylosyltransferase Proteoglycan b1-4galactosyltransferase I/ b1-4Galactosyltransferase 7 Proteoglycan b1-3galactosyltransferase II Glucuronyltransferase Proteoglycan glucuronyltransferase I b1-3Glucuronyltransferase

Abbreviated gene name

CG No.

Conventional mutants and RNAi

Identified activity (carbohydrate-structural analysis of mutants)

XylT activity (Wilson, 2002) b4GalT activity (Nakamura et al., 2002; Takemae et al., 2003; Vadaie et al., 2002) b3GalT activity (Ueyama et al., 2008)

dXylT db4GalTI/ ddb4GalT7

CG32300 CG11780

RNAi (Goda et al., 2006) RNAi (Nakamura et al., 2002; Takemae et al., 2003)

db3GalTII

CG8734

RNAi (Ueyama et al., 2008)

DmGlcAT-I

CG32775

GlcAT activity (Kim et al., 2003)

CG3881 CG6207

GlcAT activity (Kim et al., 2003) GlcAT activity (Kim et al., 2003)

DmGlcAT-BSI DmGlcAT-BSII Hereditary multiple exostoses (EXT) protein Tout-velu ttv/DEXT1

CG10117

Sister of tout-velu

sotv/DEXT2

CG8433

Brother of tout-velu

botv/DEXTL3

CG15110

Sulfotransferases Heparan N-deacetylase/Nsulfotransferase/sulfateless

sfl

sfl

GlcNAcT-II and GlcAT-II activities ttv (Bornemann et al., 2004; Han (Izumikawa et al., 2006) et al., 2004a; Takei et al., 2004) RNAi (Ueyama et al., 2008) sotv (Bornemann et al., 2004; Han GlcNAcT-II and GlcAT-II activities (Izumikawa et al., 2006) et al., 2004a; Takei et al., 2004), RNAi (Ueyama et al., 2008) botv (Han et al., 2004a; Takei et al., GlcNAcT-I and GlcNAcT-II 2004) activities (Izumikawa et al., 2006; Kim et al., 2002) sfl (Lin and Perrimon, 1999)

(carbohydrate structure of sfl (Toyoda et al., 2000a)) (continued)

Table 15.1 (continued)

CG No.

Conventional mutants and RNAi

Identified activity (carbohydrate-structural analysis of mutants)

Hs2st

CG10234

Hs2st (Kamimura et al., 2006)

Heparan sulfate 6-Osulfotransferase

dHS6ST/Hs6st

CG4451

RNAi (Kamimura et al., 2001), Hs6st (Kamimura et al., 2006)

Heparan sulfate 3-Osulfotransferase-B Pipe

Hs3st-B

CG7890

RNAi (Kamimura et al., 2004)

pip

CG9614

pip (Nilson and Schupbach, 1998; Sen et al., 1998; Zhu et al., 2005)

HS2ST activity (Xu et al., 2007), carbohydrate structure of Hs2st (Kamimura et al., 2006) HS6ST activity (Kamimura et al., 2001), carbohydrate structure of Hs6st (Kamimura et al., 2006) HS3OST activity (Kamimura et al., 2004) carbohydrate structure of pip (Park et al., 2008)

Sugar-nucleotide transporters UDP-sugar transporter/ Fringe connection

frc

CG3874

frc (Goto et al., 2001; Selva et al., 2001)

ER GDP-fucose transporter

Efr

CG3774

Efr (Ishikawa et al., 2010)

PAPS transporter Slalom

sll

CG7623

PAPS transporter 2

dPAPST2/Papst2 CG7853

RNAi (Kamiyama et al., 2003), sll (Luders et al., 2003) RNAi (Goda et al., 2006)

Transferases or transporters

Abbreviated gene name

Heparan sulfate 2-Osulfotransferase

UDP-sugar transport activity (Goto et al., 2001; Selva et al., 2001), carbohydrate structure of frc (Goto et al., 2001; Selva et al., 2001) UDP-Xyl, UDP-GlcNAc and GDP-Fuc transport activities (Ishikawa et al., 2010) PAPS transport activity (Kamiyama et al., 2003; Luders et al., 2003) PAPS transport activity (Goda et al., 2006)

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The second step of GAG biosynthesis is the formation of unique sulfated disaccharide repeats, sulfated (GlcNAca1-4GlcAb1-4)n and sulfated (GalNAcb1-4GlcAb1-3)n for HS and CS, respectively (Fig. 15.2). After synthesis of the linkage region is complete, the GAG biosynthetic pathway divides to allow the synthesis of either HS or CS. The synthesis of the HS disaccharide repeats starts with the transfer of an N-acetylglucosamine (GlcNAc) residue to the linkage tetrasaccharide structure with an a1-4 linkage by DEXTL3 (Botv) (Kim et al., 2002) (Fig. 15.2 and Table 15.1). Then elongation of the disaccharide repeats occurs. Heterocomplexes of DEXT1 (Ttv) and DEXT2 (Sotv) are thought to polymerize the HS disaccharide repeats, because neither component of the heterocomplex can synthesize these repeats by itself (Izumikawa et al., 2006). Sulfation of the polymerized HS disaccharide repeats is carried out sequentially by sulfotransferases such as N-deacetylase/N-sulfotransferase (Lin and Perrimon, 1999; Toyoda et al., 2000a), C5 epimerase, HS 2-O-sulfotransferase (Hs2st) (Kamimura et al., 2006; Xu et al., 2007), HS 6-O-sulfotransferase (Hs6st) (Kamimura et al., 2001), and HS 3-O-sulfotransferase (Hs3st) (Kamimura et al., 2004) (Fig. 15.2 and Table 15.1). Currently, one Hs2st (Xu et al., 2007), one dHS6ST/Hs6st (Kamimura et al., 2001), and one Hs3st (Kamimura et al., 2004) have been identified in Drosophila on the basis of their activities. In agreement with the activities reported in vitro, 2-Osulfated and 6-O-sulfated disaccharide structures have been identified in Drosophila (Fig. 15.1) (Lawrence et al., 2008; Toyoda et al., 2000a,b; Yamada et al., 2002). However, 3-O-sulfated disaccharide structures have not yet been detected in Drosophila, which is probably owing to the very low levels of these structures in Drosophila cells. It is likely that the synthesis of CS disaccharide repeats is performed in a similar manner to that of the HS repeats and involves the action of glycosyltransferases and sulfotransferases. The first step in the synthesis of CS disaccharide repeats is the transfer of acetylgalactosamine (GalNAc) to the linkage tetrasaccharide structure with a b1-4 linkage by chondroitin N-acetylgalactosaminyltransferase (Fig. 15.2); this enzyme has not yet been identified in Drosophila. Then elongation of the disaccharide repeats starts. Polymerization of the CS disaccharide repeats appears to be carried out by heterocomplexes of chondroitin synthase and chondroitin polymerizing factor; this enzyme complex has also not been identified yet in Drosophila. Sulfation of the polymerized CS sugar chain seems to be performed by 4-O-sulfotransferase (Fig. 15.2). At present, none of the glycosyltransferases or sulfotransferases involved in the synthesis of CS disaccharide repeats in Drosophila have been identified on the basis of their activities, although enzymes that might play these roles can be identified by their high homology to human enzymes that have already been characterized. The Drosophila pipe gene encodes a protein that exhibits amino acid sequence similarity to Drosophila Hs2st and vertebrate HS2ST. However,

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although pipe acts as a sulfotransferase (Park et al., 2008; Zhu et al., 2005), it has no HS2ST activity (Xu et al., 2007). Sugar nucleotides are high-energy donor substrates for glycosyltransferases. They are synthesized in the cytosol and transported into the lumen of the endoplasmic reticulum and Golgi apparatus where glycosylation reactions occur. The phylogenetic tree of Drosophila and human sugarnucleotide transporters, including the 3’-phosphoadenosine 5’-phosphosulfate (PAPS) transporter, is shown in Fig. 15.3. The transporter activities of five types of Drosophila sugar-nucleotide transporter, the GDP-Fuc transporter (Gfr) (Ishikawa et al., 2005; Luhn et al., 2004), GDP-Fuc/UDP-Xyl/ UDP-GlcNAc transporter (Efr) (Ishikawa et al., 2010), UDP-Gal/UDPGalNAc transporter (dmUGT) (Segawa et al., 2002), UDP-sugar transporter (FRC) (Goto et al., 2001; Selva et al., 2001), and two PAPS transporters (SLL and dPAPST2) (Goda et al., 2006; Kamiyama et al., 2003; Luders et al., 2003) have been characterized. Among them, Efr, FRC, and the two PAPS transporters have been shown to contribute to the biosynthesis of GAG (Goda et al., 2006; Goto et al., 2001; Ishikawa et al., 2010; Kamiyama et al., 2003; Luders et al., 2003; Segawa et al., 2002) (Fig. 15.3 and Table 15.1). GAG biosynthesis is regulated spatiotemporally in various ways. For example, in Drosophila embryogenesis, during establishment of the gradient UDP-GlcNAc UDP-Xyl SLC35B4 (Hm) CG14511 (Dro) PAPS

SLL (Dro)

PAPS PAPST1 (Hm)

Efr (Dro)

UDP-Xyl GDP-Fuc UDP-GlcNAc

PAPST2 (Hm) PAPS dPAPST2 (Dro)

PAPS

CG5802 (Dro) UGTrel1 (Hm) CMP-SiaT(Hm) CMP-Sia

UDP-GlcNAc UDP-Glc UDP-GalNAc UDP-GlcA 0.1

hfrc1 (Hm) UGTrel7 (Hm)

dmUGT (Dro) UDP-Gal UDP-GalNAc UDP-GlcNAcT (Hm) UDP-GlcNAc UGT (Hm) UDP-Gal UDP-GalNAc

FRC (Dro) UDP-sugars Gfr (Dro) GDP-FucT (Hm) GDP-Fuc GDP-Fuc

Hm: Human Dro: Drosophila

Figure 15.3 Phylogenetic tree of Drosophila and human sugar-nucleotide transporters. Drosophila sugar-nucleotide transporters that have been shown to contribute to the biosynthesis of glycosaminoglycan are highlighted with rectangles. The reported activities of the sugar-nucleotide transporters are shown in gray.

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of bone morphogenetic protein (BMP) activity, which is crucial for dorsal embryonic patterning, HS biosynthesis is blocked by the translational control of GAG biosynthetic enzymes through internal ribosome entry sites (IRESs; Bornemann et al., 2008).

1.3. Core proteins of proteoglycans Three types of HS proteoglycan have been reported: the glypican, Syndecan (Sdc), and Perlecan families (Table 15.2). ‘‘Division abnormally delayed’’ (Dally; Nakato et al., 1995; Tsuda et al., 1999) and ‘‘Dally-like’’ protein (Dlp) (Belenkaya et al., 2004; Khare and Baumgartner, 2000; Kirkpatrick et al., 2004; Kreuger et al., 2004) are members of the glypican family and are linked to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. Sdc contains a transmembrane domain and is present on the cell surface (Brunner et al., 2006; Fox and Zinn, 2005; Johnson et al., 2006; Spring et al., 1994; Steigemann et al., 2004). Sdc carries both HS and CS (Chanana et al., 2009), whereas Dally and Dlp carry only HS. Perlecan, which is also known as ‘‘Terribly reduced optic lobes’’ (Trol), is a secreted protein that carries HS (Datta, 1995; Datta and Kankel, 1992; Park et al., 2003; Table 15.2). The expression and presentation of HS and CS seem to be controlled spatially and temporally by the regulated expression of the core proteins (Lin, 2004). Recently, the Hox selector gene Ultrabithorax has been reported to regulate the transcription of dally and modulate Decapentaplegic (Dpp) signaling during halter development (Crickmore and Mann, 2007). In addition, the tumor suppressor genes dachsous and fat regulate the transcription of dally and dlp during imaginal disc development through the Hippo signaling pathway (Baena-Lopez et al., 2008). Table 15.2 Core proteins of Drosophila proteoglycans

Protein

Abbreviated name CG no.

Dally

dally

Dallylike

dally-like (dlp)

Syndecan sdc

Perlecan

trol

Conventional mutants and RNAi

dally (Nakato et al., 1995; Tsuda et al., 1999) CG32146 dally-like (Belenkaya et al., 2004; Khare and Baumgartner, 2000; Kirkpatrick et al., 2004; Kreuger et al., 2004) CG10497 sdc (Brunner et al., 2006; Chanana et al., 2009; Fox and Zinn, 2005; Johnson et al., 2006; Spring et al., 1994; Steigemann et al., 2004) CG33950 trol (Datta, 1995; Datta and Kankel, 1992; Park et al., 2003) CG4974

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1.4. Function of proteoglycans There have been many studies of the biological functions of HS proteoglycans in D. melanogaster. HS proteoglycans play important roles in the regulation of fundamental developmental signaling pathways; for example, Hedgehog (Hh), Dpp (a BMP-type ligand that belongs to the TGFb family), Wingless (Wg), fibroblast growth factor (FGF), and Slit/Roundabout (Slit/Robo) signaling (Lee and Chien, 2004; Tabata and Takei, 2004; Yan and Lin, 2007, 2009). In addition to HS proteoglycans, it has been reported recently that CS on Sdc is required for Slit/Robo signaling (Chanana et al., 2009). Hh, Dpp, and Wg, which are secreted signaling molecules, function as morphogens during various stages of development (Bartscherer and Boutros, 2008; Eaton, 2006; Guerrero and Chiang, 2007; O’Connor et al., 2006; Wendler et al., 2006; Zhu and Scott, 2004). Morphogens are produced by a subset of cells in a tissue and are secreted to form concentration gradients that provide positional information for cell fate specifications (Lawrence and Struhl, 1996). In particular, the wing imaginal disc, which develops into an adult wing (Fig. 15.4A), provides a good model system for studying the movement of morphogens including Hh, Dpp, and Wg. These morphogens are essential for wing pattern formation. In the wing disc of third-instar larvae, Hh is synthesized in the posterior compartment under the control of Engrailed and moves into the anterior compartment to induce various target genes including dpp. Then Dpp functions as a longrange morphogen to influence both cell growth and patterning. In contrast, Wg is expressed at the dorsal/ventral border and acts as a long-range morphogen to determine dorsal/ventral patterning (Tabata and Takei, 2004; Yan and Lin, 2009; Fig. 15.4B). In the mutants ttv, sotv, and botv, DEXT1, DEXT2, and DEXTL3, respectively, are disrupted; these are enzymes that contribute to the elongation and polymerization of HS disaccharide repeats (Izumikawa et al., 2006; Kim et al., 2002) (Table 15.1). These mutants show defects in the distribution of Hh, Dpp, and Wg and in the expression patterns of their target genes (Bornemann et al., 2004; Han et al., 2004a; Takei et al., 2004). Moreover, the distribution patterns of these morphogens depend on the expression of the EXT genes, ttv, sotv, and botv, and none of these morphogens can pass through EXT mutant cells but instead accumulate outside them. These reports prove that the movements of Hh, Dpp, and Wg are mediated by diffusion that is restricted by HS on proteoglycans. Recently, fluorescence resonance energy transfer (FRET) microscopy analysis has demonstrated that Hh forms nanometer-sized oligomers that are colocalized with HS proteoglycans on the surface of cells that are expressing Hh (Vyas et al., 2008). Mutation of a Lys residue in the predicted Hh-protomer interaction interface disrupts the formation of these oligomers, and prevents Hh interacting with HS proteoglycans or participating in paracrine signaling.

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A

A

P

Larva

A

Pupa

P

Adult

B V

A

P

D Dpp

Hh A; Anterior

P; Posterior

Wg D; Dorsal

V; Ventral

Figure 15.4 Three types of morphogen involved in Drosophila wing development. (A) A wing imaginal disc develops into an adult wing. Larvae contain wing imaginal discs that develop into adult wings. During development from the larva to the pupa, the wing porch first protrudes from the wing disc and then it develops into the wing. Arrows indicate the direction of protrusion. (B) Hedgehog, Decapentaplegic, and Wingless in wing development. Hedgehog (Hh), Decapentaplegic (Dpp), and Wingless (Wg) are essential for wing development. In third-instar larvae, Hh is synthesized in the posterior compartment under the control of Engrailed and moves into the anterior compartment to induce the expression of various target genes including dpp. Dpp acts as long-range morphogen and contributes to cell growth and patterning. Wg is expressed at the dorsal/ventral border and functions as a long-range morphogen to determine dorsal/ ventral patterning. Gray arrows indicate the direction of movement of each morphogen.

Wg and Hh are modified by lipids, which result in a high affinity of the proteins for cell surface membranes and also modulate their distribution and restricted diffusion (Bartscherer and Boutros, 2008; Eaton, 2006; Guerrero and Chiang, 2007; Nusse, 2003; Wendler et al., 2006). Wg is palmitoylated at Cys residue 77, whereas Hh is modified by both palmitate and cholesterol (Nusse, 2003). As a result of this modification, Wg and Hh associate with lipoproteins. Lipoprotein particles are spherical macromolecular particles

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that consist of a core of neutral lipids, including triglycerides and cholesterol ester, and an amphipathic shell of polar phospholipids and cholesterol (Willnow et al., 2007). Insects synthesize similar particles called lipophorins, which act as vehicles for the long-range transport of lipid-linked morphogens, such as Wg and Hh, and GPI-anchored proteins (Panakova et al., 2005), and also as providers of lipids that are required for normal patchedmediated destabilization of Smoothened (Khaliullina et al., 2009). Lipophorins also interact with HS on the GPI-anchored proteins, Dally and Dlp (Eugster et al., 2007), which suggests that restriction of diffusion by HS might partially regulate lipophorin vehicles. In addition to lipoproteins, many extracellular regulatory factors bind to HS proteoglycans and affect the distribution, restricted diffusion, and signaling of morphogens. The secreted BMP-binding protein Crossveinless 2 can bind to HS proteoglycans and enhances or inhibits BMP signaling (Serpe et al., 2008). Two HS-binding proteins, Shifted (Shf) and Interference hedgehog (Ihog), are part of the Hh signaling pathway. Shf is an ortholog of the secreted vertebrate protein Wnt Inhibitory Factor-1 (Wif1) and is required for the stability and movement of Hh (Glise et al., 2005; Gorfinkiel et al., 2005). Ihog, which is a member of the conserved Ig/ fibronectin superfamily, is a Hh-binding protein that mediates the response of cells to the active Hh signal (McLellan et al., 2006; Yao et al., 2006). As mentioned above, HS is present on various core proteins, such as Dally, Dlp, Sdc, and Perlecan (Table 15.2). Flies with mutations that affect proteoglycans show milder phenotypic defects than those with mutations that affect enzymes contributing to HS synthesis, which suggests that the spatial and temporal expressions of various core proteins modulate the spatial and temporal distributions of HS (Lin, 2004). Dally and Dlp, which are members of the glypican family, contribute to the distribution and/or stability of Dpp and Hh in wing imaginal discs (Akiyama et al., 2008; Belenkaya et al., 2004; Fujise et al., 2003; Han et al., 2004b; Hsiung et al., 2005; Zhu and Scott, 2004). Dally has been reported to retain some ability to bind Dpp in the absence of HS, which suggests that it might support the formation of low threshold signaling complexes (Kirkpatrick et al., 2006). Dally and Dlp also contribute to the formation of the Wg gradient in the wing imaginal disc (Desbordes et al., 2005; Franch-Marro et al., 2005; Han et al., 2005; Marois et al., 2006). However, Dlp shows biphasic activity with respect to Wg morphogen signaling: it represses short-range Wg signaling but activates long-range Wg signaling (Baeg et al., 2004; Kirkpatrick et al., 2004; Kreuger et al., 2004; Yan et al., 2009). Notum, which is a secreted hydrolase, also regulates the distribution of Wg through Dlp (Kirkpatrick et al., 2004; Kreuger et al., 2004). However, the biphasic activity of Dlp can be explained without the contribution of Notum by an exchange factor model: Dlp can either compete with the Wg receptor Frizzled 2 (Fz2) or provide the Wg ligand to Fz2, depending

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on the ratios of Wg, Fz2, and Dlp (Yan et al., 2009). In addition, analysis of the dynamic distribution of Dlp in living disc tissue has shown that endocytosis of Dlp is essential for its positive role in Hh signaling and that transcytosis of Dlp is required for the distribution of Wg (Gallet et al., 2008). Sdc and Dlp function in neurogenesis, for example, in axon guidance and synaptogenesis (Fox and Zinn, 2005; Johnson et al., 2004, 2006; Lee and Chien, 2004; Rawson et al., 2005; Ren et al., 2009; Steigemann et al., 2004). One signaling pathway that is important for axon guidance is Slit/Robo signaling. Slit is the ligand for the Robo receptor (Lee and Chien, 2004) and acts as the main repellent signal at the midline (Kidd et al., 1999). HS/ heparin is suggested to bind to the basic surface that extends across the Slit/ Robo interface and five HS disaccharide units are required to support Slit/ Robo signaling (Fukuhara et al., 2008). Sdc, which is modified by both HS and CS, is essential for midline axon guidance (Johnson et al., 2004; Steigemann et al., 2004). Sdc is an indispensable component of the Slit/ Robo signaling pathway and is required in Slit target cells. Dlp is also required for correct axon guidance and for the function of the visual system (Rawson et al., 2005). In addition to axon guidance, Sdc and Dlp are essential for synaptogenesis (Fox and Zinn, 2005; Johnson et al., 2006; Ren et al., 2009). The formation and plasticity of synaptic connections depend on regulatory interactions between pre- and postsynaptic cells. Sdc promotes the growth of presynaptic terminals, whereas Dlp regulates the formation and function of the active zone. Both Sdc and Dlp are high affinity ligands of the protein tyrosine phosphatase LAR, which controls both growth of the neuromuscular junction and morphogenesis of the active zone. Sdc promotes LAR activity, whereas Dlp inhibits it, through binding to LAR. The secreted proteoglycan Perlecan regulates the Hh, FGF, VEGF/ PDGF, Wg, and Dpp signaling pathways at various stages of development (Lindner et al., 2007). For example, Perlecan regulates neuroblast division in the developing nervous system by modulating both FGF and Hh signaling (Park et al., 2003). Follicle cells in the Drosophila ovary provide a good in vivo model of epithelial polarity. Perlecan is a component of the basal extracellular matrix (ECM) and dystroglycan (Dg) is an ECM receptor. Both Perlecan and Dg are required for the maintenance of epithelial polarity under energetic stress (Mirouse et al., 2009; Schneider et al., 2006). Very recently, the involvement of Dally and Dlp in the germline stem cell (GSC) niche has been described (Guo and Wang, 2009; Hayashi et al., 2009). Dally is expressed in the cap cells of the GSC niche in the Drosophila ovary, where it traps BMP and induces a high BMP response over a singlecell range to determine GSC fate in the niche (Guo and Wang, 2009; Hayashi et al., 2009). In contrast, Dlp is expressed in the hub cells of the GSC niche in the Drosophila testis and plays a major role in the maintenance of GSCs in this niche, although the signal that is responsible for this is not known (Hayashi et al., 2009).

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Sulfation is a key modification for the functions of HS and CS proteoglycans. Analyses of three types of HS sulfotransferase, N-deacetylase/Nsulfotransferase, 6-O-sulfotransferase and 3-O-sulfotransferase, have been performed using conventional mutants or RNA interference (RNAi) flies (Table 15.1). N-Deacetylase/N-sulfotransferase is encoded by sulfateless, which was identified as a segment polarity gene in Drosophila (Lin and Perrimon, 1999), and functions in Wg, Hh, Dpp, and FGF signaling (Bornemann et al., 2004; Han et al., 2004a; Lin and Perrimon, 1999; Lin et al., 1999; Takei et al., 2004). In contrast, dHS6ST/Hs6st and Hs2st are expressed in embryonic tracheal cells, and the corresponding mutants show defects that are similar to those of mutants of the Drosophila FGF receptor, breathless. This indicates that dHS6ST/Hs6st and Hs2st are required for tracheal development and that they act through the FGF signaling pathway (Kamimura et al., 2001, 2006). Recently, Hs3st-B, which is one of two putative Drosophila HS3STs/Hs3sts, was analyzed biochemically and developmentally using RNAi flies (Kamimura et al., 2004). Surprisingly, Hs3stB, which catalyzes HS 3-O sulfation, is involved in Notch signaling by affecting the stability or intracellular trafficking of the Notch protein.

2. Identification of Drosophila Glycosyltransferases and Sugar-Nucleotide Transporters that Contribute to Proteoglycan Synthesis In this section, I describe methods to clone and express genes that encode glycosyltransferases (Takemae et al., 2003; Ueyama et al., 2008) and sugar-nucleotide transporters, including PAPS transporters (Goda et al., 2006; Kamiyama et al., 2003), and to identify their glycosyltransferase or sugar-nucleotide transporter activities.

2.1. Cloning and expression of Drosophila glycosyltransferases First, a BLAST search of all available Drosophila databases is performed using the amino acid sequence of a human glycosyltransferase that is known to be involved in proteoglycan synthesis as the query sequence. This allows the identification of Drosophila glycosyltransferase homologs. The Drosophila orthologs of human glycosyltransferase are verified by constructing a phylogenetic tree for the proteins. The putative catalytic region of each candidate glycosyltransferase (with the transmembrane region deleted) is cloned using a cDNA library generated from Drosophila embryos and expressed as a secreted protein fused with

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a FLAG peptide in Sf21 insect cells using GATEWAYTM Cloning Technology (Invitrogen, Carlsbad, CA) in the following manner (Takemae et al., 2003; Ueyama et al., 2008). First, a DNA fragment that corresponds to the putative catalytic region is amplified by two-step PCR. In the first round of PCR, the glycosyltransferase cDNA is amplified from the library using the forward primer 50 -AAAAAGCAGGCT-(gene-specific sequence)-30 and the reverse primer 50 -AGAAAGCTGGGT-(gene-specific sequence)-30 . In the second PCR step, the product of the first PCR is used as the template, and it is amplified with the forward primer 50 -GGGGACAAGTTTGTACAAAAAAGCAGGCT-30 and the reverse primer 50 GGGGACCACTTTGTACAAGAAAGCTGGGT-30 . The amplified fragment is inserted into the pDONRTM201 vector (Invitrogen) by recombination. Then the insert is transferred between the attR1 and attR2 sites of pVL1393-FLAG to generate the construct pVL1393-FLAG-glycosyltransferase. pVL1393-FLAG is an expression vector derived from pVL1393 (BD Biosciences, San Diego, CA) and it contains a fragment that encodes the signal peptide of human immunoglobulin k (MHFQVQIFSFLLISASVIMSRG) and the FLAG peptide (DYKDDDDK) (Takemae et al., 2003). Then the construct pVL1393-FLAG-glycosyltransferase is cotransfected with BaculoGold viral DNA (BD Biosciences) into Sf21 insect cells, and the cells are incubated for 5 days at 25  C to produce recombinant virus. Sf21 cells are infected with the recombinant virus at a multiplicity of infection of five and incubated for 5 days to yield conditioned media that contains recombinant glycosyltransferase protein fused with the FLAG peptide. CaCl2 is added to the culture medium at a final concentration of 10 mM, and then the culture medium is mixed with Anti-FLAG M1 Agarose Affinity Gel (Sigma, St. Louis, MO). The protein–gel mixture is washed twice with TBS (50 mM Tris–HCl, pH 7.4, and 150 mM NaCl) containing 10 mM CaCl2 and eluted competitively with 100 mg/ml FLAG peptide (Sigma) in TBS containing 10 mM CaCl2. To determine the concentration of the purified glycosyltransferase protein, it is subjected to SDS–polyacrylamide gel electrophoresis followed by Western blot analysis using anti-FLAG M2-peroxidase conjugate (Sigma) and stained with Konica Immunostaining HRP-1000 (Konica, Tokyo, Japan). The amount of glycosyltransferase protein is determined by measuring the intensity of the positive band using a densitometer, and comparing the intensity to that obtained with a FLAG-BAP control protein (Sigma).

2.2. Assay of glycosyltransferase activity To measure glycosyltransferase activity, acceptor substrates that include Xyla-pNph, Xylb-pNph, GlcNAca-pNph, GlcNAcb-pNph, GlcNAcbS-pNph, Glca-pNph, Glcb-pNph, Gala-pNph, Galb-pNph, GalNAcapNph, GalNAcb-pNph, Mana-pNph, Fuca-pNph, Galb1-4GlcNAca-

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pNph, Galb1-3GalNAca-pNph, GlcNAcb1-6GalNAca-pNph, Galb1-3 (GlcNAcb1-6)GalNAca-pNph, Galb1-4Xylb1-pMph, and Galb1-3 Galb1-4Xylb1-pMph are used (Takemae et al., 2003; Ueyama et al., 2008). Using each acceptor at a concentration of 200–500 mM, the glycosyltransferase reaction is performed at 25  C for 2–4 h in 20 ml of a reaction mixture that contains the appropriate buffer (with a pH that is near the optimum pH for each glycosyltransferase reaction assayed), 10 mM MnCl2, 5 mM radiolabeled sugar nucleotide, 100–250 mM unlabeled sugar nucleotide, and 2 pmol of purified glycosyltransferase. The glycosyltransferase reaction is terminated by the addition of 400 ml of ice-cold water. After centrifugation of the reaction mixture, the supernatant is applied to a SepPakC18 column (Millipore, Billerica, MA) that has been equilibrated with water. The unreacted sugar nucleotide is washed out with water, and the product is eluted with methanol. The radioactivity of the eluate is measured using a liquid scintillation counter.

2.3. Cloning and expression of Drosophila sugar-nucleotide transporters including PAPS transporters Again a BLAST search of all available Drosophila databases is performed, in this case using the amino acid sequence of a human sugar-nucleotide transporter that contributes to proteoglycan synthesis as the query sequence, in order to identify Drosophila sugar-nucleotide transporter homologs. The Drosophila orthologs of the human sugar-nucleotide transporter are verified by constructing a phylogenetic tree for the proteins. Each candidate sugar-nucleotide transporter is cloned from a cDNA library generated from Drosophila embryos and expressed as a hemagglutinin (HA)-tagged protein in Saccharomyces cerevisiae strain W303-1a (MATa, ade2-1, ura3-1, his3-11,15, trp1-1, leu2-3,112, and can1-100) using GATEWAYTM Cloning Technology (Invitrogen) in the following manner (Goda et al., 2006; Kamiyama et al., 2003). A DNA fragment that corresponds to the full length coding region is amplified by two-step PCR. In the first round of PCR, the sugar-nucleotide transporter cDNA is amplified from the library using the forward primer 50 -AAAAAGCAGGCT-(gene-specific sequence)-30 , and the reverse primer 50 -AGAAAGCTGGGT-(gene-specific sequence)-30 . In the second PCR step, the product from the first reaction is used as the template, and is amplified with the forward primer 50 -GGGGACAAGTTTGTACAAAAAAGCAGGCT-30 , and the reverse primer 50 -GGGGACCACTTTGTACAAGAAAGCTGGGT-30 . The amplified fragment is inserted into the pDONRTM201 vector (Invitrogen) by recombination. Then the insert is transferred between the attR1 and attR2 sites of the yeast expression vector YEp352GAP-II-HA to generate YEp352GAP-II-sugar-nucleotide-transporter-HA. YEp352GAP-II-HA is a yeast expression vector derived from YEp352GAP-II (Nakayama et al.,

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2003) and contains three influenza HA epitope tags at the position that corresponds to the C terminus of the protein to be expressed.

2.4. Preparation of subcellular fractions of yeast and assay of sugar-nucleotide transport activity S. cerevisiae is used widely to express sugar-nucleotide transporters because the isolated microsomal vesicles have little sugar-nucleotide transport activity, except for that of GDP-mannose. S. cerevisiae strain W303-1a is transformed with the YEp352GAP-II-sugar-nucleotide-transporter-HA by the lithium acetate procedure. The transformed yeast cells are grown at 25  C in a synthetic defined medium, which does not contain uracil, to select transformants. Subcellular fractionation and sugar-nucleotide transport assays are performed as follows: 1. The transformed yeast cells are harvested, washed with ice-cold 10 mM NaN3, and converted into spheroplasts by incubation at 37  C for 20 min in spheroplast buffer (1.4 M sorbitol, 50 mM potassium phosphate pH 7.5, 10 mM NaN3, 40 mM 2-mercaptoethanol, and 1 mg of zymolyase 100T/g of cells). The spheroplasts are pelleted using a refrigerated centrifuge and washed twice with 1.0 M ice-cold sorbitol to remove the traces of zymolyase. 2. The spheroplasts are suspended in ice-cold lysis buffer (0.8 M sorbitol, 10 mM triethanolamine pH 7.2, 5 mg/ml of pepstatin A, and 1 mM phenylmethylsulfonyl fluoride) and homogenized using a Dounce homogenizer with 20–40 strokes. The lysate is centrifuged at 1000g for 10 min at 4  C to remove the unlysed cells and cell wall debris. 3. The supernatant is centrifuged at 10,000g for 15 min at 4  C, and the resulting pellet corresponds to the P10 membrane fraction. The supernatant is further centrifuged at 100,000g to yield a pellet that corresponds to the P100 Golgi-rich membrane fraction. 4. Each membrane fraction (100 mg of protein) is incubated in 50 ml of reaction buffer (20 mM Tris–HCl pH 7.5, 0.25 M sucrose, 5.0 mM MgCl2, 1.0 mM MnCl2, and 10 mM 2-mercaptoethanol) that contains each radiolabeled sugar-nucleotide substrate at a concentration of 1 mM at 25  C for 5 min. 5. After incubation, the radioactivity incorporated into the microsomes is trapped using a 0.45-mm nitrocellulose filter and measured using a liquid scintillation counter. The amount of radioactivity incorporated is calculated as the difference between the value for an individual sample and the background value obtained from the sample taken at 0 min for the same assay.

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6. In order to confirm the expression of the HA-tagged sugar-nucleotide transporter, each membrane fraction is suspended in 3 SDS sample buffer (New England Biolabs, Inc., Ipswich, MA) and incubated at 4  C for 16 h. The samples are then subjected to SDS–polyacrylamide gel electrophoresis and Western blot analysis. The HA-tagged sugar-nucleotide transporters are immunostained with mouse anti-HA monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and horseradish peroxidase-conjugated anti-mouse IgG.

3. Establishment and Functional Analysis of RNAi Flies for the Glycosyltransferases and Sugar-Nucleotide Transporters that Contribute to Proteoglycan Synthesis 3.1. Establishment of RNAi flies RNAi is an evolutionarily conserved phenomenon in which doublestranded RNA (dsRNA) induces gene silencing (Almeida and Allshire, 2005; Fire et al., 1998; Kavi et al., 2005). RNAi is thought to originate from an ancient endogenous defense mechanism against viral and other heterologous dsRNA (Plasterk, 2002; Waterhouse et al., 2001). Recently, RNAi has become a powerful reverse genetics tool for studying gene function in many model organisms, including plants, Caenorhabditis elegans, and D. melanogaster (Giordano et al., 2002; Ueda, 2001). In Drosophila, injection of a dsRNA that corresponds to a particular gene into an embryo can induce the degradation of its mRNA. This phenomenon might prove to be useful for the functional analysis of genes during embryogenesis (Kamimura et al., 2001; Kohyama-Koganeya et al., 2004). However, the RNAi effect is transient and is not inherited stably. The use of this method to investigate gene function in the later stages of development is limited. Therefore, we established a heritable and inducible RNAi knockdown system in Drosophila using the GAL4-upstream activating sequence (UAS) system, a scheme for which is shown in Fig. 15.5 (Nishihara et al., 2004; Takemae et al., 2003; Ueda, 2001). Two transgenic fly lines, the GAL4 driver and UAS-inverted repeat (UAS-IR), are used. The GAL4 driver fly contains a transgene that encodes the yeast transcription factor GAL4 and whose expression is controlled by a tissue-specific promoter. The UAS-IR fly has a transgene that contains an inverted repeat (IR) of the target gene ligated to the UAS, which is the target of GAL4. When these flies are crossed, the expression of dsRNA that corresponds to the target gene is controlled in the F1 generation by a tissue-specific promoter, which results in gene silencing.

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Tissue specific promoter GAL4 UAS (Upstream Inverted repeat (IR) of target gene activating sequence)

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5′ primer: 5′-AAGGCCTACATGGCCGGACCG- (cDNA 21bp)-3′ 3′ primer: 5′-AATVTAGAGGTACC- (cDNA 21bp)-3′

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Figure 15.5 Establishment of RNAi flies. (A) Schema of the heritable and inducible RNAi system. Two transgenic fly lines—GAL4 driver and UAS-IR—are used. The GAL4 driver fly contains a transgene that encodes the yeast transcriptional factor GAL4,

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The experimental procedure is as follows: (1) Approximately 500 bp of the target gene sequence is selected using dsCheck (http://dscheck.rnai.jp/); this highly sensitive search software selects sequences to avoid off-target effects that might result from dsRNA-mediated RNAi. In Drosophila, long dsRNAs efficiently induce gene-specific silencing without inducing the interferon response, whereas in mammalian cells only siRNAs (short interfering RNAs) can efficiently suppress target gene expression (Giordano et al., 2002; Ueda, 2001). To ensure that the phenotypes observed are not due to off-target effects, two different sequences are selected for each target gene and two types of UAS-IR fly line are prepared. (2) The target gene sequence is amplified by PCR from a cDNA library or from an expressed sequence tag clone (Fig. 15.5B). The following PCR primer set is used for this procedure: 50 primer, 50 -AAGGCCTACATGGCCGGACCG-(21 bp of target gene sequence)-30 , which includes a SfiI site and a CpoI site; 30 primer, 50 -AATCTAGAGGTACC-(21 bp of target gene sequence)-30 , which includes a KpnI site and an XbaI site. (3) The resulting PCR fragment is inserted as an IR sequence into the pUAST-R57 vector (accession number: AB233207) at the abovementioned restriction enzyme sites (Fig. 15.5B). pUAST-R57 is a modified pUAST vector that is used to clone IRs of the target gene sequence. A fragment that corresponds to exons 5–7 of the fly Ret oncogene is placed between the insertion sites of the IRs. (4) The resulting construct is injected into Drosophila embryos of the w1118 stock to establish the UAS-IR transgenic fly lines. Fifty nanograms of the UAS-IR vector DNA and 50 ng of helper (phs-pai) DNA are transformed into 30 fly eggs. Approximately half of the transformed eggs develop to the adult stage. The website http://www.shigen.nig. ac.jp/fly/nigfly/about/aboutRnai.jsp describes the details of the mating scheme. (5) Flies from the UAS-IR fly line are then mated with flies from the appropriate GAL4 driver fly lines. The F1 progeny are grown at 25

whose expression is controlled by a tissue-specific promoter. In contrast, the UAS-IR fly has a transgene that contains an inverted repeat (IR) of the target gene ligated to the UAS, which is the target of GAL4. When these fly lines are crossed, dsRNA that represents the target gene is expressed in a tissue-specific manner and induces gene silencing in the F1 generation. (B) Construction of the pUAST-R57 vector. pUAST-R57 is a modified pUAST vector that is used to clone IRs of the target gene sequence. A fragment that comprises exons 5–7 of the fly Ret oncogene is placed between the insertion sites of the IRs. (Modified from Ueda R, the Fly Stocks of National Institute of Genetics [NIG-FLY] website: http://www.shigen.nig.ac.jp/fly/nigfly/about/aboutRnai.jsp.)

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or 28  C, and their phenotypes are observed. Usually, stronger RNAi effects are observed in the F1 progeny grown at 28  C than in those grown at 25  C (Ichimiya et al., 2004) because the binding of GAL4 to the UAS sequence is stronger at 28  C than at 25  C. RNAi flies for the following glycosyltransferases and sugar-nucleotide transporters have been generated using the GAL4/UAS system: O-xylosyltransferase (dXylT) (Goda et al., 2006), b4GalTI (Nakamura et al., 2002; Takemae et al., 2003), b3GalTII (Ueyama et al., 2008), ttv/DEXT1 (Ueyama et al., 2008), sotv/DEXT2 (Ueyama et al., 2008), 6-O-sulfotransferase (Kamimura et al., 2006), 3-O-sulfotransferase (Kamimura et al., 2004), slalom (sll) (Kamiyama et al., 2003), PAPS transporter 2 (PAPST2) (Goda et al., 2006), and PAPS synthase (Goda et al., 2006) (Table 15.1). All these transferases and transporters are involved in proteoglycan synthesis.

3.2. Functional analyses of RNAi flies Various types of functional analysis can be performed using RNAi flies. Here viability tests, genetic interaction analysis, and the analysis of morphogenetic signals are described (Goda et al., 2006; Kamimura et al., 2001, 2004, 2006; Nakamura et al., 2002; Takemae et al., 2003; Ueyama et al., 2008). The heritable and inducible RNAi system can be used to perform tissuespecific knockdown of genes (Fig. 15.5A). When UAS-IR fly lines are crossed with the Act5C-GAL4 fly line, which expresses the yeast transcription factor GAL4 under the control of the cytoplasmic actin promoter, it is expected that gene silencing will be induced in all cells at all developmental stages in the F1 flies, Act5C-GAL4 > UAS-IR. Viability can be estimated from the proportion of F1 flies that develop to adulthood (Goda et al., 2006; Kamiyama et al., 2003; Takemae et al., 2003; Ueyama et al., 2008). Even when UAS-IR fly lines result in Act5C-GAL4 > UAS-IR flies that are not viable, tissue-specific phenotypes can be observed by crossing these UAS-IR fly lines with different GAL4 fly lines that express GAL4 under the control of a variety of tissue-specific promoters, for example glass multiple reporter (GMR)-GAL4 and eyeless (ey)-GAL4 for the eye and MS1096GAL4, A9-GAL4, 29BD-GAL4, engrailed (en)-GAL4, patched (ptc)GAL4, scalloped (sd)-GAL4, and apterous (ap)-GAL4 for the wing. Many defects are observed in the RNAi flies for the transferases and transporters that are involved in proteoglycan synthesis. To determine the in vivo function of these genes, genetic interactions between pairs of genes can be investigated, using these defects as indicators (Goda et al., 2006; Ueyama et al., 2008). For example, genetic interaction analyses for two types of Drosophila PAPS transporter gene, sll and dPAPST2, are shown in Fig. 15.6A. Double knockdown RNAi flies in which sll or dPAPST2 was knocked down in conjunction with dHS6ST, Hs3st-B, or dXylT showed

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Figure 15.6 Functional analysis of the RNAi flies. (A) Genetic interactions among the genes sll, dPAPST2, dHS6ST, Hs3st-B, and dXylT in the eye. Eyes of adult flies were observed by scanning electron microscopy. A representative eye of a fly with each genotype is shown. GMR, sll, dPAPST2, dHS6ST, Hs3st-B, dXylT, and EGFP represent GMR-GAL4, UAS-sll-IR, UAS-dPAPST2-IR, UAS-dHS6ST-IR, UAS-Hs3st-B-IR,

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enhanced rough-eye phenotypes as compared with flies in which only sll or dPAPST2 had been knocked down. This indicates that both SLL and dPAPST2 contribute to the sulfation of HS proteoglycans in vivo. In this analysis, the double knockdown RNAi flies contain two UAS-IR transgenes. If the single knockdown RNAi flies contain only one UAS-IR transgene, there will be twice as much GAL4 available to bind to the UAS sequence of the UAS-IR transgene, therefore twice as much doublestranded RNA should be produced, and the amount by which the mRNA of the target gene is reduced should be twice that by which the mRNA is reduced in the double knockdown RNAi flies. To avoid this difference, the UAS-EGFP transgene is incorporated into the single knockdown RNAi flies. Therefore the single knockdown RNAi flies now contain the same number of UAS sequences as the double knockdown flies, namely one UAS-IR and one UAS-EGFP gene. As mentioned in Section 1, three types of morphogen, Hh, Dpp, and Wg are essential for wing development (Fig. 15.4). The effect of the glycosyltransferases and transporters that are involved in proteoglycan synthesis on the distributions of these morphogens can be analyzed using RNAi flies for each gene. One example is shown in Fig.15.6B (Ueyama et al., 2008). Wg is normally expressed at the wing margin, namely, at the boundary between the dorsal and ventral sides. Expression of the enGAL4 transgene induces RNAi in the posterior region of the wing disc in third-instar larva. When UAS-(db3GalTII)-IR flies were crossed with enGAL4 flies, the progeny showed decreased amounts of extracellular Wg in the posterior region of the wing disc. This indicates that the decrease in HS proteoglycan expression, which results from decreased formation of the linkage structure, affects the distribution of extracellular Wg.

UAS-dXylT-IR, and UAS-EGFP, respectively. Double knockdown RNAi flies between sll or dPAPST2 and dHS6ST, Hs3st-B, or dXylT, that is, GMR-GAL4 > UAS-sll-IR, UAS-dHS6ST-IR; GMR-GAL4 > UAS-sll-IR, UAS-Hs3st-B-IR; GMR-GAL4 > UASsll-IR, UAS-dXylT-IR; GMR-GAL4 > UAS-dPAPST2-IR, UAS-dHS6ST-IR; GMRGAL4 > UAS-dPAPST2-IR, UAS-Hs3st-B-IR; and GMR-GAL4 > UAS-dPAPST2-IR, UAS-dXylT-IR, showed the rough-eye phenotype, whereas single knockdown RNAi flies for each gene, GMR-GAL4 > UAS-sll-IR, UAS-EGFP; GMR-GAL4 > UASdPAPST2-IR, UAS-EGFP; GMR-GAL4 > UAS-dHS6ST-IR, UAS-EGFP; GMR-GAL4 > UAS-Hs3st-B-IR, UAS-EGFP; and GMR-GAL4 > UAS-dXylT-IR, UAS-EGFP, showed no aberrant phenotypes. UAS-EGFP is used to adjust the number of UAS sequences between the double knockdown and single knockdown flies. (B) Decrease in extracellular Wingless (Wg) in the wing imaginal disc of the third-instar larva of proteoglycanb1-3galactosyltransferase II (db3GalTII) RNAi flies. en and db3GalTII-IR represent en-GAL4 and UAS-db3GalTII-IR, respectively. en-GAL4 > UAS-EGFP shows the expression of en-GAL4 in the posterior region of the wing disc. In en-GAL4 > UASdb3GalTII-IR, RNAi is induced in the posterior region of the wing disc, where extracellular Wg staining is decreased.

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In addition to the two examples described above, many other examples of functional analyses of proteoglycans using RNAi flies have also been reported, as mentioned in Section 1.

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O-GlcNAc Modification of the Extracellular Domain of Notch Receptors Yuta Sakaidani, Koichi Furukawa, and Tetsuya Okajima Contents 1. Overview 2. Preparation of Recombinant EGF Domains by Yeast Expression System 2.1. Screening 2.2. Purification of EGF20 3. In Vitro O-GlcNAc Transferase Assay 3.1. Preparation of membrane fraction proteins 3.2. O-GlcNAc transferase assay 4. Mass Spectrometry 4.1. Purification of EGF domains from S2 culture media 4.2. Reduction, S-carbamidomethylation, and trypsin digestion 4.3. MALDI-TOF-MS and MS/MS analysis 5. Detection of O-b-GlcNAc Modification Using Antibodies 5.1. Immunoblotting with CTD110.6 antibody 6. Galactosyltransferase Labeling 6.1. b4GalT-1 assay 6.2. Lectin blotting 7. Hexosaminidase Treatment 7.1. Deglycosylation of glycoproteins 7.2. Deglycosylation of glycopeptide 8. Conclusions and Future Directions Acknowledgments References

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Abstract Epidermal growth factor (EGF) domains are posttranslationally modified with unique O-linked glycans. The classical types of O-glycans on EGF domains are O-fucose and O-glucose glycans, found on many plasma glycoproteins and Department of Biochemistry II, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, Japan Methods in Enzymology, Volume 480 ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)80016-3

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signaling molecules, whose biological functions have been demonstrated especially in the context of the Notch signaling pathway. We recently discovered O-GlcNAc modification as a new modification of the EGF domain that occurs on the conserved Ser/Thr residue located between the fifth and sixth cysteine residues within the EGF domain of Notch receptors in Drosophila. Here, we describe the methods employed to detect the O-GlcNAc modification of EGF repeats of Notch receptors. These methods include mass spectrometric analysis, galactosyltransferase labeling, immunoblotting with a specific antibody, and b-N-acetyl-hexosaminidase digestion experiments. We also describe a method to detect O-GlcNAc transferase activity from crude membrane fraction proteins prepared from cultured S2 cells.

1. Overview Diverse posttranslational modifications influence the function and structure of many secreted and transmembrane glycoproteins. Besides the commonly observed types of glycosylation, several unusual glycans have been identified, which occur on certain protein domains (Shao and Haltiwanger, 2003). An example is the epidermal growth factor (EGF) domain that is characterized as a small domain of 30–40 amino acids stabilized by three intramolecular disulfide bonds between Cysteins 1 and 3, 2 and 4, and 5 and 6, thereby forming two antiparallel b-sheets at the amino and carboxyl terminal subdomains (Montelione et al., 1986). Four types of posttranslational modifications have been found at distinct sites on the EGF domains: b-hydroxylation of Asp or Asn residues (Dinchuk et al., 2002), O-linked glucose (O-glucose), and O-linked fucose (O-fucose; Harris and Spellman, 1993), and O-GlcNAc modification (Matsuura et al., 2008; Fig. 16.1A). O-Glucose was discovered as an unknown serine derivative located between the first and second cysteine residues within the first EGF domain of bovine factor VII (Hase et al., 1988; Takeya et al., 1988). The substitution of Ser by Ala at the O-glucosylation site of Factor VII implicated the functional importance of O-glucose modification in blood coagulation (Bjoern et al., 1991). O-Glucose was subsequently identified in bovine and human Factor IX (Hase et al., 1988; Nishimura et al., 1989) and Protein Z (Nishimura et al., 1989). The carbohydrate structure of the completely elongated form has been reported as the trisaccharide Glc-Xyl-Xyl (Hase et al., 1990). O-Glucose was also found in bovine thrombospondin (Nishimura et al., 1992b), Pref-1 (Krogh et al., 1997), and Notch receptors (Moloney et al., 2000b) as described in Chapter 17. O-Fucose was first discovered in human urinary plasminogen activator (uPA; Buko et al., 1991; Kentzer et al., 1990) and was implicated in uPAinduced signaling (Rabbani et al., 1992). Many plasma glycoproteins are reported to be O-fucosylated, which include human uPA (Buko et al.,

357

O-GlcNAc Modification of EGF Domains

Xyl

B

NH2 Xyl

400

Glc Fuc

GlcNAc Gal

Cys Ser or Thr

GlcNAc COOH

Sia

[3H]GlcNAc (dpm)

A

300 200 100 0

+

– Mn2+

Figure 16.1 Detection of O-GlcNAc transferase activity using membrane fraction proteins. (A) The structure of O-glycans on the EGF domains. O-fucosyltransferase 1, fringe-related genes, b-1,4-galactosyltransferase and sialyltransferase synthesize the tetrasaccharide, O-fucose-GlcNAc-Gal-Sia. O-Fucose-GlcNAc disaccharide was detected in Drosophila, and interestingly, it has recently been reported that a novel glucuronyl trisaccharide structure, GlcNAc-b1-3-(GlcAb1-4)-fucitol, was found in glycans released from glycoproteins of Drosophila embryos (Aoki et al., 2008). Three different glycosyltransferases such as O-glucosyltransferase (Acar et al., 2008), O-glucose a-1,3-xylosyltransferase (Sethi et al., 2010), and xylose a-1,3-xylosyltransferase (Ishimizu et al., 2007; Minamida et al., 1996; Omichi et al., 1997) are involved in the biosynthesis of the Oglucose trisaccharide. The consensus sequence for O-glucosylation is predicted to be C1XSXPC2 (C1 and C2 are the first and second conserved cysteines and X is any amino acid; Nishimura et al., 1992b). The consensus sequence for O-fucosylation is proposed to be C2XXX(A/G/S)S/TC3 (C2 and C3 are the second and third cysteines; X is any amino acid; Haines and Irvine, 2003). O-GlcNAc was reported as a monosaccharide and the consensus sequence for O-GlcNAc modification is currently unknown. (B) In vitro glycosylation assays. O-GlcNAc transferase activity was measured using S2 cell membrane fraction proteins, recombinant EGF20 and UDP-[3H]GlcNAc in the presence or absence of Mn2þ. The experimental values have been adjusted by subtracting the background value obtained without acceptor substrates. Bar, S.D. (n ¼ 3). This research was originally published in Matsuura et al. (2008). # The American Society for Biochemistry and Molecular Biology.

1991; Kentzer et al., 1990) and tissue plasminogen activator (tPA; Harris et al., 1991), blood clotting factors VII (Bjoern et al., 1991), factor IX (Nishimura et al., 1992a), factor XII (Harris et al., 1992), and bat salivary plasminogen activator (Gohlke et al., 1996). In contrast to Factor VII that bears only the O-fucose monosaccharide (Bjoern et al., 1991), O-fucose on Factor IX is elongated into the tetrasaccharide Fuc-GlcNAc-Gal-Sia (Harris et al., 1993; Nishimura et al., 1992a). Intriguingly, O-fucose glycans were found in components that mediate intercellular signaling, such as Notch receptors and Notch ligands; Delta and Serrate/Jagged (Moloney et al., 2000b; Panin et al., 2002); and Cripto, a membrane-bound coreceptor for

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Nodal (Schiffer et al., 2001; Yan et al., 2002). The extracellular domain of Notch contains up to 36 tandem EGF repeats, many of which are shown or predicted to be O-fucosylated (Haines and Irvine, 2003; Haltiwanger and Stanley, 2002). O-Fucose glycans are known to modulate Notch–ligand interaction (Bruckner et al., 2000; Moloney et al., 2000a; Okajima et al., 2003; Xu et al., 2007). In particular, the O-fucose at Notch EGF12, a domain responsible for ligand binding, is evolutionarily conserved and functionally important (Lei et al., 2003; Rampal et al., 2005). In contrast, O-fucose on Cripto does not appear to play a role in Nodal signaling (Shi et al., 2007). Many reports have addressed the biological importance of O-fucose glycans by analysis of glycosyltransferase gene mutants involved in their biosynthesis. These include Pofut1/Ofut1 (Okajima and Irvine, 2002; Sasamura et al., 2003; Shi and Stanley, 2003; Okajima et al., 2008; Sasamura et al., 2007), fringe-related genes (Lunatic fringe, radical fringe, and manic fringe in mammals and fringe in Drosophila; Evrard et al., 1998; Irvine and Wieschaus, 1994; Zhang and Gridley, 1998), and b4-galactosyltransferase 1 (Chen et al., 2001). For further reading on the functions of O-fucose glycans, the following review articles are available: Haines and Irvine (2003), Haltiwanger and Lowe (2004), Okajima et al. (2008), Rampal et al. (2007), Stanley (2007). O-GlcNAc modification is the most recent modification found on EGF repeats of Notch receptors (Matsuura et al., 2008). O-GlcNAcylation occurs on a serine or threonine located between the fifth and sixth cysteines within the EGF domain. This modification occurs simultaneously along with other closely positioned O-glycosylations. In this chapter, we discuss methods used to detect O-GlcNAc modification of Notch receptors and O-GlcNAc transferase (OGT) activity from membrane fraction proteins prepared from insect cell lines.

2. Preparation of Recombinant EGF Domains by Yeast Expression System To prepare unglycosylated recombinant EGF domain used for in vitro glycosylation assay, V5-hexahistidine-tagged Notch EGF20 (EGF20: V5His) was prepared using the Kluyveromyces lactis Protein Expression Kit (NEB; E1000). In brief, EGF20:V5His fragment was cloned into the pKLAC1 expression vector, and the resultant plasmid was transformed into K. lactis GG799 cells. After incubation at 30  C for 3–4 days, 96 colonies were patched onto fresh yeast carbon base (YCB) agar plates containing 5 mM acetamide. After incubation at 30  C for 3–4 days, individual clones were scraped using a sterile pipette tip and resuspended in 1 ml of YPGal (1% yeast extract, 2% peptone, 2% galactose) medium in

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a 96-well deep-well plate (Simport, T105-51). After 2 days incubation, the culture medium was analyzed for the expression of EGF20:V5His by dot blotting as shown below.

2.1. Screening Before blotting the samples, a nitrocellulose membrane (Protran BA83; Schleicher and Schuell) was marked with pencil by drawing circles (3– 4 mm diameter) to indicate the region where samples are blotted. By using a narrow-mouth pipette tip, 1–2 ml of the culture medium derived from individual colonies was spotted onto the nitrocellulose membrane at the center of the circles. To load the sample at higher concentration, the loading area of the sample was minimized by applying the sample slowly. After airdrying for 10 min, the membrane was incubated for 30 min with 1% BSA in phosphate buffered saline containing 0.05% Tween-20 (PBST) for blocking nonspecific sites. After rinsing briefly with PBST, the membrane was incubated for 30 min with peroxidase-conjugated anti-V5 (Invitrogen; 1:5000 dilution) in 1% BSA/PBST. After three 5-min washes, the membrane was incubated for 5 min with a chemiluminescent substrate (Immobilon Western; Millipore). After removing excess substrate, the blot was exposed to an X-ray film until signals were obtained. The clones expressing high levels of EGF20:V5His were stored at 70  C in 20% glycerol.

2.2. Purification of EGF20 The frozen K. lactis cells were streaked on a YCB agar plate supplemented with 5 mM acetamide, followed by incubation in 1 ml of YPGal medium for 2 days. The cell suspension was transferred to 300 ml of fresh YPGal medium and incubated for an additional 2 days to obtain a saturated culture. The culture medium was collected by centrifugation in an ST-410M rotor (Kubota) at 5000 rpm (4160g) for 15 min, filtered through a 0.45-mm filter, concentrated using Amicon Ultra-15 (5K; Millipore), and affinitypurified by mixing with 100 ml of anti-V5 affinity beads (clone V5-10; Sigma) for 2 h at 4  C. After three 10-min washes with PBS, the bound peptides were eluted five times with 0.4 ml of 100 mM glycine-HCl (pH 2.4), followed by immediate neutralization with 26 ml of 1.5 M Tris–HCl (pH 8.8). Beads were immediately washed three times with PBS containing 0.05% sodium azide and reused at least three times. The collected eluate was desalted by loading onto a Sep-Pak C18 cartridge (Waters), which was preconditioned with 1 ml of 80% acetonitrile, 0.052% trifluoroacetic acid (TFA), and was equilibrated with 1 ml of water. After washing with 5 ml of water, the bound proteins were eluted with 1 ml of 80% acetonitrile, 0.052% TFA, air-dried, and dissolved in water.

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3. In Vitro O-GlcNAc Transferase Assay To detect OGT activity toward EGF domains, membrane fraction proteins were prepared using S2 cells as an enzyme source. The reaction was performed using EGF20:V5His and UDP-GlcNAc as acceptor and donor substrates, respectively.

3.1. Preparation of membrane fraction proteins S2 cells were pelleted, washed with PBS, and resuspended in 1 ml of ice-cold PBS containing 1 mM phenylmethylsulfonyl fluoride (PMSF). The cell suspension was then placed in a nitrogen cavitation apparatus (Parr Instrument Co.), and exposed to N2 at 400 psi for 30 min. After release of the pressure, which disrupts the cells, the sample was centrifuged in an ST-720M rotor (Kubota) at 3000 rpm (1500g) for 5 min to remove nuclei and remaining whole cells. The supernatant was collected, and centrifuged in a TLS-55 (Beckman) at 40,000 rpm (100,000g) for 1 h at 4  C. The pellet was resuspended in ice-cold 50 mM HEPES–NaOH (pH 7.0), and stored at 80  C in small aliquots until used for OGT assay.

3.2. O-GlcNAc transferase assay Before the reaction, UDP-[3H]GlcNAc was dried under vacuum (Speed Vac) and resuspended in water. For the assay, 1.6 mM UDP-[3H]GlcNAc (60 Ci mmol 1; ARC), 2 mg of EGF20-V5His produced in K. lactis, and 0.2 mg of membrane fraction proteins were mixed in the glycosylation buffer (25 mM HEPES–NaOH (pH 7.0), 1 mM MnCl2, 1 mg/ml bovine serum albumin) in a total volume of 15 ml. After incubation for 2 h at 25  C, the reaction was stopped by addition of 1 ml of ice-cold 50 mM EDTA. Discovery DSC-18 SPE tubes (50 mg; Supelco) were conditioned by loading 1 ml of 80% acetonitrile, 0.052% TFA, and then equilibrated with 1 ml of H2O. The sample was loaded to the tube and washed with 5 ml of H2O, and the labeled substrates were then eluted with 2 ml of 80% acetonitrile, 0.052% TFA. Radioactivity in the eluate was measured using a liquid scintillation counter.

4. Mass Spectrometry The O-GlcNAc modification site of Notch EGF20 was demonstrated by mass spectrometric analysis of EGF20:V5His isolated from S2 cells. A plasmid encoding Notch EGF20 (pMTBip-EGF20:V5His) was transiently transfected into S2 cells using Cellfectin (Invitrogen) as described

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previously (Okajima and Matsuda, 2006). Protein expression was under the control of an inducible metallothionein promoter and was induced in Sf900 II (Invitrogen) supplemented with 0.7 mM CuSO4. After 2–3 days, the medium was collected and Notch EGF20 was purified with Ni-magnetic beads or anti-His tag beads, and O-glycosylations were analyzed by MALDI-TOF-MS as described in Fig. 16.2. To identify the O-GlcNAcylated peptide, EGF20:V5His was further purified, trypsin-digested, and separated by HPLC, followed by MALDI-TOF-MS/MS analysis.

4.1. Purification of EGF domains from S2 culture media Notch EGF20:V5His secreted in the culture media was affinity purified under native conditions using either Ni-magnetic beads (Promega) or antiHis tag beads (MBL). The beads were allowed to absorb the sample for 30 min, after which they were washed twice with 100 mM HEPES–NaOH (pH 7.5) and 10 mM imidazole, washed twice with 30% ethanol, and then the bound proteins were eluted with 100 ml of 0.1% TFA. For purification with anti-His tag beads, bound proteins were eluted with 0.1 M NH3 following the manufacturer’s instructions. The eluates were dried in a Speed Vac concentrator.

4.2. Reduction, S-carbamidomethylation, and trypsin digestion Affinity-isolated EGF20 was further purified by reverse-phase HPLC with a Cadenza CD-C18 column (2 mm  150 mm, Imtakt) at room temperature at a flow rate of 0.2 ml/min. A linear gradient formed between solvent A (H2O, 0.1% TFA) and solvent B (CH3CN, 0.1% TFA) was used for the purification. The gradient of solvent B was as follows: 0–2 min, 25%; 2– 20 min, 25–45%; 20–25 min, 45–90%; 25–28 min, 90%. Peptide separation was monitored continuously at 220 nm. HPLC-purified Notch EGF20 was neutralized by the addition of 1 M NH4HCO3 to achieve a final pH of 7.0 and concentrated to a final volume of 10 ml in a Speed Vac concentrator. For reduction and S-carbamidomethylation, 15 ml of 50 mM NH4HCO3 and 1.5 ml of 100 mM dithiothreitol (DTT) were added to the solution. After incubation for 5 min at 95  C, 3 ml of 100 mM iodoacetoamide was added, and the mixture was incubated for 30 min in the dark. Trypsin digestion was carried out at 37  C by the addition of 1 ml of 0.1 mg/ml trypsin solution (Trypsin Gold; Promega) to the mixture. After incubation for 2 h, the reaction mixture was additionally supplemented with 1 ml of trypsin solution and further incubated overnight at 30  C. Separation of the tryptic digest was performed by reverse-phase HPLC with the following gradient of solvent B: 0–2 min, 15%; 2–30 min, 15–40%; 30–35 min, 40–90%; 35–38 min, 90%.

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LESRGPFEGKPIPNPLLGLDSTRTGHHHHHH

RSPWPLD

A

EGF20

V5:His

1 2 3 4 5 6 IDECSANPCQHGGTCYDKLNAFSCQCMPGYTGQKCETN IDECSSNPCQHGGVCYDKLNAFSCQCMPGYTGQKCETN IDECSSNPCQHGGTCYDKLNAFSCQCMPGYAGQKCETN IDECSANPCQHGGVCYDKLNAFSCQCMPGYTGQKCETN IDECSANPCQHGGVCYDKLNAFSCQCMPGYSGQKCETN IDECSANPCQHGGVCYDKLNAFSCQCMPGYAGQKCETN IDECSSNPCQHGGTCYDKLNAFSCQCMPGYTGQKCETN

EGF20ΔGlc EGF20ΔFuc EGF20ΔHexNAc EGF20ΔGlc ΔFuc EGF20ΔGlc ΔFuc T38S EGF20ΔGlc ΔFuc ΔHexNAc EGF20[Wild type]

Glc

Fuc

GlcNAc

Xyl 100

(8438)

D

204

147

9083

8950

161

G

133

8879

8715 2 161

146

133

H

8950 9083 (8438) 8583 8788 133 145 PUGNAc 205 162

8933 3

203

162

Ofut1 RNAi

206

8602

164 8463

8950

8805

9082

145 132 8937

161 203

132

145

8442

132 8771 2

I 8644

ΔFuc + [M+H] 8436

8801 2

8639 1

8878 205

ΔHexNAc + [M+H] 8408

8848 3 8554 1

204

(8438) Relative intensity

8789

Relative intensity

Relative intensity

C

8585

Cont.

Relative intensity

B

ΔGlc + [M+H] 8422

8568 1 146

203 ΔGlc / ΔFuc + [M+H] 8420

8623 1

J 203

E Relative intensity

(8438) Rumi RNAi

145

8949

8788 205

161

9080

131

K

(8438)

F Relative intensity

8583

Fringe RNAi

145

8583

8786 203

8948 162

L

8800 8600 Mass (m / z)

9000

8405 1

8609 2

ΔGlc / ΔFuc / T38S + [M+H] 8406

204

202

8400

ΔGlc / ΔFuc / ΔHexNAc + [M+H] 8390

9080

132

8878

8200

8388 1

9200

8200

8600 Mass (m / z)

9000

Figure 16.2 MALDI-TOF-MS spectrum of Notch EGF20 and mutated Notch EGF20 defective in glycosylation. (A) Schematic of Notch EGF20:V5His and its derivatives wherein O-glycosylation sites are mutated. The amino acid sequences and the predicted O-glycan structure are shown below. (B–F) Notch EGF20 derivatives isolated from the culture medium of S2 cells and treated with the indicated dsRNAs or PUGNAc were subjected to MALDI-TOF-MS in the linear positive mode. Mass increments corresponding to glycosylation are indicated by double-headed arrows. The [M þ H]þ value for unglycosylated peptide with three disulfide bonds is 8438. Peaks corresponding to O-GlcNAcylated peptides are shown in red letters. (G–L) MALDI-TOF-MS analysis

O-GlcNAc Modification of EGF Domains

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4.3. MALDI-TOF-MS and MS/MS analysis For the analysis of the whole EGF20 proteins, the affinity-isolated EGF20: V5His was resuspended in 1 ml of 0.1% TFA and spotted on the sample plate; 1 ml of sinapinic acid (Sigma) in 0.05% TFA/50% CH3CN was subsequently spotted on the sample plate and air-dried. MALDI-TOFMS was performed using 4700 Proteomic Analyzer with TOF/TOF optics or Perseptive Voyager-DE Elite spectrometers (Applied Biosystems) in the linear positive ion mode. For the analysis of tryptic peptides, HPLCpurified peptides were concentrated in a Speed Vac concentrator and spotted on the sample plate. Subsequently, 1 ml of CHCA in 0.05% TFA/ 50% CH3CN was spotted and air-dried. MALDI-TOF-MS/MS was performed using 4700 Proteomic Analyzer in the positive reflector mode.

5. Detection of O-b-GlcNAc Modification Using Antibodies For detection of O-GlcNAc on Notch EGF domains, we took advantage of the fact that some O-GlcNAc antibodies have been used for the detection of intracellular O-GlcNAc modification. Several O-GlcNAc antibodies are commercially available, which include CTD110.6 (Abcam), HGAC85 (Abcam), RL2 (Abcam), 6D93 (Santa Cruz), and 10D8 (Santa Cruz). Among these antibodies, CTD110.6 and 10D8 antibodies were able to detect O-GlcNAc modification of Notch EGF repeats (N-EGF:FLAG or N-EGF:AP). CTD110.6 most effectively detected the O-GlcNAcylation of EGF repeats, and showed little cross-reactivity to other glycans such as N-glycans of Drosophila glycoproteins (Fig. 16.3). Other O-GlcNAc antibodies such as HGAC85, RL2, and 6D93 could not successfully detect O-GlcNAc modification of Notch EGF repeats.

5.1. Immunoblotting with CTD110.6 antibody Samples were loaded onto NuPAGE 3–8% Tris–acetate gel (Invitrogen) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore). The membrane was incubated for 30 min in 1%BSA in PBST (PBS containing of Notch EGF20 derivatives isolated from the culture medium of S2 cells expressing the respective EGF20 constructs. The mass/charge (m/z) values are shown in bold letters. The spectra shown are DHexNAc (G), DFuc (H), DGlc (I), DGlc/DFuc ( J), DGlc/DFuc/ DHexNAc (K), and DGlc/DFuc/T38S (L). The position corresponding to the nonglycosylated peptide is indicated by dashed lines. This research was originally published in Matsuura et al. (2008). # The American Society for Biochemistry and Molecular Biology.

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A 100 1951.8 1837.8 1748.7 1634.7 15 14 L

N

1766.7 1619.6 1532.6 1372.6 1244.5 1084.5 953.5 856.4 1563.6 1416.6 1329.5 1169.5 1041.5 881.4 750.4 653.3 13 6 12 11 10 9 8 7

A

F

4 3 299.2 446.2

S

C

5 533.3

6 693.3

Q

C

7 821.4

M

P

G

Y

y* 636.3 433.2 332.2 275.2 147.1 y 4 3 2 1 (+203) T G Q K 13 14 1530.6 1587.6 1733.7 1790.7

9 10 11 12 8 981.4 1112.4 1209.5 1266.5 1429.6

15 1715.7 1918.8

Relative intensity

2 228.1

799.4 596.3 5

b b*

+ [Peptide+H] 1862.0

y7 750.4

y7* 953.5

y8* 1084.5

y8 881.5

y4* 636.4 0 69

702

10

y8 881.5 5

y8* 1084.5 y9 1041.5

y4* 636.4

y4 433.3

1335

m/z y7* 953.5

y7 750.4

1968

y12 1416.7

y9* 1244.6 y10* 1372.6

b9 1112.5

y11 1329.7

y10 1169.6

b8 981.4

b13 or y11*

b12 1429.7

0 400

820

1240

m/z

1660

B 1903.8 1789.8 1718.7 1571.6 1484.6 1324.6 1196.5 1036.5 953.5 1700.7 1586.7 1515.6 1368.6 1281.5 1121.5 993.4 833.4 750.4 15 14 13 12 11 10 9 8 7 –48 L N A F S C Q C M P

100

2 228.1

3 299.2

4 446.2

5 533.3

856.4 799.4 653.3 596.3 6 5 G

6 7 9 10 8 693.3 821.4 981.4 1064.4 1161.5

Y

636.3 332.2 433.2 4 3 (+203) T G

11 12 1218.5 1381.6

50

Relative intensity

147.1 1

Q

K

13 14 1482.6 1539.6 1685.7 1742.7

b11 1218.4

40

275.2 2

y* y

15 1667.7 b 1870.8 b*

+ [Peptide+H] 1813.7

30 y7 750.3 20

y7* 953.4

10 b3 299.2 0 250

y9 993.4

y8 833.4

y3 332.2

y4 433.2 775

y8* 1036.4

m/z

b13* 1685.6

y10 1121.4

y11 1281.5 1300

y12 1368.4

y14 1586.5 1825

Figure 16.3 Detection of the O-GlcNAc modification site by MALDI-TOF-MS/MS analysis. MALDI-TOF-MS and MS/MS analysis of tryptic digest peptides of Notch EGF20 fractionated by HPLC. (A) HPLC-purified EGF20[26–41], a peptide spanning amino acids 26–41 of EGF20, was analyzed by MALDI-TOF MS/MS. The b and y series ions identified in the spectra are marked. Asterisks indicate the glycosylated fragment ion peaks. The peptide sequence and theoretical mass for each fragment ion are illustrated. The observed ions are depicted in bold letters. The neutral loss of the sugar group yielded a major product ion at m/z ¼ 1862.0. The mass difference relative

O-GlcNAc Modification of EGF Domains

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0.05% Tween-20) for blocking. After rinsing in PBST, the membrane was incubated with anti-O-b-GlcNAc antibody, CTD110.6 (Abcam; 1:2000– 5000 dilution in PBST). After three 10-min washes in PBST, the membrane was incubated for 60 min with peroxidase-conjugated anti-mouse IgM (Pierce; 1:30,000) in PBST. After three 10-min washes in PBST, the membrane was incubated for 5 min with a chemiluminescent substrate (Immobilon Western; Millipore). After draining excess substrate, signals were detected by exposing the PVDF membrane to X-ray films for an appropriate duration. The O-GlcNAc antibody was stripped in Restore Western blot stripping buffer (Thermo Scientific) for 30 min at 37  C. Reprobing was performed by standard procedures using either goat anti-PLAP (Santa Cruz, sc-15065; 1:1000 dilution) or rabbit anti-FLAG (Cell Signaling; 1:2000) antibodies (Fig 16.4).

6. Galactosyltransferase Labeling Alternative methods to detect O-GlcNAc rely on the specific action of b4-galactosyltransferase 1 (b4GalT-1) toward the terminal GlcNAc, thereby generating a Gal-b1,4-GlcNAc structure. The elongation with galactose was detected by the galactose-specific lectin Erythrina cristagalli lectin (ECL). Similarly, addition of GalNAc by a mutant form of b4GalT-1 (Y289L) allows to label the terminal sugar with biotin by Click-it chemistry (Invitrogen). However, it should be noted that b4GalT-1 reaction does not specifically label the O-GlcNAc, because other GlcNAc at the nonreducing end of N- and O-glycans could be similarly labeled by b4GalT-1. Below, we describe the methods to detect O-GlcNAc on EGF20:V5His bound to Ni-magnetic beads (Promega; Fig. 16.5).

6.1. b4GalT-1 assay EGF20:V5His bound to Ni-magnetic beads was equilibrated with glycosylation buffer (200 mM HEPES–NaOH (pH 7.0), 1 mM MnCl2, 1 mg/ml BSA). The beads were then incubated in glycosylation buffer containing 1 mM UDP-Gal and 0.4 U/ml bovine b4GalT-1 (Sigma). After an overnight incubation at 30  C, the reaction was terminated by washing the beads three times with 100 mM HEPES–NaOH (pH 7.5), 10 mM imidazole.

to the precursor ion was 203, indicating the presence of O-HexNAc. Asterisks indicate the glycosylated fragment ion peaks. The expanded product ion spectrum is shown below. (B) MS/MS spectrum of EGF20[26–41] with decomposed iodoacetamide-derivatized methionine (precursor ion at m/z 2016.9). Similar neutral loss by 203 amu was observed. This research was originally published in Matsuura et al. (2008). # The American Society for Biochemistry and Molecular Biology.

PNGase –

+

P Fc :A

N

-E G

F: A

P

B

–

(kDa)

+ N-EGF:AP

210

A c Fu ΔG cΔ H lc ex ΔF N uc A c T3 8S

140

WB: O -GlcNAc

95 70

ΔG

lc Δ

lc

N

G

ex

ΔH

0 F2

ΔF

EG

(kDa) 30 20 15

uc Δ

W T

A

55

* O -GlcNAc

N-EGF:AP 210

10

140 3.5 30 20 15

WB: PLAP

95

*

70

V5

10

Fc: AP

55

3.5

PNGase – 210 140 95 70

+ –

F2

2-

0 -1

+

EG

F6 EG

EG

F1

-1

0

C

32

IP: PLAP

–

+

WB: O -GlcNAc

55 (kDa) 210 140 95 70

WB: PLAP

55 IP: PLAP

Figure 16.4 Detection of O-b-GlcNAc modification by immunoblotting with a specific antibody. (A) Immunoblotting analysis for O-GlcNAcylation of EGF20, DFucDGlc, DHexNAc, DGlcDFucDHexNAc, and DFucDGlcT38S isolated from S2 cells expressing the respective EGF20 constructs. Immunoblotting was performed using the CTD110.6 and V5 antibodies. The asterisk shows nonspecific signals. (B) Immunoblotting analysis for O-GlcNAcylation of Notch EGF repeats that were C-terminally fused with PLAP (N-EGF:AP) and Fc:AP as a control (Xu et al., 2005) and that were isolated from S2 cells using the anti-PLAP antibody. Where indicated, PNGase treatment was performed to remove N-glycans. Immunoblotting was performed using the CTD110.6 and PLAP antibodies. (C) The deletion mutants of the N-EGF:AP construct (EGF1-10, EGF6-10, and EGF22-32) were analyzed as described above. This research was originally published in Matsuura et al. (2008). # The American Society for Biochemistry and Molecular Biology.

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Figure 16.5 Detection of O-b-GlcNAc modification by b4GalT-1 labeling. (A) Notch EGF20 was expressed in S2 cells, affinity-purified, and labeled with galactose by b4GalT-1 reaction. Samples were separated by SDS-PAGE, and lectin blotting was performed with ECL lectin, which recognizes a terminal galactose structure. In order to confirm the specificity of this assay, the experiments were performed without the b4GalT-1 reaction () or the preincubation of ECL lectin with lactose. Immunoblotting with the anti-V5 antibody is shown in the bottom panel. (B) Various Notch EGF20 mutants with modified O-glycosylation sites were analyzed as described in (A). This research was originally published in Matsuura et al. (2008). # The American Society for Biochemistry and Molecular Biology.

Elution was performed with 1 LDS sample buffer (Invitrogen) for 15 min at 70  C. After centrifugation for 1 min, supernatants were collected and incubated with 50 mM DTT for 10 min at room temperature.

6.2. Lectin blotting The eluted samples were separated by 17.5% SDS-PAGE. After electroblotting to a PVDF membrane (Immobilon; Millipore) and blocking with Tris– buffered saline (TBS; 100 mM Tris–HCl (pH 7.5), 150 mM NaCl) containing 0.1% Triton X-100 (TBST), the membrane was incubated with TBST containing 5 mg/ml of ECL (Vector Laboratories) for 1 h. After three 10-min washes in TBST, the blot was incubated with Vectastain ABC reagent (Vector) for 30 min. After three 10-min washes in PBS, the bound lectin was visualized in PBS containing 0.05% DAB (Sigma), 0.03% H2O2, and 2 mM NiCl2.

7. Hexosaminidase Treatment Removal of O-GlcNAc from EGF repeats of Notch was achieved by b-N-acetylhexosaminidase digestion. Deglycosylation of O-GlcNAc from the folded glycoproteins was performed under denaturing conditions using bovine b-N-acetylhexosaminidase (Sigma; Fig. 16.6).

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Figure 16.6 Removal of O-b-GlcNAc by hexosaminidase digestion. (A) N-EGF:AP treated with b-N-acetylhexosaminidase (b-HexNAcase), a-N-acetylgalactosaminidase (a-GalNAcase; NEB), or buffer alone (Cont) was subjected to immunoblotting using the CTD110.6 and PLAP antibodies. (B–C) HPLC-purified EGF20[26–41] was subjected to b-N-acetylhexosaminidase digestion, followed by MALDI-TOF-MS analysis in the positive reflector mode. The control experiment was performed without the addition of the enzyme (B). The asterisk marks the metastable ions. (C) MALDI-TOF-MS spectrum after b-N-acetylhexosaminidase treatment. This research was originally published in Matsuura et al. (2008). # The American Society for Biochemistry and Molecular Biology.

7.1. Deglycosylation of glycoproteins N-EGF:AP bound on PLAP beads was washed three times with HBSS. The collected beads were resuspended and denatured in 0.5% SDS and 40 mM DTT and incubated at 100  C for 10 min. The eluted proteins were collected after brief centrifugation. Twenty-five microliters of the sample was mixed with 5 ml of 500 mM sodium citrate (pH 4.5), 5 ml of 10% Nonidet P-40, 1 ml of b-N-acetylhexosaminidase and protease inhibitors (complete protease inhibitor cocktail; Roche) in a total volume of 50 ml, and incubated for 3 h at 37  C. Deglycosylation of N-EGF:AP was detected by immunoblotting with CTD110.6 antibody.

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7.2. Deglycosylation of glycopeptide Glycosidase digestion of glycopeptides was performed in 50 mM sodium citrate (pH 4.5) containing 2 U/ml bovine b-N-acetylhexosaminidase (Sigma). After 1 h at 37  C, the reaction solution was desalted with a C18 microcolumn (Zip Tip; Millipore) and analyzed by MALDI-TOF-MS/MS.

8. Conclusions and Future Directions In this chapter, we have summarized the methods that were employed for the detection of O-GlcNAc modification on EGF domains of Notch receptors. We have also described a method for the detection and measurement of OGT activity in crude membrane fraction proteins. Although there are a few reports that suggest the presence of O-GlcNAc in the lumen of endoplasmic reticulum (ER; Abeijon and Hirschberg, 1988) and on the cell surface (Torres and Hart, 1984), O-GlcNAc are believed to be restricted mainly to the cytoplasmic and nuclear compartments (Kearse and Hart, 1991). Notch receptors represent the first molecularly identified O-GlcNAcylated proteins in the extracellular environment in mammals. Intracellular O-GlcNAc glycosylation is catalyzed by an OGT; Haltiwanger et al., 1998; Kreppel et al., 1997). OGT is unlikely to be responsible for the O-GlcNAc glycosylation of Notch receptors since O-glycosylation of Notch EGF repeats normally occurs in the ER or Golgi apparatus. Furthermore, OGT activity was detected in the membrane fraction proteins prepared from S2 cells (Matsuura et al., 2008). Thus, it appears that O-GlcNAc modification on EGF domains occurs independently of the action of OGT in the secretory pathway, at least in Drosophila. Molecular mechanisms and biological functions of OGT-independent O-GlcNAc glycosylation are currently under investigation. It is currently unknown whether the O-GlcNAc modification is present on mammalian Notch receptors. We speculate that the answer will most likely be ‘‘yes’’ on the basis of the following reasons: (1) The Thr/Ser residues at the modification sites are highly conserved in mammalian Notch receptors and their ligand. (2) Our preliminary data demonstrated OGT activity to EGF domains in membrane fraction proteins prepared from mammalian cell lines. (3) It was previously reported that O-GlcNAc is present on the luminal face of ER (Abeijon and Hirschberg, 1988). It should be noted that in mammals, O-GlcNAc glycans on the secreted proteins could be elongated into trisaccharides (e.g., O-linked sialyl-lactosamine) due to the fact that O-GlcNAc is readily modified by b1,4-galactosyltransferase in Golgi (Matsuura et al., 2008; Whelan and Hart, 2006). Studies elucidating the structure of O-GlcNAc glycans in mammals are in progress.

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ACKNOWLEDGMENTS We thank Aiko Matsuura, Makiko Ito, Tatsuhiko Kondo, Kosuke Murakami, Daita Nadano, and Tsukasa Matsuda for their collaborations. This work is supported by grants from the Japanese Ministry of Education, Science, Sports, and Culture and the Global COE Program (to T. O. and K. F.), the Naito Foundation, the Sumitomo Foundation, the Kanae Foundation, and Human Frontier Science Program (to T. O.).

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Regulation of Notch Signaling Via O-Glucosylation: Insights from Drosophila Studies Tom V. Lee,*,1 Hideyuki Takeuchi,†,1 and Hamed Jafar-Nejad* Contents 1. 2. 3. 4.

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Overview Genetic Identification and Characterization of rumi Drosophila Strains Drosophila Culture and Husbandry 4.1. Materials 4.2. Methods A Genetic Screen to Identify New Notch Regulators 5.1. Materials 5.2. Methods Mapping and Sequencing 6.1. Methods Rescue Experiments 7.1. Materials 7.2. Methods Generation of a Protein-Null Allele of rumi Via P-Element Excision Experimental Evidence that Rumi is a Protein O-Glucosyltransferase Enzyme Assay for Protein O-Glucosyltransferase Activity 10.1. Materials 10.2. Methods Analysis of the Product Obtained from the Protein O-Glucosyltransferase Assay 11.1. Materials 11.2. Methods

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* Brown Foundation Institute of Molecular Medicine (IMM), Department of Biochemistry & Molecular Biology, The University of Texas Health Science Center at Houston, Houston, Texas, USA Department of Biochemistry and Cell Biology, Institute of Cell and Developmental Biology, Stony Brook University, Stony Brook, New York, USA 1 These authors contributed equally to this work {

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12. Detection of O-Linked Glucose on Notch EGF Repeats by Mass Spectrometry 12.1. Materials 12.2. Methods Acknowledgments References

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Abstract Recent work using Drosophila melanogaster has shown that a protein O-glucosyltransferase called Rumi regulates Notch signaling. Studies on several alleles of rumi identified in a forward genetic screen indicated that Rumi is a temperature-sensitive regulator of Notch signaling in flies. Further genetic and rescue experiments demonstrated that Rumi is a general regulator of Drosophila Notch signaling. Biochemical analyses showed that Rumi adds glucose to specific EGF repeats in the extracellular domain of Notch receptor in the Drosophila S2 cell line. Furthermore, RNAi-mediated knockdown of Rumi in this cell line resulted in a severe decrease in the level of O-linked glucose on Notch. In this chapter, we discuss the genetic and biochemical methods used to determine the role of Rumi in the regulation of Notch signaling in flies.

1. Overview Notch signaling is a vital signaling pathway required for a variety of developmental processes in animals, including cell fate specification, apoptosis, compartment boundary formation, left-right asymmetry, vertebrate somitogenesis, angiogenesis, and differentiation (Fortini, 2009; Kopan and Ilagan, 2009; Tien et al., 2009). In addition, Notch signaling is crucial for the maintenance of tissue homeostasis in adults. Therefore, aberration in Notch signaling has been linked with a variety of human pathologies such as cerebrovascular dementia (CADASIL; Joutel et al., 1996), a multiorgan developmental disorder (Alagille syndrome; Li et al., 1997; Oda et al., 1997), cancer (Bolos et al., 2007; Ellisen et al., 1991; Lee et al., 2009), aortic valve disorders (Garg et al., 2005), and pulmonary arterial hypertension (Li et al., 2009). The core components of the canonical Notch signaling pathway are the Notch receptor (in the signal-receiving cell), its ligands (in the signal-sending cell), and a transcription factor complex that regulates the expression of a variety of Notch target genes. The type I transmembrane protein Notch is synthesized in the endoplasmic reticulum (ER) and undergoes its first cleavage (S1) in the Golgi, thereby generating the heterodimeric form of Notch which is present at the plasma membrane (Logeat et al., 1998). After binding to its ligands, Delta and Serrate, the Notch receptor is cleaved again (S2) by the ADAM10 metalloproteinase Kuzbanian to generate NEXT, a membrane-bound form of Notch lacking the majority of the Notch extracellular domain (Cagavi Bozkulak and Weinmaster, 2009; Pan and

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Rubin, 1997; van Tetering et al., 2009). NEXT is subsequently cleaved twice by the g-secretase complex at sites S3 and S4 to release the intracellular domain of Notch (NICD; Schroeter et al., 1998; Struhl and Adachi, 1998). The Notch intracellular domain will then translocate to the nucleus and interact with the Suppressor of Hairless and Mastermind proteins to activate the transcription of Notch target genes (Fryer et al., 2002; Jarriault et al., 1995; Lecourtois and Schweisguth, 1995; Petcherski and Kimble, 2000; Tamura et al., 1995). The Drosophila Notch is a 300-kDa protein, with 36 epidermal growth factor-like (EGF) repeats in its extracellular domain. The Notch extracellular domain also contains three Lin12-Notch repeats and a heterodimerization domain, which together function to inhibit Notch activation in the absence of ligands (Gordon et al., 2007; Sanchez-Irizarry et al., 2004). The intracellular domain of the Drosophila Notch contains several distinct domains, including a transactivation domain, an ankyrin repeats domain that binds to Suppressor of Hairless, several nuclear localization signals, and a PEST domain that regulates Notch turnover. A variety of mechanisms are employed to regulate the spatial and temporal patterns of Notch pathway activation (Bray, 2006). These mechanisms include ubiquitination, endocytosis, intracellular trafficking, protein turnover, and glycosylation of various Notch pathway components (Bray, 2006; D’Souza et al., 2008; Le Borgne, 2006; Luther and Haltiwanger, 2009; Stanley, 2007). Protein O-glucosylation is a rare form of posttranslational modification that occurs on EGF repeats with a C1-X-S-X-P-C2 consensus motif (Harris and Spellman, 1993). The O-linked glucose is added to the serine (S) residue between cysteines 1 and 2 (C1 and C2) of the EGF repeat (X, any amino acid; P, proline). O-linked glucose on EGF repeats was originally discovered in bovine coagulation factors VII and IX, and was later shown to be present on several other proteins including other coagulation proteins and mammalian Notch1 (Hase et al., 1988; Moloney et al., 2000b; Nishimura et al., 1989, 1992). More recent studies have shown that the O-linked glucose on mammalian Notch1 primarily exists as a Xyl-a1,3-Xyl-a1,3-Glc-b1-O-serine trisaccharide (Sethi et al., 2010; Whitworth et al., 2010). However, until recently, the role of O-glucosylation in Notch signaling and the identity of enzyme(s) responsible for the O-glucosylation of Notch were not known. In this chapter, we describe the methods used for the identification and initial characterization of Rumi, the Drosophila protein O-glucosyltransferase, and its role in the regulation of Notch signaling.

2. Genetic Identification and Characterization of rumi Normal development of the Drosophila adult sensory organs is dependent on Notch signaling at two consecutive steps: lateral inhibition and asymmetric cell division. Each organ develops from an individual sensory

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organ precursor cell, which is selected from a field of equipotent cells (Hartenstein and Posakony, 1989). Notch-mediated lateral inhibition ensures that external sensory organs form a highly ordered pattern, and that several epidermal cells exist between each two neighboring sensory organs (Fig. 17.1A and B). Later during development, each sensory precursor cell undergoes a series of Notch-mediated asymmetric cell divisions to generate four different cell types that constitute the adult external sensory organs: shaft and socket cells, which are visible at the surface of the adult cuticle (Fig. 17.1A and B); sheath cell and a neuron, which reside underneath the cuticle. Loss of Notch signaling results in the transformation of the external cells of each sensory organ into neurons, and therefore causes baldness in the cuticle of the adult flies. B

C

D

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rumi79 / 79

Wild-type

A

Figure 17.1 rumi mutations induce temperature-sensitive defects in Drosophila adult sensory organ development. (A) Thorax of a wild-type fly is covered with an organized array of sensory bristles regardless of the temperature at which the fly is developed. (B) is a close-up of the open box in (A). Two external cells of each sensory organ are visible on the surface of the thorax cuticle: the round socket cell (arrowhead), and the elongated shaft cell (arrow). Each small hair (dashed oval) between the sensory bristles marks a single epidermal cell. Note that due to the Notch-mediated lateral inhibition, neighboring sensory bristles are separated by several epidermal cells. (C) When raised at 25
C, flies homozygous for the severe hypomorphic allele rumi79 show a bald cuticle phenotype, as most of the external cells in their sensory organs are transformed into neurons and therefore are not visible externally (not shown). (D) When raised at 18
C, rumi79 homozygous flies do not exhibit bristle loss any more. Note that due to a mild defect in the Notch-mediated lateral inhibition, the bristle pattern is slightly denser in these flies compared to that in wild-type animals (compare to (A)).

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By utilizing the adult sensory organ development as a model, a forward genetic screen was performed to identify novel regulators of Drosophila Notch signaling (Acar et al., 2008; Jafar-Nejad et al., 2005). One of the complementation groups identified in this screen, named rumi, showed a temperature-sensitive loss of sensory organs. At 25
C, the thorax of rumi mutant animals looks bald, because the external cells of most sensory bristles are transformed into neurons (Fig. 17.1C; Acar et al., 2008). However, when these flies were raised at 18
C, they did not show bristle loss, indicating that Notch-dependent asymmetric divisions occur normally at this temperature (Fig. 17.1D). This indicates that rumi mutations induce temperature-sensitive defects during sensory organ development. Since Notch signaling has been implicated in sensory organ development, rumi was hypothesized to regulate Notch signaling. Further genetic and marker analyses indicated that rumi is required for Notch signaling during both lateral inhibition and asymmetric cell division in sensory organ precursor cells (Acar et al., 2008). In addition to sensory organ development, rumi regulates Notch signaling in a variety of other contexts including embryonic neurogenesis, and the development of wings, eyes, and legs (Acar et al., 2008). The rumi loss-of-function phenotypes can be rescued by both a genomic rescue transgene harboring the rumi locus and via transgenic overexpression of rumi by using the UAS–GAL4 system (Brand and Perrimon, 1993). Taken together, these data indicate that rumi is a general regulator of Notch signaling. Importantly, the protein-null allele rumiD26—which lacks 95% of the Rumi coding region due to a small deletion—also shows temperature-sensitive loss of Notch signaling (Acar et al., 2008). This observation shows that the reason rumi loss-of-function phenotypes are temperature sensitive is most likely because a temperature-sensitive step during Notch signaling depends on the function of Rumi, and not due to the nature of specific rumi alleles isolated in our genetic screen.

3. Drosophila Strains (1) y w, Dr Pr/ TM3, hs-hid (heat shock hid) (2) w1118; P{GT1} Alh (3) w1118; P{GT1}BG02748 (4) w1118; P{GT1}MenBG02790 1118 (5) w ; P{GT1}cherBG02734 (6) w1118; P{GT1}BG01881 (7) w1118; P{GT1}CG5346BG02475 (8) w1118; P{GT1}BG02628 (9) y w; Ki Delta23 ry2 (10) P{EPgy2}CG7023EY00249 (The Bloomington Stock Center; (11) y w; UAS-rumi-FLAG (12) y w, rumi-FLAG rescue transgene (13) y w; FRT82B rumi79/TM6, Tb1 (14) y w; FRT82B rumi44/TM6, Tb1 (15) y w; FRT82B rumiD26/TM6, Tb1 (16) y w; UAS-FLP; C684-GAL4 FRT82B (17) y w; UAS-FLP; FRT82B Sb1/TM6, Tb1 (18) y w Ubx-FLP tub-GAL4 UAS-GFPnls-6X-Myc; FRT82B yþ tub-Gal80/TM6, Ubx (Acar et al., 2008). BG02270

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4. Drosophila Culture and Husbandry Flies can be maintained from 18 to 30
C. At 25
C and 60% humidity, the generation time from egg to emerging adult is 9–10 days. The generation times will vary according to temperature, with higher temperatures reducing the time from egg to emerging adult. To maintain healthy cultures, it is recommended that the adult flies be transferred every 14 days, and cultures be kept no longer than 18 days at 25
C (double the time in cultures kept at 18
C). To perform clean genetic crosses, virgin females are required. Since female flies will not mate within the first 8 h of adulthood, virgin females can be obtained by collecting flies once in the morning and again 7–8 h later. Newly emerged females are identified by their pale pigmentation and the dark spot on their translucent abdomen called the meconium. Once all the adult flies are cleared from the vial in the morning, then all the females collected 7–8 h later will be virgins. Using males which are 3–10 days old in genetic crosses will maximize mating efficiency. To avoid confusion in genotyping, it is preferable to transfer the parents to new vials before their progeny reach adulthood. Drosophila crosses and stocks are maintained in polystyrene vials containing approximately 5–10 ml of culture media. The culture medium recipe is as follows (the following amounts can be used to make 2 l of culture medium):

4.1. Materials Agar (Moorhead & Co) Water Molasses Corn meal Dried yeast (active baking) Propionic acid (Fisher #A258-500) Hydroxybenzoic acid methyl ester (Fisher #M245-100) (Use 20%, w/v, in 95% ethanol) Polystyrene vials (Fisher) Buzz-plugs (Fisher) Press’n SealTM Sealable Plastic Wrap Cheesecloth

120 g 1.6 l 160 ml 120 g 30 g 15 ml 10 ml

4.2. Methods 1. Dissolve agar in 1.2 l of water and cook on low heat for 1 h. 2. Increase heat to high and bring to boil. Add molasses and bring heat back to low. 3. Dissolve dried yeast in 400 ml of water, add corn meal, and mix well.

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4. Add yeast and corn meal mix to agar and cook on high heat for 30 min. 5. Turn off the heat and let the food cool down for 30 min. 6. Add propionic acid and hydroxybenzoic acid methyl ester to mixture and fill 10 ml in each vial. 7. Let the food solidify at room temperature. Cover with cheesecloth to prevent astray flies from accessing the food. 8. Once the food is solid and dry, cover with Press’n SealTM Sealable Plastic Wrap for storage. Once flies are placed in a vial, close the vial with Buzz-plug.

5. A Genetic Screen to Identify New Notch Regulators To identify novel genes involved in Notch signaling pathway, we performed a genetic screen for genes on the 3R chromosome that affect the development and specification of Drosophila external sensory organs, a process regulated by the Notch pathway (Jafar-Nejad et al., 2005). An F1 mitotic recombination screen was performed using a combination of the FLP/FRT and GAL4–UAS systems (Brand and Perrimon, 1993; Xu and Rubin, 1993). All crosses were performed on standard media and at 25
C. Approximately 50,000 F1 flies were scored and 84 lines with bristle phenotypes were established. 48 out of the 84 lines were members of 13 complementation groups with two or more alleles, with the remainder being single hits. One of these complementation groups was composed of rumi alleles.

5.1. Materials Whatman paper (Fisher) Glass milk bottles Ethyl methane sulfonate (EMS; Sigma #M0880; please read item 3 below for safety precautions) Thioglycolic acid (Sigma #T6750) Sodium hydroxide (Fisher #BP359) Sucrose (Fisher #BP220)

5.2. Methods 1. Collect isogenized y w; UAS-FLP; C684-GAL4 FRT82B males aged 2–5 days and starve them for 6–8 h in glass bottles containing Whatman #1 paper discs that cover the bottom of the bottle. To prevent severe dehydration, cover the opening of the bottle with a piece of cotton to which 300 ml of water is added.

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2. Prepare the denaturing solution by dissolving 40 g of NaOH in 500 ml of water and adding 2.5 ml of thioglycolic acid. 3. Prepare a 15 mM EMS solution by adding 0.16 ml of EMS to 100 ml of 1% sucrose solution. The flies are mutagenized by adding 1.1 ml of EMS–sucrose solution to the bottom of the bottle containing the starved flies. Let flies feed on EMS solution for 16 h. Note: EMS is a very potent mutagen. Safety precautions should be taken when using EMS. Everything that comes into contact with EMS should be decontaminated (see item 4). This includes disposable items like syringes, which should be decontaminated before being disposed of. 4. Denature the residual EMS by adding denaturing solution to all containers which have come in contact with EMS and incubate for 24 h. 5. Mutagenized males are transferred to vials containing standard media for 24 h and then mated en masse with y w; UAS-FLP; FRT82B Sb1/TM6, Tb1 females. 6. The crosses are raised at 25
C. The F1 progeny of these crosses will have large mitotic clones on the thorax that are marked with yellow – and Stubbleþ phenotypes. Both male and female F1 progeny are scored for aberrant bristle phenotypes in the thorax, and mutants with interesting phenotypes are backcrossed to y w; UAS-FLP; FRT82B Sb1/TM6, Tb1 flies to ensure germline transmission. 7. Isogenized stocks for each allele are established by crossing the sibling progeny from a single male backcross. 8. Mutant strains established from the screen are crossed to each other to establish complementation groups.

6. Mapping and Sequencing A recombination-based mapping strategy was used to map the phenotype of rumi alleles (Zhai et al., 2003). In this method, two rounds of mapping are performed to first determine the approximate location of mutations on the chromosome (rough mapping) and then to identify a small region which most likely harbors the mutations (fine mapping). By using this strategy, rumi was mapped to the cytological region 94C4. Sequencing of the genes located in this region identified mutations in the predicted gene CG31152 (CG, Computed Gene) in several rumi alleles (Acar et al., 2008; our unpublished observations).

6.1. Methods 1. y w; FRT82B rumi44/TM6, Tb1 mutant animals are crossed with various flies containing defined P-elements inserted along chromosome 3R. The molecularly mapped P-elements listed below are available from the

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4.

5. 6.

7. 8.

9.

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Bloomington Drosophila Stock Center as part of the ‘‘Baylor Mapping Kit’’ developed by Hugo Bellen’s lab (Zhai et al., 2003). These P-elements are spread along the 3R chromosome to facilitate mapping of any mutation on this chromosome arm. Similar collections for other chromosome arms are available from the Bloomington Drosophila Stock Center. The P-elements are marked by the presence of wþ marker, which provides eye color for these flies. The cytological position of insertion for each P-element is indicated in parenthesis. Note that the 3R chromosome stretches from 81 to 100. a. w1118; P{GT1}AlhBG02270 (84C1) b. w1118; P{GT1}BG02748 (85B7) c. w1118; P{GT1}MenBG02790 (87C7) d. w1118; P{GT1}cherBG02734 (89E13) e. w1118; P{GT1}BG01881 (92E2) f. w1118; P{GT1}CG5346BG02475 (94B3) g. w1118; P{GT1}BG02628 (98F5) In parallel, males harboring another allele for rumi (y w; FRT82B rumi79/ TM6, Tb1) are crossed to y w, Dr Pr/ TM3, hs-hid females. y w; FRT82B rumi79/TM3, hs-hid males from this cross will be used in the next cross. Cross the Tbþ F1 females from y w; FRT82B rumi44/TM6, Tb1, and each of the seven defined P-elements (e.g., for w, P{GT1}AlhBG02270, the correct progeny will be y w/w; FRT82B rumi44/ P{GT1}AlhBG02270) to y w; FRT82B rumi79/ TM3, hs-hid males and culture at 25
C for 48 h. Heat-shock the cross at 37
C for 1 h and place at 25
C for an additional 10 days. The hs-hid transgene induces the expression of the proapoptotic gene hid upon heat shock. Therefore, all of the progeny harboring the TM3, hs-hid chromosome will die after the heat shock. Count the number of white-eyed and red-eyed flies in the F2 generation. The recombination frequency is equal to the number of white-eyed flies divided by the total number of flies scored. Therefore, a relatively low recombination frequency indicates that a P-element is closer to rumi than a P-element with a larger recombination frequency. Repeat this series of crosses for each P-element listed. Rough mapping usually narrows down the gene of interest to a region of 1–2 megabases. For fine mapping, several additional P-elements are chosen to cover the region identified by rough mapping and the procedure is repeated. For rumi, P-elements around the 94 region were used for fine mapping. Note that since P-elements used in fine mapping are closer to the mutations, many more flies need to be scored in this step compared to rough mapping. In parallel, deficiency mapping was performed to further refine where rumi resides. In this strategy, flies containing deletions in the 94 region were crossed with y w; FRT82B rumimut/TM6,Tb1. By either scoring lethality or the presence of the rumi adult thorax phenotypes, the presence of rumi can be determined to be within the region deleted. A combination of fine mapping and deficiency mapping narrowed down rumi to a small number of genes.

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To identify the genetic lesions in the rumi alleles, sequence analysis was performed on the mutant alleles. Since rumi is homozygous viable at lower temperatures, flies were raised at 18
C, homozygous mutant animals were collected and genomic DNA was extracted using Wizard Genomic DNA Purification Kit (Promega #A1120). A series of PCR primers were designed to generate PCR products that spanned all the genes in the region of interest, including CG31152. We used the ‘‘Multiple Primer Design with Primer 3’’ Web site to design the primers (http://flypush.imgen.bcm.tmc.edu/primer/). Each of these overlapping PCR products was approximately 500 bp long. Sequencing of the PCR products showed molecular lesions in CG31152 in rumi alleles, strongly suggesting that rumi corresponds to CG31152.

7. Rescue Experiments To confirm that molecular lesions in CG31152 are responsible for the mutant phenotypes observed in rumi allele, rescue experiments were performed. For this purpose, two types of transgenes were established: UASCG31152-FLAG overexpression transgenes, and genomic CG31152 transgenes. Both of these transgenes were able to rescue the phenotypes and lethality of rumi alleles at low and high temperatures.

7.1. Materials pUAST and pCaSpeR-4 fly transgenic vectors (Drosophila Genomics Resource Center) QuikChange Site-Directed Mutagenesis Kit (Stratagene) pBluescript-SK vector (Stratagene)

7.2. Methods 1. To generate a CG31152 overexpression construct, CG31152 ORF was amplified via PCR and cloned into the pBluescript-SK vector. A FLAG tag was subsequently inserted before the KDEL motif using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). The CG31152FLAG fragment was then cloned into the pUAST expression vector. 2. To obtain a CG31152 genomic rescue construct, a 3-kb genomic fragment containing only the CG31152 gene was amplified via PCR and inserted into the pBluescript-SK vector. The following primers were used for the PCR: 50 -TTAGAGTTACGGAAGTAGGCC-30 and 50 -AATTAAAATTTAAAGAGAATTTACATAT-30 .

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3. A FLAG tag was subsequently inserted before the KDEL motif using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). The final construct containing the 3-kb fragment and the FLAG tag was then cloned into the pCaSpeR-4 vector. 4. All rescue constructs were verified by sequencing and then microinjected into preblastoderm embryos to establish transgenic stocks. 5. The ability of the CG31152 overexpression transgene to rescue rumi phenotypes was assessed by the MARCM system, which allows the overexpression of a gene of interest in clones of cells homozygous for a mutant gene (Lee and Luo, 2001). Animals with the following genotype were scored for rumi loss-of-function phenotypes, including the sensory bristle loss: y w Ubx-FLP tub-GAL4 UAS-GFPnls-6X-Myc; UASC31152-FLAG/þ; FRT82B yþ tub-Gal80/FRT82B rumimut. 6. To test the ability of the CG31152 genomic transgene to rescue the lethality and phenotypes of rumi alleles, flies with the following genotype were generated: CG31152-FLAG genomic transgene/þ; rumimut/TM6, Tb1. The stocks were placed at various temperatures and Tbþ progeny were inspected for rumi phenotypes. Also, MARCM clones of rumi alleles were generated to examine whether the genomic transgene can rescue phenotypes in mutant clones: y w Ubx-FLP tub-GAL4 UASGFPnls-6X-Myc; CG31152-FLAG genomic transgene/þ; FRT82B yþ tub-Gal80/FRT82B rumimut.

8. Generation of a Protein-Null Allele of rumi Via P-Element Excision The temperature sensitivity of the rumi alleles isolated in the screen could result from aberrant conformation of the mutant Rumi proteins at higher temperatures. Alternatively, Rumi might be involved in regulating a temperature-sensitive step during the Notch signal transduction. To differentiate between these two alternatives and to strengthen the genetic analysis, a null allele for rumi was generated. A large-scale genome disruption project has generated single, molecularly mapped transposable elements inserted throughout the Drosophila genome (Bellen et al., 2004). These strains are available from the Bloomington Drosophila Stock Center. The P-element P{EPgy2}CG7023EY00249 from this collection was identified to be inserted 238 bp upstream of the rumi locus. To generate a new amorphic allele, excision of this insertion and the surrounding DNA was performed using the following method:

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1. Females containing this P-element (y w; P{EPgy2}CG7023EY00249) are crossed to y w; Ki Delta2-3 ry2 males (the source of the transposase). The Ki (Kinked) mutation has a dominant bristle phenotype which segregates together with the Delta2-3 transposase. 2. Individual male progeny (y w; P{EPgy2}CG7023EY00249/Ki, Delta2-3 ry2) of the first cross are crossed to y w, D/TM6, Tb1 females. At least 500 crosses are set. 3. Each vial is scored for the presence of Kiþ males which have lost their eye color (y w; [deletion]/TM6, Tb1). These males have likely lost part or all of the P-element sequence and are therefore white-eyed. One whiteeyed male from each vial is backcrossed to y w, D/TM6, Tb1 females. Sibling crosses are set between the progeny to establish a [deletion]/ TM6, Tb1 stock. The Tbþ progeny of each cross are scored for rumi mutant bristle and wing phenotypes at low temperature. This strategy allowed us to isolate seven excision lines with rumi phenotypes (Acar et al., 2008). 4. To identify the molecular lesion in deletion lines with rumi phenotypes, primer pairs were designed to generate overlapping, 600-bp PCR products covering several kilobases on each side of the P{EPgy2} CG7023EY00249 insertion site. These primers were used to amplify genomic DNA isolated from each of the potential imprecise excision mutants with rumi phenotype and compared to PCR products obtained from both wild-type flies and the original P{EPgy2}CG7023EY00249 strain. PCR on genomic DNA isolated from rumiD26/D26 allele with the following primer pair generated a 344-bp band instead of the expected 1832-bp band: 50 -TGGACATCAAAAATGCATGG-30 and 50 -CGACGTTTCTTGTTGGTTTTC-30 (Acar et al., 2008; Simcox et al., 2008). Sequencing of the 344-bp PCR product confirmed that the P{EPgy2}CG7023EY00249 element along with 95% of the Rumi coding region is excised in this line.

9. Experimental Evidence that Rumi is a Protein O-Glucosyltransferase rumi encodes a soluble ER protein with an N-terminal signal sequence, a CAP10 domain, and a C-terminal KDEL ER-retrieval signal. The CAP10 domain is homologous to the product of the cryptococcal gene CAP10, mutations in which result in loss of capsule formation and virulence in Cryptococcus neoformans (Chang and Kwon-Chung, 1999; Okabayashi et al., 2007). Since the capsule of C. neoformans consists of polysaccharides, CAP10 was proposed to be a sugar-modifying enzyme such as a glycosyltransferase. However, the biochemical function of CAP10 had not been determined. Our genetic analyses showed that the target of Rumi is the extracellular domain of Notch (Acar et al., 2008).

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Three different types of O-linked sugars are found on a number of EGF repeats in the Notch extracellular domain: O-fucose and O-glucose (Moloney et al., 2000b), and O-GlcNAc (Matsuura et al., 2008; also see Chapter 16 by Okajima and colleagues). The consensus sequences for the addition of O-fucose or O-glucose to the EGF repeats of Notch proteins are evolutionarily well conserved (Haines and Irvine, 2003). The enzymes responsible for addition and elongation of O-fucose glycans to EGF repeats, Ofut1 and Fringe, affect Notch signaling in both Drosophila and mammals (Bruckner et al., 2000; Evrard et al., 1998; Irvine and Wieschaus, 1994; Moloney et al., 2000a; Okajima and Irvine, 2002; Sasamura et al., 2003; Shi and Stanley, 2003; Zhang and Gridley, 1998). However, the enzymes responsible for O-glucosylation were unknown. In order to examine whether Rumi mediates O-glucosylation, we analyzed whether knockdown of Rumi by RNAi caused a reduction in the level of Oglucose on the Notch extracellular domain produced in Drosophila Schneider 2 (S2) cells. By examining glycopeptides derived from Notch using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we observed that O-fucose glycans were unaffected by Rumi knockdown. In contrast, a significant reduction in the levels of O-glucose was detected on several Notch EGF repeats, suggesting that Rumi is required for O-glucosylation of Notch (Acar et al., 2008). Indeed, Rumi protein overexpressed in S2 cells is able to catalyze the transfer of glucose from UDP-glucose to bacterially expressed (i.e., nonglycosylated) EGF repeats containing an O-glucose consensus sequence. This activity was dependent on the concentration of Rumi protein, donor sugar nucleotide (UDP-glucose), and acceptor substrate (EGF repeat). Product analyses showed that Rumi adds a single O-linked glucose to the EGF repeats containing an O-glucose consensus sequence, as described below in detail. These results demonstrated that Rumi is a protein O-glucosyltransferase (Acar et al., 2008). Is the protein O-glucosyltransferase activity of Rumi required for Notch function? Ofut1, which adds O-fucose to specific EGF repeats of Notch, appears to have a chaperone-like function on the Notch protein independent of its fucosyltransferase activity (Okajima et al., 2005, 2008). Does Rumi play a similar nonenzymatic role in the regulation of Notch signaling in flies? DXDlike motifs (D, aspartic acid; X, any amino acid) have been implicated as the catalytic site in many glycosyltransferases (Wiggins and Munro, 1998). We identified one such motif (ERD 236–238) in fly Rumi, and mutated it to ERA and ARA to decrease the enzymatic activity of Rumi. In vitro assays showed that the O-glucosyltransferase activity of the ERA and ARA mutants was only 10% and 4% the activity of wild-type Rumi, respectively (Fig. 17.2; Acar et al., 2008). These observations indicate that the ERD motif is indeed required for the enzymatic function of Rumi. We created UAS transgenic flies harboring the ERA and ARA mutant versions of Rumi, similar to the UAS-rumi-FLAG transgenes described above. We then overexpressed the mutant versions of Rumi in rumi mutant animals to examine whether a decrease in the enzymatic

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2

Specific activity (m mol / min / mg)

ERD

1

ERA ARA

0 0

10

20 EGF (mM)

30

Figure 17.2 Mutations in a DXD-like motif (E236R237D238) of Drosophila Rumi cause a significant decrease in its protein O-glucosyltransferase activity. Purified wild-type (ERD in red) and mutated Rumi proteins (ERA in blue and ARA in black) were examined for their protein O-glucosyltransferase activities toward a recombinant human factor VII EGF repeat at different concentrations as indicated. Note that even when high levels of the recipient EGF repeat are provided, ERA and especially ARA mutants show very low levels of enzymatic activity. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this chapter.)

activity of Rumi affects its ability to rescue the mutant phenotypes. The ERA mutant was able to partially rescue the phenotypes, consistent with its 10% residual activity. However, the ARA mutant was either completely inactive (three independent transgenes) or showed a weak activity (one transgene). These data showed that the enzymatic activity of Rumi is required for the regulation of Notch signaling in vivo (Acar et al., 2008). The data do not support the presence of a chaperone-like function for the fly Rumi, although a minor chaperone function cannot be ruled out. Similar results were obtained from studies on protein stability and enzymatic activity of the Rumi-G189E mutation, which is encoded by allele rumi79 from our genetic screen. This allele shows a severe temperature-sensitive Notch phenotype. Rumi-G189E protein was detected in the extracts of the third instar larvae at similar levels as the wildtype Rumi. Similarly, when it was expressed in S2 cells, the level of RumiG189E was comparable to the level of wild-type Rumi in the extracts, suggesting that the point mutation does not affect the stability of the Rumi protein. Interestingly, Rumi-G189E did not show any detectable protein O-glucosyltransferase activity in vitro (Acar et al., 2008). These results further suggest that Rumi does not have a major chaperone-like function, and that the protein O-glucosyltransferase activity of Rumi is essential for Notch signaling in vivo.

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10. Enzyme Assay for Protein O-Glucosyltransferase Activity Protein O-glucosyltransferase activity can be detected in the extracts of cells from Drosophila to humans using UDP-glucose as donor substrate and a recombinant human factor VII EGF repeat as acceptor substrate (Shao et al., 2002). Drosophila Rumi protein expressed with a FLAG tag in Drosophila S2 cells was purified using anti-FLAG M2 monoclonal antibody-conjugated beads and used to examine whether Rumi has protein O-glucosyltransferase activity. After the product analysis as described Section 11, Rumi was shown to be a protein O-glucosyltransferase, which was the first identification of a protein O-glucosyltransferase toward EGF repeats.

10.1. Materials Expression vectors for FLAG-tagged version of Rumi (pActin5C.1-GAL4 and pUAST-rumi-FLAG) Drosophila S2 cells (Drosophila Genomics Resource Center) Schneider’s Drosophila medium (Fisher Scientific) Anti-FLAG M2 monoclonal antibody-conjugated beads (Sigma) 3FLAG peptide (Sigma) CelLytic M (Sigma) Complete Protease Inhibitor (Roche) 10 reaction buffer (500 mM HEPES, pH 6.8, 100 mM MnCl2) Recombinant human factor VII or IX EGF repeat expressed in E. coli (Moloney et al., 2000a; Rampal et al., 2005) UDP-[6-3H]glucose (60 Ci/mmol, American Radiolabeled Chemicals) 10% Nonidet P-40 100 mM EDTA, pH 8.0 SampliQ C18 cartridge (100 mg, Agilent Technologies) A vacuum manifold (VISIPREP, SUPELCO)

10.2. Methods 1. S2 cells are seeded in plates at 1  106 cells/ml. 2. pActin5C-GAL4 and pUAST-rumi-FLAG are cotransfected into S2 cells by using FuGENE-HD (Roche). 3. After 3 days, cells are spinned at 300  g for 10 min and washed with 2 PBS. CelLytic M (Sigma) plus complete protease inhibitor (Roche) is used to lyse the cells. The lysate is centrifuged at high speed to obtain the supernatant.

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4. FLAG-tagged version of Rumi is purified using Anti-FLAG M2 monoclonal antibody-conjugated beads. FLAG-tagged version of Rumi is eluted with 100 mg/ml of 3FLAG peptide. 5. Protein O-glucosyltransferase assay is performed in 1 reaction buffer containing 0.5% Nonidet P-40, 0.1 mCi UDP-[3H]glucose (60 Ci/mmol), nonradioactive UDP-glucose, and 1–10 mM recombinant human factor VII EGF in a volume of 10 ml at 37
C for 20 min. 6. To stop the reaction, 900 ml of 100 mM EDTA, pH 8.0, is added to the reaction mixture, which is then kept on ice until the next step. 7. The samples are applied to C18 cartridges in a vacuum manifold which are activated with 2 ml of 100% methanol and equilibrated with 2 ml of water. The C18 cartridges are washed with 5 ml of water and the bound samples are eluted with 1 ml of 80% methanol. 8. The eluate is mixed with scintillation liquid and radioactivity is measured using a liquid scintillation counter.

11. Analysis of the Product Obtained from the Protein O-Glucosyltransferase Assay The final structure of the sugars on the acceptor substrates must be characterized after the glycosyltransferase assays. Here, we used a series of chromatographic approaches accompanied with alkali-induced b-elimination method to analyze the structure of O-linked sugars (Moloney et al., 2000b). At first, the [3H]glucosylated product of the reaction was purified by reverse phase HPLC, where it eluted as a single radioactive peak slightly earlier than the unmodified EGF repeats (acceptor substrate; Acar et al., 2008). This observation suggested that the radioactivity was covalently attached to the EGF repeat, and that the addition of sugar made the acceptor substrate more hydrophilic. The O-linked sugars were released by alkali-induced b-elimination and separated via Superdex gel filtration chromatography. The radioactivity from the product eluted as a monosaccharide (Acar et al., 2008). The fraction containing the monosaccharide was then analyzed using a high pH anion exchange column with pulsed amperometric detection. The monosaccharide eluted as glucitol, which is the expected product from alkaliinduced b-elimination of O-glucose (Acar et al., 2008). These results indicated that Rumi transferred a single glucose to the EGF repeats in an O-linkage.

11.1. Materials Reverse phase HPLC system (PROTEIN&PEPTIDE C18 column, VYDAC) 1 M NaBH4 in 0.1 M NaOH 4 M acetic acid

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AG 50W-X8 Resin (200–400 Mesh, hydrogen form, Bio-Rad) Sep-Pak Light C18 Cartridges (Waters) Superdex Peptide HR10/30 gel filtration column (GE Healthcare) CarboPac MA1 column (DIONEX)

11.2. Methods 1. After the protein O-glucosyltransferase reaction, the product is applied to reverse phase HPLC. The column is eluted with a linear gradient from 0% to 100% solvent B (0.1% trifluoroacetic acid in 80% acetonitrile in water) in solvent A (0.1% trifluoroacetic acid in water) at a flow rate of 1 ml/min for 30 min. Eluates are monitored for absorbance at 214 nm and for radioactivity. 2. The fraction containing [3H]glucosylated human factor VII EGF repeat is evaporated using Speed-Vac. 3. For alkali-induced b-elimination, the samples are resuspended in 500 ml of 1 M NaBH4 in 0.1 M NaOH and incubated at 55
C overnight. 4. The samples are put on ice and neutralized by adding 4 M acetic acid drop by drop. 5. The neutralized samples are loaded onto 6.5 ml of 50% slurry AG 50WX8 Resin and the flow-through fraction is collected. The resin is washed with 15 ml of water and this flow-through fraction is also collected into the same tube. 6. The flow-through fraction from the AG 50W-X8 Resin is applied to Sep-Pak and the flow-through fraction is collected. The samples are lyophilized. The lyophilized samples are resuspended in 1 ml of methanol and dried by Speed-Vac. This evaporation procedure is repeated at least three times. 7. The samples are resuspended in 100 ml of water and separated on Superdex peptide gel filtration column using water as running buffer at 0.5 ml/min. 8. The radioactivity of each fraction is counted using a liquid scintillation counter. The fraction containing the radioactivity is dried by Speed-Vac. 9. The sample is resuspended in 50 ml of water and separated on CarboPac MA1 column (DIONEX). The column is eluted with a linear gradient from 0.1 to 1 M NaOH in water at a flow rate of 0.5 ml/min for 40 min. The standards are followed by pulsed amperometric detection (PAD-2 cell, settings as follows: E1 ¼ 0.00 V, T1 ¼ 4; E2 ¼ 0.80 V, T2 ¼ 3; E3 ¼ 0.25 V, T3 ¼ 6; Range ¼ 2; Response time ¼ 3 s). Fractions (0.5 min ¼ 0.25 ml) are collected and monitored for radioactivity by scintillation counting.

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12. Detection of O-Linked Glucose on Notch EGF Repeats by Mass Spectrometry We have previously established the method to analyze the sugars on Notch from cultured cells by mass spectrometric methods (Nita-Lazar and Haltiwanger, 2006a,b). We expressed a fragment of the Drosophila Notch extracellular domain (EGF repeat 7 through the transmembrane domain, EGF7-TM) in S2 cells treated with control (EGFP) or Rumi doublestranded RNAs (dsRNAs) to induce RNAi-mediated knockdown. The EGF7-TM protein was purified from the medium, reduced and alkylated, and digested with trypsin. The resulting peptides were analyzed by LC– MS/MS, and glycopeptides modified with O-fucose or O-glucose glycans were identified by neutral loss searches (Acar et al., 2008). For relative quantification, we performed a label-free approach, called differential MS, which is based on normalization strategies that enable comparison of the signal strength of the same (glyco)peptide in two distinct sample runs (Unwin et al., 2006). No changes in the level of O-fucose on peptides were detected in either the control or Rumi dsRNA samples. In contrast, a significant reduction in the level of O-glucose on several tryptic peptides was observed in the Rumi dsRNA samples compared with control (Acar et al., 2008). For example, the level of O-glucose on a peptide from EGF repeat 14 was significantly lower in the sample from Rumi-knockdown cells compared to that from control cells, while significant amounts of the nonglucosylated form of the same peptide were detected only in the Rumiknockdown sample (Acar et al., 2008). Similarly, significant reduction in the level of O-glucose was detected on peptides from EGF repeats 16, 17, 19, and 35 upon treatment of the cells with Rumi dsRNA (Acar et al., 2008). All of these EGF repeats have the consensus O-glucosylation motif. These data strongly suggest that Rumi is required for the addition of O-glucose to the extracellular domain of Notch.

12.1. Materials Expression vector for a V5- and His-tagged fragment of the Notch extracellular domain containing EGF repeat 7 through the transmembrane domain (pMT/V5-HisB-ACE-EGF7-TM). ACE encodes the signal sequence of the Drosophila acetylcholine esterase. S2 cells (Drosophila Genomics Resource Center) Serum free medium (Drosophila SFM; Invitrogen) MEGAscript T7 kit (Ambion) Fetal bovine serum (Life Technologies) CuSO4

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pUAST-Rumi-FLAG pEGFP-N1 (CLONTECH) His Bind Resin (Novagen) Formic acid Bond-Breaker TCEP solution (0.5 M TCEP, pH 7.0, PIERCE) 3 Laemmli sample buffer without reducing reagents NuPAGE gradient gel (4–12%; Invitrogen) Zinc Stain Kit (Bio-Rad) 0.1 M EDTA Agilent nanoflow LC system with HPLC-CHIP interface coupled to Agilent model 6340 3D-Ion Trap mass spectrometer (Agilent Technologies) Zorbax C8 capillary column (0.3 mm  150 mm ; Agilent Technologies)

12.2. Methods 1. To generate dsRNA for knockdown experiments, pUAST-rumi-FLAG and pEGFP-N1 vectors are first used as template to generate PCR products containing fragments of rumi and EGFP coding regions, respectively. A binding site for T7 RNA polymerase is incorporated in the beginning of both 50 and 30 primers so that the PCR products are flanked by T7 RNA polymerase binding sites on both sides. Primers 50 -TTAATACGACTCACTATAGGGGAGAGCTAGTCGAGGCTCAATAC-30 and 50 TTAATACGACTCACTATAGGGGAGAATCCTTTTTAGGCCC TATGTAA-30 are used to amplify a 481-bp fragment from exon 2 of rumi. The italicized sequence in each primer corresponds to the T7 RNA polymerase binding site. This fragment corresponds to amplicon DRSC 15177 designed against Drosophila rumi, and can be found at www.flyrnai. org. A similar, T7-flanked primer pair is used to amplify a region of EGFP for the control experiments (50 -TTAATACGACTCACTATAGGGGAGAGTGAGCAAGGGCGAGGAG-30 and 50 -TTAATACGA CTCACTATAGGGGAGACCACAACATCGAGGACGG). 2. The above-mentioned PCR products are used as template to generate dsRNA against Rumi or EGFP by using the MEGAscript T7 kit, following manufacturer’s instructions. dsRNAs are aliquoted at a final concentration of 3 mg/ml and stored at 20
C (Worby et al., 2001). 3. S2 cells are seeded onto plates (1  106 cells/ml) and transfected with the expression vector for EGF7-TM with a C-terminal V5, His-tag. 4. One day later, rumi or EGFP dsRNA is added to the transfected cells at a final concentration of 15–30 mg/ml. 5. Two days later, CuSO4 is added to the medium to a final concentration of 0.7 mM to induce expression from the metal-inducible promoter available in the pMT/V5-HisB-ACE-EGF7-TM vector.

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6. The culture medium is collected, and the His-tagged EGF7-TM protein is purified by using His Bind Resin (Novagen). 7. After acetone precipitation, the samples are resuspended in 5 ml of 50 mM Tris/HCl, pH 8.0, and mixed with 5 ml of 3 Laemmli sample buffer containing TCEP solution (50 parts 3 sample buffer and one part 0.5 M TCEP solution). The samples are heated at 100
C for 5 min and cooled to room temperature. Five microliters of 50 mM Tris/HCl, pH 8.0, containing 100 mM iodoacetamide is added to the sample. After vortexing, the samples are incubated at room temperature in the dark for 30 min. The samples are then run on a NuPAGE gradient gel (4–12%). The gel is stained using the Zinc Stain Kit from Bio-Rad, and bands of interest are excised and cut into three to four pieces no larger than 1 mm on each side. 8. In-gel digestion procedure a. Gel pieces are washed with 500 ml of 0.1 M EDTA by vortexing at room temperature for 15 min. b. Gel pieces are washed with 1 ml of 50% methanol, 20 mM diammonium phosphate, pH 8.0, by vortexing at room temperature for 15–30 min. This should be repeated at least five times. The more washes, the better. One milliliter of 50% methanol, 20 mM diammonium phosphate, pH 8.0, is added to gel pieces and vortexed at 4
C overnight. On the next day, gel pieces are washed three times with 1 ml of 50% methanol, 20 mM diammonium phosphate, pH 8.0, by vortexing at room temperature for 30–60 min. c. After aspirating the buffer, 50 ml of 100% acetonitrile should be added and vortexed for 30 s. Gel pieces are dried by Speed-Vac. d. Ten microliters of trypsin solution is added to each sample. The gel pieces are allowed to absorb the trypsin at 37
C for 15 min. Sufficient prewarmed 20 mM diammonium phosphate, pH 8.0, is added to cover gel pieces. The samples are incubated at 37
C for 4–24 h. e. Supernatant from each sample is removed and placed in new tubes. Five percent formic acid is added to the tube to cover the gel pieces. The samples are sonicated at room temperature for 20 min. The supernatant is removed and combined with the original. f. The samples are passed through spin filters and dried by Speed-Vac. 9. The samples are resuspended in 20% solvent B (0.1% formic acid in 95% acetonitrile in water) in water and sonicated for 5 min. 10. After centrifugation, the samples are injected into the mass spectrometer, fractionated by reverse phase liquid chromatography and coupled with electrospray ionization mass spectrometry using a Zorbax C8 capillary column (0.3 mm  150 mm; Agilent Technologies) with a 60-min linear gradient from 0% to 95% solvent B in solvent A (0.1% formic acid in water) at 5 ml/ml. The column effluent is sprayed into an XCT ion trap mass spectrometer (Agilent Technologies) equipped

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with a capillary electrospray source, operating in the positive ion mode, and set up to perform MS/MS on the two most intense ions in each spectrum. 11. The presence of O-fucose or O-glucose glycans is examined by neutral loss search as described (Nita-Lazar and Haltiwanger, 2006a). The relative quantities of the (glyco)peptides are analyzed by differential MS approach using the extracted ion chromatograms of each ion.

ACKNOWLEDGMENTS We thank Robert S. Haltiwanger for his support. Work in the Jafar-Nejad laboratory is funded by the Brown Foundation Institute of Molecular Medicine (start-up funds), National Institute of Health (R01 GM084135), and the March of Dimes (Basil O’Connor Award #5-FY07). H. T. is supported by the Mizutani Foundation for Glycoscience.

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O-Fucosylation of Thrombospondin Type 1 Repeats Christina Leonhard-Melief and Robert S. Haltiwanger Contents 402 407 408 408 408 408 409 413 413

1. Overview 2. Glycosylation Site Mapping by Mass Spectrometry 2.1. Materials 2.2. Stock solutions 2.3. Fresh solutions 2.4. Sample preparation 2.5. LC–MS of the digested sample Acknowledgments References

Abstract Thrombospondin type 1 repeats (TSRs) are small cysteine-rich motifs with three conserved disulfide bonds originally described as modules in the thrombospondins. Since then, TSRs have been found as tandem repeats in a wide variety of secreted and cell-surface proteins of diverse function. TSRs in many contexts are known to bind a variety of receptors and have antiangiogenic capabilities. They can be modified with O-linked fucose on serine/threonine found in the consensus, CX2–3(S/T)CX2G. Here we review what is known about O-fucosylation of TSRs and describe in detail mass spectral methods to map sites of O-fucosylation on proteins containing TSRs. These methods include techniques to identify glycosylated peptides and the relative amounts of elongated products by electrospray ionization mass spectrometry of glycopeptides.

Department of Biochemistry and Cell Biology, Institute for Cell and Developmental Biology, Stony Brook University, Stony Brook, New York, USA Methods in Enzymology, Volume 480 ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)80018-7

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2010 Elsevier Inc. All rights reserved.

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1. Overview Amino acid glycosides with the monosaccharide fucose or the disaccharide Glc-b1,3-Fuc in O-linkage to serine or threonine were first identified in human urine in 1975 by Hallgreen and coworkers (Hallgreen et al., 1975), presumably degradation products of proteins bearing these glycans. The first description of similar O-fucose glycans linked to proteins came from metabolic radiolabeling studies in rat tissues followed by release of the O-linked sugars by alkali-induced b-elimination from total cell extract (Klinger et al., 1981). It was not until 1990 that O-fucose was identified on a specific protein. Kentzer et al. (1990) found O-fucose on an epidermal growth factor-like (EGF) repeat from urokinase. EGF repeats are small cysteine-rich motifs with six conserved cysteines forming three disulfide bonds found primarily in secreted and cellsurface proteins (Campbell and Bork, 1993). In the years to follow, O-fucose was identified on EGF repeats from a growing number of proteins including tissue type plasminogen activator and human clotting factors VII, IX, and XII (Bjoern et al., 1991; Harris and Spellman, 1993; Harris et al., 1991, 1992). Comparison of the sites of O-fucosylation on these proteins led to the proposal of a consensus sequence for modification: C2XXGG(S/T)C3, where C2 and C3 are the second and third conserved cysteines of the EGF repeat, respectively (Harris and Spellman, 1993). The consensus sequence has subsequently been refined to C2XXXX(S/T)C3 (Shao and Haltiwanger, 2003). Structural analysis of the O-fucose glycans showed O-fucose monosaccharide on urokinase, tissue-type plasminogen activator and factor VII, but an O-fucose tetrasaccharide, Sia-a2,6-Gal-b1,4-GlcNAc-b1,3-Fuc, on factor IX (Harris et al., 1993). More recently, O-fucose mono- and tetrasaccharide modifications were identified on many of the tandem EGF repeats within the extracellular domain of Notch (Luther and Haltiwanger, 2009; Moloney et al., 2000; Takeuchi and Haltiwanger, 2010). Many of the EGF repeats of Notch are also modified by O-glucose glycans (Lee et al., 2010; Luther and Haltiwanger, 2009; Takeuchi and Haltiwanger, 2010). Recent studies highlight the biological significance of O-fucose in Notch function (Luther and Haltiwanger, 2009; Rampal et al., 2005; Takeuchi and Haltiwanger, 2010). Mice lacking the enzyme which adds O-fucose to EGF repeats, protein O-fucosyltransferase 1 (Pofut1), die midgestation from defects in somitogenesis, vasculogenesis, cardiogenesis, and neurogenesis (Shi and Stanley, 2003). The similarity in Notch and Pofut1 phenotypes suggests that Pofut1 is necessary for Notch receptor function. Additionally, elongation of the O-fucose monosaccharide with b3-GlcNAc by the Fringe family of b3-N-acetylglucosaminyltransferases alters Notch activity by affecting ligand binding (Bruckner et al., 2000; Luther and Haltiwanger, 2009; Moloney et al., 2000; Takeuchi and Haltiwanger, 2010; Xu et al., 2007). Recent studies in Drosophila reveal

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that elimination of the enzyme responsible for addition of O-glucose to EGF repeats, protein O-glucosyltransferase also known as Rumi, also results in Notch phenotypes (Acar et al., 2008; Lee et al., 2010). Thus, the O-glycans on EGF repeats play essential roles in the function of the Notch receptor. While these studies provided a clear emerging role for O-fucose on EGF repeats, the source of the Glc-b1,3-Fuc disaccharide originally found as an amino acid fucoside in human urine remained a mystery. Moloney et al. (1997) detected the Glc-b1,3-Fuc disaccharide on several proteins from Chinese hamster ovary (CHO) cells and proposed a branched O-fucosylation pathway for modification of EGF repeats (since the glucose and GlcNAc would compete for the 30 -hydroxyl of the fucose), but no EGF repeats were directly demonstrated to be modified with Glc-b1,3-Fuc. The mystery was solved in 2001 when Hofsteenge et al. (2001) detected a glucose–fucose (Glc–Fuc) disaccharide on all three thrombospondin type 1 repeats (TSRs) of human thrombospondin 1. The disaccharide was found serendipitously while mapping C-mannosylation sites on thrombospondin 1 TSRs. Subsequent work confirmed the linkage of the Glc–Fuc disaccharide to be b1,3 (Luo et al., 2006b). Comparison of the sites modified by Glc–Fuc disaccharide on the three TSRs revealed the common sequence CXX(S/T)CG (Hofsteenge et al., 2001). Subsequent mapping of O-fucose glycans by the same group identified Glc–Fuc on TSRs from rat f-spondin and human properdin (Gonzalez de Peredo et al., 2002; Hofsteenge et al., 2001). Alignment of the sequences surrounding these O-fucose modification sites led to the proposal of the consensus sequence WX5CX2–3(S/T) CX2G, where the cysteines are either the first and second (group 1) or second and third (group 2) cysteines in the TSR (see below for definition of group 1 and group 2 TSRs). The consensus sequence was later revised to CX2–3(S/T)CX2G based on mapping of O-fucose modification sites on ADAMTS13 and ADAMTSL1 (Ricketts et al., 2007; Wang et al., 2007). Further refinement of the consensus sequence is ongoing. While the upstream tryptophan does not appear to be necessary for glycosylation of the consensus sequence, many TSRs contain a series of upstream tryptophans which play a structural role by bending back between arginines within the folded motif and serve as potential sites for C-mannosylation (Gonzalez de Peredo et al., 2002; Hofsteenge et al., 2001; Wang et al., 2009). C-Mannose on TSRs was first identified on properdin, a member of the complement family (Hartmann and Hofsteenge, 2000). The current consensus for C-mannosylation is WXXW which occurs frequently in TSRs amino terminal to the O-fucosylation site (Furmanek and Hofsteenge, 2000). While C-mannose has been mapped to tryptophans in a number of TSRs (thrombospondin 1, properdin, and ADAMTSL1) (Gonzalez de Peredo et al., 2002; Hartmann and Hofsteenge, 2000; Hofsteenge et al., 2001; Wang et al., 2009), its function remains unknown.

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TSRs are small cysteine-rich motifs containing six conserved cysteines paired in three disulfide bonds. They are similar to EGF repeats, although larger (about 60 amino acids) with a distinct disulfide bonding pattern (Adams and Tucker, 2000). These disulfide bonds can be in two different patterns designated group 1 and group 2. Group 1 TSRs have the disulfide bonding pattern C1–C5, C2–C6, C3–C4, whereas group 2 TSRs have the bonding pattern C1–C4, C2–C5, C3–C6 (Adams and Tucker, 2000). TSRs are found in a wide array of secreted or cell-surface proteins, many of which interact with extracellular matrix (ECM) components. The number of TSRs in proteins varies from one to many; however, TSRs often occur in tandem repeats (Adams and Tucker, 2000). Searching databases for proteins that contain the current CX2–3(S/T)CX2G within the context of a TSR, we recently identified 51 proteins in the mouse genome that are predicted to have O-fucosylated TSRs (Table 18.1) (Du et al., 2010). Proteins predicted to be modified can be grouped into several families including the thrombospondins, ADAMTS (A Distintegrin And Metalloprotease with Thrombospondin type 1 repeats) and ADAMTSL (ADAMTS-like) families. Thrombospondins are well known for their antiangiogenic function and ability to increase wound healing and bind a variety of receptors including CD36 and integrins (Bornstein et al., 2004; Dawson et al., 1997; Orr et al., 2002). Interestingly, the amino acid sequence that is biologically relevant for CD36 binding coincides with the consensus sequence for O-fucosylation (Dawson et al., 1997; Yehualaeshet et al., 2000). The ADAMTS family contains secreted metalloproteinases that have diverse functions including aggrecanase and versicanase activities (ADAMTS1, 4, 5, 8, 9, 15, and 20), pro-collagen N-peptidase activity (ADAMTS2, 3, and 14), as well as von Willebrand factor protease activity (ADAMTS13) (Jones and Riley, 2005; Le Goff et al., 2006; Majerus et al., 2005). The ADAMTS-like family is similar but lacks the prodomain and metalloprotease domains characteristic of the ADAMTS family. Together, these three families of matricellular proteins likely play a critical role in creating and maintaining the dynamic equilibrium of signaling as well as composition of the extracellular space necessary for cell survival. A list of predicted targets, along with associated human diseases and knockout phenotypes for each protein can be seen in Table 18.1. Identification of the enzymes responsible for addition of O-fucose glycans to EGF repeats and TSRs has revealed the existence of two separate O-fucosylation pathways in cells (Luo et al., 2006b). As mentioned above, EGF repeats are O-fucosylated by Pofut1, an ER localized enzyme that modifies properly folded EGF repeats containing the appropriate consensus sequence (Wang et al., 2001). Protein O-fucosyltransferase 2 (Pofut2) was identified based on sequence similarity to Pofut1 and demonstrated to be the TSR-specific O-fucosyltransferase (Luo et al., 2006a,b). Like Pofut1, Pofut2 is ER localized and only modifies properly folded TSRs containing the

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Table 18.1 Human diseases and mouse knockout phenotypes of predicted Pofut2 targets Protein

Human disease/KO phenotype

TSP1

KO-viable, abnormal pulmonary homeostasis (Lawler et al., 1998) KO-viable, disordered collagen fibers, increased vasculature, increased clotting time (Kyriakides et al., 1998) KO-impaired female fertility, similarities to human congenital ureteropelvic junction obstruction (Shindo et al., 2000) Ehlers-Danlos syndrome type VIIC (Colige et al., 1999); KO-male sterility, developed defective collagen fibril morphology (Li et al., 2001) KO-viable (Glasson et al., 2004) KO-viable, stopped progression of cartilage degradation (Glasson et al., 2005) KO-early embryonic lethality, heterozygous spontaneous corneal neovascularization (Koo and Apte, 2010) Weill-Marchesani syndrome (Dagoneau et al., 2004) KO-viable, increased angiogenesis, and tumor invasion (El Hour et al., 2010) Thrombotic thrombocytopenic purpura (Zheng et al., 2002); KO-viable, strain dependent TTP (Motto et al., 2005) Weill-Marchesani-like syndrome (Morales et al., 2009) KO-belted mouse (Silver et al., 2008) Geleophysic dysplasia (Le Goff et al., 2008) Bilateral ectopia lentis (Greene et al., 2010) Embryonic lethal by E 12.5, decreased complexity of cranial cardinal vein branches (Fiore et al., 2005) KO embryonic lethal–placental vascular insufficiency (Mo et al., 2002) KO lethal immediately after birth—generalized chondrodysplasia (Ivkovic et al., 2003) KO-viable—increased neointimal hyperplasia after endothelial injury (Shimoyama et al., 2010) Progressive pseudorheumatoid dysplasia (Hurvitz et al., 1999); mature vertebral endplates (Perbal et al., 2003) KO-viable (Kimura et al., 2008) KO-viable, decreased apoptosis, and increased neurons in spinal cord (Williams et al., 2006)

TSP2

ADAMTS1

ADAMTS2

ADAMTS4 ADAMTS5 ADAMTS9

ADAMTS10 ADAMTS12 ADAMTS13

ADAMTS17 ADAMTS20 ADAMTSL2 ADAMTSL4 SEMA5A CCN1 CCN2 CCN3 CCN6

Properdin UNC5a

(continued)

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Table 18.1 (continued) Protein

Human disease/KO phenotype

No known diseases or characterized knockouts ADAMTS3, 6–8, 14–16, 18–19; ADAMTSL1, 3, 5–6; SEMA5B, Hemicentin1, ISM1, 2; XM_283765, CCN4, 5; THSD7a, b; C6, f-spondin, CILP2, Papillin, Scospondin

appropriate consensus sequence (Luo et al., 2006a). The fact that both Pofut1 and Pofut2 are ER localized (a folding compartment) and have the ability to distinguish between folded and unfolded structures (EGF repeats and TSRs, respectively) led to the suggestion that they may play a role in quality control (Luther and Haltiwanger, 2009). In support of this idea, a number of studies suggest that Pofut1 functions as a chaperone as well as a fucosyltransferase (Okajima et al., 2005). Elongation of the O-fucose is also specific to EGF repeats or TSRs. O-Fucose on EGF repeats can be elongated by the Fringe family of b3-N-acetylglucosaminyltransfersaes, while O-fucose on TSRs can be elongated by a b3-glucosyltransferase (Kozma et al., 2006; Luo et al., 2006b; Sato et al., 2006). There does not appear to be any cross-talk between these two distinct O-fucosylation pathways (Luo et al., 2006b). While there are no known diseases caused by mutations in Pofut2, mutations in the b1,3-glucosyltransferase that elongates O-fucose on TSRs lead to a human genetic disorder known as Peter’s Plus syndrome (Lesnik Oberstein et al., 2006; Sato et al., 2006). Peter’s Plus syndrome is an autosomal recessive disorder characterized by anterior eye chamber defects, short stature, developmental delay, and increased incidence of cleft lip/palate (Lesnik Oberstein et al., 2006). Analysis of properdin, a known b1,3-glucosyltransferase target, from Peter’s Plus patient serum showed that these diseasecausing mutations lead to failure to elongate the O-fucose monosaccharide (Hess et al., 2008). These findings suggest that elongation of O-fucose on TSRs plays an essential role in normal biological development. Recently, in collaboration with Dr. Bernadette Holdener’s laboratory, we have begun analyzing the effects of knocking out Pofut2 mice. These studies show that Pofut2 is necessary for early murine development, since the embryos die by E 9.5. These embryos set up proper anterior–posterior polarity but then fail to

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correctly gastrulate. Early studies suggest that Pofut2 is necessary for normal lineage specification as well as ECM homeostasis (Du et al., 2010). The embryonic lethal phenotype of the Pofut2 null mice together with Peter’s Plus syndrome strongly suggest that O-fucosylation of TSRs is essential for the function of one or more proteins bearing these glycans. We know very little about why O-fucosylation of TSRs is essential for any of the proteins predicted to be modified (Table 18.1). A potential clue comes from our prediction that Pofut2 may play a role in quality control based on the fact that it is ER localized and only modifies properly folded TSRs. Studies on the role of O-fucosylation of a subset of Pofut2 targets, including ADAMTSL1 and ADAMTS13, provide support for this concept. Loss or reduction of O-fucosylation of these proteins in cell culture leads to decreased secretion (Ricketts et al., 2007; Wang et al., 2007). Multiple methods were used to reduce O-fucosylation, including mutation of O-fucose sites on the TSRs, RNAi-mediated knockdown of Pofut2, or expression of the proteins in cells with defects in the synthesis of GDPfucose (Lec13-CHO cells) (Ricketts et al., 2007; Wang et al., 2007). These studies suggest that O-fucosylation by Pofut2 is either necessary for proper trafficking of these proteins through the secretory pathway or to mediate protein folding and exiting the ER. Another interesting possibility is that O-fucose on TSRs changes ligand binding affinity. Studies of TSP1 binding to CD36 demonstrated that the O-fucose consensus sequence coincides with the ligand binding sequence (Dawson et al., 1997; Yehualaeshet et al., 2000). Although there is no data on how glycosylation status of the TSRs effects receptor binding, it will be interesting to see how the addition of the mono- and disaccharide could possibly change binding capability or specificity. Although these are all intriguing findings, further work is needed to define the molecular mechanisms by which O-fucosylation of these proteins affects their function.

2. Glycosylation Site Mapping by Mass Spectrometry Previously, our laboratory published a method for analyzing O-fucosylated EGF repeats using an in-gel digestion prior to analysis by LC–MS/ MS methods (Nita-Lazar and Haltiwanger, 2006). While this approach is useful for some samples, the protocol is labor intensive and many large, hydrophobic peptides are not well recovered from the gel slices. To decrease the sample preparation time and to increase sequence coverage, we have developed an in-solution digest approach. The method below is highly effective and yields better sequence coverage than the in-gel digestion approach. In addition, we have added semiquantitative method known

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as multiple-reaction monitoring (MRM) to evaluate the extent of modification at a specific glycosylation site (Hess et al., 2008).

2.1. Materials Bond-Breaker TCEP (Tris[2-carboxyethyl] phosphine–HCl) solution, Pierce, cat #77720 (0.5 M TCEP, pH 7.0) Iodoacetamide (Sigma, I-1149) Acetonitrile (Optima LC/MS, Fisher Scientific) Acetone (Fisher Scientific) Formic acid (96%, Aldrich) Urea (Electrophoresis grade, Fisher Scientific) Water (HPLC grade, Pharmco-Aaper) Sequencing grade modified trypsin (Promega) Proteomics grade Asp-N, GluC, LysC (Pierce) 0.22 mm spin filter (Agilent)

2.2. Stock solutions 1 M Tris–HCl, pH 8.0 (sterile filtered) 1 M ammonium bicarbonate, pH 8.0 (sterile filtered, 1 ml aliquots stored at 20  C) 0.5 mg/ml trypsin (resuspended in buffer provided by manufacturer stored in 6 ml aliquots at 80  C) 5% formic acid Buffer A: 0.1% formic acid Buffer B: 95% acetonitrile, 0.1% formic acid

2.3. Fresh solutions 50 mM Tris–HCl, pH 8.0 8 M urea, 0.4 M ammonium bicarbonate, pH 8.0 100 mM iodoacetamide, 50 mM Tris–HCl, pH 8.0 20 mM ammonium bicarbonate, pH 8.0 0.03 mg/ml trypsin, 20 mM ammonium bicarbonate, pH 8.0

2.4. Sample preparation Precipitate 1 mg or less of protein with 4 volumes of acetone in a 1.5-ml microfuge tube. Denature and reduce the protein by resuspending in 10 ml of 8 M urea, 10 mM TCEP, 0.4 M ammonium bicarbonate, pH 8.0. Vortex, and heat to 50  C, 5 min, and then cool at room temperature. Next, add 5 ml

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100 mM iodacetamide, 50 mM Tris–HCl, pH 8.0, vortex, and incubate for 30 min in the dark at room temperature (iodoacetamide is light sensitive, so it is important to incubate in the dark). Effective reduction and alkylation is key for analysis of cysteine-rich motifs like TSRs. The reduced and alkylated sample is then diluted with 45 ml of water and vortexed to dilute the urea to a final concentration of 2 M. Trypsin (as well as most proteases) is functional at this concentration of urea. Next, check the pH using 1 ml of sample solution on pH paper. If below pH 7, add 1 M Tris, pH 8.0 (1 ml at a time) until the pH is raised to pH 8.0. Add 5 ml of enzyme solution (150 ng enzyme), vortex, and incubate at 37  C for 2 h to overnight. Stop the digestion by addition of 7 ml 5% formic acid, and sonicate in a bath sonicator for 20 min. To remove any particulates before loading on the HPLC, spin the sample through a 0.22-mm membrane spin filter.

2.5. LC–MS of the digested sample Samples are analyzed by nano-liquid chromatography/tandem mass spectrometry using an Agilent 6340 ion-trap mass spectrometer with an HPLC ChipCube interface and autosampler. One to 3 ml of sample is injected onto a Zorbax 300SB-C18 nano-CHIP with a 40-nl enrichment column and a 43-mm  75mm separation column (Agilent, 4240-62001). The sample is loaded onto the enrichment column at a flow rate of 4 ml/min at 5% Buffer B using the capillary pumps. After loading, the valve on the CHIP changes to the nanopumps, and the samples are eluted from the enrichment column and separated on the separation column at a flow rate of 450 nl/min with a 25-min linear gradient from 5% to 95% Buffer B. The effluent from the CHIP is sprayed directly into the mass spectrometer at an orifice potential of 1600–2000 V with drying gas (nitrogen) flow rate of 5 l/min with a gas temperature of 325  C. The scanning range is 300–2200 (m/z) and collision-induced dissociation (CID) fragmentation (MS2) is performed on the three most abundant ions in each spectrum. Recurring ions are excluded from MS2 for 30 s after two consecutive spectra. Ions containing O-fucose glycans are identified by neutral loss searches of the MS2 data. Neutral loss masses searched are for glucose (hexose, 162 Da), fucose (deoxyhexose, 146 Da), or the sequential loss of Glc–Fuc (308 Da). Tryptic peptides can exist in various charge states (2þ, 3þ, 4þ). For example, a neutral loss search for 102.6 Da identifies triply charged ions that have lost the Glc–Fuc disaccharide. Glycosylated peptides identified by neutral loss search are manually selected for MS/MS/MS (MS3) runs. In MS3, the most abundant ion in the MS2 spectra, usually the unglycosylated parent ion, is subjected to another round of fragmentation, yielding high intensity b and y ions which are matched to predicted fragments of the parent peptide. Automated neutral loss selection can also be performed using the Agilent 6340 mass spectrometer. During automated neutral loss runs, the instrument identifies ions in the MS2 spectra that match user input neutral loss values and then automatically subjects the major product ions to MS3.

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M = 497 WSPWSPWSACTVTCAGGIR515 + Hex-dHex m/z = 830.2

Intens. 5 MS ×10

+MS, 9.1 min #668

3+

[M + 3H] 829.6

6 4 2

829.6

0 ×106 MS / MS

3+

[M + 3H-Hex-dHex] 727.5

0.8 0.6

0.2 0.0 ×104 MS / MS / MS 3

1 0

[M + 2H-Hex-dHex]2+ 1090.9

3+

[M + 3H-Hex] [M + 3H]3+ 776.1 830.2

0.4

2

+MS2(830.2), 9.1 min

y3 345.3

y5 y4 473.6 402.5

400

y6

b4 633.5 b5 557.3 644.4

600

9 11 5 497 515 WS PW S PWSA CT VT C A G GIR 17 15 14 6 5 4 3

y142+ 2+ 768.9 y15 812.4

800

+MS3(830.2->727.5), 8.5 min 2+

y17-NH3 945.4

b11 –H2O 1328.7

b9 1085.5 1000

1200

m/z

b-ions y-ions

Figure 18.1 TSR3 of TSP2 is modified with a fucose–glucose disaccharide. Approximately 1 mg of hTSP2, generously supplied by Dr. Paul Bornstein, was reduced, alkylated, and digested with trypsin as described in the text. A neutral loss search for 102.6 (ions losing 102.6 Da during CID) identified an abundant ion eluting at 9.1 min. Top panel: MS spectrum at 9.1 min of an HPLC run. The ion 829.6 m/z corresponds to the triply charged glycopeptide M, defined at the top. Middle panel: MS/MS spectrum of 830.2 m/z from MS spectra. The major product ions indicated correspond to the sequential loss of glucose (M þ 3H-Hex) and fucose (M þ 3H-Hex-dHex) from the parent ion (M þ 3H). Bottom panel: MS/MS/MS of 727.5 m/z from the MS/MS spectra (the peptide without the fucose and glucose). The resulting spectra provides b and y ions verifying the identity of M.

An example of this type of analysis is shown in Fig. 18.1. Human thrombospondin 2 (TSP2) was digested with trypsin and analyzed as described above. A neutral loss search of the resulting data revealed a major species of m/z 829.6 in the MS spectra eluting at 9.1 min (Fig. 18.1, top panel). Fragmentation of this ion (Fig. 18.1, middle panel) results in the neutral loss of a Glc–Fuc disaccharide from the triply charged glycopeptide. Ions representing the sequential loss of the glucose ([M þ 3H-Hex]3þ, m/z 776.1) and the fucose ([M þ 3H-Hex-dHex]3þ, m/z 727.5) can be seen. The major product ion, m/z 727.5, corresponds to the triply charged form of a tryptic peptide from TSP2 containing a predicted O-fucose consensus site (WSPWSPWSACTVTCAGGIR). The identity of this peptide was confirmed by performing MS3 of the m/z 727.5 ion (Fig. 18.1, bottom panel). Similar analyses were performed to identify all other O-fucosylated peptides in the sample (data not shown). Table 18.2 provides a summary of the data identifying these glycopeptides derived from all three TSRs of TSP2, including several also bearing

Table 18.2 O-Fucosylated peptides from thrombospondin 2

TSR

Glycopeptides identified

DEGWSPWAEWTECSVTCGSGTQQRGRSC408 þ Fuc–Glc 379 DSDEGWSPW#AEWTECSVTCGSGTQQRGRSC408 þ Fuc–Glc þ Man TSR2 436QNGGWSHW#SPWSSCSVTCGVGNVTR 470 þ Fuc–Glc þ Man 436 QNGGW#SHW#SPWSSCSVTCGVGNVTR470 þ Fuc–Glc þ Man þ Man TSR3 497WSPWSPWSACTVTCAGGIR515 þ Fuc–Glc 497 WSPWSPW#SACTVTCAGGIR515 þ Fuc–Glc þ Man 497 WSPW#SPW#SACTVTCAGGIR515 þ Fuc–Glc þ Man þ Man TSR1

381

[M þ H]þ of [M þ H]þ of parent product

Predicted [M þ H]þ

Parent– product

3587.8

3277.0

3276.5

310.8

3949.0

3641.1

3640.8

307.9

3262.9

2954.5a

2970.20

308.4

3426.1

3116.5a

3132.34

309.6

2489.2 2651.5 2814.1

2180.2 2343.1 2505.1

2180.48 2342.63 2504.77

309.0 308.2 309.0

Predicted sites of O-fucosylation on peptides based on the consensus sequence, CX2–3(S/T)CX2G, are underlined. Sites of C-mannosylation are indicated by # (as determined by analysis of CID fragmentation data). Masses of singly charged parents and products were calculated by the formula [(m/z)  z]  (z  1). Predicted masses of unglycosylated peptides are based on average masses. a Product masses suggest deamidation within the peptide. b and y ions indicate deamidation of N437.

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C-mannose. Although this method does not definitively identify the Ofucosylated amino acid, it does demonstrate the peptide with the consensus sequence is modified with the Glc–Fuc disaccharide. TSRs with the O-fucose consensus sequence could potentially occur in unmodified forms, with an O-fucose monosaccharide, or with the Glc–Fuc disaccharide. While MS2/MS3 allows identification of glycosylated peptides, they do not supply information on relative amounts of unmodified, monosaccharide, and disaccharide forms of the peptide. To get a semiquantitative assessment for the relative extent of glysosylation at a single site, we use MRM (Hess et al., 2008; Unwin et al., 2006). MRM allows us to monitor the relative abundance of the individual forms of a given peptide in a sample. This method assumes equivalent ionization of different glycoforms of the same peptide. Although this may not always be the case, our experiences suggest that the addition of O-fucose or O-glucose glycans to most peptides does not significantly affect their ionization efficiency (Acar et al., 2008). As an example, we wanted to analyze the relative amounts of unmodified, monosaccharide, and disaccharide forms of the peptide described in Fig. 18.1. The specificity of the analysis relies on the ability to follow a particular parent ion (e.g., m/z 830.2 for the disaccharide form of the peptide) that generates a particular daughter, or transition ion (e.g., m/z 727.5) upon fragmentation. To follow the monosaccharide form of the peptide, the parent ion was defined as m/z 776.1 and the transition ion as m/z 727.5, and for the unmodified peptide the parent was defined as m/z 727.5 with transition ion corresponding to m/z 633.5 (y6). MRM analysis of this sample showed the disaccharide form to be the most abundant in this sample (Fig. 18.2). The monosaccharide and unglycosylated peptides were not present above background levels (Fig. 18.2). Intens. ×105

TSR3 + Fuc-Glc

1.5 1.0

TSR3 + Fuc

0.5

TSR3

0.0

1

2

3

4

5

6

7

8

Time (min)

Figure 18.2 The majority of TSP2 TSR3 is modified with a disaccharide. MRM of the disaccharide form of the peptide from TSR3 (830.2 m/z transition ion 727.5), the monosaccharide form of the peptide (776.1 m/z transition ion 727.5), and the unmodified peptide (727.5 m/z transition ion 633.5 (y6)). Peptides elute at 9.1 min with the disaccharide being the most abundant.

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Combined, these methods allow us to map glycosylation sites on predicted Pofut2 target proteins and the relative amounts of glycosylation and elongation at specific sites in our samples. Our current efforts are focused on refining the consensus sequence for O-fucosylation by Pofut2 by both mapping of glycosylation on TSRs in predicted targets as well as sitedirected mutagenesis of the current consensus sequence. The question of whether other O-fucose structures on TSRs exist as trisaccharides (or larger) still remains open. Our earlier studies show that the disaccharide is the most prevalent form of modification (Ricketts et al., 2007; Wang et al., 2007, 2009). However, the use of MRM, which allows higher specificity and sensitivity of peptide monitoring, will allow determination of whether the stoichiometries are consistent between various TSR consensus sequences and target proteins.

ACKNOWLEDGMENTS We thank Dr. Paul Bornstein for the kind gift of human thrombospondin 2 protein, Nadia Rana for her vital assistance with mass spectrometry, and members of the Haltiwanger and Holdener laboratories for helpful discussions and comments on this manuscript. This work was supported by NIH Grant CA12307101.

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Sato, T., et al. (2006). Molecular cloning and characterization of a novel human beta1, 3-glucosyltransferase, which is localized at the endoplasmic reticulum and glucosylates O-linked fucosylglycan on thrombospondin type 1 repeat domain. Glycobiology 16, 1194–1206. Shao, L., and Haltiwanger, R. S. (2003). O-fucose modifications of epidermal growth factor-like repeats and thrombospondin type 1 repeats: Unusual modifications in unusual places. Cell. Mol. Life Sci. 60, 241–250. Shi, S., and Stanley, P. (2003). Protein O-fucosyltransferase 1 is an essential component of Notch signaling pathways. Proc. Natl. Acad. Sci. USA 100, 5234–5239. Shimoyama, T., et al. (2010). CCN3 inhibits neointimal hyperplasia through modulation of smooth muscle cell growth and migration. Arterioscler. Thromb. Vasc. Biol. 30, 675–682. Shindo, T., et al. (2000). ADAMTS-1: A metalloproteinase-disintegrin essential for normal growth, fertility, and organ morphology and function. J. Clin. Invest. 105, 1345–1352. Silver, D. L., et al. (2008). The secreted metalloprotease ADAMTS20 is required for melanoblast survival. PLoS Genet. 4, e1000003. Takeuchi, H., and Haltiwanger, R. S. (2010). Role of glycosylation of Notch in development. Semin. Cell Dev. Biol. (in press) Unwin, R. D., et al. (2006). Relative quantification in proteomics: New approaches for biochemistry. Trends Biochem. Sci. 31, 473–484. Wang, Y., et al. (2001). Modification of epidermal growth factor-like repeats with O-fucose. Molecular cloning and expression of a novel GDP-fucose protein O-fucosyltransferase. J. Biol. Chem. 276, 40338–40345. Wang, L. W., et al. (2007). O-fucosylation of thrombospondin type 1 repeats in ADAMTSlike-1/punctin-1 regulates secretion: Implications for the ADAMTS superfamily. J. Biol. Chem. 282, 17024–17031. Wang, L. W., et al. (2009). Post-translational modification of thrombospondin type-1 repeats in ADAMTS-like 1/punctin-1 by C-mannosylation of tryptophan. J. Biol. Chem. 284, 30004–30015. Williams, M. E., et al. (2006). UNC5A promotes neuronal apoptosis during spinal cord development independent of netrin-1. Nat. Neurosci. 9, 996–998. Xu, A., et al. (2007). In vitro reconstitution of the modulation of Drosophila Notch–ligand binding by Fringe. J. Biol. Chem. 282, 35153–35162. Yehualaeshet, T., et al. (2000). A CD36 synthetic peptide inhibits bleomycin-induced pulmonary inflammation and connective tissue synthesis in the rat. Am. J. Respir. Cell Mol. Biol. 23, 204–212. Zheng, X., et al. (2002). ADAMTS13 and TTP. Curr. Opin. Hematol. 9, 389–394.

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Use of Glycan Microarrays to Explore Specificity of Glycan-Binding Proteins David F. Smith, Xuezheng Song, and Richard D. Cummings Contents 1. Overview 2. The Printed Glycan Microarray from the Consortium for Functional Glycomics (CFG) 3. Analysis of GBPs on the CFG Glycan Microarray 4. Defining a Glycan Motif Using Concentration-Dependent Binding of GBPs to the Printed Glycan Microarray 5. The Concentration-Dependent Binding of Sambucus nigra Agglutinin to the Printed Glycan Microarray and a Method for Ranking the Relative Binding Strengths to Glycan Ligands 6. Using Microarrays to Identify a Glycan-Binding Motif for SNA 7. Specificities of Human Galectin-8 and its Carbohydrate Recognition Domains (CRDs) 8. Challenges in Identifying Physiological Ligands for GBPs 9. Conclusion and Future Directions Acknowledgments References

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Abstract Microarrays of defined glycans represent a high throughput approach to determining the specificity of lectins, or more generally glycan-binding proteins (GBPs). The utility of a glycan microarray is directly related to the number and variety of the glycans available on the printed surface for interrogation by GBPs. The Consortium for Functional Glycomics (CFG), funded by the National Institute of General Medical Sciences (NIGMS), has generated a glycan microarray available to the public as an investigator-driven resource, where hundreds of GBPs have been analyzed. Here we describe the methods generally used by the CFG to prepare glycan arrays and interrogate them with GBPs. We also describe Department of Biochemistry, The Glycomics Center, Emory University School of Medicine, Atlanta, Georgia, USA Methods in Enzymology, Volume 480 ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)80033-3

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our new approach to normalizing glycan microarray data derived from concentration-dependent analyses of GBP binding, and the application of this approach with the plant lectin Sambucus nigra agglutinin (SNA-I) and human galectin-8. The use of glycan microarrays with this approach readily generates a prediction of the glycan determinants required for high affinity binding by a GBP.

1. Overview The printed glycan microarray evolved from a microtiter plate-based array of biotin-derivatized glycans (Alvarez and Blixt, 2006; Blixt et al., 2004) and was a logical extension of the DNA microarray for gene expression and the protein microarray for protein–protein interaction analyses. These new microarray techniques involve application of a small volume of buffer containing a glycan-binding protein (GBP) to a microarray comprised of hundreds of defined glycans, and the protein–carbohydrate interaction is detected by fluorescence of either the fluorescently labeled GBP or a secondary reagent that binds to the GBP. This approach allows investigators to accomplish in a single experiment with minimal reagents what might require months of work using hapten inhibition assays, which is one of the classical methods for defining the specificity of a GBP. The printed glycan microarray available through the Consortium for Functional Glycomics (CFG) now contains over 500 glycan targets, and is publicly available to investigators worldwide for exploring GBP specificities as part of its mission to define the paradigms by which protein–carbohydrate interactions mediate cell communication (http://www.functionalglycomics.org). This glycan microarray is produced from a large library of defined amino-functionalized glycans printed and covalently bound to N-hydroxysuccinimide (NHS)-derivatized glass slides. The glycan microarrays are available to researchers following online request and approval by the CFG Steering Committee, who evaluate specific requests, and approved requests are processed by the CFG protein– carbohydrate interaction Core (Core H), which receives samples for analysis. Through this process hundreds of GBPs have been analyzed on thousands of glycan microarrays in the time period 2004–2010, and the resulting publications represent a revolution in the process of defining the specificity of a GBP. While a variety of approaches to producing glycan microarrays are available (Blixt et al., 2004; de Boer et al., 2007; de Paz et al., 2006; Fukui et al., 2002; Kuno et al., 2005; Oyelaran and Gildersleeve, 2009; Ratner et al., 2004; Song et al., 2009; Wang, 2003), one underlying concept is clear. The utility of a defined glycan microarray is directly proportional to the number of defined glycans available for interrogation. Thus, the very large glycan microarray that has been made available to the public by NIGMS through the CFG is a discovery platform that permits examination of GBP specificities with minute

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quantities of glycans. In the microarray format, GBP specificity is predicted by inspecting its relative binding to the different glycans on the array. On the CFG glycan microarray, the initial analysis is done, if possible, at a GBP concentration of 100–200 mg/ml to determine first whether the GBP binds any glycans. If no binding is observed, there is no need to assay at higher or lower concentrations and the data are considered inconclusive. Lack of binding could be due to multiple factors, including the potential inactivity of the GBP, loss of activity during its preparation for the assay, or the absence of a glycan on the microarray that is recognized by the GBP. Ideally, all GBP preparations tested on the microarray have some independent verification that they are active, for example, binding to cells, agglutinating cells, signal transduction, etc. If binding of a GBP to glycans on the microarray is observed, however, the high protein concentrations used in the initial assay are considered irrelevant with respect to determining specificity if more than one binding motif is observed, since it is common for GBPs to display cross-reactivity for related glycans at such high concentrations. For GBPs that bind, their interactions are analyzed at decreasing GBP concentrations in order to determine the relative binding of the GBP for individual glycans available on the microarray. We describe here the methods used by the protein–glycan interaction Core H of the CFG for determining GBP specificity by inspection of binding at decreasing concentrations of GBP. Please refer Chapters 10-14 of Volume 478 of this series for additional methods.

2. The Printed Glycan Microarray from the Consortium for Functional Glycomics (CFG) Glycan Array Synthesis Core (Core D) produces the CFG glycan microarray (http://www.functionalglycomics.org/static/consortium/organization/ sciCores/cored.shtml), as previously described (Blixt et al., 2004). Figure 19.1 provides a summary of the steps involved in producing the glycan array and subsequently analyzing a GBP using a variety of fluorescent detection methods to produce data in a histogram format. The CFG array is printed on glass microscope slides that are derivatized with NHS (SCHOTT NexterionÒ Slide H, SCHOTT North America, Elmsford, NY). All glycans available to the Glycan Array Synthesis Core D of the CFG possess a primary amine on a linker attached to the reducing end of each glycan. The glycan structures and the structure of individual linkers for each version of the CFG glycan microarray are available at (http://www.functionalglycomics.org/ static/consortium/resources/resourcecoreh8.shtml). The first printed glycan microarray, available in 2005, was v2.0 and contained 264 glycan targets, and over 5 years through eight iterations it has been expanded to 511 glycans. Each glycan target is printed at the same concentration (100 mM) in replicates

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of six. Only a few nanograms of each glycan is linked to the slide in a spot diameter of
100 microns. A GAL file, which is a.txt file that identifies the location of each spot on the microarray, defines each microarray and permits glycan spot alignment with fluorescent images of the GBP binding. The printed glycan microarrays are stable and stored desiccated at room temperature.

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3. Analysis of GBPs on the CFG Glycan Microarray GBPs are dissolved at a known concentration in a binding buffer, which can be defined for the GBP being analyzed. There are wide variations allowed in the binding buffer, since the slides are stable, and the conditions for binding can vary in pH (pH 4.5–8.5) and can even include organic solvents such as dimethylsulfoxide (DMSO) and dimethylformamide (DMF). The common binding buffer used for evaluating a GBP whose glycan specificity is unknown is Tris-buffered saline containing calcium and magnesium (TSM: 0.02 M Tris–HCl, 0.15 M NaCl, 0.002 M CaCl2, 0.002 M MgCl2, 0.05% Tween 20, 1% BSA, pH 7.4). Ideally, the GBP preparation and assay buffer are ones for which there is independent evidence that it is active and stable. Prior to analysis, the microarray slide is rehydrated for 5 min in a solution of TSM plus 0.05% Tween (TSMW) at room temperature and allowed to drain by gently touching the end of the slide to a paper towel. Slides are always handled with powder-free gloves and never touched in the printed area of the slide. To conserve protein, an aliquot of the GBP (50–70 ml) is applied to the printed surface of the microscope slide (Fig. 19.1A), and a cover slip (24 mm by 50 mm) is applied to spread the sample over the entire microarray. The slide is incubated in a humidified chamber at an appropriate temperature for at least 1 h. In cases where the GBP may be available in large quantities, the microarray can be submerged in several milliliters of a GBP solution, or the 0.5–1 ml of GBP solution can be applied to the printed area of the slide defined by a hydrophobic barrier pen. After incubation and gentle removal of the cover slip, the slide is washed by dipping four times (
3–5 s each) into a Coplin jar containing 100 ml of wash buffer I (TSMW: 0.02 M Tris–HCl, 0.15 M NaCl, 0.002 M CaCl2, 0.002 M MgCl2, 0.05% Tween 20, pH 7.4) followed by washing the same way with wash buffer II (TSM, 0.02 M Tris– HCl, 0.15 M NaCl, 0.002 M CaCl2, 0.002 M MgCl2, pH 7.4). If the GBP is not directly labeled, it is detected by an indirect method, which can be any immunochemical detection by fluorescence. For example, biotinylated proteins are detected with commercially available fluorescentlabeled streptavidin (Fig. 19.1A); Fc fusion proteins with commercially available fluorescent-labeled anti-human IgG; His-tagged recombinant proteins with commercially available fluorescent-labeled anti-His antibody; GST fusion proteins with commercially available fluorescent-labeled anti-GST antibody; and other detection methods as required (Fig. 19.1B). The optimal concentration for each of the detecting reagents has to be empirically defined, and is considered to be the concentration that allows detection of specific binding of the GBP with the least possible background in the absence of specific binding by the GBP. After the secondary step, the slide is washed as described above, followed by a water wash to remove salt, which will interfere

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with the fluorescence reading. Residual water is removed by centrifugation in a slide centrifuge or by blowing the water off using a gentle stream of nitrogen. If the GBP is directly labeled with a fluorescent tag, the slide is washed in water using the same procedure. The slide is then allowed to air dry for
5 min and analyzed in a fluorescence reader such as the ProScanArray (PerkinElmer, Waltham, MA) equipped with multiple lasers to support analyses of most commercially available fluorescent probes. The software provided by the scanner manufacturer or other software used for microarray analysis (ImaGene from BioDiscovery, El Segundo, CA) processes the image using the GAL file described above to assign the glycan structures to each spot. The data are generated as an Excel file and a specific macro is designed to arrange the data in tables including an identification number, a structure, and the average relative fluorescence units (RFU) value determined as the average of the 4 RFU values after removing the high and low values from the six data points. The standard deviation, standard error of the mean, and the %CV may also be reported. The final data are presented as a histogram where the glycan identification number that can be related to the glycan structure is plotted against the average RFU value with error bars being the standard deviation (Fig. 19.1C). The data may also be sorted from high to low average RFU values to assist in identifying the bound glycans for comparison to identify the GBP specificity. Consistent with the CFG data sharing policy, the data from CFG glycan microarray slides are uploaded to the CFG database as the ImaGene.txt file; the web application manages the output presented on the CFG Web site.

4. Defining a Glycan Motif Using Concentration-Dependent Binding of GBPs to the Printed Glycan Microarray To identify the glycan-binding specificity or motif for a GBP, the pattern of its binding to the defined glycans on the microarray is inspected and the intensities of the relative fluorescent units associated with each bound glycan structure are compared. At high GBP concentrations ( 200 mg/ml), it is not uncommon to observe significant cross-reactivity with glycans bound with very low affinity. Even at these nonphysiologically relevant, high concentrations, most GBPs do not bind to all glycans on the microarray, which is a testament to the very low backgrounds observed in this technique if the washing of the slides is performed properly after incubation with the GBP and each subsequent detecting reagent. Thus, when investigating a GBP of unknown specificity or when trying to define the subtleties of a GBP motif, we have found that doing the analysis at decreasing GBP concentrations permits detection of motifs that would be missed due to the binding of

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low affinity glycan ligands, which disappear as the GBP concentration decreases. Thus, GBP binding to the immobilized, multivalent glycans is obviously related to its equilibrium dissociation constant, even though the assay is not performed at equilibrium. Examples of such analyses include recent studies on human galectins (Stowell et al., 2008a,b) and the C-type lectin human LSECtin (Powlesland et al., 2008).

5. The Concentration-Dependent Binding of Sambucus nigra Agglutinin to the Printed Glycan Microarray and a Method for Ranking the Relative Binding Strengths to Glycan Ligands Tremendous amounts of data are generated from each analysis of the CFG glycan microarray, and the prediction of a GBP glycan-binding specificity or binding motif is a laborious process of inspecting and comparing the linear sequences of monosaccharides in glycans that bind with those that do not bind or bind weakly. While a computer program was recently developed to detect preselected and defined trisaccharide determinants associated with binding glycans from microarray data (Porter et al., 2010), we have devised a simple method to rank the glycans bound by a GBP according to their relative strength of binding in terms of total fluorescence. We applied this simple ranking protocol to data available from the CFG glycan microarray on the well-characterized elderberry bark agglutinin I from Sambucus nigra agglutinin (SNA-I), which has been traditionally used to identify the Neu5Aca2-6Gal linkage (Shibuya et al., 1987b). It should be noted that Sambucus nigra contains additional related lectins to SNA-I, such as SNA-II and -III, but they have different structures and perhaps somewhat different binding specificities (Mach et al., 1991; Van Damme et al., 1997). As an example of the ranking of glycans bound by a GBP, we analyzed SNA-I binding to v4.0 of the CFG glycan microarray using biotinylated SNA (Vector Labs, Burlingame, CA) at concentrations of 1.0, 0.1, and 0.01 mg/ml. Bound lectin was detected by the fluorescence signal from Cy5-labeled streptavidin (Zymed, Carlsbad, CA) at 0.5 mg/ml. The SNA concentrations were selected from analyses where the RFU values for each analysis were within the linear range of the fluorescence scanner (0 to
60,000 RFU). The relationship of SNA binding and protein concentration is demonstrated by the differences in the RFU signals obtained at decreasing concentrations applied to the microarray as shown in Fig. 19.2A–C. To normalize the results obtained at each concentration, a rank for each glycan was obtained at each GBP concentration using the calculation, Rank ¼ 100  [RFU bound/highest RFU value in the assay]. Thus, the strongest bound

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Figure 19.2 Analysis of biotinylated Sambucus nigra agglutinin (SNA) on version 4.0 of the CFG glycan microarray. Biotinylated SNA at decreasing concentrations was incubated on the glycan array in TSM buffer under a cover slip for 1 h at room temperature. After washing, the lectin was detected by incubation with cyanine5-labeled streptavidin at 0.5 mg/ml under a cover slip in the same buffer for 1 h at room temperature. After washing, the slide was analyzed in a fluorescence scanner and the data reported as relative fluorescence units for each glycan structure identified by a number on the x-axis. The identity of individual glycans comprising version 4.0 of the CFG microarray is available at (http://www.functionalglycomics.org/static/consortium/ resources/resourcecoreh13.shtml).

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glycan in each assay at three different concentrations of GBP is assigned a rank of 100, and the ranking values decrease with decreased binding. Using the three rank values for each glycan, we calculate an average rank and sort all of the data in a descending order from highest to lowest rank as shown in Table 19.1 for the top 30 glycans. This permits a rapid selection of the bound glycans for any GBP normalized over three concentrations of GBP making the selection of binding ligands and simplifying the process of inspecting bound glycans. The entire data set is too large to be displayed here, but the list of glycan structures on the CFG microarray (v4.0) can be found on the CFG Web site (http://www. functionalglycomics.org/static/consortium/resources/resourcecoreh13.shtml). Included in Table 19.1 are all of the glycans on the microarray that possess the determinant Neu5Aca2-6Galb1-4GlcNAc with the exception of glycan number 313 (Neu5Aca2-3Galb1-4GlcNAcb1-2Mana1-3(Neu5Aca2-6Galb14GlcNAcb1-2Mana1-6)Manb1-4GlcNAcb1-4GlcNAcb-Sp12), which possesses both Neu5Aca2-6Galb1-4GlcNAc and Neu5Aca2-3Galb1-4GlcNAc termini and showed an average ranking of 8. The arrangement of data represents an average of three experiments where the highest RFU values for each analysis were 43,769 (glycan number 315), 13,083 (glycan number 353), and 2,794 (glycan number 206) for SNA concentrations 1, 0.1, and 0.01 mg/ml, respectively. Since no single glycan was ranked at 100 for all three analyses, the highest average ranking was less than 100. It is interesting to note that the average RFU for 24 of the top 30 glycans show SNA concentration dependence, and in all cases these glycan structures possess a Neu5Aca2-6Gal linkages. Four of the top 30 glycans do not show a concentration-dependence, such as glycan 193 GlcAb-Sp8, which suggests that SNA binding to such glycans is probably not by a specific sugar binding mechanism as for the other 24 glycans in the top 30. It is interesting to note that these glycans, 206 (Mana1-6(Mana1-3)Mana1-6 [Mana1-2Mana1-3]Manb1-4GlcNAcb1-4GlcNAcb), 388 (Neu5Aca2-3 (GalNAcb1-4)Galb1-4GlcNAcb1-3GalNAca), 193 (GlcAb), and 203 (Mana1-2Mana1-2Mana1-3(Mana1-2Mana1-3(Mana1-2Mana1-6)Mana16)Manb1-4GlcNAcb1-4GlcNAcb) are completely unrelated to the known determinant for SNA. Thus, glycans that do not show concentration dependence in the ranking analysis are eliminated from consideration in defining GBP specificity or binding motif.

6. Using Microarrays to Identify a Glycan-Binding Motif for SNA We were able to make some interesting observations regarding the subtle details of glycan-binding specificity by SNA by comparing the ranking of selected structures that were bound to related structures that

Table 19.1 The ranked glycans determined by glycan microarray analysis of Sambucus nigra agglutinin (SNA-I) at 3 concentrations on v4.0 of the CFG glycan microarray SNA 1 mg/ml

SNA 0.01 SNA 0.1 mg/ml mg/ml

Glycan Glycan structure (Note: alpha linkages are denoted by a; v4.0 beta linkages are denoted by b)

Avg. RFU

Avg. RFU

353 256 327

35,479 81 43,459 99 41,668 95

341 259 343 54 315 52 314 51

46

KDNa2-6Galb1-4GlcNAc-Sp0 Neu5Aca2-6Galb1-4[6OSO3]GlcNAcb-Sp8 Neu5Aca2-6Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb14GlcNAcb-Sp0 Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-3(Mana1-6)Manb14GlcNAcb1-4GlcNAc-Sp12 Neu5Aca2-6Galb1-4GlcNAcb1-3Galb1-4(Fuca1-3)GlcNAcb1-3 Galb1-4(Fuca1-3)GlcNAcb-Sp0 Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-3Manb1-4GlcNAcb14GlcNAc-Sp12 Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-3(Neu5Aca2-6Galb14GlcNAcb1-2Mana1-6)Manb1-4GlcNAcb1-4GlcNAcb-Sp13 Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-3(GlcNAcb1-2Mana16)Manb1-4GlcNAcb1-4GlcNAcb-Sp12 Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-3(Neu5Aca2-6Galb14GlcNAcb1-2Mana1-6)Manb1-4GlcNAcb1-4GlcNAcb-Sp8 Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-3(Galb1-4GlcNAcb12Mana1-6)Manb1-4GlcNAcb1-4GlcNAcb-Sp12 Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-3(Neu5Aca2-6Galb14GlcNAcb1-2Mana1-6)Manb1-4GlcNAcb1-4GlcNAcbN(LT)AVL Neu5Ac(9Ac)a2-6Galb1-4GlcNAcb-Sp8

Avg. RFU

Rank

Avg. rank

13,083 100 12,671 97 11,783 90

2248 1673 1504

80 60 54

87 85 80

42,633 97

8361

64

1279

46

69

41,634 95

8417

64

1301

47

69

41,301 94

8083

62

1274

46

67

36,250 83

9135

70

1267

45

66

43,769 100

6606

50

965

35

62

33,754 77

9467

72

831

30

60

35,848 82

7176

55

879

31

56

38,327 88

5582

43

1008

36

55

37,541 86

5539

42

1047

37

55

Rank

Rank

258 275 260 325 257 206 321 53 342 388 255 407 193 300 292 340 203 373

Neu5Aca2-6Galb1-4GlcNAcb-Sp8 Neu5Gca2-6Galb1-4GlcNAcb-Sp0 Neu5Aca2-6Galb1-4GlcNAcb1-3Galb1-4GlcNAcb-Sp0 Neu5Aca2-6Galb1-4GlcNAcb1-3Galb1-3GlcNAcb-Sp0 Neu5Aca2-6Galb1-4GlcNAcb-Sp0 Mana1-6(Mana1-3)Mana1-6[Mana1-2Mana1-3]Manb1-4Glc NAcb1-4GlcNAcb-Sp12 Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-3(Neu5Aca2-3Galb14GlcNAcb1-2Mana1-6)Manb1-4GlcNAcb1-4GlcNAcb-Sp12 Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-3(Neu5Aca2-6Galb14GlcNAcb1-2Mana1-6)Manb1-4GlcNAcb1-4GlcNAcb-Sp12 Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-6Manb1-4GlcNAcb14GlcNAc-Sp12 Neu5Aca2-3(GalNAcb1-4)Galb1-4GlcNAcb1-3GalNAca-Sp14 Neu5Aca2-6GalNAcb1-4GlcNAcb-Sp0 Neu5Aca2-6Galb1-3GlcNAcb1-3(Galb1-4GlcNAcb1-6)Galb14Glc-Sp21 GlcAb-Sp8 GlcNAcb1-2Mana1-3(Neu5Aca2-6Galb1-4GlcNAcb1-2 Mana1-6)Manb1-4GlcNAcb1-4GlcNAcb-Sp12 Galb1-4GlcNAcb1-2Mana1-3(Neu5Aca2-6Galb1-4GlcNAcb12Mana1-6)Manb1-4GlcNAcb1-4GlcNAcb-Sp12 Mana1-3(Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-6)Manb14GlcNAcb1-4GlcNAc-Sp12 Mana1-2Mana1-2Mana1-3(Mana1-2Mana1-3(Mana1-2 Mana1-6)Mana1-6)Manb1-4GlcNAcb1-4GlcNAcb-Sp12 Neu5Aca2-6Galb1-4GlcNAcb1-3GalNAc-Sp14

33,781 26,208 35,076 36,221 33,423 2737

77 60 80 83 76 6

4770 5535 4260 4756 3828 3028

36 42 33 36 29 23

916 1217 821 358 717 2794

33 44 29 13 26 100

49 49 47 44 44 43

26,481 61

2551

19

1326

47

42

25,345 58

4161

32

568

20

37

27,580 63

3263

25

516

18

35

2226 5 23,168 53 21,837 50

2268 2043 2161

17 16 17

1845 414 319

66 15 11

29 28 26

1149 3 23,101 53

1852 1649

14 13

1594 49

57 2

25 22

21,907 50

1460

11

127

5

22

24,536 56

1268

10

 62

2

21

1747

1732

13

1287

46

21

1186

9

250

9

16

4

13,530 31

For each concentration of SNA (1.0, 0.1, 0.01 mg/ml), each glycan was ranked according to their average RFU as a percentage of the average RFU of the strongest binding glycan. All of the glycans from the three analyses are ranked in order of their average rank (Avg. Rank), and the top 30 glycans are shown in this table. Alpha and beta linkages are denoted by a and b, respectively.

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were ranked low or unbound by SNA in the three assays used for the analyses. A comparison of selected glycan structures and their ranking analysis is shown in Table 19.2. It has been known for some time that SNA-I binds Neu5Aca2-6Gal terminating structures and that the galactose or N-acetylgalactosamine residues may be important in binding (Shibuya et al., 1987a,b, 1989; Van Damme et al., 1997). However, based on the simultaneous analysis of SNA binding to a variety of related glycans of defined structure, it is possible to identify the minimal SNA-I determinant to be Neu5Aca2-6Galb1-4GlcNAc based on the relative affinity of SNA for Neu5Aca2-6Galb1-4GlcNAc (glycans 258 and 257 at Average Ranks of 49 and 44) and Neu5Aca2-6Galb1-4Glc (glycan number 261 ranked at 0) indicating a strong requirement for the sequence Galb1-4GlcNAc. Thus, Table 19.2 shows the minimal SNA determinant formatted in bold in all structures where it appears. Only one glycan structure on the microarray carries Neu5Aca2-6Gal on a type 1 glycan (glycan number 407, Neu5Aca26Galb1-3GlcNAcb1-3(Galb1-4GlcNAcb1-6)Galb1-4Glc), and this had a low average Ranking of 26, supporting the concept that the minimal determinant for SNA-I binding is Neu5Aca2-6Galb1-4GlcNAc, which contains a type 2 chain. Substituting the terminal sialic acid with KDN (glycan 353) increased the average ranking almost twofold, whereas replacing the terminal sialic acid with Neu5Ac(9-O-Ac) (glycan 46) increased the average ranking slightly. Placement of a sulfate at the 6-OH of the GlcNAc (glycan 256) also increased binding. Changing the sialic acid linkage from a2-6 to a2-3 eliminated binding by SNA, as seen in glycan numbers 240 and 250, whereas substituting Neu5Ac with Neu5Gc did not change the average ranking of the minimal determinant (glycan 275). The fact that there was no significant difference in binding between glycans 260 (Neu5Aca26Galb1-4GlcNAcb1-3Galb1-4GlcNAc) and 325 (Neu5Aca2-6Galb14GlcNAcb1-3Galb1-3GlcNAc), which differ at the linkage position of the fourth monosaccharide in the sequence, also supports the hypothesis that the minimal determinant for high affinity binding by SNA-I is the trisaccharide Neu5Aca2-6Galb1-4GlcNAc, and this is consistent with previously published studies (Shibuya et al., 1987a,b, 1989; Van Damme et al., 1997). A more complex aspect of the determinant recognized by SNA-I was observed when ranking was performed on a large number of complex-type biantennary N-glycans identified as SNA-binding N-glycan ligands in the bottom half of Table 19.2. The availability of a microarray with this unique set of N-glycan structures, together with the comparison of their average ranking, permitted the identification of effects of N-glycan structure on recognition of the determinant for SNA. When the minimal determinant (Neu5Aca2-6Galb1-4GlcNAc shown in Table 19.2, formatted in bold) is located on the a3-branch of the trimannosyl core alone, as in glycan 343, compared to glycans containing incomplete a6-branched side chains (glycans 341, 315, 314), including those with more complete side chains with

Table 19.2 Selected glycans from the ranking analysis of Sambucus nigra agglutinin (SNA-I) at three concentrations on v4.0 of the CFG glycan microarray arranged for comparison to determine the glycan specificity or glycan-binding motif Glycan v4.0

Structure of selected SNA ligands

Rank

353 256 240 46 258 257 275 260 325 255 407 250 261

KDNa2-6Galb1-4GlcNAc-Sp0 Neu5Aca2-6Galb1-4[6OSO3]GlcNAcb-Sp8 Neu5Aca2-3Galb1-4[6OSO3]GlcNAcb-Sp8 Neu5Ac(9Ac)a2-6Galb1-4GlcNAcb-Sp8 Neu5Aca2-6Galb1-4GlcNAcb-Sp8 Neu5Aca2-6Galb1-4GlcNAcb-Sp0 Neu5Gca2-6Galb1-4GlcNAcb-Sp0 Neu5Aca2-6Galb1-4GlcNAcb1-3Galb1-4GlcNAcb-Sp0 Neu5Aca2-6Galb1-4GlcNAcb1-3Galb1-3GlcNAcb-Sp0 Neu5Aca2-6GalNAcb1-4GlcNAcb-Sp0 Neu5Aca2-6Galb1-3GlcNAcb1-3(Galb1-4GlcNAcb1-6)Galb1-4Glc-Sp21 Neu5Aca2-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb-Sp0 Neu5Aca2-6Galb1-4Glcb-Sp0

87 85 0 55 49 44 49 47 44 28 26 0 0

Structure of selected SNA N-glycan ligands

343 341 315 314 54 52

Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-3Manb1-4GlcNAcb1-4GlcNAc-Sp12 Mana1-6(Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-3)Manb1-4GlcNAcb1-4GlcNAc-Sp12 GlcNAcb1-2Mana1-3(Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-3)Mana1-4GlcNAcb1-4GlcNAcbSp12 Galb1-4GlcNAcb1-2Mana1-6(Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-3)Manb14GlcNAcb1-4GlcNAcb-Sp12 Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-6(Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-3) Manb1-4GlcNAcb1-4GlcNAcb-Sp13 Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-6(Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-3) Manb1-4GlcNAcb1-4GlcNAcb-Sp8

67 69 62 56 66 60 (continued)

Table 19.2 (continued) Structure of selected SNA N-glycan ligands

51 321 342 300 292 313

Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-6(Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-3)Manb14GlcNAcb1-4GlcNAcb-N(LT)AVL Neu5Aca2-3Galb1-4GlcNAcb1-2Mana1-6(Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-3)Manb14GlcNAcb1-4GlcNAcb-Sp12 Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-6Manb1-4GlcNAcb1-4GlcNAc-Sp12 Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-6(GlcNAcb1-2Mana1-3)Manb1-4GlcNAcb14GlcNAcb-Sp12 Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-6(Galb1-4GlcNAcb1-2Mana1-3)Manb1-4GlcNAcb14GlcNAcb-Sp12 Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-6(Neu5Aca2-3Galb1-4GlcNAcb1-2Mana1-3)Manb14GlcNAcb1-4GlcNAcb-Sp12

55 42 35 22 22 8

The minimal determinant for SNA binding is highlighted in bold and structural features that enhance binding are highlighted in italic while structural features that decrease binding are highlighted in bold italic. Alpha and beta linkages are denoted by a and b, respectively.

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Glycan Microarrays

a6

b4

b2 a6

a6

b4

b2

b4

b4

a3 Key

GlcNAc

Gal

Man

Neu5Ac

Figure 19.3 The glycan-binding motif of SNA on a biantennary complex N-glycan terminating in the minimal determinant defined by NeuAca2-6Galb1-4GlcNAc as determined by inspection of the ranking of glycans shown in Table 19.1 and the relative binding of SNA to selected glycan structures shown in Table 19.2.

two minimal determinants (glycans 51 and 52, one on the three-branch and another on the six-branch), no significant difference in average ranking was observed. In addition, if the a6-branch terminated in a a2-3-linked Neu5Ac (glycan 321), only a slight change in average ranking was observed. Taken together these data suggest that SNA-I recognizes the minimal determinant on the a3-branch, but not the a6-branch, of a biantennary N-glycan. These observations are in agreement with the low average rankings of N-glycans, where the minimal determinant is only on the a6-branch with the a3-branch missing (glycan 342) or partially complete (glycans 300 and 292). In addition, if the minimal determinant is on the a6-branch and the Neu5Ac on the a3-branch is linked a2-3, binding is almost completely lost, again supporting the conclusion that SNA primarily recognized Neu5Aca2-3Galb21-4GlcNAc on the a3-branch of biantennary N-glycans. Thus, SNA possesses a minimal binding motif and an extended binding motif when the determinant is located on an N-glycan as shown in Fig. 19.3. These studies demonstrate the utility of this high throughput analysis of GBP specificities and motif definition on microarrays of large numbers of defined glycans.

7. Specificities of Human Galectin-8 and its Carbohydrate Recognition Domains (CRDs) Galectin-1 was among the first animal cell lectins to be described and there are now 15 known galectins in this class of proteins that were named for the apparent specificity for galactose containing glycans (Cooper and Barondes, 1999; Leffler et al., 2004). The common method for their purification is by affinity chromatography over immobilized lactose. Early studies on the detailed glycan-binding specificity of the galectins suggested they shared a

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common and broad specificity and a similarly broad range of proposed functions (Rabinovich, 1999). With the expectation that defining the glycan-binding specificity of these proteins would provide important information regarding their function, detailed studies on the glycan-binding motifs of Galectins-1, -3, -4, and -8 were carried out at multiple protein concentrations on the CFG glycan microarray (Stowell et al., 2008a,b, 2010). Galectin-8 (Gal-8) is an example of a tandem-repeat galectin, which are generally comprised of a single polypeptide with two nonidentical CRDs joined by a linker peptide and each CRD potentially differing in glycanbinding specificity (Arata et al., 2001; Hirabayashi et al., 2002; Sato et al., 2002; Wasano and Hirakawa, 1999). To evaluate this property for Gal-8, we produced recombinant wild-type Gal-8 and truncated forms of Gal-8 containing only the N-terminal CRD (Gal-8N) or only the C-terminal CRD (Gal-8C; Stowell et al., 2008b, 2010). For these analyses, the Gal-8N and Gal-8C domains were biotinylated and stabilized with 2-mercaptoethanol (Stowell et al., 2008b) and incubated in PBS (0.02 M sodium phosphate, 0.15 M NaCl, pH 7.4) containing 0.05% Tween 20 and 14 mM 2-mercaptoethanol on the microarray under a cover slip for 1 h at 25  C. The slides were washed with PBS containing 0.05% Tween 20, drained to remove excess wash buffer, and then incubated with Alexa488- or FITC-labeled streptavidin on the microarray under a cover slip. After 1 h at room temperature in a dark humid chamber, the slides were washed in PBS/0.01% Tween 20 (three times) and in PBS (three times). The slides were then briefly rinsed with distilled water and dried under microfiltered air. An image of bound fluorescence was obtained using a microarray scanner (Scan Array Express, PerkinElmer Lifer Sciences), and the integrated spot intensities were determined using ImaGene software (BioDiscovery, El Segundo, CA). The patterns of full-length Gal-8, Gal-8C, and Gal-8N binding to version 3.0 of the CFG glycan microarray are shown in Fig. 19.4. The full-length Gal-8 (Fig. 19.4A) bound to many more glycan structures than either the Cterminal (Fig. 19.4B) or the N-terminal domain (Fig. 19.4C), but the complete profile is essentially equivalent to the composite of the C-terminal and N-terminal domain profiles. This is consistent with the C-terminal and Nterminal domains being nonidentical CRDs with different glycan-binding specificity. To identify the glycan motif of each CRD independently, we analyzed them at different concentrations between 10 and 100 mg/ml and ranked the glycans in order of their relative binding strengths as described for the analysis of SNA. Table 19.3 shows the ranking of glycans bound by Gal8N sorted by the average of individual rankings over three concentrations of protein (100, 50, and 20 mg/ml). The results indicate that Gal-8N binds sialylated glycans containing sialic acid a2-3 linked to galactose. By contrast, the results of the same analysis of the Gal-8C (100, 50, and 10 mg/ml; Table 19.4) indicated that Gal-8C has a high specificity for glycans containing the human blood group A and B glycans, with the top 10 ranked glycans

433

Glycan Microarrays

A

70

Gal-8 (20 mg/ml) N

60 50

C

40 30 20 10 0 Relative fluorescent units (⫻103)

B

1

31

61

91

121

151

181

211

241

271

301

70 Gal-8C (100 mg/ml)

60

C

50 40 30 20 10 0

C

1

31

61

91

121

151

181

211

241

271

301

70 Gal-8N (100 mg/ml)

60

N

50 40 30 20 10 0 1

31

61

91

121

151

181

211

241

271

301

Glycan number

Figure 19.4 Analysis of biotinylated recombinant galectin-8 (Gal-8) and truncated forms of Gal-8 containing only the C-terminal carbohydrate recognition domain (CRD; Gal-8C) or only the N-terminal CRD (Gal-8N) on version 3.0 of the CFG glycan microarray. Biotinylated Gal-8 at decreasing concentrations was incubated on the glycan microarray in PBS under a cover slip for 1 h at room temperature. After washing, bound galectin or CRD was detected by incubation with Alexa488-labeled streptavidin at 0.5 mg/ml under a cover slip in the same buffer for 1 h at room temperature. After washing, the slide was analyzed in a fluorescence scanner and the

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containing human blood group A or B structures or a poly-N-acetyllactosamine (-3Galb1-4GlcNAcb1-)n. The Gal-8 C-terminal domain did not bind human blood group O(H) determinants (Fuca1-2Galb1-4GlcNAc); however, the full-length Gal-8 did bind blood group O(H) determinants, shown as glycans 61–64, 69, and 70 in Fig. 19.4A. Interestingly, the Gal-8 mutant containing an inactive N-terminal domain (Stowell et al., 2008b) bound blood group O(H) determinants in addition to the blood group A and B glycans, indicating that the C-terminal domain in the full-length Gal8 mutant possessed specificity for all of the human blood groups, but that the truncated C-domain has lost some affinity for the O(H) determinants (data not shown). This is likely due to the fact that the full-length Gal-8 is a dimeric protein, whereas the dimerization of the Gal-8C domain alone might be impaired and thus might affect its avidity. Sorting of the bound glycans in order of their ranking provides a quick method for selecting candidate glycan ligands for a GBP, but this selection process should be critically interpreted, as described above for the data with SNA-I. For example, several of the glycans (glycans 197, 184, 216, 266, and 194) ranked outside of the top 10 in Table 19.4 bear no relationship to human blood group structures, but have average rankings between 3 and 8. Although it is tempting to interpret this to indicate that Gal-8C may have some affinity for these structures, this possibility is remote, because none of these glycans show a concentration-dependent binding, as can be observed by inspecting the average RFU at each concentration of protein. Note that the N-acetyllactosamine-containing glycans outside of the top 10 in Table 19.4 (glycans 148, 147, and 166), to which all galectins probably bind with low affinity, have a low ranking between 4 and 6, but demonstrate a distinct concentration-dependent binding by Gal-8C. The blood group A and B motif recognized by Gal-8C can be predicted by inspecting the rankings of some selected glycans that bind Gal-8C compared to related glycan structures that do not bind (Table 19.5). Inspection of the blood group B binding glycans indicates that Gal-8C prefers the blood group B trisaccharide, Gala1-3(Fuca1-2)Galb, linked to GlcNAc in a type 1 linkage (Galb1-3GlcNAc) as is found in glycan number 95, over a type 2 linkage (Galb1-4GlcNAc) as found in glycan numbers 97 and 98. The presence of a fucose residue, as in Lewisx (Lex) in glycan number 96, eliminates binding. The Gal-8C domain presumably requires a tetrasaccharide for strong binding, based

data reported as relative fluorescence units for each glycan structure identified by a number on the x-axis. (A) Gal-8 at 20 mg/ml; (B) C-terminal domain of Gal-8 at 100 mg/ml; and (C) N-terminal domain of Gal-8 at 100 mg/ml. The identity of individual glycans comprising version 3.0 of the CFG microarray is available at (http://www.functionalglycomics.org/static/consortium/resources/resourcecoreh10. shtml). The structures of Gal-8 and its truncated N-terminal and C-terminal domains are depicted in their respective panel.

Table 19.3 The ranked glycans determined by glycan microarray analysis of the N-terminal domain of Galectin-8 (Gal-8N) at 3 concentrations on v3.0 of the CFG glycan microarray Gal 8N 100 mg/ml Gal 8N 50 mg/ml Gal 8N 20 mg/ml Glycan v3.0 Structure

Avg. RFU

Rank

Avg. RFU

Rank

Avg. RFU

Rank

Avg. rank

240 253 226 239 30 29 249 258 220 205 284 261 223 216 224 227 33 187 234 202 212

36,907 45,056 45,805 42,408 28,358 40,803 39,653 36,458 40,445 36,095 32,350 32,037 30,349 36,022 39,777 41,406 29,444 31,699 40,061 43,193 34,761

67 82 84 77 52 74 72 67 74 66 59 58 55 66 73 76 54 58 73 79 63

42,877 22,432 41,724 26,037 21,457 17,680 41,362 35,333 37,556 39,581 42,377 38,452 45,387 31,760 20,253 27,589 33,880 13,988 22,601 12,491 23,471

94 49 92 57 47 39 91 78 83 87 93 85 100 70 45 61 75 31 50 28 52

51,030 45,713 20,102 39,807 49,032 38,347 10,876 19,217 5290 5507 5232 9382 1195 6229 13,416 3325 3790 22,743 1891 9330 2418

100 90 39 78 96 75 21 38 10 11 10 18 2 12 26 7 7 45 4 18 5

87 74 72 71 65 63 62 61 56 55 54 54 53 49 48 48 45 44 42 42 40

Neu5Aca2-3Galb1-4Glcb-Sp8 Neu5Aca2-8Neu5Aca2-3Galb1-4Glcb-Sp0 Neu5Aca2-3Galb1-3GlcNAcb-Sp8 Neu5Aca2-3Galb1-4Glcb-Sp0 [3OSO3]Galb1-4(6OSO3)Glcb-Sp8 [3OSO3]Galb1-4(6OSO3)Glcb-Sp0 Neu5Aca2-6Galb1-4Glcb-Sp0 Neu5Gca2-3Galb1-3GlcNAcb-Sp0 Neu5Aca2-3Galb1-3[6OSO3]GalNAca-Sp8 Neu5Aca2-8Neu5Aca2-8Neu5Aca2-3Galb1-4Glcb-Sp0 Neu5Aca2-3Galb1-3GlcNAcb1-3GlcNAcb-Sp0 Neu5Gca2-3Galb1-4Glcb-Sp0 NeuAca2-3Galb1-3GalNAcb1-3Gala1-4Galb1-4Glcb-Sp0 Neu5Aca2-3Galb1-3(6OSO3)GlcNAc-Sp8 NeuAca2-3Galb1-3GlcNAcb1-3Galb1-4GlcNAcb-Sp0 Neu5Aca2-3Galb1-4[6OSO3]GlcNAcb-Sp8 [3OSO3]Galb1-3GlcNAcb-Sp8 KDNa2-3Galb1-3GlcNAcb-Sp0 Neu5Aca2-3Galb1-4GlcNAcb1-3Galb1-4(Fuca1-3)GlcNAc-Sp0 Neu5Aca2-3Galb1-3GalNAca-Sp8 NeuAca2-3(NeuAca2-3Galb1-3GalNAcb1-4)Galb1-4Glcb-Sp0

(continued)

Table 19.3

(continued) Gal 8N 100 mg/ml Gal 8N 50 mg/ml Gal 8N 20 mg/ml

Glycan v3.0 Structure

Avg. RFU

Rank

Avg. RFU

Rank

Avg. RFU

Rank

Avg. rank

317 315

54,781 49,196

100 90

5576 6751

12 15

1633 2097

3 4

38 36

30,216 42,594 15,389 18,062 13,265 9734 10,755 3471 8126 3449

55 78 28 33 24 18 20 6 15 6

18,210 2731 15,391 1031 1577 3948 1364 4031 2449 2197

40 6 34 2 3 9 3 9 5 5

2149 404 8448 121 301 224 81 2974 217 4028

4 1 17 0 1 0 0 6 0 8

33 28 26 12 9 9 8 7 7 6

8499 2576 4691

16 5 9

1197 2692 897

3 6 2

139 3103 109

0 6 0

6 6 4

32 35 28 286 148 149 237 266 188 197 260 184 236

Neu5Aca2-3Galb1-3GalNAc-Sp14 Neu5Aca2-3Galb1-3(Neu5Aca2-3Galb1-4GlcNAcb1-6) GalNAc–Sp14 [3OSO3]Galb1-3GalNAca–Sp8 [3OSO3]Galb1-4[6OSO3]GlcNAcb-Sp8 [3OSO3]Galb1-4Glcb-Sp8 [3OSO3]Galb1-4[6OSO3]GlcNAcb-Sp0 Galb1-4GlcNAcb1-3Galb1-4Glcb-Sp0 Galb1-4GlcNAcb1-3Galb1-4Glcb-Sp8 Neu5Aca2-3Galb1-4GlcNAcb-Sp8 [3OSO3]Galb1-4(Fuca1-3)Glc-Sp0 KDNa2-3Galb1-4GlcNAcb-Sp0 Mana1-6(Mana1-3)Mana1-6(Mana2Mana1-3)Manb1-4GlcNAcb14GlcNAcb-Sp12 Neu5Gca2-3Galb1-4GlcNAcb-Sp0 GlcAb-Sp8 Neu5Aca2-3Galb1-4GlcNAcb-Sp0

For each concentration of Gal-8N (100, 50, and 20 mg/ml), each glycan was ranked according to their average RFU as a percentage of the average RFU of the strongest binding glycan. All of the glycans from the three analyses are ranked in order of their average rank (Avg. Rank), and the top 36 glycans are shown in this table.

Table 19.4 The ranked glycans determined by glycan microarray analysis of the C-terminal domain of Galectin-8 (Gal-8C) at three concentrations on v3.0 of the CFG glycan microarray Gal 8C 100 mg/ml Gal 8C 50 mg/ml Gal 8C 10 mg/ml Glycan v3.0 Structure

Avg. RFU

Rank

Avg. RFU

Rank

Avg. RFU

Rank

Avg. rank

95 82 81 83 79 97 149 98 146 141 197

55,092 39,940 50,898 26,098 16,623 27,467 21,511 14,539 16,402 13,519 2804

100 72 92 47 30 50 39 26 30 25 5

29,682 38,979 25,542 24,696 21,907 15,165 5986 7341 4655 3420 2053

76 100 66 63 56 39 15 19 12 9 5

28,974 25,268 21,497 9526 16,516 3661 1113 2653 2258 308 3940

100 87 74 33 57 13 4 9 8 1 14

92 87 77 48 48 34 19 18 17 11 8

2582 1737 7517 4621 1881 4758 1328

5 3 14 8 3 9 2

3161 3829 1734 1591 2221 666 1015

8 10 4 4 6 2 3

3100 2738 383 1406 2284 168 1340

11 9 1 5 8 1 5

8 7 6 6 6 4 3

184 216 148 147 266 166 194

Gala1-3(Fuca1-2)Galb1-3GlcNAcb-Sp0 GalNAca1-3(Fuca1-2)Galb1-4GlcNAcb–Sp8 GalNAca1-3(Fuca1-2)Galb1-4GlcNAcb-Sp0 GalNAca1-3(Fuca1-2)Galb1-4Glcb-Sp0 GalNAca1-3(Fuca1-2)Galb1-3GlcNAcb-Sp0 Gala1-3(Fuca1-2)Galb1-4GlcNAc-Sp0 Galb1-4GlcNAcb1-3Galb1-4Glcb–Sp8 Gala1-3(Fuca1-2)Galb1-4Glcb-Sp0 Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb–Sp0 Galb1-4GalNAca1-3(Fuca1-2)Galb1-4GlcNAcb-Sp8 Mana1-6(Mana1-3)Mana1-6(Mana2Mana1-3)Manb1-4GlcNAcb14GlcNAcb-Sp12 GlcAb-Sp8 Neu5Aca2-3Galb1-3(6OSO3)GlcNAc-Sp8 Galb1-4GlcNAcb1-3Galb1-4Glcb–Sp0 Galb1-4GlcNAcb1-3Galb1-4GlcNAcb–Sp0 [3OSO3]Galb1-4(Fuca1-3)Glc-Sp0 GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb-Sp0 Mana1-2Mana1-2Mana1-3(Mana1-2Mana1-3(Mana1-2Mana1-6) Mana1-6)Manb1-4GlcNAcb1-4GlcNAcb-Sp12

For each concentration of Gal-8C (100, 50, and 10 mg/ml), each glycan was ranked according to their average RFU as a percentage of the average RFU of the strongest binding glycan. All of the glycans from the three analyses are ranked in order of their average rank (Avg. Rank), and the top 18 glycans are shown in this table.

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Table 19.5 Selected glycans from the ranking analysis of Gal-8C at three concentrations on v3.0 of the CFG glycan microarray arranged for comparison to determine the glycan specificity or glycan-binding motif Glycan # v3.0

Structure

Blood Group B glycans 95 Gala1-3(Fuca1-2)Galb1-3GlcNAcb-Sp0 97 Gala1-3(Fuca1-2)Galb1-4GlcNAc-Sp0 98 Gala1-3(Fuca1-2)Galb1-4Glcb-Sp0 96 Gala1-3(Fuca1-2)Galb1-4(Fuca1-3)GlcNAcb-Sp0 99 Gala1-3(Fuca1-2)Galb–Sp8 290 Gala1-3(Fuca1-2)Galb–Sp18 Blood Group A glycans 79 GalNAca1-3(Fuca1-2)Galb1-3GlcNAcb-Sp0 81 GalNAca1-3(Fuca1-2)Galb1-4GlcNAcb-Sp0 83 GalNAca1-3(Fuca1-2)Galb1-4Glcb-Sp0 80 GalNAca1-3(Fuca1-2)Galb1-4(Fuca1-3)GlcNAcb-Sp0 141 Galb1-4GalNAca1-3(Fuca1-2)Galb1-4GlcNAcb-Sp8 84 GalNAca1-3(Fuca1-2)Galb-Sp8 301 GalNAca1-3(Fuca1-2)Galb-Sp18 Other glycans 146 Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb14GlcNAcb-Sp0 147 Galb1-4GlcNAcb1-3Galb1-4GlcNAcb-Sp0 197 Mana1-6(Mana1-3)Mana1-6(Mana2Mana1-3) Manb1-4GlcNAcb1-4GlcNAcb-Sp12 184 GlcAb-Sp8

Rank

92 34 18 1 0 0 48 77 48 1 11 0 0 17 6 8 8

The human blood group B trisaccharide is shown in bold and differences in the corresponding, related human blood group A-containing glycans are shown in bold italic.

on the lack of binding to trisaccharide glycans 99 and 290, which show a 0 ranking. The blood group A glycan ranking data indicate that the C-terminal domain prefers the A-blood trisaccharide, GalNAca1-3(Fuca1-2)Galb, linked to GlcNAc in a type 2 linkage (Galb1-4GlcNAc) as is found in glycan numbers 81 over a type 1 linkage (Galb1-3GlcNAc) as found in glycan number 79, and it prefers a GlcNAc over a Glc at the reducing end as seen in glycan 83. The Cterminal domain of human Gal-8 can recognize an internal blood group structure, as indicated by the lower affinity binding of the blood group A determinant extended with a Galb1-4 residue (glycan 141) and the presence of the Lex fucose, as in glycan number 80, eliminates binding to the blood group A glycan, which was observed for the blood group B structure, as well. The absence of binding to the blood group A trisaccharide (glycans 84 and 301)

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supports the conclusion that the C-terminal domain requires a tetrasaccharide for strong binding. The poly-N-acetyllactosamine-containing glycans 146 and 147 with rankings of 17 and 6, respectively, are known to bind all galectins and have a lower affinity for Gal-8 as compared to other galectins (data not shown); however, the high mannose-type N-glycan (197) and glucuronic acid (184) are not considered candidate ligands; they do not show a concentration-dependent binding and presumably interact with Gal-8 by a nonspecific mechanism.Thus, the data generated using the ranking protocol for determining the motifs of the recombinant C-terminal and N-terminal domains of human Galectin-8 indicate that their glycan binding motifs are human blood group glycans and sialylated lactose or lactosamine, respectively, as summarized in Fig. 19.5.

8. Challenges in Identifying Physiological Ligands for GBPs The results of motif analysis of a GBP on a microarray of defined glycan structures may contribute to the identification of the physiologically relevant glycan ligand for a GBP. However, one should not assume that a glycan motif based on data from a defined glycan microarray is identical to the physiological ligand or even that the glycan represents a physiological ligand, since it is currently not possible to construct a microarray of structurally defined glycans that represents the entire repertoire of the glycome of any organism, and physiological ligands have to be defined by in vivo assays. A

a3

b3/4

a3 a2

a2

Blood group A

Blood group B B

a3

b3/4

Sialyl-lactose GlcNAc

b3/4

Gal

a3

b3/4

Sialyl-lactosamine GalNAc

Man

Fuc

Key Neu5Ac

Figure 19.5 The glycan-binding motif of the carbohydrate recognition domains (CRDs) of Gal-8 as determined by inspection of the specificity of complete Gal-8, and truncated forms of Gal-8 containing only the C-terminal CRD (Gal-8C) or only the N-terminal CRD (Gal-8N) as shown in Fig. 19.4 and ranking of glycans bound by Gal8N shown in Table 19.3, ranking of glycans bound by Gal-8C shown in Table 19.4, and the relative binding of Gal-8C to selected glycan structures shown in Table 19.5. (A) Blood group A and B structures bound by Gal-8C. (B) Sialyl-lactose and sialylN-acetyllactosamine structures bound by Gal-8N.

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Nevertheless, such data may certainly provide clues to the identification of the physiologically relevant glycan ligand and subsequently to the function for a GBP. For example, the functions of galectins, which share the property of binding galactose-, lactose-, or N-acetyllactosamine- (Galb1-4GlcNAc) containing structures, have been extremely obscure, partly due to uncertainty about their endogenous glycan ligands in vivo. The observation that certain galectins (galectin-3, -8, and -4) bound to human blood group antigens (Stowell et al., 2008a,b, 2010) led to the identification of the tandem-repeat galectins (galectin-4 and -8) as innate immune lectins that are directly able to kill bacteria expressing blood group antigens. Thus, Gal-4 and Gal-8 may provide defense against pathogens expressing surface structures that do not elicit an adaptive immune response in individuals expressing the same cognate antigen (Stowell et al., 2010). However, these conclusions were based on significantly more information than just the specificity of Gal-4 and Gal-8 for binding blood group antigens. Additional information that was considered in that particular study was the evidence in regard to sites of expression of the galectins in question, which included the epithelial cells of the intestines (Huflejt and Leffler, 2004; Nagy et al., 2002), the ability of Gal-3, Gal-4, and Gal-8 to bind to Escherichia coli expressing glycan structures containing human blood group B, and the ability of Gal-4 and Gal-8, but not Gal-3, to directly kill bacteria that express blood group B antigen on their surface, along with additional studies in mice indicated that endogenous galectins specifically altered the viability of blood group B expressing bacteria in vivo (Stowell et al., 2010). Thus, these data support the conclusion that Gal-4 and Gal-8 function as innate immune lectins, and motif determination by glycan microarray analysis contributed to the underlying hypothesis. Other possible types of data that are useful in the iterative process of identifying physiological ligands of GBPs include the development of biological assays of GBP binding to biological targets at physiological concentrations, defining the class or classes of glycans being recognized; that is, glycoprotein, glycosphingolipid, glycosaminoglycans, etc., and isolation of candidate ligand(s) by affinity chromatography or pulldown experiments.

9. Conclusion and Future Directions The microarrays of structurally defined glycans are extremely powerful tools for the determination of glycan-binding specificity or glycanbinding motifs of GBPs, and the utility of a defined glycan microarray is directly related to the number of different glycans on the microarray. For example, if the human blood group antigens had not been represented

Glycan Microarrays

441

on the CFG microarray, the blood group specificity of Gal-8 would not have been defined by the analyses described here. At the same time, the current microarray has only two glycan structures that present the human B and A blood group tetrasaccharide in a type 1 configuration (Glycans 95 and 79, respectively), which makes it difficult to interpret exactly how the C-terminal domain of Gal-8 might differentiate blood group antigens presented on type 1 or type 2 chains. The ideal defined glycan microarraywould be one that represented the complete glycome of the organism of interest; however, the difficulties of complex oligosaccharide synthesis preclude production of such microarrays at present. Of course, an even better representation of the glycome would be purified glycoproteins, glycolipids, and glycosaminoglycans, which would present glycan structures in a physiologically relevant context, but the possibility of generating such a defined glycoconjugate microarray within the next decade is very remote. Thus, significant interest in the research community has focused on the ways in which glycans are presented on microarrays. There is interest in understanding the contributions of the activated solid phase on the microscope slide, the chemistry of the linker between the glycan and the solid phase, and the multivalent presentation and density of glycans on the microarray. The CFG glycan microarray is printed on slides that have been activated with NHS and the glycan derivatives are functionalized with primary amino groups making this a well defined covalent linkage based on the specificity of NHS for primary amines. Epoxy derivatized surfaces, on the other hand, are highly reactive with most amino functions and possibly with hydroxyl groups that could result in multiple orientations of glycans depending on their linker chemistries. Although the manufacturers of the activated slides do not provide the capacity or the amount of NHS/mm2 of surface, printing the glycans at an identical and constant glycan concentration provides glycan targets that are reproducibly comparable and allow the generation of concentration-dependent GBP binding data. This was confirmed in detailed empirical analysis showing that a glycan concentration of 100 mM was in a linear range of the relationship of the concentrations of glycans printed and subsequent GBP binding (Song et al., 2008). While the exact concentration of a printed glycan is difficult to calculate on a solid surface, the presentation is obviously multivalent to an extent, which in some ways may be similar to presentation in the glycocalyx, where the local concentration of sialic acid has been estimated to be in excess of 100 mM (Crocker et al., 2007). Nevertheless, while the available microarrays as described here have been highly successful, there are important issues to consider, such as the extent to which glycan presentations mimic cellular presentations, as well as issues in regard to the relationship of the binding affinity constants of GBPs to their binding properties on the microarrays.

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A key advantage of the CFG glycan microarray has been its reproducibility of printing quantities of glycans, quality control, and public availability of all data and metadata. Although there is slight variation in the lectin binding data from slide batch to slide batch, each analysis is carried out on slides from within the same batch or print date. In addition, the use of different concentrations of GBP in a linear range of signal, as described here, permits normalization of data within each analysis. These positive attributes of the CFG glycan microarray have made it extremely useful and successful in predicting glycan specificity for GBPs, and have led to the identification and a better understanding of physiological ligands for GBPs.

ACKNOWLEDGMENTS This work was carried in the protein–glycan Interaction Core (Core H) of the Consortium for Functional Glycomics supported by NIGMS Grant (GM) GM62116 and a EUREKA Grant GM GM085448 to D. F. S. The authors thank Dr Jamie Heimburg-Molinaro for editing the chapter and review.

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Huflejt, M. E., and Leffler, H. (2004). Galectin-4 in normal tissues and cancer. Glycoconj. J. 20, 247–255. Kuno, A., Uchiyama, N., Koseki-Kuno, S., Ebe, Y., Takashima, S., Yamada, M., and Hirabayashi, J. (2005). Evanescent-field fluorescence-assisted lectin microarray: A new strategy for glycan profiling. Nat. Methods 2, 851–856. Leffler, H., Carlsson, S., Hedlund, M., Qian, Y., and Poirier, F. (2004). Introduction to galectins. Glycoconj. J. 19, 433–440. Mach, L., Scherf, W., Ammann, M., Poetsch, J., Bertsch, W., Marz, L., and Glossl, J. (1991). Purification and partial characterization of a novel lectin from elder (Sambucus nigra L.) fruit. Biochem. J. 278(Pt 3), 667–671. Nagy, N., Bronckart, Y., Camby, I., Legendre, H., Lahm, H., Kaltner, H., Hadari, Y., Van Ham, P., Yeaton, P., Pector, J. C., Zick, Y., Salmon, I., et al. (2002). Galectin-8 expression decreases in cancer compared with normal and dysplastic human colon tissue and acts significantly on human colon cancer cell migration as a suppressor. Gut 50, 392–401. Oyelaran, O., and Gildersleeve, J. C. (2009). Glycan arrays: Recent advances and future challenges. Curr. Opin. Chem. Biol. 13, 406–413. Porter, A., Yue, T., Heeringa, L., Day, S., Suh, E., and Haab, B. B. (2010). A motif-based analysis of glycan array data to determine the specificities of glycan-binding proteins. Glycobiology 20, 369–380. Powlesland, A. S., Fisch, T., Taylor, M. E., Smith, D. F., Tissot, B., Dell, A., Pohlmann, S., and Drickamer, K. (2008). A novel mechanism for LSECtin binding to Ebola virus surface glycoprotein through truncated glycans. J. Biol. Chem. 283, 593–602. Rabinovich, G. A. (1999). Galectins: An evolutionarily conserved family of animal lectins with multifunctional properties; a trip from the gene to clinical therapy. Cell Death Differ. 6, 711–721. Ratner, D. M., Adams, E. W., Su, J., O’Keefe, B. R., Mrksich, M., and Seeberger, P. H. (2004). Probing protein–carbohydrate interactions with microarrays of synthetic oligosaccharides. Chembiochem 5, 379–382. Sato, M., Nishi, N., Shoji, H., Seki, M., Hashidate, T., Hirabayashi, J., Kasai Ki, K., Hata, Y., Suzuki, S., Hirashima, M., and Nakamura, T. (2002). Functional analysis of the carbohydrate recognition domains and a linker peptide of galectin-9 as to eosinophil chemoattractant activity. Glycobiology 12, 191–197. Shibuya, N., Goldstein, I. J., Broekaert, W. F., Nsimba-Lubaki, M., Peeters, B., and Peumans, W. J. (1987a). Fractionation of sialylated oligosaccharides, glycopeptides, and glycoproteins on immobilized elderberry (Sambucus nigra L.) bark lectin. Arch. Biochem. Biophys. 254, 1–8. Shibuya, N., Goldstein, I. J., Broekaert, W. F., Nsimba-Lubaki, M., Peeters, B., and Peumans, W. J. (1987b). The elderberry (Sambucus nigra L.) bark lectin recognizes the Neu5Ac(alpha 2-6)Gal/GalNAc sequence. J. Biol. Chem. 262, 1596–1601. Shibuya, N., Tazaki, K., Song, Z. W., Tarr, G. E., Goldstein, I. J., and Peumans, W. J. (1989). A comparative study of bark lectins from three elderberry (Sambucus) species. J. Biochem. 106, 1098–1103. Song, X., Xia, B., Lasanajak, Y., Smith, D. F., and Cummings, R. D. (2008). Quantifiable fluorescent glycan microarrays. Glycoconj. J. 25, 15–25. Song, X., Xia, B., Stowell, S. R., Lasanajak, Y., Smith, D. F., and Cummings, R. D. (2009). Novel fluorescent glycan microarray strategy reveals ligands for galectins. Chem. Biol. 16, 36–47. Stowell, S. R., Arthur, C. M., Mehta, P., Slanina, K. A., Blixt, O., Leffler, H., Smith, D. F., and Cummings, R. D. (2008a). Galectin-1, -2, and -3 exhibit differential recognition of sialylated glycans and blood group antigens. J. Biol. Chem. 283, 10109–10123.

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C H A P T E R

T W E N T Y

Functional Roles of the Bisecting GlcNAc in Integrin-Mediated Cell Adhesion Tomoya Isaji,* Yoshinobu Kariya,* Qingsong Xu,* Tomohiko Fukuda,* Naoyuki Taniguchi,†,‡ and Jianguo Gu* Contents 1. 2. 3. 4.

Overview Manipulation of GnT-III and GnT-V Genes in Cancer Cells Assays for Cell Spreading and Migration Construction of Various Integrin a5b1 Mutants by the Mutagenesis of Potential N-Glycosylation Sites 5. N-Glycans Differentially Regulate Integrin-Mediated Cell Adhesion and Migration 6. Identification of Important N-Glycosylation Sites for Functional Expression of a5b1 Integrin 7. Site-4 Is a Crucial N-Glycosylation Site on the a5 Subunit for GnT-III Regulation 8. Future Perspectives Acknowledgments References

446 448 449 451 452 454 455 456 456 456

Abstract N-acetylglucosaminyltransferase III (GnT-III) transfers N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to core mannose with a b1,4 linkage, so-called bisecting GlcNAc, in N-glycans. The bisecting GlcNAc is found in various hybrid and complex N-glycans. GnT-III is generally regarded as a key glycosyltransferase in N-glycan biosynthetic pathways. Introduction of a bisecting GlcNAc suppresses further processing and elongation of N-glycans catalyzed by other GlcNAc transferases to form branching structures, such as N-acetylglucosaminyltransferase V (GnT-V), since GnT-V cannot utilize the bisected oligosaccharide as a * Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, Aoba-ku, Sendai Miyagi, Japan Department of Disease Glycomics Laboratory, The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, Japan { Disease Glycomics Team, RIKEN Advanced Science Institute, Wako, Saitama, Japan {

Methods in Enzymology, Volume 480 ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)80019-9

#

2010 Elsevier Inc. All rights reserved.

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substrate. Considering that expression of the enzyme leads to a remarkable structural alteration of the N-glycans on cell surface, it has been postulated that the enzyme is associated with various biological events such as cell adhesion, migration, cell growth, cell differentiation, and tumor invasion. Integrin is a major carrier of N-glycans. In fact, overexpression of GnT-III reduced the b1,6 GlcNAc branching structures, in conjunction with the increase in the bisected Nglycans on integrins, and resulted in an inhibition of integrin-mediated cell spreading and migration, and the cellular phosphorylation levels. Conversely, knockdown of endogenous GnT-III expression resulted in increased cell migration, concomitant with an increase in b1,6 GlcNAc-branched N-glycans on integrins. Thus, N-glycan could be considered as either a positive or negative regulator for biological functions of integrin.

1. Overview Oligosaccharide structures attached to proteins (glycosylation) is conserved in eukaryotes, which is one of the most abundant posttranslational modification reactions (Apweiler et al., 1999). Glycosylation plays numerous roles in protein folding, targeting, recognition, and functions. In fact, changes in glycan structures are associated with many physiological and pathological events, including cell adhesion, migration, cell growth, cell differentiation, and tumor invasion. Oligosaccharides of glycoproteins are classified as N-glycans and O-glycans. N-glycans are linked to asparagine residues of proteins, which is a specific subset residing in the Asn-X-Ser/Thr motif, whereas O-glycans are attached to a subset of serines and threonines. Glycosylation reactions are catalyzed by the action of glycosyltransferases, which add sugar residues to various glycans on glycoproteins, glycolipids, and proteoglycans. N-acetylglucosaminyltransferase III (GnT-III) catalyzes a bisecting GlcNAc linkage, and GnT-V catalyzes the formation of b1,6 GlcNAc-branched structures (Fig. 20.1). These glycans are involved in the regulation of cellular functions including cell–cell communication and cellular signal transduction. Integrins consist of a and b subunits. Each subunit has a large extracellular region, a single transmembrane domain, and a short cytoplasmic tail (except for b4 integrin). The N-terminal domains of the a and b subunits associate to form the integrin headpiece, which contains extracellular matrix (ECM) binding site, whereas the C-terminal segments transverse the plasma membrane and mediate interactions with the cytoskeleton and signaling molecules (Fig. 20.2). Until now, 18 a- and 8 b-subunits are known to assemble into 24 integrins (Alam et al., 2007; Takagi, 2007). Integrin function has been determined through a combination of cell biological and genetic analyses. A characteristic feature of most integrins is their ability to bind a wide variety of ECMs. Conversely, many ECMs also bind to multiple integrins (Humphries et al., 2006; Takagi, 2007). Although the fact

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b 1, 6 branching GlcNAc P UD UD

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Figure 20.1 Glycosylation reactions catalyzed by the action of glycosyltransferase GnT-III and GnT-V. GnT-III transfers GlcNAc from UDP-GlcNAc to the core mannose with a b1,4 linkage to form the bisecting GlcNAc in N-glycans. GnT-V catalyzes the formation of b1,6 GlcNAc-branched structures. GnT-III could be considered to be an antagonistic of GnT-V, because bisecting GlcNAc renders the biantennary substrate inaccessible to GnT-V (Schachter, 1986). The reaction pathway represented by a dash line may not be predominant in vivo. ○, mannose; j, N-acetylglucosamine.

that the binding specificities of many of the integrin overlap, the loss of almost any integrin a or b subunit leads to biological defects in knockout mice (Hynes, 2002). The most general feature of integrin is that the interaction of integrin with its ECM can activate intracellular signaling pathways and cytoskeletal formation, so-called outside-in signaling. Another important feature of integrin is inside-out signaling, in which intracellular signals received by integrin or other receptors, in turn, activate its extracellular domain and contribute to the assembly of the ECM (Hynes, 2002; Liddington and Ginsberg, 2002). In general, integrins recognize short specific peptide sequences, such as arginine-glycine-aspartic acid (RGD) on proteins, which are found in many ECMs such as fibronectin (FN) and vitronectin (Adams and Watt, 1993; Akiyama et al., 1995; Ruoslahti, 1996). For this reason, the integrins were classified as RGD dependent and RGD independent. Integrin a5b1, a representative RGD-binding protein, specific binding to FN is fundamental for vertebrate development and is suggested to be involved in cardiovascular events and tumor invasion. The interaction between a5b1 and FN is essential for cell migration, development, as well as cell viability, since the genetic lack of integrin a5 or FN results in early embryonic lethality (George et al., 1993; Goh et al., 1997). The laminin-binding receptors, a3b1, a6b1, a6b4, and a7b1integrins are RGD-independent type of integrins (Colognato and Yurchenco, 2000). Although the precise motifs on laminins recognized by these integrins have not been determined yet, it has been well known that the interactions between integrins and laminins play crucial roles in development, wound healing, and tumorigenesis.

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Promoting cell migration ECM

-V

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Cell growth, survival, differentiation cytoskeletal organization, migration

Figure 20.2 Differential regulation of integrin functions by GnT-III and GnT-V. The interaction of integrin with its ECM can activate intracellular signaling pathways and cytoskeletal formation, and regulate cell adhesion and migration. Enhanced expression of GnT-V in various cell types results in an increase in integrin-mediated cell migration. In contrast, overexpression of GnT-III downregulates integrinmediated cell migration.

Here, we mainly focus on effects of modification of bisecting GlcNAc on a5b1 and a3b1 integrins-mediated cell adhesion and migration, and identification of important N-glycosylation sites on a5b1 integrin to address the potential roles of N-glycans in integrin-mediated biological functions.

2. Manipulation of GnT-III and GnT-V Genes in Cancer Cells Human cancer cell lines such as MKN45 cells were cultured in RPMI 1640 medium (Sigma) containing 10% fetal bovine serum (FBS; Invitrogen) and penicillin (100 U/ml), and streptomycin (100 mg/ml) under a humidified atmosphere containing 5% CO2 (Isaji et al., 2004; Zhao et al., 2006). Human GnT-V cDNA or GnT-III cDNA was inserted into a mammalian

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expression vector pCXNII. Vectors were then transfected into cells by means of LipofectAMINE (Invitrogen). Selection was performed by the addition of 500 mg/ml of G418 (Sigma). In the knockdown experiments, small interfering oligonucleotides specific for GnT-III were designed on the Takara Bio Web site and the oligonucleotide sequences used in the construction of the siRNA vector were the following: 50 -GATCCGTCAACCACGAGTTCGACCTTCAAGAGAGGTCGAACTCGTGGTTGACTTTTTTAT-30 and 50 CGATAAAAAAGTCAACCACGAGTTCGACCTCTCTTGAAGGTCGAACTCGTGGTTGACG-30 . The oligonucleotides were annealed and then ligated into BamHI/ClaI sites of the pSINsi-hU6 vector (Takara Bio). A retroviral supernatant was obtained by transfection of human embryonic kidney 293 cells using a Retrovirus Packaging Kit Ampho (Takara Bio) according to the manufacturer’s protocol. CHP134 cells, a human neuroblastoma cell line expressing a high level of endogenous GnT-III, were infected with the viral supernatant, and the cells were then selected with 500 mg/ml of G418 for 2–3 weeks (Zhao et al., 2006). Stable GnT-III knockdown clones were selected and confirmed by GnT-III activity and gene expression (Fig. 20.3).

3. Assays for Cell Spreading and Migration Cell spreading assays were performed as described previously with minor modifications (Isaji et al., 2004). Briefly, 96-well microtiter plates (Nunc, Wiesbaden, Germany) were coated with a solution of 20 nM human serum FN or 5 nM recombinant laminin-332 (LN-332) in phosphate buffer saline (PBS) overnight at 4  C and blocked with 1% bovine serum albumin (BSA) in DMEM for 1 h at 37  C. The cells were detached with trypsin containing 1 mM EDTA, washed with serum-containing DMEM and then suspended in serum-free DMEM with 0.1% BSA at 4  104 cells/ml. To confirm whether or not the cell spreading on FN or LN-332 was integrin dependent, cells were preincubated with the functional blocking mAbs against a5 (BIIG2) or a3 (P1B5) at final concentrations of 10 mg/ml at room temperature for 10 min before plating. After a 20-min incubation, nonadherent cells were removed by washing with PBS, the attached cells were fixed with 3.7% paraformaldehyde in PBS, and representative fields were then observed by phase-contrast microscopy. Transwell inserts (BD BioCoatTM Control Inserts, 8.0 mm inserts; Becton Dickinson Falcon, Bedford, MA) were coated by incubation in 20 nM FN or 5 nM LN-332 in PBS overnight at 4  C followed by an incubation with 1% BSA for 1 h at 37  C. Cells were detached with a trypsin containing 1 mM EDTA, washed once with DMEM containing 10% FBS, and

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Figure 20.3 Increased cell migration and increased GnT-V product on a3 subunit in GnT-III knockdown cells. (A) Activities of GnT-III in GnT-III-knockdown CHP134 cells. (B) Whole cell lysates were immunoprecipitated (IP) with anti-a3 antibody, the resulting immunocomplexes were subjected to 7.5% SDS-PAGE under reducing condition. The blots were respectively probed by E4-PHA (upper panel), L4-PHA (middle panel), and anti-a3 antibody (lower panel). (C) Quantification of migration of mock and GnT-III-knockdown cells on laminin-332. The number of migrated cells were quantified and expressed as the means  S.D. from three independent experiments. KD, GnT-III-knockdown cells; Ab: anti-a3 blocking antibody.

then suspended in DMEM containing 1% FBS at 1  106 cells/ml. The cell suspension (100 ml) was preincubated with the functional blocking antibody mAbs at a final concentration of 10 mg/ml for 10 min and then added to each upper side of chamber. After 3 h of incubation at 37  C, the remaining cells on the upper side of the chamber were carefully scrapped off with a cotton swab. Cells that migrated to the lower surface of the membrane were fixed with 3.7% paraformaldehyde in PBS and stained with 0.3% crystal violet for 30 min, and then observed under a phase-contrast microscope and counted as migrated cells.

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4. Construction of Various Integrin a5b1 Mutants by the Mutagenesis of Potential N-Glycosylation Sites Among the integrin superfamily, a5b1 is one of the best characterized integrins. Human integrin a5 contains 14 potential N-glycosylation sites, which are located in the extracellular segment and are well conserved in the human, mouse, and rat, as shown in Fig. 20.4A. The potential N-glycosylation sites were numbered 1–14 from N-terminus. The asparagine residues

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Figure 20.4 Identification of important N-glycosylation sites on a5b1 integrin. The various underglycosylated mutants of a5 (A) and b1 (B) subunits were constructed. The cross indicates mutation of Asn residue to Gln residue. (C) Comparison of actin stress fiber formation among various a5 unglycosylated mutants spread on FN. Cells were detached and then replated on coverslips that had been precoated with FN in PBS. After incubation for 2 h, cells were fixed, permeabilized, and then stained with Phalloidin-Alexa 549 (red). GFP tag was green. (D) The localization of WT, S4-6, and D4-6 of b1 by immunostaining. Both the WT and the S4-6 of the b1 subunit were expressed mainly on the cell surface as usual, while the D4-6 accumulated mainly in the ER colocalized with calnexin.

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within the NX(S/T) glycosylation consensus sequence in the presumed b-propeller, thigh and calf domains, respectively, were sequentially mutated to glutamine residues. The underglycosylated mutant cDNAs were transfected to CHO-B2 cells, an a5-deficient cell line, and stable cell lines for the expression of a5 integrin were selected with G418. The human integrin b1 subunit contains 12 potential N-glycosylation sites, as shown in Fig. 20.4B. Reportedly, 10 of these sites are normally N-glycosylated, while sites 2 and 11 do not normally carry N-linked glycans (Seales et al., 2005). These mutated cDNAs were then introduced into integrin b1 subunit-deficient GE11 cells. The underglycosylated mutant constructs of b1 integrin used in this study are listed in Fig. 20.4B: D1–3, D4–6, D7,8, and D9–12. These constructs correspond to the following unglycosylated sites on the PSI and in the upstream region of the hybrid domains: the I-like domain, the downstream region of the hybrid domain, EGF-repeat, and b-tail domains of the b1 subunit, respectively.

5. N-Glycans Differentially Regulate IntegrinMediated Cell Adhesion and Migration Integrin-mediated biological functions such as cell spreading and cell migration can be modulated as a consequence of an aberrant change in the N-glycosylation of integrins, which are often associated with a carcinogenic process (Bellis, 2004; Dennis et al., 2002; Gu and Taniguchi, 2004; Jasiulionis et al., 1996; Miyoshi et al., 1999). Several research groups, including our group, reported that alternations in the oligosaccharide portion of integrins, that are modulated by the expression of each glycosyltransferase gene such as GnT-III and GnT-V as well as a2,6 sialyltransferase (ST6GalI), regulate cell malignant phenotypes such as integrin-mediated cell migration and cell spreading (Gu and Taniguchi, 2008). The expression of GnT-V or b1,6 branched N-glycans is strongly associated with cancer metastasis. It has been reported that GnT-V activity and b1,6 branched N-glycan levels are increased in highly metastatic tumor cell lines (Asada et al., 1997; Pochec et al., 2003). In fact, cancer metastasis was suppressed in GnT-V knockout mice (Granovsky et al., 2000). Consistently, overexpression of GnT-V resulted in an increase in cell migration and invasion. GnT-V-null cells displayed enhanced cell adhesion to FNcoated plates with the concomitant inhibition of cell migration. The restoration of GnT-V in the null cells reversed these abnormal characteristics, indicating the direct involvement of N-glycosylation events in these phenotypic changes (Guo et al., 2002). When NIH3T3 cells were transformed with the oncogenic Ras gene, the cell spreading on FN was greatly enhanced due to an increase of b1,6 GlcNAc-branched tri- and

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tetraantennary oligosaccharides in a5b1 integrins (Asada et al., 1997). Similarly, characterization of carbohydrate moieties of integrin a3b1 from nonmetastatic and metastatic human melanoma cell lines showed that b1,6 GlcNAc-branched structures were highly expressed in metastatic cells compared with nonmetastatic cells (Pochec et al., 2003). These cancerassociated glycan chains may modulate tumor cell adhesion by affecting the ligand binding properties of these integrins. Alterations of N-glycans on integrins could also regulate their cis-interactions with membrane-associated proteins including the epidermal growth factor receptor (Kariya et al., 2010; Shigeta et al., 2006), and the tetraspanin family of proteins in microdomain (Hakomori, 2008; Hakomori Si, 2002) as well as endocytosis (Dennis et al., 2009; Taniguchi et al., 2006). In contrast to GnT-V, the overexpression of GnT-III resulted in an inhibition of a5b1 integrin-mediated cell spreading and migration, and the phosphorylation of the focal adhesion kinase (Isaji et al., 2004). The affinity of the binding of integrin a5b1 to FN was significantly reduced as a result of the introduction of a bisecting GlcNAc to the a5 subunit. In addition, overexpression of GnT-III in highly metastatic melanoma cells reduced b1,6 branching GlcNAc and increased bisected N-glycans expressed on cell surface (Yoshimura et al., 1995). Since GnT-III and GnT-V act on the same substrate (Fig. 20.1), therefore, GnT-III has been proposed as an antagonistic of GnT-V, thereby contributing to the suppression of cancer metastasis. The opposing effects of GnT-III and GnT-V have been observed for the same target protein, integrin a3b1 (Zhao et al., 2006). GnT-V stimulated a3b1 integrin-mediated cell migration, while overexpression of GnT-III inhibited GnT-V-induced cell migration. The modification of the a3 subunit by GnT-III supersedes modification by GnT-V. As a result, GnT-III inhibits GnT-V-induced cell migration. Furthermore, a knockdown of endogenous expression of GnT-III results in an increase in expression level of b1,6 branching GlcNAc as shown in Fig. 20.3. These results strongly suggest that remodeling of glycosyltransferase-modified Nglycan structures either positively or negatively modulates cell adhesion and migration. It is also worth noting that N-glycosylation can be regulated by cell–cell adhesion. We recently found that E-cadherin-mediated cell–cell interaction upregulated GnT-III expression (Akama et al., 2008; Iijima et al., 2006). A significant upregulation of GnT-III expression was observed only in epithelial cells that express basal levels of E-cadherin and GnT-III, but not in the following: the MDA-MB231 cell, an E-cadherin-deficient cell line; the MDCK cell, in which GnT-III expression is undetectable; and, fibroblasts, which lack E-cadherin (Iijima et al., 2006). The expression levels of GnT-III and the bisected N-glycans were upregulated by cell–cell interaction via the E-cadherin–catenin–actin complex. The regulation of GnT-III and E-cadherin expression may exist as a positive feedback loop. Given the

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important biological functions of GnT-III as described above, these results provide new insight into the molecular mechanism of relationships among cell–cell interaction, normal development, EMT (epithelial–mesenchymal transition), and cancer metastasis.

6. Identification of Important N-Glycosylation Sites for Functional Expression of a5b1 Integrin Although alteration of the oligosaccharide portion on integrin a5b1 could affect cis- and trans-interactions caused by GnT-III and GnT-V, as described above, the molecular mechanism remains unclear. Considering integrin a5b1 contains 26 potential N-linked glycosylation sites (14 in the a subunit and 12 in the b subunit), the determination of those crucial N-glycosylation sites for its biological function is quite important for an understanding of the underlying mechanism. It has been reported that the structures of N-glycan on integrin a5b1 may be present in a sitespecific-dependent manner (Ethier et al., 2005). We sequentially mutated either one or a combination of asparagine residues in the putative N-glycosylation sites to glutamine residues (Fig. 20.4A,C). As a result, N-glycosylation on the b-propeller domain of the a5 subunit (S3-5) is essential for its heterodimer formation and its biological functions such as cell spreading, cell migration, and cytoskeletal formation (Fig. 20.4B), as well as for the proper folding of the a5 subunit (Isaji et al., 2006). Seales et al. (2005) reported that the I-like domain on the b1 subunit, which could be the partner of the b-propeller of the a5 subunit, supporting the importance of N-glycans on the b-propeller. The crystal structure of integrin aVb3 has been successfully revealed, and the main contact between the aV and b3 subunit is the b-propeller on the aV and A domain on b3 with hydrophobic, ionic, and mixed contacts (Xiong et al., 2001, 2002). Since the a5 subunit has a 47% homology to aV, Mould et al. (2003) speculate that the structural environment of the ab interfaces could be affected by the presence of N-glycans by a homology modeling structure of a5b1. We also found three N-glycosylation sites on I-like domain of integrin b1 subunit, which are important for a5 and b1 dimmer formation and its expression on the cell surface (Fig. 20.4D; Isaji et al., 2009). Although the involvement of N-glycan in the ab interaction remains unclear, it could be explained that an unknown lectin domain may exist on the each subunit, since the lectin domain of aMb2 integrin is associated with GlcNAc on the nonreducing terminal of sugar chains on chilled platelets for its phagocytosis (Hoffmeister et al., 2003; Josefsson et al., 2005).

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7. Site-4 Is a Crucial N-Glycosylation Site on the a5 Subunit for GnT-III Regulation As described above, the N-glycans on the b-propeller domain (S3-5) of the integrin a5 subunit are essential for a5b1 heterodimerization, cell surface expression, and biological function (Isaji et al., 2006). To further investigate the underlying molecular mechanism of GnT-III-regulated biological functions, we characterized the N-glycans on the a5 subunit in detail and found that site-4 is a key site that can be specifically modified by GnT-III (Fig. 20.5; Sato et al., 2009). The intensity of E4-PHA staining in the deletion of site-3 (D3) cells was comparable with that in WT cells. However, the intensity of E4-PHA in D4 cells was substantially less than that in WT (Fig. 20.5B). Furthermore, GnT-III significantly downregulated cell spreading on FN in WT transfectants, whereas the deletion of site-4 abolished the suppression of cell spread induced by GnT-III in D4 transfectants (Fig. 20.5B). It is of interest to understand why GnT-III specifically and effectively modifies site-4 of the 14 putative N-glycosylation sites. There is currently no detailed information available regarding this observation, but several explanations could be proposed. First, N-glycosylation occurs on site-4 because it provides the easiest access for GnT-III. Second, GnT-III may associate with some other molecules, which define the specificities for protein or peptide substrates. Several studies have shown that the glycosyltransferase complex

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Figure 20.5 GnT-III selectively and functionally modifies N-glycosylation site-4 on a5 subunit. The wild type (WT), deletion of site-3 (D3: Asn297Gln), and deletion of site-4 (D4: Asn307Gln) of a5 subunit cDNAs were transfected to CHO-B2 cells. (A) The intensity of E4-PHA staining in D3 cells was less than that in WT cells, but they were comparable. However, the intensity of E4-PHA staining in D4 cells was substantially less than that in WT or D3 cells. (B), GnT-III significantly downregulated cell spreading on FN in WT transfectants, whereas the D4 abolished the suppression of cell spread induced by GnT-III.

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formation may play a crucial role in determination of both activity and substrate specificity (Ju and Cummings, 2002, 2005; Manya et al., 2004, 2007). For instance, the formation of a protein O-mannosyltransferase 1 (POMT1) and its homolog POMT2 complex is essential for POMT activity (Manya et al., 2004). Interestingly, only two peptides derived from the mucin domain of a-dystroglycan are highly O-mannosylated by POMT, but no O-mannosylation occurs in mucin tandem repeat peptides (Manya et al., 2007). Third, each glycosyltransferase may have its own pathway to operate glycosylation in Golgi apparatus. Indeed, it has been reported that caveolin-1 may colocalize with GnT-III to regulate its localization and activity (Sasai et al., 2003).

8. Future Perspectives The modification of the bisecting GlcNAc on integrins downregulates integrin-mediated cell adhesion, migration, and intracellular signaling. Although the detailed molecular mechanism remains unclear, it could be speculated that GnT-III negatively regulates integrin-mediated supramolecular complex formation on cell surface. In fact, it has been reported that the complex formation of integrin and tetraspanins is affected by the N-glycosylation of both integrin and tetraspanins, as well as by gangliosides in the microdomain (Hakomori, 2008; Hakomori Si, 2002). Recently, we found that the inhibitory effects of GnT-III could be ascribed to downregulating the formation of molecular complexes comprising ECM, integrin, and other growth factor receptors, which are linked by galectin-3 (Kariya et al., 2008, 2010). Therefore, to characterize the biological functions of N-glycan on integrin will be on the highlight of this stage of science, which might ultimately lead to novel cancer therapies.

ACKNOWLEDGMENTS These works were partly supported by Core Research for Evolutional Science and Technology (CREST), the Japan Science and Technology Agency (JST); the ‘‘Academic Frontier’’ Project for Private Universities from the Ministry of Education, Culture, Sports, Science and Technology of Japan; KAKENHI 21370059 (JSPS). The authors are deeply indebted to the outstanding related papers, which have not been cited in the present chapter, due to limited space.

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Isaji, T., Gu, J., Nishiuchi, R., Zhao, Y., Takahashi, M., Miyoshi, E., Honke, K., Sekiguchi, K., and Taniguchi, N. (2004). Introduction of bisecting GlcNAc into integrin alpha5beta1 reduces ligand binding and down-regulates cell adhesion and cell migration. J. Biol. Chem. 279, 19747–19754. Isaji, T., Sato, Y., Zhao, Y., Miyoshi, E., Wada, Y., Taniguchi, N., and Gu, J. (2006). N-glycosylation of the beta-propeller domain of the integrin alpha5 subunit is essential for alpha5beta1 heterodimerization, expression on the cell surface, and its biological function. J. Biol. Chem. 281, 33258–33267. Isaji, T., Sato, Y., Fukuda, T., and Gu, J. (2009). N-glycosylation of the I-like domain of beta1 integrin is essential for beta1 integrin expression and biological function: Identification of the minimal N-glycosylation requirement for alpha5beta1. J. Biol. Chem. 284, 12207–12216. Jasiulionis, M. G., Chammas, R., Ventura, A. M., Travassos, L. R., and Brentani, R. R. (1996). alpha6beta1-Integrin, a major cell surface carrier of beta1-6-branched oligosaccharides, mediates migration of EJ-ras-transformed fibroblasts on laminin-1 independently of its glycosylation state. Cancer Res. 56, 1682–1689. Josefsson, E. C., Gebhard, H. H., Stossel, T. P., Hartwig, J. H., and Hoffmeister, K. M. (2005). The macrophage alphaMbeta2 integrin alphaM lectin domain mediates the phagocytosis of chilled platelets. J. Biol. Chem. 280, 18025–18032. Ju, T., and Cummings, R. D. (2002). A unique molecular chaperone Cosmc required for activity of the mammalian core 1 beta 3-galactosyltransferase. Proc. Natl. Acad. Sci. USA 99, 16613–16618. Ju, T., and Cummings, R. D. (2005). Protein glycosylation: Chaperone mutation in Tn syndrome. Nature 437, 1252. Kariya, Y., Kato, R., Itoh, S., Fukuda, T., Shibukawa, Y., Sanzen, N., Sekiguchi, K., Wada, Y., Kawasaki, N., and Gu, J. (2008). N-glycosylation of laminin-332 regulates its biological functions. A novel function of the bisecting GlcNAc. J. Biol. Chem. 283, 33036–33045. Kariya, Y., Kawamura, C., Tabei, T., and Gu, J. (2010). Bisecting GlcNAc residues on laminin-332 down-regulate galectin-3-dependent keratinocyte motility. J. Biol. Chem. 285, 3330–3340. Liddington, R. C., and Ginsberg, M. H. (2002). Integrin activation takes shape. J. Cell Biol. 158, 833–839. Manya, H., Chiba, A., Yoshida, A., Wang, X., Chiba, Y., Jigami, Y., Margolis, R. U., and Endo, T. (2004). Demonstration of mammalian protein O-mannosyltransferase activity: Coexpression of POMT1 and POMT2 required for enzymatic activity. Proc. Natl. Acad. Sci. USA 101, 500–505. Manya, H., Suzuki, T., Akasaka-Manya, K., Ishida, H. K., Mizuno, M., Suzuki, Y., Inazu, T., Dohmae, N., and Endo, T. (2007). Regulation of mammalian protein Omannosylation: Preferential amino acid sequence for O-mannose modification. J. Biol. Chem. 282, 20200–20206. Miyoshi, E., Noda, K., Ko, J. H., Ekuni, A., Kitada, T., Uozumi, N., Ikeda, Y., Matsuura, N., Sasaki, Y., Hayashi, N., Hori, M., and Taniguchi, N. (1999). Overexpression of alpha1-6 fucosyltransferase in hepatoma cells suppresses intrahepatic metastasis after splenic injection in athymic mice. Cancer Res. 59, 2237–2243. Mould, A. P., Symonds, E. J., Buckley, P. A., Grossmann, J. G., McEwan, P. A., Barton, S. J., Askari, J. A., Craig, S. E., Bella, J., and Humphries, M. J. (2003). Structure of an integrin–ligand complex deduced from solution x-ray scattering and site-directed mutagenesis. J. Biol. Chem. 278, 39993–39999. Pochec, E., Litynska, A., Amoresano, A., and Casbarra, A. (2003). Glycosylation profile of integrin alpha 3 beta 1 changes with melanoma progression. Biochim. Biophys. Acta 7, 1–3. Ruoslahti, E. (1996). RGD and other recognition sequences for integrins. Annu. Rev. Cell Dev. Biol. 12, 697–715.

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Sasai, K., Ikeda, Y., Ihara, H., Honke, K., and Taniguchi, N. (2003). Caveolin-1 regulates the functional localization of N-acetylglucosaminyltransferase III within the golgi apparatus. J. Biol. Chem. 278, 25295–25301. Sato, Y., Isaji, T., Tajiri, M., Yoshida-Yamamoto, S., Yoshinaka, T., Somehara, T., Fukuda, T., Wada, Y., and Gu, J. (2009). An N-glycosylation site on the beta-propeller domain of the integrin alpha5 subunit plays key roles in both its function and site-specific modification by beta1, 4-N-acetylglucosaminyltransferase III. J. Biol. Chem. 284, 11873–11881. Schachter, H. (1986). Biosynthetic controls that determine the branching and microheterogeneity of protein-bound oligosaccharides. Adv. Exp. Med. Biol. 205, 53–85. Seales, E. C., Shaikh, F. M., Woodard-Grice, A. V., Aggarwal, P., McBrayer, A. C., Hennessy, K. M., and Bellis, S. L. (2005). A protein kinase C/Ras/ERK signaling pathway activates myeloid fibronectin receptors by altering beta1 integrin sialylation. J. Biol. Chem. 280, 37610–37615. Shigeta, M., Shibukawa, Y., Ihara, H., Miyoshi, E., Taniguchi, N., and Gu, J. (2006). beta1, 4-N-Acetylglucosaminyltransferase III potentiates beta1 integrin-mediated neuritogenesis induced by serum deprivation in Neuro2a cells. Glycobiology 16, 564–571. Takagi, J. (2007). Structural basis for ligand recognition by integrins. Curr. Opin. Cell Biol. 19, 557–564. Taniguchi, N., Miyoshi, E., Gu, J., Honke, K., and Matsumoto, A. (2006). Decoding sugar functions by identifying target glycoproteins. Curr. Opin. Struct. Biol. 16, 561–566. Xiong, J. P., Stehle, T., Diefenbach, B., Zhang, R., Dunker, R., Scott, D. L., Joachimiak, A., Goodman, S. L., and Arnaout, M. A. (2001). Crystal structure of the extracellular segment of integrin alpha Vbeta3. Science 294, 339–345. Xiong, J. P., Stehle, T., Zhang, R., Joachimiak, A., Frech, M., Goodman, S. L., and Arnaout, M. A. (2002). Crystal structure of the extracellular segment of integrin alpha Vbeta3 in complex with an Arg-Gly-Asp ligand. Science 296, 151–155. Yoshimura, M., Nishikawa, A., Ihara, Y., Taniguchi, S., and Taniguchi, N. (1995). Suppression of lung metastasis of B16 mouse melanoma by N-acetylglucosaminyltransferase III gene transfection. Proc. Natl. Acad. Sci. USA 92, 8754–8758. Zhao, Y., Nakagawa, T., Itoh, S., Inamori, K., Isaji, T., Kariya, Y., Kondo, A., Miyoshi, E., Miyazaki, K., Kawasaki, N., Taniguchi, N., and Gu, J. (2006). N-acetylglucosaminyltransferase III antagonizes the effect of N-acetylglucosaminyltransferase V on alpha3beta1 integrin-mediated cell migration. J. Biol. Chem. 281, 32122–32130.

C H A P T E R

T W E N T Y- O N E

Lectin-Based Glycoproteomic Techniques for the Enrichment and Identification of Potential Biomarkers Karen L. Abbott* and J. Michael Pierce† Contents 1. Introduction 2. Lectin Blotting and Separation of Total Glycoproteins in a Sample Using a Lectin(s) Immobilized on Paramagnetic Beads 3. Separation of Glycoproteins Using a Lectin Immobilized on Paramagnetic Beads Prior to Mass Spectrometry-Based Shotgun Proteomics 3.1. Extraction of easily solubilized glycoproteins from tissues and separation on immobilized lectin(s) prior to shotgun proteomics 3.2. Extraction of tightly membrane-associated glycoproteins from tissues and separation on immobilized lectin(s) prior to shotgun proteomics 4. Glycomics Strategy for Identifying Potential Glycoprotein Cancer Markers 5. MS/MS Data Analysis and Confirmation of Glycoprotein Glycosylation Differences When Results from Two Tissue Sources are Compared 6. Conclusions Acknowledgments References

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Abstract Glycan structures on glycoproteins are controlled by several factors such as regulated expression of glycosyltransferases and glycosylhydrolases, as well as regulation of glycoprotein expression, folding, and transport through the ER and * Complex Carbohydrate Research Center, University of Georgia Cancer Center, Athens, Georgia, USA Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA

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Methods in Enzymology, Volume 480 ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)80020-5

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2010 Elsevier Inc. All rights reserved.

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Golgi. In cancer, for example, the glycosylation of glycoproteins can be significantly altered due to changes in the expression levels of glycosyltransferases as a result of oncogene activated signaling pathways coupled with gain or loss in chromosome copy number. Cumulatively these changes result in glycoproteins exported to the cell surface and extracellular region with altered glycan structures that can lead to significant changes in cell phenotype. Therefore, it is advantageous to be able to capture and identify proteins that express particular glycans or classes of glycans. In this report, we discuss extraction methods and lectin capture methodology that can be used to enrich and identify by mass spectrometry glycoproteins that express specific glycans that change in response to disorders or diseases, such as the presence of malignancies.

1. Introduction The discovery of functions of glycans expressed on specific sites on particular proteins is at the forefront of glycobiology research (Taniguchi et al., 2009). To explore these potential functions, several analytical platforms have been developed to identify glycans whose expression change during cell differentiation and disease progression (Abd Hamid et al., 2008; Kyselova et al., 2008; Zeng et al., 2009). Moreover, since glycoproteins are frequently secreted from cells or released from cell surfaces by hydrolytic activities, glycoproteins with altered glycan expression are likely candidates for biomarkers that can predict or monitor disease states such as cancer. As a next, logical step, the discovery process requires the identities of the proteins that express the glycans of interest be determined by proteomic analysis. A common strategy to identify the proteins that express a particular glycan is to use a lectin or antibody coupled to a solid support to separate positive proteins from those that lack expression of the glycan of interest, followed by further separations and/or shotgun proteomics. But this strategy, which may seem straightforward, often yields an avalanche of spurious protein sequences for several reasons, including nonspecific binding. Delineating a methodology that would yield reproducible results resulting from specific lectin or antibody binding was, therefore, a challenge. Furthermore, although several studies have now used lectin separations to isolate and identify glycoproteins of interest, the focus has been on working with fluids such as serum and plasma, pancreatic ductal fluid, or cultured cells (Kullolli et al., 2008). A major challenge, therefore, was to focus on human tissue as the source from which particular glycoproteins were to be identified, necessitating that efficient solubilization and separation were performed under conditions compatible with subsequent proteomic analysis by mass spectrometry. Our particular focus has been on developing methodologies for detection of cancer and cancer risk based on analysis of human tumor and control tissues employing lectin separation, followed by identification of bound proteins using shotgun proteomics (Abbott et al., 2008a,b). Clearly, glycoprotein glycan structures display altered expression in diseases and disorders, in particular, oncogenic

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transformation and cancer progression (Hakomori, 1999; Kobata and Amano, 2005; Ohtsubo and Marth, 2006). Lectins or glycan-specific antibodies that are known or predicted to bind the glycan changes of interest can then be used to isolate from cancer tissues the glycoproteins that express these glycans with altered expression, and their identities established by proteomic analysis. Altered glycan expression during oncogenesis can result from oncogene activation or tumor suppressor inactivation, which can alter signal transduction pathways leading to changes in the expression levels of glycosyltransferases and glycosylhydrolases (Buckhaults et al., 1997; Pierce and Arango, 1986; Seales et al., 2003; Zhao et al., 2008). In addition, chaperones that are required for certain glycosylation reactions can be mutated or silenced (Ju and Cummings, 2002, 2005; Ju et al., 2008). Cumulatively, these changes result in glycoproteins being secreted or exported to the cancer cell surfaces with altered glycan structures that lead to alterations in functional activities, including altered receptor signaling (Gu and Taniguchi, 2008; Guo et al., 2002, 2003; Zhao et al., 2008). Therefore, these glycan changes can be exploited using reagents with glycan binding specificity to isolate glycoproteins from cancer and control tissues and determine which differ qualitatively or quantitatively in expression. These glycoproteins with cancer-specific ‘‘glycosignatures’’ are then logical candidates to test as potential biomarkers that could be developed, for example, into a diagnostic assay or targeted for delivery of a therapeutic. In this report, we will discuss lectin-based methodologies developed for identification of potential cancer biomarkers that can be used to identify glycoproteins in cells and tissues that differ based on particular glycan expression differences. These methodologies have been used to identify potential glycoprotein biomarkers for breast and ovarian cancers (Abbott et al., 2008a,b, 2010). Lectins are carbohydrate binding proteins with variable affinities for specific glycan structures (Cummings and Kornfeld, 1982a, b; Nagata et al., 1991; Wimmerova et al., 2003; Yamashita et al., 1987). Several glycan structures that have been reported to change on glycoproteins in different cancers can be targeted by certain plant lectins, listed in Table 21.1. Core fucosylation, recognized by the lectins LCA and AAL, has been reported to be elevated on glycoproteins in ovarian and liver tumors (Abbott et al., 2008a,b; Mehta and Block, 2008; Takahashi et al., 2000). The lectin L-PHA recognizes the b(1,6) branched N-acetylglucosamine extended with distal b(1,4)-linked galactose (Cummings and Kornfeld, 1982a, b). This particular glycan structure is elevated in breast carcinomas as well as several other tumor types (Abbott et al., 2008b; Dennis and Laferte, 1989; Fernandes et al., 1991; Handerson and Pawelek, 2003). The lectin DSL can recognize the b(1,6) branched glycans with distal LacNAc residues, as well as b(1,4) branched glycans, making this lectin useful for capturing multiple branched glycan structures such as tri- and tetra-antennary. GnT-IVa, the enzyme that adds b(1,4) linked N-acetylglucosamine recognized by DSL, is significantly elevated in ovarian cancer (Abbott et al., 2008a,b). The lectin E-PHA recognizes bisected complex type N-linked glycan structures (Cummings

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Table 21.1 Examples of lectins recognizing glycan structures that are frequently amplified in cancer Full name

AAL

Aleuria aurantia lectin

LCA

Len culinaris agglutinin

L-PHA

Phaseolus vulgaris leukoagglutinin

DSL

Datura stramonium lectin

E-PHA

Phaseolus vulgaris erythroagglutinin

Preferred target glycan

Asn Asn Asn

±

±

±

Asn

Asn

Abbreviation

and Kornfeld, 1982a,b), and these bisecting glycan structures are significantly amplified in ovarian cancer tissue, with very little expression in nondiseased ovarian tissue (Abbott et al., 2008a,b). First, lectin blotting experiments are useful to determine if particular glycan structures are altered when tissues undergo malignant transformation and to capture glycoproteins with these structures from nondiseased and malignant cell populations. Lectins display a wide range of binding affinities for glycans (typically mM–mM), different avidities due to multimerization, and require optimized binding conditions, such as the presence of specific divalent cations or various salt concentrations (Merkle and Cummings, 1987). Therefore, a general protocol for lectin blotting for visualization of glycoprotein bands after SDS-PAGE was developed. In addition, a procedure for the binding of lectins to intact, solubilized glycoproteins that results in efficient binding and lower nonspecific binding, at least for several lectins tested, was optimized. This affinity separation can be used to separate glycoproteins of interest prior to lectin blotting, resulting in reduced sample complexity and a more detailed comparison of samples by lectin blotting.

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2. Lectin Blotting and Separation of Total Glycoproteins in a Sample Using a Lectin(s) Immobilized on Paramagnetic Beads

188 kDa

188 kDa

98 kDa

98 kDa

62 kDa

62 kDa

49 kDa

49 kDa 1

2

3

Tumor

B Normal

Control siRNA

GnT-V siRNA

GnT-V siRNA

A

Control siRNA

To optimize the conditions for the binding of a particular lectin to its glycoprotein targets, cell lines engineered either to express or overexpress the cDNA for the glycosyltransferases required for the addition of glycans bound by the lectin or to express shRNA molecules targeting the enzymes essential for adding the glycans required for lectin binding can both be useful. Our first focus was on isolating glycoproteins expressing b(1,6) branched glycans based on L-PHA binding (Guo et al., 2007). We chose to use shRNA molecules to target the glycosyltransferase GnT-V to allow the optimization of conditions for L-PHA capture. In a sense, the shRNA expressing cells would represent nondiseased tissues with epithelial cells that do not express the glycans bound by L-PHA, while control-transfectants would represent breast carcinoma. Comparative lectin blot analysis of proteins isolated from the breast cancer cell line MDA-MB231 expressing GnT-V shRNA compared to the same cell line expressing a control shRNA revealed an abundance of glycoproteins with the b(1,6) branched glycan structures that can be detected by the lectin L-PHA (Fig. 21.1A; Guo et al., 2007). The greatly

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Figure 21.1 (A) Proteins extracted from MDAMB231 breast cancer cells expressing GnT-V shRNA or control shRNA after ConA fractionation were analyzed by lectin blot using L-PHA. Lanes 1 and 2 represent 20% the unbound ConA fractions while lanes 3 and 4 represent 20% of the ConA-bound fractions for each cell line as indicated. (B) Silver stain of L-PHA-bound proteins (10%) isolated from patient matched adjacent normal or ductal invasive breast carcinoma using the soluble protein extraction and lectin binding protocol.

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reduced levels of L-PHA reactivity with glycoproteins isolated from GnT-VshRNA expressing cells added confidence that the glycoproteins identified as interacting with L-PHA in the control shRNA cells were likely products of GnT-V (Fig. 21.1A, lanes 1 and 2). The lectin blots were similar to Western blot detection except a biotinylated lectin and streptavidin–HRP were used in place of an antibody and HRP-conjugated secondary antibody, respectively. Mono- and disaccharide ‘‘haptens’’ can, in some cases, be used to distinguish specific from nonspecific lectin binding, but in many cases the haptens bear little resemblance to the glycopeptides that show high affinity binding, for example, the use of GalNAc to inhibit L-PHA binding. One method to overcome problems with high levels of nonspecific binding on lectin blots is prior fractionation of samples using immobilized lectins prior to lectin blotting. Immobilized lectins such as WGA (binds terminal Nacetylglucosamine) and concavalin A (ConA, binds to high mannose, hybrid, and biantennary N-linked structures) can often be used to reduce sample complexity and background associated with lectin blots. The protocol detailed below uses immobilized ConA as a fractionation tool prior to L-PHA blotting. As demonstrated in Fig. 21.1A, L-PHA binds branched glycoproteins that are found in the unbound ConA fractions (lanes 1 and 2). The bands visible in the ConA-bound fractions (lanes 3 and 4) are likely due to interactions of the streptavidin reagent. In this procedure, 1% Triton X-100, which is incompatible with mass spectrometry analysis, is used to solubilize all glycoproteins in the sample prior to lectin fractionation and blotting. This procedure, therefore, is useful mainly for separation of total glycoproteins from cells or tissues and visualization of lectin binding by blotting. 1. Cells from culture are scraped into a microfuge tube (50 mg cell pellet) and resuspended in 100 ml lysis buffer (1 Tris-buffered saline/ 1% triton X-100/1 protease inhibitor cocktail, Calbiochem) for 30 min on ice. Alternatively, tissue (50 mg) can be homogenized in 100 ml lysis buffer using a micropolytron set at 3–4. 2. Lysates are centrifuged at 4  C at 10,000 rpm for 10 min. 3. Protein assay is performed using the supernatant and 50 mg of total lysate is placed in a microcentrifuge tube. The volume is adjusted to 100 ml with lysis buffer. ConA 2 mg biotinylated is added and the tubes are rotated at 4  C for 2 h. 4. Paramagnetic streptavidin magnetic beads (Promega) (50 ml) are added and the tubes are rotated 1 h at 4  C. 5. Tubes are placed on a magnetic stand to capture the ConA-bound proteins. 6. The unbound material is removed to a clean tube. 7. The ConA-bound beads are released by removal from the magnet, washed in lysis buffer, and rebound to the magnet. This washing procedure is repeated two additional times before resuspension of the released beads in 100 ml of lysis buffer.

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8. 20% of the unbound and bound fractions are mixed with laemelli loading dye before being heated and loaded on 4–12% gradient polyacrylamide gels. 9. Proteins are transferred to PVDF membrane and blocked using 3% bovine serum albumin/1 TBST overnight at 4  C. Note: It is important not to use milk as a blocking agent due to high backgrounds from milk glycoproteins. 10. The biotinylated lectin at a concentration of 2 mg/ml (Vector Labs) for detection of the desired glycan structure is diluted 1:5000 in blocking buffer and added to the blot at room temperature for 30 min. 11. The blot is washed three times for 5 min each in 1 TBST. 12. Streptavidin HRP (Vector Labs) is diluted 1:5000 in blocking buffer and incubated with the blots for 30 min at room temperature. 13. The blots are washed three times 5 min each in 1 TBST before detection of bound lectin using a chemiluminescent substrate.

3. Separation of Glycoproteins Using a Lectin Immobilized on Paramagnetic Beads Prior to Mass Spectrometry-Based Shotgun Proteomics The MDA-MB231 cell lines described above were used to optimize the binding conditions for the capture of solubilized glycoproteins with b(1,6) branched glycans in solution with L-PHA and then used to capture L-PHA reactive glycoproteins isolated from patient breast tumors and matched adjacent, nondiseased breast tissue samples (Fig. 21.1B) (Guo et al., 2007). Although nondiseased breast epithelial cells do not bind L-PHA, other cells in the tissue samples are L-PHA positive, for example, endothelia, fibroblasts, and leucocytes (Fernandes et al., 1991). Breast tumor tissue shows a significantly higher number of proteins binding to L-PHA compared to adjacent normal (Fig. 21.1B). Two methods for the extraction and enrichment of glycoproteins by lectins will be described. The first method is useful for glycoproteins that can be easily extracted using 150 mM NaCl and 0.1% NP-40 (example shown in Fig. 21.1B), which would represent glycoproteins that are soluble or not tightly membrane associated. These conditions were used to discover potential biomarkers for breast and ovarian cancers (Abbott et al., 2008a,b, 2010). The second protocol details a method recently developed for the isolation of glycoproteins more tightly membrane associated. Most membrane proteomics protocols involve gel extraction or multiple layers of chromatography prior to analysis by MS/MS to reduce lipids (endogenous detergents) and sample complexity, but these additional steps lead to potential losses of glycoproteins of interest. The method described below uses an extraction protocol that increases the depth of proteins detected

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without involving gel extraction or chromatography. It should be noted that mass spectrometry analysis is sensitive to polymers that can come from contact with various plastics; therefore, plastic tips and tubes must never be autoclaved. Furthermore, only the highest quality water and reagents must be used, limiting the exposure of samples to polymer contamination.

3.1. Extraction of easily solubilized glycoproteins from tissues and separation on immobilized lectin(s) prior to shotgun proteomics 1. Tissue (frozen 80  C) is put into a mortar and liquid nitrogen added. The pestle is chilled in liquid nitrogen as well. The tissue is ground into a fine powder and stored at 80  C in a microfuge tube until delipidation. Cells are frozen as pellets at 80  C until delipidation. 2. Delipidation is performed by mixing 2 ml of chloroform/methanol/ water (4:8:3, v/v/v) with the tissue powder or cell pellet in a glass tube. Note: No plastic can be used at this stage. Glass Pasteur pipets are necessary to pipet the chloroform/methanol/water solution and this solution is made fresh. The screw top glass tubes (13  100 mm, pyrex 99447) are placed on a tube rotator at 4  C for 2 h. 3. Tubes are centrifuged at 2000 rpm for 10 min at room temperature. 4. The liquid is removed from the tissue powder or cells and fresh chloroform/methanol/water is added again for another 2 h. Tissue that is high in fat content such as breast tissue may require three incubations while other types of tissue require only two incubations. If yellow fat is visible continue delipidation incubations until all fat is gone. 5. After the final centrifugation at 2000 rpm for 10 min add 2 ml of cold acetone 100% for 5 min on ice. 6. Centrifuge again at 2000 rpm for 10 min. Remove all acetone with glass Pasteur pipets and dry the sample under nitrogen. Delipidated tissue powder can be stored at 80  C until use. 7. Weigh out 10 mg of delipidated powder for each sample in methanol and water rinsed microcentrifuge tubes. Add 300 ml of the following lysis buffer: 50 mM Tris–HCl pH 7.5, 0.1% NP-40, 150 mM NaCl, 0.4 mM EDTA, and mini complete protease inhibitor. Sonicate 10 s pulses five times. 8. Centrifuge 10,000 rpm for 10 min. 9. Take the supernatant and dialyze overnight at 4  C into 40 mM ammonium bicarbonate using the 4000 MWCO tube-O-dialyzer (G-Biosciences). This step is essential to remove the detergent in the lysis buffer. 10. Take the dialyzed sample to a clean tube. 11. Perform a protein assay and digest 60 mg of the sample with 5 mg of sequencing grade modified trypsin (Promega) at 37  C overnight for a

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12. 13. 14. 15. 16. 17. 18. 19.

20. 21.

22. 23.

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prelectin mass spectrometry sample. Take 600 mg of protein for the lectin binding. Bring the sample volume to 500 ml with 40 mM ammonium bicarbonate after adding CaCl2 or MgCl2 if necessary for the lectin (5 mM final) and NaCl (0.15 M final). The NaCl concentration can be increased if a high degree of nonspecific binding is observed when evaluating control cell lines. Add 10 mg of biotinylated lectin (Vector Labs). Multiple lectins can be added at one time (10 mg each). Rotate tubes overnight at 4  C. Equilibrate streptavidin magnetic beads (Promega) into 40 mM ammonium bicarbonate. Add 100 ml of beads to each tube and rotate at 4  C for 2 h. Place tubes on a magnetic stand and remove the unbound to a clean tube (sample 10% for a gel). Wash the beads in 1 phosphate-buffered saline three times 5 min each. Place beads in 100 ml of 40 mM ammonium bicarbonate, remove from magnet mix, sample 10 ml for a gel. Place tubes on magnet again and remove all liquid. Make a fresh solution of 2 M urea/4 mM DTT/40 mM ammonium bicarbonate. Put 200 ml of this solution on the beads, mix, and place the tubes at 52  C for 1 h. Place tubes on magnet and remove the eluted proteins to a clean tube. Sample 20 ml for a gel. To the remaining 180 ml eluant add 180 ml of iodoacetamide (10 mg/ml) dissolved in 40 mM ammonium bicarbonate. Mix well and place in the dark for 45 min vortexing every 10 min. Sample the beads after elution by adding 100 ml of 40 mM ammonium bicarbonate, mix to suspend beads, sample 10 ml for a gel. To the tubes with the 360 ml of reduced carboxyamidomethylated proteins, add 5 mg of sequencing grade modified trypsin (Promega), and incubate the tubes overnight at 37  C. Run samples on a polyacrylamide gel and silver stain. Samples include step 14 (unbound fraction), step 16 bound, step 19 eluted, step 19 beads after elution. If glycoprotein is still present on the beads after the first elution, add a larger volume of elution buffer and repeat this step to get more protein from the beads. Pick up the protocol again from step 19. To desalt glycoproteins prior to MS/MS, add 1% TFA to the trypsin digest to have a final concentration of 0.1% TFA. Equilibrate a micro-Vydac C18 silica column (Nest Group) by adding buffer B (80% acetonitrile/0.1% formic acid) 150 ml followed by washing three times back into buffer A (0.1% formic acid) 150 ml each. Add the acidified peptides and reload the peptides to the column 3 before washing 3 with buffer A (150 ml each) and eluting the peptides in Buffer B 2 (150 ml each). Combine both

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elutions and dry the sample in a speed vac. Dried samples can be stored at 20  C until mass spectrometry analysis.

3.2. Extraction of tightly membrane-associated glycoproteins from tissues and separation on immobilized lectin(s) prior to shotgun proteomics 1. Tissue (100 mg) or cell pellet (100 mg) is resuspended in 10 mM HEPES pH 7.5 plus protease inhibitors using a polytron (1 ml volume). The slurry is placed in a glass dounce homogenizer and cells are lysed using 10 strokes each of the large and fine pestle. The solution is incubated on ice in the hypotonic solution for 1 h. 2. Nuclei are removed by transferring to a microcentrifuge tube and centrifuging at 3000 rpm for 5 min. The supernatant is removed to a fresh tube and the centrifugation is repeated twice. 3. The final supernatant is placed in a Beckman ultracentrifuge tube 1.5 ml (cat. number 357448) and centrifuged at 100,000g for 1 h at 4  C. 4. The supernatant following the ultracentrifugation is removed to a clean microcentrifuge tube and labeled as soluble fraction and stored frozen at 80  C. The pellet containing a total membrane preparation is rinsed in 40 mM ammonium bicarbonate. Do not disturb the pellet it will be very sticky, add the buffer and immediately pipet off without touching the pellet. 5. Add 300 ml of 40 mM ammonium bicarbonate/10 mM DTT and sonicate to resuspend the proteins. Sonication is essential to break up lipids. The sample is rotated at room temperature for 2 h to reduce proteins. 6. An equal volume of iodoacetamide (10 mg/ml in 40 mM ammonium bicarbonate) is added and the tubes are vortexed before incubating in the dark for 45 min. 7. The protein solution is dialyzed overnight at 4  C into 10 mM ammonium bicarbonate using 4000 MWCO tube-O-dialyzer (G-Biosciences). This step is essential to remove small lipids that were broken up by sonication. 8. Proteins are dried in the speed vacuum for long-term storage at 80  C or used directly. Digest 60 mg of membrane proteins with 5 mg of sequencing grade modified trypsin (Promega) for sample analysis before lectin binding as described above. 9. Dried proteins are resuspended in 40 mM ammonium bicarbonate or dialyzed sample is adjusted to 40 mM ammonium bicarbonate. A protein assay is performed and 600 mg are then used for lectin binding as described above starting with step 11. Alternatively, trypsin digested proteins are acidified and processed as described in step 22 above.

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4. Glycomics Strategy for Identifying Potential Glycoprotein Cancer Markers A strategy developed for the discovery of potential cancer markers with tumor-specific glycan changes is shown in Fig. 21.2. Pivotal elements of this approach are (i) quantitative real-time PCR discovery of glycosylation enzymes changing expression levels in cancer tissue compared to normal to predict glycan structures that can be targeted with lectins, (ii) use of tissue for the discovery of potential glycoprotein markers with validations using lectin capture and Western blot on patient serum, and (iii) comparison of prelectin and postlectin MS/MS analysis for each sample to determine tumor-specific glycoproteins enriched by lectin binding in >70% of the samples analyzed.

5. MS/MS Data Analysis and Confirmation of Glycoprotein Glycosylation Differences When Results from Two Tissue Sources are Compared The method we have employed to identify peptides isolated by the lectin separations is nanospray LC–MS/MS. The advantage of this method for analysis of complex peptide mixtures is the faster time for full MS scan Normal tissue

Cancer tissue

Quantitative RT-PCR to determine glycosylation enzymes changing in tumor

Lectin Blot validation of tumor-specific glycan changes

Pre Lectin MS/MS 6 E 7 9. 2 :L N 1 :VA 5 9 . 5 3 :T R 4 8 1 2# M 1 3r ] 0 0 . 5 9 0 1 - 0 0 . 5 3 1 [ 0 0. 6 3 @ 7 4. 1 4 5 2 s m ll uF d c 0.1 87

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6 E 7 9. 2 :L N 1 :VA 5 9 . 5 3 :T R 4 8 1 2# M 1 3r ] 0 0 . 5 9 0 1 - 0 0 . 5 3 1 [ 0 0. 6 3 @ 7 4. 1 4 5 2 s m ll uF d c 0.1 87

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Multilectin Capture of glycoproteins from tissue

Figure 21.2 Strategy for the identification of tumor-specific glycoprotein markers.

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and selection of ions for collision-induced dissociation (CID). Desalted samples from lectin separation experiments are resuspended in 78 ml of buffer A (0.1% formic acid) and 2 ml buffer B (80% acetonitrile/0.1% formic acid) before being filtered thru a 0.2 mm filter (PALL). The sample is then loaded off-line onto a nanospray column (75 mm  8.5 cm, New Objective) self-packed with C18 silica reverse phase resin in a nitrogen bomb for 10 min. Peptides are eluted from the column into a linear ion trap (LTQ, Thermo, San Jose, CA) equipped with a nanoelectrospray ion source. The top eight ions from a full MS (300–2000 m/z) scan are selected for CID fragmentation at 34% energy with a dynamic exclusion of two fragmented mass ions using an exclusion time of 30 s. As shown in Fig. 21.2, MS/MS data are generated for each sample before and after lectin capture to normalize the MS/MS data determining the ratio of each glycoprotein between nondiseased and tumor tissues before and after lectin capture. This comparative analysis facilitates the identification of glycoproteins whose glycosylation is altered in cancer tissue. For example, the potential markers periostin and mimecan have been identified as L-PHA reactive in breast cancer tissue (Abbott et al., 2008a,b). The ratios of peptides for periostin and mimecan between normal and tumor tissues were not significantly different prior to lectin capture. However, after lectin capture, peptides for these markers were found selectively in tumor tissue (Abbott et al., 2008a,b). Therefore, the expression of b(1,6) branched glycans (L-PH reactive) on these glycoproteins appeared to be a tumorspecific glycan alteration. This hypothesis was confirmed by L-PHA pulldown, SDS-PAGE, and subsequent blotting with antiperiostin antibody using portions of the same nondiseased and breast cancer tissues used for lectin binding and LC–MS/MS proteomic analysis. Large amounts of MS/MS data are generated that need to be filtered and aligned for this type of analysis. We use the publicly available software IDPicker which incorporates searches against a separate reverse database, probability match data from MyriMatch (also publicly available), and DeltCN scores to assemble protein lists from MS/MS data (Tabb et al., 2007). Information about these publicly available programs can be found at http://www.mc.vanderbilt.edu/msrc/bioinformatics/. Other software can be used to identify proteins and sort the data as well (Alvarez-Manilla et al., 2009). Proteins that are identified with tumor-specific glycan changes in at least 70% of the cases, analyzed are then validated using lectin capture coupled with Western blotting. Glycan changes observed for a particular glycoprotein in a specific tumor tissue, however, may not be unique for that glycoprotein found in serum (compare haptoglobin vs. periostin validation results in Fig. 21.3). Serum (5 ml) from nondiseased patients (NL1–NL3) and cancer patients (BC1–BC5) is diluted into 300 ml of the buffer described for soluble extraction and lectin binding step 7. Biotinylated lectin (10 mg) is added and

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BC1

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Lectin-Based Glycoproteomic Methods for the Identification of Biomarkers

Ppt: L-PHA WB: Periostin

Ppt: L-PHA WB: Haptoglobin

Figure 21.3 Validation of glycan changes on glycoproteins in serum. Glycoproteins were captured using the soluble extraction buffer and lectin binding procedure from 5 ml of serum. Proteins bound to magnetic beads were separated on 4–12% Bis Tris gels in 1 MES buffer before transfer to PVDF and Western blot detection using either antiperiostin or antihaptoglobin antibodies.

the samples are incubated at 4  C overnight. Lectin protein complexes are captured using magnetic streptavidin beads, as described above. The washed beads are placed in Laemelli buffer and boiled prior to separation of proteins on a gradient 4–12% polyacrylamide gel. The proteins are transferred to PVDF and proteins are detected using commercially available antibodies. Results from this type of validation analysis are shown in Fig. 21.3. The glycoprotein periostin in the top panel shows tumor-specific association with L-PHA in serum similar to the MS/MS data. However, the glycoprotein haptoglobin or haptoglobin-related protein identified from breast cancer tissue with tumor-specific L-PHA reactivity (Abbott et al., 2008a,b) shows no distinction in L-PHA reactivity between normal serum and serum from breast cancer patients. These results suggest that haptoglobin with L-PHA reactivity is released from an additional tissue(s) source and is not useful as a potential serum marker for breast cancer.

6. Conclusions Continued advances in mass spectrometry technologies, along with effective methods of tissue extraction and fractionation are increasing the success in identifying potential biomarkers useful for diagnostic, prognostic, and therapeutic targeting of cancer. The use of lectins as tools for the selective targeting of glycan structures specific to particular tumors has led to the identification of several potential glycoprotein markers for breast and ovarian cancers (Abbott et al., 2008a,b, 2010). These techniques can obviously be applied to other diseases and used with many different variations. For example, antibodies against particular glycans or glycopeptides can be coupled covalently to magnetic beads and used for affinity capture (Ingale

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et al., 2007; Teo et al., 2010). Also, animal lectins, such as the galectins and selectins, can be coupled to magnetic beads and used for affinity capture to identify potential glycoprotein ligands (Stowell et al., 2010). The tissue extraction, lectin capture, and glycoproteomics methodologies described above can be adapted to advance our knowledge of glycan changes on glycoproteins that are involved in many aspects of physiology, diseases, and disorders and which can be exploited to develop biomarkers.

ACKNOWLEDGMENTS We are grateful to Lance Wells and Jae-Min Lim for performing the proteomic analyses and useful discussions. This research was supported by these grants: NCI CA64462, CA128454, NCRR RR018502, and an award from the Georgia Cancer Coalition.

REFERENCES Abbott, K. L., Aoki, K., et al. (2008a). Targeted glycoproteomic identification of biomarkers for human breast carcinoma. J. Proteome Res. 7, 1470–1480. Abbott, K. L., Nairn, A. V., et al. (2008b). Focused glycomic analysis of the N-linked glycan biosynthetic pathway in ovarian cancer. Proteomics 8, 3210–3220. Abbott, K. L., Lim, J. M., et al. (2010). Identification of candidate biomarkers with cancerspecific glycosylation in the tissue and serum of endometrioid ovarian cancer patients by glycoproteomic analysis. Proteomics 10, 470–481. Abd Hamid, U. M., Royle, L., et al. (2008). A strategy to reveal potential glycan markers from serum glycoproteins associated with breast cancer progression. Glycobiology 18, 1105–1118. Alvarez-Manilla, G., Warren, N. L., et al. (2009). Glycoproteomic analysis of embryonic stem cells: Identification of potential glycobiomarkers using lectin affinity chromatography of glycopeptides. J. Proteome Res. 6(5), 338–343. Buckhaults, P., Chen, L., et al. (1997). Transcriptional regulation of N-acetylglucosaminyltransferase V by the src oncogene. J. Biol. Chem. 272, 19575–19581. Cummings, R. D., and Kornfeld, S. (1982a). Characterization of the structural determinants required for the high affinity interaction of asparagine-linked oligosaccharides with immobilized Phaseolus vulgaris leukoagglutinating and erythroagglutinating lectins. J. Biol. Chem. 257, 11230–11234. Cummings, R. D., and Kornfeld, S. (1982b). Fractionation of asparagine-linked oligosaccharides by serial lectin-agarose affinity chromatography. A rapid, sensitive, and specific technique. J. Biol. Chem. 257, 11235–11240. Dennis, J. W., and Laferte, S. (1989). Oncodevelopmental expression of–GlcNAc beta 1–6Man alpha 1–6Man beta 1–branched asparagine-linked oligosaccharides in murine tissues and human breast carcinomas. Cancer Res. 49, 945–950. Fernandes, B., Sagman, U., et al. (1991). Beta 1-6 branched oligosaccharides as a marker of tumor progression in human breast and colon neoplasia. Cancer Res. 51, 718–723. Gu, J., and Taniguchi, N. (2008). Potential of N-glycan in cell adhesion and migration as either a positive or negative regulator. Cell Adh. Migr. 2, 243–245. Guo, H. B., Lee, I., et al. (2002). Aberrant N-glycosylation of beta1 integrin causes reduced alpha5beta1 integrin clustering and stimulates cell migration. Cancer Res. 62, 6837–6845.

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Guo, H. B., Lee, I., et al. (2003). N-acetylglucosaminyltransferase V expression levels regulate cadherin-associated homotypic cell–cell adhesion and intracellular signaling pathways. J. Biol. Chem. 278, 52412–52424. Guo, H. B., Randolph, M., et al. (2007). Inhibition of a specific N-glycosylation activity results in attenuation of breast carcinoma cell invasiveness-related phenotypes: Inhibition of epidermal growth factor-induced dephosphorylation of focal adhesion kinase. J. Biol. Chem. 282, 22150–22162. Hakomori, S. (1999). Antigen structure and genetic basis of histo-blood groups A, B and O: Their changes associated with human cancer. Biochim. Biophys. Acta 1473, 247–266. Handerson, T., and Pawelek, J. M. (2003). Beta1, 6-branched oligosaccharides and coarse vesicles: A common, pervasive phenotype in melanoma and other human cancers. Cancer Res. 63, 5363–5369. Ingale, S., Wolfert, M. A., et al. (2007). Robust immune responses elicited by a fully synthetic three-component vaccine. Nat. Chem. Biol. 3, 663–667. Ju, T., and Cummings, R. D. (2002). A unique molecular chaperone Cosmc required for activity of the mammalian core 1 beta 3-galactosyltransferase. Proc. Natl. Acad. Sci. USA 99, 16613–16618. Ju, T., and Cummings, R. D. (2005). Protein glycosylation: Chaperone mutation in Tn syndrome. Nature 437, 1252. Ju, T., Lanneau, G. S., et al. (2008). Human tumor antigens Tn and sialyl Tn arise from mutations in Cosmc. Cancer Res. 68, 1636–1646. Kobata, A., and Amano, J. (2005). Altered glycosylation of proteins produced by malignant cells, and application for the diagnosis and immunotherapy of tumours. Immunol. Cell Biol. 83, 429–439. Kullolli, M., Hancock, W. S., et al. (2008). Preparation of a high-performance multi-lectin affinity chromatography (HP-M-LAC) adsorbent for the analysis of human plasma glycoproteins. J. Sep. Sci. 31, 2733–2739. Kyselova, Z., Mechref, Y., et al. (2008). Breast cancer diagnosis and prognosis through quantitative measurements of serum glycan profiles. Clin. Chem. 54, 1166–1175. Mehta, A., and Block, T. M. (2008). Fucosylated glycoproteins as markers of liver disease. Dis. Markers 25, 259–265. Merkle, R. K., and Cummings, R. D. (1987). Lectin affinity chromatography of glycopeptides. Methods Enzymol. 138, 232–259. Nagata, Y., Fukumori, F., et al. (1991). Crystallization and characterization of a lectin obtained from a mushroom, Aleuria aurantia. Biochim. Biophys. Acta 1076, 187–190. Ohtsubo, K., and Marth, J. D. (2006). Glycosylation in cellular mechanisms of health and disease. Cell 126, 855–867. Pierce, M., and Arango, J. (1986). Rous sarcoma virus-transformed baby hamster kidney cells express higher levels of asparagine-linked tri- and tetraantennary glycopeptides containing [GlcNAc-beta (1,6)Man-alpha (1,6)Man] and poly-N-acetyllactosamine sequences than baby hamster kidney cells. J. Biol. Chem. 261, 10772–10777. Seales, E. C., Jurado, G. A., et al. (2003). Ras oncogene directs expression of a differentially sialylated, functionally altered beta1 integrin. Oncogene 22, 7137–7145. Stowell, S. R., Arthur, C. M., et al. (2010). Innate immune lectins kill bacteria expressing blood group antigen. Nat. Med. 16, 295–301. Tabb, D. L., Fernando, C. G., et al. (2007). MyriMatch: Highly accurate tandem mass spectral peptide identification by multivariate hypergeometric analysis. J. Proteome Res. 6, 654–661. Takahashi, T., Ikeda, Y., et al. (2000). alpha1,6Fucosyltransferase is highly and specifically expressed in human ovarian serous adenocarcinomas. Int. J. Cancer 88, 914–919. Taniguchi, N., Hancock, W., et al. (2009). The second golden age of glycomics: From functional glycomics to clinical applications. J. Proteome Res. 8, 425–426.

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Teo, C. F., Ingale, S., et al. (2010). Glycopeptide-specific monoclonal antibodies suggest new roles for O-GlcNAc. Nat. Chem. Biol. 6(5), 338–343. Wimmerova, M., Mitchell, E., et al. (2003). Crystal structure of fungal lectin: Six-bladed beta-propeller fold and novel fucose recognition mode for Aleuria aurantia lectin. J. Biol. Chem. 278, 27059–27067. Yamashita, K., Totani, K., et al. (1987). Carbohydrate binding properties of complex-type oligosaccharides on immobilized Datura stramonium lectin. J. Biol. Chem. 262, 1602–1607. Zeng, Z., Hincapie, M., et al. (2009). The development of an integrated platform to identify breast cancer glycoproteome changes in human serum. J. Chromatogr. A. 6(5), 338–343. Zhao, Y. Y., Takahashi, M., et al. (2008). Functional roles of N-glycans in cell signaling and cell adhesion in cancer. Cancer Sci. 99, 1304–1310.

C H A P T E R

T W E N T Y- T W O

High-Throughput RNAi Screening for N-Glycosylation Dependent Loci in Caenorhabditis elegans Weston B. Struwe* and Charles E. Warren† Contents 478 479 480 480 482 482 482 483 486 486 489 492

1. 2. 3. 4.

Overview N-glycosylation in C. elegans RNAi in C. elegans C. elegans Strains, Culturing and RNAi Methods 4.1. Effect of tunicamycin on C. elegans phenotypes 4.2. Tunicamycin-lethality is dose dependent 4.3. Tunicamycin treatment varies among strains 4.4. Establishment of tunicamycin-hypersensitive genes 4.5. Tunicamycin does not alter RNAi effectiveness 5. Genome-wide RNAi Screen 6. Discussion References

Abstract The attachment of oligosaccharides to the amide nitrogen of asparagine side chains on proteins is a fundamental process occurring in all metazoans. This process, known as N-glycosylation, is complex and is achieved by the precise interactions of various cellular components. The initial stage of N-glycan biosynthesis is preserved among eukaryotes, and defective enzymes or components in this pathway cause congenital disorders of glycosylation type I (CDG-I) in humans. This disease is rare but exceedingly life-threatening with no known cure. Paramount to CDG treatment and care is understanding the mechanisms of N-glycosylation and factors that influence the pathology of the disease, both of which are not completely known. Here we outline a novel technique to model a CDG-I-like condition and identify genes that are vital for healthy glycosylation in Caenorhabditis elegans. C. elegans is a well-established model for understanding * National Institute for Bioprocessing Research and Training, Dublin-Oxford Glycobiology Group, Conway Institute for Biomolecular and Biomedical Sciences, University College Dublin, Belfield, Dublin, Ireland Department of Biochemistry and Molecular Biology and Program in Genetics, University of New Hampshire, Durham, NH, USA

{

Methods in Enzymology, Volume 480 ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)80021-7

#

2010 Elsevier Inc. All rights reserved.

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the complexity of glycosylation in development and disease. Although C. elegans N-glycan structures are dissimilar to that observed in higher eukaryotes, they contain over 150 gene homologs that are directly involved in glycosylation. Moreover, the annotated genome of C. elegans, its susceptibility to genetic silencing and its recognizable phenotypes, is a suitable model to dissect the complex phenomenon of glycosylation and identify genes that are required for N-glycan biosynthesis.

1. Overview N-linked glycosylation is an essential protein posttranslational modification that occurs in all eukaryotes. The early stages of N-glycan biosynthesis, namely the formation of the lipid-linked oligosaccharide (LLO) structure in the endoplasmic reticulum, is conserved among Saccharomyces cerevisiae, Caenorhabditis elegans, and vertebrates (Altmann et al., 2001; Huffaker and Robbins, 1982). The LLO consists of a Glc3Man9GlcNAc2 molecule linked to a dolichol pyrophosphate, and defects in enzymes responsible for its synthesis lead to congenital disorders of glycosylation type I (CDG-I) in humans. The biosynthetic pathway for glycoprotein formation is quite well understood and has provided a basis for establishing the etiology of CDG (Schollen et al., 2004). However, there are still cases of CDG with unknown etiology and the mechanistic consequences of defective glycoprotein synthesis are very poorly understood (Prietsch et al., 2002). Loss-of-function defects in the LLO biosynthetic pathway are embryonic lethal, patients with milder genotypes are born but are very ill, presenting with defects in virtually every body system. Because so many structures and functions are compromised, the glycoproteins that cause lethality or particular symptoms cannot be resolved. For this reason, it is vital to understand all factors that contribute to N-glycosylation that may affect the severity of CDG illnesses. Dissecting the complex biosynthesis of glycosylation is not uncomplicated, but here we present a novel approach to identify genes in C. elegans that contribute to the severity of a CDG-I-like disorder. We were able to identify genes in C. elegans that require the formation of the LLO precursor molecule to function properly and generate wild-type phenotypes. These genes products could originate from four hypothetical categories: (1) polypeptides that require glycans for function, (2) polypeptides that require glycans for structure, (3) genes responsible for glycoprotein maturation (i.e., unfolded protein response, secretion, or Golgi function), (4) genes that are involved in the lipid-linked oligosaccharide assembly pathway. To explore this hypothesis, RNAi, in combination with tunicamycin, a glycosylation inhibitor, was used in a genome-wide screen to identify genes that are hypersensitive to a mild dose of tunicamycin.

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2. N-glycosylation in C. elegans The free-living nematode C. elegans is a powerful resource for studying complex developmental topics and provides a robust model system to examine various human diseases. In addition to the ease of maintenance and manipulability C. elegans provides, it is the most understood multicellular eukaryotic animal to date. The complete cell lineage has been mapped and cell fates remain invariant between individuals (Lambie, 2002). C. elegans is a simple organism that comprises only 959 cells, but boasts of feeding, nervous, muscle, and reproductive physiological systems found in higher eukaryotes. More recently, the glycome of C. elegans has been studied and characterized (Cipollo et al., 2002, 2005; Hanneman et al., 2006; Haslam and Dell, 2003; Natsuka et al., 2002). Considering the simple anatomy of C. elegans, its N-glycan composition is extensive with over 100 structures present in the wild-type N2 Bristol strain (Paschinger et al., 2008). The annotated glycome of C. elegans, in addition to its well-characterized physiology, provides a platform to investigate the factors that affect N-glycosylation phenotypes. Much of the progress in our understanding the etiology of CDG has come from genetic model systems, particularly S. cerevisiae and mice (Aebi and Hennet, 2001). C. elegans shares many sequence similarities to mammalian genes involved in the assembly, processing, and modifications of a variety of glycans (Schachter, 2004). Specifically, the early stages of N-glycoprotein biosynthesis are conserved, making C. elegans a suitable model to identify genes that modify CDG-I phenotypes (Lehle et al., 2006). However, the later stages of glycoconjugate synthesis are much more complex in vertebrates, especially mammals. C. elegans do share similarities with vertebrate N- and Oglycans in terms of their core structures, but the majority of the differences are found in molecular size and terminal elaborations. The most notable difference between C. elegans and higher animals is the lack of sialic acid residues in C. elegans glycans (Paschinger et al., 2008). C. elegans have some biosynthetic components required for the synthesis of complex mammalian-type glycans, namely GnT-I (N-acetylglucosamine transferase-I), GnT-II, and GnT-V (Chen et al., 1999, 2002; Warren et al., 2002). Despite the identification of such enzymes, complex and hybrid Nglycans are either absent or present at low levels in C. elegans (Cipollo et al., 2002; Natsuka et al., 2002). However, the occurrence of complex glycan has been found at different developmental stages and may be only present to govern development (Cipollo et al., 2005). Additionally, C. elegans synthesize N-glycans having terminal fucose residues, similar to Lewis antigens found in humans. The C. elegans glycome predominately contains high mannose (Man5-9 GlcNAc2), paucimannose (Man3-4GlcNAc2), high-fucose (attached at the

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antennae or core), truncated complex (Man3GlcNAc3), O-methylated (OMe), and phosphorylcholine (PC)-substituted glycans (Cipollo et al., 2004, 2005; Hanneman et al., 2006; Haslam and Dell, 2003). The most abundant class of glycans in C. elegans is the high-mannose type. This is followed by paucimannose and high-fucose type structures.

3. RNAi in C. elegans The genome sequence of C. elegans was first published in 1998 and contains approximately 20,000 genes (C. elegans Sequencing Consortium, 1998). One of the most useful features of C. elegans is its susceptibility to genetic silencing, most notably, RNA-mediated interference (RNAi). Doublestranded RNA that is complementary to a gene of interest can be easily directed through feeding, soaking, or injection and results in decreased gene translation through degradation of mRNA (Fire et al., 1998). The use of systematic reverse genetics has increased the capacity to investigate functional genomics and biological functions of glycosylation in C. elegans. Several genome-wide RNAi screens have been performed to study gene function in C. elegans. These RNAi are either derived from predicted genes in C. elegans, using information from the sequenced genome, or are made from cDNA, which represent genes that are expressed in laboratory conditions (Kamath et al., 2003; Rual et al., 2004). Genome-wide RNAi screens that used the NL2099 C. elegans strain, which is hypersensitive to RNAi, showed a 23% increase in the number of genes eliciting a phenotype compared to N2 Bristol (Simmer et al., 2003). This report highlighted the variability between genome-wide RNAi-by-feeding screens and calculated the figure to be 10–30%. It is important to note that neuronally expressed genes are relatively refractory to RNAi and may not be identified as tunicamycin-hypersensitive in our screen. Most RNAi experiments aim to understand the function of individual genes and to provide insight in the homologous human gene function. In our experiment, we targeted the synthetic interaction of each gene products’ loss-of-function with decreased LLO biosynthesis via tunicamycin treatment, thus providing a list of candidate genes that require N-glycosylation to function properly.

4. C. elegans Strains, Culturing and RNAi Methods C. elegans strains NL2099 rrf-3 (pk1426), VC569 tag-179 (ok809), RE666 ire-1 (v33), and N2 (Bristol) were obtained from the Caenorhabditis Genetics Center, University of Minnesota, USA. The VC569 strain, which originally contained a genetic balancer, was outcrossed with N2 (Bristol) to

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generate a viable strain that was ok809 homozygous. The balancer was removed in the fourth generation and was confirmed by polymerase chain reaction (PCR). General methods used for cultivating, handling, and genetic manipulation of C. elegans are as described (Brenner, 1974). All strains were grown on nematode growth media (NGM) containing 3 g NaCl, 3.5 g peptone, and 18 g agar in 1 l H2O. NGM were autoclaved at 121
C for 28 min, cooled to 65
C, and supplemented with 1 ml of 5 mg/ml cholesterol (in ethanol), 2 ml of 0.5 M CaCl2, 25 ml 1 M KPO4 at pH 6, and 1 ml MgSO4 (Brenner, 1974; Hope, 1999). Where RNAi was performed, NGM were supplemented with 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) for induction of double-stranded RNA and 50 mg/ml ampicillin (500 ml from 100 mg/ml stock) for RNAi plasmid selection. Following the additions, NGM were aseptically poured into 60 and 100 mm Petri plates or 24-well cell culture plates (Nunc). For tunicamycin treatment, NGM were supplemented with concentrations ranging 0–10 mg/ml. Tunicamycin (Calbiochem) was dissolved in dimethyl sulfoxide (DMSO) to 50 mg/ml stock concentration. C. elegans strains were fed OP50 Escherichia coli or a specific E. coli strain HT115(DE3) RNAi-by-feeding clone containing a L4440 vector with the RNAi target gene flanked by two T7 promoter regions. The T7 polymerase was induced by IPTG in the NGM, resulting in the expression of double-stranded RNA (dsRNA). RNAi screening protocols were performed by feeding in the NL2099 rrf-3 (pk1426) strain, with minor adaptations for tunicamycin treatment from previous works (Simmer et al., 2003). The ORFeome v1.1 RNAi library was supplied by the Vidal lab at the Dana-Farber Cancer Institute (Rual et al., 2004). The ORFeome v1.1 library of recombinant E. coli strains, consisting of 11,942 constructs (55% of the C. elegans genome), was arrayed in 96-well microtiter plates. The ORFeome library was stored at 80
C. Bacteria were cultured in 2  YT containing 50 mg/ml ampicillin and 12.5 mg/ml tetracycline for 72 h at 22
C to ensure the presence of the L4440 plasmid (ampicillin selection) and the DE3 lysogen carrying the IPTG inducible T7 RNA polymerase (tetracycline selection). From these cultures and using a 96-plate sterile stainless steel replicator, 100 ml 2  YT supplemented with 50 mg/ml ampicillin was inoculated and grown overnight at 37
C. Fifteen microliters aliquots of the overnight culture was used to grow bacterial lawns on NGM in each well of a 24-well cluster plate supplemented with 50 mg/ml ampicillin, 1 mM IPTG, and 2 mg/ml tunicamycin. These cultures were grown at 22
C for 48 h to create a bacterial food source expressing double-stranded RNA. 2  YT/Amp/Tet was prepared as follows: 16 g tryptone, 10 g yeast extract, 5 g NaCl in 1 l distilled H2O, autoclaved at 121
C for 15 min. Once cooled to 65
C, 2  YT was supplemented with 50 ml/ml ampicillin (500 ml from 100 mg/ ml stock) and 12.5 ml/ml tetracycline (500 ml from 25 mg/ml stock). Observations were made using a Leica MS 5 stereomicroscope at 10 to

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40 magnification (Leica Microsystems). All recognizable C. elegans phenotypes were scored using a controlled vocabulary, which are accessible via Wormbase, an online bioinformatic database resource for C. elegans genetics and biology (http://www.wormbase.org).

4.1. Effect of tunicamycin on C. elegans phenotypes The first step in the genome-wide RNAi screen was to characterize the developmental consequences of increased tunicamycin treatment (i.e., CDG-I-like defects) in C. elegans. To determine the effect of tunicamycin on C. elegans phenotypes, 15 ml of synchronized L1 stage N2 Bristol larvae following egg preparation were added to 60 mm NGM plates seeded with OP50 E. coli and supplemented with 0, 3, and 5 mg/ml tunicamycin. Animals were cultivated at 20
C and phenotypes were scored after 3 days. To obtain synchronous populations of L1 hatchlings, eggs were acquired by digesting populations containing gravid hermaphrodites with an alkaline hypochlorite mixture. Egg preparation method was as follows: NGM plates containing gravid hermaphrodites were washed with 5 ml 1  M9 and pelleted. A mixture of 250 ml NaOH (10 M) and 1 ml alkaline hypochlorite was added to the pellet and vortexed every 2 min for 10 min. The lysate was pelleted, supernatant was removed, and the pellet was resuspended in 5 ml 1  M9. The 1  M9 wash was repeated three additional times until the NaOH/hypochlorite solution was sufficiently removed. C. elegans eggs were allowed to hatch overnight in 1  M9 buffer at 20
C. 1  M9 buffer was prepared as follows: 3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml MgSO4 (1 M) were autoclaved in 1 l H2O.

4.2. Tunicamycin-lethality is dose dependent To determine the lethal dose of tunicamycin during C. elegans larval development, synchronized L1 populations of N2 Bristol (n ¼ 144) following egg prep were placed in each well of a 24-well cell culture plate containing 2 ml NGM supplemented with tunicamycin (0–10 mg/ml) and seeded with OP50 E. coli. On days 3 and 6, plates were scored for death (no pumping, twitching, or movement when prodded with platinum wire) of the founder hermaphrodite or the appearance of hatched The number of animals that reached fertile adulthood at 20
C was scored. The percent of fertile live adults was plotted against the concentration of tunicamycin (Fig. 22.1).

4.3. Tunicamycin treatment varies among strains Similarly to the tunicamycin-lethality assay of N2 Bristol, separate populations of NL2099 rrf-3 (pk1426), RE666 ire-1 (v33), VC569 tag-179 (ok809), and N2 (Bristol) were tested in increasing concentrations of tunicamycin.

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Fraction of fertile adults

1.00

Day 3 Day 6

0.75

0.50

0.25

0.00 0

1

2

3 4 5 6 7 Tuicamycin (mg/ml)

8

9

10

Figure 22.1 Tunicamycin concentrations greater than 2 mg/ml induce a dose-dependent lethality during larval development in N2 Bristol strains. Each line represents the same observable population, but at days 3 and 6, respectively. Concentrations of eight or greater result in nearly complete lethality. Results are based on triplicate experiments (n ¼ 264) for each tunicamycin treatment. (Modified, with permission, from Struwe et al., 2009).

Each strain was synchronized by egg prep method as described above. Five microliters of hatchings (150 L1 larvae) in 1  M9 buffer was placed on 35 mm petri dishes containing 4 ml NGM with 0–10 mg/ml tunicamycin seeded with E. coli OP50. Animal survival was scored based on the ability of larvae to reach gravid hermaphrodites after 3 days at 20
C. The percent of gravid hermaphrodites (i.e., fertile adults) was plotted against the log scale concentration of tunicamycin (Fig. 22.2).

4.4. Establishment of tunicamycin-hypersensitive genes Table 22.1 and Figs. 22.1 and 22.2 illustrate that increased tunicamycin concentrations in NGM induce multiple phenotypes and lethality in C. elegans, as observed in CDG conditions. The key to the genome-wide screen is the ability to detect tunicamycin-hypersensitive genes, which is conditional on inducing a subphenotypic dose prior to RNAi knockdown. Otherwise, all detectable phenotypes and corresponding genes would be false positives from tunicamycin alone. Loci were scored positive if RNAi treatment in the presence of tunicamycin reproducibly caused a synthetic phenotype or unambiguously enhanced the phenotype observed in the absence of drug. From the aforementioned experiments, 2 mg/ml tunicamycin was chosen to screen a panel of 12 genes that were suspected to be tunicamycin-hypersensitive to validate the conceptual framework of the genome-wide screen. These included genes in LLO synthesis, ER-associated degradative pathway (ERAD), the unfolded protein response (UPR),

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120

% fertile adults

100 80 60 40 20 0 –2.0

–1.5

–1.0 – 0.5 0.0 0.5 (Tunicamycin) log (mg/ml)

1.0

NL2099 rrf-3(pk1426)II

VC569 tag-179(ok809)I

RE666 ire-1(v33)II

N2 (Bristol)

Figure 22.2 Tunicamycin affects postembryonic development among C. elegans strains. The percent of animals that reach fertile adulthood was measured as a function of tunicamycin concentration. Development of RE666 ire-1 (v33), which lacks a component of the unfolded protein response, is moderately affected when tunicamycin is present. Lethality is significantly increased in VC569 tag-179 (ok809), which encodes the enzyme responsible for the final step of LLO biosynthesis. Conversely, N2 (Bristol) and NL2099 rrf-3 (pk1426) strains are less affected by the presence of tunicamycin and are viable at 2 mg/ml. (Reproduced, with permission, from Struwe et al., 2009). Table 22.1 Tunicamycin treatment results in an increase in observable aberrant phenotypes [Tunicamycin] (mg/ml) Stage

Phenotype

0

3

5

Adult

W.T. Dpy Unc Sma Vab Egl Clr Dpy Unc Dpy Egl Gro Dpy Gro Let

99.81% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.05% 0.14% 0.00% 0.00% 2119

75.63% 3.39% 2.35% 2.04% 0.63% 0.57% 0.05% 0.05% 0.05% 1.51% 1.10% 9.71% 1916

1.01% 1.72% 0.20% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 14.34% 7.95% 74.77% 1974

Larval

n

Tunicamycin concentration at 3 mg/ml results in a wide variety of phenotypes among N2 adults and larvae. Concentrations at 5 mg/ml result predominantly in lethality. Clr, clear patches; Dpy, dumpy; Egl, egg-laying defective; Gro, slow growth; Let, lethality; Sma, small; Unc, uncoordinated locomotion; Vab, variably abnormal morphology; W.T., wild type. (Reproduced, with permission, from Struwe et al., 2009).

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Table 22.2 RNAi of genes involved in N-glycosylation with and without 2 mg/ml tunicamycin Function

Gene

Wormbase

 Tunicamycin

þ Tunicamycin

ERAD UPR UPR GVT GVT GVT LLO LLO LLO LLO LLO LLO

K08E3.7 C41C4.4 F46C3.1 W02D7.7 Y60A3A.9 F47G9.1 K09E4.2 C14A4.3 ZC513.5 C08B11.8 C08H9.3 T24D1.4 Vector

W.T. W.T. W.T. Gro, Unc, Pvl not tested not tested W.T. W.T. not tested W.T. W.T. W.T.

W.T. W.T. W.T. W.T. W.T. W.T. Gro W.T. W.T. W.T. W.T. W.T. W.T.

Ste, Emb, Gro Emb, Lva Gro Ste, Gro Let, Gro W.T. Emb Ste, Emb Emb, Lvl, Lva Gro Emb, Lvl, Lva Brd, Gro W.T.

RNAi of loci in the lipid-linked oligosaccharide (LLO) pathway, ER-associated degradative pathway (ERAD), unfolded protein response (UPR), or components of Golgi vesicle transport (GVT) presents no overt phenotype on NGM alone. In the presence of 2 mg/ml tunicamycin, RNAi knockdown of these genes generates multiple phenotypes. Corresponding RNAi phenotype experiments from Wormbase (http://www.wormbase.org) were are also shown for comparison. (Modified, with permission, from Struwe et al., 2009.)

and Golgi vesicular transport (GVT) (Table 22.2). Large scale RNAi was carried out on 24-well plates, where four wells containing NGM, 1 mM IPTG, and 50 mg/ml ampicillin with and without 2 mg/ml tunicamycin were inoculated with a specific RNAi expressing bacteria. Individual 24well plates contained six RNAi clones, each having four individual wells. In this manner, each gene tested provides four observable wells. Initially gravid hermaphrodite populations were synchronized via egg prep and allowed to hatch overnight at 20
C after transfer to NGM. Following egg prep, 10 NL2099 rrf-3 (pk1426) L3 animals were placed in the top row of each plate by hand using a ‘‘worm pick’’ of flattened platinum wire to generate progeny that have only existed in the presence of each RNAi construct with and without 2 mg/ml tunicamycin. After 48 h of ‘‘priming’’ at 15
C, a single gravid hermaphrodite from the top row was transferred to each of the other lawns and allowed to elaborate F1 progeny at 22
C for 72 h. Phenotypes in the four wells, including Po and F1 progeny, were scored. RNAi bacterium culture was prepared as follows: ORFeome v1.1 RNAi bacterial library was provided in 96-well microtiter plates, each well containing a specific bacterial clone. Each bacteria well was inoculated into 60 ml 2  YT/Amp/Tet in 96-well plate format using a sterile steel replicator. The inoculation plate was stored at 37
C for 18 h. Fifteen microliters aliquots of the overnight culture was used to grow bacterial lawns on

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24-well clusters. RNAi bacterial cultures were grown on NGM at room temperature for 48 h to create a bacterial food source expressing doublestranded RNA. This 24-well plate RNAi þ/ tunicamycin protocol was identical for the genome-wide screen. Our results were also compared to previously described RNAi phenotypes with an rrf-3(pk1426) mutant background of the same genes annotated in Wormbase (Kamath et al., 2003; Rual et al., 2004). The majority of RNAi clones tested correlated with previous works except in the case of sequence name C08H9.3, W02D7.7, and K09E4.2. Most important, this experiment established that tunicamycin-hypersensitive genes can be detected via RNAi in the presence of 2 mg/ml tunicamycin.

4.5. Tunicamycin does not alter RNAi effectiveness Our genome-wide screen was modeled similar to published RNAi screens in C. elegans (Simmer et al., 2003) with the exception that our NGM contained tunicamycin. Therefore the possibility that 2 mg/ml tunicamycin causes a generalized increase in the effectiveness of the RNAi-by-feeding method could not be excluded. If this was the case, then our approach would result in numerous false positives. To exclude this possibility, seven loci known to be refractory to RNAi (Asikainen et al., 2005) and that have observable mutant phenotypes were tested on 2 mg/ml tunicamycin correspondingly to the 24-well plate format described above. Briefly, bacterial constructs with RNAi targets of the seven genes were inoculated in 60 ml 2  YT with 50 mg/ml ampicillin and 12.5 mg/ml tetracycline for 72 h at 22
C. From these cultures, 100 ml 2  YT with 50 mg/ml ampicillin were inoculated and incubated for 18 h at 37
C. To each well of the 24-well plate, 20 ml of the culture was added to the surface of NGM containing 1 mM IPTG and 50 mg/ml ampicillin. Bacterial lawns were allowed to form, which expressed dsRNA specific to the seven genes tested. The NL2099 rrf-3 strain was used for the experiment as illustrated above. These results proved the legitimacy of the screen showing that tunicamycin does not elicit any phenotype from otherwise wild-type RNAi gene targets as previously reported by escalating the effect of dsRNA in C. elegans (Table 22.3).

5. Genome-wide RNAi Screen The first phase of the genome-wide RNAi screen was testing every gene from the ORFeome v1.1 library on tunicamycin with all phenotypes detected in any of the four wells being scored and cataloged. The type of phenotype was recorded and characterized as viable postembryonic phenotype (Vpep) or a nonviable phenotype (Nonv) as described (Simmer et al., 2003). The phenotypes of adults and progeny of the 10 founder NL2099 rrf-3

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RNAi Screen to Identify N-Glycosylation Dependent Loci

Table 22.3 Tunicamycin treatment does not alter RNAi effectiveness in C. elegans Gene

Locus

Mutant

Wormbase (RNAi)

þ Tunicamycin

F11C3.2 W03A3.1 C52B9.9 C17D12.2 C52E12.3 D2096.4 ZC64.3

unc-122 ceh-10 mec-18 unc-75 spv-7 spv-1 ceh-18

UNC UNC UNC UNC LET LET LET

W.T. W.T. W.T. W.T. W.T. W.T. W.T.

W.T. W.T. W.T. W.T. W.T. W.T. W.T.

Seven C. elegans strains that exhibit both a mutant knockout phenotype and wild-type RNAi phenotype were chosen to compare RNAi in the presence of 2 mg/ml tunicamycin. No phenotype was detected when tunicamycin was present, demonstrating that tunicamycin does not modify the efficiency of RNAi. (Modified, with permission, from Struwe et al., 2009.)

animals (Po) in the ‘‘priming’’ well were scored in addition to the progeny and single founder hermaphrodite in wells 1–3. The genome-wide screen required strict criteria for candidate selection for the second phase, RNAi with and without 2 mg/ml tunicamycin, to limit the accumulation of false positives. This required determining how many wells that displayed phenotypes were required to be retested in the presence and absence of tunicamycin. To establish this threshold, a set of 100 genes that gave rise to phenotypes in 0–4 observatory wells out of four were tested in the presence and absence of 2 mg/ ml tunicamycin (n ¼ 500 total genes tested). As before, any animal in each well that showed a phenotype was scored as positive. In addition to the presence of a phenotype on tunicamycin (penetrance), the significance of the phenotype (either Vpep or Nonv) was assessed. The penetrance was assessed by subtracting the number of wells showing any phenotype(s) with tunicamycin from the number in its absence (D). The greater the D value, the more dependent the phenotype penetrance is on tunicamycin; that is, a locus that induced phenotypes in all four wells containing tunicamycin but none in its absence would have a D equal to 4. The effect of expressivity was assessed similarly except D was determined using the number of wells showing Nonv. For expressivity a larger D value corresponded to a more severe phenotype when tunicamycin is present; that is, a locus that induced a ‘‘dumpy’’ phenotype in all four wells in the absence of tunicamycin but ‘‘larval arrest’’ in all wells with 2 mg/ml would have D ¼ 4. Under this analysis, false positives and loci causing single-gene phenotypes score D ¼ 0, but loci that are tunicamycin-hypersensitive score D > 0. This analysis showed that when phenotypes were present in two or more wells out of four, the distribution was nonrandom. In phase II, any gene with two positive wells was retested with and without 2 mg/ml tunicamycin. The D values for penetrance and expressivity were plotted against the percent total in each of the five sets (100 genes per set) tested (Fig. 22.3).

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Expressivity

Number of phase I positive wells 4 3 2 1 0

80

% of class

60 40 20 0 –4

–3

–2

–1

0 Δ

Penetrance

2

3

4

Number of phase I positive wells 4 3 2 1 0

60 % of class

1

40 20 0 –4

–3

–2

–1

0 Δ

1

2

3

4

Figure 22.3 D values for expressivity and penetrance were calculated by subtracting the number of wells showing any phenotype with tunicamycin from the number in its absence. Larger D values correspond to a greater dependence of the phenotype penetrance/expressivity on tunicamycin. Phenotypes in  2 of 4 possible observable wells behave nonrandomly and were candidates for further testing in phase II ( 2 mg/ml tunicamycin). D values greater than two exhibit non-Gaussian distribution and indicate tunicamycin-hypersensitive loci. Sets of 100 genes that caused observable phenotypes in 0,1,2,3, and 4 observatory wells in the initial stages of the phase I screen were used for these assays (n ¼ 500 genes tested). (Modified, with permission, from Struwe et al., 2009).

For the genome-wide screen the preparation of media and bacterial cultures were followed as stated previously. All RNAi experiments were performed on the 24-well plate format supplemented with and without 2 mg/ml tunicamycin where indicated. There were two phases of the genome-wide screen: (1) screening the ORFeome v1.1 RNAi library in the presence of 2 mg/ml tunicamycin and (2) retesting candidates from phase I with and without 2 mg/ml tunicamycin to isolate tunicamycin-hypersensitive genes. Originally the two-phase scheme was chosen for cost considerations; however, the number of phase I candidates was higher than anticipated. Encouragingly, this resulted in an additional RNAi treatment

RNAi Screen to Identify N-Glycosylation Dependent Loci

489

results (screening on 2 mg/ml tunicamycin) which ultimately reinforced the validity of the 512 tunicamycin-hypersensitive genes discovered. During phase I, genes where at least two of the three replicates displayed concordant phenotypes were regarded as candidates. Any phenotypes observed in the ‘‘priming’’ well were also scored and data collected were weighed with lesser significance when selecting phase II candidates. The presence of any phenotypes in the three remaining wells, including Po and F1 progeny, were given a value of 10 (10 for any and all phenotypes and 0 for no phenotypes). Phenotypes in the ‘‘priming’’ wells were given a score of 5. In this manner, the evaluation of expressivity and penetrance of each gene tested can easily be calculated and interpreted. For example, the gene H23N18.4, which encodes a UDP-glucuronosyl and UDP-glucosyl transferase, had an expressivity score of 10 and a penetrance score of 30. In the absence of tunicamycin, all phenotypes were wild type (Vpep ¼ 0 and Nonv ¼ 0), but on 2 mg/ml tunicamycin, the phenotypes observed were slow growing (gro) in two wells and sterile (ste) in the third, thus the score for that gene was Vpep ¼ 30 and Nonv ¼ 10. Therefore the penetrance is 30 (30-0) and expressivity is 10 (10-0). These calculations helped to characterize the extent to which each gene contributed to the presence or severity of phenotypes observed during the phase II screen. Ultimately, 512 genes were deemed tunicamycin-hypersensitive and were annotated using the criteria above (Struwe et al., 2009). All recorded phenotypes were recorded initially in Microsoft Excel, and data were analyzed using SQL queries in Microsoft Access. Concise descriptions for each tunicamycin-hypersensitive gene product were added manually using Wormbase. Moreover, the tools Net-Nglyc (http://www.cbs.dtu.dk/services/NetNGlyc) and Proteome Analyst (http://path-a.cs.ualberta.ca) were helpful to predict N-glycosylation sites and the cellular location of each gene product, respectively. The most useful tool to annotate tunicamycin-hypersensitive gene function was the implementation of clusters of orthologous groups (COGs) functional classification, specifically sequenced genomes from eukaryotic orthologous groups (KOGs). KOG analysis was more effective than gene ontology descriptions, which proved to be either too broad or too specific when annotating the 512 tunicamycin-hypersensitive genes (Fig. 22.4).

6. Discussion There is an essential and extensive requirement for N-glycosylation in animal development. To understand the variable pathology of a CDG-Ilike condition, we developed an approach to systematically dissect the interactions between a primary etiologic defect (tunicamycin treatment) and modifier loci in the genetic background (tunicamycin-hypersensitive

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Translation, ribosomal structure and biogenesis Transcription Signal transduction mechanisms Secondary metabolites biosynthesis, transport and catabolism Replication, recombination and repair RNA processing and modification Posttranslational modification, protein turnover, chaperones Nucleotide transport and metabolism Nuclear structure Lipid transport and metabolism Intracellular trafficking, secretion, and vesicular transport Inorganic ion transport and metabolism General function predictions only Function unknown Extracellular structures Energy production and conversion Defense mechanisms Cytoskeleton Coenzyme transport and metabolism Chromatin structure and metabolism Call wall/membrane/envelope biogenesis Cell cycle control, cell division, chromosome partitioning Carbohydrate transport and metabolism Amino acid transport and metabolism

49 18 33 1 9 18 27 3 1 8 20 4 37 17 7 16 5 7 3 6 3 10 4 2 0

10

20

30

40

50

60

Figure 22.4 Analysis of eukaryotic orthologous groups (KOGs) classify tunicamycinhypersensitive genes into functional sets. Of 512 genes found to be hypersensitive to tunicamycin, 308 had KOG assignments. (Reproduced, with permission, from Struwe et al., 2009).

genes) using RNAi. Our goal was to model the causes and consequences of CDG-I-like disorder in C. elegans, to perform a genetic analysis of N-glycosylation and the developmental mechanisms that depend on it. This genome-wide RNAi screen with ORFeome v1.1 library detected 512 tunicamycin-hypersensitive loci from 11,942 bacterial constructs. All RNAi screens have intrinsic limitations and for this reason we regard our approach as systemic rather than comprehensive. A major problem is that not all genes, particularly those expressed in neurons, are susceptible underscoring the need for complementary approaches such as the classical forward mutagenesis screen. Because high-throughput is necessary, only visible phenotypes were scored and it is virtually certain that several subtle traits are modulated by glycosylation that will be missed. However for our purpose, it is fortunate that RNAi generally reduces rather than eliminates expression; glycosylation cannot modulate the underlying polypeptide if it is absent, as would be the case in knockout models. Considering the C. elegans genome is roughly 20,000 genes, consideration should be given to additional RNAi screening (i.e., the remaining 8000 genes not tested in our initial screen). However, the advantage of the ORFeome RNAi library is that the RNAi constructs are generated from full coding region cDNAs; therefore the use of alternate RNAi

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RNAi Screen to Identify N-Glycosylation Dependent Loci

libraries that are derived from genomic regions encompassing predicted genes may not prove more advantageous in detecting additional tunicamycin-hypersensitive genes. For that reason, future experiments should focus on screening any additional RNAi constructs from later versions of the ORFeome library. Moreover, additional work would aim to classify the 512 tunicamycin-hypersensitive genes according to the ‘‘maturation,’’ ‘‘etiologic,’’ and ‘‘effector’’ classes outlines in Fig. 22.5. Another consideration with the genome-wide RNAi screen is the use of tunicamycin. Tunicamycin treatment cannot be disregarded when considering the 512 tunicamycin-hypersensitive genes. The rational is that RNAi of a gene responsible for tunicamycin metabolism or uptake may alter the effect of 2 mg/ ml and result in nontunicamycin-hypersensitive gene identifications. Therefore, the 512 tunicamycin-hypersensitive genes should be retested with a knockout in the LLO pathway (preferably Y60A3A.14/T08D2.2: the UDPN-acetyl-glucosamine-1-P transferase) to effectively replace drug treatment. Here we outline a proof-positive approach that models a CDG-I-like disease and identifies loci that require N-glycosylation to function properly in C. elegans. This method could be applied to other organisms or tailored to model other diseases where modifier loci are difficult to resolve. This experiment is a powerful platform for investigating the molecular pathology of aberrant N-glycosylation. Our data demonstrates the first wide-ranging screen that successfully identified genes that require N-glycosylation to function and in doing so provides a list of candidate genes that may be screened to find enhancer loci in CDG-I patients and targeted for supportive treatment to minimize the severity of the illness.

Maturation genotypes

Effector genotypes

Trafficking

Underlying polypeptide genes Tunicamycin Dolichol

Lipid-linked oligosaccharide assembly

Monosaccharides

Oligosaccharyl -transferase

Nascent glycoprotein

Oligomer ER quality assembly control

Golgi Localization function Glycan remodeling

Mature glycoproteins

Phenotypes

Etiologic genotypes Unfolded protein response

Figure 22.5 Plausible interactions between tunicamycin and modifier loci in the genetic background in C. elegans. RNAi knockdown of ‘‘effector,’’ ‘‘etiologic,’’ or ‘‘maturation’’ genes interact with a decrease of LLO donor caused by 2 mg/ml tunicamycin treatment result in visible phenotypes. (Modified, with permission, from Struwe et al., 2009).

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REFERENCES Aebi, M., and Hennet, T. (2001). Congenital disorders of glycosylation: Genetic model systems lead the way. Trends Cell Biol. 11(3), 136–141. Altmann, F., Fabini, G., Ahorn, H., and Wilson, I. B. (2001). Genetic model organisms in the study of N-glycans. Biochimie 83(8), 703–712. Asikainen, S., Vartiainen, S., Lakso, M., Nass, R., and Wong, G. (2005). Selective sensitivity of Caenorhabditis elegans neurons to RNA interference. Neuroreport 16(18), 1995–1999. Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77(1), 71–94. C. elegans Sequencing Consortium (1998). Genome sequence of the nematode C. elegans: A platform for investigating biology. Science 282(5396), 2012–2018. Chen, S., Zhou, S., Sarkar, M., Spence, A. M., and Schachter, H. (1999). Expression of three Caenorhabditis elegans N-acetylglucosaminyltransferase I genes during development. J. Biol. Chem. 274(1), 288–297. Chen, S., Tan, J., Reinhold, V. N., Spence, A. M., and Schachter, H. (2002). UDP-Nacetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I and UDP-N-acetylglucosamine:alpha-6-D-mannoside beta-1,2-N-acetylglucosaminyltransferase II in Caenorhabditis elegans. Biochim. Biophys. Acta 1573(3), 271–279. Cipollo, J. F., Costello, C. E., and Hirschberg, C. B. (2002). The fine structure of Caenorhabditis elegans N-glycans. J. Biol. Chem. 277(51), 49143–49157. Cipollo, J. F., Awad, A., Costello, C. E., Robbins, P. W., and Hirschberg, C. B. (2004). Biosynthesis in vitro of Caenorhabditis elegans phosphorylcholine oligosaccharides. Proc. Natl. Acad. Sci. USA 101(10), 3404–3408. Cipollo, J. F., Awad, A. M., Costello, C. E., and Hirschberg, C. B. (2005). N-Glycans of Caenorhabditis elegans are specific to developmental stages. J. Biol. Chem. 280(28), 26063–26072. Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., and Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391(6669), 806–811. Hanneman, A. J., Rosa, J. C., Ashline, D., and Reinhold, V. N. (2006). Isomer and glycomer complexities of core GlcNAcs in Caenorhabditis elegans. Glycobiology 16(9), 874–890. Haslam, S. M., and Dell, A. (2003). Hallmarks of Caenorhabditis elegans N-glycosylation: Complexity and controversy. Biochimie 85(1–2), 25–32. Hope, I. A. (1999). C. elegans: A practical approach. Oxford University Press, Oxford, New York. Huffaker, T. C., and Robbins, P. W. (1982). Temperature-sensitive yeast mutants deficient in asparagine-linked glycosylation. J. Biol. Chem. 257(6), 3203–3210. Kamath, R. S., Fraser, A. G., Dong, Y., Poulin, G., Durbin, R., Gotta, M., Kanapin, A., Le Bot, N., Moreno, S., Sohrmann, M., Welchman, D. P., Zipperlen, P., et al. (2003). Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421(6920), 231–237. Lambie, E. J. (2002). Cell proliferation and growth in C. elegans. Bioessays 24(1), 38–53. Lehle, L., Strahl, S., and Tanner, W. (2006). Protein glycosylation, conserved from yeast to man: A model organism helps elucidate congenital human diseases. Angew. Chem. Int. Ed. Engl. 45(41), 6802–6818. Natsuka, S., Adachi, J., Kawaguchi, M., Nakakita, S., Hase, S., Ichikawa, A., and Ikura, K. (2002). Structural analysis of N-linked glycans in Caenorhabditis elegans. J. Biochem. 131(6), 807–813. Paschinger, K., Gutternigg, M., Rendic, D., and Wilson, I. B. (2008). The N-glycosylation pattern of Caenorhabditis elegans. Carbohydr. Res. 343(12), 2041–2049.

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Prietsch, V., Peters, V., Hackler, R., Jakobi, R., Assmann, B., Fang, J., Korner, C., HelwigRolig, A., Schaefer, J. R., and Hoffmann, G. F. (2002). A new case of CDG-x with stereotyped dystonic hand movements and optic atrophy. J. Inherit. Metab. Dis. 25(2), 126–130. Rual, J. F., Ceron, J., Koreth, J., Hao, T., Nicot, A. S., Hirozane-Kishikawa, T., Vandenhaute, J., Orkin, S. H., Hill, D. E., van den Heuvel, S., and Vidal, M. (2004). Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library. Genome Res. 14(10B), 2162–2168. Schachter, H. (2004). Protein glycosylation lessons from Caenorhabditis elegans. Curr. Opin. Struct. Biol. 14(5), 607–616. Schollen, E., Kjaergaard, S., Martinsson, T., Vuillaumier-Barrot, S., Dunoe, M., Keldermans, L., Seta, N., and Matthijs, G. (2004). Increased recurrence risk in congenital disorders of glycosylation type Ia (CDG-Ia) due to a transmission ratio distortion. J. Med. Genet. 41(11), 877–880. Simmer, F., Moorman, C., van der Linden, A. M., Kuijk, E., van den Berghe, P. V., Kamath, R. S., Fraser, A. G., Ahringer, J., and Plasterk, R. H. (2003). Genome-wide RNAi of C. elegans using the hypersensitive rrf-3 strain reveals novel gene functions. PLoS Biol. 1(1), E12. Struwe, W. B., Hughes, B. L., Osborn, D. W., Boudreau, E. D., Shaw, K. M., and Warren, C. E. (2009). Modeling a congenital disorder of glycosylation type I in C. elegans: a genome-wide RNAi screen for N-glycosylation-dependent loci. Glycobiology 19 (12), 1554–1562. Warren, C. E., Krizus, A., Roy, P. J., Culotti, J. G., and Dennis, J. W. (2002). The Caenorhabditis elegans gene, gly-2, can rescue the N-acetylglucosaminyltransferase V mutation of Lec4 cells. J. Biol. Chem. 277(25), 22829–22838.

C H A P T E R

T W E N T Y- T H R E E

The Acidic Environment of the Golgi Is Critical for Glycosylation and Transport Yusuke Maeda*,† and Taroh Kinoshita*,‡ Contents 496 499

1. Introduction 2. Reporter Proteins for Transport Monitoring 3. Establishment of Parent Cells for Screening Transport Mutant Cells 4. Transport Assay of Reporter Proteins 5. Application of Transport Assay 6. Mutagenesis 7. Selection of Mutant Cells 8. Measurement of Golgi pH 9. Analysis of Glycosylation Using Lectin Staining 10. Concluding Remarks Acknowledgment References

500 501 501 504 504 504 506 506 507 507

Abstract Proteins and glycolipids are modified by various modes of glycosylation in the endoplasmic reticulum (ER) and the Golgi apparatus. It is well known that the lumen of the Golgi is acidic and compromising acidification by chemical compounds causes impaired glycosylation and transport of proteins (Axelsson et al., 2001; Chapman and Munro, 1994; Palokangas et al., 1994; Presley et al., 1997; Puri et al., 2002; Reaves and Banting, 1994; Rivinoja et al., 2006; Tartakoff et al., 1978). The mechanisms by which glycosylation and transport are regulated by an acidic pH remain largely unknown. Recent findings that the impaired regulation of an acidic environment may be implicated in the pathology of several diseases emphasize the importance of pH regulation ( Jentsch, 2007; Kasper et al., 2005; Kornak et al., 2001; Kornak et al., 2008; * Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan PRESTO, Japan Science and Technology Agency, Saitama, Japan WPI Immunology Frontier Research Center, Osaka University, Suita, Osaka, Japan

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Methods in Enzymology, Volume 480 ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)80022-9

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2010 Elsevier Inc. All rights reserved.

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Piwon et al., 2000; Stobrawa et al., 2001; Teichgraber et al., 2008). We recently established a mutant cell line in which Golgi acidification was selectively impaired and the raised luminal Golgi pH caused impaired transport and glycosylation of proteins and altered Golgi morphology (Maeda et al., 2008). As alkalinizing compounds nonselectively affect all acidic organelles including lysosomes, endosomes, and the Golgi, the mutant cell is thought to be useful in analyzing how the acidic environment of the Golgi regulates glycosylation. In this chapter, we have introduced how we established mutant cells with impaired Golgi acidification and methods for measuring Golgi pH.

1. Introduction Glycosylation is structurally complex and completed by sequential reactions that are spatially and temporally regulated. Glycosylation occurs in the endoplasmic reticulum (ER) and Golgi apparatus, with the precursors of N-glycan synthesized in the ER, which are then further modified in the Golgi with sugars such as N-acetylglucosamine, galactose, and sialic acid. A typical Golgi apparatus in mammalian cells is composed of several stacked flattened sacks called cisternae. Glycosylated proteins are transported along a secretory pathway that transverses the Golgi cisternae. The stacks most proximal and distal to the ER are called cis- and trans-cisterna, respectively, and the middle stack is called the medial-cisterna. After proteins transverse the trans-cisterna, they enter another flattened sack, called the trans-Golgi network (TGN). Hereafter, we include the TGN as part of the term ‘‘Golgi’’ with respect to glycosylation, because some glycosyltransferases are located in both the trans-Golgi and TGN. Each glycosyltransferase has a specific spatial location among the cisternae and TGN in order to execute glycosylation reactions in the correct order. If one glycosyltransferase normally expressed in the distal cisterna is ectopically expressed in the proximal cisterna, an abnormal glycan structure is generated (Ferrara et al., 2006). A ribbon-like Golgi structure is built up by connecting mini Golgi units via tubular structures, and is dependent on GM130 and GRASP65 (Marra et al., 2007; Puthenveedu et al., 2006). It was reported that GM130- and GRASP65-dependent interconnections are necessary to achieve uniform distribution of enzymes in the Golgi ribbon, thereby ensuring the optimal glycosylation (Puthenveedu et al., 2006). According to the cisterna maturation model, the cisternae themselves move in an anterograde direction together with cargo proteins, and the vesicles containing the Golgi glycosyltransferases bud from the cisternae and move back in a retrograde direction toward the ER to keep the localizations of the glycosyltransferases in the correct cisterna. This retrograde transport of glycosyltransferases requires conserved oligomeric Golgi (COG) proteins, and if they are

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defective, glycosylations are impaired most likely due to mislocalization of enzymes (Oka et al., 2005; Ungar et al., 2002). The diseases caused by defects in COG proteins are classified as congenital disorders of glycosylation (CDG) type II (Foulquier et al., 2006, 2007; Wu et al., 2004; Zeevaert et al., 2008). The mechanisms by which the localizations are determined remain largely unknown, although the protein portions critical for Golgi localization have been studied in some glycosyltransferases (Colley, 1997; Grabenhorst and Conradt, 1999; Rabouille et al., 1995; Uliana et al., 2006). In addition to these protein portions, the proper Golgi environment and structure are believed to be important for normal glycosylation. Organelles in secretory and endocytosis pathways are known to have acidic environments in the lumen (Fig. 23.1), which are believed to be critical in many biological processes (Paroutis et al., 2004; Weisz, 2003). Compromising the acidification by compounds such as monensin, bafilomycin, and ammonium chloride, which have different modes of action upon the acidic pH, results in detrimental effects including impaired protein and lipid trafficking, disorganization of organelles, as well as abnormal glycosylation (Axelsson et al., 2001; Puri et al., 2002; Rivinoja et al., 2006). These acidic environments are considered to be regulated by three factors: vacuolar-type proton ATPases, proton leaks, and counterion channels (Paroutis et al., 2004; Weisz, 2003). Vacuolar-type proton ATPase is a proton pump and thought to be the sole machinery of proton delivery into the luminal side (Glickman et al., 1983; Moriyama and Nelson, 1989; Nishi and Forgac, 2002). The presence of a proton leak is predicted by electrophysiological studies but nothing is known about these at a molecular level. The proton leak is thought to be more active in the ER and less active in the TGN, which has been considered to serve to create a pH gradient along the secretory pathway (Grabe and Oster, 2001; Wu et al., 2001). Counterion channels dissipate the positive membrane potential generated by electrogenic vacuolartype proton ATPases, and allow it to transfer more protons into the Golgi lumen (Glickman et al., 1983). Some CLC (chloride channel) family channels are localized in particular organelles of the endocytosis pathway, and are thought to function as counterion channels and to serve to acidify the organelles (Hara-Chikuma et al., 2005a, b; Jentsch, 2007; Pusch et al., 2006). Defects in these functions cause various diseases such as Dent disease, osteoporosis, and symptoms in knockout mice ( Jentsch, 2007; Kasper et al., 2005; Kornak et al., 2001; Piwon et al., 2000; Stobrawa et al., 2001; Teichgraber et al., 2008), which are thought to be partly due to the impaired acidification. Thus, counterion channels play an important role in pH regulation, but no CLC family or putative counterion channel has been identified in the Golgi. It was recently reported that loss-of-function mutations in ATP6V0A2 encoding the a2 subunit of the vacuolar-type proton ATPase caused autosomal recessive cutis laxa type II or wrinkly skin syndrome, and also exhibited abnormal glycosylation (Kornak et al., 2008). While the

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Secretory vesicle Early endosome Recyling endosome

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Figure 23.1 Each organelle in the secretory and endocytotic pathways has an acidic environment, and the pH decreases along the protein flow. From electrophysiological studies, the acidic pH is considered to be regulated by a balance among vacuolar-type proton ATPases, proton leaks, and counterion channels. In the Golgi, impaired acidification causes delayed transport, immature glycosylation, and abnormal Golgi morphology.

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precise mechanism by which dysregulation of pH impairs glycosylation remains to be solved, two possibilities have been proposed. One is that the increased pH is no longer within the optimal pH range for enzymatic activity, and therefore glycosylation is hindered. The other is that an increased pH causes incorrect localization of glycosyltransferases, leading to a loss of ability to execute reactions in the correct order. Little is known as to how the raised pH alters the location of glycosyltransferases. We recently established a mutant cell line in which Golgi acidification was selectively impaired but normal pH was maintained in the lysosomes and endosomes (Maeda et al., 2008). The mutant cells were selected by a cell sorter based on the delayed transport of reporter proteins from the ER to the plasma membrane. We identified a gene, termed GPHR (Golgi pH Regulator) that was responsible for generation of the mutant cells. It turned out that GPHR functions as a counterion conductance through anion channel activity to acidify the Golgi and that the mutant cells demonstrated delayed transport of proteins from the ER to the Golgi or through the Golgi, impaired glycosylation, and abnormal Golgi morphology as the secondary mutant phenotypes (Maeda et al., 2008). We focus on how we established the mutant cells with impaired Golgi acidification and how we measured Golgi pH.

2. Reporter Proteins for Transport Monitoring Glycosylphosphatidylinositol (GPI)-anchored and transmembrane (TM)-type reporter proteins were designed in order to obtain synchronized transport from the ER and to monitor protein trafficking (Fig. 23.2). A GPIanchored reporter protein, VSVGts(ex)-FF-mEGFP-GPI, was composed of the extracellular domain of the temperature-sensitive vesicular stomatitis virus G protein (VSVGts(ex); amino acids 1–464 of VSVGtsO45) (Doms et al., 1987; Hurtley and Helenius, 1989), a linker (GDHPPKGGGGSGGGGSGGGGSVD), a furin cleavage sequence (SRHRSKR) (Nakayama, 1997), a FLAGtag (DYKDDDDK), a linker (LSAA), an enhanced green fluorescent protein (EGFP) bearing a mutation (L221K) to reduce homotypic binding, and the GPI-attachment signal from CD59 (ENGGTSLSEKTVLLLVTPFLAAAWSLHP). A TM-type reporter protein, FLAG-VSVGts(full)-EGFP, was composed of the signal peptide sequence derived from CD59 (amino acids 1–27; MGIQGGSVLFGLLLVLAVFCHSGHSLQ), a FLAG-tag, VSVGts(full) (amino acids 17–511 of VSVGtsO45) lacking the N-terminal 16 amino acid signal peptide sequence, and EGFP. Newly synthesized VSVGts at a nonpermissive temperature (40
C) was retained in the ER due to misfolding, but once the culture was shifted to a permissive temperature (32
C), the retained

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VSVG(ex)

FLAG

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EGFP

Figure 23.2 Schematic structures of GPI-anchored and TM-type reporter proteins are shown.

VSVGts quickly refolded correctly, left the ER and was transported, enabling us to obtain a system with synchronized transport from the ER. The FLAGtag and EGFP were used to monitor surface expression and total (surface and intracellular) amounts of reporters, respectively. The GPI-anchored reporter protein has a sequence recognized and cleaved by furin in the TGN, which allows for determination of the reporter protein in post-TGN compartments due to a change in size on Western blots, and also enhances the efficient transport of the reporter to the cell surface. Both reporter genes were subcloned into the appropriate expression vectors with a stable active promoter or with a tetracycline-inducible promoter.

3. Establishment of Parent Cells for Screening Transport Mutant Cells We routinely used Chinese Hamster Ovary (CHO) cells, as we observed excellent frequency of appearance of mutant cells due to pseudohemizygote-like gene expression characteristics, well-preserved genomic stability, and good transfection efficiency. Both reporter genes described above were cloned into the pTRE2pur vector (Clontech), in which gene expression was regulated under a tetracycline-inducible promoter so that

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the reporter proteins were induced and accumulated in the ER, as needed, without preexisting protein background. FF8 parent cells were established by stable transfection of pTRE2pur-VSVGts(ex)-FF-mEGFP-GPI with pUHrT 62-1 (a gift from Dr. W. Hillen, Erlangen University), which encodes a tetracycline-dependent transcriptional activator (Urlinger et al., 2000). Cells that had a low level of background expression of the reporter in the absence of doxycycline (Dox), a derivative of tetracycline, and that well responded to Dox and induced high amount of the reporter were sorted by a FACSVantage (BD) and cloned by limiting dilution.

4. Transport Assay of Reporter Proteins Two different protocols were used depending on whether the cells were transfected with the reporter gene stably or transiently. In the case of stably transfected cells, Dox (1 mg/ml) was added 1 day after plating, moved to an incubator adjusted at 40
C, and cultured for 1 day. Cells were quickly removed from the plate by routine trypsin/EDTA treatment followed by the addition of prewarmed complete culture medium, divided and transferred to 1.5 ml microcentrifuge tubes (1  106 cells per tube), and incubated at 32
C for the indicated time periods. The cells were stained with an M2 anti-FLAG antibody (Sigma) followed by PE-conjugated goat antimouse immunoglobulin (BD) and analyzed using a flow cytometer (Fig. 23.3). In the case of transiently transfected cells, cells were transfected with expression plasmids for the reporter proteins driven under a constitutively active promoter using a lipofection reagent. The temperature was shifted to 40
C 10–12 h after transfection and the cells cultured for a further 24 h. Cells were then incubated at 32
C for the indicated time periods, stained, and analyzed by a flow cytometer as described above. When the flow cytometry data were analyzed, it was important to set the second gating to select cells expressing a similar amount of reporter protein, which was evaluated by a similar FL1 intensities, in addition to the first gating with FSC and SSC (Figs. 23.3 and 23.4). This enabled us to analyze the data quantitatively despite different transfection efficiencies and spontaneous loss of reporters in stably transfected cells.

5. Application of Transport Assay The transport assay used reporter proteins in combination with flow cytometry and was very sensitive and quantitative for analyzing the functions of genes of interest. The assay was used to study the function of PGAP1, which is a deacylase that removes an acyl chain linked to an inositol

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Reporter-inducible cells (FF8)

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Figure 23.3 A schematic for the transport assay. Reporter proteins are induced by either doxycycline under an inducible promoter or by transient transfection of the plasmid at a nonpermissive temperature (40
C) for 1 day in order for accumulation to occur in the ER. Then, after the cells are dissociated from the plates, the temperature is shifted to 32
C, and synchronized transport commences from the ER. After the desired time period, the cells are placed on ice and stained with an M2 anti-FLAG antibody and PE-conjugated secondary antibody. The second gate that indicates the cells with a similar amount of total reporter protein expressed is critical for the quantitative analysis. Dot plot in right bottom indicates transient transfection of reporter plasmids.

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Figure 23.4 The transport assay was applied to analyze the significance of PGAP1 in the transport of GPI-anchored proteins. Plasmids for the expression of GPI-anchored (VSVGts(ex)-FF-mEGFP-GPI) and TM-type (FLAG-VSVGts(full)-EGFP) reporter proteins were transiently transfected into wild-type and PGAP1-deficient mutant cells. Dot plots (left) representing the wild-type cells transfected with the TM-type reporter are shown as an example. Histograms were obtained from the cells within the second gating (R2). Times indicate the periods the cells were incubated at 32
C.

residue of GPI-anchored proteins in the ER. It was previously reported that the transport of GPI-anchored proteins from the ER was delayed in PGAP1 mutant cells (Tanaka et al., 2004). The transport assay with transient transfection of reporter plasmids confirmed that the transport of GPI-anchored but not TM-type reporter proteins was delayed in PGAP1-deficient cells (Fig. 23.4). We also reported that the p24 family of proteins were required for the efficient transport of GPI-anchored proteins from the ER using this transport assay system in combination with the knockdown of p24

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expression (Takida et al., 2008). Yet another example of the successful use of the transport assay involved the identification of a new protein, PGAP5, which is involved in the removal of a phosphoethanolamine from GPIanchored proteins (Fujita et al., 2009).

6. Mutagenesis Approximately 1  107 FF8 parent cells were incubated with 400 mg/ml ethyl methanesulfonate (EMS) for 18–24 h (Maeda et al., 2008). Cells were then washed and cultured for at least 1 week before analysis by the transport assay. Caution: EMS is a strong mutagen and volatile and harmful if inhaled, ingested, or absorbed through the skin. Use it with extreme caution. Discard all materials containing or contacted with EMS as hazardous wastes according to the regulation.

7. Selection of Mutant Cells The transport kinetics of the reporter proteins were evaluated by the transport assay described above. After mutagenesis with EMS, the cells were cultured for 1 week with the cell number maintained at least at 3  107 cells. The cells showing the delayed transport of the reporter protein in transport assay were enriched by several rounds of cell sorting (Fig. 23.3). Often, the majority of sorted cells were found to be defective in known genes involved in protein transport. Such cells had to be eliminated by cell sorting in a transport assay with transient transfection of the known genes. An alternative way to prevent generation of cells that are defective in known genes and expected to be enriched by transport assay if mutated under EMS-treatment was to stably introduce such genes into the parent cells in advance. The cells harboring the mutation in the reporter gene were also frequently enriched by several rounds of cell sorting. Such cells should be eliminated by transient transfection of the intact reporter gene followed by transport assay and cell sorting.

8. Measurement of Golgi pH A pH-sensitive green fluorescence protein was very useful in measuring the pH of organelles, because the localization of the protein was easily controlled. A pHluorin (a gift from Dr. G. Miesenbock, Yale University and Dr. J. Rothman, Columbia University) has a biphasic excitation spectra

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and pH is measured based on the ratio of emission intensities at different wavelengths (Miesenbock et al., 1998). In order to construct pH sensor proteins localized at the Golgi, amino acids 1–107 of rat cis-Golgi protein GPP130 or amino acids 1–99 of human N-acetylglucosaminyltransferase I (GnTI) were fused with the amino-terminus of pHluorin. To localize the pHluorin at the TGN, the carboxy-terminus of pHluorin was fused with 56 amino acids of rat TGN38 at the C-terminal (Fig. 23.5). Cells expressing pH sensors were plated on glass-bottom dishes (Matsunami) for 1–2 days before pH measurement. We used a FluoView FV1000 laser scanning confocal microscope (Olympus) equipped with an LD laser 405 (405 nm) and a multiline argon laser (458, 488, and 515 nm) for measurement of pH. After preincubation of the cells in an adequate

Golgimembrane Lumen GPP130 (N-terminal 1–107 a.a.)

pHluorin TGN38 (C-terminal 56 a.a.)

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7.5 7.0

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Figure 23.5 The pH-sensor proteins, pHluorin fused with Golgi-targeting peptide sequences derived from Golgi-resident proteins are expressed and used to measure the pH in the lumen of different Golgi cisternae. The standard curve was determined by incubating the cells in calibration buffers at different pHs. A standard curve obtained from pHluorin fused with GPP130 peptide (lower panel) is shown here.

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solution, such as Na-Ringer buffer (140 mM NaCl, 2 mM CaCl2, 1 mM MgSO4, 1.5 mM K2HPO4, 10 mM glucose, 10 mM MES, and 10 mM HEPES, pH 7.3) or culture medium without phenol red, pHluorin-fused sensor proteins were consecutively excited at 405 and 458 nm, and the emitted fluorescence captured through a spectral slit for wavelengths of 500–600 nm or through a 505–540 nm filter. To obtain the standard curve for pH calibration, cells expressing each pHluorin-fused protein were incubated in calibration buffer (140 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 1.5 mM K2HPO4, 10 mM glucose, 10 mM nigericin, 10 mM monensin, 10 mM MES, and 10 mM HEPES) adjusted to various pHs (5.5–7.5) for 5 min, then analyzed as described above. Regions of interest (ROIs) were automatically created using the MetaMorph software (Molecular Devices) by setting a threshold in the captured pictures and the emission intensities in the ROIs were calculated using the region transfer and measurement functions. The data were imported into Microsoft Excel for calculation of the intensity ratios (405 nm/458 nm) and further analyses.

9. Analysis of Glycosylation Using Lectin Staining SDS-PAGE is the easiest way to screen for abnormalities in glycosylation. The raised pH impairs the elongation of glycan, resulting in the apparent protein size on an SDS-PAGE gel to appear smaller than normal. Alternatively, lectin staining in combination with flow cytometry is a feasible and quantitative method to analyze glycosylation. Because sialylation seems to be impaired most severely, peanut agglutinin (PNA) that binds preferentially to a structure such as galactosyl (b-1,3) N-acetylgalactosamine (T-antigen) is useful. Soybean agglutinin (SBA), Helix pomatia (HPA), and Griffonia simplicifolia lectin II (GSL II) (Saveriano et al., 1981) also showed the enhanced staining in GPHR-deficient CHO and mouse embryonic fibroblast cells (Fig. 23.6). Antibodies against specific glycosphingolipids may also be useful, as is the case with an antibody against GM3 in the CHO mutant cells (Fig. 23.6).

10. Concluding Remarks The mechanisms by which Golgi luminal pH controls the trafficking and glycosylation of proteins and by which the localizations of glycosyltransferases are regulated by Golgi luminal pH are barely understood.

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Figure 23.6 The acidification-impaired mutant cells stably transfected with empty vector (solid) or with the gene responsible for the defect (green line) were stained with various FITC-conjugated lectins (SBA, PNA, and HPA) or with an antibody against GM3 ganglioside.

Further analyses of mutant cells and the functions of GPHR would be very useful for revealing the underlying mechanisms.

ACKNOWLEDGMENT This work was supported by PRESTO, the Japan Science and Technology Agency.

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Doms, R. W., Keller, D. S., Helenius, A., and Balch, W. E. (1987). Role for adenosine triphosphate in regulating the assembly and transport of vesicular stomatitis virus G protein trimers. J. Cell Biol. 105, 1957–1969. Ferrara, C., Brunker, P., Suter, T., Moser, S., Puntener, U., and Umana, P. (2006). Modulation of therapeutic antibody effector functions by glycosylation engineering: Influence of Golgi enzyme localization domain and co-expression of heterologous beta1,4-N-acetylglucosaminyltransferase III and Golgi alpha-mannosidase II. Biotechnol. Bioeng. 93, 851–861. Foulquier, F., Vasile, E., Schollen, E., Callewaert, N., Raemaekers, T., Quelhas, D., Jaeken, J., Mills, P., Winchester, B., Krieger, M., Annaert, W., and Matthijs, G. (2006). Conserved oligomeric Golgi complex subunit 1 deficiency reveals a previously uncharacterized congenital disorder of glycosylation type II. Proc. Natl. Acad. Sci. USA 103, 3764–3769. Foulquier, F., Ungar, D., Reynders, E., Zeevaert, R., Mills, P., Garcia-Silva, M. T., Briones, P., Winchester, B., Morelle, W., Krieger, M., Annaert, W., and Matthijs, G. (2007). A new inborn error of glycosylation due to a Cog8 deficiency reveals a critical role for the Cog1–Cog8 interaction in COG complex formation. Hum. Mol. Genet. 16, 717–730. Fujita, M., Maeda, Y., Ra, M., Yamaguchi, Y., Taguchi, R., and Kinoshita, T. (2009). GPI glycan remodeling by PGAP5 regulates transport of GPI-anchored proteins from the ER to the Golgi. Cell 139, 352–365. Glickman, J., Croen, K., Kelly, S., and Al-Awqati, Q. (1983). Golgi membranes contain an electrogenic Hþ pump in parallel to a chloride conductance. J. Cell Biol. 97, 1303–1308. Grabe, M., and Oster, G. (2001). Regulation of organelle acidity. J. Gen. Physiol. 117, 329–344. Grabenhorst, E., and Conradt, H. S. (1999). The cytoplasmic, transmembrane, and stem regions of glycosyltransferases specify their in vivo functional sublocalization and stability in the Golgi. J. Biol. Chem. 274, 36107–36116. Hara-Chikuma, M., Wang, Y., Guggino, S. E., Guggino, W. B., and Verkman, A. S. (2005a). Impaired acidification in early endosomes of ClC-5 deficient proximal tubule. Biochem. Biophys. Res. Commun. 329, 941–946. Hara-Chikuma, M., Yang, B., Sonawane, N. D., Sasaki, S., Uchida, S., and Verkman, A. S. (2005b). ClC-3 chloride channels facilitate endosomal acidification and chloride accumulation. J. Biol. Chem. 280, 1241–1247. Hurtley, S. M., and Helenius, A. (1989). Protein oligomerization in the endoplasmic reticulum. Annu. Rev. Cell Biol. 5, 277–307. Jentsch, T. J. (2007). Chloride and the endosomal-lysosomal pathway: Emerging roles of CLC chloride transporters. J. Physiol. 578, 633–640. Kasper, D., Planells-Cases, R., Fuhrmann, J. C., Scheel, O., Zeitz, O., Ruether, K., Schmitt, A., Poet, M., Steinfeld, R., Schweizer, M., Kornak, U., and Jentsch, T. J. (2005). Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration. EMBO J. 24, 1079–1091. Kornak, U., Kasper, D., Bosl, M. R., Kaiser, E., Schweizer, M., Schulz, A., Friedrich, W., Delling, G., and Jentsch, T. J. (2001). Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man. Cell 104, 205–215. Kornak, U., Reynders, E., Dimopoulou, A., van Reeuwijk, J., Fischer, B., Rajab, A., Budde, B., Nurnberg, P., Foulquier, F., Lefeber, D., Urban, Z., Gruenewald, S., et al. (2008). Impaired glycosylation and cutis laxa caused by mutations in the vesicular HþATPase subunit ATP6V0A2. Nat. Genet. 40, 32–34. Maeda, Y., Ide, T., Koike, M., Uchiyama, Y., and Kinoshita, T. (2008). GPHR is a novel anion channel critical for acidification and functions of the Golgi apparatus. Nat. Cell Biol. 10, 1135–1145.

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Takida, S., Maeda, Y., and Kinoshita, T. (2008). Mammalian GPI-anchored proteins require p24 proteins for their efficient transport from the ER to the plasma membrane. Biochem. J. 409, 555–562. Tanaka, S., Maeda, Y., Tashima, Y., and Kinoshita, T. (2004). Inositol deacylation of glycosylphosphatidylinositol-anchored proteins is mediated by mammalian PGAP1 and yeast Bst1p. J. Biol. Chem. 279, 14256–14263. Tartakoff, A., Vassalli, P., and Detraz, M. (1978). Comparative studies of intracellular transport of secretory proteins. J. Cell Biol. 79, 694–707. Teichgraber, V., Ulrich, M., Endlich, N., Riethmuller, J., Wilker, B., De OliveiraMunding, C. C., van Heeckeren, A. M., Barr, M. L., von Kurthy, G., Schmid, K. W., Weller, M., Tummler, B., et al. (2008). Ceramide accumulation mediates inflammation, cell death and infection susceptibility in cystic fibrosis. Nat. Med. 14, 382–391. Uliana, A. S., Giraudo, C. G., and Maccioni, H. J. (2006). Cytoplasmic tails of SialT2 and GalNAcT impose their respective proximal and distal Golgi localization. Traffic 7, 604–612. Ungar, D., Oka, T., Brittle, E. E., Vasile, E., Lupashin, V. V., Chatterton, J. E., Heuser, J. E., Krieger, M., and Waters, M. G. (2002). Characterization of a mammalian Golgi-localized protein complex, COG, that is required for normal Golgi morphology and function. J. Cell Biol. 157, 405–415. Urlinger, S., Baron, U., Thellmann, M., Hasan, M. T., Bujard, H., and Hillen, W. (2000). Exploring the sequence space for tetracycline-dependent transcriptional activators: Novel mutations yield expanded range and sensitivity. Proc. Natl. Acad. Sci. USA 97, 7963–7968. Weisz, O. A. (2003). Organelle acidification and disease. Traffic 4, 57–64. Wu, M. M., Grabe, M., Adams, S., Tsien, R. Y., Moore, H. P., and Machen, T. E. (2001). Mechanisms of pH regulation in the regulated secretory pathway. J. Biol. Chem. 276, 33027–33035. Wu, X., Steet, R. A., Bohorov, O., Bakker, J., Newell, J., Krieger, M., Spaapen, L., Kornfeld, S., and Freeze, H. H. (2004). Mutation of the COG complex subunit gene COG7 causes a lethal congenital disorder. Nat. Med. 10, 518–523. Zeevaert, R., Foulquier, F., Jaeken, J., and Matthijs, G. (2008). Deficiencies in subunits of the Conserved Oligomeric Golgi (COG) complex define a novel group of congenital disorders of glycosylation. Mol. Genet. Metab. 93, 15–21.

C H A P T E R

T W E N T Y- F O U R

Enzymatic Synthesis of Lacto-N-Difucohexaose I Which Binds to Helicobacter pylori Tatsuo Miyazaki,* Takeshi Sato,† Kiyoshi Furukawa,† and Katsumi Ajisaka* Contents 1. Overview 2. Synthesis of Lacto-N-triose II Using b-1,3-N-Acetylglucosaminyltransferase 2.1. Materials 2.2. Methods 3. Synthesis of Lacto-N-tetraose by Transglycosylation Using b-1,3-Galactosidase 3.1. Materials 3.2. Methods 4. Preparation of Recombinants FUT1 and FUT3 4.1. Preparation of recombinant baculoviruses 4.2. Expression of recombinants FUT1 and FUT3 5. Synthesis of Lacto-N-fucopentaose I and Lacto-N-difucohexaose I with the Aid of Fucosyltransferases 5.1. Synthesis of Fuca1!2Galb1!3GlcNAcb1!3Galb1!4Glc (lacto-N-fucopentaose I) using recombinant FUT1 5.2. Synthesis of Fuca1!2Galb1!3[Fuca1!4] GlcNAcb1!3Galb1!4Glc (lacto-N-difucohexaose I) using FUT3 Acknowledgments References

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* Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Akiha-ku, Niigata, Japan Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Japan

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Methods in Enzymology, Volume 480 ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)80023-0

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2010 Elsevier Inc. All rights reserved.

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Abstract Helicobacter pylori is known to bind with sugar chains possessing Lewis b structure. We are trying to combine oligosaccharides containing Lewis b sugar chain to water insoluble polysaccharide through some linker. Lacto-N-difucohexaose I (LNDFH I; Fuca1!2Galb1!3[Fuca1!4]GlcNAcb1!3Galb1!4Glc) fits for that purpose, since it consists of Lewis b tetrasaccharide and lactose whose D-glucose residue can be utilized as a linker. We thus developed a method to synthesize this hexaose enzymatically. First, b-1,3-N-acetylglucosaminyltransferase (b-1,3-GnT) was partially purified from bovine blood by an established method. Using this enzyme preparation, D-GlcNAc was attached to the D-galactose residue of lactose with a b-1,3-linkage to produce lacto-N-triose II at 44% yield. The low yield was thought to be due to contaminating N-acetylglucosaminidase that would have hydrolyzed the product, lacto-N-triose II. Next, D-galactose was attached by transglycosylation using ortho-nitrophenyl b-D-galactopyranoside as a donor with the aid of recombinant b-1,3-galactosidase from Bacillus circulans to generate lacto-N-tetraose (LNT) at 22% yield. L-Fucose was then linked to the D-galactose residue of LNT via an a-1,2-linkage using recombinant human fucosyltransferase I (FUT1) expressed in a baculovirus system (71% yield). The obtained pentasaccharide was subsequently incubated with GDP-b-L-fucose and commercial fucosyltransferase III (FUT3) to attach L-fucose to the D-GlcNAc residue of LNT with an a-1,4-linkage. After purification with an activated carbon column chromatography, 1.7 mg of LNDFH I was obtained (85% yield). We thus produced LNDFH I over four enzymatic steps with a yield of 6%.

1. Overview Helicobacter pylori was first isolated by Warren and Marshall (1983) and is a spiral, urease-positive, and Gram-negative flagellated bacterium. This microorganism has been implicated as the principal cause of gastritis, gastric ulcer, and duodenal ulcer and is thought to be associated with gastric cancer (Cover et al., 2001). H. pylori expresses adhesins that bind to carbohydrates in the gastric mucosa (Bore´n et al., 1993). In addition, several kinds of carbohydrate-binding specificities for H. pylori (Evans and Evans, 1995; Ilver et al., 2003; Karlsson, 2000, Walz et al., 2005) including sialic acid conjugates, sulfatide, heparan sulfate, Lewis b conjugates, and glycosphingolipids have been reported. At present, two adhesins of H. pylori, blood group antigen-binding adhesin (BabA) and sialic acid-binding adhesin (SabA), have been identified (Ilver et al., 1998; Mahdavi et al., 2002). BabA, which belongs to a family of H. pylori outer membrane proteins, mainly recognizes the Lewis b and H type 1 antigen but not Lewis a, Lewis X, Lewis Y, or H type 2 (Bore´n et al., 1993, 1994). SabA recognizes sialyl dimeric Lewis X (Mahdavi et al., 2002) and complex gangliosides containing NeuAca2!3Gal structure (Johansson et al., 1999; Roche et al., 2001, 2004). Most disease-associated H. pylori strains express BabA

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(Aspholm-Hurtig et al., 2004). Recently there have been two reports on milk substances that inhibit the binding of H. pylori to the ligands. In the first of these, Gustafsson and coworkers revealed that porcine milk having the carbohydrate epitopes Lewis b and sialyl Lewis X can inhibit H. pylori binding in vitro and colonization in vivo (Gustafsson et al., 2005; 2006). In a second study, Xu and coworkers reported that the binding activity of H. pylori to Lewis b antigen is reduced by goat milk with a transgenic introduction of a1,3/4-fucosyltransferase (Xu et al., 2004). Hence, milk oligosaccharides containing the Lewis b structure appeared to have potential therapeutic effects against H. pylori infection. In this regard, lacto-N-difucohexaose I (Fuca1!2Galb1!3 [Fuca1!4]GlcNAcb1!3Galb1!4Glc), a representative human milk oligosaccharide having Lewis b structure, was thought likely to be useful compound to investigate as a H. pylori adhesin. This hexasaccharide has previously been chemically synthesized on a preparative scale to investigate the binding of Lewis b with BabA and to utilize for the purification of the BabA via Lewis b based affinity chromatography (Chernyak et al., 2000; Lahmann et al., 2004). Chemical synthesis of the lacto-N-difucohexaose I has also been achieved using glycal assembly method (Randolph and Danishefsky, 1994). However, chemical synthesis involves multistep synthetic manipulations and tedious processes and is thus laborious and time-consuming. However, enzymatic synthesis using glycosyltransferases is a powerful approach to building oligosaccharides with regio- and stereo-selectivity, although these enzymes are hard to obtain and sugar nucleotides are expensive. Enzymatic synthesis using glycosidase fits for the low-cost production of oligosaccharides because both the enzyme and donor substrates are relatively inexpensive. Therefore we attempted enzymatic synthesis of the lacto-N-difucohexaose I using both glycosyltransferases and glycosidase in our current study. The convenient synthetic route is shown in Scheme 24.1.

2. Synthesis of Lacto-N-triose II Using b-1,3-N-Acetylglucosaminyltransferase For the synthesis of lacto-N-triose II, b-1,3-N-acetylglucosaminyltransferase (b-1,3-GnT, EC 2.4.1.149) was used to transfer an N-acetyl-bD-glucosamine (D-GlcNAc) residue to lactose. This enzyme has been detected in serum (Kawashima et al., 1993; Piller and Cartron, 1983; Yates and Watkins, 1983), milk (Hosomi et al., 1984), and in tissues from various animals including human (Tsuji et al., 1996). Recently, Murata et al. (1999) synthesized lactoN-triose II in a preparative scale using b-1,3-GnT prepared from bovine serum. Our current procedure for the synthesis of lacto-N-triose II essentially follows the method with minor modifications of the purification steps and the reaction conditions. In our present synthesis approach, the reaction yield was not satisfactory and did not exceed 44%. We speculate this may be due to the

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HO OH

OH O

HO

O

O HO OH

OH

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UDP-a -D-GlcNAc b -1, 3-GnT

HO HO

OH

OH

O

O O OH

AcHN

OH O

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GlcNAcb 1→ 3Galb1→ 4Glc (Lacto-N-triose II)

Galb1→4Glc

HO

Galb-oNP b -1, 3-galactosidase

HO

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OH O HO O

HO

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O O OH

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OH

OH O

O HO

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Galb 1→ 3GlcNAcb1→ 3Galb1→ 4Glc (Lacto-N-tetraose) HO HO

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OH O HO O

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GDP-b-L-Fuc FUT1

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OH O

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GDP-b-L-Fuc FUT3

OH OH HO O O

HO HO

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Me HO

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OH O

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O OH

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O OH

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Fuca 1 → 4 Fuca 1→ 2Galb1→ 3GlcNAcb 1→ 3Galb1→ 4Glc (Lacto-N-difucopentaose I)

Scheme 24.1 Enzymatic Synthesis of Lacto-N-difucohexaose I.

hydrolysis of lacto-N-triose II by contaminating N-acetyl-b-D-glucosaminidase as soon as it is formed. Hence, the yield may be improved by eliminating N-acetyl-b-D-glucosaminidase from partially purified enzyme preparation.

2.1. Materials Bovine blood (kindly supplied by the Niigata Meat plant Foundation, Niigata, Japan) UDP-a-D-GlcNAc, 2Na (Yamasa Co., Tokyo, Japan) b-Galactosidase from Aspergillus oryzae (Sigma) Minimate TFFÒ ultrafiltration system (Pall Life Science Co., Tokyo, Japan)

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2.2. Methods Bovine serum (675 ml) was obtained from 1.36 l of heparinized fresh bovine blood by centrifugation at 8000 rpm for 30 min at 4  C. Ammonium sulfate (99.3 g) was added to this serum to generate a 25% (w/v) ammonium sulfate saturation. The mixture was stirred for 2 h at 4  C and centrifuged at 8000 rpm for 30 min at 4  C. The pellet was then discarded and ammonium sulfate (104 g) was added to the supernatant to achieve a 50% (w/v) saturation. The mixture was then stirred for 2 h at 4  C and centrifuged at 8000 rpm for 30 min at 4  C. The pellet was subsequently suspended in 140 ml of 150 mM Tris–HCl buffer (pH 7.5), and this solution was dialyzed three times against 5 l of the same buffer. Finally, a partially purified b-1,3-GnT solution was obtained after concentration to approximately 10 ml with a Minimate TFFÒ ultrafiltration system containing a membrane with a molecular weight cutoff of 10 kDa. Lactose (239 mg, 0.7 mmol) and UDP-a-D-GlcNAc (36.8 mg, 0.07 mmol) were dissolved in 1 ml of 150 mM Tris–HCl buffer (pH 7.5) containing 0.1 M MnCl2 and 0.1% NaN3. After the addition of the concentrated partially purified b-1,3-GnT (1 ml), the solution was incubated at 37  C for 7 days. The time course of the reaction was monitored once or twice a day with the Dionex DX-500 Bio-LC system attached with CarboPac PA-1Ò column. After 7 days, the enzyme was filtered through a membrane with molecular weight cutoff of 20 kDa and this filtrate was diluted 50-fold with 150 mM Tris–HCl buffer (pH 7.0). b-Galactosidase from A. oryzae (5 U) was then added to the diluted solution which was incubated for 16 h at 37  C to hydrolyze the remaining lactose. The reaction mixture was next treated using an activated carbon column chromatography (2 cm f  45 cm). The column was eluted with a gradient of 0–40% (v/v) aqueous ethanol solution for 240 min with a flow rate of 2 ml/min, and fractions of 5 ml were collected. The sugar concentration in each fraction was then examined by phenol–sulfuric acid method (Dubois et al., 1956) and fractions 34–48 (peak B in Fig. 24.1) were collected and concentrated in vacuo to give 17.1 mg of lacto-N-triose II (44% yield).

3. Synthesis of Lacto-N-tetraose by Transglycosylation Using b-1,3-Galactosidase To attach D-galactose residue to lacto-N-triose II with a b-1,3-linkage to obtain lacto-N-tetraose (LNT), Galb1!3GlcNAcb1!3Galb1!4Glc, the transglycosylation activity of glycosidase (Ajisaka and Yamamoto, 2002;

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Absorbance (495 nm)

1.5 B 1

0.5

A

0 0

10

20 30 Fraction No.

40

50

Figure 24.1 Elution profile of lacto-N-triose II fractionated by an activated carbon column. Hundred microliters of each fraction was treated using the phenol–sulfuric acid method and the absorption at 495 nm was plotted. Peak A, a mixture of monosaccharides; peak B, lacto-N-triose II.

Crout and Vic, 1998; Yamamoto et al., 2004) was employed. We utilized a b-1,3-galactosidase from Bacillus circulans ATCC 31382 the cDNA for which has previously been cloned and expressed in Escherichia coli (Ito and Sasaki, 1997). The resulting recombinant b-1,3-galactosidase showed a high specificity for the hydrolysis of b-1,3-linkages and high regioselectivity in the synthesis of b-1,3-linked oligosaccharides (Fujimoto et al., 1998; Miyasato and Ajisaka, 2004). This enzyme has thus been utilized for the enzymatic synthesis of various biologically important galactosyl oligosaccharides (Naundorf et al., 1998). In our present study, we used this recombinant b-1,3-galactosidase for the synthesis of LNT. In this transglycosylation reaction, we employed ortho-nitrophenyl-b-D-galactopyranoside, Galb-oNP, as the donor and lacto-N-triose II as the acceptor. The time course of the reaction was monitored by HPLC, and at a Galb-oNP peak height of almost zero, the reaction was stopped by inactivating the enzyme. The produced LNT was then isolated using an activated carbon column chromatography and the amount of reducing sugars in each fractionated solution was examined by phenol–sulfuric acid method and the elution profile is shown in Fig. 24.2. Peak A in Fig. 24.2 represents unreacted lacto-N-triose II and peak B was found to represent a mixture of LNT and its regio-isomer, Galb1!6GlcNAcb1!3Galb1!4Glc. This regio-isomer was subsequently hydrolyzed with the aid of b-galactosidase from E. coli which has a high specificity for Galb-1,6-linkages. The hydrolyzate was then treated with an activated carbon column chromatography and pure LNT was obtained at 22% yield.

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3.1. Materials Galb-oNP (Sigma) Recombinant b-1,3-galactosidase from B. circulans (donated from Meiji Dairies Co., Tokyo, Japan)

3.2. Methods Lacto-N-triose II (10.9 mg, 0.02 mmol) and Galb-oNP (15.1 mg, 0.03 mmol) were dissolved in 400 ml of 100 mM sodium acetate buffer (pH 5.5) containing 10% dimethylformamide (DMF). After the addition of b-1,3-galactosidase from B. circulans (150 mU), the solution was incubated at 37  C. After 3 h, the reaction was stopped by heating in a boiling water bath for 5 min. The solution was then diluted to 10 ml and applied to an activated carbon column chromatography (1.5 cm f  15 cm). After washing the column with 100 ml water followed by 100 ml of 7.5% (v/v) aqueous ethanol solution with a flow rate of 0.4 ml/10 min, the column was eluted for 10 h with a gradient of 7.5–35% (v/v) aqueous ethanol solution with a flow rate of 0.5 ml/min. Each 3 ml fraction was collected and the sugars in each fraction were detected using the phenol–sulfuric acid method (Fig. 24.2). Lacto-N-triose II (peak A, 5 mg) was recovered, and a mixture (peak B, 3.7 mg) of LNT and its Galb-1,6-isomer was also obtained. The contents of peak B were dissolved in 100 mM potassium phosphate buffer (5 ml) and 100 mU of b-galactosidase from E. coli was added. After 1.5

Absorbance (495 nm)

A B

1

0.5

0 0

10

20

30

40 50 Fraction no.

60

70

80

Figure 24.2 Elution profile of oligosaccharides fractionated by an activated carbon column. Hundred microliters of each fraction was treated using the phenol–sulfuric acid method and the absorption at 495 nm was plotted. Peak A, recovered lacto-N-triose II; peak B, mixture of lacto-N-tetraose and Galb1!6GlcNAcb1!3Galb1!4Glc.

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incubation for 3 h at 37  C, the solution was applied to an activated carbon column chromatography (1.5 cm f  15 cm). After washing the column with water (50 ml) followed by a 7.5% (v/v) aqueous ethanol solution (50 ml), the products were eluted with a gradient of 7.5–35% (v/v) aqueous ethanol (total 200 ml), and fractions (2 ml) were collected. Finally, 2.8 mg of pure LNT was obtained (22% yield) after concentration of the oligosaccharide fractions. The physical data were identical to those of the corresponding commercially available compounds.

4. Preparation of Recombinants FUT1 and FUT3 Lewis b carbohydrate antigen contains the Fuca1!2Gal and Fuca1!4GlcNAc groups. In mammalian cells, the a-1,2-Fuc residue is synthesized by a-1,2-fucosyltransferases, FUT1 and FUT2 (Kelly et al., 1995; Larsen et al., 1990). Kinetic study showed that FUT1 possesses a higher activity toward LNT than FUT2 (Sarnesto et al., 1992). In the case of the a-1,4-Fuc residue, FUT3 has been shown to transfer L-fucose from GDP-b-L-Fuc to acceptors in an a-1,4-linkage (Weston et al., 1992). Here, we describe the methods to prepare recombinants FUT1 and FUT3 for the synthesis of Lewis b carbohydrate antigen.

4.1. Preparation of recombinant baculoviruses 4.1.1. Materials pFASTBAC1 donor vector (Invitrogen) MAXEFFICIENCY DH10BAC competent cells (Invitrogen) Spodoptera frugiperda (Sf)-9 cells (Invitrogen) Sf-900II serum free medium (Sf-900II SFM) (Invitrogen) Fetal calf serum (FCS; Japan Bioserum) CELLFECTIN reagent (Invitrogen) 4.1.2. Methods For the synthesis of Lewis b carbohydrate antigen, recombinants human FUT1 (GenBank accession no. M35531) and human FUT3 (GenBank accession no. M81485) were used as enzyme sources. The 50 -truncated cDNA fragments encoding the stem regions and catalytic domains of human FUT1 and FUT3 were amplified by PCR [(98  C, 10 sec; 60  C, 0.5 min; and 72  C, 1.5 min)  40] (Guo et al., 2001). For construction of the donor vector, the cDNA fragments encoding honeybee melitin signal peptide and FLAG peptide were introduced into a pFASTBAC1 donor vector, and then the human truncated FUT1 or FUT3 cDNA was ligated

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into the 30 -flanking region of the FLAG fragment in the vector. The bacmid DNA containing the FUT1 or FUT3 cDNA was obtained by transformation of DH10BAC competent cells with the donor vector. Sf-9 cells were grown at 27  C in Sf-900II SFM containing 10% heatinactivated FCS. To obtain the recombinant baculoviruses containing FUT1 or FUT3 cDNA, the recombinant bacmid DNA (1 mg each) was then transfected into Sf-9 cells (1  106 cells) with 6 ml of a CELLFECTIN reagent. The virus stocks with high titers were obtained after five-successive amplifications in Sf-9 cells (Kitamura et al., 2005).

4.2. Expression of recombinants FUT1 and FUT3 4.2.1. Materials Amicon Ultra centrifugal filter, Ultracel-10K (Millipore) GDP-b-L-[3H]Fuc (17.2 Ci/mmol) (PerkinElmer Life and Analytical Sciences) Galb1!3GlcNAcb1!pNP (enzymatically synthesized by Drs. K. Ajisaka and T. Miyazaki) Fuca1!2Galb1!3GlcNAcb1!3Galb1!4Glcb1!PA (Takara Bio) Sep-Pak C18 cartridge (Waters) 4.2.2. Methods Sf-9 cells were placed at 4  105 cells/ml in 25 ml of Sf-900II SFM containing 3% heat-inactivated FCS in a 250 ml Erlenmeyer flask, and cultured at 27  C with shaking at 90 rpm. For expression of the protein, suspension-cultured cells with a cell density of 2  106 cells/ml were infected with 100–500 ml of viral stocks. After 6–8 days of culture, the spent medium was collected and subsequently concentrated by 10- to 20-fold with an Amicon Ultra centrifugal filter, and then used for transferase assays as an enzyme source. FUT activities were determined in a reaction mixture containing 50 mM 3-(Nmorpholino) propanesulfonic acid buffer (pH 7.4) containing 10 mM ATP, 100 mM GDP-b-L-[3H]Fuc, 1 mM Galb1!3GlcNAcb1!pNP or 2 mM Fuca1!2Galb1!3GlcNAcb1!3Galb1!4Glcb1!PA, 20 mM MnCl2, and enzyme preparation in a total volume of 50 ml. After incubation, the product was isolated by using a Sep-Pak C18 cartridge, and radioactivity incorporated into the product was determined (Sato et al., 1998). The results showed that specific activities of the FUT1 and FUT3 toward Galb1!3GlcNAcb1!pNP and Fuca1!2Galb1!3GlcNAcb1!3Galb1!4Glcb1!PA are approximately 65 and 1.3 nmol L-fucose transferred/h/ml-enzyme sources, respectively. Therefore, the recombinants FUT1 and FUT3 can be used for the synthesis of Lewis b carbohydrate antigen as enzyme sources.

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5. Synthesis of Lacto-N-fucopentaose I and Lacto-N-difucohexaose I with the Aid of Fucosyltransferases Lacto-N-difucohexaose was synthesized by firstly attaching successive residues to the D-galactose residue of LNT with an a-1,2-linkage and then to the D-GlcNAc residue with an a-1,4-linkage using our recombinant FUT1 preparation and commercially obtained FUT3, respectively. The activity of our recombinant FUT3 proved to be insufficient for this preparative scale synthesis of lacto-N-difucohexaose and a commercial FUT3 preparation from Glycogene Co., was thus used. This fucosyltransferase stock from Glycogene is provided as a solution of recombinant human fucosyltransferases III (FUT3) containing a FLAGÒ tag. We thus immobilized this FUT3 preparation to an anti-FLAG affinity resin when it was used for the synthesis of fucosyl oligosaccharides. The formation of lacto-N-pentaose I and lacto-N-difucohexaose I was monitored by TLC and products were successfully purified via an activated carbon column chromatography.

L-fucose

5.1. Synthesis of Fuca1!2Galb1!3GlcNAcb1!3Galb1!4Glc (lacto-Nfucopentaose I) using recombinant FUT1 5.1.1. Materials GDP-b-L-Fuc, 2Na (Toyobo) Galb1!3GlcNAcb1!3Galb1!4Glc (LNT) Recombinant FUT1 Phosphatase, alkaline from bovine calf intestine (CIAP; Sigma) Albumin, from bovine serum (Sigma) Silica Gel 60N, 40–50 mm, spherical, neutral (Kanto Chemical) 5.1.2. Methods Fucosylation was performed by incubating a solution consisting of 2.5 ml of 200 mM 3-(N-morpholino) propanesulfonic acid buffer (pH 7.4), 3.5 mg (4.95 mmol) of LNT, 7.9 mg (12.5 mmol) of GDP-b-L-Fuc, 500 ml of recombinant FUT1, 20 mM MnCl2, NaN3 (1 mg/ml), CIAP (50 U), and BSA (1 mg/ml). After 24 h at 37  C, an additional 3.2 mg (5.0 mmol) of GDP-b-L-Fuc and 100 ml of FUT1 were added and the incubation was continued for a further 2.5 days. The reaction was monitored by TLC on silica plates using 2-propanol/H2O/conc. NH3 ¼ 14/7/1 (v/v) as the solvent system and spots were detected by spraying 5% H2SO4 in MeOH

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followed by heating. The Rf values of the product and acceptor were 0.18 and 0.20, respectively. At the end of the reaction, the mixture was dried by evaporation under reduced pressure. Purification of the residue was performed using silica gel column chromatography with 2-propanol/H2O/ conc. NH3 ¼ 14/7/1, (v/v) as the eluent and subsequent gel permeation column chromatography (Sephadex G-15, water). The yield of lacto-Nfucopentaose I was 71% (3.0 mg, 3.51 mmol). The physical data were identical to those reported previously (Bush et al., 1985; Kuhn et al., 1956).

5.2. Synthesis of Fuca1!2Galb1!3[Fuca1!4] GlcNAcb1!3Galb1!4Glc (lacto-N-difucohexaose I) using FUT3 5.2.1. Materials Fuca1!2Galb1!3GlcNAcb1!3Galb1!4Glc (lacto-N-fucopentaose I) FUT3 stock (Glycogene) ANTI-FLAGÒ M2-Agarose from mouse (Sigma) Charcoal, Activated for Chromato (Wako) 5.2.2. Methods The FUT3 stock (1 mU, 12 ml) was thawed at 4  C. NaN3 (0.05%, w/v), NaCl (150 mM), and ANTI FLAGÒ M2-Agarose (500 ml, 50% slurry in 50 mM, pH 7.0 Tris–HCl buffer, containing 150 mM NaCl) were then added to this enzyme solution. The resin was then equilibrated in accordance with the manufacturer’s instructions before use. The stock solution containing the resin was next incubated for about 24 h at 4  C with gentle mixing in Nutator orbital mixer to capture the FLAG fusion proteins. After completion of the binding step, the resin was collected by centrifugation (1000  g for 5 min) at 4  C and washed twice with 1 ml of 25 mM Tris–HCl buffer (pH 7.0) containing 150 mM NaCl to remove all of the nonspecific proteins. The resin was again collected by centrifugation (1000  g for 5 min) at 4  C and 100 mM Tris–HCl buffer (pH 7.0) was added to the affinity gel to produce a 50% slurry. The activity of the immobilized FUT3 was 1 mU per 500 ml of affinity resin (50% slurry) and this preparation was used in the subsequent fucosylation reaction described below. Fucosylation was carried out by incubating a solution containing 1 ml of 100 mM Tris–HCl buffer (pH 7.0), 1.7 mg (1.99 mmol) of lacto-N-fucopentaose I, 3.2 mg (5.01 mmol) of GDP-b-L-Fuc, 75 ml of immobilized FUT3 (0.3 mU), 20 mM MnCl2, NaN3 (2 mg/ml), CIAP (20 U), and BSA (2 mg/ml) for 5 days at 37  C. The reaction was monitored by TLC on silica plates, developed twice with 2-propanol/H2O/conc. NH3 ¼ 14/7/1 (v/v) as the solvent system, and the spots were detected by spraying 5% H2SO4 in

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MeOH followed by heating. The Rf values of the product and acceptor were 0.15 and 0.23, respectively. At the end of the reaction, the mixture was filtered to remove the immobilized FUT3, and then diluted up to 8 ml. The solution was then applied to an activated carbon column (0.8 cm f  10 cm). After washing with 100 ml water to remove the Tris–HCl, MnCl2, and BSA, the product was eluted with a 10–20% (v/v) aqueous ethanol solution. Each 2 ml fraction was collected and the products in each fraction were detected by TLC analysis as described above. The fractions containing hexasaccharide were collected and dried by evaporation under reduced pressure. Gel permeation column chromatography (Sephadex G-15, water) was used to purify lacto-N-difucohexaose I (1.7 mg, 85% yield). The physical data were identical to those published for the commercially supplied compound.

ACKNOWLEDGMENTS This work was supported by the Research Promotion Fund from the Japan Science Technology Agency (2007–2009).

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Miyasato, M., and Ajisaka, K. (2004). Regioselectivity in b-galactosidase-catalyzed transglycosylation for the enzymatic assembly of D-galactosyl-D-mannose. Biosci. Biotechnol. Biochem. 68, 2086–2090. Murata, T., Inukai, T., Suzuki, M., Yamagishi, M., and Usui, T. (1999). Facile enzymatic conversion of lactose into lacto-N-tetraose and lacto-N-neotetraose. Glycoconj. J. 16, 189–195. Naundorf, A., Caussette, M., and Ajisaka, K. (1998). Characterization of the immobilized b-galactosidase C from Bacillus circulans and the production of b-1,3-linked disaccharides. Biosci. Biotechnol. Biochem. 62, 1313–1317. Piller, F., and Cartron, J. P. (1983). UDP-GlcNAc:Galb1-4-Glc(NAc)b1-3-N-acetylglucosaminyl transferase. J. Biol. Chem. 258, 12293–12299. Randolph, J. T., and Danishefsky, S. J. (1994). An interactive strategy for the assembly of complex, branched oligosaccharide domains on a solid support: A concise synthesis of the Lewisb domain in bioconjugatable form. Angew. Chem. Int. Ed. 33, 1470–1473. Roche, N., Larsson, T., A˚ngstro¨m, J., and Teneberg, S. (2001). Helicobacter pylori-binding gangliosides of human gastric adenocarcinoma. Glycobiology 11, 935–944. Roche, N., A˚ngstro¨m, J., Hurtig, M., Larsson, T., Bore´n, T., and Teneberg, S. (2004). Helicobacter pylori and complex gangliosides. Infect. Immun. 72, 1519–1529. Sarnesto, A., Kolin, T., Hindsgaul, O., Thhurin, J., and Blaszczyk-Thurin, M. (1992). Purification of the secretor-type b-galactoside a1-2fucosyltransferase from human serum. J. Biol. Chem. 267, 2734–2744. Sato, T., Furukawa, K., Bakker, H., van den Eijnden, D. H., and van Die, I. (1998). Molecular cloning of a human cDNA encoding a b-1,4-galactosyltransferase with 37% identity to mammalian UDP-Gal: GlcNAc b-1,4-galactosyltransferase. Proc. Natl. Acad. Sci. USA 95, 472–477. Tsuji, T., Urashima, T., and Matsuzawa, T. (1996). The characterization of a UDP-N-acetyl glucosamine: Galb1-4Glc(NAc)b1-3-N-acetylglucosaminyltransferase in fluids from rat rete testis. Biochim. Biophys. Acta 1289, 115–121. Walz, A., Odenbreit, S., Mahdavi, J., Bore´n, T., and Ruhl, S. (2005). Identification and characterization of binding properties of Helicobacter pylori by glycoconjugate arrays. Glycobiology 15, 700–708. Warren, J. R., and Marshall, B. (1983). Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet 321, 1273–1275. Weston, B. W., Nair, R. P., Larsen, R. D., and Lowe, J. B. (1992). Isolation of a novel human a(1,3)fucosyltransferase gene and molecular comparison to the human Lewis blood group a(1,3/1,4)fucosyltransferase gene. Syntenic, homologous, nonallelic genes encoding enzymes with distinct acceptor substrate specificities. J. Biol. Chem. 267, 4152–4160. Xu, H.-T., Zhao, Y.-F., Lian, Z.-X., Fan, B.-L., Zhao, Z.-H., Yu, S.-Y., Dai, Y.-P., Wang, L.-L., Niu, H.-L., Li, N., Hammarstro¨m, L., Bore´n, T., et al. (2004). Effects of fucosylated milk of goat and mouse on Helicobacter pylori binding to Lewis b antigen. World J. Gastroenterol. 10, 2063–2066. Yamamoto, Y., Saito, T., and Ajisaka, K. (2004). Study of the regioselectivity in the transglycosylation to D-galactose derivatives using b-galactosidase of various origins. J. Appl. Glycosci. 51, 335–339. Yates, A. D., and Watkins, W. M. (1983). Enzymes involved in the biosynthesis of glycoconjugates. A UDP-2-acetamido-2-deoxy-D-glucose: b-D-Galactopyranosyl (1-4)-saccharide (1-3)-2-acetamido-2-deoxy-b-D-glucopyranosyltransferase in human serum. Carbohydr. Res. 120, 251–268.

C H A P T E R

T W E N T Y- F I V E

The Drosophila 7-Pass Transmembrane Glycoprotein BOSS and Metabolic Regulation: What Drosophila Can Teach Us About Human Energy Metabolism Ayako Kohyama-Koganeya and Yoshio Hirabayashi Contents 1. Introduction 1.1. Drosophila as a model system for studying metabolism 1.2. Glucose is a key molecule for the maintenance of energy (glucose and lipid) homeostasis 2. BOSS: Drosophila Orphan Membrane Receptor 3. BOSS Responds to Extracellular Glucose 4. Sugar and Lipid Metabolism Is Impaired in boss Null Mutants 5. boss Mutant Flies Are Sensitive to Starvation 6. Biochemical Techniques for Detecting TAG 6.1. Measurement of circulating (hemolymph) TAG levels 6.2. Measurement of total body TAG levels (TLC assay) 6.3. Oil-Red-O and BODIPY staining References

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Abstract Glucose is a key carbohydrate for the majority of living organisms. In animals, plasma glucose levels must be strictly regulated and maintained at proper levels. Abnormal upregulated glucose levels lead to various human metabolic disorders such as diabetes or obesity. In the diabetic state, protein glycation occurs, producing nonenzymatic products that are thought to be causative compounds for the disease. During evolution, animals developed sensing and regulatory mechanisms to maintain constant levels of body glucose levels. How organisms respond to extracellular glucose and how glucose controls nutrient homeostasis, however, have remained uncertain. Recently, we identified Laboratory for Molecular Membrane Neuroscience, Brain Science Institute, RIKEN, Japan Methods in Enzymology, Volume 480 ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)80024-2

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2010 Elsevier Inc. All rights reserved.

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bride of sevenless (BOSS) in Drosophila as a glucose-responding membrane receptor. In this chapter, we summarize the utility of Drosophila as a model organism for studying conserved mechanisms of glucose and triacylglycerol (energy) homeostatic metabolism through the 7-pass transmembrane glycoprotein BOSS, which carries N-linked carbohydrates.

1. Introduction 1.1. Drosophila as a model system for studying metabolism Drosophila is a unique and ideal model organism for conducting energy metabolism research for numerous reasons. The major advantages of Drosophila are its sophisticated genetics, small genome size, high fecundity, low cost, and short generation time. Furthermore, many of the genes implicated in human metabolic diseases exist in Drosophila. Thus, Drosophila is a genetically tractable lower organism that can expand and deepen our understanding of the metabolic processes underlying obesity and diabetes. Through studies of Drosophila, insights relating not only to energy homeostasis but also to life span, body size, reproduction, and immune function have been gained. Drosophila constantly adapts its energy needs to its nutritional status through metabolic regulation, that is, sugar and lipid homeostases. The emerging picture of the Drosophila system is that of a simple and wellbalanced integrated system existing across various tissues, each with distinct physiological roles in maintaining energy homeostasis. The fat body, which shares properties with adipose tissue; oenocytes, which correspond to mammalian hepatocyte-like cells; and certain types of central neurons in the brain are examples of tissues that play a key role in metabolic regulation and energy homeostasis in Drosophila (Bader et al., 2007; Canavoso et al., 2001; Gutierrez et al., 2007; Fig. 25.1). 1.1.1. The fat body The fat body is the main tissue where fuel molecules such as glycogen and triacylglycerides (TAG) are stored. Recently, the fat body has increasingly received attention, because it secretes soluble factors that modulate metabolism, reminiscent of mammalian adipocytokines (Rosen and Spiegelman, 2006). The fat body secretes the fly ortholog of vertebrate insulin-like growth factor (IGF)-binding protein acid-labile subunit (ALS) (Arquier et al., 2008). Like human ALS, which forms a ternary complex with IGFbinding protein 3 and insulin-like growth factor-1 (IGF-1), Drosophila ALS (dALS) binds to Drosophila insulin-like peptides (dILPs) and has been proposed to modulate insulin action. Interestingly, Drosophila Imp-L2 (dlmp-L2) forms a ternary complex with dALS and dILPs, analogous to

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Sugar metabolism

AKH Ring gland dILP

Heart NPY Esophagus

Hugin

dlmp-L2

dALS

Fat body(= liver, adipose tissue)

Brain

TAG

Feeding behavior

TAG Lipid metabolism Oenocyes(= Hepatocyte-like cells)

Figure 25.1 Schematic diagram of Drosophila tissues and organs showing the various pathways responsible for maintaining energy metabolism. The fat body, oenocytes, and some neurons of the brain function as nutrient sensors that coordinate energy metabolism and feeding behavior.

mammalian IGF-binding proteins (Arquier et al., 2008; Honegger et al., 2008). In addition, the fat body stores glycogen. Thus, the fat body is also considered to share some functions performed by the mammalian liver. Moreover, it uses many of the similar enzymes that regulate glycogen synthesis and breakdown. 1.1.2. Oenocytes Oenocytes have been shown to accumulate lipids under starvation (Gutierrez et al., 2007). Oenocytes express many of the same genes found in hepatocytes, such as those encoding enzymes required for processing fatty acids, cell surface proteins for the uptake of lipoprotein particles, and orthologs of hepatic transcription factors such as hepatocyte nuclear factor 4-a and chick ovalbumin upstream promoter transcription factor (Gutierrez et al., 2007). Thus, oenocytes are currently thought to regulate the processing of lipids released by the fat body. 1.1.3. The brain In mammals, fat store levels are regulated by brain centers that control food intake and metabolism (Sandoval et al., 2008). Drosophila brain also contains regions that are highly specialized for controlling food intake and metabolism. Both mammals and Drosophila use many of the same families of peptides to affect energy homeostasis and feeding behavior. For example, Neuropeptide Y or F (NPY/NPF) and neuromedin-U (hugin) have been identified (Bader et al., 2007; Wu et al., 2005). Very recently, neurons that

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regulate fat deposition and feeding behavior have been identified (Al-Anzi et al., 2009). Further studies of the neuronal circuitry of feeding behavior will provide new insights revealing remarkable parallels or similarities between humans and Drosophila.

1.2. Glucose is a key molecule for the maintenance of energy (glucose and lipid) homeostasis Glucose is utilized by all cells of the body as a source of metabolic energy and in diverse biosynthetic reactions. Glucose is especially essential for the brain, because the brain has a unique and absolute dependence on glucose for ATP synthesis. Moreover, hypoglycemia is a major threat for its functioning and survival. Indeed, chronic exposure to hyperglycemia leads to cellular dysfunction that may become irreversible over time, a process that is termed glucose toxicity. Glucose toxicity is brought about by accelerated glycation and increased oxidative stress. An accelerated glycation response produces advanced glycation end products (AGEs), which occur as a result of nonenzymatic glycation and oxidation of proteins, lipids, and nucleic acids (Ramasamy et al., 2005). AGEs are implicated in the pathogenesis underlying the damaging complications seen in diabetes. Thus, maintaining glucose homeostasis is critical for all living animals for maintaining healthy states and survival. Besides being a nutrient, glucose is also a signal detected by several glucose-sensing organs, such as the hypothalamus in the brain, b-cells in the pancreas, and the gut. Thus, detecting and sensing glucose are critical for all organisms, if they are to survive and remain healthy throughout their life. Next, we summarize our current understanding of glucose-sensing mechanisms in yeast and other organisms. 1.2.1. Glucose-sensing mechanisms 1.2.1.1. Yeast G-protein-coupled receptor (GPCR) for glucose sensing Glucose-sensing mechanisms have been extensively studied in yeast. The plasma membrane proteins Snf3, Rgt2, and Gpr1 are involved in glucose-sensing mechanisms (Fig. 25.2A; Gancedo, 2008). Both Snf3 and Rgt2 are 12-transmembrane proteins highly similar to the glucose transporter Hxt. Because Snf3 and Rgt2 are required for the induction of the transcription of HXT genes, these proteins are thought to be sensing receptors for extracellular glucose. Gpr1 is a glucose-sensing GPCR coupled to the Gprotein Gpa2 (Xue et al., 1998; Yun et al., 1997). Eukaryotic GPCR systems are well known for their ability to detect and mediate rapid responses to extracellular signals, such as lipid and glucose metabolites, hormones, pheromones, odorants, neurotransmitters, light, and different taste compounds. The Gpr1–Gpa2 couple is responsive to glucose and to sucrose but not to other sugars such as fructose, 2-deoxyglucose, or xylose (Rolland et al., 2000).

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A Snf3/Rtg2 signaling pathway

Gpr1 signaling pathway Glucose

Snf3/Rtg2

Gpr1

Gpa2 Cyr1

ATP

Signal

Induction of HXTs (glucose transporters) transcription

cAMP

PKA

Switch from respirative/gluconeogenic metabolism to fermentation B

Glucose Receptor (GPCR etc.) GLUT

Signal

Metabolism gene expression

Energy (glucose & lipid) Homeostasis

Figure 25.2 (A) Plasma membrane glucose-sensing proteins in yeast. The 12-transmembrane proteins Snf3 and Rtg2, and GPCR Gpr1 function as extracellular glucose sensors in yeast. (B) Model showing the mechanisms underlying glucose sensing at the plasma membrane surface in multicellular organisms. Receptors (GPCR or others) or transporters respond to extracellular glucose levels to change metabolism states and gene expression.

When glucose is present, Gpr1 interacts with Gpa2 and generates cAMP (cyclic adenosine monophosphate) which activates PKA (protein kinase A) and results in the stimulation of growth and pseudohyphal differentiation, loss of stress resistance, mobilization of trehalose and glycogen, and in reduced lifespan. Although there is no direct evidence for the binding of glucose to

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Gpr1, Gpr1 mutant analysis suggests that Gpr1 is a glucose-sensing receptor. Moreover, Gpr1 mutants do not exhibit glucose-induced cAMP increases (Lemaire et al., 2004). In yeasts, the presence of the glucose-sensing GPCR Gpr1 indicates the possible presence of glucose-sensing GPCRs in higher organisms. However, Gpr1 belongs to a separate GPCR family that is only very distantly related to the other GPCR subfamilies (Graul and Sadee, 2001). 1.2.1.2. Glucose sensing in multicellular organisms Glucose-sensing pathways also trigger metabolic signaling cascades that regulate various aspects of fuel and energy metabolism and influence cell size, growth, proliferation, and survival in multicellular organisms. Current knowledge about the upstream sensing mechanisms in glucose regulatory pathways is limited (Fig. 25.2B). In mammals, glucose transporters (GLUTs) have been identified as glucose sensors in some tissues. Glut4 translocates to the plasma membrane in response to insulin stimulation (Huang and Czech, 2007). Although there is no direct evidence demonstrating the existence of glucose-sensing membrane receptors, a sugar-sensing GPCR similar to the yeast GPCR (Kraakman et al., 1999; Rolland et al., 2001) that acts through the cAMP pathway has been proposed to exist in intestinal epithelial cells (Dyer et al., 2003). Furthermore, hypothalamic orexin neurons have been proposed to harbor cell surface receptors that respond to extracellular glucose (Burdakov et al., 2006). However, the precise molecular characteristics of such receptors remain unclear. Very recently, we identified a novel glucose-responding membrane receptor, Drosophila bride of sevenless (BOSS), which can provide us with another clue toward deciphering glucose-sensing mechanisms in vertebrates.

2. BOSS: Drosophila Orphan Membrane Receptor BOSS was first identified as a ligand for sevenless tyrosine kinase, which is involved in eye differentiation in Drosophila melanogaster (Hart et al., 1990). The long N-terminal region (498 amino acid residue) of BOSS functions as a SEVENLESS ligand. While this N-terminal residue is not evolutionarily conserved, the 7-transmembrane (7TM) and C-terminal regions of BOSS show high sequence identity with the orphan receptor GPRC5B of human (28%) and other species (Fig. 25.3). BOSS and GPRC5Bs (from frog to human) have a potential N-glycosylation site. Indeed, mouse GPRC5B has N-linked type carbohydrate chains. BOSS/GPRC5B falls into family C, which consists of metabotropic glutamate, Ca2þ-sensing, and GABA receptors (Josefsson, 1999; Graul and

Figure 25.3 Sequence alignment of the GPCR motif of BOSS and various GPRC5Bs. ClustalW alignment of BOSS and BOSS orthologs from other species. The BOXSHADE program was used to highlight identical and similar residues. The program checks every residue to determine whether it is identical or similar to the consensus of the corresponding alignment column. Residues that are identical to the columnconsensus are highlighted in black, whereas residues that are not identical but similar to the column-consensus are highlighted in gray. Putative transmembrane regions (TM1–7) are indicated by heavy lines located above the aligned residues. (Partly adapted from Kohyama-Koganeya et al., 2008.)

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Sadee, 2001). All these GPCRs play important physiological roles particularly in the central nervous system, strongly indicating that BOSS also may play yet uncharacterized but important physiological roles. Indeed, we have found that BOSS functions as a glucose-responding receptor that regulates both glucose and lipid metabolism (Kohyama-Koganeya et al., 2008).

3. BOSS Responds to Extracellular Glucose A reporter gene assay can be used to detect GPCR activation and signaling in response to stimulation. Reporter gene assays are based on the ability of GPCR-mediated secondary messengers, such as calcium (Ca2þ) or cAMP, to activate or inhibit a responsive element placed upstream of a minimal promoter, which in turn regulates the expression of a selected reporter protein. In our experiment, an NFAT-luciferase reporter gene assay was introduced to measure Gq-mediated intracellular Ca2þ release. We expressed BOSS in HEK293 cells and then examined changes in intracellular Ca2þ concentrations by measuring luciferase activity. Glucose evoked GPCR signaling in a dose-dependent fashion at 1–10 mM concentrations (Fig. 25.4A). This response was very specific to glucose; BOSS did not respond to other sugars such as trehalose or sucrose (Fig. 25.4B). When GPCRs transduce information provided by extracellular stimuli into cells, the internalization of GPCRs occurs (Tsao et al., 2001). Likewise, BOSS expressed in the fat body internalizes in response to glucose stimulation (Kohyama-Koganeya et al., 2008). When larvae were fasted for 30 min and then were fed with 20% glucose, rapid and excess internalization of BOSS was observed just 5 min after the initiation of glucose feeding (Fig. 25.4C(b)). The same response to glucose stimulation was also observed in HEK293 cells expressing the boss ortholog GPRC5B (Fig. 25.4C(d)). By contrast, internalization of GPRC5D, which belongs to the same subfamily as GPRC5B but shares less identity with BOSS, did not occur (Fig. 25.4C (f)). These results suggest that BOSS functions as a glucose-responding receptor.

4. Sugar and Lipid Metabolism Is Impaired in boss Null Mutants From analysis of biochemical phenotypes of boss null mutant flies that do not respond to extracellular glucose, we found that hemolymph sugar (glucose and trehalose) concentrations were elevated in boss1 larvae (boss KO) (Fig. 25.5A), suggesting that BOSS is involved in the glucose homeostatic system. Hemolymph lipid measurements revealed that boss1 larvae had

533

Glycoprotein BOSS and Metabolic Regulation

C

Luciferase activity (fold of basal level)

A 7 6 5 4 3 2 1 0

c

d

e

f

BOSS

0.1

Luciferase activity (fold of basal level)

Fed (Glucose) b

Mock Boss-myc

GPRC5B

1 10 Glucose (mM)

B 6 5 4 3 2 1 0

Fasted a

*

Mock Boss-myc

GPRC5D

Glucose

Galactose Maltose

Sucrose

Trehalose

Figure 25.4 BOSS responds to extracellular glucose levels. (A) Glucose-induced reporter activity in BOSS-expressing HEK293 cells is concentration dependent. (B) BOSS responded only to glucose but not to other sugars. Data are means  SEM; *P < 0.05. (C) Glucose induces internalization of BOSS and GPRC5B. BOSS, GPRC5B-GFP, or GPRC5D-GFP was overexpressed in the larval fat body, and larvae were stimulated with glucose. BOSS localization to plasma membranes and internalized vesicles (*) were visualized with anti-BOSS antibody or anti-GFP antibody. Scale bar, 25 mm. (Partly adapted from Kohyama-Koganeya et al., 2008.)

significantly increased TAG levels (Fig. 25.5B), indicating that lipid metabolism in these mutants was also impaired. Supporting this observation are data showing that changes in circulating sugar levels are linked to alterations in lipid metabolism (Saltiel and Kahn, 2001). As we mentioned above, oenocytes function as hepatocyte-like cells that accumulate lipid droplets only during starvation (Gutierrez et al., 2007). The oenocytes of boss KO larvae contained lipid droplets, regardless of whether the larvae were fed a standard diet (Fed) or were food deprived for 14 h (fasted) (Fig. 25.5D). All these results demonstrate the effects of BOSS on metabolic control.

5. boss Mutant Flies Are Sensitive to Starvation TAG is stored predominantly in the fat body as stored energy (Van der Horst, 2003) and is critical for supporting an animal during starvation. The survival rate of boss1 flies during starvation was very low (Fig. 25.6A).

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Ayako Kohyama-Koganeya and Yoshio Hirabayashi

B Control Boss KO Boss rescued

300 * 200

* *

100 0 Total

Glucose

D Fed state

* 200

100

0

Trehalose

C

300 Relative TAG level (%)

Sugar contents (mg/ml)

A

Control

Control

Boss KO Boss rescued Boss KO

Boss rescued

Fasted state

TAG ( ) is stored in lipid droplets at the fat body Fed Fat body

Oenocyes

Fasted

TAG is retrieved by oenocytes (starvation signal)

Figure 25.5 Sugar and lipid metabolism is impaired in boss1 larvae. (A) Total circulating sugar (glucose and trehalose) levels in hemolymph from control, boss null mutant (boss KO), and boss-rescued L3 larvae. Expression of boss reversed the hyperglycemia exhibited by boss KO larvae (n > 10 each). (B) Circulating TAG levels (normalized to total protein) in control, boss KO, and boss-rescued L3 larvae. Experiments were performed in triplicate (n ¼ 60/genotype). Data are means  SEM; P ¼ 0.047. (C) Under the fed condition, oenocytes do not contain TAG (dark gray circle). However, under the fasted condition, TAG accumulates in the oenocytes. (D) Oenocytes of boss KO L3 larvae contain Oil-Red-O-positive lipid droplets when they are fed a standard diet, but the oenocytes of control and boss-rescued larvae do not. (Partly adapted from Kohyama-Koganeya et al., 2008.)

Thus, we examined TAG levels in these flies by BODIPY staining, which detects fat droplets. The intensity of BODIPY staining in the fat body of boss1 flies was weaker than that in wild-type (wt, control) flies after starvation (Fig. 25.6B). To confirm these results, we developed a thin layer chromatography (TLC) assay to quantitate the total body TAG levels, as described below. We found that TAG stores dropped more rapidly in boss1 flies than in wt flies (Fig. 25.6C and D). In other words, the lipid consumption rate was higher in boss1 flies than in wt flies (Fig. 25.6C and D). All these experiments show that boss1 flies are sensitive to starvation and that the regulatory mechanisms modulating lipolysis (lipid expenditure) do not function properly in boss1 flies. Our findings are the first to demonstrate the presence of a glucoseresponding membrane receptor that regulates glucose and lipid metabolism in a multicellular organism. Nevertheless, many unanswered questions remain. Does glucose directly bind or interact with BOSS to activate

535

Glycoprotein BOSS and Metabolic Regulation

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B 120

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Control Boss KO

Boss KO Boss rescued

Fed

Survival rate (%)

Boss rescued 80 Fast (8h) 40 Fast (24h)

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12

24

36

24 (h)

0

8

48

60 72

84 (h)

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D 0

8

24(h)

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8

24(h)

Control Boss KO Boss rescued

TAG decay rate (%)

120%

2 Control

Boss KO

Boss rescued

4 (m g)

TAG

80%

40%

0%

8

24 (h)

Figure 25.6 BOSS mutant flies are sensitive to starvation. (A) Survival curves demonstrating the starvation sensitivity of boss1 mutants. Experiments were performed in triplicate (n ¼ 60/genotype). Data are means  SEM. (B, C, D) Starvation-induced TAG consumption is accelerated in boss KO flies. BODIPY staining (B) shows the decay of lipid storage droplets in the fat body. Scale bar, 100 mm. Whole-fly TAG content during starvation is shown in thin layer chromatograms (C) and decay curves (D). TAGs were collected from starved (0, 8, and 24 h) flies, analyzed by TLC assay, and then the intensity of TAG bands (arrowhead) was measured. (Partly adapted from Kohyama-Koganeya et al., 2008.)

intercellular signaling cascades? How does BOSS control whole body metabolism? Is it through cell-autonomous functions in the fat body? Are some other BOSS-expressing tissues or cells involved? Future research must be directed toward the development of experimental approaches to reveal the more precise roles of glucose-responding GPCRs in energy homeostasis and its mechanisms of action.

6. Biochemical Techniques for Detecting TAG The primary type of stored fat in Drosophila is TAG, which is stored in so-called fat droplets in the fat body. TAG-containing droplets can be visualized through staining with hydrophobic dyes such as BODIPY or Oil-Red-O. For the quantitation of total TAG levels, we developed

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Ayako Kohyama-Koganeya and Yoshio Hirabayashi

an assay using TLC. TAG reagent kits are also commercially available for measuring TAG in hemolymph.

6.1. Measurement of circulating (hemolymph) TAG levels 6.1.1. Materials 1. 2. 3. 4.

0.05% Tween 20 in H2O Protease inhibitor (Sigma inhibitor cocktail; Sigma) Triglyceride reagent (Sigma) Bio-Rad protein assay reagent (Bio-Rad)

Batches of 20 larvae or 10 flies of the desired genotypes are homogenized in 0.05% Tween 20 in the presence of protease inhibitor in H2O, on ice. The homogenates are spun at 5000 rpm on a tabletop centrifuge for 1 min, and 500 ml of supernatant is transferred into a fresh tube void of debris. The supernatant is then spun at 14,000 rpm for 3 min on a tabletop centrifuge at 4  C. In order to measure circulating TAG, 80 ml of the resulting supernatant is combined with 1 ml of triglyceride reagent and incubated for 10 min at 37  C. The OD520nm is measured and compared to a standard curve. In order to measure protein levels, 100 ml of the final supernatant is combined with 700 ml of H2O and 200 ml of Bio-Rad Protein Assay Reagent and incubated for 3 min at room temperature. The OD595nm is measured and compared to a standard curve. This method is a rapid and convenient method for measuring TAG.

6.2. Measurement of total body TAG levels (TLC assay) 6.2.1. Materials Chloroform–methanol (2:1, v/v) Chloroform–methanol–acetic acid (98:1:1, v/v/v) TLC (Silica gel 60 plate; Merck, Darmstadt, Germany) Charring reagent (Ceric ammonium nitrate/ H2SO4 solution): Dissolve 12 g of hexaammonium heptamolybdate tetrahydrate and 0.4 g of ceric ammnonium nitrate in 100ml of 10% H2SO4 solution 5. TAG purified from Drosophila larvae (TAG standard) 1. 2. 3. 4.

For the TLC assay, lipids from the sample (5 flies or larvae) are extracted with 0.5 ml of chloroform–methanol (2:1, v/v). Aliquots of the extract (2 ml) are applied onto a TLC plate, and the plate is developed with a solvent system of chloroform–methanol–acetic acid (98:1:1, v/v/v). The TLC plate is then sprayed with charring reagent and heated at 180  C. TAG levels are estimated with a LAS 3000 (Fujifilm, Tokyo, Japan) set to digitized mode.

Glycoprotein BOSS and Metabolic Regulation

537

6.3. Oil-Red-O and BODIPY staining 6.3.1. Materials 1. Oil-Red-O (Sigma): Prepare fresh when needed. Mix 6 ml of 0.1% OilRed-O in isopropanol and 4 ml distilled water, then pass the dye through a 0.45-mm syringe filter 2. BODIPY 493/503 (Molecular Probes, Carlsbad, CA): Dissolve BODIPY 493/503 in ethanol at 1 mg/ml, and then add to PBS to a final concentration of 2 mg/ml 3. 4% paraformaldehyde in PBS (Phosphate buffered saline) 4. Glycerol To visualize fat stored in droplets, tissues are stained with dyes (Oil-Red-O or BODIPY 493/503) that detect these droplets. Adult flies or dissected larvae are fixed in 4% paraformaldehyde for 25 min at room temperature and washed four times in PBS. Next, tissues are stained with Oil-Red-O or BODIPY493/ 503 solution. After incubation for 30 min to 1 h, specimens are washed three times with PBS, and then mounted in glycerol. For Oil-Red-O, the stained droplets are photographed with a digital camera and scored. It is also possible to detect Oil-Red-O-stained signals using epifluorescence and a Texas Red excitation filter (540–580 nm) (Koopman et al., 2001). For BODIPY, the droplets are analyzed using a confocal microscope, such as LMS5 PASCAL (Zeiss).

REFERENCES Al-Anzi, B., et al. (2009). Obesity-blocking neurons in Drosophila. Neuron 63, 329–341. Arquier, N., et al. (2008). Drosophila ALS regulates growth and metabolism through functional interaction with insulin-like peptides. Cell Metab. 7, 333–338. Bader, R., et al. (2007). Genetic dissection of neural circuit anatomy underlying feeding behavior in Drosophila: Distinct classes of hugin-expressing neurons. J. Comp. Neurol. 502, 848–856. Burdakov, D., et al. (2006). Tandem-pore Kþ channels mediate inhibition of orexin neurons by glucose. Neuron 50, 711–722. Canavoso, L. E., et al. (2001). Fat metabolism in insects. Annu. Rev. Nutr. 21, 23–46. Dyer, J., et al. (2003). Glucose sensing in the intestinal epithelium. Eur. J. Biochem. 270, 3377–3388. Gancedo, J. M. (2008). The early steps of glucose signalling in yeast. FEMS Microbiol. Rev. 32, 673–704. Graul, R. C., and Sadee, W. (2001). Evolutionary relationships among G protein-coupled receptors using a clustered database approach. AAPS PharmSci. 3, E12. Gutierrez, E., et al. (2007). Specialized hepatocyte-like cells regulate Drosophila lipid metabolism. Nature 445, 275–280. Hart, A. C., et. al., (1990). Induction of cell fate in the Drosophila retina: the bride of sevenless protein is predicted to contain a large extracellular domain and seven transmembrane segments. Genes Dev. 4, 1835–1847.

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Honegger, B., et al. (2008). Imp-L2, a putative homolog of vertebrate IGF-binding protein 7, counteracts insulin signaling in Drosophila and is essential for starvation resistance. J. Biol. 7, 10. Huang, S., and Czech, M. P. (2007). The GLUT4 glucose transporter. Cell Metab. 5, 237–252. Josefsson, L. G. (1999). Evidence for kinship between diverse G-protein coupled receptors. Gene 239, 333–340. Kohyama-Koganeya, A., et al. (2008). A Drosophila orphan G protein-coupled receptor BOSS functions as a glucose-responding receptor: Loss of boss causes abnormal energy metabolism. Proc. Natl. Acad. Sci. USA 105, 15328–15333. Koopman, R., et al. (2001). Optimisation of oil red O staining permits combination with immunofluorescence and automated quantification of lipids. Histochem. Cell Biol. 116, 63–68. Kraakman, L., et al. (1999). A Saccharomyces cerevisiae G-protein coupled receptor, Gpr1, is specifically required for glucose activation of the cAMP pathway during the transition to growth on glucose. Mol. Microbiol. 32, 1002–1012. Lemaire, K., et al. (2004). Glucose and sucrose act as agonist and mannose as antagonist ligands of the G protein-coupled receptor Gpr1 in the yeast Saccharomyces cerevisiae. Mol. Cell 16, 293–299. Ramasamy, R., et al. (2005). Advanced glycation end products and RAGE: A common thread in aging, diabetes, neurodegeneration, and inflammation. Glycobiology 15, 16R–28R. Rolland, F., et al. (2000). Glucose-induced cAMP signalling in yeast requires both a G-protein coupled receptor system for extracellular glucose detection and a separable hexose kinase-dependent sensing process. Mol. Microbiol. 38, 348–358. Rolland, F., et al. (2001). Glucose-sensing mechanisms in eukaryotic cells. Trends Biochem. Sci. 26, 310–317. Rosen, E. D., and Spiegelman, B. M. (2006). Adipocytes as regulators of energy balance and glucose homeostasis. Nature 444, 847–853. Saltiel, A. R., and Kahn, C. R. (2001). Insulin signalling and the regulation of glucose and lipid metabolism. Nature 414, 799–806. Sandoval, D., et al. (2008). The integrative role of CNS fuel-sensing mechanisms in energy balance and glucose regulation. Annu. Rev. Physiol. 70, 513–535. Tsao, P., et al. (2001). Role of endocytosis in mediating downregulation of G-proteincoupled receptors. Trends Pharmacol. Sci. 22, 91–96. Van der Horst, D. J. (2003). Insect adipokinetic hormones: Release and integration of flight energy metabolism. Comp. Biochem. Physiol. Biochem. Mol. Biol. 136, 217–226. Wu, Q., et al. (2005). Regulation of aversion to noxious food by Drosophila neuropeptide Y- and insulin-like systems. Nat. Neurosci. 8, 1350–1355. Xue, Y., et al. (1998). GPR1 encodes a putative G protein-coupled receptor that associates with the Gpa2p Galpha subunit and functions in a Ras-independent pathway. EMBO J. 17, 1996–2007. Yun, C. W., et al. (1997). G-protein coupled receptor from yeast Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 240, 287–292.

Author Index

A Aaronson, S. A., 236 Abate-Shen, C., 358 Abbott, K. L., 461–474 Abd Hamid, U. M., 462 Abedin, M., 184 Abeijon, C., 250, 369 Abe, S., 248–249, 255 Abiatari, I., 53 Abrams, J., 236 Abramson, J., 202, 231–232 Acar, M., 357, 379, 382, 386–388, 390, 392, 403, 412 Adachi, A., 273, 377 Adachi, J., 479 Adams, E. W., 418 Adams, J. C., 404, 447 Adams, S., 497 Adang, M. J., 143 Addae-Mensah, K. A., 13 Adema, G. J., 132 Aderca, I., 53 Adkins, H. B., 358 Aebi, M., 141–148, 183–184, 193, 479 Aggarwal, P., 92 Ahmad, N., 246, 248, 271 Ahmed, Y. A., 65–81 Ahorn, H., 478 Ahrend, M., 312 Ahringer, J., 480–481, 486 Aigaki, T., 334, 358 Ai, X., 52–53 Ajisaka, K., 513–524 Ajuebor, M. N., 212 Akama, R., 453 Akama, T. O., 256 Akasaka, K., 53 Akasaka-Manya, K., 456 Akimoto, Y., 521 Akiyama, S. K., 447, 452 Akiyama, T., 334 Alam, N., 446 Alamowitch, S., 376 Al-Anzi, B., 530 Al-Awqati, Q., 497 Albiges-Rizo, C., 12 Albini, A., 17 Alcais, A., 256

Alexandre, C., 334 Alexopoulou, A. N., 10 Ali-Ahmed, D., 145 Allan, D. S., 202 Allison, J. P., 200 Allshire, R. C., 340 Almazan, T., 220 Almeida, R., 340 Al-Mohsen, I., 256 Alnemri, E. S., 236 Altmann, F., 304, 478 Altraja, S., 514 Alvarez, M., 202, 224, 231 Alvarez-Manilla, G., 472 Alvarez, R. A., 134, 142, 167–168, 418, 419 Amano, J., 463 Amano, M., 230 Amano, T., 187 Ambasta, R. K., 52 Ambavane, A., 300 Ammann, M., 423 Amoresano, A., 452–453 Anderson, A., 201 Andre, S., 246, 248, 271 Andrews, B. J., 128 Andrews, H. K., 379 Anelli, T., 183 Angstrom, J., 514 Anjos, S., 256–257 Annunziata, I., 52 Antonicek, H., 316 Antonopoulos, A., 255, 257 Antunes, E., 248–249, 256 Anumula, K. R., 302 Ao, J., 142 Aoki, A., 202, 271 Aoki, K., 14, 297–321, 328, 330, 357, 462–464, 467, 472–473 Appelmelk, B., 130 Appenzeller-Herzog, C., 183 Apte, S. S., 405 Apweiler, R., 446 Arango, J., 463 Arani, R. B., 277 Aratani, S., 187 Arata, Y., 183, 190, 246–248, 250, 257, 279, 432 Arboleda-Velasquez, J. F., 358 Archer, M., 331 Ardman, B., 230

539

540

Author Index

Ariki, S., 142 Arnaout, M. A., 454 Arnqvist, A., 514 Aroian, R. V., 143 Arquier, N., 528–529 Arron, J., 152 Arthur, C. M., 142, 204, 246–247, 250, 282–283, 423, 432, 434, 440, 474 Arthur, L. O, 274, 286 Asada, M., 452– 453 Asano, M., 317 Asawa, R., 282 Ashikari-Hada, S., 6 Ashkenazi, A., 53 Ashline, D., 302–303, 479–480 Ashton, P. D., 126 Asikainen, S., 486 Askari, J. A., 454 Aspord, C., 152 Assmann, B., 478 Aster, J. C., 377 Atkinson, C. E., 167 Atochina, O., 119 Atrih, A., 71, 73 Audit, M., 272 Aulehla, A., 358, 387 Austin, C. L., 386 Avanesov, A.., 334 Avci, F. Y., 76 Avner, R., 187 Avula, R., 53 Awad, A. M., 312, 479–480 Axelrod, D., 12 Axelsson, M. A., 495, 497 Aya, H., 221 Ayukawa, T., 328, 330, 358 B Babini, E., 497 Bachert, C., 495–497 Bachmann, E., 356 Bader,. R., 528–529 Baeg, G. H., 36, 39, 43, 328, 330, 334 Baena-Lopez, L. A.., 331 Baer, H. H., 523 Bai, X. M., 53 Bakker, H., 357, 377, 521 Bakker, J., 497 Bakkers, J., 205 Balanzino, L., 310 Balch, W. E., 499 Baldwin, R. J., 53 Balfour, H. H. Jr., 272 Ballabio, A., 52 Banchereau, J., 152 Bank, C. M., 130, 134 Banta, A. B., 376

Banting, G., 495 Baonza, A.., 331 Barbeau, B., 287 Baribaud, F., 270 Barnes, R., 376 Barnes, S., 305 Barondes, S. H., 270–272, 431 Baron, U., 501 Barre, A., 423, 428 Barrett, A. L., 335 Barrio, M. M., 167, 172, 174 Barrionuevo, P., 220 Barr, M. L., 496–497 Barrows, B. D., 143 Barton, S. J., 454 Bartscherer, K., 332–333 Basa, L. J., 357 Bass, M. D., 7, 14 Bastid, C., 273 Bates, P., 270 Baude, G., 357 Bauer, M., 53 Baumeister, S., 129 Baumgartner, S., 34, 331, 335 Baum, L. G., 142, 201, 204–205, 220, 224, 228–231, 233, 235, 246–248, 271–272 Bayat, V., 379, 382 Bays, N. W., 182 Beachy, P. A., 334 Beaulieu, A. D., 270 Beg, A. A., 152 Behrendt, C. L., 142 Beigier-Bompadre, M., 220 Beilman, G. J., 269 Belenkaya, T. Y., 33–49, 327, 331–332, 334, 336 Bella, J., 454 Bellen, H. J., 357, 376, 379, 382, 385 Bellis, S. L., 452 Benveniste, R. E., 286 Berg, D. E., 514 Bergemann, C., 271 Berger, E. G., 497 Bergeron, M. G., 279 Berkefeld, C., 125–126, 129 Berkhout, B., 279 Bernasconi, R., 184, 188, 192 Bernfield, M., 5, 53, 67, 317, 326, 331 Bernier, S. M., 356 Bertozzi, C. R., 52 Bertsch, W., 423 Bessia, C., 376 Bess, J. W. Jr., 274, 276, 283 Bettelli, E., 234 Beverley, S. M., 328, 330 Beznoussenko, G., 496 Bhamidipati, A., 182 Bhanot, P., 43 Bhaumik, P., 271

Author Index

Bianchi, M. E., 271 Bianco, G. A., 201–202, 205, 219–220, 224, 228–229, 231–233, 235, 247 Bianco, I., 203 Bickle, Q. D., 120, 123, 126, 128–129 Bide`re, N., 229 Bielmann, R., 182 Bienvenu, K., 216 Bierhuizen, M. F., 298 Bigge, C., 302 Bilder, D., 358, 387 Bilska, M., 277 Binley, J. M., 269 Birchler, J. A., 340 Bird, J., 376 Bi, S., 230, 247 Bishop, J. R., 53, 67 Bistrup, A., 53–54 Bitegye, C., 233 Bitton, N., 270 Bjoern, S., 356–357, 402 Blach, M., 229 Blackburn, J., 78 Blacklow, S. C., 377 Blair, S. S., 332–334 Blanchard, H., 282 Bland, K. S., 335 Blaser, C., 201, 271–272 Blaser, M. J, 514 Blaszczyk-Thurin, M., 520 Bleuler-Martinez, S., 141–148 Blixt, O., 134, 167–168, 246–247, 250, 283, 418–419, 423, 432, 440 Blobe, J. C., 4 Block, T. M., 463 Blois, S. M., 202, 231 Blomberg, M. A., 369 Bluestone, J. A., 200 Blumenthal, R., 269 Bobardt, M. D., 270 Boden, D., 269 Boer, A. R., 418 Bogitsh, B. J, 128 Bohks, S. M., 270 Bohorov, O., 497 Boisson-Dupuis, S., 256 Bolivar, I., 356 Bolon, B., 53 Bolos, V., 376 Bolton, A. E., 172 Bonifaz, L., 217–219 Bonnyay, D., 217–219 Boon, T., 248–249 Bordallo, J., 182 Boren, T., 514–515 Borissenko, L. V., 52 Bork, P., 402 Bornberg-Bauer, E., 142

541 Bornemann, D. J., 316, 327, 331–332, 336 Bornstein, P., 404, 410 Borregaard, N., 204 Bosl, M. R., 495–497 Boudreau, E. D., 483–485, 487–488, 490–491 Boudreau, N., 53 Bouillet, P., 229 Boulay, F., 204 Bounou, S., 271, 275 Bou-Reslan, H., 53 Boutros, M., 332–333 Bowers, K., 212 Bowie, A. G., 152 Bowman, M. J., 70, 74, 76 Boyden, S., 16 Braakman, I., 183 Brady, J. P., 170 Brand, A. H., 379, 381 Brandwijk, R. J., 53, 205 Bravo, A., 202, 224, 231 Bray, S. J., 377 Breiderhoff, T., 496–497 Breimer, M. E., 515 Breloy, I., 299, 309 Brenchley, J. M., 269 Brenner, S., 481 Brentani, R. R., 452 Brewer, C. F., 201, 229, 246–248, 271 Brickman, Y., 67 Bringmann, P., 357 Brittle, E. E., 497 Brockhausen, I., 273 Brock, J., 229 Brodsky, J. L., 183 Broekaert, W. F., 423, 428 Bronckart, Y., 440 Brou, C., 376–377 Brown, J. K., 142 Bruce, J. A., 302 Bruckner, K., 358, 387, 402 Bruijns, S. C. M., 121, 133–136, 153 Brunker, P., 496 Brunner, A., 331 Bryan, M. C., 167, 419 Buchler, M. W., 53 Buckhaults, P., 463 Buckley, P. A., 454 Budde, B., 495–497, 499, 504 Buddecke, E., 67 Buerger, E., 188 Buettner, F. F., 357, 377 Buff, E. M., 336 Bugarcic, A., 282 Bujard, H., 501 Buko, A. M., 356–357 Bukrinskaya, A. G., 279 Bullard, D. C., 234 Bulow, L., 515

542

Author Index

Burchert, M., 202 Burdakov, D., 532 Burda, P., 182–183 Burdett, I., 271 Burgess, A. W., 356 Burnette, W. N., 172 Burn, T. C., 356 Burridge, K., 14 Burton, D. R, 269 Buschhorn, B. A., 182, 188–189 Bush, C. A., 523 Bustamante, J., 256 Butler, W. T., 281 Butschi, A., 141–148 Butterfield, J. E., 446 Byron, A., 446 C Cabral, C. M., 183 Cabrera, P. V., 229 Cagavi Bozkulak, E., 376 Calanca, V., 188 Calarese, D., 167, 419 Caldwell, M. C., 331, 335 Callejo, A., 334 Callewaert, N., 497 Camby, I., 440 Cameron, P., 270 Campagna, L., 229, 235 Campbell, I. D., 402 Campbell, R. E., 26 Campbell, R. M., 256 Camphausent, R. T., 167 Canavoso, L. E., 528 Cantin, R., 269–270, 273, 279 Cantu, R., 298–300, 302–305 Cao, T. V., 152, 203–204, 208, 212 Cao, Y., 382 Cao, Z., 202 Capurro, M. I., 174 Carano, R. A., 53 Carlis, J., 269 Carlson, J. W., 385 Carlson, R. W., 95 Carlsson, S., 431, 440 Carrier, Y., 234 Cartron, J. P., 515 Carvalho, M., 334 Carvalho, P., 182, 191 Casbarra, A., 452–453 Cash, H. L., 142 Cassiman, J. J., 53 Castagna, L. F., 202, 229, 271–272 Castillejo-Lopez, C., 335 Castronovo, V., 270 Caulfield, J. P., 126–128 Caussette, M., 518

Cecillion, M., 376 Cerchiaro, G., 203–204, 208, 212 Cernuda-Morollo´n, E., 202, 232 Ceroni, A., 76, 124–125, 303 Ceron, J., 480–481, 486 Cervantes, S., 53–57 Chabriat, H., 376 Chai, W., 167, 418 Chalasani, S., 53 Chammas, R., 452 Chanana, B., 331–332 Chan, D. C., 269 Chandraratna, D., 334 Chandrasekharappa, S. C., 376 Chang, M. H., 220 Chang, Y. C., 386 Chan, J., 229 Chapgier, A., 256 Chapman, J., 184 Chapman, R. E., 495 Cha, S. K., 248 Chatterjee, D., 126–128 Chatterton, J. E., 497 Chau, V., 187 Chavant, L., 142, 145 Chen, C., 142 Cheng, X., 453 Chen, H. L., 246, 248–249, 252–253, 255, 258–259, 261 Chen, I. J., 246–250, 252–254, 256, 258–262 Chen, J., 326–327, 343, 358, 387, 389 Chen, L.-F., 161 Chen, W., 202, 231–232 Chen, Y. W., 312 Cheresh, D. A., 6 Chermann, J. C., 286 Chernajovsky, Y., 203–204, 212 Chernyak, A., 515 Chertova, E., 274, 276 Chesebro, B., 277 Chet, I., 142 Cheung, P., 248–249, 452 Chiang, C., 332–333 Chiba, A., 456 Chiba, Y., 255, 257, 328–330, 336, 338, 343, 456 Chien, C. B., 332, 335 Chien, J. R., 53 Chilton, K., 203–204, 208, 212 Chin, D. R., 53 Chitsulo, L., 119 Chittenden, T. W., 9 Cho, G., 78 Choi, K. S., 142 Choi, Y., 152 Cho, M., 142, 204, 281–282, 284 Chou, D. K. H., 316 Choux, R., 273 Christensen, M., 356–357

543

Author Index

Christianson, J. C., 182, 187–188, 192 Christie, M., 212 Christoforou, C. P., 335 Christophe, T., 204 Chu, C. C., 202, 232 Chui, D., 256 Chung, C. D., 249 Chuzhanova, N., 256 Cipollo, J. F., 312, 479–480 Citores, L., 423, 428 Ciucanu, I., 105 Claessen, J. H., 187 Clark, T., 377 Clausen, H., 250, 312, 358, 387, 454 Clements, J. E., 277 Clerc, S., 183–184, 193 Coath, C., 67 Cochran, J., 376 Coddeville, B., 310 Cohen, S. M., 36, 39, 312, 331, 334, 358, 387 Colau, D., 248–249 Colige, A., 405 Colley, K. J., 497 Collins, C. C., 376 Collins, F. S., 376 Colognato, H., 447 Colucci, J. K., 282 Colvin, J. S., 68, 79–80 Comer, F. I., 305 Commodaro, A. G., 231–232 Connolly, J., 152 Conradt, H. S., 497 Conroy, E., 134, 168 Conyers, S. M., 129 Coombs, P. J., 170–171, 174 Cooper, D. N. W., 199–239, 270–272, 431 Coppolino, M., 258 Cordo-Russo, R., 202, 231 Corigliano, A., 201, 229, 231 Cormier, J. H., 193 Cornelissen, I. L., 134, 270 Corpechot, C., 376 Correale, J., 201, 224, 228–229, 231, 233, 235, 247, 284 Correa, S. G., 203 Cosma, M. P., 52 Costa, T., 376 Costello, C. E., 70, 74, 76, 125, 312, 479–480 Cottrell, J. S., 177 Couchman, J. R., 11–12, 18 Coulier, F., 68, 79–80 Courtoy, P. J., 248–249 Covacci, A., 514 Cover,T. L., 514 Cowman, M. K., 100 Craig, N. M., 142 Craig, S. E., 454 Creasy, D. M., 177

Cremer, P. S., 143 Crickmore, M. A., 331 Crise, B. J., 276 Croci, D. O., 199–239, 247, 284 Crocker, P. R., 78, 246, 441 Croen, K., 497 Cronin, S. R., 182 Crozatier, M., 334 Cruciat, C. M., 187, 191 Cummings, R. D., 72, 119, 126–128, 130, 142, 201, 204, 246–247, 250, 252, 270, 282–284, 324, 329, 417–442, 456, 463–464 Cunningham, A. L., 270 Cunningham, J. M., 53 Cunningham, M. R., 356 Curwen, R. S., 126 Czech, M. P., 532 D Dabrowski, U., 312–313 Da’dara, A. A., 119 Dagoneau, N., 405 Dahlgren, C., 204 Dahms, N. M., 182 Dai, M., 187 Dai, Y.-P., 52, 515 Damian, R. T., 126–128 Damon, B., 358 Danishefsky, S. J., 515 Dann, S. M., 142 Danos, O., 272 Daroqui, M. C., 201, 229, 231 Das, A. M., 212 DasGupta, R., 46, 334 D’Atri, L. P., 205 Datta, S., 331, 335 David, G., 53, 269–270 Dawson, D. W., 404, 407 Day, S., 423 Deakin, J. A., 67, 70 De Angelis, D. A., 505 de Beaucoudrey, L., 256 DeBose-Boyd, R., 128 De Castro, C., 89–114 Decker, J. M., 277 Dedhar, S., 258 Deelder, A. M., 126, 418 Deepa, S. S., 324–325, 329 Deguchi, M., 221 Dejima, K., 142 de la Pompa, J. L., 376 de la Rosa, G., 271 Delegeane, A., 272 Delenda, C., 272 Dell, A., 76, 124–128, 143, 167, 170–172, 174, 177–178, 255–257, 302–303, 316, 423, 479–480

544 Delling, G., 495, 497 del Pozo, M. A., 24 De Matteis, M. A., 496 Demetriou, M., 234, 245–262, 452–453 De Miguel, M., 202 Demotte, N., 248–249 Demotz, S., 233 den Dunnen, J., 131, 152–153, 155–157, 159, 161–163 Deng, W. M., 335 Deng, Y., 376 Denic, V., 182, 189 Dennis, J. W., 234, 246–250, 252, 254–262, 452–453, 463 Dennis, R. D., 120–121, 123–129, 312–314, 316 Dennissen, M. A., 53–55 De Oliveira-Munding, C. C., 496–497 de Paz, J. L., 418 Deprez, P., 183 Desai, C. J., 304 Desaire, H., 72 Desbordes, S. C., 334 Detraz, M., 495 Dettin, L., 202, 271 Dhodapkar, M., 217–219 D’Hooge, R., 53 Dhoot, G. K., 52–53 Dias-Baruffi, M., 142, 204, 282–283, 432, 440 Diaz-Cano, S., 202 Di Campli, A., 496 Dick, M., 52 Diefenbach, B., 454 Dierks, T., 52–53, 69–70, 72–73 Diez-Roux, G., 52 Di Guglielmo, G. M., 248–249 Dimitroff, B. D., 135, 331, 334 Dimitrov, A., 269 Dimopoulou, A., 495–497, 499, 504 Dinchuk, J. E., 356 Ding, J. L., 142 Dings, R. P., 205 Di, P. T., 152 Distler, U., 122 Ditto, D., 255–257 Di Tullio, G., 496 Dixon, J. E., 393 Dlugosz, M., 358 Do, A. T., 52–53 Doenhoff, M. J., 120–121, 123–129 Dohmae, N., 456 Domenga, V., 376 Domon, B., 74, 76, 125 Doms, R. W., 270, 499 Dong, B., 298–300, 303, 305, 308–310, 316, 357 Dong, Y., 480, 486 Donner, P., 357 Doolman, R., 187 Dornadula, G., 273

Author Index

Douek, D. C., 269 Dow, J. M., 91 Draguhn, A., 496–497 Dreisewerd, K., 122 Dreyfuss, J. L., 6 Drickamer, K., 131, 134, 165–178, 270, 423 Drinnan, M., 506 Driver, S. E., 340, 480 D’Souza, B., 377 Duan, L., 269 Dubendorff, J. W., 144 Dubois, M., 517 Duckworth, A. M., 331, 335 Ducros, A., 376 Du, 404, 407 Dumax-Vorzet, A. F., 71, 73 Duncan, D. M., 41 Duncan, J. E., 316, 327, 332, 336 Dunker, R., 454 Dunn, J. J., 144 Dunn, K. W., 495 Dunoe, M., 478 Durbin, R, 480, 486 Durell, S., 269 Dyer, J., 532 Dykxhoorn, D. M., 497 Dzierszinski, F., 236 E Earl, L. A., 230, 247 Eaton, S., 312, 332–334 Ebel, H., 52 Ebe, Y., 418 Eckmann, L., 142 Egeblad, M., 53 Egge, H., 312–313 Egge, J., 314 Egger-Adam, D., 335 Egusa, N., 327, 329, 332 Ekuni, A., 452 Elbein, A. D., 257 Elfenbein, A., 3–28 El Hour, M., 405 Elkahloun, A. G., 376 Ellgaard, L., 183 Ellisen, L. W., 376 Elola, M. T., 167, 172, 174 Elson, C. O., 234 Emerson, C. P. Jr., 52–53 Endlich, N., 496–497 Endo, T., 343, 452–453, 456 Engel, D., 496–497 Engels, D., 119 Engstrand, L., 514 Ens, W., 454 Epstein, E., 331, 335 Erbs, G., 91

545

Author Index

Eriksson, A. U., 220 Ernst, L. K., 520 Esko, J. D., 53, 67–68, 72, 79, 298, 316, 324, 329 Esser, M. T., 274 Estes, J. D., 269 Estrella, R. P., 74 Ethier, M., 454 Etienne, A. T., 302 Eugster, C., 334 Evans, C. A., 392 Evans, D. G., 514 Evans, D. J. Jr., 514 Evans-Holm, M., 385 Evrard, Y. A., 358, 387 F Fabini, G., 478 Fainboim, L., 201–202, 224, 229, 231 Fairclough, S., 184 Fais, M., 78 Faissner, A., 316 Falk, P., 514 Fan, B.-L., 515 Fang, J., 478 Fan, Y. Y., 143 Fata, J., 452 Fathman, C. G., 200 Fay, J. W., 152 Fazio, F., 167, 419 Feasley, C. L., 282 Feinberg, H., 134, 167–168, 270 Feinberg, J., 256 Feizi, T., 167, 270, 418 Feldrappe, S., 202 Felix, K., 53 Fellert, S., 331, 335 Feng, Y., 334 Fernandes, B., 463, 467 Fernandez, H. R., 340 Fernando, C. G., 472 Fernig, D. G., 67, 78, 81 Ferrara, C., 496 Ferrari do Outeiro-Bernstein, M. A., 17 Ferrero, M., 202 Field, R., 68, 78 Fieschi, C., 256 Fife, B. T., 200 Figdor, C. G., 130, 132, 134, 270 Filipe-Santos, O., 256 Fimmel, C. J., 495, 497 Finger, A., 182 Finnegan, C. M., 269 Fiore, R., 405 Fire, A., 340, 480 Firkus, C., 334 Fischer, B., 495–497, 499, 504 Fisch, T., 170–171, 423

Fitch, F. W., 233 Fitzgerald, M. L., 53 Flanagan, J. G., 331, 335 Flitsch, S., 78 Florian, J. A., 13 Flower, R. J., 212 Focht, R. J., 356 Foley, S., 358 Folks, T., 273 Follin, P., 204 Ford, M., 67 Ford-Perriss, M., 67 Forgac, M., 497 Forsberg, L., 514 Fortin, J. F., 270, 273–274, 279 Foster, D. C., 356–357 Fouchier, R. A., 269 Foulquier, F., 495–497, 499, 504 Fournier, D., 142, 145 Fouts, T. R., 269 Fox, A. N., 331, 335 Fox, B., 300, 324–325, 327, 329, 331, 334 Franch-Marro, X., 39, 41, 43, 332–334 Frantz, G. D., 53 Frasch, C. E., 99 Fraser, A. G., 480–481, 486 Frech, M., 454 Fredman, P., 300 Freeman, S. D., 53 Freeze, H. H., 256, 497 French, A. T., 142 French, D. M., 53 Frese, M. A., 52 Frick, I.-M. ., 514 Friedl, A., 53 Friedrich, W., 495, 497 Friese, K., 271 Friess, H., 53 Fritsch, C., 335 Fryer, C. J., 377 Fuertes, M. B., 201 Fuhrmann, J. C., 495, 497 Fujii, R., 187 Fujii, T., 248, 250, 259–260 Fujimoto, H., 518 Fujise, M., 39, 334 Fujita, H., 187 Fujita, K., 53 Fujita, M., 324–325, 329, 504 Fukase, K., 357 Fukuda, M., 229–230, 250, 272, 298 Fukuda, M. N., 256 Fukuda, T., 445–456 Fukuhara, N., 78, 335 Fukui, S., 418 Fukumori, F., 463 Fukushima, K., 142 Fulcher, J. A., 220

546

Author Index

Furia, M., 340 Furmanek, A., 403 Furukawa, K., 326–327, 343, 355–369, 387, 452–453, 513–524 Furukawa, T., 377 Futai, M., 246–248, 250, 257, 279, 432 Futch, T. A., 331 Fuwa, T. J., 305 G Gabius, H. J., 202, 229, 232, 246, 248, 271 Gabrilovich, D., 216–218 Gajewski, T. F., 233 Galanos, C., 94 Gallagher, J. T., 52–54, 66–68, 70, 73, 76, 79–80 Gallay, P. A., 269–270 Gallet, A., 335 Galli, C., 188 Gallo, S. A., 269 Galluzzi, L., 236 Galustian, C., 418 Galvan, M., 230 Gancedo, J. M., 530 Gan, L., 358, 387 Gao, F., 277 Gao, G., 68, 79–80, 234, 247–250, 252, 254, 256–257, 259–262 Gao, W., 234 Garbani, M., 141–148 Garcia, M., 202, 231 Garcı´a-Vallejo, J. J., 131, 166 Garczynski, S. F., 143 Gardner, R. G., 182 Garg, V., 376 Garin, M. I., 202, 232 Garman, S. C., 183 Garrity-Park, M. M., 53 Gasiunas, N., 52–53, 76 Gauhe, A., 523 Gauss, R., 182, 189 Gaver, R. C., 124 Gebhard, H. H., 454 Ge, C., 358 Geffner, J. R., 219–220, 228 Gehring, W. J., 299 Geijtenbeek, T. B. H., 130–132, 134, 151–163, 270 Geisbrecht, B. V., 334 Gendelman, H. E., 273 Gengyo-Ando, K., 142 Genin, A., 376 George, E. L., 447 Georges-Labouesse, E. N., 447 Georgiou, S. A., 389 Gerardy-Schahn, R., 357, 377 Gertz, A., 256 Gerwig, G. J., 105 Ge, X., 335

Geyer, E., 129 Geyer, H., 76, 117–136, 303 Geyer, R., 76, 117–136, 303, 312, 314 Ghirlando, R., 334 Ghose, A., 331, 335 Ghosh, P., 182 Ghosh, S., 155 Gibbs, L., 212 Giese, T., 53 Giguere, D., 281 Gilbert, C., 270 Gilbert, P., 277 Gil, C. D., 203 Gildersleeve, J. C., 418 Gilles, K. A., 517 Ginsberg, M. H., 447 Ginsburg, V., 122 Giordana, E., 340 Giorgi, D., 520 Giraldez, A. J., 331, 334 Giraudo, C. G., 497 Girbes, T., 423, 428 Gitt, M. A., 270 Glasson, S. S., 405 Glickman, J., 497 Glickman, M. H., 183 Glise, B., 334 Glossl, J., 423 Glushka, J., 357 Goda, E., 327–328, 330, 336, 338 Godelaine, D., 248–249 Goder, V., 182 Goel, H. L., 446 Goepfert, P., 277 Goetz, J. G., 248–249 Goh, K. L., 447 Gohlke, M., 357 Goldfarb, M., 68, 79–80 Goldstein, I. J., 142, 423, 428 Golshayan, D., 202, 232 Goltz, J. S., 328, 330 Goltzman, D., 356 Gomez, C. M., 304 Go´mez, R. M., 205 Gonzalez de Peredo, A., 403 Goodman, C. S., 304, 335 Goodman, R. M., 334 Goodman, S. L., 454 Gopal, S., 11 Gordon, W. R., 377 Gore, B. B., 53 Gorfinkiel, N., 334 Goridis, C., 316 Go¨rnig, M., 202 Gorovoy, M., 9 Goswami, D., 332 Goto, S., 328–330, 336, 338, 340, 343 Gotta, M., 480, 486

547

Author Index

Gotte, M., 53 Gourdine, J. P., 142, 432, 440 Grabe, M., 497 Grabenhorst, E., 497 Grabitzki, J., 312 Graham, S. A., 167, 170–172, 174 Granger, D. N., 216 Granovsky, M., 248–249, 252, 255, 257–258, 452 Grant, D., 53 Graul, R. C., 532 Greenbaum, M. P., 382 Greenberg, P. D., 248–249, 256 Greene, D. K., 13 Greene, E. L., 53 Greene, K. M., 277 Greene, V. B., 405 Greene, W. C., 161, 268 Green, J. M., 230 Green, R., 256 Greferath, U., 67 Grego-Bessa, J., 376 Gregorio, G., 270 Greijer, A. E., 279 Greis, K. D., 305 Grewal, P. K., 246 Gridley, T., 358, 387 Griffioen, A. W., 205 Griffitts, J. S., 143 Grigorian, A., 245–262 Grimes, M. K., 274 Grimm, C., 121, 129 Gringhuis, S. I., 131–132, 151–163 Grinstein, S., 497 Grobe, K., 67 Grompe, M., 52 Grossfeld, P. D., 376 Grossmann, J. G., 454 Grove, K., 369 Gruenewald, S., 495–497, 499, 504 Gru¨nert, M., 5 Grunow, D., 357 Gruppi, A., 201–202, 271 Guan, K., 157 Guay, G., 248–249 Guenneau, S., 76 Guerardel, Y., 310 Guerrero, I., 332–334 Guerrero, R. B., 53 Guggino, S. E., 497 Guggino, W. B., 497 Guillot, J., 145 Guimond, S. E., 6, 65–81 Guiral, E. C., 53 Gu, J., 445–456, 463 Gunther, W., 496–497 Guo, H. B., 452, 463, 465, 467 Guo, S., 520 Guo, X., 146

Guo, Y., 134, 167–168 Guo, Z., 335 Gustafsson, A., 515 Gustafsson, M. K., 52–53 Gutierrez, E., 528–529, 535 Gutternigg, M., 479 Guzzetta, A. W., 357 Gyback, H., 515 H Haab, B. B., 423 Haase, A. T., 269–270 Habuchi, H., 67, 328–329, 336, 343 Hacker, U., 316, 328, 330 Hackler, R., 478 Hadari, Y., 440 Hafmans, T., 53 Haggarty, B. S., 270 Hagiwara, K., 248–249, 255 Hahn, H. P., 220 Haier, J., 122 Haines, N., 326–327, 343, 357–358, 387 Hakomori, S. I., 118, 128, 453, 456, 463 Halbisen, M. A., 334 Hallgreen, P., 402 Haltiwanger, R. S., 356–359, 377, 379, 382, 386–390, 392, 395, 401–413 Hamilton, J. K., 517 Hammarstrom, L., 515 Hammes, A., 334 Hampton, R. Y., 182 Hamshou, M., 142 Han, C., 35–36, 38–39, 327, 331–332, 334, 336 Hancock, W. S., 462 Handerson, T., 463 Handjiski, B., 202, 231 Hanisch, F.-G., 299, 309 Hanneman, A. J., 302–304, 479–480 Hannier, S., 233 Hannon, R., 212 Hansson, G. C., 495, 497 Han, T., 53 Hao, T., 480–481, 486 Haq, M., 356 Hara-Chikuma, M., 497 Harigaya, K., 330, 358, 387 Harjacek, M., 202 Harn, D. A., 119 Harrelson, A. L., 304 Harrington, L. E., 234 Harris, A. K., 13 Harrison, R., 170 Harris, R. J., 356–357, 377, 402 Hart, A. C., 532 Hartenstein, V., 378 Hart, G. W., 305, 369 Hartmann, S., 403

548 Hartwig, J. H., 454 Hasan, M. T., 501 Hascall, V. C., 67 Hasegawa, A., 386 Hase, S., 356–357, 377, 479 Hashidate, T., 246–248, 250, 257, 279, 432 Hashimi, S. T., 220 Hasilik, A., 182 Haslam, S. M., 76, 124–126, 143, 178, 255, 257, 303, 316, 479–480 Hassink, G., 187 Hassler, C., 187 Hata, Y., 432 Hatton, R. D., 234 Hauptrock, B., 248–249, 256 Hauri, H. P., 183 Hause, B., 142 Hawiger, D., 217–219 Hayashida, K., 10 Hayashi, N., 452 Hayashi, S., 328, 330 Hayashi, Y., 335 Hayden, M. S., 155, 157 Hayes, B. K., 305 Head, S., 167, 419 He, B., 53, 57, 59 Hebert, D. N., 183, 193 Hebrok, M., 53, 57 Hedlund, M., 431, 440 Heeringa, L., 423 Heimburg-Molinaro, J., 142, 432, 440 Heinz, E., 119 He, J., 230 Helenius, A., 183, 499 Helling, F., 312–313 Hellsten, U., 184 Helms, W. S., 234 Helwig-Rolig, A., 478 Hel, Z., 269 Hembd, C., 316 Hemmerich, S., 52–53, 55 Henderson, L. E., 276, 286 Henderson, N. L., 356 Hendrickson, W. A., 270 Hengartner, M. O., 141–148 Henkel, B., 312 Henkin, J., 356 Hennet, T., 143, 479 Henrissat, B., 143 Henttinen, T., 53 Hermjakob, H., 446 Hernandez, J. D., 201, 224, 228–231, 233, 235, 247, 284 Herscovics, A., 193 Heslip, T. R., 331, 335 Hess, D., 406, 408, 412 Heuser, J. E., 497 Hiesinger, P. R., 382, 385

Author Index

Higashi, M. E., 331, 335 Higashi, S., 328, 330, 358 Hikawa, N., 282 Hilbink, F. J., 98 Hildreth, J. E., 270, 277 Hill, B., 269 Hill, D. E., 480–481, 486 Hillen, W., 501 Hillman, P. R., 335 Hincapie, M., 462 Hindsgaul, O., 250, 520 Hirabayashi, J., 190, 202–204, 208, 212, 229, 231–232, 235, 246–250, 255, 257, 270–271, 279, 284, 418, 432 Hirabayashi, Y., 305, 324–325, 329, 340, 527–539 Hirakawa, Y., 432 Hirashima, M., 201, 246–248, 250, 257, 279, 432 Hirata, R., 70 Hiroi, T., 282 Hirota, K., 202, 232 Hirozane-Kishikawa, T., 480–481, 486 Hirschberg, C. B., 250, 369, 479–480 Hirsch, C., 182–183 Histen, G., 377 Hitchcock, A. M., 70, 74, 76 Hitchen, P. G., 167, 172, 174, 178 Hivroz, C., 248–249 Hjerpe, A., 70 Ho, B., 142 Hoeppner, M. P., 142 Hoet, R. M., 53 Hoffmann, G. F., 478 Hoffmeister, K. M., 454 Hofsteenge, J., 403 Hogan, C., 269 Hohenester, E., 78, 335 Hojo, H., 357 Hojrup, P., 356 Hokke, C. H., 119, 126, 418 Holgersson, J., 515 Holme, A. D., 53 Holst, C. R., 53 Holst, O., 89–114 Holt, C. E., 78 Honegger, B., 529 Hong, K., 328, 330 Honjo, T., 377 Honke, K., 448–449, 453, 456 Hood, J. D., 6 Hooper, L. V., 142 Hope, I. A., 481 Hope, T. J., 270 Hopwood, J. J., 70, 80 Horie, H., 282 Hori, M., 452 Hori, T., 313 Horn, S. C., 182

549

Author Index

Horowitz, A., 269 Horton, J. R., 423, 432, 434, 440 Hoskins, R. A., 385 Hosokawa, N., 181–193 Hosomi, O., 515 Hosono-Fukao, T., 53–54, 57, 61 Hossain, M. M., 53–54, 57, 61 Hou, X., 358 Hovingh, P., 70 Howitt, J. A., 78, 335 Hoxie, J. A., 270 Hronowski, X., 358 Hsiung, F., 334 Hsu, D. K., 142, 229, 247, 271, 279 Hsu, K. L., 235 Huang, C. L., 248, 376 Huang, R. T., 312 Huang, S., 532 Huang, Y., 308 Huber, R. M., 356 Hu, D., 187, 190 Huffaker, T. C., 478 Huflejt, M. E., 167, 419, 440 Hufnagael, L., 41 Hughes, B. L., 483–485, 487–488, 490–491 Hughes, R. C., 270–272 Hughson, F. M., 497 Hui, N., 497 Hu-Li, J., 233 Hulinsky, R. S., 326–327 Hultberg, A., 515 Humphrey, M., 331 Humphries, J. D., 446 Humphries, M. J., 446, 454 Hung, H. C., 269–270 Hunte, F., 497 Hunter, W. M., 172 Hurley, A., 269 Hurtig, M., 514 Hurtley, S. M., 183, 499 Hurvitz, J. R., 405 Hussain, S. A., 78, 335 Hyde, P., 68, 79 Hynes, R. O., 6, 331, 447 I Iacobelli, S., 201, 271 Ibanez, C., 334 Ibraheem, A., 26 Ibrani, D., 357, 379, 382, 386–388, 390, 392 Ichikawa, A., 479 Ichimiya, T., 343 Ideo, H., 142 Idris, M. A., 121, 125–126, 129 Iglesias, M. M., 201–202, 229, 271–272 Ihara, H., 453, 456 Ihara, Y., 183, 453 Iijima, J., 453

Ikeda, R., 187 Ikeda, T., 316 Ikeda, Y., 248, 250, 259–260, 452, 456, 463 Ikehara, S., 221 Ikekita, M., 521 Ikenaka, T., 356, 377 Ikura, K., 479 Ilagan, M. X., 376 Ilarregui, J. M., 199–239, 247, 284 Ilver, D., 514 Imanari, T., 70 Imberty, A., 120–121, 123, 133–134 Inaba, K., 217–219, 221 Inagaki, F., 313 Inamori, K., 448–449, 453 Inazu, T., 456 Incecik, E. T., 514 Ingale, S., 473–474 Inomata, M., 521 Inoue, H., 282 Inoue, M., 317 Inukai, T., 515 Ioannidis, J. P., 256–257 Iobst, S. T., 167 Ioffe, E., 256 Iozzo, R. V., 6 Ipe, U., 330 Irimura, T., 515 Irvine, K. D., 300, 326–327, 343, 357–358, 366, 387, 389 Isaac, N. A., 454 Isaji, T., 445, 456 Ishida, H. K., 202, 248–249, 255, 305, 340, 456 Ishida, N., 328, 330 Ishii, S., 190 Ishikawa, D., 122, 129 Ishikawa, H. O., 328, 330, 358, 387 Ishikawa, Y., 282 Ishimaru, T., 53 Ishimizu, T., 357 Isogai, Y., 184 Isomoto, H., 53 Israel, A., 376–377 Isturiz, M. A., 220 Itoh, S., 316, 448–449, 453, 456 Ito, M., 356–358, 363, 365–369, 387 Itonori, S., 299, 313 Ito, Y., 183, 190, 518 Ivanovic, V., 202 Ivkovic, S., 405 Iwaki, D. D., 334 Iwakura, Y., 317 Iwamoto, K., 142 Iwanaga, S., 356–357, 377 Iwata, S., 324–325, 329 Iyoda, T., 217–219 Izumikawa, T., 327, 329, 332 Izumi, S., 53, 326–329, 334, 336–338, 340, 343 Izutsu, K., 376

550

Author Index J

Jablons, D. M., 53, 57, 59 Jackle, H., 331–332, 335 Jackson, J. B., 272 Jackson, K. K., 53 Jackson, M. C., 335 Jackson, S. S., 201, 231 Jacobsen, T. L., 386 Jacobs, L., 247 Jaeken, J., 256, 497 Jafar-Nejad, H., 357, 375–395, 402 Jahn, H., 496–497 Jahn, R., 496–497 Jakob, C. A., 182–183 Jakobi, R., 478 Jamieson, S. W., 376 Janeway, C. Jr., 131 Jang-Lee, J., 126 Jan, L. Y., 304 Jann, K., 95–96 Jan, Y. N., 304 Jaques, A., 302 Jarosch, E., 182 Jarriault, S., 376–377 Jarvis, D. L., 326–327 Jasiulionis, M. G., 452 Jayson, G., 67 Jegalian, A. G., 142 Je´gouzo, S. A. F., 170 Jenniskens, G. J., 53–55, 57, 61 Jentsch, T. J., 495–497 Jeschke, U., 271 Jeyaretnam, B. S., 95 Jigami, Y., 338, 456 Joachimiak, A., 454 Johansson, L., 514 Johansson, P., 514 Johns, M., 331, 335 Johnson, K. G., 331, 335 Johnson, P., 247, 271 Johnson, R. L., 358, 387 Johnston, S. H., 358, 387, 389 Jones, A. S., 96 Jones, G. C., 404 Jones, K. A., 377 Josefsson, E. C., 454 Josefsson, L. G., 532 Joshi, B, 248–249 Joutel, A., 376 Juarez, C. P., 202 Jungalwala, F. B., 316 Jurado, G. A., 463 Juszczynski, P., 202, 224, 231–232 Ju, T., 142, 432, 440, 456, 463 K Kabat, D., 277 Kacskovics, I., 515

Kadoya, T., 282 Kaffashan, A., 358 Kahn, C. R., 535 Kaiser, E., 495, 497 Kakuda, S., 317 Kaltner, H., 246, 248, 271, 440 Kalus, I., 53, 69–70, 72–73 Kaluza, V., 331 Kamar, M., 452 Kamath, R. S., 480–481, 486 Kamerling, J. P., 105 Kameyama, A.., 453 Kamimura, K., 328–329, 331, 334, 336, 343 Kamiya, D., 182–184, 190 Kamiyama, S., 327–330, 336, 338, 343 Kamiya, Y., 181–193 Kanai, M., 358 Kanapin, A., 480, 486 Kane, C., 236 Kankel, D. R., 331 Kanneganti, T. D., 152 Kannicht, C., 357 Kantelhardt, S. R., 120, 123–124, 126–128 Kappes, J. C., 277 Kapsenberg, M. L., 130 Karamanos, N. K., 70 Karamanska, R., 78 Kariya, Y., 445, 448–449, 453, 456, 458 Karlsson, A., 204 Karlsson Hedestam, G. B., 269 Karlsson, K.-A., 120, 514 Karlsson, N. G., 74, 495–497 Karmakar, S., 204, 282–283 Kasai, K. I., 183, 190, 202–204, 208, 212, 229, 246–248, 250, 257, 270, 279, 432 Kasai Ki, K., 432 Kasper, D., 495, 497 Kato, K., 181–193 Kato, M., 67 Kato, R., 456 Kaufmann, M., 201, 271 Kaufmann, S. H., 53 Kavi, H. H., 340 Kavvoura, F. K., 256–257 Kawabata, S., 142, 356, 377 Kawaguchi, M., 479 Kawahara, K., 187 Kawakami, H., 521 Kawakita, M., 328, 330 Kawamura, C., 453, 456 Kawar, Z. S., 126 Kawasaki, N., 190, 316, 448–449, 453, 456 Kawasaki, T., 316–317 Kawashima, H., 515 Kayed, H., 53 Kearse, K. P., 369 Keino-Masu, K., 52 Keldermans, L., 478 Keller, D. S., 499

Author Index

Keller, M., 312–314 Kelley, J. A., 356 Kellokumpu, I., 495–497 Kellokumpu, S., 495–497 Kelly, R. J., 520 Kelly, S., 497 Kempf, A., 299 Kentzer, E. J., 356–357, 402 Kerek, F., 105 Kerner, D., 312 Kersulyte, D., 514 Kessler, D. S., 53 KewalRamani, V. N., 134, 270 Khalil, A. A., 335 Khaliullina, H., 334 Khare, N., 34, 331 Khodoun, M., 327, 331–332, 334, 336 Khokha, R., 452 Khoo, K.-H., 126–128, 302 Khoruts, A., 269 Kidd, T., 335 Kidwell, D. A., 129 Kieckbusch, R., 497 Kierjoffe, E. B., 201, 229, 231 Kikkert, M., 187–188 Kikuchi, N., 328–330, 336, 338, 343 Kilby, J. M., 277 Kimata, K., 53–54, 57, 61, 67, 328–329, 336, 343 Kimble, J., 377 Kim, B. T., 317, 326–327, 329, 332 Kim, C., 182 Kim, J. J., 182 Kim, P. S, 269 Kimura, Y., 405 Kim, W., 182 Kim, Y. M., 142 Kim, Y. S., 70, 73, 78 King, I. N., 376 King, N., 184 Kinoshita, A., 70 Kinoshita, T., 495–507 Kinoshita-Toyoda, A., 300, 305, 324–325, 328–330, 336, 338, 343 Kintner, C., 377 Kirk, M., 305 Kirkpatrick, C. A., 39, 41, 331, 334–345 Kisiel, W., 356–357, 377 Kisslinger, J. A., 377 Kitada, T., 452 Kitagawa, H., 70, 317, 326–327, 329, 332 Kitagawa, M., 330, 358, 387 Kita, M., 67 Kitamura, N., 521 Kitazawa, T., 53 Kittelberg, R., 98 Kizuka, Y., 316 Kjaergaard, S., 478 Kjeldsen, L., 204

551 Kjelle´n, L., 4, 66 Klagsbrun, M., 68, 79 Klambt, C., 330 Klechevsky, E., 152 Kleeff, J., 53 Klemm, E. J., 187 Klinger, M. M., 402 Kloetzel, P. M., 188 Klunder, R., 129 Knight, P. A., 142 Knoblich, J. A., 379 Knox, S. M., 334 Kobata, A., 452–453, 463 Kobayashi, S., 335 Kobelt, P., 202, 231 Kobialka, S., 9 Kogure, T., 515 Kohatsu, L., 142 Koh, T. W., 382 Kohyama-Koganeya, A., 340, 533– 537 Koike, S., 52 Ko, J. H., 452 Kokkonen, N., 495–497 Kolarich, D., 331 Kolatkar, A., 167 Kolb-Maurer, A., 257 Kolin, T., 520 Komiyama, Y., 356–357 Kondo, A., 448–449, 453 Kondo, T., 356–358, 363, 365–369, 387 Koning, F., 188 Konse, T., 308 Koo, B. H., 405 Koopman, R., 539 Kopan, R., 376–377 Kopcow, H. D., 202, 229 Kopito, R. R., 182 Koreth, J., 480–481, 486 Kornak, U., 495–497, 499, 504 Kornberg, T. B., 334 Korner, C., 478 Kornfeld, R., 246 Kornfeld, S., 182, 246, 250, 252, 463–464, 497 Korn, T., 234 Koseki-Kuno, S., 418 Kosik, K. S., 358 Kostas, S. A., 340, 480 Kostova, Z., 182–183, 188–189 Koul, O., 316 Koutsoukos, M., 277 Koyama, N. S., 283 Koyama, T., 328–329, 336, 343 Kozma, K., 406 Kozono, Y., 248–249, 255 Kozutsumi, Y., 316 Kraakman, L., 532 Krantz, I. D., 376 Kreppel, L. K., 369

552

Author Index

Kreuger, J., 6, 39, 41, 331, 334 Krieger, E., 130 Krieger, M., 497 Krin-Safran, C., 7 Krishnamoorthy, L., 283 Krogh, T. N., 356 Krokhin, O., 454 Krufka, A., 68, 79 Kruse, J., 316 Kruse, N., 257 Krylov, V. B., 357, 377 Kuball, J., 248–249, 256 Kuchroo, V. K., 234 Kuenzler, M., 143, 146 Kuhmann, S. E., 277 Kuhn, R., 523 Kuijk, E., 480–481, 486 Kullolli, M., 462 Kumano, K., 376 Kuno, A., 235, 248–249, 255, 284, 418 Ku¨nzler, M., 141–148 Kuo, A., 184 Kuo, W. L., 376 Kurata, S., 142 Kurihara, H., 316 Kuro, O. M., 248 Kurosu, H., 248 Kusche-Gullberg, M., 52–53, 66, 327, 329, 332 Kusumoto, S., 357 Kutok, J. L., 202, 231–232 Kwon-Chung, K. J., 386 Kwon, D. S., 134, 270 Kyriakides, T. R., 405 Kyselova, Z., 462 L Labosky, P. A., 53 Laemmli, U. K., 144 Laferte, S., 463 Lagana, A., 248–249 Lahmann, M., 515 Lahm, H., 440 Lai, J. P., 53 Laine, R. A., 312 Lajoie, P., 248–249 Lakso, M., 486 Lal, A., 256 La, M., 203–204, 208, 212 Lamanna, W. C., 52–53 Lamar, E., 377 Lamari, F. N., 70 Lambert, A. A., 270 Lambie, E. J., 479 Lamkanfi, M., 152 Lamontagne, G., 270 Landa, C. A., 202, 229, 271 Lander, A. D., 67

Languino, L. R., 446 Lanneau, G. S, 463 Lanni, F., 496 Lannoo, N., 142 Lanzetta, R., 91 Laremore, T. N., 76 Larregina, A. T., 220 Larsen, R. D., 520 Larson, M., 269 Larsson, T. A, 514 Lasanajak, Y., 418, 441 Laskowska, A., 330 Lasky, J. A., 53 Lau, J. M., 74, 76 Lau, K. S., 246–250, 252, 254–256, 259–260, 262, 453 Laurent, N., 78 Lawler, J., 405 Lawrence, P. A., 332 Lawrence, R., 72, 324, 329 Lawson, A. M., 418 Leahy, D. J., 334 Leary, J. A., 52, 72, 76 Leathers, R., 376 LeBail, O., 376 Le Borgne, R., 377 LeBot, N., 480, 486 Lechler, R. I., 202, 232 Leclerc, J. E., 275 Lecourtois, M., 377 Ledeen, R. W., 229, 232 Lederkremer, G. Z., 183–184 Leder, P., 68, 79 Le, D. T., 255, 257 Lee, B., 220 Lee, C., 229, 248 Lee, I., 452, 463 Lee, J. S., 332, 335 Lee, S. S., 298–300, 303, 305, 308–310, 316, 357 Lee, S. U., 247–250, 252, 254, 256, 259–262 Lee, S. Y., 376 Lee, T. V., 375–395, 402–403 Lefeber, D., 495–497, 499, 504 Leffler, H., 246–247, 250, 270–271, 283, 423, 431–432, 440 Legendre, H., 440 Le Goff, C., 404–405 Lehle, L., 479 Lehr, S., 299, 309 Lehr, T., 123 Lei, L., 357–358, 366 Lemaire, K., 532 Lemjabbar-Alaoui, H., 51–62 Lemosy, E. K., 328, 330 Lenardo, M. J., 229 Lennon, G. G., 520 Leonard, C. K., 357 Leonhard-Melief, C., 401–413

553

Author Index

Leontein, K., 103 Leppanen, A. M., 128 Lercher, D. M., 334 Le Roy, C., 248–249 Leroy, Y., 310 Leslie, G. J., 270 Lesnik Oberstein, S. A., 406 Letunic, I., 184 Leung, Y., 202 Levery, S. B., 122, 128, 312 Levi, G., 201 Levinson, S. R., 256 Levis, R. W., 385 Levitzki, A., 170 Lewis, K. A., 5 Lewis, L. A., 249 Leymarie, N., 74, 76 Lian, Z.-X., 515 Liao, G., 385 Liao, Y. F., 256 Liao, Z., 277 Li, A. S., 389 Li, B., 234, 248–249, 257 Liberman, A., 231–232 Li, C. F., 249, 252, 255 Liddington, R. C., 447 Lifson, J. D., 276 Li, H., 300 Li, J., 53–54 Li, L., 376 Lilley, B. N., 187–188 Li, M., 277 Lim, J. M., 298–300, 302–305, 463, 467, 473 Lim, P. G., 165–178 Li, N., 515 Lincecum, J., 53, 317, 326, 331, 335 Lindahl, U., 4, 52, 66–68, 327, 329, 332 Lindberg, B., 103 Lindner, B., 92 Lindner, J. R., 335 Lin, G., 270 Ling, E., 142 Ling, V. T., 357 Linhardt, R. J., 70, 72–74, 76–78, 328, 330 Linker, A., 70 Link, J., 356 Lin, S. C., 334 Linstedt, A. D., 495–497 Lin, X., 33–49, 316, 327, 331–332, 334, 336 Liotta, L. A., 17 Li, Q., 269 Li, S. C., 312 Li, S. W., 405 Litjens, M., 131, 152–153, 155–157, 159, 161–163 Littman, D. R., 134, 270 Litynska, A., 452–453 Liu, B., 271

Liu, F. T., 142, 201, 229, 239, 246–247, 250, 271, 279 Liu, H., 72, 277, 331, 334 Liu, J. J., 72, 328–330, 358 Liu, K., 217–219 Liu, S. D., 231 Liu, Y., 183 Li, X., 53, 376 Li, Y. T., 312 Lochnit, G., 119–121, 123–124, 126–129, 312 Locke, R., 356–357, 377, 387, 390 Loganathan, D., 70, 73, 78 Logeat, F., 376–377 Lohse, D. L., 70, 73, 78 Lollike, K., 204 Long, J. M., 256 Longley, R. L., 12 Lo¨nngren, J., 101, 103 Lopez-Diego, R. S., 228 Lopez, P. H., 118 Loserth, S., 257 Lougarre, A., 145 Lough, T., 340 Loverde, P., 119 Lowary, T. L., 305 Lowe, J. B., 256, 358, 520 Lozynska, O., 52 Luban, J., 192 Lu¨deritz, O., 94 Luders, F., 328, 330 Luescher, I. F., 248–249 Lu, F. M., 356–357, 377, 387, 390 Luhn, K., 330 Lu, K., 52 Lu, L., 453 Lum, D. H., 53 Lum, L., 334 Luna, J. D., 202 Lun, Y., 358, 387 Luo, L., 385 Luo, Y. E. C., 358, 389, 403–404, 406 Lupashin, V. V., 497 Lu, Q., 53 Luther, K. B., 358, 377, 389, 402, 406 Lu¨thy, P., 141–148 Lu, Z. H., 229, 232 Lyon, M., 53–54, 67, 70 M Maass, K., 76, 123–125, 303 Macaluso, F., 246, 248, 271 Macarthur, C. A., 68, 79–80 Maccarana, M., 67 Maccari, F., 70 Maccioni, H. J., 497 MacDonald, A. S., 119, 236 Machen, T. E., 497

554 Mach, L., 423 Macias, J., 376 Maciazek, J., 376 MacLeod, V., 52 Madianos, P. N., 204 Maeda, Y., 338, 495–507 Maes, E., 310 Magnani, J. L., 122, 126, 128 Mahal, L. K., 235, 283 Mahdavi, J., 514 Mahmoud, A., 334 Mahnke, K., 217–219 Mailhammer, R., 316 Majerus, E. M., 404 Makaaru, C. K., 126–128 Makino, A., 376 Makita, A., 120 Malaver, E., 205 Maldonado, C. A., 202, 271 Malina, V., 248–249, 256 Malinda, K. M., 17 Mallucci, L., 201, 271 Mamat, U., 92 Manabe, T., 317 Mangan, P. R., 234 Mann, D. L., 286 Mann, R. S., 331 Manonmani, A., 332 Manya, H., 343, 456 Marcantonio, E. E., 13 Marchand, O., 334 Marcu, O., 331, 335 Marechal, E., 376 Marek, K. W., 256 Margolis, R. U., 456 Mariappan, M., 52 Markert, U. R., 202, 231 Markowitz, M., 269 Marois, E., 334 Marra, P., 496 Marr, M., 184 Marshall, B., 514 Marsh, J. L., 331, 335 Marth, J. D., 246, 248, 255–258, 463 Martini, R., 316 Martin, M. A., 269, 273 Martin, R., 227–228 Martinsson, T., 478 Marz, L., 304, 423 Mascola, J. R., 277 Massa, S. M., 271 Masu, M., 52, 328–329, 336, 343 Matani, P., 304 Mathieson, W., 126 Matsuda, M., 14 Matsuda, T., 356–358, 361, 363, 365–369, 387 Matsuhisa, A., 142 Matsui, A., 52

Author Index

Matsui, T., 152 Matsumoto, A., 376, 453 Matsumoto, M., 142 Matsumoto, N., 187, 190 Matsuno, K., 328, 330, 358, 387 Matsuo, I., 183 Matsuo, T., 334 Matsuura, A., 356–358, 363, 365–369, 387 Matsuura, N., 452 Matsuura, S., 53 Matsuzawa, T., 515 Matta, K. L., 356–357, 377, 387, 390 Mattapallil, J. J., 269 Matthews, H., 9, 14 Matthews, T. A., 53 Matthijs, G., 478, 497 Maughan, M. F., 277 Maurer, M., 257 Maxfield, F. R., 495 Maynard, C. L., 200 Mayo, K. H., 205 Mayor, S., 332, 495 Mazar, A. P., 356 Ma, Z. M., 269 McAuliffe, J., 250 McCracken, A. A., 183 McDermott, S., 331, 335 McDonald, D., 270 McDonald, J., 119 McDowell, R. A., 302 McEver, R. P., 204, 282–283 McEwan, P. A., 454 Mcewen, D. G., 68, 79–80 McGhee, J. R., 269 McGlamry, K. H., 298–300, 303, 305, 308–310, 316, 357 McGraw, T. E., 495 McGuire, S. E., 39 McKerlie, C, 248–249, 252, 254, 256 McLellan, J. S., 334 McManus, M., 53, 57 McNeely, M., 328–329, 336, 343 McQuistan, T., 256 Mechref, Y., 308, 462 Medicherla, B., 182, 188–189 Medzhitov, R., 131, 152 Mehandru, S., 269 Mehta, A., 463 Mehta, P., 246–247, 250, 282–283, 423, 432, 440 Mehta, S. Q., 379 Mehul, B., 272 Mellman, I., 182 Mello, C. C., 340, 480 Meltzer, P. S., 376 Mendelsohn, R., 247–250, 252, 254, 259–262 Menges, H., 314 Menon, G., 356–357 Mercier, S., 271–273, 278–280, 286–287

Author Index

Meredith, T. C., 92 Merkle, R. K., 464 Merry, A., 302 Merry, C. L., 53 Mestecky, J., 269 Methot, S., 269 Metsikko, K., 495 Metzler, M., 256 Meyer, K., 53 Meyer, S., 120–121, 123, 133–134 Miatkowski, K., 358 Miceli, M. C., 201, 231, 246–249 Michelson, A. M., 336 Michiels, K., 142 Middel, J., 134, 270 Miersch, O., 142 Miesenbock, G., 505 Mikami, K., 190 Miki, Y., 316 Militsopoulou, M., 70 Millan, J. L., 256 Miller, C. A., 74, 76, 334 Miller, C. J., 269 Miller, H. R., 142 Miller-Podraza, H, 514 Miller, R. L., 65–81 Mills, P., 497 Milz, F., 52 Minamida, S., 357 Miner, J., 204 Min, H., 100 Minoguchi, S., 377 Minowa, M. T., 248, 255, 258 Mironov, A. Jr., 496 Mirouse, V., 335 Mitani, S., 142 Mitchell, D. A., 134, 168, 270 Mitchell, E., 463 Mitoma, J., 229, 298 Miura, N., 187 Miyamoto, A., 377 Miyamoto, M., 317 Miyamoto, T., 53 Miyasato, M., 518 Miyashita, F., 358, 387 Miyata, T., 356, 377 Miyaura, S., 53 Miyazaki, K., 448–449, 453 Miyazaki, T., 513–524 Miyoshi, E., 358, 448–449, 452–453 Mizuno, M., 456 Mobley, H. L. T., 518 Mochizuki, S., 13 Modesti, N. M., 202, 229, 272 Mo, F. E., 405 Molinari, M., 183–184, 188, 192–193 Molinaro, A., 89–114 Molinaro, R. J., 142, 432, 440

555 Molinder, K. M., 231 Molinero, L. L., 201 Molitor, A., 331, 335 Moloney, D. J., 356–358, 377, 387, 389–390, 402–403 Mondala, T., 167, 419 Mondor, I., 269–270 Montelione, G. T., 356 Montgomery, M. K., 340, 480 Monti, S., 202, 231–232 Montoya, D. P., 53 Montresor, A., 119 Moon, J. J., 13 Moore, H. P., 497 Moore, J. P., 269 Moore, W. M., 53 Moorman, C., 480–481, 486 Morales, J., 405 Moran, M., 249 Morcock, D. R., 276 Mordoh, J., 167, 172, 174, 202, 224, 231 Moreland, M., 299–300, 312–314 Morelli, A. E., 220 Moremen, K. W., 192–193, 256 Moreno, S., 480, 486 Morgan, M. R., 6, 8, 11, 15 Morgan, R., 234, 248–249, 257 Morimoto-Tomita, M., 52–55 Morita, R., 152 Moriyama, T., 182 Moriyama, Y., 497 Morri, H. R., 302 Morris, H. R., 76, 126–128, 143, 255–257, 316 Moser, C. D., 53 Moser, D. R., 53 Moser, M., 53 Moser, S., 496 Mosesson, Y., 10 Mostafapous, K., 423, 428 Motran, C. C., 231 Motto, D. G., 405 Mould, A. P., 454 Mourad, M., 248–249 Mouton, P., 376 Movchan, A. B., 76 Mozaffar, T., 248–249, 252, 254, 256 Mrksich, M., 418 Mueller, B., 187–188, 192 Muin˜o, J. C., 202 Mu¨ller, C., 201, 271 Muller, W. E., 246–248, 250, 257, 432 Muller, W. J., 452 Mulloy, B., 143 Mundlos, S., 53 Munevar, S., 13 Munro, S., 182, 387, 495 Murakami, K., 356–358, 363, 365–369, 387 Murakami, M., 5

556

Author Index

Muramatsu, S., 221 Muraoka, M., 328, 330 Murata, T., 515 Murgia, A. R., 497 Murphy, M., 67 Murugesan, S., 76 Muth, A. N., 376 Mu¨thing, J., 119, 122, 129 Mythreye, K., 4 N Nabi, I. R., 246, 248–249, 258, 453 Naccache, S. N., 8 Nadano, D., 356–358, 363, 365–369, 387 Nagamine, S., 52 Nagasawa, K., 182 Nagashima, K., 283 Nagashima, T., 70 Nagata, K., 182, 190, 193 Nagata, Y., 463 Nagorney, D. M., 53 Nagy, N., 440 Nahm, M. H., 278 Nairn, A. V., 462–464, 467, 472–473 Nair, R. P., 520 Nakabeppu, Y., 205 Nakagawa, H., 256 Nakagawa, K., 356 Nakagawa, T., 448–449, 453 Nakahara, Y., 357 Nakajima, T., 188 Nakakita, S., 479 Nakamura, A., 316 Nakamura, K., 202, 232 Nakamura, M., 300, 316, 340 Nakamura, O., 142 Nakamura, T., 14, 246–248, 250, 257, 279, 432 Nakamura, Y., 326–327, 343 Nakao, S., 358, 387 Nakato, H., 34–35, 53, 67, 326–329, 336–338, 340, 343, 345 Nakayama, J., 256, 298 Nakayama, K., 338, 499 Nakayama, M., 328, 330 Nakaya, S., 453 Nakazaki, K., 376 Nannya, Y., 376 Narimatsu, H., 235 Narisawa, S., 256 Narita, K., 53 Nass, R., 486 Natoli, C., 201, 271 Natsuka, S., 357, 479 Naundorf, A., 518 Nawroth, R., 53, 57 Nayak, M., 331, 335 Needham, L. K., 122

Neff, N. T., 356 Negrotto, S., 205 Nei, M., 186 Nelson, N., 497 Nesmelova, I., 205 Neuber, O., 182 Neumann, C. J., 36 Neumeister, B., 147 Neutz, S., 312 Newbold, R. F., 52 Newell, J., 497 Newman, M.-A., 91 Ng, D. T., 182 Ng, P. M., 142 Nguyen, A., 256–257 Nguyen, H. T., 53 Nguyen, J. T., 204, 230 Nguyen, K., 298–300, 303, 305, 308–310, 316, 357 Nguyen, P. L., 269 Niamy, H., 74, 76 Nicaise, J., 248–249 Nice, E. C., 356 Nickel, W., 271–272 Nicot, A. S., 480–481, 486 Niehrs, C., 187 Nieminen, J., 248, 271, 279, 284 Nifantiev, N. E., 357, 377 Ni, H., 270 Nilson, L. A., 328 Nilsson, T., 495, 497 Nishihara, S., 305, 323–346 Nishikawa, A., 453 Nishikawa, M., 190 Nishimura, H., 356–357, 377 Nishimura, S., 256, 324–325, 329 Nishimura, Y., 269 Nishi, N., 246–248, 250, 257, 432 Nishi, T., 497 Nishiuchi, R., 448–449, 453 Nita-Lazar, A., 358, 389, 392, 395, 407 Nita-Lazar, M., 182 Niu, H.-L., 515 Nobauer, K., 146 Noda, K., 358, 452 Nolo, R., 36, 41 Nomura, K., 142 Nomura, T., 202, 232 Nores, G. A., 312–313 Norga, K. K., 382 Norling, L. V., 204–205, 209, 212 North, S. J., 178, 316 Noti, C., 418 Nottet, H. S., 134, 270 Nourshargh, S., 213 Novina, C. D., 497 Novotny, M. V., 308 Nsimba-Lubaki, M., 423, 428

557

Author Index

Nuck, R., 357 Nugent, M. A., 6, 66 Nu´n˜ez, G., 152 Nurcombe, V., 67 Nurnberg, P., 495–497, 499, 504 Nussenzweig, M., 217–219 Nusse, R., 333 Nyame, A. K., 119, 126, 128, 130 Nyfeler, B., 183 Nyholm, P.-G., 514 O O’Brien, D. A., 256 O’Connor, M. B., 331–332, 334–335 Oda, Y., 190 Odenbreit, S., 514 Oggier, D. M., 183 Ogren, J., 514 Oh-Eda, M., 256 Ohkura, T., 248–249, 255 Ohmae, Y., 327, 336–337, 343, 345 Ohto, T., 52 Ohtsubo, K., 248, 255, 257–258, 463 Ohyama, C., 256 Oida, T., 202, 232 Okabayashi, K., 386 Okada, Y., 324–325, 329 Okajima, K., 376 Okajima, T., 326–327, 343, 355–369, 387, 406 Oka, S., 316–317 Oka, T., 246–248, 250, 257, 432, 497 Okawa, K., 182 Oka, Y., 142 O’Keefe, B. R., 418 Okubo, A., 70 Okubo, R., 326–330, 336–338, 340, 343 Olden, K., 447 Olfat, F. O., 514 Oliani, S. M., 203–204, 208, 212 Olieric, V., 146 Olson, D. J., 334 Olson, S. K., 72, 324, 329, 331 Olwin, B. B., 68, 79 Omichi, K., 357 O’Neill, L. A. J., 152 Ono, K., 317 Onozato, M. L., 316 Oosterhof, A., 53 Opoka, R. J., 331, 334 Oppenheim, J. J., 271 O’Quinn, D. B., 234 Orentas, R. J., 270 Ori, A., 67, 81 Oriol, R., 310 Orkin, S. H., 480–481, 486 Ornitz, D. M., 5, 68, 79–80 Orr, A. W., 404

Orsal, A. S., 202, 231 Ortega, B., 248 Osawa, T., 515 Osborn, D. W., 483–485, 487–488, 490–491 Oscarson, S., 515 Oshima, E., 340 Osmond, D. G., 506 Oster, G., 497 Ostman, N., 53 Ota, S., 376 Ota, Y., 376 Othmer, H. G., 332, 334 Ouaaz, F., 152 Ouellet, M., 267, 271–273, 278–279, 286–288 Ouellet, N., 279 Oukka, M., 234 Ounissi-Benkalha, H., 256–257 Ouwendijk, J., 495, 497 Ouyang, J., 202, 231–232 Ovich, G. A., 202 Oyelaran, O., 418 Ozawa, M., 453 Ozawa, Y., 327, 332, 336 P Pace, K. E., 142, 220, 229, 248 Pacienza, N., 205 Packer, N. H., 74 Padva, M., 53 Paganelli, R., 201, 271 Pai, A., 256 Paine-Saunders, S. E., 52–53, 331 Palokangas, H., 495 Palucka, A. K., 152 Panakova, D., 334 Pan, D., 376 Pang, M., 220, 229, 247, 271–272 Pan, H., 357, 379, 382, 386–388, 390, 392 Panico, M., 126, 256 Panin, V. M., 300, 316, 357–358, 387, 389 Panitch, M. M., 523 Panjwani, N., 202 Pankov, R., 8, 14–15 Panneerselvam, K., 256 Pappin, D. J., 177 Papworth, G. D., 220 Paquereau, L., 142, 145 Paraskeva, C., 67 Parekh, R., 302 Parenti, G., 52 Parfitt, K., 331, 335 Park, K. I., 142 Park, P. W., 53 Park, T. J., 76 Park, Y., 328, 330–331, 335 Paroutis, P., 497 Parrilli, M., 89–114

558 Parry, S., 167, 170, 172, 174 Parsa, P., 376 Partovian, C., 9 Partridge, E. A., 246–250, 252, 254–256, 259–260, 262, 452 Partridge, M. A., 13 Paschinger, K., 146, 312, 331, 479 Pascual, V., 152 Pate, J. A., 142 Patel-King, R. S., 447 Patel, N. H., 304 Patel, T. P., 302 Patel, V. P., 249 Patnaik, S. K., 298 Patterson, R. J., 250 Paulson, J. C., 246, 441 Pawelek, J. M., 463 Pawling, J., 234, 248–249, 252, 254, 256–257, 452 Payan, M. J., 273 Pearce, E. J., 119, 236 Pear, W. S., 377 Pector, J. C., 440 Pedersen, A. H., 356–357 Pedersen, J. W., 312 Pedersen, L. C., 328–330 Peeters, B., 423, 428 Peggs, K. S., 200 Pelletier, I., 271, 278–279 Peluso, I., 340 Pemberton, A. D., 142 Pemberton, S., 170 Peng, J., 52 Penman, M., 497 Pepe, S., 52 Perbal, B., 405 Perez, L., 331, 334, 358, 387 Perillo, N. L., 204, 229 Perkins, D. N., 177 Perkins, H. M., 446 Perlman, M., 298–300, 302–305 Perone, M. J., 220 Perreault, H., 454 Perretti, M., 199–239 Perrimon, N., 39, 316, 327–330, 334, 336, 358, 379, 381, 387 Perry, T. L., 335 Pertel, T., 192 Pesoa, S. A., 199–239 Petcherski, A. G., 377 Peter-Katalinic, J., 122, 312–314 Peters, V., 478 Petitou, M., 53–55 Petros, A., 356 Peumans, W. J., 142, 423, 428 Philipsberg, G. A., 369 Phin, S., 331 Phogat, S., 269 Piccoli, D. A., 376

Author Index

Picollo, A., 497 Piddini, E., 334 Pieffers, M., 53–55 Pielage, J., 330 Pierce, J. D., 257 Pierce, J. M., 461–474 Pierce, M., 452, 463 Pierpont, M. E., 376 Pike, B. L, 376 Piller, F., 515 Pincus, D., 184 Ping, J.-P., 68 Piper, M., 78 Pipirou, Z., 165–178 Pircher, H., 201, 271 Pitts, J., 300 Piwon, N., 496–497 Pizette, S., 312 Planells-Cases, R., 495, 497 Plasterk, R. H., 340, 480–481, 486 Platt, E. J., 277 Plemper, R. K., 182 Ploegh, H. L., 187 Plummer, T. H. J., 304 Pochec, E., 452–453 Podhajeer, O. L., 202, 224, 231 Poet, M., 495, 497 Poetsch, J., 423 Po¨hlmann, S., 170–171, 270, 423 Poirier, F., 201, 205, 224, 228–229, 231, 233, 235, 247, 431, 440 Poles, M. A., 269 Polonis, V. R., 277 Poltl, G., 312 Polychronakos, C., 256–257 Pomerantz, R. J., 273 Ponath, A., 257 Ponce, M. L., 17 Pope, M., 270 Popova, E., 304 Popplewell, J. F., 78 Porter, A., 423 Porterfield, M., 298–300, 303, 305, 308–310, 316, 357 Posakony, J. W., 378 Postel, R., 205 Poulin, G., 480, 486 Poulton, J., 335 Powell, A. K., 66–68, 73, 76–78 Powell, J. T., 283 Powlesland, A. S., 165–178, 423 Pownall, M. E., 53 Pozner, R. G., 205 Prasadarao, N., 316 Presley, J. F., 495 Preston, A. B., 283 Preusser, A., 52 Price, D. A., 269

559

Author Index

Prietsch, V., 478 Puntener, U., 496 Puri, A., 269 Puri, S., 495–497 Pusch, M., 497 Puthenveedu, M. A., 496 Puvirajesinghe, T. M., 65–81 Pye, D. A., 68, 79 Q Qian, Y., 283, 431, 440 Qi, M., 376 Qin, S. Y., 190 Quaggin, S., 248–249, 252, 255, 257–258 Quan, E. M., 182–183, 190 Quelhas, D., 497 Quezada, S. A., 200 Quintero-Martinez, A., 165–178 R Raasi, S., 183 Rabbani, S. A., 356 Rabiet, M. J., 204 Rabinovich, G. A., 166, 199–239, 246–247, 249, 271–272, 432 Rabouille, C., 497 Rabson, A., 273 Racz, P., 269 Raemaekers, T., 497 Raetz, C. R., 91 Rahbe, Y., 145 Rajab, A., 495–497, 499, 504 Rajan, A., 357, 376, 379, 382, 386–388, 390, 392 Ralston, A., 334 Ra, M., 504 Ramakrishnan, S., 53 Ramasamy, R., 530 Ramhorst, R. E., 201, 229, 231 Ramirez-Weber, F. A., 334 Rampal, R., 358, 389, 402 Rana, N. A., 357–358, 379, 382, 386–388, 390, 392 Rancourt, A., 279 Randolph, J. T., 515 Randolph, M, 465, 467 Ranganath, H. A., 332 Rangel, C., 331, 335 Ransom, J. F., 376 Ranzinger, R., 124 Rapoport, T. A., 182–183, 188 Rapraeger, A. C., 52, 68, 79 Ratcliffe, N. A., 142 Ratner, D. M., 418 Ratzka, A., 53 Rauch, U., 5 Ravetch, J., 217–219 Raviv, Y., 269

Rawat, S. S., 269 Rawson, J. M., 331, 334–335 Rayburn, H., 447 Ray, R. P., 335 Razi, N., 167, 419 Razzazi-Fazeli, E., 143 Reason, A. J., 302 Reaves, B., 495 Rebers, P. A., 517 Reggio, H., 273 Reilly, C., 269 Reimer, T., 271 Reinhold, V. N., 246–250, 252, 254–256, 259–260, 262, 300, 302, 479–480 Reis e Sousa, C., 218 Reizes, O., 53 Rendic, D., 479 Rendina, R., 340 Ren, Y., 335 Repnikova, E., 300 Reuter, G., 299 Reutter, W., 357 Reynders, E., 495–497, 499, 504 Reynolds, M. M., 331, 335 Reynolds, T. C., 376 Rhodes, J. M., 328–329, 336, 343 Ricardo, S., 334 Rice, K. G., 70, 73, 78 Richard, M., 270 Ricketts, L. M., 403, 407, 413 Ridley, B. L., 95 Rieckmann, P., 257 Riedel, F., 334 Riera, C. M., 202–203, 229, 271–272 Riethmuller, J., 496–497 Rietschel, E. T., 91, 111, 113, 119 Rietveld, A., 312 Riley, E. M., 201, 224, 228–229, 231, 233, 235, 247 Riley, G. P., 404 Rine, J., 182 Riul, T. B., 204 Rivera-Marrero, C., 142, 432, 440 Rivinoja, A., 495–497 Rizzo, L. V., 231–232 Robbins, P. W., 250, 478–480 Roberts, C. E., 128 Roberts, D. L., 182, 187 Roberts, L. R., 53 Robertson, E. J., 205 Robinson, J. E., 269 Roche, N., 514 Rodig, S. J., 202, 231–232 Rodrigues, L. C., 142, 204, 432, 440 Rodriguez, I., 331 Roederer, M., 269 Rohowsky-Kochan, C., 229, 232 Roitelman, J., 187

560

Author Index

Rolland, F., 530, 532 Romani, N., 221 Romero, M. D., 202 Ron, D., 188 Roos, J. W., 277 Rosa, J. C., 302, 479–480 Rosenberg, A. H., 144 Rosenblatt, K. P., 248 Rosen, E. D., 528 Rosen, S. D., 51–62 Rosetti, F., 202 Rosignoli, G., 203 Rossio, J. L., 274 Roth, J., 183, 299 Roth, K. A., 514 Rothman, J. E., 505 Rouge, P., 423, 428 Rouquier, S., 520 Rourke, T., 269 Roy, J., 271 Royle, L., 462 Roy, R., 246, 248, 271, 281 Roy, S., 423, 428 Rual, J. F., 480–481, 486 Rubin, G. M, 36, 376, 381, 385 Rubinstein, N., 201–202, 224, 229, 231, 271 Ruether, K., 495–497 Ruff, L. E., 269 Ruhl, S., 514 Ruoslahti, E., 447 Rupar, M. J., 356 Russell, D., 78 S Saad, O. M., 52, 72, 76 Saag, M. S., 277 Sabesan, S., 246, 248, 271 Sado, Y., 328, 330 Sagman, U., 463, 467 Said, J., 229 Saigo, K., 326–330, 336–338, 340, 343 Saito, T., 518 Saitou, N., 186 Sakagami, J., 317 Sakaguchi, S., 232 Sakaidani, Y., 355–369, 387 Sakai, T., 316, 377 Sakamoto, N., 53 Sakata-Yanagimoto, M., 376 Saksela, O., 5 Sakura, Y., 67 Salatino, M., 219–220, 228–229, 235 Salmen, B., 53 Salmivirta, K., 53 Salmivirta, M., 53 Salomon, D., 358 Saltiel, A. R., 535

Salvaterra, P. M., 304 Salvatore, L., 496 Salyan, M. E., 128 Sampaio, A. L., 205, 212 Sanada, M., 376 Sanai, Y., 328, 330 Sanchez-Irizarry, C., 377 Sanderson, R. D., 52, 67 Sandhu, D. S., 53 Sandoval, D., 529 Sanford, G. L., 283 Sango, K., 282 Sanicola, M., 358 Sano, K., 357 Sanson, B., 334 Sansoucy, B. G., 446 Santer, V., 506 Sanzen, N., 456 Saphire, A. C., 269–270 Sarin, V. K., 356–357 Sarkar, M., 256 Sarnesto, A., 520 Sasai, K., 248, 250, 259–260, 456 Sasaki, K, 250 Sasaki, N., 305, 358, 387 Sasaki, S., 497 Sasaki, T., 518 Sasaki, Y., 452 Sasamura, T., 330, 340, 358, 387 Sasisekharan, R., 52 Satijn, S., 205 Sato, K., 70 Sato, M., 282, 327, 332, 336, 432 Sato, N., 248–249, 255, 257 Sato, S., 248, 267–294 Sato, T., 235, 248–249, 255, 257, 406, 513–524 Sato, Y., 317, 453–455 Sattentau, Q. J., 269–270, 274 Saunders, S., 52–53 Saveriano, N., 506 Savioli, L., 119 Sawyer, T. K., 446 Scanlan, C., 167, 419 Schachner, M., 316 Schachter, H., 246, 256, 304, 312, 447, 479 Schacker, T. W., 269 Schaefer, J. R., 478 Schaefer, L., 4 Schaefer, R. M., 4 Schares, G., 129 Schattner, M., 205 Schatz, P. J., 170 Schauer, R., 299 Scheel, O., 495, 497 Scheraga, H. A., 356 Scherf, W., 423 Schiffer, S. G., 358 Schluterman, M. K., 376

Author Index

Schmid, K. W., 496–497 Schmidt, A., 67 Schmidt, B., 52 Schmitt, A., 495, 497 Schmitz, D., 53 Schnaar, R. L., 118–120, 122, 310 Schneider, D. K., 274 Schneider, M., 335 Schoeb, T. R., 234 Schollen, E., 478, 497 Schrader, J. W., 256 Schreiber, G., 202 Schroeter, E. H., 377 Schuksz, M., 53, 67 Schulenburg, H., 142 Schulz, A., 495, 497 Schulze, A., 188 Schulze, K. L., 382, 385 Schulze, S., 271 Schupbach, T., 328 Schwake, M., 496–497 Schwarz, T. L., 331, 335 Schweingruber, H., 299–300, 312–314 Schweisguth, F., 377 Schweizer, M., 495–497 Schwientek, T., 299, 309 Scott, D. L., 454 Scott, M. G., 278 Scott, M. P., 332, 334 Scott, S. A., 282 Seales, E. C., 463 Seeberger, P. H., 146, 418 Seeger, M., 188 Seelig, L., 182 Segawa, H., 328, 330 Segawa, K., 452–453 Seidah, N. G., 376 Seigner, C., 273 Seiki, T., 316 Seilhamer, J. J., 272 Sekiguchi, K., 448–449, 453, 456 Seki, M., 432 Sellakumar, G., 256 Selleck, S. B., 67, 300, 316, 324–325, 327, 329, 331–332, 334–336 Selva, E. M., 328, 330, 334 Sengelv, H., 204 Sen, J., 328, 330 Seppo, A., 299–300, 304, 310, 312–314, 316 Serpe, M., 334 Serra, H. M., 231–232 Seta, N., 478 Sethi, M. K., 357, 377 Seya, T., 142 Shair, L. H., 356–357, 377, 387, 390 Shaler, T. A., 182 Shao, L., 356–358, 387, 389, 402 Sharma, P., 332 Sharon, N., 446

561 Sharrow, M., 304 Shashidhara, L. S., 332 Shaw, G. D., 167 Shaw, G. M., 277 Shaw, K. M., 483–485, 487–488, 490–491 Shaw, S., 11 Shen, M. M., 358 Sher, A., 131 Shetty, S., 273 Shevach, E. M., 200 Shibata, Y., 188 Shibukawa, Y., 453, 456 Shibuya, N., 91, 423, 428 Shigeta, M., 453 Shimmi, O., 334 Shimonishi, Y., 356–357, 377 Shimoyama, T., 405 Shimuta, M., 317 Shindo, T., 405 Shin, H., 187 Shinya, T., 91 Shipp, M. A., 202, 231–232 Shirane, K., 520 Shire, A. M., 53 Shi, S., 358, 387, 402 Shi, X., 74, 76 Shoji, H., 432 Shridhar, V., 53 Shriver, Z., 52 Shufesky, W. J., 220 Shukla, D., 328–329, 336, 343 Shworak, N. W., 328–329, 336, 343 Siegfried, E., 331 Sierra, J., 334 Sifers, R. N., 183, 192 Silipo, A., 91 Silver, D. L., 405 Silvescu, C. I., 246–250, 252, 254–256, 259–260, 262 Simard, M., 279 Simcox, A. A., 386 Simmer, F., 480–481, 486 Simmons, G., 270 Simons, K., 312 Simons, M., 3–28 Simonson-Leff, N., 393 Singer, M. S., 53, 57, 59 Singh, R. R., 220 Singh, S., 303 Sirois, S., 281 Sitia, R., 183 Sjostrom, R., 515 Skaletz-Rorowski, A., 67 Skar, J., 376 Skidmore, M. A., 69–73 Skjodt, K., 356 Slanina, K. A., 246–247, 250, 283, 423, 432, 434, 440 Sleno, B., 193

562 Smagghe, G., 142 Smith, D. F., 122, 126–128, 142, 170–171, 246–247, 250, 283, 418, 423, 432, 434, 440–441 Smith, D. I., 53 Smith, F, 417–442 Smith, K. J., 357 Smith, S. D., 376 Smith, W. D., 142 Snow, P. M., 304 Sodroski, J., 269 Sohrmann, M., 480, 486 Soltwisch, J., 122 Somehara, T., 455 Somers, W. S., 167 Sommer, I., 316 Sommer, T., 182–183 Sonawane, N. D., 497 Sonden, B., 514 Song, D., 328–330 Song, X., 282, 417–442 Song, Z. W., 423, 428 Sonnenberg, A., 6 Soreng, A. L., 376 Sospedra, M., 227–228 Sotomayor, C. E., 202–203, 229, 271–272 Sotomayor, E. M., 216–218 Souady, J., 122 Sowder, R. C. 2nd, 286 Sowdhamini, R., 332 Spaapen, L., 497 Spana, E., 358, 387 Sparrow, C. P., 271 Spear, E. D., 182 Spear, P. G., 328–329, 336, 343 Spellman, M. W., 356–357, 377, 402 Spiegelman, B. M., 532 Spiegel, Y., 142 Spiess, M., 170 Spinner, N. B., 376 Spitalnik, S. L., 122 Spooner, E., 187 Spradling, A. C., 385 Spring, J., 331 Sprong, H., 334 Squifflet, J. L., 248–249 Srivastava, D., 376 Staatz, W. D., 316, 327, 331–332, 334, 336 Staccini-Lavenant, L., 335 Stamatatos, L., 277 Standera, S., 188 Standiford, D. M., 52–53 Standing, K. G., 454 Stanley, P., 256, 298, 326–327, 343, 358, 377, 387, 389, 402 Staples, G. O., 70, 74, 76 Staub, J. K., 53 Steel, D. M., 495, 497

Author Index

Steele, R. E., 72, 324, 329 Steet, R. A., 497 Stehle, T., 454 Steigemann, P., 331–332, 335 Stein, D., 328, 330 Steinfeld, R., 495, 497 Steinman, R. M., 152, 217–219, 221 Stevens, D., 167 Stevens, J., 167, 419 Stevens, L., 328, 330 Stewart, P. L., 229, 248 Stiernagle, T., 147 Stillman, B. N., 229, 247, 271 St Johnston, D., 335 Stobrawa, S. M., 496–497 Stolz, D. B., 220 Stossel, T. P., 454 Stowell, C. J., 282 Stowell, S. R., 142, 204, 246–247, 250, 282–283, 285, 418, 423, 432, 434, 440, 474 St-Pierre, C., 267–288 Strahl, S., 479 Strand, M., 126, 128 Strasser, A., 229 Strecker, G., 310 Streilein, J. W., 202 Strigini, M., 36, 39 Strochlic, L., 78 Strome, S. E., 53 Strominger, J. L., 202 Strom, T. B., 234 Strong, R., 248–249, 256 Stroobant, V., 248–249 Struhl, G., 332, 377 Struwe, W. B., 477–491 Studier, F. W., 144 Suda, T., 328–330, 336, 338, 343 Sudo, K., 317 SueyoshI, T., 377 Sueyoshi, T., 356 Sugahara, K., 70, 317, 324–327, 329, 332 Sugaya, N., 53–54, 57, 61 Sugden, B., 277 Sugihara, K., 256 Sugimoto, N., 202, 232 Sugita, M., 299, 313 Su, H. C., 229 Suh, E., 423 Su, J., 418 Sullivan, D. E., 53 Sullivan, M. L., 220 Sumiyoshi, N., 53 Sun, B., 304 Sun, M., 335 Sunryd, J. C., 193 Sun, W., 52–53 Suryanarayana, K., 274 Suter, T., 496

563

Author Index

Sutton-Smith, M., 177, 256 Suzuki, E., 340 Suzuki, M., 250, 328–330, 336, 338, 343, 515 Suzuki, N., 248–249, 255 Suzuki, R., 376 Suzuki, S., 432 Suzuki, T., 184, 456 Suzuki, Y., 142, 456 Svennerholm, L., 300 Svensson, S., 101 Swann, M. J., 78 Swarbrick, G. M., 182 Sweeley, C. C., 124 Sweet, R., 269 Swulius, M. T., 192 Symonds, E. J., 454 Szathmary, R., 182 T Tabata, T., 327, 332, 336 Tabb, D. L., 472 Tabei, T., 453, 456 Tagawa, H., 316 Taguchi, R., 504 Tajiri, M., 455 Takagi, J., 446 Takahashi, H., 317 Takahashi, K., 343 Takahashi, M., 448–449, 453, 463 Takahashi, N., 183 Takahashi, T., 463 Takamatsu, S., 248, 255, 257–258 Takamori, S., 496–497 Takao, T., 356–357, 377 Takashima, S., 418 Takechi, E., 53 Takei, Y., 327, 332, 336 Takemae, H., 326–327, 336–338, 340, 343, 345 Takeo, S., 334 Takeshita, K., 282 Takeuchi, H., 357–358, 375–395, 402 Takeuchi, M., 248, 255, 258 Takeya, A, 515 Takeya, H., 356 Takeyama, K., 202, 231–232 Takida, S., 504 Takio, K., 316 Taki, T., 122, 129 Tamura Ji, J., 327, 329, 332 Tamura, K., 377 Tamura, T., 193 Tanaka, K., 184 Tanaka, S., 503 Tang, E., 157 Tang, J., 167 Tang, R., 52–54, 57, 61 Taniguchi, F., 327, 329, 332

Taniguchi, M., 328, 330 Taniguchi, N., 120, 248, 250, 255, 257, 259–260, 358, 445, 456, 462–463 Taniguchi, S., 453 Taniguchi, Y., 377 Tan, J., 53, 256 Tanner, W., 479 Tapanadechopone, P., 18 Tarentino, A. L., 304 Tarner, I. H., 200 Tarr, G. E., 423, 428 Tartakoff, A., 495 Tashima, Y., 503 Tateno, H., 190, 235 Tauchi, M., 327, 332, 336 Tavernarakis, N., 312 Tawada, A., 67 Taylor, J. H., 269 Taylor, M. E., 131, 134, 165–178, 423 Taylor, P. B., 302 Tazaki, K., 423, 428 Tefsen, B., 123 Teichberg, V. I., 201 Teichgraber, V., 496–497 Teisner, B., 356 Ten Dam, G., 53 Teneberg, S., 514 Tenner-Racz, K., 269 Tenney, A. P., 331, 335 Teo, C. F., 474 Teodorovic, P., 515 Terayama, K., 316 Termine, D. J., 192 Teshima, H., 357 Tessier-Lavigne, M., 53 Tessier, M. C., 256–257 Tewary, P., 271 Thanawiroon, C., 74 Thanedar, S., 187 Thellmann, M., 501 Theobald, M., 248–249, 256 Theocharis, A., 70 Therond, P. P., 335 Thhurin, J., 520 Thiele, C., 334 Thijissen, V. L., 205 Thim, L., 356–357 Thistlethwaite, P. A., 376 Thompson, R. D., 213 Thomson, A. W., 200, 220 Tian, W., 247–250, 252, 254–255, 259–262 Tiemeyer, M., 297–321, 357 Tien, A. C., 376 Tinari, N., 201, 271 Tirado, I., 202, 231 Tissot, B., 76, 126, 170–171, 423 Titz, A., 143, 146 Tkachenko, E., 5, 7, 9, 15, 20

564

Author Index

Todeschini, A. R., 118 Togayachi, A., 235, 248–249, 255 Toida, T., 70 Tojo, A., 316 Tolosa, E., 11 Tometten, M., 202, 231 Torensma, R., 132, 134, 270 Torgersen, D., 167 Torossian, S., 246 Torres, C. R., 369 Toscano, M. A., 201–202, 205, 219–220, 224, 228–229, 231–233, 235, 247, 249, 284 Totani, K., 463 Touret, N., 497 Toyoda, H., 70, 300, 305, 316, 324–325, 327– 330, 336–338, 340, 343, 345 Toyoshima, K., 142 Trask, B. J., 376 Travassos, L. R., 452 Tremblay, L. O., 193 Tremblay, M. J., 267–294 Tretter, V., 304 Trigueros, V., 142, 145 Trinchieri, G., 131 Trkola, A., 269 Trucco, A., 496 Tsai, B., 183 Tsai, C. M., 99 Tsang, G., 385 Tsao, P., 534 Tsay, D., 53, 57, 59 Tsien, R. Y., 497 Tsuchida, K., 317, 326 Tsuchimochi, K., 187 Tsuda, M., 331 Tsuda, T., 248, 250, 259–260 Tsuji, S., 142 Tsuji, T., 515 Tsunoda, Y., 248–249, 255 Tsurugaya, T., 53 Tucker, R. P., 404 Tummler, B., 496–497 Tun, T., 377 Turbachova, I., 377 Turco, S. J., 328, 330 Turek, D., 515 Turley, E. A., 12 Turnbull, J. E., 6, 53, 65–81 Turville, S., 270 Tyler, R. E., 182 Tzanakakis, G. N., 70 U Uchida, H., 52 Uchida, S., 497 Uchida, T., 357 Uchimura, K., 51–62

Uchiyama, N., 235, 418 Uchiyama, T., 202, 232 Ueda, R., 305, 326–330, 336–338, 340, 343, 345 Uehori, J., 142 Ueno, H., 152 Ueno, M., 324–325, 329 Ueyama, M., 327–328, 330, 336–338, 343, 345 Ugolini, S., 269–270 Uittenbogaart, C. H., 204, 272 Ujita, M., 250 Uliana, A. S., 497 Ulrich, M., 496–497 Umana, P., 496 Umulis, D., 332, 334 Ungar, D., 497 Uno, T., 330, 338 Unutmaz, D., 270 Unwin, R. D., 392, 412 Uozumi, N., 452 Urade, R., 184 Urano, T., 326–327, 343 Urashima, T., 246–248, 250, 257, 279, 432, 515 Urban, Z., 495–497, 499, 504 Urlinger, S., 501 Usui, T, 515 V Vaananen, K., 495 Vadaie, N., 326–327 Vahedi, K., 376 Valdembri, D., 9 van Anken, E., 183 van Beijnum, J. R., 205 Van Damme, E. J., 142, 423, 428 van den Berghe, H., 53 van den Berghe, P. V., 480–481, 486 van den Born, J., 53 Vandenborre, G., 142 van den Broek, M., 121, 133–136 van den Eijnden, D. H., 70, 521 Vandenhaute, J., 480–481, 486 van den Heuvel, S., 480–481, 486 van der Flier, A., 6 Van der Horst, D. J., 535 van der Linden, A. M., 480–481, 486 van der Schueren, B., 53, 269–270 Van Der Smissen, P., 248–249 van der Vlist, M., 153, 163 van der Wall, E., 377 van Die, I., 70, 117–136, 324–325, 329, 521 van Diest, P., 377 van Duijnhoven, G. C, 130, 132, 134, 270 van Echten-Deckert, G., 120, 122 van Halbeek, H., 357 Van Ham, P., 440 van Heeckeren, A. M., 496–497 van het Hof, B., 131, 152–153, 155–157, 159, 161–163

565

Author Index

van Kooyk, Y., 120–121, 123, 130–136, 152–153, 155–157, 159, 161–163, 166, 201, 270 van Kuppevelt, T. H., 51–62 Vanky, P., 70 Van Leuven, F., 423, 428 van Liempt, E., 120–121, 123, 132–134 van Reeuwijk, J., 495–497, 499, 504 van Stijn, C. M. W., 117–136 van Tetering, G., 377 Van Vactor, D., 331, 335 van Venrooij, W. J., 53 van Vliet, S. J., 130–132, 134, 270 van Voorden, S., 187–188 van Wamel, J., 279 van Zante, A., 53, 57, 59 Vardar-Ulu, D., 377 Varki, A., 246, 298, 441 Varki, N. M., 256 Vartiainen, S., 486 Vasile, E., 497 Vasquez, G. M., 274 Vassalli, P., 495 Vasta, G. R., 142, 201, 231, 271 Vayssiere, C, 376 Veerkamp, J. H., 53–55 Vembar, S. S., 183 Venkataraman, G., 52 Ventura, A. M., 452 Verhoef, K., 279 Verhofstad, N., 205 Verity, A., 212 Verkman, A. S., 497 Verlaan, I., 377 Vermeulen, M. E., 219–220, 228 Versteeg, E. M., 53–55 Verstreken, P., 382 Vestweber, D., 330 Viard, M., 269 Vicente-Manzanares, M., 7 Vidal, M., 480–481, 486 Viebahn, C., 53 VijayRaghavan, K., 332 Villa, F., 328–329, 336, 343 Vincent, A., 334 Vincent, J. P., 332–334 Vives, R. R., 68, 79 Viviano, B. L., 52–53 Vliegenthart, J. F. G., 105 Vocadlo, D. J., 377 Voglmeir, J., 78, 331 Vogt, G., 256 Vogt, T. F., 358, 387, 389 Volpi, N., 70 von der Lieth, C. W., 124 von Figura, K., 52–53, 182 von Kurthy, G., 496–497 Vooijs, M., 377

Vorbruggen, G., 331–332, 335 Vortkamp, A., 53 Voss, R. H., 248–249, 256 Vriend, G., 130 Vuillaumier-Barrot, S., 478 Vyas, N., 332 W Wada, I., 182, 193 Wada, Y., 455–456 Wadstrom, T., 514 Wahl, S. M., 234 Wait, R., 202, 232 Wallis, R., 166 Wall, K. A., 257 Walti, M. A., 146 Walz, A., 514 Walz, D. A., 357, 377 Walzel, H., 229, 271 Wandall, H. H., 312 Wang, B., 334 Wang, C.-Y., 157 Wang, D., 418 Wang, H., 70 Wang, H. M., 67, 70, 73, 78 Wang, J. L., 229, 232, 250 Wang, L.-L., 72, 142, 324, 329, 515 Wang, L. W., 403, 407, 413 Wang, M. B., 142, 340 Wang, S., 53, 220 Wang, W., 230 Wang, X., 304, 453, 456 Wang, Y. Q., 53, 57, 59, 256, 269, 358, 387, 389, 404, 497 Wang, Z., 335 Warren, C. E., 477–491 Warren, G., 497 Warren, J. R., 514 Warren, N. L., 277, 472 Warrior, R., 72, 316, 324, 327, 329, 331–332, 336 Wasano, K., 432 Wasternack, C., 142 Watanabe, A., 327, 332, 336 Watanabe, M., 328–330, 336, 338, 343 Watanabe, T., 142, 386 Waterhouse, P. M., 340 Waters, M. G., 497 Watkins, S. C., 220 Watkins, W. M., 515 Watt, F. M., 447 Weaver, C. T., 200, 234 Wehrly, K., 277 Weibezahn, J., 182 Weiner, H. L., 200, 228, 234 Weiner, J., 3rd., 142 Weinmaster, G., 376–377

566 Weiss, J. B., 126, 128 Weissman, D., 270 Weissman, J. S., 182 Weiss, R. A., 268 Weis, W. I., 131, 134, 166–168, 270 Weisz, O. A., 497 Wei, X., 277 Weix, D. J., 182 Welchman, D. P., 480, 486 Weller, M., 496–497 Wells, L., 298–300, 302–305 Wells, V., 201, 271 Welsh, C. J., 331, 335 Wendler, F., 332–333 Weng, A. P., 377 Wennerberg, K., 14 Wen, X., 220 Werb, Z., 52–55 Wernecke, H., 316 Weske, B., 312–313 West, A. P., 155 Westbrook, M. J., 184 West, D. C., 376 Weston, B. W., 520 Westphal, O., 94–96 Wevers, B., 153 Whelan, S. A., 369 Whetton, A. D., 392 Whitelock, J. M., 74 Whitfield, C., 91 Whitham, C. V., 142 Whitlock, K. E., 304 Whitman, M., 358 Whitney, P. L., 283 Whitworth, G. E., 377 Wiberg, F. C., 356–357 Wiegandt, H., 299, 312–314, 316 Wiertz, E., 187–188 Wieschaus, E., 358, 387 Wiese, S., 257 Wiest, I., 271 Wiggins, C. A. R., 387 Wikswo, J. P., 13 Wild, M. K., 330 Wilhovsky, S., 182 Wilker, B., 496–497 Wilkins, J. A., 454 Wilkinson, J., 270 Wilkinson, M. C., 67, 81 Willey, R., 273 Williams, M. E., 405 Willnow, T. E., 334 Wilson, I. B., 143, 146, 312, 326–327, 331, 478–479 Wilson, R. A., 126, 142, 358, 387, 389 Wiltrout, T. A., 274 Wimmerova, M., 463

Author Index

Winchester, B., 497 Winter, H. C., 142 Wirtz-Peitz, F., 379 Wise, S., 334 Wodarz, A., 335 Wolf, D. H., 182–183, 188–189 Wolfe, H., 202 Wolfenstein-Todel, C., 202, 229, 272 Wolfert, M. A., 474 Wolfl, M., 248–249, 256 Wollina, U., 202 Wong, C. H., 167 Wong, G., 486 Wong, K., 53 Wong, N. K., 256 Woodard, R. W., 92 Worby, C. A., 393 Wormald, M. R., 302 Wrana, J. L., 248–249 Wright, A., 78 Wu, G., 229, 232 Wuhrer, M., 120–121, 123–129, 418 Wu, L., 270 Wu, M. M., 497 Wu, Q., 529 Wu, T., 272 Wuthrich, K., 356 Wu, X., 277, 497 Wu, Y., 33–49, 192, 277, 334 Wyatt, R. T., 269 Wynn, R., 356 Wynshaw-Boris, A., 256 X Xia, B., 142, 418, 432, 440–441 Xia, J., 356–357, 377, 387, 390 Xie, J., 72 Xie, W., 340 Xiong, J. P., 454 Xu, A., 358, 366, 387, 402 Xu, D., 328–330 Xue, Q., 53, 57, 59 Xue, Y., 530 Xu, H.-T., 515 Xu, J., 68, 79–80 Xu, Q., 445–458 Xu, S., 340, 480 Xu, T., 36, 381 Y Yagi, F., 246–248, 250, 257, 432 Yagishita, N., 187 Yamada, K.M., 190, 447 Yamada, S., 70, 324–325, 329 Yamagishi, M., 515

567

Author Index

Yamaguchi, D., 187, 190–191 Yamaguchi, Y., 183, 504 Yamamichi, Y., 356 Yamamoto, G., 376 Yamamoto, K., 183, 187, 190, 515 Yamamoto, S., 317 Yamamoto, T., 53 Yamamoto, Y., 515–518 Yamasaki, S., 187 Yamashita, K., 142, 463 Yamashita, S., 357 Yamazaki, H., 52 Yamazaki, S., 70 Yanagishita, M., 67 Yan, D., 40–44, 46, 331–332, 334 Yang, B., 497 Yang, D., 271 Yang, F., 157 Yang, J. T., 447 Yang, K., 256 Yang, R. Y., 201, 246 Yang, T., 143 Yang, Y., 52 Yan, S., 170 Yan, Y. T., 358 Yao, S., 334 Yasuyama, K., 304 Yates, A. D., 515 Yates, E. A., 67, 69–73, 76–78 Yates, J. L., 277 Yates, K. E., 70, 74 Yayon, A., 5, 68, 79 Yazdanbakhsh, M., 119 Yeaton, P., 440 Yehualaeshet, T., 404, 407 Yeo, C. Y., 358 Ye, Y., 183, 188 Ye, Z., 256 Yokota, A., 70 Yoshida, A., 248, 255, 258, 456 Yoshida, H., 305, 327, 336–338, 343, 345 Yoshida, K., 53, 67 Yoshida, Y., 184 Yoshida-Yamamoto, S., 455 Yoshimura, M., 453 Yoshinaka, T., 455 Yoshioka, T., 282 Young, R. E., 213 Young, S. L., 184 Yuan, J. X., 376 Yu, C., 53 Yue, T., 423 Yue, X., 52–53 Yun, C. W., 188, 530

Yurchenco, P. D., 447 Yu, S.-Y., 515 Yu, X. Q., 142, 269–270 Z Zagorevski, D. V., 76 Zahner, H., 129 Zahorchak, A. F., 220 Za¨hringer, U., 119 Zaia, J., 70, 74, 76 Zako, M., 53 Zarif, M. J., 446 Zdebik, A. A., 496–497 Zecca, M., 36 Zeevaert, R., 497 Zeitz, O., 495, 497 Zeng, Z., 357, 377, 462 Zhai, R. G., 382–383 Zhang, H., 273 Zhang, L., 187 Zhang, N., 358, 387 Zhang, R., 454 Zhang, X., 376 Zhang, Z., 72, 269–270, 277, 328, 330 Zhao, Y.-F, 448–449, 453, 515 Zhao, Y. Y., 463 Zhao, Z.-H., 515 Zheng, X., 334, 405 Zheng, Y., 152 Zhi, Z., 76, 78 Zhou, B., 312 Zhou, X., 26 Zhu, A. J., 332, 334 Zhu, H., 204 Zhu, X., 328, 330 Zhu, Y., 142 Zicha, D., 16 Zichittella, A. E., 358 Zick, Y., 440 Zifarelli, G., 497 Zigmond, S. H., 16 Zimmermann, C., 201, 271 Zimmermann, P., 8 Zinn, K., 304, 331, 335 Zipperlen, P., 480, 486 Zolotarjova, N. I., 356 Zoran, M. J., 300 Zou, H., 53 Zuberi, R. I., 279 Zuiderweg, E. R., 356 Zun˜iga, E., 201–202, 271 Zwirner, N. W., 201–202, 224, 228–229, 231, 233, 235, 247

Subject Index

A Acanthamoeba castellanii, 147–148 Acidic polysaccharides, 100 Acquired immunodeficiency syndrome (AIDS) HIV-1 infection, 268 NIH AIDS Research & Reference Reagent Program enfuvirtide (T-20), 286 LuSIV and TZM-bl cell line, 277 p24 ELISA, 274–275 primary cells, 274 prevention education, 269 Acticlean ETOX endotoxin-removing gels, 280–281 Aedes aegypti, 145 Affinity-isolated EGF20, 361, 363 AIDS. See Acquired immunodeficiency syndrome Anion-exchange chromatography, 121 Arginine-glycine-aspartic acid (RGD), 447 B Bacillus circulans, 518–519 Bacillus subtilis, 222 Bisecting GlcNAc cell spreading and migration assays, 449–450 cytoskeleton and signaling molecules, 446, 448 ECM, 446–447 glycosylation reactions, 446–447 GnT-III and GnT-V genes, 448–449 integrin a5b1 functional expression, 454 mutants, 451–452 integrin-mediated cell adhesion and migration carcinogenic process, 452 E-cadherin-mediated cell–cell interaction, 453 GnT-V/b1,6 expression, 452–453 integrin a3b1, 453 molecular mechanism, 454 oligosaccharide structures, 446 RGD-binding protein, 447 a5 subunit, GnT-III regulation, 455–456 Blood group antigen-binding adhesin (BabA), 514–515 Bone morphogenetic protein (BMP) binding protein, 334

Dally, 335 Dpp, 332 gradient establishment, 330–331 C Caenorhabditis elegans, high-throughput RNAi screening CDG-I, 478, 489, 491 genome sequence, 480 genome-wide RNAi screen COGs, 489 dumpy phenotype, 487 H23N18.4 gene, 489 KOGs, 489–490 NL2099 strain, 480 ORFeome v1.1 library, 486 phases, 488 types of phenotype, 486–487 D values, 487–488 knockout models, 490 LLO, 478, 480 maturation, etiologic and effector genes, 491 N-glycosylation animal development, 489 biosynthetic components, 479 glycome and anatomy, 479 high-mannose, paucimannose and highfucose type structures, 479–480 molecular pathology, 491 strains and culturing dose dependent tunicamycin-lethality, 482 N2 (Bristol), 480 ok809 homozygous, 480–481 ORFeome v1.1 library, 481, 490 RNAi effectiveness, 486–487 tunicamycin effect, 482 tunicamycin-hypersensitive genes, 483–486 tunicamycin treatment, 482–483 Wormbase, 482 Caenorhabditis Genetics Center (CGC), 147 Capsular polysaccharide (CPS) crude extract purification, 96–98 O-specific polysaccharide, 92 Carbohydrate analysis acetylated methyl glycosides elution order, 101 fragmentation pattern, 101–102 method, 102–103

569

570 Carbohydrate analysis (cont.) oxonium ion, 101 reagents and equipment, 102 carbohydrate absolute configuration, 105 monosaccharide absolute configuration, 103–104 monosaccharides branching points AAPM, 105 derivatization protocol, 105–106 methods, 107–109 molecular ion, 105–106 reagents and equipment, 107 uronic acids, 105 Carbohydrate recognition domains (CRDs) A-blood trisaccharide, 438 affinity chromatography, 431 Alexa488-/FITC-labeled streptavidin, 432 cell surface glycosylation affinity column generation, 172–173 cell fractionation, affinity columns, 173–175 flow diagram, proteomic and glycomic analysis, 172–173 gel analysis, 175–176 glycomics, 177–178 informatics, 177 purified ligands analysis, 176 tryptic peptides, 176–177 engineering glycan-binding specificity galactose-binding activity, 167–168 Gal-MBP, 167, 169 glycan arrays, 168–171 solid-phase binding assays, 171 tryptophan, 167 fucose residue, 434 full-length Gal-8 patterns, 432–433 galectin galectin-1, 235, 431–432 structures, 201, 270–271 glycan specificity/glycan-binding motif, 434, 438–439 human blood group A and B, 434 N-acetyllactosamine-containing glycans, 434 probes, glycan detection, 171–172 ranked glycans, 432, 434–437 recombinant C- and N-terminal domains, 439 Carbohydrate signaling, dendritic cells acetyltransferases, 160–161 DC-SIGN signaling, 157–160 LPS and ManLAM, 153–155 NF-kB activation, 156–157 RNA interference, 155–156 T helper (TH) cell differentiation, 152 TLRs, 152–153 transcriptional regulation, p65 acetylation, 161–162 Caspase-recruiting domain (CARD), 152 Cation dependent M6P receptor (CD-MPR), 182, 187

Subject Index

CBP. See CREB-binding protein CDG-I. See Congenital disorders of glycosylation type I CD-MPR. See Cation dependent M6P receptor Cell surface glycoproteins. See Galectin–glycoprotein lattice Cell surface glycosylation affinity column generation, 172–173 cell fractionation, affinity columns, 173–175 gel analysis, 175–176 glycomics, 177–178 informatics, 177 purified ligands analysis, 176 tryptic peptides, 176–177 Ceramide monohexoside (CMH), 119, 121, 128 CFG. See Consortium for Functional Glycomics Chemotaxis assay, 207–208 Chloride channel (CLC), 497 Clostridium perfringens, 233 CLRs. See C-type lectin receptors Clusters of orthologous groups (COGs), 489 CMH. See Ceramide monohexoside Collision-induced dissociation (CID), 72, 303 Congenital disorders of glycosylation type I (CDG-I), 478, 489, 491 Conserved oligomeric Golgi (COG) proteins, 496–497 Consortium for Functional Glycomics (CFG), 169–170 analysis, 420–422 binding buffer, 421 fluorescent detection methods, 419–420 GAL file, 420 Glycan Array Synthesis Core D, 419 protein–carbohydrate interaction Core H, 418 protein–glycan interaction Core H, 419 Steering Committee, 418 Coomassie blue stain, 175 Coprinopsis cinerea, 145 Counterion channels, 497 CPS. See Capsular polysaccharide CRDs. See Carbohydrate recognition domains CREB-binding protein (CBP) acetyltransferase activity, 160–161 HAT, 157, 159, 163 CTD110.6 antibody immunoblotting, 365–366 PBST, 363, 365 PVDF membrane, 363, 365 reprobing, 365 Restore Western blot stripping buffer, 365 Tris–acetate gel, 363 C-type lectin receptors (CLRs), 131–134 D Dally-like protein (Dlp), 331, 334–335 DCs. See Dendritic cells

Subject Index

Decapentaplegic (Dpp) signaling, 331–336 Deglycosylation glycopeptide, 369 glycoproteins, 368 b-N-acetylhexosaminidase digestion, 367–368 Dendritic cells (DCs) carbohydrate signaling acetyltransferases, 160–161 DC-SIGN signaling, 157–160 LPS and ManLAM, 153–155 NF-kB activation, 156–157 RNA interference, 155–156 T helper (TH) cell differentiation, 152 TLRs, 152–153 transcriptional regulation, p65 acetylation, 161–162 receptors human monocyte-derived immature dendritic cells, 131–132 immature dendritic and K562 cells adhesion, 132–134 immune system, 130–131 modulation, 134–136 Derivatization protocol, 105–106 4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sindacene-3-propionic acid (BODIPY), 71–72 Dimethylsulfoxide (DMSO) hydroxyl fatty acids, 113 monosaccharides branching points, 107–109 Dithio-bis-sulphosuccinimydylpropionate (DTSSP), 251, 253 Division abnormally delayed (Dally), 331, 334–335 Dlp core protein Dlp/Fz2 ratio co-immunoprecipitation, 46–48 luciferase reporter assay, 46 Wg interaction cell binding experiment, 43–44 co-immunoprecipitation, Wg-GFP, 45–46 cultured S2 cells, immunostaining, 44–45 pUAST-dlp(-HS)-GFP and stable cell line generation, 43 Wg conditioned medium preparation, 43 Wg morphogen signaling gradient formation, 39 immunofluorescent staining, imaginal discs, 41–42 transgenic line generation, 40–41 UAS-dlp and UAS-dlp(-HS) expression, 41 DMEM. See Dulbecco’s modified eagle medium DMSO. See Dimethylsulfoxide Drosophila glypican, Wg/Wnt signaling and distribution Dally and Dlp loss-of-function experiments, 36–38 null alleles generation, 35–36 overexpression, 38–40

571 Dlp core protein Dlp/Fz2 ratio, 46–48 Wg interaction, 42–46 Wg morphogen signaling, 39–42 Drosophila melanogaster cell–cell and cell–matrix adhesion, 316 culture and husbandry, 380–381 GAG biosynthetic pathway, 326–328 b4GalTI and b3GalTII, 326 GlcAb1–3Galb1–3Galb1–4Xylb1-O-Ser, 325–326 HS biosynthesis, 331 OXylT, 325 phylogenetic tree, 330 structures, 324–326 sugar nucleotides, 330 sulfated disaccharide formation, 329 sulfation, 329 GlcA, 299–300 glucuronyltransferase, 317–318 glycan analysis CID, 303 HRP-epitopes, 304–305 LacdiNAc, 305 a6-linked Fuc residue, 304 MSn fragmentation, 305, 307 PNGaseF and PNGaseA, 303–304 terminal modification, N-linked glycans, 305–306 TIM, 303, 305 glycomic strategy, 298–299 glycopeptide preparation, 302 glycosaminoglycan chains, 316 glycosyltransferases assay, 337–338 cloning and expression, 336–337 GSL DEAE Sephadex chromatography, 311–312 iatrobead chromatography, 310–311 isolation and fractionation, 310 NSI-MSn data-dependent acquisition, 312–315 QAE Sephadex chromatography, 311 300-kDa protein, 377 N-linked glycan release, 302 Notch intracellular domain, 377 notch regulators, genetic screen, 381–382 O-linked glucose detection materials, 392–393 methods, 393–395 O-linked oligosaccharide release b-elimination, 308 glucuronylated O-Fuc trisaccharide, 310 glucuronylated O-linked glycans, 308–309 HexNAc-HexA disaccharides, 310 incubation and lyophilization, 305 b4-linkage, 309

572 Drosophila melanogaster (cont.) permethylation, 302 prodigious genetic armamentarium, 298 proteins and lipids extraction, 300–301 proteoglycans core proteins, 331 Dally and Dlp, 334–335 dHS6ST/Hs6st and Hs2st, 336 dystroglycan and Perlecan, 335 germline stem cell (GSC), 335 HS-binding proteins, 334 lipophorins, 334 lipoprotein particles, 333–334 morphogens, 332–333 N-deacetylase/N-sulfotransferase, 336 Sdc and Dlp function, 335 Slit/Robo signaling, 332 sulfation, 336 RNAi flies experimental procedure, 342–343 gene silencing, 340, 343 genetic interactions, 343–345 heritable and inducible RNAi system, 340–341, 343 morphogens, 345 tissue-specific promoter, 340 viability tests, 343 yeast transcription factor, 340, 343 rumi genetic identification, 377–379 mapping and sequencing, 382–384 protein-null allele generation, 385–386 protein O-glucosyltransferase (see Protein O-glucosyltransferase) rescue experiments, 384–385 strains, 379 sugar-nucleotide transporters cloning and expression, 338–339 subcellular fraction preparation, yeast, 339–340 type I transmembrane protein Notch, 376 Drosophila 7-pass transmembrane glycoprotein BOSS BOSS mutant flies, starvation BODIPY staining, 536 boss1 flies, 536–537 glucose-responding GPCRs, 537 TAG, 535–536 extracellular glucose, 534–535 glucose, energy homeostasis maintenance, 530–532 model system, metabolism brain, 529–530 fat body, 528–529 oenocytes, 529 orphan membrane receptor glucose-responding receptor, 534 sequence alignment, 532–533

Subject Index

SEVENLESS ligand, 532 sugar and lipid metabolism boss1 larvae, 534, 536 hemolymph lipid measurements, 534 oenocytes function, 535 TAG detection circulating (hemolymph) TAG level measurement, 538 fat droplets, 537 Oil-Red-O and BODIPY staining, 539 total body TAG level measurement, 538 Drosophila pipe gene, 329 DTSSP. See Dithio-bissulphosuccinimydylpropionate Dulbecco’s modified eagle medium (DMEM) multicellular migration assay, 26 proteoglycan chimera oligomerization, 20–21 Rac1 pull-down assay, 25 E EGF. See Epidermal growth factor Electrospray ionization mass spectrometry (ESI-MS), 122, 125 ELISA. See Enzyme-linked immunosorbent assay Endoplasmic reticulum-associated degradation (ERAD) ER lumen, 191 NHK degradation, 191–192 pulse-chase analysis, 192–193 Endoplasmic reticulum quality control (ERQC), 183 Endotoxin, 281 Enhanced green fluorescent protein (EGFP), 499–500 Enzymatic hydrolysis method, 97–98 reagents and equipment, 96–97 Enzyme-linked immunosorbent assay (ELISA), 54–55 Epidermal growth factor (EGF) affinity-isolated EGF20, 361, 363 bovine factor VII, 356 EGF20:V5His, 365 extracellular domain, 358 b-N-acetylhexosaminidase, 365–366 Notch EGF20 MALDI-TOF-MS spectrum, 361–362 plasmid encoding, 360 purification, 361 O-GlcNAc transferase (OGT) activity, 360 O-GlcNAcylation, 358, 363 O-glycans, 356–357 TSRs, O-fucosylation, 402–403 yeast expression system, 358–359 ERAD. See Endoplasmic reticulum-associated degradation

573

Subject Index ER-degradation enhancing a-mannosidase-like proteins (EDEMs), 193 Escherichia coli carbohydrate-binding, 143 cDNA, 518 Coprinopsis cinerea, 145 plasmid pACYC-184, birA gene, 170 protein expression, solubility, and toxicity, 145–146 soluble protein (SP), 144 Xerocomus chrysenteron, 145 Ethyl methanesulfonate (EMS), 504 Eukaryotic orthologous groups (KOGs), 489–490 Extracellular matrix (ECM), 446–447 Ex vivo sulf assay materials, 59, 61 method, 60–61 F FAC. See Frontal affinity chromatography Fatty acids compositional analysis hydroxyl fatty acids, 113–114 methanolysis C-3 and C-2 hydroxylated fatty acid, 109 14-carbon atom fatty acid, 110 fatty acid methyl ester, 110–111 b-hydroxyl function, 111 methods, 112 reagents and equipment, 112 simple and saturated fatty acids, 109 O-linked fatty acid, 112–113 Fetal calf serum (FCS), 207, 221–225 Fibroblast growth factor (FGF) signaling assay, 79–80 Flavobacterium, 70 Flow chamber assay human PMN and lymphocyte interactions, 208 in vitro equipment, 211 HUVECs, 209 leukocyte recruitment, 212 parallel plate flow chamber, 210 Poiseuille’s law, 210 wall shear stress, 210–211 Flow cytometry-based sulf assay materials, 55, 57 method, 57–59 Fluorescence resonance energy transfer (FRET) microscopy, 332 Fo¨rster resonance energy transfer (FRET), 14 Frontal affinity chromatography (FAC), 190–191 Fruiting body lectins, biotoxicity assays Acanthamoeba castellanii, 147–148 Aedes aegypti, 145 Caenorhabditis elegans, 147 Escherichia coli carbohydrate-binding, 143

Coprinopsis cinerea, 145 protein expression, solubility, and toxicity, 145–146 soluble protein (SP), 144 Xerocomus chrysenteron, 145 lectin-mediated defense, 142 nematotoxic lectins and toxins, 143 statistics, 148 G GAGs. See Glycosaminoglycans b-Galactoside, 271 Galb-oNP, 518–519 Galectin-1 cognate ligand–receptor interactions, 201 CRD, 201 double-edge sword effect, 237 endotoxin-free recombinant protein generation, 237 expression, 271 galectin–glycan lattices (see Galectin–glycan lattices) and galectin-3 purification, 279–280 gene and protein therapy, 202 glycan-binding specificity, 271 immune cell trafficking, recruitment, and chemotaxis antiinflammatory dose, 204 buffering effect, 205 cell counting, 207 chemotaxis assays, 207–208 cremaster preparation, 215–216 endogenous lectin, 203 flow chamber assay, 208–212 human blood leukocyte isolation, 206 intravital setup, 215 IVM, 214, 216–218 leukocyte migration and extravasation, 202–203 LPS-stimulated macrophages, 203 lymphocyte isolation, 206–207 mesentery preparation, 215 microvascular beds, 214 mouse peritonitis, experimental model, 212–214 peritonits induction, 213 platelet/leukocyte interactions, 205 PMN, 203–204, 207 primary HUVEC isolation and culture, 208–209 recruited cell harvesting, 213 tonic inhibitor, 205–206 inhibition of lectin activity dominant-negative form, 284–285 sugar antagonists, 283–285 multidisciplinary approach, 237–238 multiple regulatory pathways, 200

574 Galectin-1 (cont.) proinflammatory and tolerogenic microenvironments, 239 proof-of-concept data, 238 quality control, 280–281 release/secretion, 271–272 S-carboxyamidomethylation dithiothreitol (DTT), 281–282 hemagglutination assay, 283 2-mercaptoethanol, 282 oxidation, 281 structures, 270–271 Galectin–glycan lattices DC physiology control adoptive transfer experiments, 226 cell surface phenotypic analysis, 223 EAE induction and assessment, 227–229 endocytosis assays, 223 galectin-1-binding assays, 223 glycan-binding protein, 219 human allogeneic mixed leukocyte reaction, 224–225 human DC differentiation and maturation, 221 immune cell tolerance and homeostasis, 216 immunoregulatory circuit linking galectin-1 signaling, 219 mouse allogeneic MLR, 225–226 mouse DC differentiation and maturation, 221–223 myeloid and plasmacytoid DCs, 217 predominant immunogenic function, 218 real-time quantitative RT-PCR, 224 receptor segregation, 223–224 recombinant galectin-1 preparation, 220–221 tolerogenic effects, 220 Treg cell expansion, 218–219 tumor protection assays, 226–227 Western blot analysis, 224 T helper cell fate Ab-mediated blockade, 232 apoptotic mechanisms, 229 cell death assays, 235–236 differential glycosylation, 231 gene expression profiles, 232 glycophenotype analysis, 234–235 human T helper cell polarization, 233–234 mouse T helper cell polarization, 234 N- and O-glycan biosynthesis, 229–230 a2,6-sialyltransferase 1 (ST6Gal1), 230 T-cell susceptibility, 229–230 Th1- and Th17-differentiated cells, 231 Th1, Th2, and Th17 cell polarization, 232–233 in vivo assays, 236–237 Galectin–glycoprotein interactions disruption

Subject Index

galectin binding, competitive inhibition, 257–259 gene-targeted deletions, 255–256 N-X-S/T site mutation, 256–257 N-glycan branching measurement coimmunoprecipitation, 251–252 flow cytometry, 252–253 glycoprotein clustering and colocalization, 253 Golgi enzyme assays, 254 inhibitors, 257 mass spectroscopy, 253–254 mRNA half-life/stability measurement, 255 real-time PCR, 254 strengthening golgi branching enzymes overexpression, 258 metabolic supplementation, hexosamine pathway, 259–260 Galectin–glycoprotein lattice cytoplasm, 250 galectin–glycoprotein interactions disruption, 255–258 N-glycan branching measurement, 251–255 strengthening, 258–260 galectins and N-glycan ligands, 246–247 genetic and metabolic regulation, 249–250 glycoprotein concentration regulation, 248–249 T cell activation and proliferation, 261–262 homeostasis and self-tolerance, 249 TCR signaling, 260–261 Gas chromatography-mass spectrometry (GC-MS). See Fatty acids compositional analysis GEMs. See GM1-enriched membrane microdomains Genome-wide RNAi screen COGs, 489 dumpy phenotype, 487 H23N18.4 gene, 489 KOGs, 489–490 NL2099 strain, 480 ORFeome v1.1 library, 486 phases, 488 types of phenotype, 486–487 D values, 487–488 Germline stem cell (GSC), 335 GlcA. See Glycans carry glucuronic acid GlcAT-I, 317 GlcAT-P, 316–317 GlcAT-S, 316–317 Glucose-sensing mechanisms multicellular organisms, 532 yeast GPCR cAMP, 531–532 GPCR family, 532 Gpr1–Gpa2 couple, 530 PKA activation, 531

Subject Index

plasma membrane proteins, 530–531 transcription of HXT gene, 530 Glycan antigens ELISA, 129–130 overlay technique, 128–129 Glycan arrays binding competition assays, 170–171 biotin-tagged protein, 170 human asialoglycoprotein receptor, 170 oligomeric proteins, 168 screening, 167, 169 sugar-Sepharose column, 169 Glycan Array Synthesis Core D, 419 Glycan-binding protein (GBP). See Printed glycan microarray Glycans carry glucuronic acid (GlcA) arthro-series, 312–313 biosynthesis, 316 Gal residue, 312, 314 GlcA-extended At5Cer, 313 glycosaminoglycan chains, 316 glycosaminoglycan family, 300 b4-linkage, 309 N-linked glycans, 305 sialic acid, 299–300 standard Core 1 disaccharide, 308 terminal extension, 312 Glycogene, 522 Glycomics profiling. See Heparan sulfate Glycophenotype analysis, 234–235 Glycosaminoglycans (GAGs) biosynthetic pathway, 326–328 chain cleavage, 18 charge distribution and steric structure, 6 disaccharide chain sequences, 4 extracellular accumulation, 10 b4GalTI and b3GalTII, 326 gel electrophoresis, 17 GlcAb1–3Galb1–3Galb1–4Xylb1-O-Ser, 325–326 HS biosynthesis, 331 HPLC and mass spectrometry, 76 linear polysaccharides, 66 rapid purification, 69 ionic interactions, growth factors, 5 OXylT, 326 phylogenetic tree, 330 steric effects, 7 structures, 324–326 sugar nucleotides, 330 sulfated disaccharide formation, 329 sulfation, 329 types of, 4–5 Glycosphingolipid (GSL) DEAE Sephadex chromatography, 311–312 iatrobead chromatography, 310–311 isolation and fractionation, 310

575 NSI-MSn data-dependent acquisition At6 and At9 cores, 313 ceramide heterogeneity, 314 endoglycosylceramidase, 312, 314 GlcA-extended At5Cer, 313 glucuronylated O-linked GSL, 312–313 MSn fragmentation, 314–315 neutral arthro-series core, 312 QAE Sephadex chromatography, 311 Schistosoma mansoni (see Schistosoma mansoni glycosphingolipids) Glycosylphosphatidylinositol (GPI)-anchored reporter proteins, 499–500 Glycosyltransferases. See also Drosophila melanogaster assay, 337–338 cloning and expression, 336–337 lectin-based glycoproteomics, 465 GM1-enriched membrane microdomains (GEMs), 249, 253 Golgi acidification CLC family, 497 COG proteins, 496–497 glycosylation analysis, lectin staining, 506–507 Golgi pH measurement FluoView FV1000 laser scanning confocal microscope, 505 MetaMorph software, 506 pHluorin, 505–506 pH-sensitive green fluorescence protein, 504 pH sensor, 505 ROIs, 506 GPHR functions, 499 mutagenesis, 504 mutant cell selection, 504 organelles, 497–498 parent cells establishment, 500–501 TGN, 496–497, 500, 505 transport assay, 501–504 transport monitoring, reporter proteins, 499–500 vacuolar-type proton ATPases, proton leaks, and counterion channels, 497 Golgi pH regulator (GPHR) functions, 499 gp120, 269, 285 GPRC5Bs, 532–533 Gram-negative bacterial lipopolysaccharides carbohydrate analysis acetylated methyl glycosides, 101–103 carbohydrate absolute configuration, 105 monosaccharide absolute configuration, 103–104 monosaccharides branching points, 105–109 enzymatic hydrolysis method, 97–98 reagents and equipment, 96–97 fatty acids compositional analysis hydroxyl fatty acids, 113–114

576

Subject Index

Gram-negative bacterial lipopolysaccharides (cont.) methanolysis, 109–112 O-linked fatty acid, 112–113 glycerophospholipid bilayer, 92 host–bacterium interactions, 91 hydrophilic heteropolysaccharide, 91 LPS and LOS extraction hot phenol/water extraction, 95–96 microbial carbohydrate, 93 PCP extraction, 94 phenol/TEA/EDTA extraction, 95 protocols, 93 polysaccharide moiety, 92 SDS-PAGE acidic polysaccharide detection, 100 Kittelberg and Hilbink protocol, 98–99 Tsai and Frasch protocol, 99–100 GSL. See Glycosphingolipid H Hank’s balanced salt solution (HBSS), 155, 278 HAT. See Histone acetyltransferase Hedgehog (Hh), 332–336 HEK. See Human embryonic kidney Helicobacter pylori BabA, 514–515 fucosyltransferases fuca1!2Galb1!3[Fuca1!4] GlcNAcb1!3Galb1!4Glc (lacto-N-difucohexaose I) synthesis, 523–524 fuca1!2Galb1!3GlcNAcb1!3Galb1 !4Glc (lacto-N-fucopentaose I) synthesis, 522–523 Glycogene, 522 lacto-N-triose II synthesis b–1,3-GnT, 515 materials, 516 methods, 517–518 N-acetyl-b-D-glucosamine (D-GlcNAc), 515 N-acetyl-b-D-glucosaminidase, 516 LNT synthesis Escherichia coli, 518 b–1,3-galactosidase, 518 Galb1!3GlcNAcb1!3Galb1!4Glc, 517 materials, 519 methods, 519–520 phenol–sulfuric acid method and elution profile, 518–519 milk oligosaccharides, 515 recombinant FUT1 and FUT3 preparation, 520–521 SabA, 514 synthetic strategy, 515–516 Helix pomatia, 235

Heparanome, 68, 81 Heparan sulfate (HS) decoding structure–function, 68 disaccharide composition profiling BODIPY fluorescent tag, 71–72 CID, 72 fluorescence HPLC analysis, 72–73 fluorophores, 70 high sensitivity detection, 70 offline and online mass spectrometry, 72 multifunctional cell regulators, 67–68 oligosaccharide profiling FGF signaling assay screening, 79–80 LC-ESI MS, 74–76 oligosaccharide library production, 77–79 online mass spectrometry, 74 partial cleavage, HS chains, 73 SEC fractionation, 77 software tools, oligosaccharide characterization, 76–77 structure–activity relationships (SAR), 77 tissue oligosaccharides, 74 RIP method, proteoglycan isolation, 69–71 structure and biosynthesis, 66–67 Heparan sulfate proteoglycans (HSPGs), 53 core proteins, 66 cultured cells, 67 structural selectivity, 68 High-performance thin-layer chromatography (HPTLC), 122 Histone acetyltransferase (HAT) anacardic acid (AA), 160–161 anti-Ac-lysine rabbit pAb, 159 CREB-binding protein (CBP), 157, 159, 163 lysis and assay buffer, 160 Raf inhibitors, 162 HIV-1. See Human immunodeficiency virus-1 HPTLC. See High-performance thin-layer chromatography HS. See Heparan sulfate HSPGs. See Heparan sulfate proteoglycans Human embryonic kidney (HEK), 192, 273–274 Human immunodeficiency virus-1 (HIV-1) AIDS, 268 antiretroviral therapy, 269 dendritic cells, 270 host membrane proteins, 270 immunosuppression, 268 infection assay, 287–288 CD4þ T lymphocytes, 278 cell activation, 278–279 LuSIV cell line, 277 MDM, 278 PBMCs, 277–278 TZM-bl cell line, 277 precautions, 272 production

577

Subject Index

AT-2 inactivation, 276–277 concentration, ultracentrifuge, 275 HEK 293 T cells, 273–274 p24 ELISA, 274–275 primary cells, 274 TM purification, Optiprep , 275–276 relentless progression, 268 virus attachment assays luciferase activity, binding, 286–287 p24, binding, 287 virion-associated gp120, 286 Human umbilical vein endothelial cells (HUVECs) isolation and culture, 208–209 PMN, 204, 211 I Intravital microscopy (IVM) inflamed microcirculation, 204 leukocyte trafficking, 214 Lgals1/ mice, 205 microvascular beds, 214 parameter analysis, 216–218 Iodine staining, 122 Isothiocyanate, 168, 170 IVM. See Intravital microscopy K Kanamycin, 144, 147 KOGs. See Eukaryotic orthologous groups L Lacto-N-tetraose (LNT) synthesis Escherichia coli, 518 b–1,3-galactosidase, 518 Galb1!3GlcNAcb1!3Galb1!4Glc, 517 materials, 519 methods, 519–520 phenol–sulfuric acid method and elution profile, 518–519 Lacto-N-triose II synthesis b–1,3-GnT, 515 materials, 516 methods, 517–518 N-acetyl-b-D-glucosamine (D-GlcNAc), 515 N-acetyl-b-D-glucosaminidase, 516 LC-ESI MS. See Liquid chromatography electrospray mass spectrometry LC-MS. See Liquid chromatography-mass spectrometry Lectin activity, 281 Lectin-based glycoproteomics affinity separation, 464 cancer detection, 462 cancer-specific glycosignatures, 463 carbohydrate binding proteins, 463

glycan identification, 462 glycan structures, 463–464 glycoprotein separation easily solubilized glycoprotein extraction, 468–470 L-PHA reactive glycoproteins, 467 membrane proteomics protocols, 467 nondiseased breast tissue samples, 465, 467 polymer contamination, 468 tightly membrane-associated glycoprotein extraction, 470 lectin blotting and separation glycosyltransferases, 465 GnT-V, 465–466 immobilized lectins, 466 lectin binding visualization, 466–467 L-PHA binding, 465–466 mono- and disaccharide haptens, 466 shRNA molecules, 465 MS/MS data analysis advantage, 471 haptoglobin/haptoglobin-related protein, 473 linear ion trap, 472 L-PHA reactivity, 472–473 mimecan, 472 periostin, 472–473 validation analysis, 473 potential glycoprotein cancer markers, 471 Lipid-linked oligosaccharide (LLO), 478, 480 Lipopolisaccharide (LPS), 203. See also Gramnegative bacterial lipopolysaccharides Liquid chromatography electrospray mass spectrometry (LC-ESI MS), 74–76 Liquid chromatography-mass spectrometry (LC-MS) ion fragmentation, 410 ion identification, 409 MRM analysis, 412 Pofut2 target proteins, 413 semiquantitative assessment, 412 thrombospondin 2 (TSP2) fucose–glucose disaccharide, 410 O-fucosylated peptides, 410–411 tryptic peptides, 409 LLO. See Lipid-linked oligosaccharide LNT synthesis. See Lacto-N-tetraose synthesis Luciferase reporter assay, 46 LuSIV cells, 277, 288 Lycopersicon esculentum agglutinin (LEA), 235 M Macrophage galactose-type C-type lectin (MGL), 132 Mannose 6-phosphate receptor homology domain-containing lectins N-glycan, 182

578

Subject Index

Mannose 6-phosphate receptor homology domain-containing lectins (cont.) OS-9 and XTP3-B (see OS-9 and XTP3-B lectins) Saccharomyces cerevisiae, 182 synthesized glycoproteins, quality control, 183–184 Mass spectrometric analysis, 123–124 Matrix-assisted laser-desorption mass spectrometry (MALDI-MS), 176–178 MDM. See Monocyte-derived macrophages Membrane glycoproteins, 173 MOG. See Myelin oligodendrocyte glycoprotein Monocyte-derived macrophages (MDM), 274, 278 Monosiga brevicollis, 184, 187 Multiple-reaction monitoring (MRM), 408, 412–413 Mycobacterium tuberculosis, 153, 226 Myelin oligodendrocyte glycoprotein (MOG), 228 N N-acetyl-b-D-glucosamine (D-GlcNAc), 515 b–1,3-N-Acetylglucosaminyltransferase. See Lacto-N-triose II synthesis Nanoliquid chromatography, 176 Nematode growth media (NGM), 147 NF-kB activation, 156–157 phosphorylation and acetylation, 157–160 N-glycans branching measurement coimmunoprecipitation, 251–252 flow cytometry, 252–253 glycoprotein clustering and colocalization, 253 Golgi enzyme assays, 254 inhibitors, 257 mass spectroscopy, 253–254 mRNA half-life/stability measurement, 255 real-time PCR, 254 integrin-mediated cell adhesion and migration carcinogenic process, 452 E-cadherin-mediated cell–cell interaction, 453 GnT-V/b1,6 expression, 452–453 integrin a3b1, 453 molecular mechanism, 454 oligosaccharides, 446 N-glycosylation sites identification, 454 mutagenesis, 451–452 a5 subunit, GnT-III regulation, 455–456 NHS. See N-hydroxysuccinimide N-hydroxysuccinimide (NHS), 418–419, 441 Non-small cell lung cancer (NSCLC), 57, 59

Notch EGF20 MALDI-TOF-MS spectrum, 361–362 plasmid encoding, 360 purification, 361 NSCLC. See Non-small cell lung cancer Nucleotide binding oligomerization domain (NOD), 152 O O-fucosylation, TSRs. See Thrombospondin type 1 repeats O-GlcNAc modification, notch receptors galactosyltransferase labeling b4GalT-1 assay, 364, 367 lectin blotting, 367 hexosaminidase treatment, deglycosylation glycopeptide, 369 glycoproteins, 368 b-N-acetylhexosaminidase digestion, 367–368 mass spectrometry MALDI-TOF-MS and MS/MS analysis, 363 Notch EGF20, 360–362 protein expression, 361 reduction, S-carbamidomethylation, and trypsin digestion, 361 S2 culture media, 361 Notch–ligand interaction, 358 O-b-GlcNAc modification detection immunoblotting, CTD110.6 antibody, 363, 365–366 MALDI-TOF-MS/MS analysis, 363–364 O-fucose, 356–358 O-glucose, 356 posttranslational modifications, 356 recombinant EGF domain preparation, 358–359 in vitro O-GlcNAc transferase assay, 360 O-glycans, 446 O-linked fatty acid, 112–113 Orcinol staining, 122 Ortho-nitrophenyl-b-D-galactopyranoside, 518 OS-9 and XTP3-B lectins ERAD ER lumen, 191 NHK degradation, 191–192 pulse-chase analysis, 192–193 glycoproteins, quality control, 183 membrane-embedded ubiquitin ligase HRD1/synoviolin, 187 immunoprecipitation and Western blotting, 188–190 ubiquitylation, 188 sugar recognition specificity, 190–191 and Yos9p primary structures, 184–187

579

Subject Index P Partially methylated and acetylated alditol (AAPM), 105–106, 109 Pathogen recognition receptors (PRRs), 152 PBMCs. See Peripheral blood mononuclear cells PBST. See Phosphate buffered saline Tween-20 p24 ELISA, 274–275, 287 Peripheral blood mononuclear cells (PBMCs), 276–278, 287 PEtn. See Phosphoethanolamine PGAP1 cells, 501, 503 Phage display antibody. See Sulfs enzymatic activity Phaseolus vulgaris, 252 Phenylmethanesulfonylfluoride (PMSF), 144 pHluorin, 505–506 Phosphate buffered saline Tween-20 (PBST), 363, 365 Phosphoethanolamine (PEtn) At6 and At9 cores, 313 biosynthetic precursors, 314 zwitterionic character, 312 pH-sensitive green fluorescence protein, 504 pH sensor, 505 Pofut1. See Protein O-fucosyltransferase 1 Polymorphonuclear neutrophils (PMN) degranulated mast cells, 203 human transmigration, 203–204 inhibitory properties, 205 isolation, 207 phosphatidylserine (PS) exposure, 204 preincubation, 204 Polyvinylidene difluoride (PVDF) membrane, 363, 365 Primary cells, 288 Printed glycan microarray advantage, 442 CFG binding buffer, 421 fluorescent detection methods, 419–420 GAL file, 420 GBP analysis, 420–422 Glycan Array Synthesis Core D, 419 protein–carbohydrate interaction Core H, 418 protein–glycan interaction Core H, 419 Steering Committee, 418 complex oligosaccharide synthesis, 441 concentration-dependent binding glycan motif definition, 422–423 SNA (see Sambucus nigra agglutinin) human galectin-8, CRDs A-blood trisaccharide, 438 affinity chromatography, 431 Alexa488-/FITC-labeled streptavidin, 432 fucose residue, 434 full-length Gal-8 patterns, 432–433 galectin-1, 431–432

glycan specificity/glycan-binding motif, 434, 438–439 human blood group A and B, 434 N-acetyllactosamine-containing glycans, 434 ranked glycans, 432, 434–437 recombinant C- and N-terminal domains, 439 NHS, 418–419, 441 physiological ligands identification, 439–440 protein–protein interaction analysis, 418 Proprionibacterium acnes, 222 Protein O-fucosyltransferase 1 (Pofut1), 402, 404, 406 Protein O-fucosyltransferase 2 (Pofut2), 404–407 Protein O-glucosyltransferase. See also Rumi CAP10 domain, 386 chaperone-like function, 388 enzyme assay, 389–390 mutations, DXD-like motif, 387–388 O-fucose, O-glucose and O-GlcNAc, 387 product analysis, 390–391 Rumi-G189E protein, 388 in vitro assays, 387 Proteoglycans biological functions Dally and Dlp, 334–335 dHS6ST/Hs6st and Hs2st, 336 dystroglycan and Perlecan, 335 GSC, 335 HS-binding proteins, 334 lipophorins, 334 lipoprotein particles, 333–334 morphogens, 332–333 N-deacetylase/N-sulfotransferase, 336 Sdc and Dlp function, 335 Slit/Robo signaling, 332 sulfation, 336 core proteins, 331 Proteoglycan signaling networks autonomous signaling limitations, proteoglycan chimera model, 8 nonimmune IgG, 7 PKCa, 7 Rho GTPases, 7–8 RNAi-mediated genetic knockdown, 9 signal transduction, 6, 8 cell adhesion fluorescent membrane dyes, 11 force mapping, 13 live-cell microscopic imaging, 12 matrix attachment and migration, 10–11 mechanisms, 11 microdeformities/shifts, 13 TIRFM, 12–13 chimera oligomerization, 19–20 downregulation, 9–10 endocytosis assays cellular sensitivity, 23 cytoplasmic protein markers, 24

580

Subject Index

Proteoglycan signaling networks (cont.) experimental methods, 21, 23 flow cytometry, 23–24 live-cell microscopy, 24 quantification and internalization, 21–23 RFPECs, 21 growth factor signaling, 5–6 immunoblotting, heparan sulfate proteoglycans cell lysates, 18 GAG chain, 17–18 luminance-/fluorescence-based methods, 19 nonspecific membrane binding sites, 18 TBST and TBS, 18–19 Western blot analysis, 17 integrin interactions, 6 migration Boyden and Dunn chambers, 16 FRET, 14 multicellular migration studies, 16–17 single-cell migration assays, 16 thermal regulation, 15 three-dimensional models, 17 multicellular migration assay, 26–27 Rac1 pull-down assay, 24–26 structure, nomenclature and function, 4–5 two-chambered migration assay, 27–28 PVDF membrane. See Polyvinylidene difluoride membrane R Rac1 pull-down assay, 24–26 Rapid isolation of proteoglycans (RIP) method, 69–71 Rat fat pad endothelial cells (RFPECs), 20–21 RB4CD12 epitope ELISA, 54–55 ex vivo sulf assay materials, 59, 61 method, 60–61 flow cytometry-based sulf assay materials, 55, 57 method, 57–59 Red blood cells (RBCs), 281 Regions of interest (ROIs), 506 Relative fluorescence units (RFU), 420, 422–423, 425 Reversed-phase chromatography, 120 RFPECs. See Rat fat pad endothelial cells Rumi genetic identification lateral inhibition and asymmetric cell division, 377–378 loss-of-function phenotypes, 379 mutations, 378–379 shaft and socket cells, 378 sheath cell and neuron, 378 temperature-sensitive loss, sensory organs, 379

mapping and sequencing methods, 382–383 PCR primers, 384 protein-null allele generation, 385–386 protein O-glucosyltransferase CAP10 domain, 386 chaperone-like function, 388 enzyme assay, 389–390 mutations, DXD-like motif, 387–388 O-fucose, O-glucose and O-GlcNAc, 387 product analysis, 390–391 Rumi-G189E protein, 388 in vitro assays, 387 rescue experiments, 384–385 Rumi-G189E protein, 388 S Saccharomyces cerevisiae, 182–183 Salmonella typhosa, 152 Sambucus nigra agglutinin (SNA) biotinylated analysis, 423–424 bound lectin, 423 concentration-dependence, 425 elderberry bark agglutinin I, 423 lectin cytometry, 235 Neu5Aca2–6Gal linkages, 423, 425 ranked glycans, 425–427 RFU, 423, 425 Schistosoma mansoni egg antigen (SEA), 222, 231, 236–237 Schistosoma mansoni glycosphingolipids cercariae transform, 119 chronic helminth infection, 118 dendritic cell receptors human monocyte-derived immature dendritic cells, 131–132 immature dendritic and K562 cells adhesion, 132–134 immune system, 130–131 modulation, 134–136 glycolipids, 118 immunochemical characterization ELISA, 129–130 overlay technique, 128–129 isolation and purification anion-exchange chromatography, 121 carbohydrate moiety, 119 desalting, reversed-phase chromatography, 120 fractionation, silica gel chromatography, 121 glycosphingolipid extraction, 120 lipid contaminant saponification, 120 structural characterization ceramide moiety, 124–126 HPTLC, 122

581

Subject Index

mass spectrometric analysis, 123–125 survey, 126–128 SDS-PAGE. See Sodium dodecyl sulfate polyacrylamide gel electrophoresis SEA. See Schistosoma mansoni egg antigen SEC. See Size exclusion chromatography Sialic acid-binding adhesin (SabA), 514 Size exclusion chromatography (SEC), 77–78, 80 SNA. See Sambucus nigra agglutinin Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) acidic polysaccharides, 100 galectins quality control, 280 Kittelberg and Hilbink protocol, 98–99 Tsai and Frasch protocol, 99–100 Sulfs enzymatic activity ELISA, 54–55 ex vivo sulf assay materials, 59, 61 method, 60–61 flow cytometry-based sulf assay materials, 55, 57 method, 57–59 formylglycine and N-linked glycan modification, 52 HSPGs, 53 lysosomal sulfatases, 52–53 N-sulfation, 2-O-sulfation, and 6-O-sulfation, 53 QSulf-1, 52 SWISSPROT database, 177 Syndecan (Sdc), 331–332, 334–335

ligand binding affinity, 407 O-fucose glycans, 402–404, 409 O-glucose glycans, 402, 412 Peter’s Plus syndrome, 406–407 Pofut1 functions, 406 predicted Pofut2 targets, 404–406 properdin analysis, 406 TLC assay, 538 Toll-like receptors (TLRs), 131, 134–136 Total internal reflection fluorescence microscopy (TIRFM), 12–13 Total ion mapping (TIM), 303, 305 Trans-Golgi network (TGN), 496–497, 500, 505 Transmembrane (TM)-type reporter proteins, 499–500 Transport assay, 501–504 Triacylglycerides (TAG) BOSS mutant flies, starvation, 535–536 detection circulating (hemolymph) TAG level measurement, 538 fat droplets, 537 Oil-Red-O and BODIPY staining, 539 total body TAG level measurement, 538 Tryptophan, 167 TSP2. See Thrombospondin 2 TSRs. See Thrombospondin type 1 repeats Two-chambered migration assay, 27–28 TZM-bl cell line, 277

T

V

TAG. See Triacylglycerides T cell receptor (TCR) signaling, 260–261 Tetramethylbenzidine (TMB), 130 Thrombospondin 2 (TSP2) fucose–glucose disaccharide, 410 O-fucosylated peptides, 410–411 Thrombospondin type 1 repeats (TSRs) CD36 and integrins, 404 C-mannose, 403, 412 cysteine-rich motifs, 404 EGF repeat, 402–403 Glc-b1,3-Fuc disaccharide, 403 glycosylation site mapping in-gel digestion, 407 LC–MS, digested sample, 409–413 materials, 408 MRM, 408 sample preparation, 408–409 stock and fresh solutions, 408

U UDP-GlcNAc biosynthesis, 259–260

Vacuolar-type proton ATPases, 497 Vesicular stomatitis virus G protein (VSVGts(ex)), 499–500 W Western blot analysis, 224 Western blotting, 472 Whole-cell extract (WCE) bacterial suspension, 145 BL21(DE3) control transformant, 144 La¨mmli sample buffer, 144 soluble protein (SP), 144, 146 Wingless (Wg), 332–336 X Xenopus laevis, 191 Xerocomus chrysenteron, 145