Advances in Carbohydrate Chemistry and Biochemistry, Volume 35

Advances in Carbohydrate Chemistry and Biochemistry Volume 35

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Advances in Carbohydrate Chemistry and Biochemistry Editors R. STUART TIPSON

DEREK HORTON

Board of Advisors LAURENSANDERSON STEPHENJ. ANGYAL GUY G. S. DUTTON ALLAN B. FOSTER DEXTER FRENCH

BENCT LINDBERC HANS PAULSEN NATHANSHARON MAURICESTACEY ROY L. WHISTLER

Volume 35 1978

ACADEMIC PRESS

New York San Francisco London

A Subsidiary of Harcourt Brace Jovanovich, Publishers

COPYRIGHT @ 1978, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONlC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSlON IN WRITING FROM THE PUBLISHER.

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United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON)LTD. 24/28 Oval Road, London N W I 7DX

LIBRARY OF CONGRESS CATALOG CARD NUMBER: 45-11351 ISBN 0-12-007235-1 PRINTED IN THE UNITED STATES OF AMERICA

CONTENTS LIST OF CONTRIBUTORS. . . . . . . . . . . . . . PREFACE . . . . . . . . . . . . . . . . . . . . .

............ ............

vii ix

Edmund Langley Hirst (1898-1975) MAURICESTACEY Text

AND

DAVIDJ . MANNERS

...................................

1

Carbohydrate Boronates ROBERTJ . FERRIER I . Introduction ............................ I1 Synthesis of Boronates ....................... I11. Structures of Carbohydrate Boronates .............. IV Boronates in Chemical Reactions . . . . . . . . . . . . . . . . . . . . . V Separations of Carbohydrates by Use of Their Boronates VI Mass Spectrometry of Boronates . . . . . . . . . . . . . . . . . VII . Nuclear Magnetic Resonance Spectroscopy ofBoronates . . . . . . VIII . Borinates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1X.Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X.Addendum . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . .

31 37 41 48 57 65 70 70 71 80

....... ....... ....... . . . . . . . .......

81 82 102 122 122

. . . .

.. . .

Biosynthesis of Sugar Components of Antibiotic Substances HANS GRISEBACH

..................... I . Introduction I1. Branched-chain Sugars ................ I11. Aminocyclitol Antibiotics ............... IV Amino Sugars Not Occurring in Aminocyclitol Antibiotics V Nucleoside Antibiotics ................

. .

The Lectins: Carbohydrate-binding Proteins of Plants and Animals IRWIN J . GOLDSTEINAND COLLEENE . HAYES I . Introduction ............................ I1. D-Mannose(D-Glucose)-binding Lectins . . . . . . . . . . . . . . . . I11. 2-Acetamido-2-deoxy-~-glucose-binding Lectins . . . . . . . . . . . . IV . 2-Acetamido-2-deoxy-Dgalactose-binding Lectins . . . . . . . . . . . V . D-Galactose-binding Lectins . . . . . . . . . . . . . . . . . . . . . VI . L-Fucose-binding Lectins ...................... VII . Other Lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII . Cell-surface. Lectin-reactive Glycoproteins . . . . . . . . . . . . . . IX.Tables ............................... V

128 150 206 226 254 277 291 317 334

CONTENTS

vi

Biochemistry of Plant Galactomannans PRAKASH M . DEY

. .

I Introduction ...... I1 Biosynthesis ...... I11. Biochemical Degradation IV Function . . . . . . . .

.

...................... ...................... ......................

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341 352 356 375

Bibliography of Crystal Structures of Polysaccharides. 1975 PUDUPADI R.

. .

SUNDARARAJAN AND

ROBERT H . MARCHESSAULT

I Introduction ................ I1. Amylose and Other a-D-Glycans . . . . . . I11 Cellulose and Other p-D-Glycans ..... IV. Glycosaminoglycans (Amino Polysaccharides)

............ 377 . . . . . . . . . . . . . 378 . . . . . . . . . . . . . 379 . . . . . . . . . . . . . 381 AUTHOR INDEX FOR VOLUME 35 . . . . . . . . . . . . . . . . . . . . . . 387 SUBJECTINDEXFOR VOLUME35 . . . . . . . . . . . . . . . . . . . . . . 415 CUMULATIVE AUTHOR INDEX FOR VOLUMES 31-35 . . . . . . . . . . . . . 429 CUMULATIVE SUBJECT INDEX FOR VOLUMES 31-35 . . . . . . . . . . . . . 431 ERRATUM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434

LIST OF CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin. PRAKASH M. DEY,Department of Biochemistry, Royal Holloway College, University

of London, Egham Hill, Egham, Surrey TW20 OEX, England (341) ROBERT J. FERRIER,Department of Chemistry, Victoria University of Wellington, Private Bag, Wellington, New Zealand (31)

J. GOLDSTEIN,Department of Biological Chemistry, The university of Michigan, Ann Arbor, Michigan 48109 (127)

IRWIN

HANS GRISEBACH,Biologisches Znstitut 11, Biochemie der Pjlanzen, Universitat Freiburg i. Br., D 7800 Freiburg i m Breisgau, Germany (81) COLLEEN E. HAYES, Immunobiology Research Center, University of Wisconsin, Madison, Wisconsin 53706 (127)

DAVIDJ. MANNERS,Department of Brewing and Biological Sciences, Heriot-Watt University, Chambers Street, Edinburgh EHl I H X , Scotland (1) ROBERTH . MARCHESSAULT,Department of Chemistry, University of Montreal, P. 0. Box 6210, Succursale A , Montreal, Quebec H3C 3V1, Canada (377) MAURICE STACEY,Department of Chemistry, The University of Birmingham, P. 0. Box 363, Birmingham B15 ZTT,EngZand (1) SUNDARARAJAN, Xerox Research Centre of Canada, 2480 Dunwin Drive, Mississauga, Ontario L5L 119, Canada (377)

PUDUPADI R.

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PREFACE

In 1961, Ferrier (Wellington, N. Z.) began a study of the condensation of phenylboronic acid with the diol groupings of various glycosides, and since then, as a result of research by him and many other workers, the subject has developed rapidly and has afforded information that is of great potential value to synthetic carbohydrate chemists; Ferrier now provides us with a fascinating account of the progress made to date in the study of carbohydrate boronates. This unifying picture of the use of these cyclic, protected sugar derivatives should afford considerable help to the chemist searching for novel approaches in synthesis by use of these versatile protecting groups. In an article that focuses particularly on some of the more-recent developments, Grisebach (Freiburg im Breisgau) discusses the biosynthesis of sugar components of antibiotic substances, a field that has shown major advances since the time of the article by Dutcher in Volume 18 of Advances. The chapter constitutes an integrating complement to the articles on aminocyclitol antibiotics by the Umezawas (Volume 30). Goldstein (Ann Arbor, Michigan) and Hayes (Madison) contribute a monumental chapter on lectins, the specific carbohydrate-binding proteins present in both plant and animal species. The remarkable specificity of certain carbohydrate-protein interactions has farreaching implications in biochemistry, the full significance of which is only just beginning to be properly understood. As this subject has not previously been treated in depth in Aduances, these authors have written a comprehensive history of the subject, starting with Stillmark’s discovery of plant agglutinins in 1888, and proceeding to 1977; this article brings together in one place an enormous amount of information scattered throughout the literature, and should constitute the definitive treatment of lectins for many years to come. Another biochemical topic, the biochemistry of plant galactomannans, is discussed by Dey (Egham, Surrey); the article rounds out aspects of the field that are complementary to those treated by Gorin and Spencer in Volume 23 (on fungal polysaccharides) and Dea and Morrison in Volume 31 (on the chemistry and interactions of seed galactomannans). In a continuation of the series of bibliographic articles on the structures of polysaccharides as established by X-ray crystallographic methods, Sundararajan (Mississauga, Ontario) and Marchessault ix

X

PREFACE

(Montreal) present the information recorded in the literature during 1975, thus updating their article in Volume 33. Since the latter was written, SI units have become generally adopted; the time-honored Angstrom unit (A) has now fallen into disuse, and so it will no longer be employed in this Series. The death of our friend and mentor Sir Edmund Hirst was briefly noted in the Preface to Volume 32. A full account of his career and wide-ranging achievements is given here by Stacey (Birmingham) and Manners (Edinburgh). The Subject Index was compiled by Dr. L. T. Capell. Kensington, Maryland Columbus, Ohio February, 1978

R. STUART TIPSON DEREK HORTON

Advances in Carbohydrate Chemistry and Biochemistry Volume 35

z<*-%K. 1898 - 1975

EDMUND LANGLEY HIRST* 1898- 1975 Edmund Langley Hirst was the elder son of the Reverend Sim Hirst, a Baptist minister, and Elizabeth Hirst (nke Langley). He was born in Preston, Lancashire, on 21st July, 1898, and he had a somewhat unsettled childhood, due to the many changes in his father’s ministry. His father’s family had been long established in the town of Clayton, near Bradford, Yorkshire, where his grandfather and several uncles worked in the woollen mills, and where other uncles were shopkeepers. All the family were stout Nonconformists and loyal to the local Baptist church. The Reverend Sim was born in Clayton in 1856, the son of John Hirst, a weaver, and Martha Hirst. He graduated B.A. from the University of Durham, was trained for the ministry at Rawdon College near Leeds, Yorkshire, and later gained the Bachelor of Divinity degree of St. Andrews University. He was for various periods Minister at churches in Stoke-on-Trent, St. Andrews (for three different periods), Durham, Preston, Burnley, and Ipswich; he died suddenly in 1923. Hirst’s mother (Elizabeth), to whom Edmund was greatly attached, was born in 1869, the daughter of Joseph and Mary Langley of Liverpool, where Joseph was a successful flour merchant and baker, and she was educated at private schools. She was of mixed Welsh and North Country stock, and her family had farmed land in the English Lake District for generations. They had married in 1897 and she, living to the age of 86, survived her husband by 32 years. The marriage was particularly happy, for, although she belonged to the Church ofEngland, she was an understanding and loyal Baptist Minister’s wife. She made sure that Edmund and his younger brother Sim had a sound schooling, despite the many changes of homes. Hirst first attended a kindergarten school in Burnley, and then had private tutoring in other towns, and a governess in Ipswich where, at the age of 8, he attended Ipswich Municipal Secondary School. The following year, he suffered a sharp set-back due to a severe mastoid illness. In 1910, he was more settled in St. Andrews, where after a considerable personal effort, he succeeded in gaining entrance to the

* The authors are indebted to Lady Hirst for her advice during the preparation of this article, and to Walter Bird for the photograph. 1

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MAURICE STACEY A N D DAVID J. MANNERS

famous Madras College in that ancient city. He received a sound grounding in Greek and mathematics, and finished school at the age of 16, as Dux (Head Boy) of the School, winning entrance to St. Andrews University with a i40-a-year bursary and a Carnegie Scholarship. In his first year, he studied mainly mathematics, Greek, and Latin, at which he did so well that he almost changed over to take a degree in Classics. It was the advent ofworld War I that turned him to Chemistry. In the summer vacation of 1915,he volunteered to assist the war effort by joining a group in the Chemistry Department led by Professor (later Principal Sir James) Irvine and Dr. (later Sir Norman) Haworth, who were making such chemicals as local anesthetics, previously obtained from Germany. He was able to continue this work during the following summer, when he gained excellent experience in preparative chemistry and had his first taste of carbohydrate chemistry by the preparation of D-galactose and galactitol (dulcitol) from lactose. In 1917, he was called up for military service, but was immediately seconded back to the University for urgent work on the large-scale preparation of mustard gas. In 1918, he volunteered for the Special Branch of the Royal Engineers, and was despatched to Northern France. The following February, he was demobilized and was able to return to complete his studies. His performance in examinations was brilliant, for he gained his Bachelor’s degree with distinction in chemistry, mathematics, and natural philosophy, and was awarded First Class Honours in mathematics and natural philosophy in his Master’s degree. He gained many medals during his degree courses. Following discussions with Haworth, he decided to join him in order to follow a research career in carbohydrate chemistry. He was awarded a Carnegie Teaching Fellowship to study the structure of cellobiose. He was immediately successful and gained his Ph.D. in 1921, after only two years of research. When Haworth moved to a Chair at Armstrong College, Newcastle (part of the University of Durham), Hirst continued to work on cellulose in collaboration with Sir James Irvine, and he also began independently his important studies on the ring structures of sugars, particularly xylose. At this time, Irvine became Principal of St. Andrews University, and Robert Robinson was appointed Professor of Chemistry. Robinson’s stay, however, was brief, for he moved to the Manchester Chair in 1922, and in the following year, Robinson invited Hirst to join his staff. From 1924 to 1926, Hirst was a lecturer at Armstrong College, Newcastle, first with Haworth and then with H. V. A. Briscoe. Haworth moved to Birmingham in 1925, and was joined by Hirst in 1927, when their famous partnership really began. There was already in Birmingham a strong staff, a number of whom, including H. D. K. Drew, C. E. Wood, C. R. Porter, S. R. Carter, and E.

OBITUARY-EDMUND

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3

G. Cox, were brought into the carbohydrate team to join the research students, including Stanley Peat, whom Haworth had brought from Newcastle. Hirst was appointed Lecturer and Assistant Director of Research, and also undertook a heavy teaching load. He was, however, spared administrative and University committee work. His years at Birmingham were intensive and highly productive of important results in the carbohydrate field, including the vitamin C work, as will be described later. He was awarded his Birmingham D.Sc. in 1929, and was made Reader in the Chemistry of Natural Products in 1934,the year he was elected a Fellow ofthe Royal Society. In 1936, he was appointed to the Alfred Capper Pass Chair of Organic Chemistry in the University of Bristol, in succession to Professor F. Francis. He took with him to Bristol Dr. (later Professor) J. K. N. Jones and Dr. G. T. Young, who assisted in the direction of his research groups. With Bristol graduates, they quickly built up a vigorous research team working on numerous carbohydrate problems, which included starches, pectic substances, and certain plant gums. This work was suspended early in 1939, for the Bristol Laboratories, of which Professor W. E. Garner was the Head, were turned over for urgent Government work on explosives. Garner and Hirst and their staff supervised a group of workers from the Research Branch of Woolwich Arsenal. Hirst, already heavily involved in Civil Defence work as Senior Gas Adviser to the South West Region, engaged in the work with his usual vigor. He regularly visited various ordnance factories concerned with manufacturing and filling. He was a member of several committees of the Scientific Advisory Council of the Ministry of Supply and of other Committees on explosives. J. K. N. Jones was the senior assistant for this work, which now occupied 90%of Hirst’s time. Important help, especially for undergraduate teaching, came from Professor G. M. Bennett and his staff, who were evacuated from King’s College, London, to Bristol. In 1944, Hirst was appointed to succeed Alexander Todd (now Lord Todd, P.R.S.) in the Sir Samuel Hall Chair of Organic Chemistry at Manchester University. He went there in January, 1945, taking with him J. K. N. Jones, as Senior Lecturer, and some members ofthe Bristol team, to complete the explosives researches. He immediately became involved with Professor Michael Polanyi in much reorganization and in planning new laboratories to cope with the large intake of ex-Service students. H e was also in great demand for public service, especially as Chairman of the Research Section of a Working Party reporting on the state ofthe cotton industry of Lancashire. He also gave great help to the Shirley Institute for Cotton Research as a member of their research committees. In 1947, he was delighted to accept an invitation to return to his

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MAURICE STACEY AND DAVID J. MANNERS

beloved Scotland. This came from Professor James Kendall of the University of Edinburgh, who had created the Forbes Chair of Organic Chemistry, which Hirst moved to occupy in that year. He remained in Edinburgh for the rest of his life, making during the 21 years of his Professorship a great impact on that University, on Scottish higher education, and on the applications of science to Scottish industry and agriculture. On the personal side, Hirst was always a charming companion, and a valued friend to many people, particularly his former students. He was a good listener and always had sound advice to give. His early domestic life in Birmingham had not been easy. In 1925, he married Beda Winifred Ramsay, daughter of Frank and Mrs. Ramsay of Glasgow, who were friends of the family. Early in their married life, his wife developed a mental illness which necessitated continuous hospital care until her death. The marriage was dissolved in 1948, and, in 1949, he married Kathleen (Kay) Jennie Harrison. Her father was a well known Birmingham headmaster, and her two brothers were chemistry graduates of Birmingham University. Kay was one of His Majesty’s Inspectors of Schools, and later became well known for her charitable interests. This marriage was ideally happy, and Kay gave Edmund the fullest support in all his endeavors. She took part, as a Committee member, in the many activities of the University Ladies Club and the Women Students’ Union. She was involved in work for crippled children, the Queensberry House Hospital for old people, the Edinburgh College of Domestic Science (now Queen Margaret College), and the Moray House College of Education, and was on the Council, and Chairman for two years, of an Edinburgh Training School for nurses. Edmund was immensely proud of Kay, who understood him so well, and together they made a fine team. Hirst’s first personal research venture into the structural carbohydrate field showed remarkable insight. He selected xylose for investigation, and discovered that mild oxidation of the methylated sugar with bromine-water formed the methylated lactone which, on stronger oxidation with nitric acid, gave a number of methylated dibasic acids, the identification of which could be used to decide the size of the ring in the parent sugar. With A. Carruthers in 1922, and C. B. Purves in 1923, he showed that, on oxidation with bromine-water, crystalline tri-0methyl-D-XylOSe gave 2,3,4-tri~-methy~-D-xy~ono~actone which, on further oxidation with nitric acid, yielded “i-xylo-trimethoxyglutaric acid” (tri-0-methylxylaric acid). This proved that the original xylose possessed a six-membered ring. Before going to Birmingham he made, with a succession of research

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5

students, important studies on methylation, on the structure of cellulose, on other monosaccharides, and on raffinose. The methylationoxidation techniques were applied systematically and with great vigor and success in Birmingham during the ten years of the Haworth-Hirst partnership. The ring-structures of most of the sugars known at that time, mono-, di-, tri-, oligo-, and poly-saccharides, including the difficult problem presented by sucrose, were elucidated. A number of innovations speeded the work considerably, one of the most important being the introduction into Britain by Drew and Haworth, in 1925, of the Kuhlmann balance and Pregl’s methods of microanalysis. Another was the appreciation that physicochemical methods could be used to solve structural problems; such tools as U.V. absorption spectra, optical rotatory dispersion, and X-ray crystallographic analysis, introduced by E. G. Cox (now Sir Gordon), were pioneered. The research workers studying the relationship between carbohydrate structure and optical rotatory power included R. S. Tipson. The Haworth-Hirst partnership, aided by a succession of able and enthusiastic research students, including many from overseas, resulted in over one hundred publications and the establishment of the Haworth system of furanose and pyranose nomenclature and of perspective and conformational depictions of sugars. Both Haworth and Hirst believed in team-work, and this was seen to advantage in the research that settled the notable controversy with C. S. Hudson over the structure of D-mannose. This was concluded at a famous meeting with Haworth, Hudson, and Purves in 1930, which was fully described by Hirst in The Hudson Memorial Lecture [J. Chem. SOC., 4042-4058 (1954)l. Hirst always had great admiration for Hudson’s researches on the optical properties of carbohydrates, and conducted extensive personal experimental work (with C. E. Wood) on optical rotatory dispersion. Numerous other fascinating projects were in progress at this time, notably studies on novel forms of stereochemistry, the beginnings of conformational analysis, crystalline sugar carbonates, furanose forms of sugars, and structural studies on polysaccharides, including glycogens and numerous mold and bacterial polysaccharides. A major step forward in polysaccharide chemistry was the demonstration of cellobiose residues in cellulose. From these investigations and the numerous methylation studies, a valuable collection of reference compounds was synthesized and generously supplied to workers all over the world. All of this work was set aside in 1932, when Haworth formed most of the carbohydrate workers into a team (or “syndicate”) to investigate the structure of Szent-Gyorgyi’s “hexuronic acid,” which proved to b e vita-

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MAURICE STACEY A N D DAVID J. MANNERS

min C, and was later named ascorbic acid. These were exciting times, and the rapid and successful outcome of the work was due in large measure to Hirst’s remarkable intuition and his real inspiration to the team. Consolidation of the ascorbic acid work was achieved in close collaboration with F. Smith and J. K. N. Jones who, during the next few years, produced a fine series of papers on analogs of ascorbic acid. A number of pioneer studies pointed the direction to future work in Bristol, Manchester, and Edinburgh. Among these were researches on the carbohydrates of grasses and barley leaves, and on hexuronic acids, particularly the “aldobionic acid” from gum arabic on which F. Smith was working with such vigor. Inspiration came from a visit in 1936 to England by Michael Heidelberger, who impressed on Hirst the close relationship between gums and immunopolysaccharides. The move to Bristol, and thence to Manchester, as already indicated, brought Hirst additional duties away from chemistry, and, without the devoted help given by J. K. N. Jones at both Manchester and Bristol, and by G. T. Young at Bristol and T. G. Halsall at Manchester, his contributions to carbohydrate chemistry would have been lessened. It is a remarkable fact that the Bristol and Manchester periods resulted in over 70 papers in which the constitutions of a whole range of plant gums, pectic substances, and mucilages were determined. Amongst these were damson gum, slippery-elm mucilage, and various arabinans and galactomannans. During this period, chromatographic methods for the separation of sugars were being developed, and, with J. K. N. Jones and also Leslie (now Professor) Hough, Hirst played a full part in this work. Methods for the separation and identification of sugars, especially methylated sugars, by paper chromatography were devised, and in a series of three papers, they described standardized methods for their quantitative determination. Their methods were reported in a Discussion of the Faraday Society in 1949, and their application to the analysis of Sterculia setigera gum was published in the same year. These methods, and subsequent improvements, were later widely used in the Edinburgh laboratories. When Hirst returned to Scotland in 1947, he knew that the Edinburgh laboratories already contained a very active research school in carbohydrate chemistry that had been built up by E. G. V. Percival, whose work was mainly concerned with the structure of plant mucilages and seaweed polysaccharides. These interests were complementary to Hirst’s own recent studies in Manchester on the structure of polysaccharides, particularly the plant gums, and the next few years saw a renewal of the successful collaboration of the Birmingham period 1930-1933, until the untimely death of Percival in 1951.

OBITUARY-E DMU N D LANGLEY HI RST

7

Several papers resulted from this collaboration, some of which represented the first stages ofcontinuing investigations later completed with other members of the staff, particularly G. 0. Aspinall and Elizabeth Percival. With S. K. Chanda, structural analyses of the xylan and an arabinose-rich hemicellulose from esparto grass, and of a xylan from pear cell-walls, were carried out. Preliminary studies on the overall molecular structure of the two mannans from ivory nuts, the reserve p-D-glucan from Laminaria cloustoni, the alginic acid from Laminaria digitata, and the seed mucilage from Plantago arenaria were reported. During this period, two laboratories in Britain were particularly interested in polymers of D-fructose. D. J. Bell in Cambridge had made progress with various samples of inulin and plant levans [see Adv. Carbohydr. Chem. Biochem., 30,l-8 (1974)],whilst, in Edinburgh, his personal friends Hirst and Percival reported similar results with inulin from dahlia tubers, and with a fructan from Dactylis glomerata. Both laboratories confirmed the presence of chains of (2+1)- or (2+6)-linked p-D-fmctofuranosyl residues, which were terminated at the potential reducing end by a sucrose residue. This latter structural feature had not been revealed in earlier investigations, and had an important bearing on the biosynthesis of the D-fructans from sucrose by transfructosylation reactions. During the early 1950s, Hirst appointed several new members of staff having interests in certain specialized areas of carbohydrate chemistry. These included D. M. W. Anderson, G . 0. Aspinall, C. T. Greenwood, D. J. Manners, Elizabeth Percival, D. A. Rees, and J. C. P. Schwarz who, with their respective research students, and visiting post-doctoral fellows from many parts of the world, together helped to constitute a large and successful research school. Collectively, substantial progress was made, not only in most branches of polysaccharide chemistry but also with many topics in monosaccharide chemistry. The carbohydrate staff held regular meetings to discuss the progress oftheir work, and the significance of papers in the current literature, especially those from the laboratories of Hirst’s former colleagues, Professors Maurice Stacey and Stanley Peat at Birmingham and Bangor, respectively. There were times when the monthly issues of the Journal of the Chemical Society were renamed the “Book(s) of Revelation.” For many of us, these were days full of incident and interest, before the advent of the current spectrometric techniques, when research workers still prepared crystalline derivatives of monosaccharides, and when the successful identification of these derivatives by the measurement of appropriate physical constants gave immense satisfaction to research student and supervisor alike. We recall the interest and excitement generated by the isolation of 6-0-methyl-~-galactosefrom a native polysaccharide, the

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MAURICE STACEY AND D,4VID J. MANNERS

synthesis by Peter Schwarz OfD-xylOSe containing sulfur in the ring, the confirmation by Elizabeth Percival of the presence of the hitherto rare L-guluronic acid as a constituent of alginic acid, and, later, the first applications by Dai Rees of conformational analysis to polysaccharide structure and functions. During those days, there was a constant flow of visitors from other carbohydrate laboratories, particularly external examiners for the numerous Ph.D students. The other noticeable product of this era was a steady stream of publications, mainly to theJourna1 of the Chemical Society, but also to the Biochemical Journal, to Chemistry and Industry, and later, to Carbohydrate Research. The atmosphere of this period is conveyed in several review lectures given by Hirst; for example, the fourteenth Pedler lecture to the Chemical Society (1955) on “Some Problems in the Chemistry of the Hemicelluloses,” the Presidential Addresses to the Chemical Society (1957 and 1958) on “Some Aspects of the Chemistry of the Fructosans” and “Polysaccharides of the Marine Algae,” respectively, on “Plant Gums,” at the IVth International Congress of Biochemistry, Vienna (1958), and the Bakerian Lecture to the Royal Society (1959) on “Molecular Structure in the Polysaccharide Group.” These lectures were delivered with a quiet authority, and the published manuscripts show meticulous attention to detail. In the structural analysis of polysaccharides, the importance of methylation, introduced by Purdie and Irvine, and developed by Haworth, is widely accepted, and was naturally fully utilized by Hirst and his many collaborators. Perhaps less widely known is his development of methods based on periodate oxidation for the analysis of a wide range of polysaccharides. With F. Brown, T. G. Halsall, and J. K. N. Jones, the first end-group assays of starches and glycogens were carried out. The experimental conditions for the analysis of starches were later refined, with Anderson and Greenwood. In these days of micro-scale methylation analyses using g.1.c.-m.s. techniques, the real advance represented by the periodate oxidation methods has tended to be overlooked. The latter methods lessened the amount of material required, from the gram scale to the decigram or even milligram scale, and the overall time of analysis from weeks to days. Periodate oxidation was also used to examine the nature of the inter-chain linkages in starches and related polysaccharides. The absence of periodate-resistant residues was evidence for the presence of glucosyl residues triply linked at C-1, C-4, and C-6, whilst the presence of some periodate-resistant residues could, assuming that the oxidation was complete, indicate the presence of other types of linkage. The fine structure of the (1-*3)-p-~glucan from Laminaria cloustoni was examined by other periodate-

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9

oxidation methods, involving estimation of the production of formaldehyde from the borohydride-reduced glucan, and hence, giving the degree of polymerization, and b y the application of the Barry and Smith methods of degradation. These latter provided evidence for the presence of ( 1 ~ 6 ) - ~ - g l u c o s i dinter-chain ic linkages rather than (1+6)inter-residue linkages, as had been suggested by other workers. The complex nature of the molecular architecture of the plant gums and mucilages had long been a major challenge to Hirst. Throughout the Edinburgh period, first with Aspinall, and later with Anderson, he published more papers on this than any other group of polysaccharides, the last appearingin 1968. Systematic studies on various exudate gums, involving determination of the composition, partial acid hydrolysis, methylation, and periodate oxidation studies (including multiple Smith degradations) led to the recognition of distinct groups of gums. The largest group contain a core of D-galactopyranosylresidues united by (1+3)- and (l+6)-linkages, to which are attached various proportions of D-glucuronic acid, 4-O-methy~-D-glucuronicacid, L-arabinofuranose, and L-rhamnopyranosyl residues. The central, galactan core showed multiple branching. Examples include gum arabic (Acacia senegal gum), and the gums fromA. pycnantha andA. arabica; Dr. (now Professor) A. S. Perlin was amongst those involved in the early studies of this group, as were two visitors from South Africa, Dr. A. J. Charlson and Dr. (now Professor) A. M. Stephen. A second group of gums contains a main backbone of alternating, 4-0-substituted D-glucosyluronic acid and 2-0-substituted D-mannOSyl residues, to which side chains containing various D-galaCtOSe, L-arabinopyranose, and D-glucuronic acid residues are attached. Gum ghatti (Indian gum), examined with Dr. (now Professor) A. Wickstrdm and (Mrs.) B. J. Auret, is a good example of this type. Gums of the third group contain interior residues of D-galacturonic acid and L-rhamnose, to which various side chains are attached, and include Khaya grandifolia gum and certain Sterculia gums, which show characteristic differences in fine structure from the Khaya gums. In many cases, the overall complexity ofthe gums was so great that only partial structures could be proposed. Full details of much of this work have been given by Aspinall [Adv. Carbohydr. Chem. Biochem., 24, 333-379 (1969)l. A second major interest during the Edinburgh period was the nature of the reserve and structural polysaccharides of the marine algae. This work paralleled the establishment ofthe Institute of Seaweed Research at Musselburgh, near Edinburgh. Hirst was a member of the Board of Governors, and Chairman of the Research Committee. A full exchange

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of information, samples, and materials operated to the mutual benefit of all concerned. Hirst’s first paper on alginic acid was published in 1939, and this, and later work in 1952, had established the presence of (1+4)-linked D-mannuronic acid residues. However, Fischer and Dorfell obtained evidence for the presence of L-guluronic acid residues, and this was confirmed by the isolation of L-threaric acid from a hydrolyzate of alginic acid after oxidation with periodate and then with bromine. Subsequent examination of reduced alginic acid gave a partial, acid hydrolyzate containing L-gulose, 4-O-~-D-mannosy~-L-gu~ose, and 4-0-p-D-mannosyl-D-mannose, thus showing the existence of both kinds of uronic acid residues in the same chain. Chemical and enzymic studies were carried out on the reserve p-Dglucans from various species oflaminaria, and from a mixed culture of diatoms. Although they all contained chains of (l+3)-linked residues, some of the algal samples contained 2-3% of mannitol residues which terminated a proportion of the chains; certain samples also showed a low degree of branching involving C-6. Other seaweed polysaccharides that were examined included those from Cladophora rupestris and Chaetomorpha spp.; these proved to be complex sulfated heteropolysaccharides containing residues of D-galactOSe, L-arabinose, and D-XylOSe. Floridean starch from the red seaweed Dilsea edulis was also characterized; it was found to resemble the amylopectin component of starch in some, but not all, respects. Hirst had a wide interest in the plant kingdom, as an examination of his personal library has shown, and his researches included studies of polysaccharides from a large number of different plants. With Aspinall, the hemicellulose A of beechwood and the egalactan from larch were investigated. With Rees, he surveyed the polysaccharide components of mustard seeds, with special reference to their embryos and their role in germination. The polysaccharide components of certain mosses were examined. With N. B. Chanda, the constitutions of the a-D-glucan and P-D-gluCan from Iceland moss (Cetraria islandica) were established; both are linear polysaccharides containing various proportions of both (1+3)- and (1*4)-linkages. By contrast, reindeer moss (Cladonia alpestris) synthesizes a highly branched galactoglucomannan. The constitution of starch had been a major research interest since the Birmingham days, and Hirst continued this work in Edinburgh. In collaboration with the Brewing Industry Research Foundation, for (1) F. G . Fischer and H. Dbrfel, Hoppe-Seyler’s Z. Physiol. Clzem., 302, 186-203 (1955).

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whom he acted as a consultant for many years, h e obtained the first evidence of the structural differences between the starches from malted barley and that from the ungerminated cereal, which had previously been examined by MacWilliam and E. G. V. Percival. As an organic chemist, Hirst tended to be somewhat suspicious of enzymic methods of analysis, and, during this period, when new starchdegrading enzymes were being “discovered,” and later, when the existence of some was refuted, these reservations were understandable. How many now remember the first published reports of isophosphorylase, Z-enzyme, R-enzyme, D-enzyme, and T-enzyme, and the years of endeavor that followed in various laboratories, before their specificity and properties were finally clarified? However, long before Hirst retired, he came to accept the view that, when used with appropriate caution, enzymic methods really are valuable tools! For many years, Hirst was a member of the Board of Governors of the Rowett Research Institute, Aberdeen, where he took a keen interest in the work of the Institute as a whole, and particularly that of Dr. A. E. Oxford and the microbiology department. This led to collaboration on the nature of the reserve carbohydrate synthesized by Cycloposthium and by the holotrich ciliates present in sheep’s rumen. Both protozoal polysaccharides were shown to be amylopectin in type. The work was continued with other protozoa, in collaboration with J. F. Ryley, and the presence of starch or amylopectin-type polysaccharides was established in Chilomonas paramecium, Haemotococcus pluuialis, and Tetraselmis carteriiformis. Finally, Hirst’s later contributions to monosaccharide chemistry must not be overlooked. With E. G. V. and Elizabeth Percival, four different trimethyl, 3,4- and 4,5-dimethyl, and the 4-methyl ethers of D-fructose were synthesized from isopropylidene derivatives having well established structures. Certain methyl ethers of D-mannuronic acid and D-ghcuronic acid were also prepared, and their periodate oxidation was compared with that of related ethers of D-galacturonic acid. With Elizabeth Percival, he also contributed articles on the methyl ethers of mono- and di-saccharides, and on glycofuranosides from cyclic carbonates, to Methods in Carbohydrate Chemistry, Vol. 2 (1963). Although this obituary is primarily concerned with Hirst’s achievements in carbohydrate chemistry, it must b e emphasized that the Edinburgh laboratories contained research groups active in other branches of organic chemistry. The largest of these dealt with polycyclic, aromatic hydrocarbons, and was directed by Dr. (later Professor) Neil Campbell, who, throughout the period, was an effective lieutenant in

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the Department, a loyal colleague, and a close friend. Many chemists who were not involved with carbohydrates have obtained senior positions in other universities, or in the chemical industry, and Hirst did not fail to give them his advice, help, and encouragement, as appropriate. Hirst had an extensive knowledge of organic chemistry as a whole, as visiting lecturers on non-carbohydrate subjects sometimes realized from the shrewd questioning at the end of a lecture. He was much in demand as an external examiner in other universities. The department of Professor Wesley Cocker at Trinity College, Dublin, was a special favorite of his, and he enjoyed returning to Birmingham, Manchester, and St. Andrews. Hirst was always concerned with both the theoretical and experimental aspects of organic chemistry; he had little time for students who could recite the detailed mechanisms of the nitration of aromatic compounds, but did not know the products of the nitration of phenol! When Hirst arrived in Edinburgh, Professor James Kendall, F. R. S., was the Head of the Department. On Kendall’s retirement in 1959, Hirst took over this major responsibility, and helped to plan and co-ordinate the steady growth of the Department, in terms of numbers of staff and students, and in respect of accommodation, including the adaptation of parts of the original building. Within the University of Edinburgh, Hirst played an extremely full part in its committee work. From 1959 to 1962, h e served as Dean of the Faculty of Science, and, in 1963, was elected to the University Courtthe most senior body, with financial and legal responsibilities for the administration ofthe University. He continued to be a member ofcourt from 1965, when he became one of the Curators of Patronage (a combined committee of the University and the City of Edinburgh having particular responsibilities towards appointments to certain chairs), until his retirement. Within the City of Edinburgh, he played important roles with other academic institutions and with the Royal Society of Edinburgh. H e was elected a Fellow ofthe last in 1948, and held a number ofoffices, before serving as President during 1959-1964. In 1965, he received the Gunning Victoria Jubilee Prize of the Society, for the period 1960-1964. He was a member ofthe Board of Governors ofthe East of Scotland College of Agriculture, and, until 1968, was convenor of the Appointments Committee. He represented Edinburgh University on the Governing Body of the Heriot-Watt College, and, following its elevation to University status in 1966, he became a member of its University Court, where his experience and judgment were invaluable. He became a

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member of the Watt Club (a graduate club of the Heriot-Watt), and served as President in 1967. Hirst received the Honorary Fellowship of the Heriot-Watt College in 1964, and the D.Sc. degree (honoris causa) in 1968; both of these awards were greatly appreciated by him. In Scotland as a whole, Hirst made many important contributions to the academic and scientific worlds. He served as Chairman of the Academic Advisory Committee that was involved in the transformation of the Royal Technical College, Glasgow, into the University of Strathclyde. This demanding work, and a subsequent appointment as a member of Court from 1965 to 1970 gave him special satisfaction and pleasure. He was a member of the Council of Management of the Macaulay Institute for Soil Research, Aberdeen, where his specialist knowledge was particularly welcomed by Dr. J. S. D. Bacon and his coworkers. Until 1968, h e was a member of the Governing Body, and chairman of the Research Committee, of the Hill Farming Research Organisation, Edinburgh; his continued involvement with the Rowett Research Institute and the Institute of Seaweed Research has already been noted. In 1964, h e was elected an Honorary Fellow of the Royal Scottish Society of Arts. However, Hirst’s contributions were by no means confined to Scotland, and, in fact, his major service to Britain and its scientific community resulted from his regular visits to London. This service was recognized by the award of a Coronation Medal in 1953, and h e was made a C.B.E. in 1957 and a Knight Bachelor in 1964. Hirst was elected a Fellow of the Chemical Society in 1922; he served on various committees, including the Council (from 1939 to 1942), and was elected President for 1956-1958. H e gave the Tilden Lecture in 1940, the Hugo Muller Lecture in 1948, and the C. S. Hudson Memorial Lecture in 1954, and was awarded the Longstaff Medal of the Society in 1957. He was a respected member cf the British Carbohydrate Nomenclature Committee. H e was elected Fellow of the Royal Institute of Chemistry in 1934, and served on several committees, including the Council (from 1942 to 1944), becoming Vice- President for 1967-1969. From 1934, h e was an active member of the local sections of the Society of Chemical Industry in Bristol, Manchester, and Edinburgh, respectively, serving on the Council as Chairman of the last in 1955-1956. H e was also a long-standing member of the Faraday Society, the Biochemical Society, and the Society for Experimental Biology. Hirst was elected a Fellow ofthe Royal Society in 1934. H e served on the Council from 1945to 1947 and again from 1962 to 1964, and was, for some years, a member of the Chemistry Sectional Committee and the

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Government Grant Board. H e received the Davy Medal of the Royal Society in 1948. For many years, he was a member of the select Royal Society Dining Club. Following the end of the 1939-1945 war, he continued his work for the Scientific Advisory Council of the Ministry of Supply, especially with the Chemical Engineering Committee. For a period, he was a member of the Plants and Soils Committee of the Agricultural Research Council, and was a member of the Blackman Committee which reported on research and related matters for the natural-rubber industry. His work for the Department of Scientific and Industrial Research (D.S.I.R., later the Science Research Council) was of special importance; here, he served on the Studentships Committee and as Chairman of the Chemistry Sub-Committee, and, during 19501955, he served as Chairman of the Chemistry Research Board. For several years, he acted as D.S.I.R. visitor to the Jute Industries Research Association, Dundee, and he was for a time a member of the Forest Products Research Board. From his early days as a research student, Hirst was actively interested in the British Association for the Advancement of Science. In 1922, he read a paper on esparto cellulose, under the Presidency of Professor J. C. Irvine. He regularly attended the annual meeting, being President of Section B in Birmingham in 1950, and was elected a member of the Council for 1964-1968. Hirst’s scientific achievements, and services to the academic world were recognized by the award of honorary degrees by the Universities of St. Andrews, Aberdeen, Birmingham, Strathclyde, and Trinity College, Dublin, and as already mentioned, the Heriot-Watt University. Hirst’s scientific contributions extended far beyond the United Kingdom. Many will know ofhis membership ofthe Editorial Advisory Boards ofAdvances in Carbohydrate Chemistry, of Carbohydrate Research (which published a special issue in July, 1968, to honor his 70th birthday”, and-a special interest-of the encyclopedic Rodd’s Chemistry of Carbon Compounds. Formany years, he was a member of the American and Swiss Chemical Societies; from 1959, he was an Honorary Member ofthe Polish Chemical Society; and, in 1967, h e was elected an Honorary Member of the Royal Irish Academy. Hirst enjoyed travelling, and visited many countries either to attend an international conference or as a visiting lecturer. His most important conference was the International Union meeting at Likge in 1930, at which the historic discussions with Haworth, Hudson, and Purves (referred to earlier) took place. He visited the U.S.A. in 1951, to attend (2) For a Memorial Issue, dedicated to Sir Edmund, see Carbohydr. Res., 57 (1977).

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an American Chemical Society meeting in New York and to participate in a Starch Round Table, and again, in 1966, to visit several institutions. In 1961, he visited Canada, to attend the International Union meeting in Montreal, and visit his old colleague and friend, J. K. N. Jones at Kingston. His own biographical notes record visits to conferences in several cities in Europe, including Stockholm (1953), Hamburg (1956),Galway (1958),Vienna (1958),and Warsaw (1959),and he was a visiting lecturer at various universities in Norway and West Germany, in 1955 and 1956, respectively. His lectures and personal discussions with other carbohydrate chemists are long remembered. The foregoing account describes many aspects of Hirst’s scientific career, but is by no means complete. His advice was widely sought by University Vice-Chancellors, by other professors of chemistry, and by the senior officials of Government bodies and Scientific Societies. Throughout, his work was characterized by thorough preparation, and when verbal comment was required, this was succint. He was a quiet but effective chairman of meetings, but could be very firm when the occasion demanded it. On his retirement, it was typical of Hirst that he retained a room in the Chemistry Department, and he used this regularly during the following years. He had a substantial correspondence with former students and colleagues, who still continued to seek his help and advice, and, as always, this was freely given. Many of his former colleagues now hold high positions in the academic and industrial worlds, and all readily acknowledge the great debt that they owe him. Despite his many heavy commitments, he was never too busy to discuss scientific and academic matters with his colleagues, and he showed a kindly interest in their well-being and that of their families. Many of the children of the staff received unexpected Christmas presents from Edmund and Kay, to their mutual pleasure. Although Hirst tended to be somewhat reserved on public occasions, he was good company in smaller groups. He developed a special rapport with Neil Campbell and the late Dr. Mowbray Ritchie, the Reader in Physical Chemistry, which was characterized by a sense of gentle good humor. If a junior colleague should ever show signs of brashness, this was soon halted by effective leg-pulling. The numerous commitments in England and Scotland, especially during the years of the Second World War, involved Hirst in extensive travel by railway. Whilst this was tedious to others, he enjoyed it, and he possessed a detailed knowledge ofthe railways and their timetables. Once, while waiting in a train in a station in Glasgow to return home, a large express train drew into the neighboring platform, whereupon Hirst glanced at his watch and startled his companions with the remark

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“That will be the 1.00 p.m. from Euston.” And it was! His return to the Department from a visit usually brought forth some amusing anecdote about the meeting or his companions, or some detail of the railway journey, or of the countryside that he enjoyed so much. One particular railway journey paid handsome dividends. Conversation with a fellow traveller eventually led to the award of a $23,650 grant to the Department from the U.S. Department ofAgriculture-which, nearly 20 years ago, was a not inconsiderable sum. Away from the Department, Edmund Hirst enjoyed many hobbies. He was a keen and knowledgeable photographer, both with blackand-white and color films; with Kay’s help, he kept a vegetable and fruit garden, although he preferred growing flowers rather than vegetables. In his early days, he and a companion, usually E. G. Cox, climbed in the Welsh hills, and toured-often on foot, but once on a tandem bicycle-a great part of rural England; he was a member of the Cyclists Touring Club until his death. He and Kay regularly attended, for many years, the Scottish National Orchestral Concerts in the Usher Hall, Edinburgh. His taste was exclusively classical and, preferably, preBartok, although he derived great pleasure from the operas of Gilbert and Sullivan, and the music of Johann Strauss the elder and his sons. He read widely, but his taste in novels was classical-Scott, Dickens, Jane Austen, the Bronte sisters, and Trollope. Hirst adored dogs, and, during the last few weeks of his life, he sat for hours in his arm-chair reading “Pickwick Papers,” with Jennie, a little Cairn terrier, curled up on his lap. In 1973, Hirst developed Hodgkin’s disease, and his health gradually deteriorated until his death on 29th October, 1975. From 1949 on, Hirst was supported in all his work by Kay. Visitors, especially from abroad, always received a warm welcome at their home, and the special kindness which they showed to individual guests will not be forgotten. In presenting Hirst for an honorary degree at Strathclyde University, the orator included this sentence: “Quiet and unassuming, but decisive in thought and action, his wise guidance and judgments have been of the greatest value to us.” We offer these words now, as atribute to a man who achieved so much for carbohydrate chemistry, and who is sadly missed by so many of us.

MAURICE STACEY DAVIDJ. MANNERS

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APPENDIX The following is a chronological list of the scientific publications of Sir Edmund Hirst and his colleagues. “The Constitution ofthe Disaccharides. Part V. Cellobiose (Cellose),” W. N. Haworth and E. L. Hirst,J. Chem. Soc., 119, 193 (1921). “The Labile Nature of the Halogen Atom in Organic Compounds. Part V. The Action of Hydrazine on the Halogen Derivatives of Some Esters and Substituted cycloHexanes,” E. L. Hirst and A. K. Macbeth,J. Chem. Soc., 121,2169 (1922). “Methylation of Xylose,” A. Carruthers and E. L. Hirst,J. Chem. Soc., 121, 2299 ( 1922). “The Labile Nature of the Halogen Atom in Organic Compounds. Part VI. The Action of Titanous Chloride and of Ammonia on Representative Halogen Compounds,” I. A. Black, E. L. Hirst, and A. K. Macbeth,J. Chem. Soc., 121,2527 (1922). “The Constitution of Polysaccharides. Part VI. The Molecular Structure of Cotton Cellulose,” J. C. Irvine and E. L. Hirst,J. Chem. Soc., 123, 518 (1923). “The Structure of the Normal Monosaccharides. Part I. Xylose,” E. L. Hirst and C. B. Purves,]. Chem. Soc., 123, 1352 (1923). “The Constitution ofRaffinose,” W. N. Haworth, E. L. Hirst, and D. A. RuellJ. Chern. Soc., 123,3125 (1923). “The Action of Highly Concentrated Hydrochloric Acid on Cellulose and on Some Derivatives of Glucose and of Xylose,” E. L. Hirst and D. R. Momson,]. Chem. Soc., 123,3226 (1923). “Constitutional Studies in the Monocarboxylic Acids Derived from Sugars. Part 111. The Isomeric Tetramethyl Galactonolactones and Trimethyl Arabonolactones,” J. Pryde, E. L. Hirst, and R. W. Humphreys,J. Chem. Soc., 127,348 (1925). “The Constitution ofthe Normal Monosaccharides. Part 11.Arabinose,” E. L. Hirst and G. J. Robertson, J. Cheni. Soc., 127, 358 (1925). “The Structure of the Normal Monosaccharides. Part 111. Rhamnose,” E. L. Hirst and A. K. Macbeth,J. Chem. SOC., 22 (1926). “The Structure of the Normal Monosaccharides. Part IV. Glucose,” E. L. Hirst, J. Chem. Soc., 350 (1926). “The Structure of Fructose, y-Fructose and Sucrose,” W. N. Haworth and E. L. Hirst, J. Chern. Soc., 1858 (1926). “The Structure of Normal Fructose: Crystalline Tetramethyl p-Methyl-fructoside and Crystalline Tetramethyl Fructose (1:3:4:5),” W. N. Haworth, E. L. Hirst, and A. Learner, J. Chem. Soc., 1040 (1927). “The Constitution of the Disaccharides. Part XIII. The y-Fructose Residue in Sucrose,” W. N. Haworth, E. L. Hirst, and V. S. Nicholson,J. Chem. Soc., 1513 (1927). “The Constitution of the Disaccharides. Part XV. Sucrose,” J. Avery, W. N. Haworth, and E. L. Hirst,J. Chem. Soc., 2308 (1927). “The Ring Structure in Normal Galactose. Oxidation of Tetramethyl 6-Galactonolactone,” W. N. Haworth, E. L. Hirst, and D. I. Jones,J. Chem. Soc., 2428 (1927). “1:3:4:6-TetramethyI (y-) Fructose and 2:3:5-Trimethyl (y-) Arabinose. Oxidation ofdand Z-Trimethyl y-Arabonolactone,” W. N. Haworth, E. L. Hirst, and A. LearnerJ. Chem. Soc., 2432 (1927). “The Structure of the Normal and y-Forms of Tetramethyl Glucose. Oxidation of Tetramethyl S and y-Gluconolactones,” W. N. Haworth, E. L. Hirst, and E. J. Miller, I. Chem. Soc., 2436 (1927).

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“Derivatives of Orcinol. Part I,” E. L. Hirst,]. Chem. Soc., 2490 (1927). “Ring Structure and Optical Relationships in the Mannose-Rhamnose-Lyxose Series of Sugars. Isolation of a New Form of Lyxose,” W. N. Haworth and E. L. Hirst,]. Chem Soc., 1221 (1928). “Polysaccharides. Part 11. The Acetylation and Methylation of Starch,” W. N. Haworth, E. L. Hirst, and J. I. Webb,]. Chem. Soc., 2681 (1928). “Organic Chemistry. Part I . (Aliphatic Division),” W. N. Haworth and E. L. Hirst, Annu. R e p . Prog. Cheni., Chem. S O C . London, 25,67 (1928). “Polysaccharides. Part IV. The Constitution ofxylan,” H. A. Hampton, W. N. Haworth, and E. L. Hirst,]. Chem. Soc., 1739 (1929). “The Structure of Normal Monosaccharides, Part VI. 2:3:4-Trimethyl Rhamnonolactone,” J. Avery and E. L. Hirst,]. Chem. Soc., 2466 (1929). “The Development ofa Novel Form ofStereoisomerism in the Sugar Series. Part I. The Third Variety of Triacetyl Methylrhamnoside,” W. N. Haworth, E. L. Hirst, and E. J. Miller,]. Chem. SOC., 2469 (1929). “Polysaccharides. Part V. Glycogen,” W. N. Haworth, E. L. Hirst, and J. I. Webb, J. Chem. Soc. 2479 (1929). “Organic Chemistry. Part I. (Aliphatic Division),” W. N. Haworth and E. L. Hirst, Annu. R e p . Prog. Chem., Chem. Soc. London, 26,74 (1929). “Crystalline cu-Methylmannofuranoside (y-Methylmannoside). Part 11,” W. N. Haworth, E. L. Hirst, and J . I. Webb,]. Chem. Soc., 651 (1930). “Derivatives of Lyxofuranose,” H. G . Bott, E. L. Hirst, and J. A. B. Smith,]. Chem. Soc., 658 (1930). “The Development of a Novel Form of Isomerism in the Sugar Series. Part 11. The Third Variety of Tetra-acetyl Methylmannoside,” H. G. Bott, W. N. Haworth, and E. L. Hirst, J . Chem. SOC., 1395 (1930). “The Conversion of 1:2:3:4-Tetra-acetyl P-d-Glucose into 2:3:4:6-Tetra-acetyl P-Methylglucoside,” W. N. Haworth, E. L. Hirst, and (Miss) E. G. Teece,]. Chem. Soc., 1405 (1930). “The Structure of Carbohydrates and their Optical Rotatory Power. Part I. General Introduction,” W. N. Haworth and E. L. Hirst,]. Chem. Soc., 2615 (1930). “The Structure of Carbohydrates and their Optical Rotatory Power. Part 11. 4-Glucosido-cu-niannose and its Derivatives,” W. N. Haworth, E. L. Hirst, H. R. L. Streight, H. A. Thomas, and J. I. Webb,]. Chem. Soc., 2636 (1930). “The Structure of Carbohydrates and their Optical Rotatory Power. Part 111. 4-Galactosido-c~-mannoseandits Derivatives,” W. N. Haworth, E. L. Hirst, (Miss) M. M. T. Plant, and R. J. W. Reynolds,]. Cheni. S O C . , 2644 (1930). “The Structure of Carbohydrates and their Optical Rotatory Power. Part IV. Derivatives ofa- and P-Methylmannopyranoside,” H. G. Bott, W. N. Haworth, E. L. Hirst, and R. S. Tipson, J . Chem. Soc., 2653 (1930). “The Structure ofcarbohydrates and their Optical Rotatory Power. Part V. The Optical Rotatory Powers of Methylated Lactones Derived from the Simple Sugars,” W. N. Haworth, E. L. Hirst, and J. A. B. Smith,]. Chem. Soc., 2659 (1930). “The Existence of the Cellobiose Residue in Cellulose,” W. N. Haworth, E. L. Hirst, and H. A. Thomas, Nature, 126, 438 (1930). “The Compound Uronic Acids. Structure ofthe Aldobionic Acid from Gum Arabic,” S . W. Challinor, W. N. Haworth, and E. L. Hirst,]. Chem Soc., 258 (1931). “Polysaccharides. PartVI. Trimethyl Cellulose,” W. N. Haworth, E. L. Hirst, and H. A. Thomas,]. Chem. SOC., 821 (1931). “Polysaccharides. Part VII. Isolation of Octamethyl Cellobiose, Hendecaniethyl Cel-

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lotriose, and a Methylated Cellodextrin (Cellotetrose?) as Crystalline Products of the Acetolysis of Cellulose Derivatives,” W. N. Haworth, E. L. Hirst, and H. A. Thomas, J . Chem. Soc., 824 (1931). “The Structure of Glucal,” E. L. Hirst and C. S. Woolvin,J. Chem. Soc., 1131 (1931). “The Structure of Carbohydrates and their Optical Rotatory Power. Part VI. 4-Glucosido-mannose and its Methylated Derivatives,” W. N. Haworth, E. L. Hirst, and H. R. L. Streight,J. Chem. Soc., 1349 (1931). “The Structure of Carbohydrates and their Optical Rotatory Power. Part VII. 4-Galactosido-niannose and its Methylated Derivatives,” W. N. Haworth, E. L. Hirst, and (Miss) M. M. T. Plant,]. Chem. SOC., 1354 (1931). “Conversion of 2:3:4-Triacetyl a-Methylglucoside into 3:4:6-Triacetyl 2-Methyl a-Methylglucoside,” W. N. Haworth, E. L. Hirst, and (Miss) E. G. Teece,J. Chem Soc., 2858 (1931). “The Third Variety ofTriacety1 Methyl-rhamnoside,” W. N. Haworth, E. L. Hirst, and H. Samuels,J. Chem. Soc., 2861 (1931). “Walden Inversion in the a-Glucoheptose Series. The Preparation ofNew Derivatives and the Determination of the Structure of Methyl-a-glucoheptoside,” W. N. Haworth, E. L. Hirst, and M. Stacey,J. Chem. Soc., 2864 (1931). “The Rble of Tobacco-smoking in the Production of Cancer,” E. A. Cooper, H. G. Bott, H. D. Cheeseworth, R. S. Tipson, F. W. Lamb, E. Sanders, and E. L. Hirst, J . Hyg., 32,293 (1932). “Optical Rotatory Dispersion in the Carbohydrate Group. Part I,” T. L. Harris, E. L. Hirst, and C . E. Wood,]. Chem. Soc., 2108 (1932). “Polysaccharides. Part XII. Acetolysis Products of Cellulose,” W. N. Haworth, E. L. Hirst, and 0. Ant-Wuorinen,J. Chenz. Soc., 2368 (1932). “Polysaccharides. Part XIV. The Molecular Struchne of Amylose and of Aniylopectin,” E. L. Hirst, (Miss) M. M. T. Plant, and (in part) (Miss) M. D. Wilkinson,J. Chem. Soc., 2375 (1932). “Polysaccharides. Part XV. The Molecular Structure of Inulin, ” W. N. Haworth, E. L. Hirst, and E. G. V. Percival, J . Chem. Soc., 2384 (1932). “Methylation of Monocarboxylic Acids Derived from Aldoses. Structure of Pentamethyl a-Gluco-heptono-y-lactone,” W. N. Haworth, E. L. Hirst, and M. Stacey, J . Chem. Soc., 2481 (1932). ‘‘Formation of Furfural from Methylated Pentoses,” H. G. Bott and E. L. Hirst, J . Chem. Soc., 2621 (1932). “Hexuronic Acid as the Antiscorbutic Factor,” E. L. Hirst and R. J. W. Reynolds, Nature, 129,576 (1932). “The Absorption Spectrum of Hexuronic Acid,” R. W. Herbert and E. L. Hirst, Nature, 130,205 (1932). “Constitution of Vitamin C,” E. G. Cox and E. L. Hirst, Nature, 131,402 (1933). “Constitution of Ascorbic Acid,” E. L. Hirst, E. G. V. Percival, and F. Smith, Nature, 131,617 (1933). “The Structure of Ascorbic Acid,” E. L. Hirst, J . Soc. Chem. Znd. London, 52, 221 (1933). “Synthesis of Ascorbic Acid,” W. N. Haworth and E. L. Hirst, J . SOC. Chem. Znd. London, 52, 645 (1933). “Ascorbic Acid as the Antiscorbutic Factor,” E. L. Hirst and S. S. Zilva, Biochem.J., 27, 1271 (1933). “The Constitution ofAscorbic Acid,” R. W. Herbert, E. L. Hirst, E. G. V. Percival, R. J. W. Reynolds, and F. Smith,]. Chem. Soc., 1270 (1933).

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“Synthesis of d- and l-Ascorbic Acid and of Analogous Substances,” R. G. Ault, D. K. Baird, H. G. Carrington, W. N. Haworth, R. W. Herbert, E. L. Hirst, E. G. V. Percival, F. Smith, and M. Stacey,]. Chem. SOC.,1419 (1933). “Optical Rotatory Dispersion in the Carbohydrate Group. Part 11. Ascorbic Acid,” R. W. Herbert, E. L. Hirst, and C. E. Wood,]. Chem. Soc., 1564 (1933). “The Molecular Structure of Polysaccharides,” W. N. Haworth and E. L. Hirst, Truns. Furuduy SOC.,29, 14 (1933). “Sucrose and Other Disaccharides. Sir James Irvine’s ‘Correction’,” H. C. Carrington, W. N. Haworth, and E. L. Hirst,].Am. Chem. SOC.,55, 1084 (1933). “Ascorbic Acid and Synthetic Analogues,” D. K. Baird, W. N. Haworth, R. W. Herbert, E. L. Hirst, F. Smith, and M. Stacey,]. Chem. Soc., 62 (1934). “Isolation of a Crystalline Dimethyl Anhydromethyl-hexoside. Characterisation of 3:4:6-Trimethyl Glucose,” W. N. Haworth, E. L. Hirst, and L. Panizzon,]. Chem. Soc., 154 (1934). “Malta1 and 4-a-Glucosidomannose,” W. N. Haworth, E. L. Hirst, and R. J. W. Reynolds, J. Chem. SOC.,302 (1934). “Polysaccharides. Part XVII. The Constitution and Chain Length of Levan,” S. W. Challinor, W. N. Haworth, and E. L. Hirst,]. Chem. Soc., 676 (1934). “Optical Rotatory Dispersion in the Carbohydrate Group. Part 111. Tetra-methyl a-Methylglucopyranoside and Tetramethyl a-Methylmannopyranoside,” R. W. Herbert, E. L. Hirst, and C. E. Wood,]. Chem. Soc., 1151 (1934). “Physiological Activity of Synthetic Ascorbic Acid,” W. N. Haworth, E. L. Hirst, and S. S. Zilva,]. Chem. SOC.,1155 (1934). “Synthesis of Ascorbic Acid and its Analogues: The Addition of Hydrogen Cyanide to Osones,” W. N. Haworth, E. L. Hirst, J. K. N. Jones, and F. Smith,J. Chem. SOC.,1192 ( 1934). “Methyl Ethers ofAscorbic Acid,” W. N. Haworth, E. L. Hirst, and F. Smith,]. Chem. SOC., 1556 (1934). “The Carbohydrates of Grass. Isolation of a Polysaccharide of the Levan Type,” S. W. Challinor, W. N. Haworth, and E. L. Hirst,]. Chem. SOC., 1560 (1934). “Preparation ofArabinose from Gum Acacia (Gum Kordofan),” H. C. Carrington, W. N. Haworth, and E. L. Hirst,]. Chem. SOC., 1653 (1934). “The Constitution of Ascorbic Acid. Action of Sodium Hypochlorite on a-Methoxyacid Amides,” R. G. Ault, W. N. Haworth, and E. L. Hirst,J. Chem. Soc., 1722 (1934). “Optical Rotatory Dispersion in the Carbohydrate Group. Part IV. Tetramethyl-y-mannonolactone,” T. L. Harris, E. L. Hirst, and C. E. Wood, J. Chem. Sac., 1825 (1934). “Polysaccharides. Part XVIII. The Constitution ofXylan,” W. N. Haworth, E. L. Hirst, and E. Oliver,]. Chem. Soc., 1917 (1934). “The Primary Product of the Synthesis of Ascorbic Acid and its Analogues,” W. N. Haworth and E. L. Hirst, Helv. Chim. Actu, 17, 520 (1934). “Organic Chemistry, Part I (Aliphatic Division),” H. D. K. Drew, E. L. Hirst, R. S. Morrell, and S. Peat, Annu. Rep. Prog. Chem., Chem. Soc. London, 31, 143 (1934). “Polysaccharides. Part XIX. The Molecular Structure of Waxy Maize Starch,” W. N. Haworth, E. L. Hirst, and M. D. Woolgar,]. Chem. Soc., 177 (1935). “Optical Rotatory Dispersion in the Carbohydrate Group. Part V. Tetramethyl y-Gluconolactone,” R. W. Herbert, E. L. Hirst, H. Samuels, and C. E. Wood,]. Chem. SOC.,295 (1935). “Preparation of d-Mannuronic Acid and its Derivatives,” R. G. Ault, W. N. Haworth, and E. L. Hint,]. Chem. SOC., 517 (1935). “Acetone Derivatives of Methylglucosides,” R. G. Ault, W. N. Haworth, and E. L. Hirst,]. Chem. Soc., 1012 (1935).

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“Polysaccharides. Part XX. The Molecular Size of Amylose and the Relationship Between Amylose and Starch,” D. K. Baird, W. N. Haworth, and E. L. Hirst,]. Chem. Soc., 1201 (1935). “Polysaccharides. Part XXI. The Constitution and Chain Length of Some Starch Dextrins,” W. N. Haworth, E. L. Hirst, and M. M. T. Plant, J . Chem. Soc., 1214 (1935). “Polysaccharides. Part XXII. Constitution and Molecular Structure of a-Amylodextrin,” W. N. Haworth, E. L. Hirst, and A. C. Waine, J . Chem. Soc., 1299 (1935). “Absorption Spectra in Relation to the Constitution of Derivatives of Isatin and Carbostyril,” R. G. Ault, E. L. Hirst, and R. A. Morton,]. Chem. Soc., 1653 (1935). “Optical Rotatory Dispersion in the Carbohydrate Group. Part VI. The Amide Rotation Rule,” T. L. Harris, E. L. Hirst, and C. E. Wood,]. Chem. Soc., 1658 (1935). “Absorption Spectra of the Metabolic Acids of Penicillium charlesii and Their Relationship to the Absorption Spectrum of Ascorbic Acid,” R. W. Herbert and E. L. Hirst, Biochem. ]., 29,1881 (1935). “Organic Chemistry. Carbohydrates,” E. L. Hirst and S. Peat, Annu. Rep. Prog. Chein., Chem. Soc. London, 32, 272 (1935). “Optical Rotatory Dispersion in the Carbohydrate Group. Part VII. The Glucal Series,” T. L. Harris, R. W. Herbert, E. L. Hirst, C. E. Wood and H. Woodward,]. Chem. Soc., 1403 (1936). “Organic Chemistry. Carbohydrates,” E. L. Hirst and S. Peat, Annu. Rep. Prog. C l z e n . , Chem. Soc. London, 33, 245 (1936). “The Chemistry of the Carbohydrates and the Glucosides,” W. N. Haworth and E. L. Hirst, Annu. Reu. Biochem., 5, 81 (1936). “Gluco-ascorbic Acid,” W. N. Haworth, E. L. Hirst, and J. K. N. Jones,]. Chem. Soc., 549 (1937). “Polysaccharides. Part XXIII. Determination of the Chain Length of Glycogen,” W. N. Haworth, E. L. Hirst, and F. A. Isherwood,]. Cheni. Soc., 577 (1937). “The Ring Structure ofXylal,” W. N. Haworth, E. L. Hirst, and C. S. Woolvin,]. Chem. Soc., 780 (1937). “Degradation of Methylated Inulin to Hexamethyl Difructosan,” W. N. Haworth, E. L. Hirst, and F. A. Isherwood,]. Chem. Soc., 782 (1937). “Polysaccharides. Part XXIV. Yeast Mannan,” W. N. Haworth, E. L. Hirst, and F. A. Isherwood,]. Chem. Soc., 784 (1937). “Polysaccharides. Part XXV. a-Amylodextrin,” W. N. Haworth, E. L. Hirst, H. Kitchen, and S. Peat,]. Chem. Soc., 791 (1937). “Acetone Derivatives of Gluconic Acid,” W. N. Haworth, E. L. Hirst, and K. A. Chamberlain, J . Chem. Soc., 795 (1937). “Isomerisation of 2:3-Dimethyl Ascorbic Acid,” W. N. Haworth, E. L. Hirst, F. Smith, and W. J. Wilson,]. Chem. Soc., 829 (1937). “Optical Rotatory Dispersion in the Carbohydrate Group. Part VIII. Tetramethyl 8-Gluconolactone and Tetramethyl 6-Galactonolactone,” T. L. Harris, E. L. Hirst, and C. E. Wood, J . Chem. Soc., 848 (1937). “Polysaccharides. Part XXVI. Xylan,” R. A. S. Bywater, W. N. Haworth, E. L. Hirst, and S. Peat,”]. Cheni. Soc., 1983 (1937). “A Water-soluble Polysaccharide from Barley Leaves,” W. N. Haworth, E. L. Hirst, and R. R. Lyne, Biochem. ]., 31, 786 (1937). “The Chemistry of the Carbohydrates and the Glycosides,” W. N. Haworth and E. L. Hirst, Annu. Reo. Biochem., 6, 99 (1937). “Pectic Substances. Part I. The Araban and Pectic Acid ofthe Peanut,” E. L. Hirst and J. K. N. Jones,]. Chem. Soc., 496 (1938).

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“Analogues of Ascorbic Acid Containing Six-membered Rings,” W. N. Haworth, E . L. Hirst, and J. K. N. Jones,]. Chem. SOC., 710 (1938). “The Constitution of Damson Gum. Part I. Composition of Damson Gum and Structure of an Aldobionic Acid (Glycuronosido-2-mannose) Derived from it,” E. L. Hirst and J. K. N. Jones,]. Chem. SOC., 1174 (1938). “Polysaccharides. Part XXVIII. The ‘End-group’ Method as Applied to Starch. An Improved Method for the Estimation of Tetramethyl Glucose in Admixture with Trimethyl Glucose,” E. L. Hirst and G. T. Young,]. Chem. Soc., 1247 (1938). “The Ring Structure of Methylgalactofuranoside,” W. N. Haworth, E. L. Hirst, D. I. Jones, and H. Woodward,]. Chem. Soc., 1575 (1938). “Methylation of a-Methylglucoside by Thallous Hydroxide and Methyl Iodide,” C. C. Barker, E. L. Hirst, and J. K. N. Jones,]. Chem. Soc., 1695 (1938). “Estimation of Uronic Anhydride Residues in Polysaccharides,” E. L. Hirst and G. T. Young, Nature, 142, 912 (1938). “The Chemistry of Ascorbic Acid (Vitamin C) and its Analogues,” W. N. Haworth and E. L. Hirst, Ergeb. Vitam.Hormonforsch., 160, 191 (1939). “The Structure and Synthesis of Vitamin C (Ascorbic Acid) and its Analogues,” E. L. Hirst, Fortschr. Chem. Org. Naturst., 2, 132 (1939). “Structure of Alginic Acid,” E. L. Hirst, J. K. N.Jones, and W. 0. Jones, Nature, 143, 857 (1939). “Methyl Ethers ofArabo-ascorbic Acid and their Isomerism,” E. G. E. Hawkins, E. L. Hirst, and J. K. N. Jones,]. Chem. SOC., 246 (1939). “Pectic Substances. Part 11. Isolation ofan Araban from the Carbohydrate Constituents of the Peanut,” E. L. Hirst and J. K. N. Jones,J. Chem. Soc., 452 (1939). “Pectic Substances. Part 111.Composition ofApple Pectin and the Molecular Structure of the Araban Component of Apple Pectin,” E. L. Hirst and J. K. N. Jones,]. Chem. Soc., 454 (1939). “Polysaccharides. Part XXXI. Constitution of Wheat Starch and Horse-chestnut Starch,” E. L. Hirst and G . T. Young,]. Chem. Soc., 951 (1939). “Constitution ofthe Mucilage from the Bark of Ulmusfuloa (Slippery Elm Mucilage). Part I. The Aldobionic Acid Obtained by Hydrolysis of the Mucilage,” R. E. Gill, E. L. Hirst, and J. K. N. Jones,]. Chem. Soc., 1469 (1939). “Polysaccharides. Part XXXII. The Molecular Constitution of Rice Starch,” E. L. Hirst and G. T. Young,]. Chem. Soc., 1471 (1939). “The Constitution of Damson Gum. Part 11. Hydrolysis Products from Methylated Degraded (Arabinose-free) Damson Gum,” E. L. Hirst and J. K. N. Jones,]. Chem. Soc., 1482 (1939). “Pectic Substances. Part IV. Citrus Araban,” G. H. Beaven, E. L. Hirst, and J. K. N. Jones,]. Chem. SOC., 1865 (1939). “2:3:4-Trimethyl Mannose,” W. N. Haworth, E. L. Hirst, F. A. Isherwood, and J . K. N. Jones, J . Chem. SOC., 1878 (1939). “The Structure of Alginic Acid. Part I,” E. L. Hirst, J. K. N. Jones, and (Miss) W. 0. Jones,]. Chem. Soc., 1880 (1939). “Polysaccharides. Part XXXIII. The Methylation of Cellulose in Air and in Nitrogen,” W. N. Haworth, E. L. Hirst, L. N. Owen, S. Peat, and (in part) F. J. AverillJ. Chem. Soc., 1885 (1939). “Polysaccharides. Part XXXV. Hydrocellulose,” H. C. Carrington, W. N. Haworth, E. L. Hirst, and M. Stacey,]. Chem. Soc., 1901 (1939). “Polysaccharides. Part MXVIII. The Constitution of Glycogen from Fish Liver and Fish Muscle,” W. N. Haworth, E. L. Hirst, and F. Smith,]. Chem. Soc., 1914 (1939). “The Nature of the Bonds in Starch,” C. E. H. Bawn, E. L. Hirst, and G. T. Young, Trum. Faraday Soc., 36, 880 (1940).

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“The €-Galactan of Larch Wood,” E. L. Hirst, J. K. N. Jones, and W. G. Campbell, Nature, 147,25 (1941). “Linkage Between the Repeating Units in the Starch Molecule,” C. C. Barker, E. L. Hirst, and G. T. Young, Nature, 147, 296 (1941). “Recent Progress in the Chemistry of the Pectic Materials and Plant Gums” (Tilden Lecture), E. L. Hirst,]. Chem. Soc., 70 (1942). “Nitrogenous Substances Synthesized by Moulds,” A. H. Campbell, M . E. Foss, E. L. Hirst, and J. K. N. Jones, Nature, 155, 141 (1945). “Application of New Methods of End-group Determination to Structural Problems in the Polysaccharides,” F. Brown, Sonia Dunstan, T. G. Halsall, E. L. Hirst, and J. K. N. Jones, Nature, 156, 785 (1945). “The Constitution of Damson Gum. Part 111. Hydrolysis Products from Methylated Damson Gum,” E. L. Hirst and J. K. N. Jones,]. Chem. Soc., 506 (1946). “Methylation of P-Methylglucopyranoside and cup-Methylxylopyranosides by Thallous Hydroxide and Methyl Iodide,” C. C. Barker, E. L. Hirst, and J. K. N. Jones, J. Chem. Soc., 783 (1946). “Constitution of the Mucilage from the Bark of Ulmusfuloa (Slippery Elm Mucilage). Part 11. The Sugars Formed in the Hydrolysis of the Methylated Mucilage,” R. E. Gill, E. L. Hirst, and J. K. N. Jones,]. Chem. Soc., 1025 (1946). “The Chemistry of Pectic Materials,” E. L. Hirst and J. K. N. Jones, Adv. Carbohydr. Chem., 2,235 (1946). “Structure of Starch and Cellulose,” T. G. Halsall, E. L. Hirst, and J. K. N. Jones, Nature, 159, 97 (1947). “Quantitative Estimation of Mixtures of Sugars by the Paper Chromatogram Method,” A. E. Flood, E. L. Hirst, and J. K. N. Jones, Nature, 160, 86 (1947). “Structure of Starch: Mode of Attachment of the Side-chains in Amylopectin,” T. G . Halsall, E. L. Hirst, J. K. N. Jones, and A. Roudier, Nature, 160, 899 (1947). “The Chemistry of Some Plant Gums and Mucilages,” E. L. Hirst and J. K. N. Jones, J. Soc. Dyers Colour., 63,249 (1947). “The Reaction of 1-Nitropropane with Formaldehyde and Ammonia,” E. L. Hirst, J. K. N. Jones, (Mrs) A. Minahan, F. W. Ochynski, A. T. Thomas, and T. Urbanski,]. Chem. Soc., 924 (1947). “The Quantitative Determination of Galactose, Mannose, Arabinose and Rhamnose,” E. L. Hirst, J. K. N. Jones, and E. A. Woods,]. Chem. Soc., 1048 (1947). “The Synthesis of 3-Methyl and 3:5-Dimethyl I-Arabinose,” E. L. Hirst, J. K. N. Jones, and (Miss) E. Williams,]. Chem. Soc., 1062 (1947). “The Constitution of Egg-plum Gum. Part I,” E. L. Hirst and J. K. N. Jones,]. Chem. Soc., 1064 (1947). “Pectic Substances. Part VI. The Structure ofthe Araban fromArachis hypogea,” E. L. Hirst and J. K. N. Jones,J. Chem. Soc., 1221 (1947). “Pectic Substances. Part VII. The Constitution ofthe Galactan from Lupinus albus,” E. L. Hirst, J. K. N. Jones, and (Mrs) W. 0. Walder,]. Chem. Soc., 1225 (1947). “The Structure of Glycogen. Ratio of Non-terminal to Terminal Glucose Residues,” T. G. Halsall, E. L. Hirst, and J. K. N. Jones,]. Chem. Soc.,1399 (1947). “Oxidation of Carbohydrates by the Periodate Ion,” T. G. Halsall, E. L. Hirst, and J. K. N. Jones,]. Chem. Soc., 1427 (1947). “The Galactomannan of the Lucerne Seed,” E. L. Hirst, J. K. N. Jones, and (Mrs)W. 0. Walder,]. Chem. Soc., 1443 (1947). “Separation and Identification of Methylated Sugars on the Paper Chromatogram,” F. Brown, E. L. Hirst, L. Hough, J. K. N. Jones, and H. Wadman, Nature, 161,720 (1948). “The Structure of Starch. The Ratio of Non-terminal to Terminal Groups,” F. Brown, T. G. Halsall, E. L. Hirst, and J. K. N. Jones,]. Chem. Soc., 27 (1948).

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“The Structure of Egg-plum Gum. Part 11.The Hydrolysis Products Obtained from the Methylated Degraded Gum,” E. L. Hirst and J. K. N. Jones,]. Chem. Soc., 120 (1948). “The €-Galactan ofLarch Wood(Larixdeciduu),” W. G. Campbell, E. L. Hirst, and J. K. N. Jones,]. Chem. Soc., 774 (1948). “The Galactomannan of Carob-seed Gum (Gum Gatto),” E. L. Hirst and J. K. Jones, J . Chem. Soc., 1278 (1948). “The Structure of Almond-tree Gum. Part I. The Constitution of the Aldobionic Acid Derived from the Gum,” F. Brown, E. L. Hirst, and J. K. N. Jones,]. Chem. Soc., 1677 (1948). “Quantitative Analysis of Mixtures of Sugars by the Method of Partition Chromatography. Part I. Standardisation of Procedure,” A. E. Flood, E. L. Hirst, and J. K. N. Jones, J . Chem. Soc., 1679 (1948). “Structure of Acorn Starch,” E. L. Hirst, J. K. N. Jones, and A. J. Roudier,]. Chem. Soc., 1779 (1948). “Pectic Substances. Part VIII. The Araban Component of Sugar-beet Pectin,” E. L. Hirst and J. K. N. Jones,]. Chem. Soc., 2311 (1948). “The Amylose Content of the Starch Present in the Growing Potato Tuber,” T. G. Halsall, E. L. Hirst, J. K. N. Jones, and F. W. Sansome, Biochem. ]., 43, 70 (1948). “La Structure chimique de l’Amidon: Mode de Liaison. Des Chaines Laterales de l’hylopectine,” T. G . Halsall, E. L. Hirst, J. K. N. Jones, and A. Roudier, Mkm. Sera Chim. Etat, 34 (1948). “Composition of the Gum of Sterculia setigera: Occurrence of D-Tagatose in Nature,” E. L. Hirst, L. Hough, and J. K. N. Jones, Nature, 163, 177 (1949). “Chromatographic Analysis. The Application of Partition Chromatography to the Separation of the Sugars and Their Derivatives,” E. L. Hirst and J. K. N. Jones, Discuss. Furaday Soc., No. 7, 268 (1949). “Pear Cell-wall Cellulose,” E. L. Hirst, F. A. Ishenvood, M. A. Jermyn, and J. K. N. Jones,]. Chem. Soc., S182 (1949). “The Occurrence and Significance ofthe Pentose Sugars in Nature, and their Relationship to the Hexoses,” E. L. Hirst,]. Chem. Soc., 552 (1949). “Quantitative Analysis of Mixtures of Sugars by the Method of Partition Chromatography. Part 11. The Separation and Determination of Methylated Aldoses,” E. L. Hirst, L. Hough, and J. K. N. Jones,]. Chem. Soc., 928 (1949). “The Polysaccharides of the Florideae. Floridean Starch,” V. C. Barry, T. G. Halsall, E. L. Hirst, and J. K. N. Jones,]. Chem. Soc., 1468 (1949). “Quantitative Analysis of Mixtures of Sugars by the Method of Partition Chromatography. Part 111.Determination ofthe Sugars by Oxidation with Sodium Periodate,” E. L. Hirst and J. K. N. Jones,]. Chem. Soc., 1659 (1949). “The Constitution of Egg-plum Gum. Part 111. The Hydrolysis Products Obtained from the Methylated Gum,” F. Brown, E. L. Hirst, and J. K. N. Jones,]. Chem. Soc., 1757 (1949). “Cholla Gum,” F. Brown, E. L. Hirst, and J. K. N. Jones,]. Chem. Soc., 1761 (1949). “The Structure of Sterculia setigera Gum. Part I. An Investigation by the Method of Paper Partition Chromatography of the Products of Hydrolysis of the Gum,” E. L. Hirst, L. Hough, and J. K. N. Jones,]. Chem. Soc., 3145 (1949). “The Action of PAmylase on Amylopectin and on Glycogen,” T. G. Halsall, E. L. Hirst, L. Hough, and J. K. N. Jones,]. Chem. Soc., 3200 (1949). “The Industrial Utilisation of Agricultural Products and of Seaweed,” E. L. Hirst, Proc. Colloq. R. Inst. Chem., Third Session, Dublin (1949). “Chemical Constitution of Slippery Elm Mucilage: Isolation of 3-Methyl D-Galactose

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from the Hydrolysis Products,” E. L. Hirst, L. Hough, and J. K. N. Jones, Nature, 165,34 (1950). “Sir Norman Haworth, F.R.S.: Obituary Notice,” E. L. Hirst, Nature, 165,587 (1950). “Modern Development in Carbohydrate Chemistry,” E. L. Hirst, Adu. Sci., No. 26, September (1950). “On the Structure of Knudsen’s Base and of Related Compounds. Part I,” M. E. Foss, E. L. Hirst, J. K. N. Jones, H. D. Spr,ingall, A. T. Thomas, and T. Urbanski,]. Chem. SOC., 624 (1950). “The Constitution ofXylan from Esparto Grass (Stipatenacissima, L.),” S . K. Chanda, E. L. Hirst, J. K. N. Jones, and E. G. V. Percival,]. Chem. Soc., 1289 (1950). “Studies in Fructosans. Part I. Inulin from Dahlia Tubers,” E. L. Hirst, D. I. McGilvray, and E. G. V. Perciva1,J. Chem. Soc., 1297 (1950). “On the Structure of Knudsen’s Base and of Related ComDounds. Part 11,” M. E. Foss. E. L. Hirst, J K. N. Jones, H. D. Springall, A. T. Thomas, an2 T. Urbanski,j. Chem. Soc.; 1691 (1950). . , “Grapefruitand Lemon Gums. Part I. The Ratio of Sugars Present in the Gums and the Isolated by Graded Structure of the Aldobionic Acid (4-~-Glucuronosido-~-galactose) Hydrolysis of the Polysaccharide,” J. J. Connell, (Miss) Ruth M. Hainsworth, E. L. Hirst, and J. K. N. Jones,]. Chem. Soc., 1969 (1950). “The Constitution of Laminarin. Part I. An Investigation on Laminarin Isolated from Laminaria cloustoni,” J. J. Connell, E. L. Hirst, and E. G. V. Percival,]. Chem. Soc., 3494 (1950). “Constitution of the Mucilage from the Bark of Ulmusfulua (Slippery Elm Mucilage). Part 111. The Isolation of 3-Monomethyl D-Galactose from the Products of Hydrolysis,” E. L. Hirst, L. Hough, and J. K. N. Jones,J. Chem. Soc., 323 (1951). “The Constitution of a Pear Cell-wall Xylan,” S. K. Chanda, E. L. Hirst, and E. G. V. Percival,]. Chem. Soc., 1240 (1951). “Walter Norman Haworth, 1883-1950. Obituary Notice,” E. L. Hirst,]. Chem. Soc., 2790 (1951). “Wood Starches. Part I,” W. G. Campbell, J. L. Frahn, E. L. Hirst, D. F. Packman, and (the late) E. G. V. Percival,]. Chem. Soc., 3489 (1951). “The Chemistry ofplant Gums and Mucilages,” E. L. Hirst,En&aoour, 10,106 (1951). “The Chemistry of the Plant Gums,” E. L. Hirst and J. K. N. Jones, Research, 4,411 (1951). “Walter Norman Haworth, 1883-1950. Obituary Notice,” E. L. Hirst, R. Soc. London, Obituary Notices, 7, (Nov) 373 (1951). “Obituary Notice. Dr. E. G. V. Percival,” E. L. Hirst, Nature, 168, 855 (1951). “Walter Norman Haworth, 1883-1950,” E. L. Hirst, Adu. Carbohydr. Chem., 6, 1 (1951). “Obituary Notice. Edmund George Vincent Percival,” E. L. Hirst,]. Chem. Soc., 1557 (1952). “The Structure of Alginic Acid. Part 11,” S. K. Chanda, E. L. Hirst, (the late) E. G. V. Percival, and A. G. Ross,]. Chem. Soc., 1833 (1952). “Obituary Notice. W. G. Campbell,” E. L. Hirst, Nature, 169,524 (1952). “Sir James Irvine, K.B.E., F.R.S., 1877-1952,” E. L. Hirst,]. Am. Chem. SOC., 75,253 (1953). “Studies on Fructosans. Part IV. A Fructosan from Dactylis glomerata,” G. 0. Aspinall, E. L. Hirst, (the late) E. G. V. Percival, and R. G. J. Telfer,]. Chem. Soc., 337 (1953). “The Hemicelluloses of Esparto Grass (Stipa tenacissima L.).The Arabinose-rich

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Fraction,” G. 0. Aspinall, E. L. Hirst, R. W. Moody, and (the late) E. G. V. Percival,

J. Chem. SOC., 1631 (1953). “Protozoal Polysaccharides. Structure of the Polysaccharides Produced by the Holotrich Ciliates Present in Sheep’s Rumen,” G. Forsyth and E. L. Hirst, J. Chem. SOC., 2132 (1953). “Protozoal Polysaccharides. Structure of a Polysaccharide Produced by Cycloposthium,” G . Forsyth, E. L. Hirst, and A. E. Oxford,J. Chem. SOC., 2030 (1953). “The Structure of Karaya Gum (Cochlospermum gossypium),” E. L. Hirst and Sonia Dunstan,]. Chem. SOC., 2332 (1953). “Syntheses of Methyl Ethers o f Fructose,” E. L. Hirst, W. E. A. Mitchell, Elizabeth E. Percival, and (the late) E. G. V. Perciva1.J. Chem. SOC., 3170 (1953). “The Mannans o f Ivory Nut (Phytelephas macrocarpa). Part I. The Methylation o f Mannan A and Mannan B,” G. 0.Aspinall, E. L. Hirst, (the late) E. G. V. Percival, and I. R. Williamson,J. Chem. SOC., 3184 (1953). “Schools of Chemistry in Great Britain and Ireland-VII. The University of Edinburgh,” E. L. Hirst and M. RitchieJ. R . Znst. Chem., 77 (November), 505 (1953). “SirJames ColquhounIrvine, K.B.E., D.C.L.,LL.D., F.R.S.: ObituaryNotice,”R. SOC. Edinburgh, Year Book, 1951-1952, 22 (1953). “James Colquhoun Irvine, 1877-1952,” E. L. Hirst, Adv. Carbohydr. Chem. 8, xi (1953). “Studies on Seed Mucilages. Part VI. The Seed Mucilage ofPlantago arenaria,” E. L. Hirst, (the late) E. G. V. Percival, and Clare B. Wylam,J. Chem. SOC., 189 (1954). “Multiple Branching in Amylopectin,” E. L. Hirst and D. J. Manners, Chem. Ind., (London), 224 (1954). “Hemicellulose A of Beechwood(Fagus sylvatica),” G. 0.Aspinall, E. L. Hirst, and R. S . Mahorned,]. Chem. Soc., 1734 (1954). “The Gum of Acacia pycnantha,” E. L. Hirst and A. S. Perlin, J. Chem. SOC., 2622 (1954). “The Hudson Memorial Lecture. Claude Silbert Hudson,” E. L. Hint,]. Chem. SOC., 4042 (1954). “Physicochemical Studies on Starches. Part 11. The Oxidation o f Starches by Potassium Metaperiodate,” D. M. W. Anderson, C. T. Greenwood, and E. L. Hirst, J. Chem. SOC., 225 (1955). “The Alkali-soluble Polysaccharides o f the Lichen Cladonia ulpestris (Reindeer Moss),” G . 0. Aspinall, E. L. Hirst, and (Mrs.) Margaret Warburton,J. Chem. SOC., 651 (1955). “Gum Ghatti (Indian Gum). The Composition o f the Gum and the Structure of Two Aldobiouronic Acids Derived from It,” G. 0. Aspinall, E. L. Hirst, and A. Wickstrdm, J. Chem. SOC., 1160 (1955). “The Synthesis of Methyl Ethers of Mannuronic and Glucuronic Acid, and their Reaction with Periodate,” R. A. Edington, E. L. Hirst, and Elizabeth E. Percival, J . Chem. SOC., 2281 (1955). “Edmund George Vincent Percival, 1907-1951,” E. L. Hirst and A. G. Ross, Ado. Carbohydr. Chem. 10, xiii (1955). “Some Problems in the Chemistry of the Hemicelluloses,” E. L. Hint,]. Chem. SOC., 2974 (1955). “The Constitution o f a Modified Starch from Malted Barley,” G. 0. Aspinall, E. L. Hirst, and W. McArthur,J. Chem. SOC., 3075 (1955). “The Analysis o f Plant Gums and Mucilages,” E. L. Hirst and J. K. N. Jones, in “Modern Methods of Plant Analysis,” K. Paech and M. Y. Tracey, eds. SpringerVerlag, Berlin, 1955, Vol. 11, p. 275.

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27

“Plant Gums of the Genus Khaya. The Structure of Khaya grandifolia Gum,” G. 0 . Aspinall, E. L. Hirst, and N. K. Matheson,]. Chem. SOC., 989 (1956). “a-l:4-Glucosans. Part IV. A Re-examination of the Molecular Structure of Floridean Starch,” I. D. Fleming, E. L. Hirst, and D. J. Manners,]. Chem. SOC.,2831 (1956). “Carbohydrates,” E. L. Hirst, in “Perspectives in Organic Chemistry,” A. Todd, ed., Interscience, New York, 1956,p. 214. “Some Aspects of the Chemistry of the Fructosaus,” E. L. Hirst, Proc. Chem. Soc. London, 193 (July, 1957). “A Comparison of Isolichenin and Lichenin from Iceland Moss (Cetraria islandica),” N. B. Chanda, E. L. Hirst, and D. J. Manners,]. Chem. Soc., 1951 (1957). “Periodate Oxidation of Laminarin,” F. B. Anderson, E. L. Hirst, and D. J. Manners, Chem. Ind. (London), 1178 (1957). “Changes in Organic Acid Content of Perennial Rye-grass During Conservation,” E. L. Hirst and S . Ramstad, J. Sci. Food Agric., 8, 727 (1957). “Introductory Remarks on the Chemical Reactivity of Sucrose,” E. L. Hirst, C . R . Assemblie Comm. Intern. Tech. Sucrerie, lo“,Londres, 12 (1957). “Gum Ghatti (Indian Gum). Part 11. The Hydrolysis Products Obtained from the Methylated Degraded Gum and the Methylated Gum,” G. 0. Aspinall, (Mrs) Barbara J. Auret, and E. L. Hirst,]. Chem. Soc., 221 (1958). “The Constitution of Larch +Galactan,” G. 0.Aspinall, E. L. Hirst, and Else Ramstad, J. Chem. Soc., 593 (1958). “The Structure of Brachychiton diversifolium Gum (Sterculia caudata),” E. L. Hirst, Elizabeth Percival, and R. S. Williams,]. Chem. SOC., 1942 (1958). “The Constitution of Laminarin. Part 111.The Fine Structure of Insoluble Laminarin,” F. B. Anderson, E. L. Hirst, D. J. Manners, and A. G. Ross,]. Chem. SOC., 3233 (1958). “Gum Ghatti (Indian Gum). Part 111. Neutral Oligosaccharides Formed on Partial Acid Hydrolysis of the Gum,” G. 0.Aspinall, (Mrs) Barbara J. Auret, and E. L. Hint,]. Chem. SOC., 4408 (1958). “The Gums and Mucilages of Plants,” E. L. Hirst and J. K. N. Jones, Handb. Pjlanzenphysiol., 6,500 (1958). “Structural Chemistry of Plant Polysaccharides with Reference to their Colloidal Character,” E. L. Hirst, Verhandlungsber. Kolloid-Ges., 18, 104 (1958). “Barry Degradation of Laminarin,” E. L. Hirst, J. J. O’Donnel1,and Elizabeth Percival, Chem. Ind. (London), 834 (1958). “The Presence of L-Guluronic Acid Residues in Alginic Acid,” D. W. Drummond, E. L. Hirst, and Elizabeth Percival, Chem. Ind. (London), 1088 (1958). “Chemical Structure in the Hemicellulose Group,” E. L. Hirst, Fundamentals Papermaking Fibers, Trans. Symp. Cambridge, Engl. 1957, 93 (1958). “Polysaccharides ofthe Marine Algae” (Presidential Address), E. L. Hirst, Proc. Chem. Soc. London, 177 (July, 1958). “Seaweed Mucilages,” E. L. Hirst,Abstr. Int. Seaweed Symp., 3rd, Galway, 52 (1958). ‘‘Analytical Studies on the Carbohydrates of Grasses and Clovers. IX. Changes in Carbohydrate Composition during the Growth oflucerne,” E. L. Hirst, D. J. Mackenzie, and Clare B. Wylam,]. Sci. Food Agric., 10, 19 (1959). “The Structure ofAcacia pycnantha Gum,” G. 0.Aspinall, E. L. Hirst, and A. Nicholson,J. Chem. Soc., 1697 (1959). “Molecular Structure in the Polysaccharide Group,” E. L Hirst, Proc. R. SOC. London, Ser. A , 252,287 (1959). “Studies on Uronic Acid Materials. 11. The Variation in Composition of Gum Nodules from Combreturn leonense,” D. M. W. Anderson, E. L. Hirst, and N. J. King, Talanta, 3,

118 (1959).

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“Plant Gums,” E. L. Hirst. Proc. Int. Congr. Biochem., 4th, Vienna, 1958,1,31(1959). “Studies on the Metabolism of the Protozoa. Part VIII. The Molecular Structure of a Starch-type Polysaccharide from Chilomonas paramecium,” A. R. Archibald, E. L. Hirst, D. J. Manners, and J. F. Ryley,]. Chem. Soc., 556 (1960). “Studies on the Metabolism of the Chrysophyceae. Comparative Structural Investigations on Leucosin (Chrysolaminarin) Separated from Diatoms and Laminarin from the Brown Algae,” Anne Beattie, E. L. Hirst, and Elizabeth Percival, Biochem. J., 79, 531 (1961). “The Structure of Polysaccharides,” E. L. Hirst, Biochem. SOC.Symp., No. 21, 45 ( 1962). “The Constitution of Alginic Acid,” D. W. Drummond, E. L. Hirst, and Elizabeth Percival,]. Chem. SOC.,1208 (1962). “The Position ofMannit01 in Laminarin,” W. D. Annan, E. L. Hirst, and D. J. Manners, Chem. Ind. (London), 984 (1962). “The Acidic Sugar Components ofCochlospemnum gossypium Gum,” G. 0.Aspinall, E. L. Hirst, and (Miss) Margaret J. Johnston, J. Chem. SOC.,2785 (1962). “The Chemical Structure of the Hemicelluloses,” E. L. Hirst, Pure Appl. Chem., 5,53 (1962). “Structural Studies ofAlginic Acid,” E. L. Hirst, E. Percival, and J. K. Wold,Chem. Ind. (London), 257 (1963). “The Location of L-Rhamnopyranose Residues in Gum Arabic,” G. 0. Aspinall, A. J. Charlson, E. L. Hirst, and R. Young,]. Chem. Soc., 1696 (1963). “Methyl Ethers of Mono- and Disaccharides,” E. L. Hirst and Elizabeth Percival, Methods Carbohydr. Chem., 2, 145 (1963). “Glycofuranosides from Cyclic Carbonates,” E. L. Hirst and Elizabeth Percival, Methods Carbohydr. Chem., 2,349 (1963). “Professor John Read, F.R.S.,” E. L. Hirst, Nature, 198,336 (1963). “Professor John Read, 1884-1963 (Obituary Notice),” E. L. Hirst. Proc. Chem. SOC. London, 353 (1963). “John Read, 1884-1963,” E. L. Hirst, Biogr. Mem. Fellows R. SOC.,9,237 (1963). “The Structure of Alginic Acid. Part IV. Partial Hydrolysis of the Reduced Polysaccharide,” E. L. Hirst, Elizabeth Percival, and J. K. Wold,J. Chem. Soc., 1493 (1964). “The Constitution of Laminarin. Part IV. The Minor Component Sugars,” W. D. Annan, Sir Edmund Hirst, and D. J. Manners,J. Chem. Soc., 220 (1965). “The Constitution of Laminarin. Part V. The Location of 1,6-Glucosidic Linkages,” W. D. Annan, Sir Edmund Hirst, and D. J. Manners,]. Chem. SOC., 885 (1965). “The Structure of Alginic Acid. Part V. Isolation and Unambiguous Characterization of Some Hydrolysis Products of the Methylated Polysaccharide,” Sir Edmund Hirst and D. A. Rees,]. Chem. SOC.,1182 (1965). “The Water-soluble Polysaccharides of Cladophora rupestris and of Chaetomorpha spp., Part 11. The Site of Ester Sulphate Croups and the Linkage between the Galactose Residues,” Sir Edmund Hirst, W. Mackie, and Elizabeth Percival,]. Chem. Soc., 2958 (1965). “Seed Polysaccharides and their Role in Germination. A Survey of the Polysaccharide Components ofMustard Seeds with Special Reference to the Embryos,”E. L. Hirst, D. A. Rees, and N. G. Richardson, Biochem. I. 95,453 , (1965). “The Role of Sugars as Energy Reserves in Nature,” Sir Edmund Hirst,]. R. Soc. Arts, 64,290 (1966). “Studies on Uronic Acid Materials. Part XVII. Some Structural Features of Acacia senegal Gums (Gum Arabic),” D. M. W. Anderson, Sir Edmund Hirst, and J. F. Stoddart, /. Chem. Soc., 1959 (1966).

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“Review of the Chemistry of Water-soluble Gums,” Sir Edmund Hirst, in Monograph No. 24 (“The Chemistry and Rheology of Water-soluble Gums and Colloids”), The Society of Chemical Industry, London, 1966, p. 3. “Studies on Uronic Acid Materials. Part XVIII. Light-scattering Studies on Some Molecular Weight Fractions fromAcacia senegal Gum,” D. M. W. Anderson, Sir Edmund Hirst, S. Rahman, and G. Stainsby, Carbohydr. Res., 3,308 (1967). “Studies on Uronic Acid Materials. Part XXI. Some Structural Features of Acacia arabica Gum,” D. M. W. Anderson, Sir Edmund Hirst, and J. F. StoddartJ. Chenz. Soc., 1476 (1967). “The Ongins of Chemistry,” by Robert P. Multhauf (book review), J . R . Soc. Arts, 116,75 (1967). “Studies on Uronic Acid Materials. -11. Some Structural Features of the Gum Exudate from Acacia seyal Del.,” D. M. W. Anderson, I. C. M. Dea, and Sir Edmund Hirst, Carbohydr. Res., 8, 460 (1968). “Characterisation of Polysaccharide Structures by Glycoside Stabilisation with Toluene-p-sulphonates: Model Experiments with Dextran,” D. A. Rees, N. G. Richardson, N. J. Wight, and Sir Edmund Hirst, Carbohydr. Res., 9, 451 (1969). “Stanley Peat,” Sir Edmund Hirst, Biogr. Mem. Fellows H. Soc., 16,441 (1970). “a-(1+4)-~Glucans. Part XXI. The Molecular Structure of Starch-type Polysaccharides from Haemutococcus pluvialis and T e t r a s e h i s carteri$omis,” Sir Edmund Hirst, D. J. Manners, and I. R. Pennie, Carbohydr. Res., 22, 5 (1972). “Paul Karrer-Obituary Notice,” R. Soc. Edinburgh, Year Book, 1971-72,50 (1972). “R. C. Menzies-Obituary Notice,” R. Soc. Edinburgh, Year Book, 1973, 42 (1973).

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CARBOHYDRATE BORONATES BY ROBERTJ . FEFWER Department of Chemistry. Victoria University of Wellington.Wellington.New Zealand

I . Introduction .......................................................... I1. Synthesis of Boronates ................................................ 1. Direct Condensation of Carbohydrates with Boronic Acids . . . . . . . . . . . . 2 . From Borinates .................................................... I11. Structures of Carbohydrate Boronates .................................. 1. Alditol Boronates .................................................. 2 . Sugar Boronates .................................................... 3 . Boronates of Glycosides, Nucleosides. and Related Compounds . . . . . . . IV . Boronates in Chemical Reactions ...................................... 1. Boronates in Aqueous Systems ...................................... 2 . Removal of Boronate Groups ........................................ 3. Stability of Boronates during Chemical Reactions .................... V Separations of Carbohydrates by Use of Their Boronates ................ 1. Use of Isolated Boronates ........................................... 2 . Use in Paper Chromatography ...................................... 3 Use in Electrophoresis ............................................. 4 . Use in Column Chromatography .................................... 5 . Use in Gas-Liquid Chromatography ................................. VI . Mass Spectrometry of Boronates ....................................... VII . Nuclear Magnetic Resonance Spectroscopy of Boronates ................ VIII . Borinates ............................................................ IX . Tables ............................................................... X . Addendum ...........................................................

.

.

31 37 37 39 41 42 43 45 48 48 52 53 57 57 58 62 63 65 65 70 70 71 80

I . INTRODUCTION Condensation between D-ghCOSe and boric acid in acetone. in the presence of a strong-acid catalyst. gives the discrete. cyclic ester 1.2-0 . isopropylidene-a-D-glucofuranose3.5.boratel (1). which may be used in the preparation of 6-substituted derivatives of D.glucose . Such instances of the isolation and syntheti.c application of specific carbohydrate borates are. however. few. because of the complexity of boric acid as an esterifying agent As well as affording the 3.5.cyclic ester 1. the aforementioned reaction could have led to ( a ) a dimeric

.

(1) L . Vargha. Ber., 66. 704-707 (1933). 31

32

ROBERT J. FERRIER

product containing boron-oxygen-boron linkages: (b) various dimers linked by one boric acid unit, or (c) an intramolecular triester. With a view to exploiting the clear potential of boric acid derivatives in preparative, carbohydrate chemistry, but with reagents not subject to such diverse means of reaction, the present autho9 began, in 1961, a study of the condensation undergone between phenylboronic acid [PhB(OH),I and the diol systems of various glycosides. At that time, it had been found that free sugars interact with this acid in aqueous soluti0n,4-~and several boronates of free sugars’ and alditols8.9 had been isolated, but, despite initial efforts,s no discrete glycoside derivatives had been reported, no successful structural work had been carried out, and the esters had not found application in carbohydrate chemistry. In the ensuing fifteen years, a wide range of specific boronates, for example, 1,2-O-isopropylidene-a-D-g~ucofuranose 3,5phenylboronate (2), have been prepared, and many workers have

1 R=OH 2 R=Ph

shown how useful they can be as suitable crystalline derivatives, as intermediates in synthesis, in a range of separatory techniques, and as reaction catalysts. The present article surveys the progress made during this time. The role of boronic acids in carbohydrate biochemistry is not dealt with, although, in plants, they have been found to influence the storage of polysaccharides and aspects of their growth5 (2)W.Gerrard, M.F. Lappert, and B. A. Mountfield,J. Chem. SOC., 1529-1535 (1959);N. N. Greenwood, in “Comprehensive Inorganic Chemistry,” J. C. Bailar,H. J. Eniekus, R. Nyholm, and A. F. Trotman-Dickenson,eds., Pergamon Press, Oxford, 1973,Vol. 1, p. 895. (3)R. J. Ferrier,]. Chem. SOC., 2325-2330 (1961). (4)K. Torssell, Ark. Kemi, 10, 541-547 (1957);Chem. Abstr., 52, 14,555(1958). (5)K. Torssell, J. H. McClendon, and G. F. Somers,Acta Chem. Scand., 12,1373-1385 (1958). (6)J. P. Lorand and J. 0. Edwards,]. Org. Chem., 24,769-774 (1959). (7)M.L.Wolfrom and J. Solms,J. Org. Chem., 21,815-816 (1956). (8)H.G. Kuivila, A. H. Keough, and E. J. Soboczenski, J. Org. Chem., 19, 780-783 (1954). (9)J. M.Sugihara and C. M. Bowman,]. Am. Chem. SOC., SO, 2443-2446 (1958).

CARBOHYDRATE BORONATES

33

Boron, having the electronic configuration ls22s22p,has 3 valence electrons, and forms planar, tricovalent derivatives that are electron deficient, and which, as Lewis acids, accept two electrons from bases to complete the boron outer-shell octet and give tetrahedral adducts. Boric acid exemplifies this behavior by ionizing, in aqueous solution, not by direct deprotonation, but by hydration and subsequent ionization, to give the symmetrical borate anion:

B(OH)B+ H 2 0

-

B(OH)4+ H+.

Wide attention has been given to the interaction of carbohydrates with these ions,l’Jdespite the complexity of the systems produced; reaction of a conformationally unrestricted, contiguous trio1 could, for instance, result in the formation of many species exemplified by the following.

B B O H

PfoH ’ ‘OH

0 ‘

With boronic acids, formation of dimeric species is essentially precluded, but cyclic products having different ring-sizes and electronic configuration at boron can b e formed, and it is here suggested that isomerization of certain cyclic boronates may consequently occur much more readily than has been recognized. In the case the five-membered boronates (4) could of the general triols (3, conceivably rearrange to the six-membered structures (5) by way of accessible ionic intermediates (6),and thus 4 and 5 may be thought of as (10) J. Boeseken,Adu. Carbohydr. Chem., 4,189-210 (1949);A. B. Foster, ibid.,12,81115 (1957); T. E. Acree,Adu. Chem. Ser., 117, 208-219 (1973); J. Dale,./. Chem. SOC., 922-930 (1961); E. W. Malcolm, J. W. Green, and H. A. Swenson, ihid., 4669-4676 (1964).

ROBERT J. FERRIER

34

4

5

6

tautomers, as well as isomers, bearing the same relationship to each other as do furanose and pyranose forms of free sugars, and thereby presenting classically difficult problems of structural analysis. In the chemistry of alditol boronates, this issue is potentially of wide significance (see, for example, the inconsistencies reported in the chemistry of glycerol phenylboronate; Section III,l), but fortunately, boronates of cyclic carbohydrates are not susceptible (except, conceivably, in such compounds as ribopyranoside esters), and most of the work described in this Chapter relates to discrete compounds of tricovalent boron-all of which can, however, form tetrahedral complexes with Lewis bases. The relevant bond-lengths and bond-angles associated with trigonal and tetrahedral boron in boronates are as shown. Consequently, both

can be accommodated in strainless, six-membered rings formed from l,Sdiols, whereas five-membered, cyclic boronates are free from angle strain only when the boron is tetrahedral. Thus, esters formed from vicinal diols are more strained,ll and exhibit a stronger tendency to react with bases12 (which frequently leads to relatively easy hydroly s i P ) , than their six-membered counterparts. These characteristics (11) A. Finch, P. J. Gardner, P. M. McNamara, and G. R. Wellum,J. Chem. Soc., A, 33393345 (1970). (12) A. Finch and J. C. Lockhart,J. Chem. SOC., 3723-3726 (1962). (13) R. A. Bowie and 0. C. Musgrave,J. Chem. SOC., 3945-3949 (1963).

CARBOHYDRATE BORONATES

35

therefore parallel, as expected, those of five- and six-membered, cyclic borates.14 Apart from this generalization, however, the question of stabilities of carbohydrate boronates is complex, because, not only are they dependent upon ring size but also upon the carbon-bonded radicals (ring strain in alkylboronates being substantially higher than in aryl analogs1'), and, notably, upon the presence and type of substituent groups on the ester rings and elsewhere in the molecules. Groups that inhibit coordination of water with boron stabilize the esters towards hydrolysis,l3 as, especially, do groups which can themselves act as fourth ligands. As, in carbohydrate boronic esters, oxygen atoms are frequently available for this function, this specific means of stabilization is of great importance; for example, in determining the degree of complexing of polyhydroxy compounds in aqueous systems and, consequently, their mobility as boronates on chromatograms or electrophoretograms (see Section V). Specific solvation, such as occurs15 in the complex 7, is also likely to influence ester stability to some extent.

Cyclic boronic esters can be formed readily from simple, acyclic 1,2-, 1,3-, and 1,4-diolsYand five-, six-, and, to a lesser extent, sevenmembered esters are, consequently, frequently encountered. Initial attempts9 to obtain an eight-membered, cyclic product from 1,5pentanediol were not successful, presumably because linear diesters or macrocyclic products preponderated, but it has been shown by massspectrometric methods16 that some of the cyclic phenylboronate can be formed and, furthermore, 1,6-hexanediol affords some nine-membered, cyclic phenylboronate. In model, cyclic systems, cis-lY2-diols on cyclopentane and cyclohexane rings readily give bicyclic boronates, whereas trans-related analogs react with two molar equivalents of boronic acids, to give seven-membered, cyclic diesters (for example, 8 (14) A. J . Hubert, B. Hartigay, and J. Dale,J. Chem. SOC., 931-936 (1961). (15)J. D. Morrison and R. L. Letsinger,J. Org. Chem., 29, 3405-3407 (1964). (16)E. J. Bourne, I. R. McKinley, and H. Weigel, Carbohydr. Res., 35,141-149 (1974)

36

ROBERT J. FERRIER

and 9) of diboronic acids.9 In the case of 1,3- and 1,4-cis-cyclohexanediols, forcing conditions permit the synthesis of cyclic boronate s.l 7

8 n=l 9 n=2

Esters may be prepared from a range of boronic acids that vary widely in their properties, including their aciditiesls (boric acid K, 6.53 x 10-10, phenylboronic acid, 13.7 x and butylboronic acid, 0.18 x 10-10), but most of the known carbohydrate boronates are phenyl esters, although substituted-aryl compounds have received some slight attention. Butyl, ethyl, and methyl analogs, although seldom crystalline compounds, unlike the majority of carbohydrate phenylboronates, have been utilized mainly because of their enhanced volatility and their consequent value in gas-liquid chromatography and mass spectrometry. Naming of carbohydrate boronates may be based on three principles: ( a ) ,the central use of the ester heterocyclic rings: 1,3,2-dioxaborolane (five-membered), lY3,2-dioxaborinane(six-membered), and 1,3,5,2,4trioxadiborepane (seven-membered, for example, 8 and 9); (b),the use of radical prefixes: “borylene” (Chem. Abstr.) or “boranediyl” (Z.U.P.A.C.) for )BH; or (c), ester terminology. Thus, for example, according to these respective procedures, the glycerol derivative 10

may be named ( a ) 5-hydroxy-2-phenyl-l,3,2-dioxaborinane, ( b ) 1,3-0(phenylborylene)glycerol,or 1,3-0-(phenylboranediyl)glycerol,or ( c ) glycerol lY3-phenylboronate.In this Chapter, the third method will be adopted, and, in accordance with current, Chemical Abstracts usage, (17) W. V. Dahlhoff and R. Koster,Justus Liebigs Ann. Chem., 1625-1636 (1975). (18) G. E. K. Branch, D. L. Yabroff, and B. Bettman,]. Am. Chem. SOC., 56,1850-1857 (1934).

CARBOHYDRATE BORONATES

37

alkyl- and aryl-boronate (rather than alkane- and arene-boronate) terminology will b e used. A brief summary is included of carbohdyrate borinates-acyclic esters derived from borinic acids (R,BOH)-which have received less attention than the related boronates. Earlier, short reviews have dealt with m o n o ~ a c c h a r i d eand ~~ nucleoside phenylboronates,20 and the application of phenylboronic acid in carbohydrate chemistry.21 11. SYNTHESIS OF BORONATES 1. Direct Condensation of Carbohydrates with Boronic Acids

Boronic acids readily undergo self-condensation to give cyclic anhydrides, and either these trisubstituted boroxins (ll),or the acids themselves, react spontaneously, but not necessarily completely, in solution, with suitable diols, to give cyclic boronates 12 (see Scheme l),

\ /

12

11

Scheme 1

for the isolation of which the water and solvent have simply to be removed. Frequently, a suitable procedure involves treatment of the carbohydrate with the required number of molar equivalents of the acid or anhydride in boiling benzene, and azeotropic removal of the water formed (which can conveniently be collected in a Dean-Stark distillation head, and, provided that sufficient quantities are evolved, measured, in order to monitor the progress of the reaction). Methyl p-Dxylopyranoside (16.8 g) treated in this way with triphenylboroxin (10.6 g, 0.33 mol. equiv.) liberates water (1.8ml) during conversion into its (19) A. M. Yurkevich and S. G. Verenikina, Vitam. Vitam.Prep., 247-256 (1973);Chem. Abstr., 80, 48,2474 (1974). (20) I. I. Kolodkina and A. M. Yurkevich, Vitam. Vitam. Prep., 257-268 (1973);Chem. Abstr., 79, 146,774h (1973). (21) R. J . Ferrier, Methods Carbohydr. Chem., 6,419-426 (1972).

38

ROBERT J. FERRIER

2,4-phenylboronate7 which may be isolated in excellent yield by removal of most of the solvent and addition of dry, light petroleum.22 Some carbohydrate derivatives are too insoluble in benzene to be so esterified, and, for them, a suitable, alternative solvent is 174-dioxane, the water azeotrope of which may, by careful, fractional distillation be removed before the solvent, and the water evolved in the reaction determined, if desired, by the Karl Fischer method. Whereas methyl a-D-glucopyranoside can be esterified by the benzene procedure, the analogous a-D-mannoside ester must be prepared by use of 1,Cdioxane or by some other variation.3 A further method applicable to benzene-insoluble carbohydrates involves the addition of the compounds, in water, to triphenylboroxins (preferably in methanoP), and, under these conditions, some boronic esters have been found to precipitate. Otherwise, the reactants may be fused under vacuum7 (a procedure not in current use, and not recommended) or treated in such solvents as acet0ne,~*23,24 or 2methoxyethanol.25~28Particularly for the formation of nucleoside boronates, pyridine has often been used as the solvent, and the reactants have been heated at the boiling point, or in sealed tubes27-30; on occasion, N7N-dimethylformarnide2s*31has been employed. A feature of the use of pyridine is the occasional isolation-not surprisingly-of the products as pyridine complexes.32 A slight modification of the reaction in acetone incorporates use of sulfuric acid as a catalyst and provides, from the corresponding, free sugars, moderately efficient, one-step procedures for obtaining 1,2-0isopropylidene-a-D-xylofuranose3,5-phenylboronate and the D-glU(22) R. J. Ferrier, D. Prasad, A. Rudowski, and I. Sangster,]. Chern. SOC.,3330-3334 (1964). (23) E. J. Bourne, E. M. Lees, and H. Weigel,]. Chem. Soc., 3798-3802 (1965). (24) A. M. Yurkevich, S. G . Verenikina, E. G . Chauser, and N. A. Preobrazhenskii, Zh. Obshch. Khim., 36,1746-1749 (1966). (25) S. G. Verenikina, A. M. Yurkevich, and N. A. Preobrazhenskii, Zh. Obshch. Khim., 37,2181-2184 (1967). (26) A. S. Guseva, S. G . Verenikina, S. F. Dymova, and A. M. Yurkevich, Zh. Obshch. Khim., 44,2327-2331 (1974). (27) A. M. Yurkevich, L. S. Varshavskaya, I. I. Kolodkina, and N. A. Preobrazhenskii,Zh. Obshch. Khim., 37,2002-2006 (1967). (28) A. M. Yurkevich, I. I. Kolodkina, L. S. Varshavskaya, V. I. Borodulina-Shvetz, I. P. Rudakova, and N. A. Preobrazhenskii, Tetrahedron, 25,477-484 (1969). (29) J. J. Dolhun and J. L. Wiebers,J. Am. Chem. SOC.,91, 7755-7756 (1969). (30) S. A. Ermishkina and A. M. Yurkevich, Zh. Obshch. Khim., 40,652-655 (1970). (31) A. M. Yurkevich, V. I. Borodulina-Shvetz, I. I. Kolodkina, and N. A. Preobrazhenskii, Zh. Obshch. Khim., 37,21762180 (1967). (32) P. J. Wood and I. R. Siddiqui, Carbohydr. Res., 36,247-256 (1974).

39

CARBOHYDRATE BORONATES

cose analog, 1,2-0-isopropylidene-~-~-arabinopyranose 3,4-phenylboronate and the a-D-galactose analog, and 2,3-0-isopropylidene-Dmannohranose 5,6-~henylboronate.~~ Koster and Dahlhoff introducedsa a variation by using, as the esterifying agent, the ethylboronic anhydride derivative (Me,CCO,BEt),O. 2. From Borinates

1,2-Ethanediol undergoes reaction at 380” with trimethylborane, to give 2-methyl-1,3,2-dioxaborolane34 (14), presumably by way of the intermediate dimethylborinate (13) (see Scheme 2), and Russian workers unexpectedly encountered an analogous reaction during CH,OH

I CH,OH

+

-

H,COBMe,

Me,B

I

CH,OH 13

-Hzlo\BMe H, 0’ 14

Scheme 2

heating of ribonucleosides in pyridine with equimolar amounts of isobutyl diphenylborinate, as, instead of the borinic esters anticipated (presumably 5’), they obtained almost quantitative yields of the 2’,3’phenylboronate~.35-~* The procedure may be used for preparative purposes, and has the advantage of not producing water as the byproduct. German workers independently found that 1,2- 1,3-, and some 1,4diols can be converted into bis(diethy1borinates) which, on heating to -200°, lose triethylboron to give cyclic ethylboronates. Otherwise, these boronates can be prepared directly from the diols by heating with triethylboron in the presence of diethylboryl pivalate, or by dismutation from equimolar proportions of diols and their bis(diethylbori(33) B. E. Stacey and B. Tierney, Carbohydr. Res., 49, 129-140 (1976). (33a) R. Koster and W. V. Dahlhoff, Justus Liebigs Ann. Chem., 1925-1936 (1976). (34) D. W. Wester, F. Longcor, and L. Barton, Synth. Inorg. Met.-Org. Chem.,3,115-123 (1973). (35) A. M. Yurkevich, L. S. Varshavskaya, and I . I. Kolodkina, Zh. Obshch. Khim., 38, 21 15 (1968). (36) I . I. Kolodkina, A. S. Guseva, E. A. Ivanova, L. S. Varshavskaya, and A. M. Yurkevich, 2%. Obshch. Khim., 40,2489-2493 (1970). (37) V. N. Rekunova, I. P. Rudakova, and A. M. Yurkevich,Zh. Obshch. Khim., 44,11821187 (1974). (38) V. N. Rekunova, I. P. Rudakova, and A. M. Yurkevich, Tetrahedron Lett., 281 1-2814 (1973).

ROBERT J. FERRIER

40

nates), and these procedures have been used to obtain ethylboronates of simple diols,17 triols and tetrols,39 alditols,40-42 and the cis-isomers of 1,2-, 1,3-, and 1,4-cyclohexanediol.~~ Pyrolysis of acyclic 1,4- and 1,5diols as their diethylborinates did not give seven- and eightmembered, cyclic products but, instead,l7 the macrocycles 15 and 16.

Et

15 n = 4 l6n=5

In the case of the alditols, the per(diethylboriny1) derivative may be selectively converted into mixed borinate-boronates, the D-mannitol derivative giving D-mannitOl 1,2,5,6-tetrakis(diethylborinate) 3,4ethylboronate (17), which can be isolated prior to its conversion into the 1,2:3,4:5,6-triboronate,4'and the galactitol ester giving galactitol 1,6-bis(diethylborinate) 2,3:4,5-bis(ethylboronate)(18) prior to the formation of galactitol 176:2,3:4,5-tris(ethylboronate) (19) having an

%

BEt,

%

Et,BOCH,

,OBEt,

Et,BOCH,

,OBEt,

18

17

Ef 19

(39) W. V. Dahlhoff and R. Koster, Justus Liebigs Ann. Chem., 1914-1925 (1975). (40) W. V. Dahlhoff and R. Koster,Justus Liebigs Ann. Chem., 1926-1933 (1975). (41) W. V. Dahlhoff, W. Schussler, and R. Koster,Justus Liebigs Ann. Chem., 387-394 (1976). (42) W. V. Dahlhoff and R. KosterJ. Org. Chem., 41, 2316-2320 (1976).

CARBOHYDRATE BORONATES

41

unusual, nine-membered ring.42 Selective deborination provides a means of obtaining, from compounds 17 and 18, D-mannitol 3,4e t h y l b ~ r o n a t and e ~ ~ galactitol 2,3:4,5-bi~(ethylboronate).~* Koster and Dahlhoff have reviewed their work with carbohydrate ethylboron esters.qa From this work, it follows that boronates of polyhydric alcohols can be prepared directly by heating with trialkyl- or triaryl-boranes. Galactitol heated with triphenylborane in refluxing toluene gives the same tris(phenylboronate), in 93%yield22 as can be prepared by use of phenylboronic acid.9 It seems probable that a fundamental distinction may exist between the two methods available for the synthesis of carbohydrate boronates, in that, under dehydration conditions, esterifications may be reversible, whereas boronate formation from borinates is not. In the first, therefore, products may be thermodynamically controlled, whereas kinetic control will operate in the second. It is noteworthy, however, that products obtained from several glycopyranosides (a) by use of an ethylboronate derivative, and (b) by way of the per(diethy1borinates) had the same

111. STRUCTURES OF CARBOHYDRATE BORONATES Chemical methods for determining boronate structures usually involve the substitution of unesterified hydroxyl groups, and the characterization of the products, either by examination of the compounds obtained after removal of the boronate groups, or by independent synthesis of the fully substituted compounds by boronation of known derivatives. Occasionally, the positions of sulfonic ester groups in fully substituted boronates have been determined by means of nucleophilic displacement-reactions. Although these methods are often well suited to derivatives containing one boronate group, they cannot always be applied unambiguously if more than one boronate group is present. The most useful physical methods of structural analysis, apart from X-ray diffraction, are nuclear magnetic resonance (n.m.r.) spectroscopy and mass spectrometry. Newer applications of 13C- and 'lB-n.m.r. spectroscopy (see Section VII) indicate their value in the determination of the ring size of boronates, and mass spectrometry (see Section VI) is of importance largely for the same reason. Proton n.m.r. (p.m.r.) spectroscopy is of

(42a) R . Koster and W. V. Dahlhoff,Am. Chem. SOC. Symp. Ser., 39, 1-21 (1977).

42

R O B E R T J. FERRIER

great, general significance, and on occasion, infrared spectroscopy has been a useful structural tool, as it can offer means of determining the nature of the intramolecular hydrogen-bonding in which unsubstituted hydroxyl groups may be engaged, and this may permit structural assignment.

1. Alditol Boronates For some properties of alditol boronates, see Table V. Glycerol phenylboronate (m.p. 75.5-76.5') is obtainable in high yield,l3 and was assigned the 1,2-cyclic structure on examination of the glycerol N-phenylcarbamate formed from it by treatment with phenyl isocyanate, followed by removal of the boronate ring.23 Consistent with this conclusion are the characteristics of hydrolysis of the parent ester13 and the fact that phenylboronic acid complexes more strongly with 1,2- than with 1,3-diols.6 However, a re-inve~tigation~~ of the glycerol phenylcarbamate revealed that it reduces 0.84 molar equivalent of periodate, instead of the 1 molar equivalent expected, which was taken to indicate that it was contaminated with some of the unoxidized, 2-substituted isomer. A new procedure for structural analysis was then developed to check this conclusion. Glycerol phenylboronate was methylated with diazomethane in dichloromethane containing boron trifluoride etherate, the boronate ring was removed, and the resultant diols were acetylated, to give two mono-0methylglycerol diacetates which were characterized, after g.1.c. separation, by mass spectrometry. Surprisingly, this method revealed that the initial phenylboronate was a mixture comprising the sixmembered ester as the main product. In the present author's opinion, the control experiment conducted on the critical methylation step within the foregoing procedure may not have been sufficiently rigorous, on the grounds that methylation of a-Dglucofuranose 1,2:3,5-bis(phenylboronate)with diazomethane-boron trifluoride may not establish that all boronates react without change under these conditions. As was indicated in the Introduction, chemical methods may be seriously deficient for characterizing such compounds, and techniques that do not disturb delicate chemical states may have to be used in just such cases. However, when applied to glycerol phenylboronate, "B-n.m.r.-spectral analysis did not unambiguously resolve the issue either, because, at 20', the boron chemicalshift indicated the presence of a six-membered ring, whereas, at 80', it moved to a position indicative of a five-membered ring.26 Chemical procedures have to be used with the greatest care, and (43) I. R. McKinley and H. Weigel, Carbohydr. Res., 31, 17-26 (1973).

CARBOHYDRATE BORONATES

43

McKinley and Weigel's results43 with other triols should also be considered with this in mind. By applying the diazomethane methylation procedure, they also investigated the phenylboronates 4-deoxy-~-erythritol,and obtained from 3-deoxy-~~-glycero-tetritol, 1,5-dideoxy-~-arabinitol,-ribitol, and -xylitol, and found that all except that derived from the last trio1 were mixtures. Their conclusion was that, where six-membered rings that do not have axial substituents can be formed, their production is favored (glycerol, 3-deoxy-~~-glycerotetritol, 4-deoxy-~-erythritol,and 175-dideoxyribitol);in other cases, substantial proportions of five-membered ring esters are produced. However, other points that appear to increase suspicion of the validity of the methylation procedure are: (i) the inconsistency between the periodate and the methylation results, as applied to glycerol phenylboronate, ( i i ) the finding of the formation, from some triols, of some thermodynamically unfavored, five-membered, cyclic products, and ( i i i ) the conclusion that the phenylboronate obtained from 4deoxy-L-erythritol comprises all three possible isomers, despite its being a sharp-melting compound. Glycerol ethylboronate produced by the borinate method39 has the five-membered, cyclic structure, consistent with its being a kinetically controlled product, whereas that obtained from 3-deoxy-D~-glycerotetritol contains a six-membered ring. From these findings with triols, it follows that, apart from the expectation that formation of five- and six-membered rings would be favored (see, however, the exceptional compound 19), no general conclusions can be drawn regarding the structures of boronates derived from more-complex polyhydric alcohols. In Table V, alditol boronates are listed with structures when these can be concluded either from the method of synthesis, from physical studies, or by deduction (as with the 1,2:5,6-diesters formed from 3,4-di-O-substituted mannitols).

2. Sugar Boronates For some properties of boronates of sugars, see Table 111. Although boronates of sugars were amongst the first such carbohydrate esters to be prepared,' little structural work was performed until the advent of n.m.r.-spectroscopic and mass-spectrometric methods. The high-yielding condensations undergone by D-xylose and Larabinose with phenyl- and butyl-boronic acid have thus been shown to give a-D-xylose 1,2:3,5-bis(phenyl-and butyl-boronate) (20)and p-Larabinose 1,2:3,4-bis(phenyl- and butyl-boronate)44 (21). (44) P. J. Wood and I. R. Siddiqui, Curbohydr. Res., 33,97-104 (1974).

ROBERT J. FERRIER

44

20

R

= Bu or Ph

21

In 1956, a ribose diphenylboronate (map.140-142") was prepared in modest yield by a fusion method,' but it was later shown26 that an almost quantitative yield of a mono-ester was obtainable by conducting the condensation in hot 2-methoxyethanol, and this was a-D-ribose 2,4phenylboronate (22), as indicated by p.m.r. and "B-n.m.r. and conversion by selective substitution into the S-p-toluenesulf~ n a t eIt. ~is ~here suggested that the 2,4-ester (22) might be very sus-

22

ceptible to structural change, as it could give the 1,2-, 1,3-, 2,3-, and 3,4-isomers by a set of tautomeric rearrangements by way of the accessible, boronate anions (23 and 24).

23

24

Condensation of D-ribose with two molar equivalents of phenylboronic acid in 2-methoxyethanol gave a diester to which was assigned the 1,5:2,3-P-furanose structure (25) on the basis of as-yet-unreported n.m.r. data.26 (45) M. G. Edelev, T. M. Filippova, V. N. Robos, I. K. Shmyrev, A. S. Guseva, S. G. Verenikina, and A. M. Yurkevich, Z h . Obshch. Khim., 44,2321-2327 (1974). (46) A. S. Guseva, I. P. Rudakova, S. G. Verenikina, and A. M. Yurkevich, Zh. Obshch. Khim., 44, 1187-1193 (1974).

CARBOHYDRATE BORONATES

45

D-Glucose gives a crystalline 172:3,5-bis(phenylboronate)(26) Ph

phAFGy &H

0

b-hPh

B Ph 25

26

( ~ . m . r . ~and * * 13C-n.m.r.45 ~~ evidence) which can b e used to prepare 6substituted esters32.4' and ethers** of the sugar. Likewise, 1,2-0isopropylidene-a-D-glucose gives a crystalline 3,5-phenylboronate724. 32*33 and, with two molar equivalents of the boronating agent, a further crystalline derivative, characterized as the 5,6-dib0ronate.~~ When a substituent is present at C-3, as in 3-deoxy-3-fluoro-1,2-O-isopropylidene-a-~-glucofuranose,4~ direct boronation takes place, as expected, at the 5,6-diol. D-Fructose reacts in the P-pyranose form, to give the 2,3:4,5bis(phenylboronate)32 (27), and 6-deoxy-a-~-galactoseaffordP the stereochemically related 1,2:3,4-die ster (28).

Phb-0

27 R' = H, R2 = CH,OH 28 R' = Me. Rz = H

Little structural work has been performed on derivatives of other hexoses, or higher sugars. 3. Boronates of Glycosides, Nucleosides, and Related Compounds

For some properties of boronates of glycosides, nucleosides, and related compounds, see Tables IV, VI, and VII. Glycosides and related compounds tend to give boronates easier to (47) L. G. Mogel and A. M. Yurkevich, Zh. Obshch. Khim., 39, 1882-1886 (1969). (48) E. J. Bourne, I. R. McKinley, and H. Weigel, Carbohydr. Res., 25,516-517 (1972). (49) A. B. Foster, R. Hems, and J. M . Webber, Carbohydr. Res., 5,292-301 (1967).

ROBERT J. FERRIER

46

characterize than corresponding derivatives of alditols and sugars, because of the specific diol systems they present to the boronating reagents. Most interestingly, cis-173-related diols on pyranoid rings condense to give cyclic boronates [for example, methyl a-D-xylopyranoside 2,4-phenylboronate (29) and N-(p-bromopheny1)-a-D-ribopyronosylamine 2,4-phenylboronate (30)] which, in the ~ y l o s e ~ ~ , ~ ~ ~

Ph 29

Ph 30

and ribose51 series, afford useful means for obtaining 3-substituted derivative~2~*~~.52 and glycosid-3-uloses.53 The specific complexing that occurs between boronic acids and contiguous cis,cis-triols (as in ribopyranosides; see Section V) is postulated as resulting from stabilization of the cyclic esters formed from 1,3-related7axial hydroxyl groups by coordination from the oxygen atom of the central hydroxyl groups. However, although the crystal structure of N-(p-bromopheny1)-a-D-ribopyranosylamine 2,4-phenylboronate (30) indicates that the pyranoid ring is in the expected IC4conformation with the ester oxygen bonds axial, it also reveals that the boron is trigonal and that 0 - 3 is too far from it (305 pm; 3.05 A)* for coordination. The propensity for this oxygen atom to co-ordinate is, however, satisfied by hydrogen bonding with the N-bonded proton of an adjacent molecule.54 The a r a b i n o p y r a n ~ s i d e sand ~ ~ ~lyxopyranosides are esterified, as expected, at the 3,4- and 2,3cis-diols7 respectively5’; in the ribofuranosyl series, esterification occurs at 0-2’,0-3’, and a range of nucleoside phenylboronates is known, mainly through the work of Yurkevich and his associates (see Table VII). In the hexopyranoside series, 4,6-phenylboronates [for example, methyl a-D-glucopyranoside 4,6-phenylboronate (31)l are obtainR. J. Ferrier, D. Prasad,and A. Rudowski, Chern. lnd. (London), 1260-1261 (1964). R. J. Ferrier and D. Prasad,J. Chem. Soc., 7425-7428 (1965). R. J. Ferrier and D. Prasad,J. Chern. Soc., 7429-7432 (1965). B. Lindberg and K. N. Slessor, Curbohydr. Res., 1,492-493 (1966); Actu Chern. S c u d . , 21, 910-914 (1967). (54) H. Shimanouchi, N. Saito, and Y. Sasada, Bull. Chern. SOC.Jpn., 42, 1239-1247 (1969).

(50) (51) (52) (53)

*Calculated by Dr. J. H. Johnston of this Department.

CARBOHYDRATE BORONATES

47

able,3.33a,55 and residual diols may react further, to lead to fully substituted compounds [for example, methyl a-D-glucopyranoside (32)l. Chemical 4,6-phenylboronate 2,3-(diphenyl~yclodiboronate)~*~~

PCHa

p""'

OHOMe

PhBQ

O,

Ph\&PhB

Me

b-B, 31

Ph

32

analyses55 indicated that the galactopyranosides likewise give 4,6esters in preference to the five-membered-ring derivatives that might have been formed from the cis-related 3,4-diols. However, the situation appears to be delicately balanced in compounds containing such 3,4,6-triol groupings, as some undergo reaction to give 4,6-esters (33), whereas others (presumably different in flexibility and timeaveraged conformation) afford mainly the five-membered derivatives (34). Later work, based on mass spectrometry,56 suggested that the esterifications of the methyl galactopyranosides are not so unambiguous as suggested by the chemical methods,55and revealed that 3,4- as well as 4,6-esters are produced. Here, again, it is possible that facile isomerization of the initial products might have occurred during their isolation or subsequent analysis, and, for this reason, the available information should be considered with reservation.

"0

Phl3/OcH,

R'

h.1

Rs

R4 R' H OH H OMe OH H H OMe H H H OMe O H H H H H H H H R2

33

R' H

R2 RS R4 H OMe H

34

(55) R. J. Ferrier, A. J . Hannaford, W. G. Overend, and B. C. Smith, Carbohydr. Res., 1, 38-43 (1965). (56) V. N. Reinhold, F. Wirtz-Peitz, and K. Biemann, Carbohydr. Res., 37, 203-221 (1974).

ROBERT J. FERRIER

48

Methyl a-D-mannopyranoside appears to give mixed monoesters (presumably 2,3- and 4,6-), but reacts readily to afford the 2,3:4,6d i e ~ t e P . ~ (35) ~ * ~with ~ * two ~ ' molar equivalents of acid, whereas, in the 6-deoxyhexoside series, reaction occurs at vicinal, cis-diol sites (2,3 for methyl 6-deoxy-a-~-mannopyranoside,58 and 3,4 for methyl 6-deoxy-a~-galactopyranoside),5Bbut, again, a compound having a cis-related, diol grouping at C-2 and C-4 gives a six-membered, cyclic ester. For methyl 6-deoxy-/3-~-allopyranoside,this is somewhat surprising, in view of the diaxial relationship between the groups at C-1 and C-5 in the product58,60 (36).Methyl 6-deoxy-a-glucopyranosidelikewise gives59 the 2,4-ester (37).

/b

ph\

@

Q o

dB,o

Me

Me

-B' 35

Ph B/O

36

Ph

37

With the 1,6-anhydrohexopyranoses,condensation occurs at vicinal cis-diols or, in the case of 1,6-anhydro-/3-~-glucopyranose, at the diaxial 2,4-~ites,~l despite the observation that this anhydride does not readily complex with the acid (see Section V,2).

Iv. BORONATES IN CHEMICAL REACTIONS 1. Boronates in Aqueous Systems

a. Interaction Between Carbohydrates and Boronic Acids in Aqueous Media.-Initial1 y, Torssel14 detected interaction between phenylboronic acid and D-fructose, and, by potentiometric titration with sodium hydroxide solution, determined a formation constant for a 1:1 complex: 0 OH PhB(OH),

+ D-fructose * (D-fructose)/ \ I

(57) D. S. Robinson, J. Eagles, and R. Self, Carbohydr. Res., 26,204-207 (1973). (58)J. S.Brimacombe, F. Hunedy, and A. Husain, Carbohydr. Res., 10,141-151 (1969). (59)J. S.Brimacombe, A. Husain, F. Hunedy, and M. Stacey,Ado. Chem. Ser., 74,56-69 (1968). (60) J. S. Brimacombe and D. Portsmouth,]. Chem. SOC., C , 499-501 (1966). (61) F. Shafizadeh, G.D. McGinnis, and P. S. Chin, Carbohydr. Res., 18,357-361(1971).

CARBOHYDRATE BORONATES

49

With colleagues,5 he then proceeded to examine this complexing for a range of substituted phenylboronic acids (as part of a study of their influence in certain aspects of plant biochemistry), and found that, as expected, electron-withdrawing groups on the aromatic ring increased the stability of the complexes formed. By examining pH depressions, other workers6 determined formation constants for the phenylboronate complexes formed with various diols and polyhydroxy compounds, and found that, of several sugars examined, D-frUCtOSe formed much the most stable complex. Fourteen years later, S. A. Barker and his colleagues62 conducted a detailed, polarimetric analysis of the complexing undergone between Dglucose, D-mannose, and D-fructose (separately) and phenylboronic acid and its m-nitro and p-methoxy derivatives, respectively, and showed that the favored complexing with the ketose is pH-dependent. Their observations indicated that, with phenylboronic acid, D-glucose is uncomplexed up to pH 6, and fully complexed beyond pH 9, whereas D-fructose begins to form a complex near pH 5 and, in the experiment reported, is -30% complexed before D-glucose begins to react. The ketose is also fully complexed at, and above, pH 9. Similar effects were observed for the substituted acids, the pH ranges within which partial complexing occurs going to lower values with the m-nitrated acid and to higher values with the p-methoxy compound. It was then demonstrated63 that these arylboronic acids displace, in favor of the ketose, the D-glucose-D-mannose-D-fructose pseudoequilibrium that is established in alkaline solution, so that the usual, single-step, conversion efficiency of D-glUCOSe to D-fructose of -30% may be increased to as high as 81%. A detailed investigation of the effects of pH, temperature, and concentration on this phenomenon was undertaken with a view to optimizing a commercial preparation of Dfructose. This displacement may also be effected with polymeric arylboronic acids which, as expected, also show differential binding of the ketose.64 Yurkevich and coworkers,65 using the pH-depression procedure, reported complexing constants of nucleosides and nucleotides, and showed that the depressions are themselves pH-dependent, adenosine exhibiting maximal effects near pH 7.8, 7.3, and 8.2 on mixing with S. A. Barker, A. K. Chopra, B. W. Hatt, and P. J. Somers,Carbohydr. Res., 26,33-40 (1973). S. A. Barker, B. W. Hatt, and P. J. Somers, Carbohydr. Res., 26,41-53 (1973). S. A. Barker, B. W. Hatt, P. J. Somers, and R. R. Woodbury, Carbohydr.Res., 26,5564 (1973). E. A.Ivanova, I. I. Kolodkina, and A. M. Yurkevich,Zh. Obshch. Khim., 41,455-459 (1971).

50

ROBERT J. FERRIER

phenylboronic acid and its p-nitro and p-methyl derivatives, respectively. Here, it may safely be assumed that the 2’,3’-diol is involved in complex-formation; in all of the other work referred to in this Section, no specific evidence is available regarding the structures of the complexes.

b. Hydrolysis of Boronates-There is very little good information available on the stability to hydrolysis of an adequate range of carbohydrate boronates. Some evidence indicates that at least certain esters are stable in aqueous systems: phenylboronates of sugars and alditols may be isolated by crystallization from aqueous m e t h a n ~ l ~ , ~ ~ ; p-tolylboronates of various diols and of 1,2-0-(trichloroethylidene)-aD-glucofuranose can be precipitated by acidification of aqueous alkaline solutions66; and 1,3-O-benzylidene-~-arabinitol gives a phenylboronate that is apparently unchanged on heating in aqueous sodium hydr0xide.6~It has been suggested9 that the first of these points does not establish the stability, but rather the insolubility of the esters in water, and the second point conceivably reflects the insolubility of trigonal esters relative to their tetrahedral, anionic analogs. In the case of the L-arabinitol derivative, after the hydrolysis, the solution was extracted continuously with chloroform for several hours, and it is here suggested that, during this treatment, phenylboronic acid and the benzylidene acetal may have been separately taken into the chloroform, to recondense during the subsequent removal ofthis solvent. The finding that phenylboronic acid could be removed at room temperature by chromatographic separation on an anionic resin or on neutral alumina adds support to the conjecture that hydrolysis had occurred in the alkaline medium. It is, therefore, suggested that none of this evidence establishes the stability of any ofthe esters in aqueous media. All other evidence indicates that carbohydrate boronates readily undergo hydrolysis on addition of water to their solutions in organic solvents. Thus, early in the history of the compounds, it was found that very mild hydrolysis of L-arabinose bis(phenylboronate)7 and Dmannitol tri~(pheny1boronate)~ gave the respective starting-materials, which could readily be separated from each other. In all cases, when water has been added to solutions of boronates in dry solvents, optical rotational changes in magnitude occur in directions consistent with their being caused by hydrolysis. In this way, Hannaf0rd5~468 (66)Brit. Pat. 885,766(1960);Chem. Abstr., 56, 15,546f(1962). (67)A.B.Foster, A. H. Haines, T. D. Inch, M. H. Randall, and J. M. Webber, Carbohydr. Res., 1, 145-155 (1965). (68)A. J. Hannaford, Ph.D. Thesis, University of London, 1964.

CARBOHYDRATE BORONATES

51

calculated the equilibrium constants for the reaction

0 Carbohydrate

/ \ \ / 0

BPh

+ H 2 0 * carbohydrate + PhB(OH),

to be 0.2 k O . 1 for methyl a- and P-D-glucopyranoside and their 2,3-diO-methyl derivatives and for D-glucal. This means that -4% of water caused complete hydrolysis of the esters dissolved (2%)in 1,cdioxane. Similar, semiquantitative work on methyl xyloside phenylboronates revealed much more variable susceptibilities, the percentages of water needed to cause complete hydrolysis ofthe esters (29,38,39,and 40) of OMe

JpQ( 0 \/O

OH 39 R’ = H. R* = OMe 40 R1 = OMe, RZ = H

Ph

38

the a- and p-pyranoside and a- and p-furanoside (1%, in 174-dioxane) therefore, the highly being 1,3,9, and 30, r e s p e c t i ~ e l yAs . ~ expected, ~ strained, pyranoside derivatives are highly susceptible to hydrolysis, whereas the furanoside analogs, particularly the p anomers, are appreciably more stable. Although the trans-relationships of the groups at C-1 and C-2 of the p-furanoside (40) make it thermodynamically the more stable, it does not seem likely that its marked hydrolytic stability can be attributed to this factor alone (see later). Other compounds examined by this type of procedure are the 2’,3’phenylboronates of nu~leosides,~7~28~3~ and the butyl- and phenylboronates of D-xylose and L-arabinose,” and these, also, are hydrolytically unstable. It is here concluded that carbohydrate boronic esters should normally be treated as being readily susceptible to hydrolysis and, presumably, alcoholysis, and that their occasional isolation in crystalline form from aqueous solvents**23.70or ethanol67 should be (69) R. J. Ferrier, D. Prasad, and A. Rudowski,J. Chem. SOC.,858-863 (1965). (70)R. J. Ferrier and L. R. Hatton, Carbohydr. Res., 5, 132-139 (1967).

52

ROBERT J. FERRIER

viewed as evidence of their insolubility and not of their stability in these solvents. Although it might have been expected that such solvents as pyridine could stabilize the esters by co-ordination with the boron atoms, this does not appear to be so44; however, intramolecular co-ordination by suitably oriented oxygen atoms does stabilize them considerably, and it is probably this factor that is responsible for the observed character (see earlier) of methyl /3-D-xylofuranoside 3,5phenylboronate (40), in which 0 - 1 can specifically bond to boron. Although no formal studies of hydrolyses of carbohydrate derivatives stabilized in this way appear to have been reported, their stability as revealed by their behavior in chromatography and electrophoresis is well recognized; this is discussed in Section V,2 and 3. Products of the partial hydrolysis of complex esters have not often been isolated, but the very labile, seven-membered ring of the diphenyldiboronate 32 can be selectively removed, to give methyl aD-ghcopyranoside 4,6-phenylboronate (31)in good yield.3 In their work with alditol derivatives containing ethylboronate and diethylborinate groups, Dahlhoff and K o ~ t e r ~ O found - ~ ~ that the latter may be selectively removed by treatment with either methanol or 2,4pentanedione and, in the case of xylitol 2-diethylborinate 1,3:4,5bi~(ethylboronate),4~ they were able to remove the six-membered ring selectively. This is against expectations (see Section I), and may result from intramolecular stabilization of the smaller ring by co-ordination from 0-2.

2. Removal of Boronate Groups When boronic esters are utilized as protecting groups in the synthesis of specifically substituted, or otherwise modified, carbohydrate derivatives, the cleavage of the esters must be followed by removal of the by-products liberated. With esters of strongly hydrophilic, carbohydrate compounds, the boronic acids can be specifically extracted from aqueous into organic solvents, and, in this way, alditolss and free sugars' have been recovered from their phenylboronates, but a more widely applicable procedure involves exchanging the boronic acids from the carbohydrate to 1,e-ethanedio140,41*71 or, more usually, 1,3-propanediol,22*58~72 with which they condense to give volatile, cyclic derivatives. Addition of 1,3-pro(71)A. A. Amagaeva, A. M. Yurkevich, I. P. Rudakova, L. V. Khristenko, I. M. Kustanovich, and N. A. Preobrazhenskii, Khim. Prfr. Soedfn., 4, 304-307 (1968); Chem. Abstr., 70, 115,471s(1969). (72)L.G.Mogel and A. M. Yurkevich, Zh. Obshch. Khim., 40,708(1970).

CARBOHYDRATE BORONATES

53

panediol to a solution of a carbohydrate boronate in acetone, and removal of the volatile products, usually offers a very efficient method of deboronation. Column separations of the products of hydrolysis of carbohydrate boronates by use of anionic resins provides an alternative, efficient means of deb0ronation,~~,67,69 and other similar separations have used columns of cellulose,48 alumina,67 and, for carbonyl-containing compounds, anion-exchange resins in the hydrogensulfite In special cases, e l e c t r o p h o r e ~ i sand ~ ~ direct crystallizationz3 have been employed. An interesting, alternative procedure, applicable at least to the removal of phenylboronic acid, involves its conversion, by treatment with bromine-water, into bromobenzene and boric acid, and the removal of these by distillation-the latter as its trimethyl ester.43

3. Stability of Boronates during Chemical Reactions a. Esterificatioa-Successful acetylation of unsubstituted hydroxyl groups in carbohydrate boronates has been reported on several occasions. Acetyl chloride in pyridine has usually been used as the acetylating agent, and the products have frequently been distilled prior to crystallization. Acetates of boronates of glycosides,3~22*51*~5~~9 a l d i t o l ~and , ~ ~ nucleosides3" have been reported. Benzoylation is, likewise, usually a satisfactory process, but the products are insufficiently volatile for distillation. Fully substituted aldito1,41,67 and nucleoside36 esters have been prepared, and, sometimes, the boronate groups have been removed without isolation of the (benzoylated) first products. An instance of the use of an insoluble poly(styry1boronic acid) in the preparation of methyl 2,3-di-0-benzoy~-a-D-ghcopyranoside and -galactopyranoside has been reported.72aUsually, benzoyl chloride is the reagent used, but, with this, methyl a-D-xylopyranoside 2,4phenylboronate gave only 37% of the 3-benzoate7 whereas the yield was doubled by use of benzoic anhydride.22 p-Toluenesulfonylation of methyl a-D-glucopyranoside 4,6-phenylboronate did not give an isolable product, although the bis-ptoluenesulfonate could be prepared by boronation of the appropriate Dglucoside derivatives; othersZ3 have reported failure to esterify galactitol bis( phenylboronate). Despite these findings, there is little reason to doubt that p-toluenesulfonylation of boronates can be satisfactorily achieved. Thus, direct products have been obtained from (72a) E. Seymour and J. M. J. Frechet, Tetrahedron Lett., 1149-1152 (1976).

54

ROBERT J . FERRIER

alditol67 and sugar47 boronates having free primary-hydroxyl groups, and secondary p-toluenesulfonates from 1,6-anhydroaldohexose boronates61 (although the D-gdo compound was slow to react); D-ribose 2,4-phenylboronate gave,"6 selectively, the 3-p-toluenesulfonate at -10". Several reports have appeared on the preparation of 5'-pbut toluenesulfonates of nucleoside 2',3'-phenylb0ronates,37,7~*73*74 these still remain poorly characterized derivatives. The preparation of N-phenylcarbamates from incompletely substituted, carbohydrate boronates is, perhaps, the least destructive of esterifying reactions, requiring only that the compounds be heated with phenyl isocyanate in dry benzene or toluene. Several reports have sugar,47and appeared on successful application to aldito123 derivatives. By using the 2',3'-phenylboronate protecting-group, Yurkevich and his colleagues prepared 5'-phosphates of adeno~ine~28.75-77 and g ~ a n o s i n e Usually, .~~ 2-rnorpholino-1,luridine, 27,76 cytidineYz7 diphenylpyrophosphorochloridate has been used as the phosphorylating reagent, but the morpholinophosphorodichloridate and 2-cyanoethyl phosphate-N,N'-dicyclohexylcarbodiimide procedures have also been employed.28 Nucleoside 2',3'-phenylboronates may also be used in the synthesis of dinucleoside phosphates.30 Chloroacetylation can be effected by use of chloroacetic anhydride in pyridine, and, in this way, the 6- and 3-esters of l,%O-isopropylidene-a-D-glucofuranose were prepared, in good yield, by using the 3,5phenylboronate and 5,6-(diphenylcyclodiboronate), respectively, as starting materials, and, from the chloroacetates, trimethylammonioacetyl salts were prepared.24In further work, connected with pangamic was synthesized by use of acid, 6-O-(N,N-dimethylglycyl)-~-glucose N,N-dimethylglycine and N,N'-dicyclohexylcarbodiimide in acetonitrile-pyridine,25 and pangamolactone [6-0-(N,N-dimethylglycyl)-~glucono-1,4-lactone] by a similar procedure from D-glucono-1,4lactone 3,5-phenylboronate.78 (73) A. M. Yurkevich, A. A. Amagaeva, I. P. Rudakova, and N. A. Preobrazhenskii, Zh. Obshch. Khim., 39,434-440 (1969). (74) I. P. Rudakova,T. A. Pospelova, and A. M. Yurkevich,Zh. Obshch. Khim., 40,24932499 (1970). (75) A. M. Yurkevich, I. I. Kolodkina, and N. A. Preobrazhenskii, Dokl. Akad. Nauk S S S R , 164,828-830 (1965);Chem. Abstr., 64,3661g (1966). (76) I. I. Kolodkina, L. S. Varshavskaya,A. M. Yurkevich, and N. A. Preobrazhenskii,Zh. Obshch. Khim., 37,1996-2002 (1967). (77) A. M. Yurkevich, I. I. Kolodkina, G. S. Evdokimova, E. T. Bazhanova, and N. A. Preobrazhenskii, Khim. Org. Soedin. Fosfora, Akad. Nauk SSSR, Otd. Obshch. Tekh. Khim., 215-220 (1967);Chem. Abstr., 69, 10,667m (1968). (78) K. Murase and M. Murakami, Yamanouchi Seiyaku Kenkyu Hokoku, 2, 62-65 (1974); Chem. Abstr., 83, 179,446p (1975).

CARBOHYDRATE BORONATES

55

Methacrylic anhydride in pyridine, applied to 1,6-anhydro-~glucose 2,4-phenylboronate, gave the 3-methacrylate7from which an addition polymer was prepared.79

b. Etherification.-Successful methylation of hydroxyl groups in partially substituted carbohydrate boronates has not often been reported. The first attempt, in which methyl iodide, silver oxide, and a drying agent were applied to methyl a-D-glucopyranoside 4,6phenylboronate, gave,3 after distillation of the product, a poor yield of the diether, chromatographic evidence being obtained that some triether (but no tetraether) was formed during the reaction. Applied .to methyl P-D-xylopyranoside 3,5-phenylboronate in N,N-dimethylformamide, the same reagent gavez2only 18% of the 2-ether7 which could be purified by sublimation. Other workers have reported no success on attempted methylation of galactitol bi~(pheny1boronate)~~ and methyl 6-deoxy-/3-~-allopyranoside2,4-phenylboronate.60 The first, satisfactory methylation appears to have been effected by Bourne and his colleague^,^* who prepared 6-0-methyl-D-glucose in high yield from the 1,2:3,5-bis(phenylboronate),using diazomethane and boron trifluoride etherate in dichloromethane for methylation, and a final separation on a column of cellulose powder. It is this procedure which the same group used for structural analysis of trio1 phenylboronate^^^ (see Section 111,l); others60have attempted to use it, but without success. The phenylboronate group has been shown to be stable under Koenigs-Knorr glycosylation conditions, and, from benzyl p-D-xylopyand 3ranoside 2,4-phenylboronate73-0-~-D-g~ucopyranosy~-D-xy~ose 0 - a - and p-D-xylopyranosyl-D-xylose have been synthesized52by using nitromethane as the solvent. The anomer of the initial boronate was less reactive under these conditions, conceivably because its 3hydroxyl group cannot become hydrogen-bonded intramolecularly and thereby gain enhanced nucleophilicity. Tritylation of the nucleoside 2’,3’-phenylboronate has been used to obtain 5’-O-trityladenosine,’T and trimethylsilyl ethers of glycoside phenyl-, methyl-, and butyl-boronates have been produced by use of chlorotrimethylsilane and trifluorobis(trimethy1silyl)acetamide in pyridine for mass-spectrometric studies56 (see Section VI). c. Nucleophilic Displacement Reactions-Several reports have appeared on the introduction of nucleophilic groups into carbohydrate (79) S . P. Valueva, E. P. Cherneva, V. A. Kargin, and N. M. Merlis, Vysokomol. Soedin. Ser., 13, 468-470 (1971); Chern. Abstr., 75, 152,153~(1971).

56

ROBERT J. FERRIER

derivatives carrying boronic ester groups. In the most straightforward, D-glucose 1,2:3,5-bis(phenylboronate)was separately treated with triphenylphosphine in bromoform and carbon tetrachloride, and the initial products were deboronated, to give 6-bromo- and 6-chloro-6deoxy-D-glucose in 81 and 79% yield, r e ~ p e c t i v e l y .Although ~~ displacements of the sulfonyloxy groups of 1,3-O-benzylidene-5-OO-ptolylsulfonyl-L-arabinitol 2,Cphenylboronate and 6-deoxy-3,4-0-isopropylidene-5-0-p-tolylsulfonyl-~-mannitol 1,2-phenylboronate did not occur so readily when the esters were heated in refluxing N , N dimethylformamide with sodium benzoate and sodium azide, respectively, direct substitution did occur, to give products whose structures were used to establish the sites of bor0nation.~7 Yurkevich and coworkers46 used 3-0-p-tolylsulfonyl-D-ribose 2,4phenylboronate to prepare a cobalt-containing carbohydrate compound which, without reported evidence, they claimed has a C-3cobalt bond and the D-XY~O configuration. Similarly,74 they prepared, from the 5’-p-toluenesulfonates, nucleoside derivatives having C-5’cobalt bonding, and, in related work, they obtained nucleoside derivatives bonded directly to cobalt at C-5’ by use of an intermediate which, on the basis of carbon and hydrogen analytical data, they proposed had structure 41 (R = H). Synthesis of this compound was a~hieved71.8~ by treatment of 5’-O-p-tolylsulfonyladenosine2’,3’phenylboronate with lithium bromide in acetic anhydride at 100’conditions that would be expected81to yield the acetylated analog (41, R = Ac). As such a compound would have carbon and hydrogen compositions similar to those of the monoboronate (41, R = H), the two NHAc

I

b

b

PhBOR, Ac

41

(80) I. P. Rudakova, V. I. Sheichenko, T. A. Pospelova, and A. M. Yurkevich,Zh.Obshch. Khim., 37, 1748-1753 (1967). (81) B. C. Maiti, 0.C. Musgrave, and D. Skoyles,]. Chem. SOC. Chem. Commun., 244245 (1976).

CARBOHYDRATE BORONATES

57

would not be readily distinguishable on this basis. The same 5’-bromo5‘-deoxy intermediate was also used to obtain 5’-thioadenosine derivatives .73

d. Oxidation Reactions.-The phenylboronate group has been found to be stable during acetic anhydride-dimethyl sulfoxide oxidation of hydroxyl groups, and this has made possible53the synthesis of methyl a-D-erythro-pentopyranosid-3-ulose in 50-60% yield from methyl Q-Dxylopyranoside 2,4-phenylboronate (29); the P-glycosidulose was obtained similarly from the p-ester 38 in 80% yield. Oxidations were effected at 40°, and the stability of the esters during the reactions was indicated by polarimetry, as the first product obtained from the boronate 29 is levorotatory, whereas the derived, deboronated ketone is dextrorotatory. Furthermore, the oxidations were highly specific, indicating that the readily oxidized, ketonic products remained protected during the reactions. The oxidized boronates were not, however, isolated, the de-esterified products being obtained by column chromatography. Other worker@ reported the unreactivity of methyl 6-deoxy-P-D-allopyranoside2,4-phenylboronate under these conditions. Oxidation of phenylboronates with the periodate ion has been used in structural analysis,3*22*23,56as, in aqueous 1,4-dioxane7 they are hydrolyzed to release diols that are susceptible to oxidation only when they are vicinally related. Thus, the esters of mOnO-O-aCetyl-D-glUCal and -D-galactal were found to reduce 0.09 and 1.07 molar equivalents of periodate, respectively, and were, therefore, assigned55 the structures 42 and 43. Whereas, in our lab0ratory,3*2~*~5 we have found it unnecessary in these analyses to correct for oxidation of reaction components other than the carbohydrates, other workers23 have reported the use of substantial corrections. $!H,OAc

42

43

v. SEPARATIONS OF CARBOHYDRATES BY USE OF THEIRBORONATES 1. Use of Isolated Boronates As only the cis isomers of 1,2-, 1,3-, and 1,4-cyclohexanediols can form simple, cyclic boronates, whereas the trans compounds give

58

ROBERT J. FERRIER

polymeric diesters, the epimeric diols may be separated by distillation of their boronates, and this procedure has been applied by using butylboronates prepared directly with butylboronic acid,82 and ethylboronates obtained by thermal cyclization of bis(diethy1borinates).17 The anomeric methyl 3,5-O-isopropylidene-~-xylofuranosides are separable by distillation,83 and, therefore, the corresponding 3,5cyclic boronates (for example, 39 and 40) could also afford means of separating the methyl xylofuranosides by this method. This procedure has, apparently, not yet been attempted, but the intramolecular hydrogen-bonding responsible for making a anomers in this series relatively volatile makes them, also, very much more soluble in nonpolar solvents, and this characteristic has been usefully applied to the isolation of the anomeric methyl D - x y l o f u r a n o s i d e ~Their . ~ ~ ~ ~3,5~ phenylboronates prepared from a glycoside mixture rich in furanosides can be fractionally crystallized from light petroleum [solubilities (g/lOO ml): a, 17.3; /3, 0.641, thus providing a means of obtaining the unsubstituted a- and P-glycosides in 21 and 29% yield, respectively. With the methyl D-xylopyranosides, it is the ester (38) of the P anomer that has the intramolecular, strongly hydrogen-bonded hydroxyl group, making it the more soluble anomer [solubilities50 (g/lOO ml in light petroleum): a,0.07; P, 531. By virtue of this great difference, methyl a-D-xylopyranoside 2,4-phenylboronate can readily be separated from its isomers, to provide a convenient means of isolating this glycoside, which is otherwise difficult to obtain.69Methyl a-and /3-D-~ylopyranoside-5-~~0 have both been prepared by application of this procedure,83aand the stereoisomers of cis-3,4-thiolanediol l-oxide have been separated as their p h e n y l b ~ r o n a t e s . ~ ~ ~ 2. Use in Paper Chromatography

It has been shown by two groups of w0rkers84-8~that phenylboronic acid incorporated into paper-chromatographic solvents specifically (82) H. C. Brown and G . Zweifel,]. Org. Chem., 27,4708-4709 (1962). (83) B. R. Baker, R. E. Schaub, J. P. Joseph, and J. H. Williams,]. Am. Chem. Soc., 76, 4044-4045 (1954). (83a) W. D. Hitz, D. C. Wright, P. A. Seib, M. K. Hoffman, and R. M. Caprioli, Carbohydr. Res., 46, 195-200 (1976). (83b) J. E. McCormick and R. S. McElhinney,]. Chem. Soc. Perkin Trans. 1 , 25332540 (1976). (84) H. M. Wall, M.Sc. Thesis, University of London, 1964. (85) R. J. Ferrier, W. G. Overend, G. A. Rafferty, H. M. Wall, and N. R. Williams, Proc. Chem. Soc., 133 (1963). (86) E. J. Bourne, E. M. Lees, and H. Weigel,]. Chrornatogr., 11,253-257 (1963).

CARBOHYDRATE BORONATES

59

TABLEI Enhancement Factors for Paper-chromatographic Mobility of Free Sugars by Phenylboronic Acid

sugar GIycerose Erythrose Threose Ribose Arabinose Xylose Lyxose Allose Altrose Glucose Mannose Gulose Idose Galactose Talose

Enhancement factora 1.05: 2.7b 1.7,b 2.0,b 0.9,b 1.0: 1.0:

1.0'

1.0' 2.0e 0.7' 0.7" 0.8' 1.4" 0.95" 1.0,* 0.75" l . l , b 0.7' 2.l,* 2.1' 1.8: 2.0' 1.3: 1.0" 2.7'

Sugar G1ycerone glycero-Tetrulose erythro- Pentulose threo-Pentulose Fructose Sorbose gluco- Heptulose 2-Deoxy-erythro-pentose 2-Deoxy-ribo- hexose 2-Deoxy-arabino-hexose 2-Deoxy-lyxo-hexose 3-Deoxy-ribo-hexose 3-Deox y-xylo-hexose 4-Deoxy-xylo-hexose 6-Deoxytalose

Enhancement factor" 1.oc 1.0'

1.8= 2.3" 1.1: 0.9' 1.6b 2.2' 0Bc 0.8' 0.8e 1.3c

0.9' 1,4c 0.8''

2.0e

"Ratios of RF values in solvents (ii) and (i).The enhancement factors determined in the butanol-containing solvents are frequently somewhat less than unity, presumably because the solvents differed slightly in their ethanol content (see footnote c). bDescending chromatograms with solvents (i) 9:2:2 ethyl acetate-acetic acid-water, and (ii) the same, but with phenylboronic acid (0.55%) added.86'Descending chromatograms with solvents (i) butanol-ethanol-water (4:1:5, upper phase), and (ii) the same, but with phenylboronic acid (5%) added. This caused slight phase-separation, and so ethanol (2.5%) was then also added.84

enhances the mobilities of compounds that possess trio1 systems from which particularly stable, boronic esters are formed (see Tables I and 11). From work with six-membered-ring compounds, it was concluded that this stabilization arises from contiguous cis&-triols on such rings, which use 1,3-related, axial hydroxyl groups to give six-membered, cyclic esters (44) stabilized to hydrolysis by the intervening, equatorial

44

ROBERT J. FERRIER

60

TABLEI1 Enhancement Factorsa for Paper-chromatographic Mobility of Alditols, Inositols, and Anhydro Compounds by Phenylboronic Acid Enhancement factop

Inositols and anhydro compounds

Enhancement factop

Glycerol Erythritol Ribitol Arabinitol Xylitol Allitol Alhitol

1.1; 0.85' 1.4; 1.6' 3.4," 2.85' 3.6,b3.3c 3.2,b4.2' 2.5' 3.2b

2.7b 4.0b 0.7b 1.06 1.@ LO* 1.0; 0.9'

Glucitol

5.6,b7.8'

Mannitol

5.4," 4.6c

Galactitol

6.7," 5.7'

2-Deoxy-erythropentitol 1-Deoxygalactitol

1.46 2.26

1,6-Dideoxygalactitol

1.5b

d o - Inositol epi-Inositol chiro-Inositol muco-Inositol myo-Inositol scyZZo- Inositol l,6-Anhydro-Paltropyranose 1,6-Anhydro-pglucopyranose 1,6-Anhydro-Pmannopyranose 1,6-Anhydro-Pgulopyranose 1,6-Anhydro-Pgalactopyranose Methyl 3,B-anhydro-aglucopyranoside Methyl 3,6-anhydro-Pglucopyranoside

Alditol

1 . 0 , b 0.9"

1.2: 2.w 1.0; 1.w l.lb

1.2" 1.2'

aFootnotes as for Table I.

hydroxyl group. Of the pentoses, ribose alone has such a triol grouping (P-D-lyxopyranose is excluded, because of its instability in the lC4 conformation) and is specifically affected (see Table I and Fig. 1).In the aldohexose series, allose, gulose, and talose show enhancements, presumably because, in the pyranose modifications, they contain the required cis,cis-trio1 groupings, but mannose does not, and is thus analogous to lyxose. Idose does not possess a cis,cis-trio1 grouping but shows marked boronate enhancement, suggesting that either its boronate is formed at 0-2,O-4 of a pyranose form, and the ester is stabilized by the ring-oxygen atom, or else a furanoid form presents a suitable triol grouping to the reagent. Other than glycerol, the alditols (see Table 11) all form strong complexes, so the technique is usually suitable for separations of free sugars from their reduction products.86 Removal of oxygen atoms from these derivatives, as expected, lessens their association with the acid.

CARBOHYDRATE BORONATES

61

FIG. 1.-Paper Chromatograms of the Aldopentoses (1, Arabinose; 2, Lyxose; 3, Ribose, and 4, Xylose). [Solvent: A, butanol-ethanol-water (4:1:5, upper phase); B, the same, but containing 2% of phenylboronic acid.]

Evidence of complexing was obtained with the anomeric methyl 3,6anhydro-D-glucopyranosides(see Table II), which suggests that the oxygen atoms on the pyranoid ring can play a role in ester stabilization (45); but, with 1,6-anhydro-/3-~-glucopyranose (46), no complex was

45

62

ROBERT J. FERRIER

formed, because of the splayed relationship of its carbon-oxygen bonds at C-2 and C-4. The 0-2-0-4 distances in these compounds87 of 276 and 330 pm (2.76 and 3.30 A) are in agreement with these observations, but it should be noted that the 1,6-anhydride can be converted into an unstable 2,4-phenylb0ronate.~l The complexes formed by the methyl 3,6-anhydroglucosides and 1,6anhydro-P-mannopyranose (see Table 11) establish that cyclic systems other than that depicted in formula 44 may give rise to mobility enhancements, but, nevertheless, this chromatographic technique can be used in certain cases to detect cis&-triols and, thereby, in configurational analysis, The configurations of 2-C-methyl-~-arabinose and -L-ribose have thus been ascertained by determination of the mobilities of the free sugars and their methyl P-pyranosides. The epimer and the glycoside that showed enhanced mobilities in solvents containing phenylboronic acid were assigned the ribo structure.88 3. Use in Electrophoresis

Sulfonylated phenylboronic acid (mainly the ortho isomer) shows selective, complexing abilities with carbohydrates, and may thus be used in electrophoresis to permit separations of alkali-sensitive compounds at neutral pH values .89 Complexing occurs with all alditols other than glycerol, that is, they migrate on the electrophoretograms, and the relative degrees of complexing are related to the enhancement values found during chromatography with phenylboronic acid (see Tables I and 11). (For example, relative mobilities are: ribitol, 0.3; arabinitol, 0.6; xylitol, 0.9; glucitol, 1.3; mannitol, 1.0; and galactitol, 1.0). Observations with free sugars indicate, however, that the two procedures are basically different, because all aldopentoses, for example, migrate on the electrophoretograms (mobilities relative to glucose as unity: ribose, 4.7; arabinose, 2.4; xylose, 1.8; and lyxose, 2.3), whereas only ribose shows chromatographic enhancement. Likewise, fructose showed no complexing during chromatography, but underwent an extremely strong interaction with sufonylated phenylboronic acid. With the information available at present, the two methods cannot be accurately compared; both, however, appear to be selective for cis&-trio1 groupings on six-membered rings, and the (87) Borje Lindberg, Bengt Lindberg, and S. Svensson,Actu Chem. Scand., 27,373-374 (1973). (88) R. J. Fernier, W. G. Overend, G.A. Rafferty, H. M. Wal1,andN. R. Williams,J. Chem. SOC.,C, 1091-1095 (1968). (89) P. J. Garegg and B. Lindberg, Actu Chem. Scund., 15, 1913-1922 (1961).

63

CARBOHYDRATE BORONATES

electrophoresis results also reveal interaction with cis-1,2-diol groupings in furanoid systems.

4. Use in Column Chromatography From the known, differential complexing between boronic acids and polyhydroxy compounds, it follows that carbohydrate mixtures may be separated b y column-chromatographic methods that exploit the differences. Nucleoside and nucleotide boronates have been separated on columns of anion-exchange resinsFOand sugars and alditols have been shown to be differentially retained on such resins in the sulfonated phenylboronic acid form,64but perhaps the best uses of column chromatography in this connection have incorporated the resolving powers of insoluble polymers to which boronic acid groups have been covalently bonded. Such insoluble forms of boronates have been synthesized either by substitution of polysaccharide derivatives, or by polymerization of suitable arylboronic acids. In the first approach, O-(carboxymethy1)cellulose (47) has been converted into the acyl azide (48) and thence, by treatment with maminophenylboronic acid, into the borylated form 49, to give a polymer

47

48

49

on columns of which the alditols had the relative retention-times: erythritol, 51; ribitol, 56; arabinitol, 76; xylitol, 93; glucitol, 182; mannitol, 109; and galactitol, 111(eluted with acetate buffer, pH 7.5).91 These values indicate that the complexing that results in enhancements of paper-chromatographic mobilities of alditols when phenylboronic acid is added to solvents (see Table 11) operates in a closely similar fashion, to determine their retention by the borylated cellulose. In the same work, it was showng1that nucleosides could be separated on this stationary phase, and that they have retention volumes that depend on the pH and ionic strengths of the buffers used, and on the heterocyclic bases, but, especially, on the presence ofthe 2’,3’-diols. At high pH-values, binding of the purine ribonucleosides was so strong (90) I. I. Kolodkina, E. A. Ivanova, and A. M. Yurkevich, Khim. Prir. Soedin., 6,612-616 (1970); Chem. Abstr., 74, 112,363e (1971). (91) H. L. Weith, J. L. Wiebers, and P. T. Gilham, Biochemistry, 9,4396-4401 (1970).

64

ROBERT J. FERRIER

that they could not be eluted. Yurkevich and coworkers prepared similar column-materials (50)by treatment of 0-(2-diethylaminoethyl)-

so

Sephadex and O-(2-diethylaminoethyl)cellulose with a-bromotolylboronic acid, and studied the separation of free sugarse2~93and nucleosides and nucleotidess3~94on them at various pH values and with different eluants. Compounds containing cis-172-diol groupings were, again, the most strongly bound. Solms and Deuelg5initially prepared a wholly synthetic, borylated polymer by using m-phenylenediamine, p-aminophenylboron dichloride, and formaldehyde, and they investigated carbohydrate separations on it, but addition polymers have usually been favored. Thus, ribonucleosides and deoxyribonucleosides have been efficiently separated on a column of a mixed copolymer of the methacroyl derivativesee 51 and 52, and the method has been extended to

oligonucleotides, including free transfer-ribonucleic acids (t-RNA). The strong complexing that again occurs with compounds containing 2’,3’cis-diol groupings makes it possible to retain ribonucleoside 5’phosphates, 3‘-ribonucleoside-terminatedoligo(deoxynucleotides), and t-RNA’s on the columns, while deoxynucleotides and their oligomers, ribonucleoside 2’-or 3’-phosphates and aminoacyl t-RNA’s (92) E. A. Ivanova, S. I. Panchenko, I. I. Kolodkina, and A. M. Yurkevich, Zh. Obshch. Khim., 45,208-212 (1975). (93) A. M. Yurkevich, I. I. Kolodkina, E. A. Ivanova, and E. I. Pichuzhkina, Carbohydr. Res., 43, 215-224 (1975). (94)E. A. Ivanova, I. I. Kolodkina, and A. M. Yurkevich,Zh.Obshch. Khim., 44,430-434 ( 1974). (95) J. Solms and H. Deuel, Chimia, 11,311 (1957). (96) H. Schott, Angew. Chem. Int. Ed. Engl., 11,824-825 (1972).

CARBOHYDRATE BORONATES

65

are eluted, and this represents97 a significant development in techniques available for the purification of t-RNA’s. Similar polymers have been used by S . A. Barker a n d his colleague^.^^ Radical-induced copolymerization of iminodiethyl (4vinylphenyl)boronate, divinylbenzene, and ethylvinylbenzene gave a polymer on which free sugars can be differentially eluted with distilled water. It is noteworthy that ribose was specifically bound by the polymer (compare, Table I). Separations-particularly those of Dglucose and D-fructose-were shown to be temperature- and pHdependent. 5. Use in Gas-Liquid Chromatography

Separations were first achieved with butylboronates of sugars and alditols, many of which were shown to be separable at 200”. In the earliest report,f’8 it was recognized that products obtained by direct esterification of some sugars in pyridine were chromatographically homogeneous, whereas others were not-glucose giving, for example, three resolvable products. A concurrent study,99 however, showed that glucose gave a single ester if the butylboronation was allowed to proceed to equilibrium, and, similarly, lyxose, fucose, arabinose, xylose, fructose, galactose, and mannose gave essentially homogeneous products (eluted in that order); ribose, rhamnose, erythritol, arabinitol, xylitol, and glucitol did not give single esters. This workggdiffered from that reported in the first paper98 by trimethylsilylation of the unsubstituted hydroxyl groups of the esters prior to g.1.c. examination. A fuller report,loOwhich included mass-spectral results on the main products obtained from D-glUCOSe, D-mannose, D-galactose, and D-fructose showed them to be bis(buty1boronate) trimethylsilyl ethers. Similar substitutions, involving methylboronation followed by trimethylsilylation, have been performed in a g.1.c.-m.s. investigationS8 of the structures of esters of several glycosides and sugars (see Section VI). VI. MASSSPECTROMETRY OF BORONATES

Although, in the mass spectrometer, several fragmentationpathways are followed by carbohydrate boronates, those which result (97) H. Schott, E . Rudloff, P. Schmidt, R. Roychoudhury, and H. Kossel, Biochemistry, 12,932-938 (1973). (98) F. Eisenberg, Carbohydr. Res., 19, 135-138 (1971). (99) P. J. Wood and I. R. Siddiqui, Carbohydr. Res., 19,283-286 (1971). (100) P. J. Wood, I. R. Siddiqui, and J. Weisz, Carbohydr. Res., 42, 1-13 (1975).

ROBERT J. FERRIER

66

in the formation ofthe cyclic, boron-containing ions shown in Scheme 3

‘p):’

‘PR

Bu

Ph

84

126

146

98

140

160

’ 0 ‘

Jb

R’

Ion masses

R = Me

i t k 3 R (R‘=H)

Scheme 3

are of the greatest value in structural analysis. The formation, structures, and masses of such ions formed from five- and six-membered methyl-, butyl-, and phenyl-boronates are shown, together with those of the ions derived from analogous a-diol cyclodiboronates. In the case of the six-membered species, loss of hydrogen atoms can readily occur, to give intense, conjugated ions of one mass unit less than those given in Scheme 3. Boronates of vicinal diols are, therefore, recognizable by the ions derived by direct excision of the two carbon atoms involved, together with their ester substituents, whereas boronates of 173-related diols give ions having a neighboring group (R’) attached. In terminal systems of this type (derived, for example, from 4,6-diols of aldopyranosides), ions having R’ = H are produced, and are, therefore, readily recognized by having the mle values given in Scheme 3. The bis(pheny1boronates) of the tetritols and pentitols produced by the dehydration procedure all gave ions with mle 159 and 160, and were therefore assigned 173:2,4-bicylostructures; for the D-arabinitol derivative, however, the 2,4:3,5-isomeric structure cannot be excluded.101On the other hand, the bis(boronate) obtained from xylitol per(diethy1borinate) was shown in this way to have the 1,2:3,5-structure.40 With the bis(pheny1boronates) of D-glucitol, galactitol, and D-mannitol, the first two were each determined to contain two-fused, six-membered (101) I. R. McKinley and H. Weige1,J. Chem. Soc. Chem. Commun., 1051-1052 (1972).

CARBOHYDRATE BORONATES

67

ring-systems (and a five-membered ring), whereas the third gave no ions indicative of a similar structure.101 Applied on a micro scale, and without isolation of the esters, to products derived from mixtures of phenylboronic acid and methyl a-D-glucopyranoside, methyl a-D-galactopyranoside, and methyl a-Dmannopyranoside, the procedure indicated that the D-ghcoside and D-galactoside derivatives contained 4,6-ester rings (mle 160) and 2,3-(diphenylcyclodiboronate)systems (mle 250), whereas the Dmannoside gave the 2,3:4,6-bis(phenylboronate)structure (mle 160; mle 146; no rnle 250).57 Molecular-ion masses confirmed these conclusions, which are in agreement with the results ofchemical analysis (see Section 111). Analogous results were obtained from the eight methyl amino-4,6-O-benzylidenedeoxy-a-~-hexopyranosides~~~; when the hydroxyl and amino groups at C-2 (C-3) and C-3 (C-2) were cis-related, 2-phenyl-1,3,2-oxazaborolane(53) rings were formed, whereas 2,4diphenyl-1,3,5,2,4-dioxazadiborepaneproducts (54) were obtained from the trans-related glycoside derivatives (compare Section I),so the method provides a means for determining the relative configurations of a-diols and vicinal amino-alcohol groups on cyclic structures.

53

54

For free sugars, the procedure has been applied with isolated est e r ~ ? and ~ * with ~ ~ samples prepared on a micro-scale and purified by g.l.c.563esL-Arabinose gave a bis(buty1boronate) and a bis(pheny1boronate) displaying ions mle 126 and 146, respectively, indicative of their containing five-membered ester groups.44 No ions of mle 140 or 160 were detected (indicating the absence of six-membered rings), and thus, 1,2:3,4-structures were assigned. On the other hand, D-XylOSe bis(buty1boronate) and bis(pheny1boronate) gave ions having rnle 126 and 146 respectively, and also others having mle 140 (139) and 160 (159), respectively, indicating their 1,2:3,5-furanoid structures. Care has to be taken, however, with analytical work of this type, as is illustrated by the spectra given by the bis(methy1boronates) of these two sugars?e which are distinguishable mainly on the basis of ion inten(102) I. R. McKinley, H. Weigel, C. B. Barlow, and R. D. Guthrie, Carbohydr. Res., 32, 187-193 (1974).

68

ROBERT J . FERRIER

sities. All of the structurally significant fragment-ions appear in both spectra. Biemann and coworkers56 extended the g.1.c.-mass spectrometric analysis of boronates by studying the trimethylsilyl ethers of compounds which, after methylboronation, still retain unsubstituted hydroxyl groups. On such treatment, D-fUCOSe gives a product showing a molecular ion mle 212, establishing that it contains two boronic ester and no trimethylsilyl groups, whereas the isomer L-rhamnose is converted into a derivative having one cyclic ester group and two ether substituents (mle 332). The esterification process therefore occurred at one diol site only. In this way, the xylose and arabinose results (see the preceding) were confirmed, and the diesters respectively formed from D-ghcose, D-mannose, and D-galaCtOSe were shown to have the 1,2:3,5, 2,3:4,6, and 1,2:3,4 structures, respectively. Similar work was performed with several methyl aldopyranosides and, with an excess of methylboronic acid, methyl a-D-mannopyranoside gave the 2,3:4,6diester, as expected, and, with limiting amounts of the reagent, a mixture of the 2,3- and 4,g-esters (compare Section 111,3).Methyl a - ~ galactopyranoside was shown to be esterified to give both the 3,4- and 4,6-cyclic esters, in contrast to the conclusion drawn from chemical evidence (see Section 111,3). One feature of this analysis that merits comment is the failure to detect evidence for trioxadiborepane ringstructures with such compounds as methyl a-D-glucopyranoside, despite the use of a 20-fold molar excess of the reagent. A study of the 3-0-a-D-glucopyranosyl-, 3-0-P-D-glucopyranosyl-, 3-0-a-D-galactopyranosyl-, 3-0-~-D-ga~actopyranosy~-, and 3 - 0 - m ~ mannopyranosyl-L-glycerols as phenylboronates, prepared in submilligram quantities, illustrates ( a ) the value of the method in structural analysis, and ( b ) the diversity of the fragmentation reactions that can occur.1o3In Scheme 4, the full degradation pattern of the derivative of 3-0-a-D-glucopyranosy~-L-glycero~ is illustrated; all except the mannosy1 isomer gave the same ions, but having different intensities. Initial work with nucleoside 2’,3’-phenylboronates and their 5’trimethylsilyl ethers was conducted by Dolhun and Wiebers,l04 who identified the main fragmentation-pathways as including rupture of the C-l‘-N and C-4’-C-5’ bonds. They then showed29 that phenylboronated and then trimethylsilylated dinucleotides undergo fragmentation in a way that identifies their sequence, and the method has potential for characterizing the 3’-terminal base of oligo(ribonucleotides). (103) S. G . Batrakov, E. F. Il’ina, B. V. Rozynov, V. L. Sadovskaya,and L. D. Bergel’son, Izu. Akad. Nauk S S S R , Ser. Khim., 821-828 (1975). (104) J. J. Dolhun and J. L. Wiebers, Org. Mass Spectrom., 3,669-681 (1970).

CARBOHYDRATE BORONATES

69 OH

pq 146

159

353

I P

h

t q

160

phB%+-

O?

4 51

250

Ph 306

phQ 410

459

1

Ph ss5

1 Ph

Ph 141

Ph 305

438

409

334

Scheme 4

Carbohydrate phosphates have been examined as the methyl- and butyl-boronates of their dimethyl esters.105 Procedures involved methylation of the enzymically prepared phosphates, and conversion into the dimethyl esters with diazomethane, followed by the usual boronation. Fragmentation patterns were identified for the following: 6-(dimethyl phosphate), a-D-glucofuranose 172:3,5-bis(butylboronate) a-D-galaCtOpyranOSe 1,2:3,4-bis(butylboronate) 6-(dimethyl phosphate), P-D-fructopyranose 2,3:4,5-bis(butylboronate)1-(dimethyl phosphate), and methyl D-gluconate 2,3:4,5-bis(butylboronate) 6-(dimethyl phosphate). In the case of the D-mannose &(dimethyl phosphate), the bis(buty1boronate) obtained was assigned the 1,2:3,5furanoid structure, which contrasts with the 2,3:4,6-structure of boronates derived from the unsubstituted ~ u g a r . ~ 6 With the boronate phosphates,l05 provided that stereochemical factors permit, the phosphate groups stabilize species formed by loss of (105) J. Wiecko and W. R. Sherman, Org. Mass Spectrom., 10, 1007-1020 (1975).

70

ROBERT J. FERRIER

the radicals attached to the boron atoms. Similarly, in acetates of sugar boronic ester~,105~ the acetyl groups stabilize ions produced from neighboring boronate rings, and this behavior can be used, for example, to distinguish between a-D-glucofuranose 1,2-butylboronate 3,smethylboronate and the isomeric 3,5-butylboronate 1,2-methylboronate; an acetyl group on 0-6 interacts only with the boron of the adjacent 3,5-ester. VII. NUCLEARMAGNETIC RESONANCE SPECTROSCOPY OF BORONATEs Several proton-, W - , and l'B-n.m.r. studies have been reported, and all provide means for elucidating the structures of boronates. Results from p.m.r. work26.32,33.44.45.108 were interpreted in the conventional way, and gave information on the molecular structure and the conformation of the carbohydrate portions of the molecules. The 13C shifts of the ortho-aromatic carbon atoms of phenylboronates may provide a means of determining the ester ring-sizes,32 but, on the basis of studies of five compounds, Yurkevich and his ~olleagues4~ pointed out that all three aromatic-carbon resonances (the signal from C-1 is not observed, because of relaxation caused by bonding to boron) occur at lower fields (0.5-1 p.p.m.) in the case of five-membered boronates. Following the work of Cragg and Lockhart,l07 they also demonstrated that broad-line, "B-n.m.r. spectra allow distinction between five- and six-membered, cyclic phenylboronates.26 The former give resonances 4 p.p.m. upfield of those of the latter, and similar findings have been reported for e t h y l b o r o n a t e ~ . ~This ~*3~ observation has assisted in the structural analysis of cyclic ethylboronates of ~ y l i t o lD-mannitol>l ,~~ and galactit01.4~ VIII. BOFUNATES The reaction undergone by alcohols with trialkyl- and triarylboranes in the presence of pivalic acid, to give borinic esters, and the thermal cyclization of bis(dialky1borinates) to boronates, are discussed briefly in Section 11. Many borinates have been prepared in quantitative yield from mono-, di-, oligo-, and poly-sa~charides,3~~J~* and mixed (105a) J. Wiecko and W. R. Sherman,J. Am. Chem. Soc., 98,7631-7637 (1976). (106) A. B. Foster, R. Hems, and L. D. Hall, Can. J. Chem., 48,3937-3945 (1970). (107) R. H. Cragg and J. C. Lockhart,J. Inorg. Nucl. Chem., 31,2282-2284 (1969). (108) R. Koster, K.-L. Amen, and W. V. Dahlhoff,Justus Liebigs Ann. Chem., 752-788 (1975).

CARBOHYDRATE BORONATES

71

borinic-boronic esters have been reported.17*33a*40-42 Treatment of all of these compounds with methanol or 2,4-pentanedione causes deesterification, the borinic esters of mixed compounds being selectively removable, to provide a means, for example, of preparing D-mannitol 3,4-ethylboronate41 and galactitol 2,3:4,5-bi~(ethylboronate)~ (see also, Ref. 33a). At pH 10, diphenylborinic acid gives a tetrahedral anion that complexes with various diol systems, and thus it can be used in electrophoresis like borate.lo9 I n a more detailed study of such complexing,llO diols were examined by 13C-n.m.r. spectroscopy, before and after addition of sodium diphenylborinate, and complexes were detected, and their spectra observed, for a variety of carbohydrate derivatives. 1,2-Diol groupings in acyclic and cis-cyclic compounds, lY3-relateddiols at C-4,C-6 of hexopyranosides, the 3,s-diols of glucofuranoses, and 2,4-diols of the anomeric methyl 3,6-anhydro-~glucopyranosides were all found to react. No interaction occurred with 1,6-anhydro-/3-~-glucopyranose (compare Section V,2).

IX. TABLES The following Tables record some physical properties of boronates of sugars, glycosides, C- and N-glycosyl compounds including nucleosides, alditols, and anhydro sugars,

(109) P.J. Garegg and K. Lindstrom, Acta Chem. Scand., 25, 1559-1566 (1971). (110) P.A. J. Gorin and M. Mazurek, C a n . J .Chem., 51,3277-3286 (1973).

TABLEI11 Boronates of Sugars Melting point sugar

Arabinopyranose, 8-L1,2-0-isopropylidene3,4-O-isopropylidene2-Deoxy-~erythro-pentose 1-0-p-tolylsulfonylFructopyranose, 8-D14-benzoylGalactopyranose, a - ~ 1,2-0-isopropylideneGalactopyranose, a - ~ 6-deoxyGlucohranose, Q-D6-0-benzoyl1,24-isopropylidene3-deoxy-3-fluoro6-0-(N-phenylcarbamoyl)6-0-p- tolylsulfonyl6-0-(N-phenylcarbamoy1)6-0-p- tolylsulfonyl1,2-0-(trichloroethylidene)-

Boronate

(“C)

-

[QI, (degrees)

+19

Rotation solvent

1,2:3,4bis(butyl1,2:3,4bis(phenyl3,4phenyll,&phenyl3,4-phenyl3,Pphenyl2,3:4,5-bis(phenyl2,3:4,5-bis(phenyl-

166 130-131 80-82 146147 117-1 18 89-98 139-142

-25.4 +77.4 - 17 -26

3,4-phenyl-

143-145

-

-

+24 +29 +22 +81 + 14

CHCl, C,H,

-2

CHCl,

+24.4

CHCl,

+42.4

CHCl, -

1,2:3,4-bis(butyl1,2:3,4-bis(phenyl1,2:3,5bis(phenyl1,2:3,5-bis(phenyl3,s-phenyl5,6-(diphenylcyclodi5,gphenyl3,Sphenyl3,Sphenyl1,2:3,5-bis(phenyl1,2:3,5bis(phenyl3,5-p-tolyl-

108.5-109.5 161-162 142-144 116-1 17 4648 115-116 6669 93-94 78-80 120-122 95

+8.5

-

-

-

CHCI3 CsH6

References

c6b

44 7.44 33 33 26 46 32 32

-

33

-

CHC13 CHCl, CsHa

c 6 b

CsHs CHCI,

-

-

32 7,32 23,25,32,48 32 24,32,33 24 49 47 47 47 47 66

P

sm

s 4

?

m P

z!

!L

Lyxose, D Mannofuranose, a-D 2,3OisopropylideneMannose, L6-deoxyRibofuranose, 8-D Ribopyranose, a-D3-09- tolylsulfonylRibose, D Xylofuranose, a-D1,20isopropylidene3,5-di-O-methylXylose, D, dibutyl acetal diethyl a c e d ethylene acetal di-isobutyl acetal dimethyl acetal dipropyl acetal

7

bis(pheny1-

109-110

-60.4

5,Gphenyl-

171-173

-

bis(pheny11,5:2,3-bis(phenyl2,4phenyl2,4-phenylbis(pheny11,2:3,5-bis(butyl1,2:3,5-bis (phenyl3,Sphenyl1,Bphenyl-

107.5 124-126 90-92 57-58 140-142 142-142.5 126-127 54-55

+87 +82.8 -44.1 -6.9 +116 +34 -10 - 14 -9

CHCll C6Kl 1,Cdioxane 1P-dioxane

7,44 32,33,69 69

bis(pheny1bis(pheny1bis(pheny1bis(pheny1bis(pheny1bis(pheny1-

114-115 150-151 180-181 130-132 168-169 135-136

+25 +28 +1 +23 +30 +22

l,.l-dioxane 1,4dioxane 1,4-dioxane 1,Pdioxane 1,Pdioxane 1,4-dioxane

70 70 70 70 70 70

-

C&

CiHs CHCl, CHCI, CHCl, c6&

33 7 26 26 46 7

44

TABLEIV

2

Boronates of Glycosides and C-and N-Glycosyl Compounds Glycoside or glycosyl compound Allopyranoside, methyl p-D 6deoxy3-O-(N-phenylcarbamoyl)Arabinopyranoside, methyl p-L2 0benzoylGalactopyranoside, methyl a-D 2,3-di-O-acetyl6-deoxyGalactopyranoside, methyl p-D 2,Sdi-O-acetyl2,3-di-O- benzoylGlucopyranoside, benzyl p-D 2,3,6-tri-O-acety1-4-0(2,3-di-O-acetyl-p-~ glucopyranosy1)Glucopyranoside, methyl a-D

2,3-di-O-acetyl2,3-di-O-benzoyl6-deoxy2,3-di-O-methyl2,3-di-0-p- tolylsulfonyl-

Melting point Boronate

(“C)

[ffID

(degrees)

2,4phenyl2,Pphenyl3,4-phenyl3,4-ethyl3,4-phenyl4,6-phenyl4,Gphenyl3,4phenyl4,6-phenyl4,gphenyl4,Gphenyl4,Cphenyl-

145-146 154-155 73-74

176-177 145 161-162 177- 178

-76 -74 +117 + 27 +184 +147 +232 -28 +75 +125 -91

4‘,6’-phenyl4,6-p-chlorophenyl3,4-ethyl4,6-m-nitrophenyl4,g-phenyl4,6-phenyl 2,3(diphenylcyclodi4,6-phenyl4,6-phenyl2,4-phenyl4,6-phenyl4,g-phenyl-

199-200 164-165 46-47 168-169 166-167 162-163 116-117 203-204

-

119-120 166-167

-

-

120-122 180-181

Rotation solvent

References

1,4-dioxane 1,Cdioxane 1,4-dioxane 1,4-dioxane

58,60 58 51 33a 51 55 55 59 55 55 55 3

-58.9 +59 + 80 +49 +59

CHC& 1,4-dioxane 1,4-dioxane 1P-dioxane 1,Pdioxane

111 3 33a 3 3

-31 +74 +94 +61 - 15

1P-dioxane 1,4-&oxane 1,Pdioxane 1,Cdioxane 1,4dioxane

3 3 3 59 3 3

GH, 1,Pdioxane 1,4-dioxane MezSO 1,4-dioxane 1,4-dioxane I,4-dioxane

-

Glucopyranoside, methyl p-D2,3-di-O-acetyl2,3-di-O-benzoyl Hex-2-enopyranoside,p-nitrophenyl 2,3-dideoxy-a-~erythroHexopyranoside, methyl 2-deoxy-a-~arabinoHexopyranoside, methyl 2-deoxy-a-~-lyxo6-0-acetylHexopyranoside, methyl 2-deoxy-P-~-lyxo3-0-acetylLyxopyranoside, methyl a-D 4-0-acetylMannopyranoside, methyl a-DMannopyranoside, methyl a - ~ 6-deoxy4-0-(N-phenylcarbamoyl)Psicofuranoside, methyl p-D l-chloro-l-deoxyRibofuranoside, methyl p-D Benzene 2,4,6-trimethoxy-l-p--~ ribofuranosylRibopyranoside, methyl p-D3-0-acetyl3-0-(N-phenylcarbamoyl)-

4,6-phenyl4,6-phenyl-2,3(diphenylcvclodi4,g-phenyl4,Gphenyl-

188-189

-82

1,4-dioxane

3,55

185- 186 123-124 123-124

-127 -99 -2.6

l,4-dioxane 1,4-dioxane 1,Pdioxane

55 55

4,6-phenyl-

180-181

+265

1,4-dioxane

3

4,g-phenyl-

142-143

+63

1,4-dioxane

3

3,4phenyl3,4-phenyl-

159 132-133

+114 +39

1,Cdioxane 1,4-dioxane

55 55

4,g-phenyl4,g-phenyl2,3-phenyl2,3-phenyl2,3:4,Gbis(phenyl2,3:4,6-bis(ethyl-

188 131 66-69 116-117 -

-71 -87 +36 +13 - 118 - 39

1,Pdioxane 1,4-dioxane 1P-dioxane 1,4-dioxane 1,Pdioxane CCl,

55 55 51 51

2,3-phenyl2,3-phenyl3,4-phenyl3,4-phenyl2,3-phenyl-

184-185 123-124 95-96 87.5

-34 + 13 - 136 -111.8 -64.6

1,4-dioxane 1,4-dioxane C& -

-

58 58 112 113 113

2,3-phenyl2,4-phenyl2,4-phenyl2,4-phenyl-

136-137 149-150 82-83 163-164.5

-113 -118 -94

1,4-dioxane 1,4-dioxane 1,4-dioxane

114 51 51 51

55

3 33a

(Continued)

8

4

TABLEIV (Confinued) Glycoside or glycosyl compound Ribopyranosylamine,N-(pbromopheny1)-a-DXylofuranoside, ethyl 1-thio-a-D Xylofuranoside, ethyl 6-Dl-thioXylofuranoside, methyl a - ~ 2-0-(N-phenylcarbamoyl)Xylofuranoside, methyl p-D20acetylXylopyranoside, benzyl a - ~ Xylopyranoside, benzyl p-D 3-0- (tetra-0-acety l-p-D glucopyranosy1)Xylopyranoside, ethyl CY-D l-thioXylopyranoside, ethyl l-thioP-D-

Xylopyranoside, methyl a-D 3-0-acetyl3-0-benzo ylXylopyranoside, methyl p-D3-0-acetyl3-0-benzoyl3-0-methyl3-0-(N-phenylcarbamoyl)-

Melting point Borunate

("C)

2,4-phenyl-

-

3,Sphenyl3,Sphenyl3,5-phenyl3,Sphenyl3,5-phenyl3,Sphenyl3,5-phenyl2,4-phenyl2,Pphenyl-

102-104 157-158 83-84 215-216 122-123 99-100 152-153 77-78

2,4phenyl2,4-phenyl2,4phenyl-

151-152 137-138 143-145

2,4-phenyl2,4-phenyl2,4-phenyl2,Pphenyl2,Pphenyl2,4-ethyl2,Pphenyl2,4-phenyl2,4phenyl2,4-phenyl-

109-110 175- 176 119-121 138-140 85-86 122-123 99-100 82-84 146-147

110-111

0)

1.13. (degrees)

Rotation solvent

References

-

-

54

+27 - 146 -254 +21 +97 - 158 -88 -4 - 144 -97 +11 +79.5 -233

+ 10 + 13 + 18

- 104 - 113 - 127 -82 -114 -90

1,4-dioxane 1,Pdioxane 1,Pdioxane 1,4-dioxane 1,4-dioxane 1,4dioxane 1P-dioxane 1,Pdioxane 1,4-dioxane

115 116 116 50,69 69 50,69 69 52 52

1,cdioxane 1,4dioxane 1,4-dioxane

52 116 115

1,4-dioxane 1,4dioxane 1,4-dioxane 1,4-dioxan e 1,Cdioxane CCl, 1P-dioxane 1,4-dioxane 1,Pdioxane 1,cdioxane

115 22,50 22 22 22,50 33a 22 22 22 22

5

TABLEV Boronates of Alditols Melting point Boronate

Alditol L-Arabinitol 5-0-benzoyl1,3-O-benzylidene5-0-ptolylsulfonylErythritol 1,3-O-butylideneDErythritol l-deoxyGalactitol

bis(pheny12,4-phenyl2,4-phenyl2,4-phenylbis(phenyl2,4-phenyl-

2,5-di43-(N-phenylcarbamoyl)-

DGlucitol 2,4di-0- benzoyl2,402-butenylidene2 , 4 0 butylidene1,3:2,4-dia-ethylidene2,4-O-furylidene-

phenyl2,3:4,5-bis(ethyl1,6:2,3:4,5tris(ethyl1,3:4,6-bis(phenyltris(pheny1bis(pheny1tris(pheny11,3:5,&bis(phenyl1,3:5,6bis(phenyl1,3:5,6bis(phenyl5,6phenyl1,3:5,6-bis(phenyl-

(“C) 114-1 16 141-142 120-121 141-142 88 60.5-62

DI.[ (degrees)

-

+12 +ll + 16 +28

Rotation solvent

-

23

CHCl, CHCl, CHC13

67 67 67 23 117

-

CHCl,

75-76 97-98

-

125-130 162-163 223-224 187-190 199 129-131 82-84.5 88 177- 178

+39.6 -

+18.6 -6.1 -

+40.5

References

CHCI, CHC&

-

CHCl,

~~~

(111) H. Kuzuhara and S. Emoto, Agric. Biol. Chem., 30,122-125 (1966);Chem. Abstr., 64, 19,736h (1966). (112) H. Hiehnhecky and J. FarkaS, Collect. Czech. Chem. Commun., 39, 1093-1106 (1974). (113) J. Farkas’, Collect. Czech. C h e p . Commun., 31, 1535-1543 (1966). (114) L. Kalvoda, J. Farkag, and F. Sorm, Tetrahedron Lett., 2297-2300 (1970). (115) R. J. Femer, L. R. Hatton, and W. G. Overend, Carbohydr. Res., 6,87-96 (1968). (116) R. J. Femer and L. R. Hatton, Carbohydr. Res., 6,75-86 (1968).

43 42 42 23,42 9,42 23 8,9,23 23 118

119 23 120 ~

(Continued)

4

-l

4

TABLEV (Continued)

Alditol Glycerol DMannitol

1,2,5,6-tetra-O-acetyl1,6-di-O-benzoyl3,4-di-O-benzoyl1,3-O-butylidene3,4-0- butylideneL-Mannitol 6-deoxy3,4-O-isopropylidene5-0-ptolylsulfonylPentaerythritol Xylitol 4-0-acetyl4-0- benzoyl3,4,5-tri-O-acetyl-

Boronate phenyl3,4-ethyltris(p-bromophenyltris(p-chlorophenyl1,2:3,4:5,6-tris(ethyl1,2:3,4:5,6tris(phenyltris(p-tolyl3,4-ethylbis(pheny11,2:5,6bis(phenylphenyl1,2:5,6bis(phenyl-

Melting point (“C) 76-78 128 204-205 184-185

-

134-135 162-164

-

150 149-150 116118 69-72

W

Iff1 (degrees) +28.2 +35.1 +45.4 +9.7 +53.4 +45.9 +48.6 -

-

-10.8 +45

Rotation solvent

CCI, -

CCI, -

-

CCl,

-

CHCl, CHCI,

References 13,23,26,43 41 8 8 41 8,9,23,123 8 41 23 9 121 121

!=

m

!= 4 ?

m P 1,e-phenyl1,e-phenylbis(pheny11,2:3,5bis(ethyl1,2:3,5-bis(ethyl1,e-ethyl1,2:3,5-bis(ethyl1,e-ethyl-

78-80 124-126 207-208 -

-

-27 -23

-

-

(117) T. G. Bonner, E. J. Bourne, and D. Lewis,]. Chem. Soc., 7453-7458 (1965). (118) T. G. Bonner, E. J. Bourne, and D. Lewis, J . Chem. Soc., 3375-3381 (1963). (119) T. G. Bonner, E. J. Bourne, S. E. Harwood, and D. Lewis,J. Chem. SOC., 121-126 (1965). (120) T. G. Bonner, E. J. Bourne, S. E. Harwood, and D. Lewis,]. Chem. SOC., C , 2229-2233 (1966). (121) T. G. Bonner, E. J. Bourne, D. G. Gillies, and D. Lewis, Carbohydr. Res., 9,463-470 (1969).

CHCl, CHCl,

-

-

-

67 67 8 40 40 40 40 40

22

m

!=

TABLEVI Boronates of Anhydro Sugar Derivatives (Including a Lactone)

Anhydro compound

Boronate

Altropyranose, 1,6-anhydro-p-~ 3,4-phenyl2-0-p- tolylsulfonyl3,4-phenylGalactitol, 1,5-anhydro-~4,Gphenyl4,6-phenyl-2,3-(diphenylcyclodi2,3-diOacetyl4,6-phenylGalactopyranose, 1,6-anhydro3,4-phenylP-D2-0-p- tolylsulfonyl3,4-phenylGlucitol, 1,5-anhydro-~4,g-phenyl4,6-phenyl-2,3-(diphenylcyclodiD-Glucono-l,4-lactone 3,5-phenylGlucopyranose, 1,6-anhydrop-D2,4-phenylGulopyranose, 1,6-anhydro-p-~- 2,3-phenyl4-0-p- tolylsulfonyl2,3-phenylHex-1-enitol, 1,5-anhydr0-2deoxy-Darabino- (D-glucal) 4,Gphenyl3-0-acetyl4,6-phenylHex-1-enitol, 1,5-anhydro-2deoxy-Dlyxo- (D-galactal) 3,4-phenyl3,4-phenyl6-0-acetylHexitol, 1,5-anhydro-2-deoxy4,g-phenylD-lyX04,6-phenyl3-0-acetylMannopyranose, 1,6-anhydrop-D2,3-phenyl4-0-p- tolylsulfonyl2,3-phenyl-

Melting point (“C)

166-167 173- 175 141-142

[&I, (degrees)

Rotation solvent

- 129.6

-

References

-112 +69

1,4-dioxane

61 61 55

201-203 143-144

+ 177

1,4-dioxane 1,Cdioxane

55 55

169- 170 135-136 176- 177

-114 +50 -80

1,4-dioxane

61 61 3

188-189

-121

1,4-dioxane

3 78

-

61 61 61

-

122-124 212-216 140-141

+177

-

-70.4 +43.7 +83.9

-

128 88-89

-53 -90

1,4-dioxane 1,4-dioxane

104 98-99

-87 -74

1,4-dioxane 1P-dioxane

55

114-115 89

+ 172

+60

1,Cdioxane 1,4-dioxane

55

149-150 157-158

- 104.9 - 104

-

-

55

55 55

55 61 61

03

TABLEVII

0

Boronates of Nucleosides Nucleoside Adenosine N6-benzoyl5'-phosphate 5'4-p-tolylsulfonyl5'4-t~itylCytidine N-acetylN-benzoyl5'-O-p-tolylsulfonylGuanosine N- benzoylInosine 5'-O-p-tolylsulfonylUridine 5'-O-p-tolylsulfonyl-

Boronate 2',3'-phenyl2',3'-m-nitrophenyl2',3'-p-tolyl2',3'-phenyl2',3'-phenyl2',3'-phenyl2',3'-phenyl2',3'-phenyl2',3'-phenyl2',3'-phenyl2',3'-phenyl2',3'-phenyl2',3'-phenyl2',3'-phenyl2',3'-phenyl2',3'-phenyl2',3'-phenyl-

Melting point ("C)

[ffl (degrees)

Rotation solvent

223-224 235-237 226229 187- 188 170

-

78 140 110 175-177

-

270 160,168-170 196, 178-179

-

221-222

-

References 28,35,36,75,77 36 36 122 36 71,80 36 27,28 36 30 74 35,36 35,36 28,35,36 37 27,28 73

(122) S. G. Verenikina, E. G. Chauser, and A. M. Yurkevich, Zh. Obshch. Khim., 41, 1630-1632 (1971).

X. ADDENDUM An X-ray structural study of D-mannitol1,2:3,4:5,6-tris(phenylboronate) has been reported.123 (123) A. Gupta, A. Kirfel, G. Will, and G. Wulff,Acta Crystallogr., Sect. B, 33,637-641 (1977).

BIOSYNTHESIS OF SUGAR COMPONENTS OF ANTIBIOTIC SUBSTANCES BY HANSGRISEBACH Biologisches Znstitut IZ der Uniuersitat Freiburg i . Br., Lehrstuhl f u r Biochemie der Pflanzen, D 7800 Freiburg i . Br., Germany

. . . . . . . . . . . . . . . . . . , , . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Methyl-branched Sugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Sugars Having a Two-Carbon Branch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. Introduction

11. Branched-chain Sugars

3. General Conclusions on the Biosynthesis of Methyl-branched .. Sugars and on Sugars Having a Two-Carbon Branch . . . . . . . 4. Sugars Having a Formyl or Hydroxymethyl Branch: .. .. L-Streptose and L-Dihydrostreptose . . . . . . . . . . . . . . . . . . . . . . 111. Aminocyclitol Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . , , 1. Streptomycins and Bluensomycin . . . . . . . . . . . . . . . . .. . . . . . . . . 2. Gentamicins . . .. . ., .. .. . . .. . . , . .. .. . . . . 3. Neomycin., . . . . . . . . . . .. . . . , . . . . . . . . . .. . . .. . .. . . . . .. . , 4. Spectinomycin .. . . . . .. . . . . . . . . . .. . . . . .. . . . . . . . . .. 5. Validamycin . . . . . . . . . , . , . . . .. .. . . . . . . . . . . . . . . . . . . IV. Amino Sugars Not Occurring in Aminocyclitol Antibiotics . . . . , . . , , . . . . . 1. Desosamine and Mycaminose.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . V. Nucleoside Antibiotics.. . . . . . . . . . . . . . .. . . . . . . , .. . ... . ..

. .. . .. .. . .. .... .. . . . .. . . . . .. . . . . .. .. . . . .. . . . . . . . . . . . ... . . . . . . .. .. . .. .. .. . . . .. . . .. . .. .. . .. .. . . . . . . . . . . .. .. . . .. . . . . .. . .. .. . . . .. . .. .. . . . .. . . .. .. . . . . I

81 82 82 89 97 98 102 102 110 115 118 120 122 122 122

I. INTRODUCTION The extensive research on the chemistry of antibiotics has led to the discovery of numerous new sugars, many of which possess unusual structures. Carbohydrate-containing antibiotics may be classified into two large groups: those which are entirely carbohydrate in nature, for example, the aminocyclitol antibiotics,’ and those which contain sugars as glycosidic components, for example, the large family of “macrolide” antibiotics.2 Dutchel.3 has classified the glycosidic antibiotics into five subgroups according to the nature of the aglycon. (1) S. Umezawa, Adu. Carbohydr. Chem. Biochem., 30,111-182 (1974);MTP Znt. Rev. Sci.: Org. Chem. Ser. Two, 7 , 149-200 (1976). ( 2 ) W. Keller-Schierlein, Fortschr. Chem. Org. Naturst., 30,314-460 (1973). (3) J. D. Dutcher, Adu. Carbohydr. Chem., 18,259-308 (1963). 81

82

HANS GRISEBACH

Inspired by the partly exotic structures of antibiotic sugars, a number of chemists and biochemists have tried to unravel their biosynthesis. As in other biosynthetic studies, labelling patterns obtained with isotopically labelled precursors dominated the earlier phases of this work, and this approach is still being used. Only recently has it been possible in some cases to study the reactions at the enzymic level. The present article concentrates mainly on recent developments. Where it is necessary for a better understanding, the biosynthesis of related sugars that have not yet been found in antibiotics is also considered. The author also does not hesitate to include some speculations on the biosynthesis of sugars for which experimental results are still lacking, because these hypotheses might inspire future work in this field. 11. BRANCHED-CHAINSUGARS

More than a dozen branched-chain, deoxy sugars have now been discovered as components of antibiotics. One review on their biosynthesis4 and ~ w o on ~ , the ~ chemistry and biochemistry of branched-chain sugars have appeared. These sugars can be divided into two groups according to their biosynthesis. Group 1 includes methyl-branched sugars and sugars having a twocarbon branch. These sugars arise by transfer of a C, or C2 unit from appropriate donors to nucleotide-bound hexosuloses. Group 2 consists of sugars having a hydroxymethyl or formyl branch. These sugars are formed by intramolecular rearrangement of nucleotide-bound hexosuloses, with ring contraction and expulsion of one carbon atom. 1. Methyl-branched Sugars

a. L-Mycarose, L-Cladinose, and L-Noviose.-Early tracer studies on the biosynthesis of L-mycarose (l),L-cladinose (2), and L-noviose (3) led to the initially surprising observation that the C-methyl branch of these sugars originates from the S-methyl group of L-methionine. The hexose portion of 1,2, and 3 was shown to be formed from D-glucose without inversion or rearrangement of the hexose chain. In the case of mycarose and noviose, it was also shown, with the aid of ~-[methyl-’~C, methyl-2H,]methionine that the C-methylation proceeds by way of (4) H. Grisebach, Helo. Chim. Acta, 51, 928-939 (1968). (5) H. Grisebach and R. Schmid, Angew. Chem. Int. Ed. Engl., 11,159-173 (1972). (6) H. Grisebach, “Biosynthetic Patterns in Microorganisms and Higher Plants,” Wiley, New York, 1967, pp. 66-101.

SUGAR COMPONENTS OF ANTIBIOTIC SUBSTANCES

83

transfer ofintact CD,groups. This work has been covered in the earlier

I

H,NCO II

I

OH

0

1 L-Mycarose R = H 2 L-Cladinose R = Me

3 L-Noviose

The results mentioned led to the hypothesis that C-methylation occurs with S-adenosyl-L-methionine (AdoMet, “active methionine”) as the methyl donor and a nucleotide-bound aldosulose as the acceptor.6 This assumption has now been confirmed with a cell-free system from Streptomyces rimosus that produces the antibiotic tylosin, which contains L-mycarose. When a cell-free extract from mycelia of S. rimosus was incubated with dTDP-D-glucose, S-adenosyl-~-[methyl-’~C]methionine,and NADPH, a new radioactive product was formed that contained at least two dTDP-sugars.‘ One of these was identified as dTDP-mycarose (6, see Scheme 1).The second product has not yet been identified, but its properties are very similar to those of dTDP-mycarose. Kuhn-Roth oxidation of the mixture of the dTDP-sugars gave acetic acid that contained over 90% of the carbon-14; this proved that both sugars must bear a C-methyl branch originating from [14C]AdoMet. The same products were obtained when dTDP-6-deoxy-~-xylo-hexos-4-ulose(4, see Scheme 1)was used instead of dTDP-D-glucose as the substrate. The enzyme dTDP-Dglucose 4,gdehydratase (EC 4.2.1.46), which catalyzes the conversion ofdTDP-D-glucose into 4, was also shown to be present in extracts from S. rimosus.8The D configuration of 4 formed by use of the enzyme from S. rimosus was proved in the following way.8 Reduction of 4 with sodium borohydride, and subsequent hydrolysis of the product, gave 6deoxyglucose and fucose. The former could be oxidized with D-glucose oxidase (EC 1.1.3.4); as this enzyme is specific for D-glucose, the configuration in 4 must also be D. Besides the methylation step, the following reactions are necessary for the synthesis of dTDP-L-mycarose from dTDP-D-glucose: reduction at C-2, inversion of configuration at C-5, and (possibly as the last (7) H. Pape and G. U. Brillinger, Arch. Mikrobiol., 88,25-35 (1973). ( 8 ) H. Matem, G. U. Brillinger, and H. Pape, Arch. Mikrobiol., 88, 37-48 (1973).

84

HANS GRISEBACH

step) stereospecific reduction of the carbonyl group at C-4, leading to the L-rib0 configuration (see Scheme 1).

dTDP-0-glucose

-

0-

0

dTDP

OH

M

H

Ci+

e

O

O OdTDP

J

I

4

H,C

H

5

R

H,C 6

SCHEME 1.-Postulated Reaction-sequence for the Biosynthesis of dTDP-L-mycarose (6) from dTDP-D-glucose.

This sequence of reactions therefore requires several enzymes that have not yet been separated from each other. As the methylation step is NADPH-dependent,’ it may be postulated that reduction at C-2 occurs before methylation at C-3. In Scheme 1,the 3,4-enediol of the hexos-4ulose 5 is shown as the nucleophilic acceptor for the methyl group from AdoMet, but the existence of such an enediol has not been proved. dTDP-D-glucose 4,gdehydratase activity increases during the stationary-growth phase of S . rimosus, together with production of tylosin and the dTDP-mycarose-synthesizing system.8This is in accord with the assumption that these enzymes are involved in the biosynthesis of tylosin. The 0-methyl group of L-cladinose (2) is also derived from Lmethionine. The O-methylation step does not, however, take place at the level of the “nucleotide-sugar,” but it occurs when the substrate is erythromycin C, which contains L-mycarose and D-desosamine as glycosidic componentsg (see Scheme la). The 0-methylation of the Lmycarose moiety of erythromycin C by a partially purified enzyme from Streptomyces erythreus was described by Alpine and Corcoran.loJ1 The reaction catalyzed is shown in Scheme la. (9) W. Hofheinz and H. Grisebach, 2. Naturforsch. Teil B , 17, 852 (1962). (10) T. S. McAlpine and J. W. Corcoran, Fed. Proc. Fed. Am. SOC. E x p . B i d . , 30, 1168 ( 197 1). (11) J. W. Corcoran, Methods EnzymoZ., 43, 487-498 (1975).

SUGAR COMPONENTS O F ANTIBIOTIC SUBSTANCES

85

ErythromycinA R, = OH, R, = Me Erythromycin C R, = OH, R, = H

[

:Lo" OQ . Hz

+

AdoMet

- [ :Lo" O Q M :;

+ AdoHcy

H,C SCHEME la.-Reaction Catalyzed by S-Adenosy1methionine:ErythromycinC 0Methyltransferase. (D = desosamine.)

The enzyme converts erythromycin C into erythromycin A in the presence of AdoMet. Evidence was obtained that the enzyme is associated with the microsomal fraction. The enzyme showed a very high degree of substrate specificity. Aside from erythromycin C, it failed to catalyze the methylation of any other L-mycarosyl moiety tested. Erythromycin A and S-adenosyl-L-homocysteine (AdoHcy) were potent inhibitors of the enzyme, and it was assumed that the AdoM e t AdoHcy ratio could be a major regulatory factor of the final step in the formation of erythromycin A.

b. L-Vinelose.-Cytidine 5'-(~-vinelosyl diphosphate) (CDP-6deoxy-3-C-methyl-2-O-methyl-~-talose)~~-~~ (7)and its 4-(O-methylglycolyl) ester15(8)were isolated from Acetobacter vinelandii strain 0 . Although L-vinelose has not yet been found in an antibiotic substance, (12) S. Okuda, N. Suzuki, and S. Suzuki,]. Biol. Chem., 242, 958-966 (1967). (13) J. S. Brimacombe, S. Mahmood, and A. J. Rollins,]. Chem. SOC. Perkin Trans. 1, 1292-1297 (1975). (14) M. Funabashi, S. Yamazaki, and J. Yoshimura, Carbohydr. Res., 44,275-283 (1975). (15) S. Okuda, N. Suzuki, and S. Suzuki,]. Biol. Chem., 243,6353-6360 (1968).

HANS GRISEBACH

86

the work on its biosynthesis is discussed here, because it complements the results on the enzymic formation of L-mycarose.

Hb

bMe

7 CDP-L-vinelose, R = H 8 CDP- (0-rnethylglycoly1)L-vinelose, R = MeOCH,CO

First, the incorporation of ~-[methyl-'~C]methionineinto CDPvinelose was investigated with cells of a methionine-requiring mutant ofA, vineZandii.lsThe degradation of the radioactive CDP-vinelose by the procedure shown in Scheme 2 showed that about half of the radioactivity was present in the 0-methyl group (isolated as chloromethane), and the other half was in the C-methyl group (isolated as p bromophenacyl acetate). CH,OH

9

HO

OH

OCDP H + + HO CH3

HO

oie

HO

NaBH,

I

HCdH, .I H,CCOH

I

HCOH I HOCH

I

olke

CH, BC13

HYHO HF0,H B r G C O C H , B r

CqOH I

HCOH 10;

HYO.,H +

Br + o c H , - y ~ H ,

0 CHO

I

cH3

&,C1

.I

H,CCOH HCOH I I

HOCH I

CH, of CDP-L-vinelose Synthesized by A. uinelandii in the

SCHEME2.-Degradation Presence of ~-[Methyl-"C]rnethionine. ( 0 , Isotope from ~-[methyl-'~C]methionine.)

When a crude enzyme-preparation from A. vinelandii was incubated with CDP-D-[U-14C]glucoseand AdoMet, and the incubation mixture (16) Y. Eguchi, M. Takagi, F. Uda, K. Kimata, S. Okuda, N. Suzuki, and S. Suzuki,]. B i d . Chem., 248,3341-3352 (1973).

SUGAR COMPONENTS OF ANTIBIOTIC SUBSTANCES

87

was then digested with phosphate diesterase and phosphatase, three new radioactive sugars (Xl, X,, and X,) were detected on paper chromatograms.ls One of the products (XI) was also formed in the absence of AdoMet, and was identified as 6-deoxy-~-xylo-hexos-4ulose. With the aid of Ad~[methyl-~H]Met, it was shown that the other two products were methylated sugars. The corresponding CDP-sugars were extremely labile, and were degraded to the glycosyl phosphates immediately after the mixture was applied to the paper. All of the evidence so far obtained indicates that one of the sugars (X,) obtained by hydrolysis of the 1-phosphate was a 6-deoxy-3-C-methyl-hexos-4ulose (9, see Scheme 3). The evidence for this structure was educed mainly from the series of reactions shown in Scheme 3. Reduction of 9 with sodium borohydride gave a pair of alditols (lo), indicating the reduction of a keto group in the original sugar. Structure 9 was also consistent with the isotope distribution found in the degradation products. When, for example, a sample of r3H, 3-'4c-jx, (obtained from incubation of CDP-6-deoxy-~-xy~o-[3-~~C]hexos-4-ulose with [methyl14C]AdoMet)was reduced with sodium borohydride, and the product decomposed with sodium periodate, an acetic acid having the same ratio of ,H to 14Cas that of the original sugar was obtained. This result proved the attachment of the rH]methyl group to C-3.

/-

\

10

[I;:] CH,CO,H C6,H I CHOH I CH3

HCHO

+

HC+O,H H,CCO,H

+

HC0,H CAO

I

CH3

SCHEME3.-Reactions Performed in the Structural Assignment ofthe Intermediate in the Biosynthesis of L-Vinelose.

88

HANS GRISEBACH

When the crude enzyme-preparation was separated into five fractions by stepwise precipitation with ammonium sulfate, a partial separation of the X2-formingactivity from the X,-forming activity was achieved. It was also found that one of the fractions catalyzes the conversion of CDP-X2 into CDP-X,. Although it has not yet been possible to assign a definite structure to sugar X,, the experimental evidence strongly suggests that it is an isomer of sugar X,. On the basis of these results, the authorsi6 proposed the reaction sequence shown in Scheme 4 for the biosynthesis of CDP-L-vinelose from CDP-D-glucose. As with L-mycarose, the 6-deoxyhexos-4-ulose derivative (11) is the substrate for the methylation step leading to the CDP-6-deoxy-3-C-methylhexos-4-ulose (12). The corresponding dTDP-sugar might, therefore, be one of the unidentified reactionproducts in the bias ynthesis of dTDP-L-mycarose.

SCHEME4.-Proposed Pathways for the Conversion of CDP-D-glucose into CDP-Lvinelose. (The configuration of 12 is still speculative.)

For the conversion of CDP-D-glucose into CDP-L-vinelose, the following reactions must be considered: ( a ) reduction at C-6, (b) methylation at C-3, (c) inversion of configuration at C-3 and C-5, (d)

SUGAR COMPONENTS OF ANTIBIOTIC SUBSTANCES

89

stereospecific reduction at C-4, and (e) methylation of the 2-hydroxyl group. In Scheme 4,the epimerization at C-3 and C-5 is postulated to occur after the methylation step. This assumption has, however, not been proved true, as the configuration of 12 is still unknown. A 3,5epimerase was found to be involved in the biosynthesis of dTDP-Lrhamnose from dTDP-~-glucose.''~'~ This epimerase converts its substrate dTDP-6-deoxy-~-xyZo-hexos-4-uloseinto enzyme-bound dTDP-6-deoxy-~-lyxo-hexos-4-ulose.By analogy, it could be assumed that, in the biosynthesis of L-vinelose, epimerization of 11 at C-3 and C5 takes place before methylation at C-3. Methylation at 0-2 must occur late in the sequence, because X, (9) and X3 do not possess an 0-methyl group.

2. Sugars Having a Two-Carbon Branch a. D-A1dgarose.-Aldgamycin E, from cultures of Streptomyces ZavenduZae, is a macrolide antibiotic's*20 containing, besides Dmycinose, the (1-hydroxyethy1)-branched sugar D-a1dgarose2'(13).The stereochemistry at C-7 of aldgarose has been clarified in connection with a total synthesis of this s ~ g a r . ~ ~ J ~

0 13

For studies on the biosynthesis of D-aldgarose, growing or resting cells of S . lavendulae were used. ~-[methyl-'~C]Methionine, L-[ethyl''C]ethionine, [1-14C]acetate, D-[U-'4C]glUCOSe, [2-'4C]pyruvate, and [3-14C]pyruvate were t e ~ t e d as ~ ~potential ,~~ precursors for the 1(17) R. W. Gaugler and 0. Gabriel,]. Biol. Chem., 248, 6041-6047 (1973). (18) A. Melo and L. Glaser,]. Biol. Chem., 243, 1475-1478 (1968). (19) H. Achenbach and W. Karl, Chem. Ber., 108,759-771 (1975). (20) H. Achenbach and W. Karl, Chem. Ber., 108,780-789 (1975). (21) G. A. Ellestad, M. P. Kunstmann, J. E. Lancaster, L. A. Mitscher, and G. Morton, Tetrahedron, 23,3893-3902 (1967). (22) H. Paulsen and H. Redlich, Chem. Ber., 107,2992-3012 (1974). (23) J. S. Brimacombe, C. W. Smith, and J. Minshall, Tetrahedron Lett., 2997-3000 (1974). (24) R. Schmid, H. Grisebach, and W. Karl, Eur. J. Biochem., 14,243-252 (1970). (25) R. Schmid and H. Grisebach, Z. Naturforsch. Teil B , 25, 1259-1263 (1970).

90

HANS GRISEBACH

hydroxyethyl branch of 13. The degradation reactions outlined in Scheme 5 were used for localization of 14C-activity.Methanolysis of aldgamycin E yielded methyl aldgaroside and methyl mycinoside, which were separated by thin-layer and gas-liquid chromatography. Methyl aldgaroside was treated with sodium hydroxide solution to saponify the cyclic carbonate. Periodate oxidation of the free sugar yielded acetaldehyde from the 1-hydroxyethyl branch (containing C-7 and C-8) and ~-(-)-3-hydroxybutanoic acid from C-3 to C-6 of aldgarose. The latter acid could be identified by oxidation with D-( -)-3hydroxybutanoic acid dehydrogenase (EC 1.1.1.30).Direct periodate oxidation of aldgamycin C (see Scheme 5 ) also yielded acetaldehyde and the butanoic acid from aldgarose, leaving the rest of the molecule intact. This was proved by a mass-spectrometric i n v e ~ t i g a t i o n . ~ ~

SCHEMES.-Chemical Degradation of Aldgamycin E.

SUGAR COMPONENTS OF ANTIBIOTIC SUBSTANCES

91

By application of these degradation reactions, the following results were obtained with the precursors already mentioned. L-Methionine, L-ethionine, acetate, and Dglucose did not function as precursors of the hydroxyethyl branch of aldgarose. In all cases, the incorporation of carbon-14 into C-7 and C-8 of aldgarose was negligible. By contrast, 4445% of the radioactivity of aldgarose was located in C-7 and (2-8, with [2-14C]- and [3-14C]pyruvateas the source of radioactive carbon. The distribution of carbon-14 in the two-carbon branch was determined by Kuhn-Roth oxidation of the acetaldehyde (2,4-dinitrophenyl)hydrazone, followed by Schmidt degradation of the acetic acid. In this way, it was found that 88% of the carbon-14 was located in C-7, with [214C]pyruvateas the precursor, and 93% in C-8, with [3-‘4C]pyruvate as the precursor, proving the specific incorporation of C-2 and C-3 of pyruvate into the hydroxyethyl branch. D-[U-14C]Glucosewas almost exclusively incorporated into the hexose portion of D-aldgarose, with equal distribution of activity in the hexose chain. No radioactivity was found in the cyclic carbonate group with the aforementioned precursors, but 45% of carbon-14 (related to aldgamycin E) from sodium hydrogen[14C]carbonatewas located in this group. It may, therefore, be assumed that a carboxylation reaction is involved in the formation of the cyclic carbonate. As aldgamycin C is also a fermentation product of Strepomyces lauendulae,21formation of the cyclic carbonate probably occurs at the stage of aldgamycin C. On the basis of these results, it was postulated24that 1-(hydroxyethy1)thiamine pyrophosphate (HTPP, “active acetaldehyde”) is the actual precursor for the hydroxyethyl branch of aldgarose. Reaction of HTPP with a hexos-8ulose would lead to the intermediate 14 (see Scheme 6). Stereospecific reduction of the carbonyl group in 14 would then lead to 15. Strong support for the formation of a methylcarbonyl sugar such as 14 has come from work on the biosynthesis of the quinocycline sugars (see Section II,2,b). However, experiments with oxythiamine and pyrithiamine, which are antivitamins of thiamine pyrophosphate , showed no inhibition of the formation of aldgamycin E. The mechanism of the attachmant of the two-carbon branch is, therefore, still undecided (see also, Section 11,2,b).

b. Sugars of the Quinocycline Complex.-The anthracycline antibiotics 16, namely, quinocyclines A and B and isoquinocyclines A and B from Streptomyces aureofaciens (FD 11188 Pfizer) contain as glycosidic components the (1-hydroxyethy1)-branched sugar 17 [2,6dideoxy-4-C-(1-hydroxyethy1)-L-xylo-hexopyranose] in the A compo-

HANS GRISEBACH

92 OH

I

H,C--C-R'

6

H,C -C

HO

OH

OR

14

15

SCHEME6.-Hypothesis for the Introduction of the Two-carbon branch of Aldgarose. (R' = thiamine pyrophosphate or group of an enzyme.)

nents, and the methylcarbonyl-branched sugar 18 [2,6-dideoxy-4-Cacetyl-~-xy20-hexopyranose]in the B components.2s

The structural identification of the sugar from the B components as a C-acetyl-branched sugar constituted strong support for the conclusion reached from work on the biosynthesis of D-aldgarose (see Section II,2,a) that a C-acetyl-branched sugar is the primary product in the attachment of the two-carbon branch (see Scheme 6). The incorporation of [2-'4C]pyruvate and [l-'*C]acetate into sugars 17 and 18 was investigated." Oxidation of the methyl glycosides of sugar 17 with periodate yielded acetaldehyde from the l-hydroxyethyl branch. The acetaldehyde (2,4-dinitrophenyl)hydrazonewas further oxidized by Kuhn-Roth oxidation to acetic acid, which was degraded by the Schmidt reaction to methylamine and carbon dioxide. Periodate oxidation of the methyl glycosides of sugar 18 produced acetic acid from the C-acetyl branch. The acetic acid was isolated, and purified as l-acetamidonaphthalene. The following results were obtained with these degradation reactions. [2-14C]Pyruvate was specifically incorporated into the 1hydroxyethyl branch of 17 and into the C-acetyl branch of 18. In each instance, -90% of the radioactivity of the methyl glycosides was (26) U. Matern, H. Grisebach, W. Karl, and H. Achenbach, Eur. J . Biochem., 29, 1-4 (1972). (27) U. Matern and H. Grisebach, Eur. J. Biochem., 29, 5-11 (1972).

SUGAR COMPONENTS OF ANTIBIOTIC SUBSTANCES

93

16

17

18

Structures of the Sugar Components (17 and 18) of the Quinocycline Complex and of the Aglycon of Isoquinocyclines (Isoquinocycline A 16, with R = 17; isoquinocycline B 16, with R = 18.)

located in the two-carbon branch. Further degradation proved that carbon-14 was present only in C-7 of this branch. In contrast, no incorporation of radioactivity from [l-14C]acetate into the two-carbon branch was found. [‘4C]Quinocycline B (obtained from [l-14C]acetate)containing sugar 18 was efficiently converted into [14C]quinocyclineA by growing cells of Streptornyces uureofuciens. Under the same conditions, only a very low conversion of quinocycline A into quinocycline B took place. It may, therefore, be concluded that quinocycline B is the precursor of quinocycline A. Neopyrithiamine, which is a competitive inhibitor of thiamine, had no influence on the incorporation of [2-’4C]pyruvate into the twocarbon branch. On the basis of these results, the following, metabolic sequence leading to quinocycline B and quinocycline A was postulated: XDP2,6-dideoxy-~-erythr-hexos-4-ulose(X = nucleoside residue) reacts with an activated, two-carbon species originating from pyruvate, to form a “nucleotide sugar” of 18. Sugar 18 is then transferred, with inversion of configuration at C-1, to the aglycon to form quinocycline B, and the carbonyl group in sugar 18 is reduced, to yield quinocycline A. Further insight into the reaction sequence leading to the two-carbonbranched sugars was obtained with a cell-free extract from S. uureofuciens.28 Incubation of dTDP-[U-14C]glucosewith this extract, (28) U. Matem and H. Grisebach, 2. Naturforsch. Teil C, 29,407-413 (1974).

HANS GRISEBACH

94

and subsequent hydrolysis of the “nucleotide sugars” led to the formation of four radioactive products (I-IV). Addition of pyruvate to the incubation caused an increase in the amount of 111, and a decrease in that of 11. Product I was identified as 6-deoxy-~-xyZo-hexos-4-ulose by its absorption spectrum after addition of sodium and by reduction with sodium borohydride to give 6-deoxyglucose and 6deoxygalactose. Incubation of dTDP-6-deoxy-~-xyEo-[U-’~C]he~0~-4-~lo~e with the cell-free extract led to the same products 11-IV as with dTDP-D-[U14C]glucose as substrate. Product I11 was isolated as the methyl glycoside from an incubation performed on a preparative scale. The n.m.r. spectrum of I11 was very similar to that of the methyl glycoside of 18.The signal of H-3 was, however, shifted by 0.82 p.p.m. to lower field compared with the corresponding signal of 18,and had a large coupling-constant of 11 Hz, indicating trans-diaxial coupling. Furthermore, the H-5 signal was shifted by 0.42p.p.m. to higher field. These data were consistent with the assumption that, in 111, the 3hydroxyl group is equatorial (not axial, as in 18).This conclusion was supported by the fact that I11 formed a borate complex (with the hydroxyl groups at C-3 and C-4),whereas 18 did not form such a complex. On the assumption that I11 belongs, as does 18, to the L series, structure 19 may be proposed for the enzymic product from dTDP-6deoxy-~-xy2o-hexos-4-ulose and pyruvate. On reduction with sodium borohydride, product I1 yielded a pair of radioactive products that were tentatively identified as 2-deoxy-~-Zyxohexose and 2,6-dideoxy-~-urubino-hexose. This result, together with the chromatographic properties of 11, and its reaction on chromatograms with vanillin-perchloric acid to give a blue color, are consistent with the structure of 2,6-dideoxy-~-Zyxo-hexos-4-ulose (20)for 11. This structure is also consistent with the concept that I1 (20) is, very probably, the precursor of I11 (19).

19

20

(29) R.Okazaki, T. Okazaki, J. L. Strominger, and A. M. Michelson,]. Biol. Chem., 237, 3014-3026 (1962).

SUGAR COMPONENTS OF ANTIBIOTIC SUBSTANCES

95

When a mixture of dTDP-~-[3-~H]glucoseand dTDP-D-[U''C]glucose was incubated with the cell-free extract, the 3H/'4Cratio in the 6-deoxy-~-xyZo-hexosuloseremained unchanged. In contrast, there was no tritium in products 19 and 20. This result is strong evidence for the participation of a 3,5-epimerase13,14in a reaction converting dTDP6-deoxy-~-xylo-hexos-4-ulose into an enzyme-bound dTDP-6-deoxy~-Zyxo-hexos-4-ulose(21, see Scheme 7) which is then reduced at C-2 to yield dTD P-2,6-dideoxy-~-lyxo-hexos-4-ulose. The mechanism for the introduction of the deoxy function at C-2 is still undecided. Preliminary experiments showed no influence of pyridoxamine 5'phosphate on the conversion of dTDP-~-[U-'~C]glucoseinto 19. (Pyridoxamine 5'-phosphate is a cofactor in the 3-deoxygenation step in the biosynthesis of CDP-3,6-dideoxyhe~oses.~~) On the basis of the results discussed and of studies on the timedependence of formation of product from dTDP-D-glucose, the sequence of reactions shown in Scheme 7 was postulatedz8 for the biosynthesis of the C-acetyl-branched sugar 19. It is at present unknown why the sugar formed in the cell-free system is the C-3 epimer of sugar 18 from quinocycline B.

SCHEME7.-Reactions Proposed for the Biosynthesis of the Oxoethyl-branched dTDP-sugar 19 from dTDP-D-glucose in a Cell-free System from S. uureofuciens.

Further experiments were undertaken to investigate the possible participation of thiamine pyrophosphate in the carboligase reaction.z8 Addition of TPP, Mgz+,and pyruvate to the incubation mixture gave no (30) P. Gonzalez-Porque and J. L. Strominger,J. Biol. Chem., 247,6748-6756 (1972).

96

HANS GRISEBACH

increase of product I11 (19).The cofactor for the ligase reaction could be partially removed by gel filtration through Sephadex G-25,but no restoration of activity by TPP and Mg2+ was obtained. Finally, incubation of dTDP-D-glucose and l-([l-'4C]hydroxyethyl)thiamine pyrophosphate (HETPP) gave no radioactive product 111. Despite these negative results, a participation of HETPP cannot be completely excluded, because HETPP cannot be regarded as a genuine cofactor, as it must first be transformed into the carbanion by enzyme catalysis.31 c. Pil1arose.-Pillaromycin A, an antitumor, antibiotic substance from cultures of Streptomycesflavovireus,is composed of a tetracyclic aglycon 22, and a branched-chain monosaccharide residue named pillarose. The structure of a 2,6-dideoxyhexos-4-ulosehaving a glycoloyl branch at C-2 (23)was originally proposed for p i l l a r o ~ e . ~ ~ However, crystallographic and mass-spectral evidence,= as well as synthetic led to a revised structure of pillarose; it was identified as 2,3,6-trideoxy-4-C-(2-hydroxyacetyl)-~-threo-aldohexose

(24). H OH i

HO

OH

0

0

22

C=O I

CH,OH

23

24

Structures of Pillaromycin (22, R = 24 -H on 0-1) and Pillarose (24), and Structure (23) Previously Proposed for Pillarose

(31) J. Ullrich and A. Mannschreck, Eur. J. Biochem., 1, 110-116 (1967). (32) M. Asai, Chem. Pharm. Bull., 18, 1713 (1970). (33) J . 0. Pezzanite, J . Clardy, P. Y. Lau, G . Wood, D. L. Walker, and B. Fraser-Reid, J . Am. Chem. SOC., 97,6250-6251 (1975). (34) D. L. Walker and B. Fraser-Reid,J. Am. Chem. SOC., 97,6251-6253 (1975).

SUGAR COMPONENTS OF ANTIBIOTIC SUBSTANCES

97

Although no biosynthetic studies on pillarose have as yet been published, the close structural relationship of pillarose to sugar 19 from quinocyline B (see Section II,2,b) permits postulation of the following reactions. Linkage of an activated glycolaldehyde to a 2,3,6-trideoxyhexos-4-ulose leads directly to 24. Glycolaldehyde may originate from hydroxypyruvate with the enzyme hydroxypyruvate d e c a r b ~ x y l a s e ~ ~ (EC 4.1.1.40) and “active glycolaldehyde” from hydroxypyruvate or a ketose donor with the enzyme transketolase (EC 2.2.1.1). 3. General Conclusions on the Biosynthesis of Methyl-branched Sugars and on Sugars Having a Two-Carbon Branch

The foregoing results permit some general conclusions regarding the biosynthesis of methyl-branched sugars and of sugars having a twocarbon branch. In each example investigated, the acceptor for the

M * O O X D P

XDP-a-glucose

L-noviose

I

(a) C- and O-methylation (b) carbamoylation

(a) C -methylation C-2 and C-4 CHS

‘ Q HO O X D P ] OH

(a) C-methylation b) reduction at C-2and C-4

D-Evermicose

L-mycarose

L-olivomycose

SCHEME8.-General Scheme for the Biosynthesis of Methyl-branched Sugars. [(i) C Methylation by AdoMet, with inversion of configuration; (ii) C-methylation with retention of configuration.] (35) J. L. Hedrick and H. J. Sallach, Arch. Biochem. Biophys., 105,261-269 (1969).

98

HANS GRISEBACH

branch is a nucleotide-bound hexos-4-ulose in which the carbon atoms adjacent to the 0x0 group are activated for attachment of the electrophilic methyl group from S-adenosyl-L-methionine or an activated, two-carbon fragment. In most cases, C-methylation takes place at C-3 (as in L-mycarose), but it may also occur at C-5 (as in Lnoviose). An exception is L-garosamine (see Section III,2) having a4-Cmethyl group. All other known examples of methyl-branched sugars5 can be assumed to be derived from XDP-6-deoxy-~-xy2o-hexos-4-ulose by the action of known enzymes, as shown in Scheme 8. Obviously, Cmethylation can occur with retention, or inversion, of configuration. Other variations arise by deoxygenation at C-2, and by reduction of the keto group at C-4 (leading to different stereochemistry). 4. Sugars Having a Formyl or Hydroxymethyl Branch: L-Streptose and

L-Dihydrostreptose The work of Baddiley and coworkers and of Bruton and Horner with labelled precursors in connection with the biosynthesis of L-streptose (25) from streptomycin has been r e v i e w e d . 4 ~It ~ *was ~ ~ found that the aldehyde branch of streptose originates from C-3 of glucose, and that the hexose unit as a whole is incorporated into the streptose molecule (see Scheme 9).

OH

OH

HO 25

SCHEME 9.-Positions of the Carbon-14 label in L-Streptose (25) from D-Glucose Labelled at C-1 (A), C-3 (o), or C-6 (m).

On the basis of the observation that dTDP-D-mannose and dTDP-Lrhamnose occur in Streptornyces g r i s e ~ s , ~and ' that cell-free preparations from this organism can convert both dTDP-D-glucose and dTDPD-mannose into dTDP-~-rhamnose,~* it was suggested that the biosynthesis of streptose and that of L-rhamnose are related. (36) J. Walker, Lloydia, 34,363-371 (1971). (37) N. L. Blumson and J. Baddiley, Blochem. J., 81, 114-124 (1961). (38) J. Baddiley, N. L., Blumson, A. Di Girolamo, and M. Di Girolamo, Biochim. Biophys. Acta, 50,391-393 (1961).

SUGAR COMPONENTS OF ANTIBIOTIC SUBSTANCES

99

Further insight into the biosynthesis of streptose was gained in experiments in which dTDP-~-[U-'~C]glucose was incubated with a cell-free extract from a streptomycin-producing strain of S. g r i s e ~ sIn .~~ the presence of NADPH, and after subsequent hydrolysis of the " nucleotide sugars," a new radioactive product was formed that was identified as dihydrostreptose (26, see Scheme 10) by paper chromatography, paper electrophoresis, gas chromatography of the trimethylsilyl derivative, and complex-formation with phenylboronic acid in molybdate b ~ f f e r . 3Besides ~ dihydrostreptose, rhamnose and a 6-deoxyhexos-4-ulose were formed during the incubation. A compound having the properties of the dTDP derivative of dihydrostreptose was apparently very labile, because it decomposed readily to a compound tentatively identified as dihydrostreptosyl phosphate. The latter was also very labile, and gave free dihydrostreptose upon attempted paper chromatography. dTDP-dihydrostreptose and dTDP-L-rhamnose were also obtained when dTDP-6-deoxy-~-xyZo-hexos-4-ulosewas used as the substrate. Only NADPH was needed as a cofactor for the reaction. NADH had -50-60% of the activity of NADPH. No dihydrostreptose or streptose could be detected when NADPH was replaced by NADP+ or NAD+. The formation of dihydrostreptose could be completely inhibited b y the addition of 1 mM p-(chloromercuri)benzoate, whereas formation of L-rhamnose was not inhibited by this reagent. As Streptomyces contain high proportions of proteases, a protease inhibitor, diphenylcarbamoyl chloride, was added to the buffer during purification of the "dTDP-dihydrostreptose synthase." A partially purified, enzyme preparation from S. griseus could be obtained by removal of nucleic acids with streptomycin and fractionation with ammonium sulfate.4o However, when this enzyme preparation was subjected to gel filtration on a column of Sephadex G-100, enzyme activity was completely lost. By combining certain fractions of the column eluate, enzyme activity could be partially restored. It was, therefore, assumed that separation into two, or more, active protein-fractions had occurred on the Sephadex column. As the biosynthesis of dTDP-L-rhamnose and dTDP-L-dihydrostreptose are related,37*39 and as, moreover, a dTDP-L-Zym-4-hexulose3,5-epimerase is necessary for the formation of dTDP-~-rhamnose,"*'~ the Sephadex G-100 fractions were assayed for the presence of the 3,5-epimerase by (39) R. Ortmann, U. Matern, H. Grisebach, P. Stadler, V. Sinnwell, and H. Paulsen, Eur. J . Biochem., 43,265-271 (1974). (40) P. Wahl, U. Matern,and H. Grisebach,Biochem. Biophys. Res. Commun., 64,10411045 (1975).

HANS GRISEBACH

100

determining the loss of tritium from dTDP-6-deoxy-~-[3-~H]xyZohe~os-4-ulose.'~ A sharp peak of epimerase activity was found; this was cleanly separated from a second protein fraction that catalyzed the synthesis of dTDP-dihydrostreptose from TDP-6-deoxy-~-xylo-hexos4-ulose in the presence of NADPH and the 3,5-epimerase. These results, taken together with the results already discussed, proved that the biosynthesis of dTDP-L-dihydrostreptose from dTDP-D-glucose requires three enzymes: dTDP-D-glucose 4,6-dehydrataseYdTDP-LZyxo-4-hexulose 3,5-epimeraseYand an NADPH-dependent dTDP"dihydrostreptose synthase" (see Scheme 10).

dTDP-o-glucose

dTDP-o-Clucose

4 , 6 - dehy dratas e

I

OH

dTDP- ~ - " r l i k y d ~ o

ntrrptos e synlkasr "

HO

OH 21

SCHEME10.-Biosynthesis

OH

HO 26

of dTDP-L-dihydrostreptose from dTDP-D-glucose.

By analogy to results obtained in studies on the biosynthesis of and GDP-~-fucose,~l it dTDP-L-rhamnose,'* dTDP-6-deoxy-~-talose,'~ seems very likely that dTDP-6-deoxy-~-Zyxo-hexos-4-ulose (21), formed by the 3,5-epimerase reaction, remains enzyme-bound. The biosynthesis of dTDP-dihydrostreptose from dTDP-D-glucose shows a close similarity by way of dTDP-6-deoxy-~-xyZo-hexos-4-ulose to the biosynthesis of UDP-D-apiose" from UDP-D-glucuronic acid in higher plants5 (see Scheme 11). In both cases, the hydroxymethyl branch originates by ring contraction of a 4-ketoseYwith expulsion of (41)V. Ginsburg,J. B i d . Chem., 236,2389-2393(1961). (42)R. R. Watson and N. S. Orenstein, Adv. Carbohydr. Chem. Biochem., 31,135-184 (1975).

SUGAR COMPONENTS OF ANTIBIOTIC SUBSTANCES

101

C-3. However, for apiose, the formation of the enzyme-bound glycos-4ulose i n t e m ~ e d i a t e ,and ~ ~ its rearrangement and reduction at the branched carbon atom, are catalyzed by one enzyme. In experiments in which sodium borohydride was added to the enzyme incubation mixture of the apiose/xylose synthase, no reduction products could be found that would arise from epimerization at C-3 of the 4-ketose intermediate.44In the synthesis of apiose, ring contraction therefore seems to take place with the ~-threo-pentos-4-ulose27 (see Scheme ll),and not with the L-erythro intermediate, which would be analogous to the ring contraction in the biosynthesis of L-dihydrostreptose.

QOUDP

OH UDP-~-glucuronic acid

HA

bH

UDP-D-apiose

SCHEME11.-Biosynthesis of UDP-D-apiose from UDP-D-glucuronicAcid.

When the dihydrostreptose synthase reaction was conducted in the presence of either (4-pr0-R-~H) NADPH or (4-pro-SPH)NADPH, label in dihydrostreptose appeared44ain both cases. This apparent lack of stereospecificity for the hydride transfer needs clarification. The unexpected result that dihydrostreptose, but not streptose, was formed under all conditions, either in the crude extract of S . griseus or (43) C. Gebb, D. Baron, and H. Grisebach, Eur. J . Biochem., 54,493-498 (1975). (44) U. Matern and H. Grisebach, Eur. J . Biochem., 74,303-312 (1977). (44a) H. P. Wahl, unpublished results.

102

HANS GRISEBACH

in the partially purified synthase was understood only later, when the fermentation products of S . griseus were investigated more closely.45 The products formed were separated by paper chromatography and electrophoresis, and detected by bio-autography. It could be shown that dihydrostreptomycin is a normal product of a streptomycinproducing strain of s. griseus. At all stages of fermentation, only dihydrostreptomycin was found inside the mycelium, whereas dihydrostreptomycin and streptomycin were present in the medium. From these results, it was concluded that dihydrostreptomycin is the primary product in the biosynthesis of streptomycin, and that dihydrostreptomycin is oxidized to streptomycin.

111. AMINOCYCLITOL ANTIBIOTICS The aminocyclitol antibiotics-gentamicins, kanamycins, neomycins, paramomycins, spectinomycins, streptomycins, and tobramycins-constitute a group of basic oligosaccharides that have a broad, antibacterial Instead of describing the biosynthesis of the individual sugar components of these antibiotics, it is advantageous to review the biosynthesis of the total molecule. A review on the biosynthesis of aminocyclitol (“aminoglycoside”) antibiotics has appeared?’

1. Streptomycins and Bluensomycin Streptomycin and dihydrostreptomycin are45normal fermentationproducts of S . griseus (see Section 11,4). Certain Streptomyces strains produce mannosido-streptomycin and hydroxystreptomycin (see 28). Bluensomycin (29) and glebomycin are apparently identical4*;29 is a monoguanidinated analog of dihydrostreptomycin in which a carbamoyl group replaces4s the guanidino group at C-1. The biosynthesis of the dihydrostreptose moiety has already been described. The biosynthesis of the streptidine and 2-deoxy-2-(methylamino)-L-glucose moieties, and their assembling to give the antibiotic, will now be discussed. Furthermore, the biosynthesis of the bluensidine moiety of bluensomycin will be compared with that of streptidine. (45)S. Maier, U. Matern, and H. Grisebach, FEBS Lett., 49,317-319 (1975). (46)R. Reiner, “Antibiotika,” G. Thieme Verlag, Stuttgart, 1974,pp. 136-147. (47)K. L.Rinehart, Jr., and R. M. Stroshane,J. Antibiot., 29,319-353 (1976). (48)M. Okanishi, H. Koshiyama, T. Ohmari, M. Matsuzaki, S. Ohashi, and H. Kawaguchi,J. Antibiot., Ser. A, 15, 7-13 (1962). (49)C. B. Barlow and L. Anderson,J. Antibiot., 25,281-286 (1972).

SUGAR COMPONENTS OF ANTIBIOTIC SUBSTANCES

103

YH2

C=NH I I1 H,N -C--NH

d R2H,CQ

H 28 Variations in the Structure of Streptomycins (28) (Streptomycin, R' = CHO; R* = H;Rs = OH. Dihydrostreptomycin, R' = CH,OH, Rz = H, R s = OH. Mannosidostreptomycin, R1 = CHO, Rz = H , Rs = p-D-mannosyl. Hydroxystreptomycin, R' = CHO, R2 = OH, Rs = OH.)

II

H,N-C-NH

0

Bluensomycin 29

a. Streptidine 6-Phosphate.-Extensive investigations by Walker and coworkers have shown that the streptidine moiety of streptomycin is synthesized from D-glucose 6-phosphate by way ofmyo-inositol; this

104

HANS GRISEBACH

work has been covered in two review^,^^*^^ and is therefore only summarized here. The sequence of reactions that are now assumed to occur in the formation of streptidine 6-phosphate from D-ghCOSe 6phosphate is shown in Scheme 12. It involves two analogous sequences of five enzymic reactions each, operating in series: a dehydrogenation, transamination, phosphorylation, transamidation, and dephosphorylation. The individual enzymic reactions have been described by Walker.51D-Glucose 6-phosphate is cyclized to ~ - m y o inositol l-phosphate (30),as has been observed with enzymes from higher and lower plants, as well as from mammals.52After dephosphorylation, myo-inositol (31)is oxidized to 2-keto-scyllo-inositol (32), which by transamination gives l-amino-l-deoxy-scyllo-inositol (33). After phosphorylation of 33 by a kinase, to give l-amino-l-deoxy-scylloinositol4-phosphate (34),an amidinotransferase transfers the amidine residue from arginine to the amino group of the inosamine, to afford N-amidino-O-phosphono-scy llo-inosamine (35).After dephosphorylation of 35 to give 36,oxidation at C-3 leads to N-amidino-3-keto-scylloinosamine (37),which is transformed in a second transamination step into N-amidinostreptamine (38).The latter is rephosphorylated, to give 39,which undergoes further amidination, to yield streptidine 6-phosphate (40). For certain pairs of corresponding reactions in the two sequences, it is known that different enzymes are involved, although some have overlapping substrate-specificities. Additional information on some of these enzymes was obtained in studies on the biosynthesis of bluensidine (see Section III,l,b). Rinehart and have pointed out that earlier analysis did not allow an unequivocal correlation between the labelling pattern in streptidine obtained with differently labelled D-glUCOSe and the label of the D-glucose precursors. As they found that biosynthesis of deoxystreptamine in S . fradiae follows53an alternative pathway (see Scheme 20 and Section 111,3), the biosynthesis of streptidine was reinvestigated with D-[613C]glucoseas the The 13C-n.m.r.spectrum of streptomycin showed resonances for each ofthe 21 carbon atoms.55The streptomycin (50) A. L. Demain and E. Inamine, Bacteriol. Rev., 34, 1-19 (1970). (51) J. B. Walker, Methods Enzymol., 43,429-470 (1975). (52) F. Pittner, W. Fried, and 0.Hohann-Ostenhof, Hoppe-Seyler’s Z. Physiol. Chem., 355,222-224 (1974), and references cited therein. (53) K. L. Rinehart, Jr., J . M. Malik, R. F. Nystrom, R. M. Stroshane, S. G. Truitt, M. Taniguchi, J. P. Rolls, W. J. Haak, and B. A. RuffJ. Am. Chem. Soc., 96,2263-2265 (1974). (54) M. H. G. Munro, M. Taniguchi, K. L. Rinehart,Jr., D. Gottlieb, T. H. Stoudt, and T. 0. Rogers,]. Am. Chem. SOC., 97,4782-4783 (1975). (55) M. H. G . Munro and K. L. Rinehart, Jr.,J. Am. Chem. Soc., in press.

OH

OH

OH 31

30

NH 1 I

NH

33

32

NH

NH

II

II

HO’ 36

HO’

OH

OH 37

OH

36

NH

NH

OH 34

35

NH

m

cn

OH 3s

40

ODSBA Dihydrostreptomycin

SCHEMEl2.-Sequence of Reactions Leading from D-Glucose 6-Phosphate to Skeptidine 6-Phosphate (40), or Bluensidine (41, R = H). (Abbreviations: DSBA, dihydrostreptobiosamine;Gln, Glutamine; aKGN, a-keto-glutaramate;Om, ornithine; phosphate and pyr, pyruvate.) Arg, arginine; Ala, alanine; 08,

41

tt Bluensomycin 29

r 0

u1

HANS GRISEBACH

106

isolated from the experiment with D-[6-'3C]glucose displayed three enhanced resonances, at 13.4, 61.2, and 72.4 p.p.m. from tetramethylsilane, which are the respective signals for C-5' of streptose, C-6" of 2deoxy-2-(methylamino)-~-glucose, and C-6 of streptidine. Labelling of C-6 of streptidine b y ~-[6-'~C]glucoseis consistent with path A (see Scheme 13), proposed earlier on the basis of labelling studies and enzymic work (compare Scheme 12),but not with path B, which leads to deoxystreptamine.

OOH = Q= Q CH,OH

HO

HO OH

HO OH

OH

OH

32 NHR

NHR

I

OH

OH

SCHEME 13.-Biosynthetic Conversion of D-Glucose into Streptidine by way of Ketoscyllo-inositol 32 (compare Scheme 12), with C-6 of D-Glucose Labelling C-6 of Streptidine by Path A. (Path B is analogous to the pathway found for formation of deoxystreptamine by S. frudiue.)

Support for the assumption that streptidine or its phosphorylated derivative is an intermediate in the synthesis of streptomycin came through studies with a mutant of S . griseus which is presumably blocked in the biosynthesis of streptidine or streptidine phosphate.56 This mutant only produced antibiotics when the medium was supplemented with streptidine dihydrogensulfate. The maximal amount of antibiotic (530 pg/ml, calculated as streptomycin) was produced in the presence of 1.000 p g of streptidine per ml. Two substances having antibiotic activity were produced; they respectively had chromatographic mobilities identical with those of streptomycin and mannosidostreptomycin. Several other aminocyclitols and guanidocyclitols were also tested for their ability to support production of new antibiotics by the mutant. Only with 2-deoxystreptidine was a new antibiotic produced; this was named streptomutin A. Presumably, (56) K. Nagaoka and A. L. Demain,]. Antibiot., 28,627-635 (1975).

SUGAR COMPONENTS OF ANTIBIOTIC SUBSTANCES

107

streptomutin A is the streptomycin analog having a 2-deoxystreptidine moiety, but the structure has not yet been proved.

b. B1uensidine.-Bluensidine (41,R = H;see Scheme 12) is the monoguanidinated inositol moiety of bluensomycin (29).The biosynthesis of bluensidine was studied with cell-free extracts of S. glebosus, and compared with the biosynthesis of ~treptidine.~' The following enzymic reactions were observed. Dialyzed extracts of S. glebosus catalyzed the conversion of rnyo-[U-'4C]inositol (31, Scheme 12)to aminodeoxy-scyllo-[U-14C]inositol (33)when both NAD+ and an amino donor were provided. NAD+was presumably required in (32),and the amino donor (for order to form ket~-scyllo-[U-'~C]inositol example, L-glutamine) was required in order to convert 32 into 33.The transamination reaction was confirmed in a separate assay. Assays for reactions H and I (see Scheme 12) with S. glebosus were negative. The substrate specificity of S. glebosus amidinotransferase could not be distinguished from the substrate specificity of the amidinotransferase of streptomycin producers. In summary, extracts of S. glebosus could catalyze reactions C, D, F, and G, but not reactions H and I; these reactions are apparently catalyzed by enzymes similar to those involved in the biosynthesis of the first guanidino group of the diguanidinated inositol (streptidine) moiety of dihydrostreptomycin. The scheme for the biosynthesis of bluensidine predicts a labelling pattern from D-glucose 6-phosphate, different from that observed for the biosynthesis of streptidine. Whereas C-5 and C-6 of streptidine arise from C-1 and C-6 of D-glucose 6-phosphate, r e s p e ~ t i v e l y Scheme , ~ ~ ~ ~ ~13 predicts that C-2 of bluensidine would be derived from C-6 O f D-glucose 6-phosphate. This is the labelling pattern that has been found in the biosynthesis of deoxystreptaminea (see Section 111,3). c. 2-Deoxy-2-(methylamino)-~-glucose.-Notmuch information is available on the biosynthesis of the 2-deoxy-2-(methylamino)-~glucose moiety (42),which is common to all streptomycins and to bluensomycin. CH,OH

HO 42

(57) J. B. Walker,]. Biol. Chent., 249,2397-2404 (1974). (58) R. M. Bruce, H. S. Ragheb, and H. Weiner, Biochim. Biophys. Acta, 158,499-500 ( 1968).

108

HANS GRISEBACH

Tracer studies have s ~ o w ~that~ the ~ individual , ~ ~ , carbon ~ ~ atoms of D-glucose are incorporated into the corresponding carbon atoms of 42. It must, therefore, be assumed that inversion at all four chiral centers of D-glucose takes place during the biosynthesis. Experiments with 2deoxy-2-(methylamino)-~-[’~C]glucoseand 2-amino-2-deoxy-~-[ll4 C]glucose gave predominant incorporation into 42, although it is not clear if the amino group stays attached to the hexose moiety during this conversion. Experiments with 15N-labelledprecursors are, therefore, needed, in order to clarify this question. ~-[‘~C]Glucose does not seem to be a precursor of 42. Epimerization at C-2 of a “sugar nucleotide” has been described. An enzyme from Escherichia coli catalyzes the epimerization of UDP-2-acetamido-2-deoxy-~-glucoseto UDP-2-acetamido-2-deoxy-~m a n n o ~ e Such . ~ ~ an epimerization, together with the 3,5-epimerase reaction of a 4-ketose and stereospecific reduction at C-4, could lead to inversion at all of the chiral centers of, for instance, 2-amino-2-deoxy-~glucose. L-Methionine has been shown to be the source of the N-methyl group. The following results led Heding and coworkers to the conclusion that the last step in the biosynthesis of streptomycin is N methylation of N-demethylstreptomycin. By adding the methylation inhibitor ethionine to the culture medium of S . griseus, it was possible to isolate N-demethylstreptomycin.60 Addition of ~-[rnethylJ~C]methionine and N-demethylstreptomycin to a culture led to labelled streptomycin, which could have been formed either by de no00 synthesis or by methylation of the N-demethylstreptomycin.61 To decide between these two possibilities, N-demethyldihydrostreptomycin was used as the potential methyl-acceptor, because it was assumed that this compound could be not a natural substrate. It was found that [14C]dihydrostreptomycinwas formed in the presence of the labelled methionine. N-Demethyldihydrostreptomycin also seemed to inhibit de novo biosynthesis of streptomycin. Also, because, in the presence of a larger excess of N-demethylstreptomycin and N demethyldihydrostreptomycin, both were converted into the corresponding methylated products in the presence of L-methionine, it was concluded that radioactivity had been incorporated by N-methylation of these substrates, and not by de no00 biosynthesis.61The interpreta(59)T. Kawamura, N. Ichihara, N. Eshimoto, and E. Eto, Biochem. Biophys. Res. Commun., 66, 1506-1512 (1975). (60) H. Heding, Acta Chem. Scand., 22, 1649-1654 (1968). (61) H.Heding and K. Bajpai,]. Antibiot., 26,725-727 (1973).

SUGAR COMPONENTS OF ANTIBIOTIC SUBSTANCES

109

tion of these results is, however, not unambiguous. Attempts to demonstrate methylation of N-demethyldihydrostreptomycin with Sadenosyl-~-['~C]methionine in a cell-free extract from S. griseus have thus far been

d. Assembling of the Streptomycin Molecule.-Walker and coWorkerP postulated that L-streptose and 2-deoxy-2-(methylamino)-~glucose are transferred from the corresponding "nucleotide sugars" to streptidine 6-phosphate and O-a-~-streptose-(144)-streptidine 6phosphate, respectively, to form streptomycin 6-phosphate. With the possibility of preparing dTDP-~-[U-'~C]dihydrostreptose~~ (see Section II,4),61a*62 the enzymic transfer of the dihydrostreptose moiety to streptidine 6-phosphate could now be tested experimentally.63 Streptidine phosphate was incubated with dTDP-~-[U-'~C]dihydrostreptose, obtained in s i t u from dTDP-~-[U-'~C]glucosewith a cellfree extract from s. griseus. Two new, positively charged, radioactive products were obtained which, upon hydrolysis, gave dihydrostreptose as the only radioactive product. Comparison with a synthetic sample of 0-a-L-dihydrostreptose-(1+4)-streptidine (43,see Scheme 14) proved that one of the products was identical with this pseudodisaccharide, and the second product was identified as the corresponding 6-phosphate (44). It was concluded that the phosphorylated product had been partially hydrolyzed during the incubation by streptomycin 6-phosphate phosphatase present in the cell-free extract. No transfer-products were formed in controls in which ( a ) denatured extract was used, ( b )streptidine phosphate was absent, or(c) streptidine was substituted for streptidine phosphate. From these results, it may be concluded that the pseudo-disaccharide 44 is the first intermediate in the assembling of the three components of dihydrostreptomycin. The fact that no transfer-product was observed with streptidine is in agreement with the conclusions from a number of studies in which the importance of phosphorylated intermediates in the biosynthesis of streptomycin was established.36,64,65

(61a) S. Maier and H. Grisebach, unpublished results. (62) M. S. Walker and J. B. Walker,J. B i d . Chem., 246, 7034-7040 (1971). (63) B. Kniep and H. Grisebach, FEBS Lett., 65,44-46 (1976). (64) J. B. Walker and M. Skorvaga,]. BioZ. Chem., 248,2441-2446 (1973). (65) 0.Nimi, H. Kiyohara, T. Mizoguchi, Y. Ohata, and R. Nomi,Agric. B i d . Chem., 34, 1150-1156 (1970).

HANS GRISEBACH

110

NH II

0

-t

I

+

dTDP

I ,

HO

I

OH

OR

HO

OH 43 R = H 44 R =-POSH,

SCHEME14.-Formation of 0-a-L-Dihydrostreptose-(1+4)-streptidine 6-Phosphate (44) from dTDP-L-dihydrostreptose and Streptidine 6-Phosphate with a Cell-free Extract from S . griseus.

2. Gentamicins The gentamicin complex, produced by Micromonospora sp., consists66of the three major components, gentamicins C,, C2,and C,a and a larger number of minor component^.^' The major C components differ only in their degree of methylation, and are closely related to the kanamycins. Minor components closely related to the C components are CZb,a 6’-(aminomethy1)gentamicinCla, and C2a,a 6’-methyl epimer of gentamicin C,. As shown, gentamicins Cla, C2,and C, respectively possess one C-methyl and one N-methyl group, two C-methyl groups and one N-methyl group, and two C-methyl and two N-methyl groups. When ~-[methyl-’~C]methioninewas added to a growing culture of Micromonospora purpurea, a high incorporation of radioactivity into gentamicins was observed.68 As expected, the radioactivity incorporated into gentamicins increased with the number of C-methyl and N-methyl groups in the molecule (Cia 3, C, 18, and C, 60% of the total incorporation). (66) D. J. Cooper, M. D. Yudis, R. D. Guthrie, and A. M. Prior,J. Chem. SOC., C. 960963 (1971). (67) P. J. L. Daniels, in “Drug Action and Drug Resistance on Bacteria,” S. Mitsuhashi, ed., Tokyo Univ. Press, Tokyo, 1975, pp. 77-111. (68) B. K. Lee, R. T. Testa, G. H. Wagman, C. M. Lin, L. McDaniel, and C. Schaffner, J . Antibiot., 26,728-731 (1973).

SUGAR COMPONENTS OF ANTIBIOTIC SUBSTANCES

111

OH Structures of Some Components of the Gentamicin Complex R' R2 R3 Gentamicin C, -NHMe H -Me -Me H -NH, Gentamicin C, H Gentamicin C,, H -m, Gentamicin C,, H H -NHMe Gentamicin C,, -Me -NH, H

Later studies, with ~-[methyl-'~C]methionine,in which the gentamicin C components were separated and then hydrolyzed to the individual sugars supported the earlier results.60 Further information on the origin of the methyl groups was obtained'O from experiments with ~-[methyl-'~C]methionineand L[methyl-2H,]methionine, and analysis of the n.m.r. and mass spectra of gentamicins C,, C2,and Cia. The 13C-n.m.r.spectrum of, for instance, gentamicin C, showed enrichment of the two C-methyl and two N methyl groups, proving the origin of all four methyl groups by transmethylation from L-methionine, probably by way of S-adenosyl-Lmethionine (see Section II,l,a,b). High-resolution, mass spectroscopy using the [M + 13' peak gave the following incorporation of deuterium in gentamicin C, : 1CD, 15.5%;2 CD, 3.7%; 3 CD, -1%; 4 CD, <0.5%. No peaks corresponding to the presence of CHDz or CHDz + CD3 groups in gentamicin could be detected. These results showed that methyl groups from methionine are incorporated substantially intact into all four positions of gentamicin C,. (69) B. K. Lee, R. G. Condos, A. Murawski, and G . H. Wagman,J.Antibiot., 28,163-166 (1975). (70) P. J. L. Daniels, Bloomfield, New Jersey, personal communication.

112

HANS GRISEBACH

The sequence of reactions shown in Scheme 15 may be postulated for the biosynthesis of L-garosamine (compare Section 1,3). A nucleotide-bound pentos-4-ulose (probably enzyme-bound) that could arise from the “nucleoside diphosphate (NucDP) D-glucuronic acid” (compare Scheme 11) is methylated at C-4. Transamination and N methylation then gives NucDP-L-garosamine.

Nucleoside 5 ‘-(D- glucosyluronic acid diphosphate)

OH

OH NucDP- L-garosamine

SCHEME 15.-Proposed Biosynthetic Sequence for L-Garosamine. [( 1) Transamination; (2) N-methylation.]

Studies on the biosynthetic relationships of the various gentamicins were conducted with mutants blocked in regard to formation of gentamicin. A mutant of Micromonospora purpurea blocked in synthesis of gentamicin p r o d ~ c e d ‘ the ~ pseudodisaccharide paromamine (see Scheme 16). The bioconversion of several gentamicins and related compounds isolated from the fermentation of M . purpurea was investigated to (71) R. T. Testa and B. C. Tilley,J. Antibiot., 29, 140-146 (1976).

SUGAR COMPONENTS OF ANTIBIOTIC SUBSTANCES

113

determine which, if any, of these may be precursors of the gentamicin C group. The products formed by these transformations indicated71that the mutant was able to carry out the enzymic steps leading to the gentamicin C components from gentamicin A (see Scheme 16).On the basis of similar studies with a deoxystreptamine-negative mutant of Micromonospora inyoensis, a biosynthetic scheme for sisomycin (45), a dehydrogentamicin, was proposed.72 Biotransformation studies also indicated that transformation of gentamicin X p (46) into antibiotic G418 (47) and gentamicin C,requires cobalt ions, and that, therefore, the 6’-methylation is a cobalt-ion-dependent step.73Furthermore, the step in the biosynthetic pathway from gentamicin A (48) to gentamicin X p (46), involving 4”-methylation and hydroxyl epimerization, is also cobalt-ion-de~endent.’~

F2

-6

R’

-R3

/ 1

0 I

kH2

OH

OH Sisomycin

45

46 Gentamicin X,

R’ = H , R2 = H ,

R3 = O H , R4 = CH,, R5 = OH 47 Antibiotic G-418 R1 = O H , R2 = CH,, R3 = H , R4 = CH,, R5 = O H R’ = H , Rz = H , R3 = O H , 48 Gentamicin A R4 = O H , R5 = H

The data obtained in these biotransformation studies have to be interpreted with some degree of caution. For example, the possibility that gentamicin A might be broken down to paromamine, or even to 2deoxystreptamine, before incorporation into later products has not been excluded. It is known that micro-organisms do degrade their own secondary metabolites. Scheme 16 would, for instance, imply that Lgarosamine is not formed as a nucleotide-bound sugar as depicted in Scheme 15, but by modification of the a-D-xylosyl group of gentamicin (72) R. T. Testa and B. C. Tilley,]. Antibiot., 28,573-579 (1975). (73) B. C. Tilley, M. Sc. Thesis, Department of Biology, Seton Hall University, South Orange, N. J., 1976.

114

HANS GRISEBACH

SUGAR COMPONENTS OF ANTIBIOTIC SUBSTANCES

t

115

C-methylation, with inversion of configuration, at C-4"

Gentamicin X,

Il-

I

l

C-methylation at C-6', with inversion of configuration

1

I

t Antibiotic G-418 amino substitution at C-6'

t Antibiotic JI-20-A

t-

-4 t

Antibiotic JI-20-B

3', 4 '-dehydroxylation

c

Gentamicin C,,

Gentamicin C,, epimerization at C-6'

i

Gentamicin C,

N-methylation at C-6'

Gentamicin C,,

-I

Gentamicin C,

SCHEME 16.-Postulated Pathway from the Building Units of Gentamicin A2 to Various Gentamicins, as Concluded from Transformation Studies with Mutants.

A2. However, by analogy with the biosynthesis of other methylbranched sugars (see Section II,l), C-methylation of a nucleotidebound pentos-4-ulose seems more likely. From the results thus far obtained, parallel pathways to gentamicins A and C , instead of the sequential pathway shown in Scheme 16, cannot be excluded. Labelling experiments and, even more important, studies of enzymic transformations in cell-free systems are therefore needed in order to clarify the biosynthetic relationships between the gentamicins.

3. Neomycin The biosynthesis of neomycin (see Scheme 17) has been studied by Rinehart and ~ o w o r k e r s .Early ~ ~ incorporation studies with ~-[1-

116

HANS GRISEBACH

14C]glucose,D-[6-14C]glucose, and 2-amino-2-deoxy-D-[l-14C]g~ucose led to some ambiguities in interpretation of the biosynthetic pathway due to the lack of a satisfactory degradation scheme for deoxystreptamine (cited in Ref. 53). The incorporation O f D-glucose and 2-amino2-deoxy-D-glucose was, therefore, reexamined, employing carbon-13 were labels.s3D-[6-13C]Glucoseand 2-amino-2-deoxy-D-[l-13~]glucose administered in separate experiments to cultures of Streptomyces fradiae. Neomycin was isolated 5 days after addition of the precursor. Neomycin B was separated from small proportions of neomycin C and neamine, and was then converted into hexa-N-acetylneomycin. The 13C-n.m.r. spectrum of the acetyl derivative showed that [6J3C]glucose had labelled C-6 of the neosamine moieties, C-5 of the D-ribosyl residue, and C-2 of deoxystreptamine, whereas 2-amino-2-deoxy-[11 3 C l g l ~ ~ olabelled se C-1 of each of the subunits (see Scheme 17).

r

Deoxystreptamine D -Ribose

I

m*

Neosamine B (C') Neomycin B

SCHEME 17.-Labelling pattern of Neomycin B After Feeding of ~-[6-'~C]Ghcose and 2-Amino-2-deoxy-~-[l-'~C]glucose. (The incorporation of 2-amino-2-deoxy-D-gluco~e probably occurs by way of D-[l-'3C]glucose; X = H, Y = CHzNHz.)

The results indicated a specific conversion of C-6 of D-glUCOSe into C-5 of D-ribose, explicable by the hexose monophosphate pathway, and suggested conversion of C-1 of 2-amino-2-deoxy-D-g~ucoseinto C-1 of D-ribose, which could be explained by the conversion of 2amino-2-deoxy-D-glucose into D-glucose, and the operation, in S. frudiue, of some version of the glucuronate pathway for the removal of C-6 of D-glucose.

SUGAR COMPONENTS OF ANTIBIOTIC SUBSTANCES

117

The labelling pattern in deoxystreptamine was unexpected, because it differed from that found for ~ t r e p t i d i n e(see ~ ~ Section II1,l). The labelling of C-2 (rather than of C-6) of deoxystreptamine by ~ - [ 6 '3C]glucose was explained by a new biosynthetic pathway (see Scheme 18, pathway B) involving cyclization of D-glucose to a cyclitol, perhaps deoxyinosose (my0-[2-'~C]inositolwas not incorporated into neomyin^^), followed by amination at the carbonyl carbon atom. From this stage on, the results could be explained if synthesis of deoxystreptamine involves oxidation and subsequent amination at the &carbon atom in the direction opposite to that reported for the biosynthesis of streptamine.

___t

HO OH Streptamine

OH

OH Deoxystreptamine

SCHEME18.-Proposed Biosynthetic Pathway from an Intermediate Inosamine to (A) Streptamine (compare Scheme 13) and (B) Deoxystreptamine. (For pathway A, the intermediate inosamine has X = OH, and, for pathway B, X = H or OH.)

The incorporation of 2-amino-%deoxy-D-[l-'3C]glucose into deoxystreptamine is probably not direct, but takes place after deamination to D-[ 1-W]glucose. Experiments with mutant strains of S. fradiae that only produce neomycin in the presence of added deoxystreptamine have been , ~ ~will not be discussed here. reviewed b y Rinehart and S t r ~ s h a n eand [l-14C]Deoxystreptaminewas also incorporated into the deoxystrepta. ~ ~the other hand, no incorporation of [lmine moiety of n e ~ m y c i nOn 14C]neosamineC into neomycin was found, probably because this sugar cannot be activated as the ester of a n ~ c l e o t i d e . ~ ~ In a study on the effect of nitrogen compounds on the production of neomycin, some phosphoramido-amino sugar antibiotics were isolated . ~ ~compounds were separated, and characterized as from S . f r ~ d i a eThe neomycin B pyrophosphate, neomycin C pyrophosphate, and neomycin C dipyr~phosphate.'~ From the analytical data, it may be concluded (74) M. K. Majumdar and S. K. Majumdar,]. Antibiot., 32, 174-175 (1969). (75) M. K. Majumdar and S . K. Majumdar, Biochem.]., 120,271-278 (1970).

HANS GRISEBACH

118

that the pyrophosphate groups are linked to nitrogen as pyrophosphoramido derivatives. Together with the finding that phosphatases that can hydrolyze phosphoramidoneomycins are present in S . fradiae, and that enzyme activity is related to antibiotic production,'6 it appears that these phosphate derivatives might be biosynthetic intermediates. 4. Spectinomycin Spectinomycin (see Scheme 19) has a tricyclic structure, and contains actinamine. In aqueous solution, the carbonyl group is present in hydrated form. The biosynthesis of spectinomycin was examined by feeding various, labelled precursors to Streptomyces flavopersicus.77 The percent incorporation of some potential precursors is listed in Table I. TABLEI

Incorporation of Potential Precursorsa into Spectinomycin Compound administered ~-[Methyl-'~C]methionine ~[6-~H]Glucose rny0-[2-'~C]Inositol [2-14C]Actinamine

Time isolated

(h)

Isotope incorporated (%)

48 48 24 72

39 3.5 47 6.6

aThe labelled precursors were added after 69 h, and the antibiotic was isolated after the time period shown.

[2-14C]Acetate and D-[U-'4C]galactose gave no significant incorporation, although galactose considerably stimulated the production of spectinomycin. After recrystallization of the antibiotic substance to constant specific activity, it was degraded according to Scheme 19. Label from my0-[2-'~C]inositol was found exclusively in the actinamine moiety. Label from ~-[methyl-'~C]methioninewas also found was an only in the N-methyl groups of actinamine. ~-[6-~H]Glucose excellent precursor, and its radioactivity was almost equally distributed between rings A and C of the molecule. Essentially all of the tritium in ring C was associated with the C-methyl group of spectinomycin. (76) M. K. Majumdar and S. K. Majumdar, Biochern. J., 122, 397-404 (1971). (77) L. A. Mitscher, L. L. Martin, D. R. Feller, J. R. Martin, and A. W. Goldstein, Chern. Cornrnun., 1541-1542 (1971).

SUGAR COMPONENTS OF ANTIBIOTIC SUBSTANCES

Hu NHMe I

NHMe

NHMe

I

I

Actinamine

+ I

CO,H

1

Spectinomycin

119

Actinospectinoic acid

2 CH,NH,

CH,CH=CHCCH,OH I1 0

+ "A

L-2

CrO,

CH,CO,H

SCHEME19.-Chemical Degradation of Spectinomycin for Localization of the Label. (Derivatization of degradation products is not shown.)

Radioactive actinamine prepared by cleavage of labelled spectinomycin formed biosynthetically from myo-[2-'*C]inositol was incorporated lnto spectinomycin to a considerably lower extent than myoinositol itself. The interpretation of the authors7?that this result argues against actinamine's being a direct precursor of spectinomycin is not in agreement with a mutant study in which it was shown that a mutant unable to produce the antibiotic could do so in the presence of a ~ t i n a m i n eEvidence .~~ was also presented that N-methylation is the last step in the biosynthesis of a ~ t i n a m i n e . ~ ~ As the label from D-[6-3H]glucose could not be located in actinamine by the procedure of Scheme 19, Rinehart and coworkers undertook feeding experiments with D-[6-13C]gl~~ose.79 In confirmation of the results of Mitscher and coworkers,77 enrichment in carbon-13 was found at (2-6' of actinospectose, and at C-6 of actinamine (see Scheme 20). The label at C-6 of actinamine is similar to the labelling44 of streptidine with D-[6-'3C]glucose (see Scheme 13). From the mother liquors of the crystallization of spectinomycin was isolated a compound that was identified as dihydrospectinomycinsO (see Scheme 21). The n.m.r. spectrum of the pentaacetyl derivative (78) L. Slechta and J. H. Coats,AAbstr.Interscience Conf. Antirnicrob. Agents Chemother. 14th, Sun Francisco, Calif., Sept. 1974, 294. (79) R. M. Stroshane, M. Taniguchi, K. L. Rinehart, Jr., J. P. Rolls, W. J. Haak, and B. A. R ~ i f f , j Am. . Chem. Soc., 98,3025-3027 (1976).

HANS GRISEBACH

120

o’/ SCHEME20.-Carbon Atoms of Spectinomycin Labelled by ~-[6-~~C]Glucose.

proved that the epimer produced by Streptomyces spectabilis was that represented by the structure depicted in Scheme 21. In Scheme 21, a sequence of reactions is shown in which dihydrospectinomycin is the precursor of spectinomycin.sOHowever, the conversion of dihydrospectinomycin into spectinomycin, or vice versa, has not yet been shown.

5. Validamycin Validamycins A, C, D, E, and F contain validoxylamine A (49) as a common moiety in their molecule, but differ from one another in at least one of the following characteristics: the configuration of the anomeric center of the D-glucoside, the position of the D-glucosidic linkage, and the number of D-glucosyl groups. Kameda and coworkerss1studied the HOH,C

HOH,C,

)=-7

Q

HO

HO

I

NH

OH

Validoxy lamine

Validamycin A

49

50

(80) H. Hoeksema and J. C. Knight,./. Antibiot., 28, 240-241 (1975). (81) Y. Kameda, S. Horii, and T. Yamano,J. Antibiot., 28, 298-306 (1975).

SUGAR COMPONENTS OF ANTIBIOTIC SUBSTANCES

121

+

"@OH

OH

J OH

by way of enediol

OH

I

YHMe

NHMe I

HO Spect inomy c in

SCHEME2l.-Postulated

OH

Dihydrospectinomycin

Pathway for the Biosynthesis of Spectinomycin by way of

Dihydrospectinomycin.

incorporation of D-[U-14C]glucose into validamycin A (50). High incorporation (- 10%) and approximately uniform distribution of radioactivity were observed between the three moieties (validamine, valienamine, and D-glucose) of the antibiotic. When [14C]validoxylamine was added to a culture of Streptomyces hygroscopicus var.

122

HANS GRISEBACH

limonues, all radioactivity was found in the validoxylamine A moiety, and virtually no label was found in the D-glucose portion. It was, therefore, assumed that the D-glucose moiety is added last in the biosynthesis of validamycin A. Support for this assumption came from experiments in which the pD-glucosylation of validoxylamine A to give validamycin A was shown by use of certain yeast strains in a medium containing cellobiose as the p-D-glucosyl donor.s1 In this way, the semi-synthesis of several validamycins was achieved by using validoxylamine A as the acceptor and a disaccharide as the glycosyl donor. However, it is more likely that, in the biosynthesis of validamycins, not a disaccharide but a nucleoside 5‘-(a-D-glucosyldiphosphate) acts as the D-glucosyl donor.

I v . AMINOSUGARS NOT OCCURRING IN AMINOCYCLITOL ANTIBIOTICS 1. Desosamine and Mycaminose D-Desosamine (51) and D-mycaminose (52) occur in macrolide antibiotics.2 Early studies with tracers showed that the hexose portion of these sugars can be derived from D-glucose without inversion or breakdown of the sugar chain.B2*83 The N-methyl groups are derived from r n e t h i ~ n i n e .No ~ ~further . ~ ~ work on the biosynthesis of these sugars has been reported.

I

I

OH

OH 51

52

V. NUCLEOSIDE ANTIBIOTICS Nucleoside antibiotics contain a number of unusual sugar components. The chemistry, biosynthesis, and biochemistry of these antibiotics has been described in a book by Suhadolniks5in which the literature is covered up to 1969. In Table 11, significant results on the (82) H. Achenbach and H. Grisebach, Z. Naturforsch. Teil B , 19,561-568 (1964). (83) J. C. Butte and J. W. Corcoran,Fed. Proc. Fed. Am. SOC. E x p . Biol., 21,89 (1962). (84) H. Grisebach, H. Achenbach, and W. Hofheinz, Tetrahedron Lett., 234-237 (1961). (85) R. J. Suhadolnik, “Nucleoside Antibiotics,” Willey-Interscience, New York, 1970.

TABLEI1 Biosynthesis of Sugar Components from Nucleoside Antibiotics Antibiotic Cordycepin (3'deoxyadenosine)

3'-Amino-3'deoxyadenosine

Sugar moiety

Experimental evidence for biosynthesis ~-[6-'~C]glucose + incorporation into sugar portion D-[ l-14C]ribose+ no incorporation [U-'4C]adenosine + incorporation into cordycepin without change in the adeninekibose activity ratiosga Enzymic mechanism by which reduction of the 3'-hydroxyl group occurs is not known [U-"C]adenosine + incorporation into 3'-amino-3'-deoxyadenosinewithout cleavage of the ribosyl carbonnitrogen bondsgb

Psicofuranine [6-amino-g-(PD-pSiCOfUran0syl)purine]

~-[U-'~C]glucose+ direct precursor for psicose moiety ~-[1-'~C]glucosewas converted, in a cell-free extract of S . hygroscopicus in the presence of ATP and Mg2+, into psicosesgc

Decoyinine [6-amino-9-(6deoxy-P-& erythro-&hex5-enulofuranosy1)purinel

[U-'4Cbsicofuranine was converted into [U-14C]decoyininewithout cleavage of the N-glycos ylic bondsgd

Arabinos yl nucleosides

9-p-D-arabinofuranosyladenine can b e formed by direct epimerization at C-2' of adenosinesa (see text)

(85a) R. J. Suhadolnik, G. Weinbaum, and H. P. Meloche,J. Am. Chem. Soc., 86,948949 (1964). (85b) B. M. Chassy and R. J. Suhadolnik, Biochim. Biophys. Acta, 182,316-321 (1969). (85c) R. J. Suhadolnik and T. Sugimori, Fed. Proc. Fed.Am. SOC. E x p . Biol., 25, 525 (1966). (85d) B. M. Chassy, T. Sugimori, and R. J. Suhadolnik, Biochim. Biophys. Acta, 130,1218 (1966). (86) P. B. Farmer, T. Uematsu, H. P. C. Hogenkamp, and R. J. Suhadolnik,]. Biol. Chem., 248, 1844-1847 (1973).

124

HANS GRISEBACH

biosynthesis of the sugar moieties of nucleoside antibiotics are summarized. For each example, a leading reference is given. Since the appearance of Suhadolnik’s book, very little new information on the biosynthesis has become available. Farmer and coworkers have further investigated the biosynthesis of ~-P-Darabinofuranosyladenine by Streptomyces antibioticus.86Nitrogen-15 and carbon- 14 from [6-amino-l5N,U-14C]adenosine were incorporated into the arabinosyladenine with -17% efficiency. The 15N:14Cratios of arabinosyladenine and of the precursor were the same. To show that there is no hydrolysis of the N-ribosyl bond of adenosine, the percent distribution of carbon-14 in the adenine of arabinosyladenine was compared with that in the adenine of [U-14C]adenosineadded to the culture filtrates. The percent of carbon-14 in the adenine residue of the two compounds was the same. On the basis of these results, and others not discussed here, it was thus proved that the amino nitrogen atom of adenosine is retained as the amino nitrogen atom of arabinosyladenine, and that the N-ribosyl bond is not cleaved during conversion into arabinosyladenine. It was further shown that 2’-deoxy-[U-14C]adenosine is not an intermediate in the conversion of adenine into arabinosyladenine. To determine the fate of the 2’- and 3’-hydrogen atoms of adenosine in the conversion of adenosine into arabinosyladenine, experiments with [2’-3H, U-14C]adenosine and D-[3-3H, U-14C]ribose were performed. Arabinosyladenine derived from [2’-3H,U-14C]adenosine had lost all of its tritium, whereas adenosine isolated from the RNA of the organism had about the same 3H:14Cratio as the added adenosine. In the D-[3-3H, U-14C]ribose experiment, the proportion of tritium in arabinosyladenine was only 33% of that present in the adenosine from

RNA. The results indicated that, during the epimerization of adenosine to arabinosyladenine, oxidation to a 2’-keto-nucleoside takes place. The low percentage of tritium in the arabinosyladenine from D-[3-3H, U14C]ribosemay be explained by the formation of an enolic intermediate. The reversibility of the adenosine-arabinosyladenine reaction could not be shown in z)iz)o, because [U-l4C]arabinosy1adenine was hydrolyzed by S. antibioticus to adenine. Six novel, cytosine nucleosides were isolated from the fermentation broth of S. griseochromogenes, which produces blasticidin S (55, see Scheme 22). The structural relationship between these nucleosides and cytosinine, the nucleoside moiety of blasticidin S, was investigated.” One of these compounds was identified as l-(P-D-glucopyranosyluronic acid)cytosine (53). The presence of 53 was taken as evidence (87) H. Seto, K. Furihata, and H. Yonehara,J. Antibiot., 29, 595-596 (1976).

SUGAR COMPONENTS OF ANTIBIOTIC SUBSTANCES

125

that this compound is the direct precursor of a hypothetical intermediate (54 in Scheme 22), and that oxidation of the hydroxymethyl group of the hexose moiety takes place prior to oxidation of the hydroxyl group at C-4. g glucose

+

Cytosine

HO

RHN OH

OH

53

SCHEME22.-Postulated

54

55

Pathway for Biosynthesis of Blasticidin S (55, R

= blastidyl).

Interestingly, the sugar moiety of the postulated intermediate 54 is identical to that of the intermediate in the biosynthesis of UDP-Dapiose from UDP-D-glucuronic acid (27, see Scheme 11). The polyoxins (56) formed by S. cacaoi contain a 5-amino-5-deoxy-Dallofuranosyluronic acid residue. The biosynthesis of the polyoxins 0 II

O=CHN-CH HCOH I HOCH

I

HO

OH

CH,OCNH,

8

Polyoxin L 56

126

HANS GRISEBACH

TABLEI11 Distribution of Carbon-14 in Polyoxin Having Various Potential Precursorss8 ______

~~

Compound added

[U-14C]Uridine &[l-'4C]Ribose D[ l-'4C]Allose D-[l-'4C]Ghcose D-[6-14C]Glucose D[3,4-14C2]Glucose [1,3-14C2]Glycerol

______

Distribiition of 14C in nucleoside Pyrimidine base (%)

Uronic acid (%)

100 13 0 31 28 11 35

0 87 0 69 72 89 65

~~

Distribution of '4C in uronic acid C-1'-5'

C-6'

(%I

(%)

-

-

95

5

-

-

78 79 98 80

22 21 2 20

was investigated with 14C-labelled precursors.88 The distribution of carbon-14 (from the labelled compounds) incorporated into the uracil and 5-amino-5-deoxy-~-a~~ofuranosyluronic acid residues is shown in Table 111. The results obtained make it unlikely that the glycosyluronic acid residue is formed directly from either D-glucose, D-allOSe, or Dribose. Although the [14C]uracil residue from [U-14C]uridine was incorporated into the polyoxins, the ~-['~C]ribosylresidue did not contribute to the biosynthesis of the uronic acid by the addition of a one-carbon unit. D-[l-'4C]Glucose and D-[6-'4C]glucose gave the same distribution of carbon-14 in C-1 and C-6 of the uronic acid. This distribution of carbon-14 favors the theory of an aldolase pathway, followed by resynthesis of a hexose, and subsequent oxidation to the uronic acid. However, further investigations are needed in order to clarify the biosynthesis of the sugar portion of the polyoxins.

(88) K. Isono and R. J. Suhadolnik, Arch. Biochem. Biophys., 173,141-153 (1976).

THE LECTINS: CARBOHYDRATE-BINDING PROTEINS OF PLANTS AND ANIMALS*

BY IRWINJ . GOLDSTEIN AND COLLEEN E . HAYES Department of Biological Chemistry. The University of Michigan. Ann Arbor. Michigan 48109; lmmunobiology Research Center. University of Wisconsin. Madison. Wisconsin 53706

I . Introduction .......................................................... 1. Detection of Lectins ............................................... 2. Isolation and Purification of Lectins ................................. 3. Carbohydrate-binding Specificity of Lectins ......................... 4. Nomenclature of Lectins ........................................... 5. Function of Lectins ................................................ 11. D-Mannose(D-Glucose)-binding Lectins ................................ 1. Concanavalin A of the Jack Bean (Canaualia ensiformis) ............. 2. Lens culinaris syn . esculenta (Lentil) ................................ 3. Pisum sativum (Pea) .............................................. 4. Viciafaba (Fava Bean) ............................................. I11. 2-Acetamido-2-deoxy-~-glucose-binding Lectins ........................ 1. Bandeiraea simplicifolia I1 ......................................... 2. Cytisus sessilifolius ................................................ 3. Solanum tuberosum (Potato) ........................................ 4. Triticum uulgaris (Wheat Germ) .................................... 5. Ulex europeus I1 (Gorse or Furze Seed) ............................. IV . 2-Acetamido-2-deoxy-~-galactose-binding Lectins ....................... 1. Dolichos bijlorus (Horse Gram) ..................................... 2. Glycine max (Soybean) ............................................. 3. Helix pomatia (Edible Snail) ....................................... 4. Phaseolus lunatus syn. limensis (Lima Bean) ........................ 5. Sophora japonica (Japanese Pagoda Tree) ........................... V. D-Galactose-binding Lectins ............................................ 1. Abrus precatorius (Jequirity Bean) .................................. 2. Arachis hypogaea (Peanut) ......................................... 3. Bandeiraea simplicifolia I .......................................... 4. Maclura pomifera syn. aurantica (Osage Orange) .................... 5. Ricinus communis (Castor Bean) ....................................

128 133 136 139 145 146 150 150 190 196 201 206 206 208 210 214 224 226 226 231 239 243 250 254 254 257 262 267 270

* Writing of this article was supported. in part. by a grant (AM-10171) from the National Institutes of Health . The authors are grateful for the assistance of the Editors and of Paula Kane and Peggy Rogers in the preparation of this Chapter . 127

128

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

VI. L-Fucose-binding Lectins ...................... .......... 1. Anguilla anguilla (Eel Serum) ...................................... 2. Lotus tetragonolobus (Asparagus Pea) . ........................... 3. UZex europeus I (Gorse or Furze Seed) .............................. VII. Other Lectins ....... ............................................. 1. Phaseolus vulgaris (Red Kidney-bean) ............. 2. Vicia graminea .................................................... 3. Miscellaneous Lectins ........ .................................. VIII. Cell-surface, Lectin-reactive Glyc teins ............... ...... 1. Erythrocytes and Platelets .......................................... 2. Lymphocytes ............. .................................... 3. Neuronal Cells ............................................ 4. Tumor Cells ....................................................... IX. Tables . . . ................................ ...............

277 277 282 289 291 291 302 304 317 3 18 325 326 327 334

I. INTRODUCTION Stillmark’s discovery,’ in 1888, that castor-bean extracts agglutinate animal erythrocytes, marked the initiation of studies on plant agglutinins. The hemagglutinating activity of seed extracts was traced to the presence of discrete proteins (glycoproteins), variously termed agglutinins, hemagglutinins, phytohemagglutinins ,and, most recently, lectins. Boyd and Shap1eigh2a3coined the term lectin (Latin, legere, to select or pick out), based on their observation that some seed extracts could distinguish among human b l o o d - g r ~ u p s . ~Specifically, -~ Boyd and Reguera4 discovered that lima-bean (Phaseolus lunatus) extracts selectively agglutinate type A erythrocyte^.^,^ Almost simultaneously, Renkoned made the same observation, reporting several bloodgroup-specific seed-extracts among 57 species belonging to 28 different genera. These discoveries stimulated renewed interest in the isolation and characterization of lectins, and initiated a seemingly endless number of applications of these fascinating plant-proteins to biological studies. Lectins have played an important role in the development of immunology. In 1891,Ehrlich7 showed that specific immunity to the toxic (1) H. Stillmark, “Uber Rizin, ein gifiiges Ferment aus dem Samen von Ricinus communis L. und einigen anderen Euphorbiaceen,” Inaug. Diss., Dorpat. 1888. (2) W. C. Boyd and E. Shapleigh,]. Immunol., 73,226-231 (1954). (3) W. C. Boyd and E. Shapleigh, Science, 119, 419 (1954). (4) W. C. Boyd and R. M. Reguera,]. Immunol., 62,333-339 (1949). (5) W. C. Boyd, “Introduction to Immunochemical Specificity,” Wiley-Interscience, New York, 1962, pp. 1-158. (6) K. 0. Renkonen, Ann. Med. Exp. Biol. Fenn., 26,66-72 (1948). (7) P. Ehrlich, Dtsch. Med. Wochenschr., 17, 976-979, 1218-1219 (1891).

LECTINS: CARBOHYDRATE-BINDING PROTEINS

129

lectins ricin (Ricinus communis) and abrin (Abrus precatorius) could be achieved by repeated injection of small amounts of these antigens into white mice. The first quantitative determination of an antibody in vitro was performed by Ehrlich8 in 1897; immune serum inhibited ricin-red blood-cell agglutination. Furthermore, abrin was employed by Landsteinergin an early demonstration of the reversibility of the antibodyantigen reaction; abrin was displaced from agglutinated erythrocytes by incubating at 50°, and then centrifuging the cells. Currently, lectins find application in serological laboratories for typing blood and determining secretor s t a t ~ s , ’ ~ separating -*~~ leucocytes from erythrocytes,1sand agglutinating cells from blood in the preparation of plasma.16 Moreover, lectins serve as reagents for the detection, isolation, and characterization of carbohydrate-containing macromolecules, including blood-group Enormous interest centers at present on probing the nature and distribution of membrane-bound, carbohydrate-containing structures by using lectins The distribution and mobility of cellof defined specificity. surface glycoproteins on normal and malignant cells has been investigated25by employing lectins labelled with ferritin, fluorescein, or 16325-27

(8) P. Ehrlich, Fortschr. Med., 15,41-43 (1897). (9) K. Landsteiner, Wien. Klin. Wochenschr., 14, 713-714 (1901). (10) G. W. G. Bird, Br. Med. Bull., 15, 165-168 (1959). (11) W. C. Boyd, Vox Sang., 8 , l - 3 2 (1963). (12) W. C. Boyd and E. Shapleigh,J. Lab. Clin. Med., 44,235-237 (1954). (13) W. C. Boyd and E. Shapleigh, Blood, 9, 1195-1198 (1954). (14) R. R. Race and R. Sanger, “Blood Groups in Man,” Blackwell, Oxford and London, 6th Edition, 1975, pp. 1-659. (14a) 0. Prokop and G. Uhlenbruck, “Human Blood and Serum Groups” (Translated by J. L. Raven), Maclaren, London, 2nd Edition, 1965, pp. 1-891. (15) J. G. Li and E. E. Osgood, Blood, 4,670-675 (1949). (16) M. Dorset and R. R. Henley,J. Agric. Res. (Washington, D.C.), 6,333-339 (1916). (17) I. J. Goldstein, Methods Carbohydr. Chem., 6, 106-119 (1972). (18) H. Lis and N. Sharon, Annu. Rev. Biochem., 42,541-574 (1973). (19) T. Osawa, Biochim. Biophys. Acta, 115, 507-510 (1966). (20) 0. Makela, Nature, 184, 111-113 (1959). (21) W. M. Watkins and W. T. J. Morgan, Nature, 169, 825-826 (1952). (22) W. T. J. Morgan and W. M. Watkins, Br. J . E x p . Pathol., 34,94-103 (1953). (23) W. T. J. Morgan and W. M. Watkins, Nature, 177,521-522 (1956). (24) W. M. Watkins, in “Glycoproteins,” A. Gottschalk, ed., Elsevier, Amsterdam, 2nd Edition, 1972, Part B, pp. 830-891. (%a) T. Kristiansen, Methods Enzymol., 34 (Part B), 331-341 (1974). (25) G. L. Nicolson, Znt. Rev. Cytol., 39, 89-190 (1974). (26) N. Sharon and H. Lis, Methods Membrane B i d . , 3,147-200 (1975). (27) N. Sharon, in “Extracellular Matrix Influences on Gene Expression,” H. C. Slavkin and R. C. Greulich, eds., Academic Press, New York, 1975, pp. 479-487.

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IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

isotopes. Certain lectins distinguish normal from malignant cells18,25-31; the possible use of lectins in cancer chemotherapy has been proIn their interaction with saccharides, lectins serve as models for carbohydrate-specific antibodies, with the important advantage that it is possible to purify lectins in gram quantities. Furthermore, lectin combining-sites appear to be homogeneous and noninteracting, in contrast to those of immune antibodies. Nowell’s that extracts of red kidney-bean stimulate lymphocyte division in vitro led to enormous interest in mitogenic lectins as tools for studying the biochemical events accompanying cellular growth, differentiation, and d i ~ i s i o n . ~ ~ - ~ ~ ~ Investigations using lectins to probe membrane-bound hormonereceptors have been spurred by the observation that certain lectins (for example, wheat-germ agglutinin, concanavalin A, and lima-bean and lentil lectins) are as effective as insulin in enhancing transport of D-glUCOSe and in inhibiting epinephrine-stimulated lipolysis in isolated adipocytes. 38-40 Finally, lectins are being studied with respect to toxic properties that may affect the nutritional value of b e a n ~ . ~ l - ~ ~ (28) J. C. Aub, C. Tieslau, and A. Lankester, Proc. Natl. Acad. Sci. U.S.A.,50,613-619 (1963). (29) J. C. Auh, B. H. Sanford, and M. N. Cote, Proc. Natl. Acad. Sci. U.S.A.,54,396-399 (1965). (30) M. M. Burger and A. R. Goldberg, Proc. Natl. Acad. Sci. U.S.A.,57,359-366 (1967). (31) M. Inhar and L. Sachs, Proc. Natl. Acad. Sci. U.S.A., 63, 1418-1425 (1969). (32) J.-Y.Lin,K.-Y.Tsemg, C.-C. Chen, L.-T. Lin, andT.-C. Tung,Nature, 227,292-293 (1970). (33) H. Lin, W. R. Bruce, and M. J. Walcroft, Cancer Chemother. Rep., 59, 319-326 (1975). (34) P. C. Nowell, Cancer Res., 20,462-466 (1960). (33) C. K. Naspitz and M. Richter, Prog. Allergy, 12, 1-85 (1968). (36) N. R. Ling, “Lymphocyte Stimulation,” North-Holland, Amsterdam, 1968, pp. 1-290. (37) G. Moller, Transplant. Rev., 11, 1-216 (1972). (37a) N. Sharon, in “Mitogens in Immunobiology,” J. J. Oppenheim and D. L. Rosenstreich, eds., Academic Press, New York, 1976, pp. 31-41. (37b) H. Lis and N. Sharon, in “The Antigens,” M. Sela, ed., Academic Press, New York, 1977, Vol. 4, pp. 429-529. (38) P. Cuatrecasas and G. P. E. Tell, Proc. Natl. Acad. Sci. U.S.A.,70,485-489 (1973). (39) M. P. Czech and W. S. Lynn, Biochim. Biophys. Acta, 297, 368-377 (1973). (40) H. M. Katzen and D. D. Soderman, Biochemistry, 14,2293-2298 (1975). (41) H. J. H. de Muelenaere, Nature, 206,827-828 (1965). (42) I. E. Liener,]. Agric. Food Chem., 22, 17-22 (1974). (43) A. T. Andrews and D. J. Jayne-Williams, Br. I . Nutr., 32, 181-188 (1974). (44) W. G. J d b , in “Toxic Constituents of Plant Foodstuff,” I. E. Liener, ed., Academic Press, New York, 1969, pp. 69-101. (45) I. E. Liener, Annu. Reo. Plant Physiol., 27,291-319 (1976). (46) R. H. Turner and I. E. Liener,]. Agric. Food Chem., 23,484-487 (1975).

LECTINS: CARBOHYDRATE-BINDING PROTEINS

131

Definition of the term lectin is a difficult problem in limiting the scope of this article. By employing the term lectin, Boyd and Shapleigh2-3,swished to call attention to the serological specificity of certain plant-seed agglutinins. After it became apparent that the activity of many hemagglutinins is inhibited by simple sugars,21*22 these proteins were commonly referred to as plant-seed, carbohydrate-binding proteins. However, the discovery of hemagglutinins in such diverse l i ~ h e n s ,fish ~ ~ r, ~ ~ e:~-~~ sources as b a ~ t e r i a , ~ ~ * ~ * eels,21*64-66 and even mammal^^'-^^^ has resulted in a broadening of the term “lectin” to include carbohydrate-binding proteins (glycoproteins) without regard to their origin. For the purpose of this article, carbohydrate-binding proteins of known enzymic function, carbohydrate-transport proteins, and carbohydrate-specific antibodies are not considered to be lectins. Moreover, we have included only those lectins that have been purified to homogeneity, and studied with regard to their biophysical, biochemical, and carbohydrate-binding specificity. This arbitrary decision regrettably necessitated the exclusion of an enormous number of interesting studies conducted with crude seed-extracts. (47) E. Neter, Bacteriol. Reu., 20, 16G-188 (1956). (48) N. Gilboa-Garber, Biochim. Biophys. Acta, 273, 165-173 (1972). (49) W. W. Ford,]. Pharmacol. E x p . Ther., 2,285-318 (1910-11). (50) M. Coulet, J. Mustier, and J. Guillot, Rev. Mycol., 35, 71-89 (1970). (51) H. J. Sage and S. L. Connett,]. Biol. Chem., 244, 4713-4719 (1969). (52) Y. Fujita, K. Oishi, K. Suzuki, and K. Imahori, Biochemistry, 14,4465-4470 (1975). (53) E. Estola and K. 0. Vartia, Ann. Med. E r p . Biol. Fenn., 33,392-395 (1955). (54) M. L. Howe and J. T. Barrett, Biochim. Biophys. Acta, 215,97-104 (1970). (55) 0. Prokop, D. Schlesinger, and G. Geserick, Z. Immunitaetsforsch. Allerg. Klin. Immunol., 132,491-494 (1967). (56) G. Uhlenbruck and 0. Prokop, Vox Sang., 12,465-466 (1967). (57) D. J. Anstee, P. D. J. Holt, and G. I. Pardoe, Vox Sang., 25, 347-360 (1973). (58) 0. Prokop, A. Rackwitz, and D. Schlesinger,]. Forensic Med., 12, 108-110 (1965). (59) A. Rackwitz, D. Schlesinger, and 0. Prokop, Acta Biol. Med. Ger., 15, 187-190 (1965). (60) 0. Prokop, D. Schlesinger, and A. Rackwitz, Z. Immun. Allergieforsch., 129, 402-412 (1965). (61) G. Uhlenbruck and 0. Prokop, Vox Sang., 11,519-520 (1966). (62) W. C. Boyd and R. Brown, Nature, 208, 593-594 (1965). (63) S. Hammarstrom and E. A. Kabat, Biochemistry, 8,2696-2705 (1969). (64) B. Jonsson, Acta Pathol. Microbiol. Scand., Suppl., 54,456-464 (1944). (65) R. Grubb, Acta Pathol. Microbiol. Scand., Suppl., 84,5-72 (1949). (66) P. R. Desai and G. F. Springer, Methods Enzymol., 28, Part B, 383-388 (1972). (67) R. J, Stockert, A. G. Morell, and I. H. Scheinberg, Science, 186,365-366 (1974). (67a) R. L. Hudgin, W. E. Pricer, Jr., G. Ashwell, R. J. Stockert, and A. G. Morell,]. Biol. Chem., 249,5536-5543 (1974). (6%) T. Kawasaki and G. Ashwell,]. Biol. Chem., 251, 1296-1302 (1976). (67c) A. de Waard, S. Hickman, and S. Kornfeld,]. Biol. Chem., 251,7581-7587 (1976). (67d) H. Den and D. A. Malinzak,]. Biol. Chem., 252,5444-5448 (1977).

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The reader is referred to a number of r e ~ i e w s ~ . ~ ~(espe. ~ ~ * ~ * ~ ~ cially those published18*37b,45,72,75,76a,b since 1970) and treatise~,~~-~O as well as to valuable compendia dealing with the screening and serological properties of thousands of plant-seed extracts.2,5*6,68*77-91a Lectins deriving from micro-organisms and invertebrates were discussed in a volume.76The cell-binding and biological properties of lectins have also been reviewed.18*25,26*g2.92a Full proceedings of two conferences on plant and invertebrate agglutinins%and ong4concanavalin A have been published. (68) G. W. G. Bird, Acta Chir. Belg., 53, 33-40 (1954). (69) W. C. Boyd,J. Immunol., 85,221-229 (1960). (70) I. E. Liener, Econ. Bot., 18,27-33 (1964). (71) J. M. Dechary, Vox Sang., 15,401-409 (1968). (72) N. Sharon and H. Lis, Science, 177,949-959 (1972). (73) W. C. Boyd, Ann. N.Y. Acad. Sci., 169, 168-190 (1970). (74) G. C. Toms and A. Western, in “Chemotaxonomy of the Leguminosae,” J. B. Harbome, D. Boulter, and B. L. Turner, eds., Academic Press, London and New York, 1971, pp. 367-462. (75) J. A. Callow, Curr. Adu. Plant Sci., 18, 181-193 (1975). (76) E. R. Gold and P. Balding, “Receptor-Specific Proteins, Plant and Animal Lectins,” American Elsevier, New York, 1975, pp. 1-440. (76a) E. Kottgen, Klin. Wochenschr., 55,359-373 (1977). (76b) N. Sharon, Sci. Am., 236, 108-119 (1977). (77) M. Kriipe, “Blutgruppenspezifische Pflanzliche Eiweisskorper (Phytagglutinine),” Ferdinand Enke Verlag, Stuttgart, 1956, pp. 1-131. (78) 0. Makela, Ann. Med. E x p . Biol. Fenn. Suppl. 1 1 , 35, 1-156 (1957). (79) 0. Makela, “Studies on Hemagglutinins of Leguminosae Seeds,” Weilin and Goos, Helsinki, 1957, pp. 1-133. (80) J. Tobiska, “Die Phythaemagglutinine,” Akadeniie Verlag, Berlin, 1964,pp. 1-302. (81) P. Cazal and M. Lalaurie, Acta Haematol., 8, 73-80 (1952). (82) G. W. G. Bird,]. lmmunol., 69, 319-320 (1952). (83) G. W. G. Bird, Br. J . E x p . Pathol., 35,252-254 (1954). (84) G. W. G. Bird, Army Med. C o r p s J . , 11, 17-25 (1955). (85) W. C. Boyd, E. Waszczenko-Zacharczenko, and S. M. Goldwasser, Transfusion (Philadelphia), 1, 374-382 (1961). (86) L. Brilliantine and N. K. Allen,]. Zmmunol., 86, 575-577 (1961). (87) L. Brilliantine, L. H. Aranda, D. M. Foster, and N. K. Allen,]. Zmmunol., 92, 555558 (1964). (88) T. Martin and G. Bomchil, Vox Sung., 11,54-58 (1966). (89) A. A. Hossaini, Vox Sang., 15,410-417 (1968). (90) N. K. Allen and L. Brilliantine,]. Immunol., 102, 1295-1299 (1969). (91) G. W. G. Bird and J. Wingham, Vox Sang., 24,48-57 (1973). (91a) J. Tobiska, Z . Immunitaetsforsch. E x p . Ther., 117, 156-163 (1959). (92) K. D. Noonan, in “Virus Transformed Cell Membranes,” C. Nicolau, ed., Academic Press, London, 1976. (92a) A. M. C. Rapin and M. M. Burger, Ado. Cancer Res., 20,l-91 (1974). (93) E. Cohen, Ann. N.Y. Acad. Sci., 234, 1-412 (1974). (94) T. K. Chowdhury and A. K. Weiss, Ado. E x p . Med. Biol., 55, 1-360 (1975).

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In the ensuing discussion, we have classified lectins according to their carbohydrate-binding specificity. Thus, D-mannose(D-glucose)binding lectins (concanavalin A and the agglutinins from the pea, lentil, and fava bean) are dealt with first, followed by 2-acetamido-2-deoxyD-glucose-binding lectins. Although this grouping is arbitrary, it brings a semblance of order to an extremely active and burgeoning field of investigation. Moreover, it readily permits a comparison of properties and data on carbohydrate-binding specificity within a group of agglutinins. As already stated, emphasis will be placed on chemical and physicochemical aspects. The literature on biological properties and activity of lectins is now so enormous as to require special treatment. The interested reader is referred to the many reviews cited that deal with various cellular and molecular-biological aspects of lectins. Finally, we have summarized (1) the physicochemical properties of purified lectins, (2) their carbohydrate-binding and blood-group specificity, and (3) equilibrium-dialysis, binding data in the three Tables at the end of this Chapter (Section IX). These Tables should provide the reader with a synopsis of the most important and distinguishing characteristics of lectins. We also present several glycopeptide structures (Figs. 14-16) showing the carbohydrate-binding loci with which various lectins interact.

1. Detection of Lectins Inasmuch as the first biological activity to be recognized for lectins was their capacity to agglutinate erythrocytes, most investigators have detected lectins by hemagglutination, using a panel of freshly drawn, animal or human erythrocytes, or both.78,9s-g7 Red blood-cells digested with papain, trypsin, neuraminidase, or other enzymes have also been employed; such treatment often renders cells more sensitive to Other types of animal cells have also been ~ ~ e Blood-group-specific d . ~ ~ ~ ~ lectins ~ , are ~ identified ~ with the aid of a panel of typed erythrocytes. Hemagglutination tests are generally conducted at room temperature by adding a drop of a 2 to 3% erythrocyte suspension to tubes or wells containing serially diluted lectin. After incubation for 1.5-2 h, the (95) E . A. Kabat and M. M. Mayer, “Experimental Immunochemistry,” Charles C. Thomas, Springfield, Ill., 2nd Edition, 1961, pp. 1-905. (96) M. M. Burger, Methods Enzymol., 32, 615-621 (1974). (97) H. Lis and N. Sharon, Methods Enzymol., 28, Part B, 360-365 (1972). (98) G. I. Pardoe and G. Uhlenbruck,]. Med. Lab. Technol., 27,249-263 (1970). (99) J. A. Gordon, N. Sharon, and H. Lis, Biochim. Biophys.Acta, 264,387-391 (1972). (100) 0 . Prokop, G. Uhlenbruck, and W. Kohler, Vox Sang., 14,321-333 (1968).

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agglutination is observed by the naked eye or by microscope. Activity is expressed as titer, the reciprocal of the maximal dilution of lectin that gives visible aggregation. An error o f f 1 tube is commonly accepted, that is, a titer of 64 could range from 32 to 128. Occasionally, a lectin will not agglutinate red blood-cells unless they are suspended in a medium more viscous than physiological saline, for example, poly(vinylpyrrolidinone), serum albumin, and serum.78Such agglutinins have been termed "incomplete" lectins, in that they require the presence ofaccessory factors in order to cause a g g l ~ t i n a t i o n . ~ ~ A spectrophotometric assay was introduced by Liener.'O' It is based on the observation that rabbit erythrocytes sediment at a rate proportional to the concentration of the hemagglutinin. Hemagglutinating activity is calculated from absorbance measurements at 620 nm of the unsedimented cell-suspension after a given time.97*'0' Hemagglutinins may be of several types: (a) nonspecific lectins that agglutinate cells without regard to their origin (either species, or blood type), ( b ) lectins that preferentially agglutinate the cells of one or several kinds of animals, or (c) blood-group-specific lectins. As additional knowledge of the properties of lectins became available, more-refined screening-procedures for detecting them were developed. Naturally occurring g l y ~ o p r o t e i n s , ~ ~ ,synthetic ~~~*~~-~~~ carbohydrate-protein (and carbohydrate-phloroglucinol) conjugates,112-1'6and polysa~charides'~~~~~~-'~~ have been employed in (101) I. E. Liener, Arch. Biochem. Biophys., 54, 223-231 (1955). (102) J. B. Sumner and S. F. Howell,]. Bacteriol., 32, 227-237 (1936). (103) W. C. Boyd, E. Shapleigh, and M. McMaster,Arch. Biochem. Biophys., 55,226234 (1955). (104) G. W. G. Bird, Vox Sang., 4,307-313 (1959). (105) J. H. Morse, Immunology, 14,713-724 (1968). (106) H. Harris and E. B. Robson, Vox Sang., 8,348-355 (1963). (107) S. Nakamura, K. Tanaka, and S. Murakawa, Nature, 188, 144-145 (1960). (108) M. E. Etzler and E. A. Kabat, Biochemistry, 9,869-877 (1970). (109) N. M. Young and M. A. Leon, Biochim. Biophys. Acta, 365,418-424 (1974). (110) S. Yachnin,]. Immunol., 108,845-847 (1972). (111) S. Yachnin,]. Exp. Med., 141,242-256 (1975). (112) I. J. Goldstein and R. N. Iyer, Biochim. Biophys. Acta, 121, 197-200 (1966). (113) R. N. Iyer and I. J. Goldstein, Immunochemistry, 10,313-322 (1973). (114) W. T. Shier, Proc. Natl. Acad. Sci. U.S.A.,68,2078-2082 (1971). (115) J.-P. Privat, F. Delmotte, and M . Monsigny, F E B S Lett., 46, 224-228 (1974). (116) A. E. Clark, R. B. Knox, and M. A. Jermyn,]. Cell Sci., 19, 157-167 (1975). (117) J. A. Cifonelli, R. Montgomery, and F. Smith,]. Am. Chem. Soc., 78,2485-2488 (1956). (118) G. F. Springer,]. Zmmunol., 76,399-407 (1956). (119) G. W. G. Bird, Nature, 187,415-416 (1960). (120) I. J. Goldstein and L. L. So, Arch. Biochem. Biophys., 111, 407-414 (1965).

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detecting lectins and in studying their specificity. Formation of a precipitate between a lectin and carbohydrate-containing macromolecules, either in liquid (capillary tubes) or semisolid (agar gel) media, is indicative of precipitating lectin activity. Moreover, it can provide information regarding lectin specificity and anomeric preference, as well as on the constituent sugars and glycosidic linkages ofthe macromolecule. Sugar-protein conjugates in which p-diazophenyl glycosides were or to phloroglucinol,116provided coupled to a protein model substrates for detecting lectins and studying their specificity. In fact, a second phytohemagglutinin, a 2-acetamido-2-deoxy-~glucopyranosyl-binding protein, was first detected in Bundeiruea simplicifolia seeds by using a p-azophenyl 2-acetamido-2-deoxy-P-~glucopyranoside-bovine serum albumin conjugate.12s Precipitate formation between multivalent, model substrates and lectins must be cautiously interpreted. Nonspecific interactions must be distinguished by sugar inhibition-tests; for example, addition of maltose to the precipitate formed between a maltose-protein conjugate and seed extract should dissolve the precipitate, whereas lactose should be ineffective.lZ0Furthermore, the carbohydrate-protein linkage of the synthetic substrate may have a marked influence on the precipitation reaction, and may even give rise to artifactual precipitates. It is, therefore, preferable to employ a di- or tri-saccharide conjugate, in which the nonreducing, terminal glycosyl group is glycosidically bound to a second sugar residue rather than to an aromatic aglycon. A new class of synthetic sugar-protein conjugates has been introduced to circumvent this problem.1z6An aldonic acid was joined, through peptide bonds, to a protein, and the product was used for screening and specificity studies. HofejSi and Kocourek devised a new procedure for the detection of lectins which they termed l Z 7 “affinity electrophoresis.” This technique (121) I. J. Coldstein, C. E . Hollerman, and J. M. Merrick, Biochim. Biophys. Actu, 97, 68-76 (1965). (122) M. Paulovi, M. Tichi, G . Entlicher, J. KoStii, and J. Kocourek,FEBS Lett., 9,345347 (1970). (123) N. M . Young, M. A. Leon, T. Takahashi, I. K. Howard, and H. J. Sage,]. Biol. Chem., 246, 1596-1601 (1971). (124) J. P. Van Wauwe, F. C. Loontiens, and C. K. D e Bruyne, Biochim. Biophys. Actu, 313,99-105 (1973). (125) P. N. Shankar Iyer, K. D. Wilkinson, and I. J. Coldstein,Arch. Biochem. Biophys., 177,330-333 (1976). (126) J. Lonngren, I. J. Coldstein, and J. E. Niederhuber,Arch. Biochem. Biophys., 175, 661-669 (1976). (127) V. Hoiejs’i and J. Kocourek, Biochim. Biophys. Actu, 336, 338-343 (1974).

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combines the principles of affinity chromatography and electrophoresis; proteins are subjected to electrophoresis on a matrix formed by copolymerization of alkenyl glycosides with acrylamide. Proteins having combining sites complementary to the ligand are retarded, whereas other proteins undergo a normal separation. By determining the electrophoretic mobility of a lectin in the presence of several concentrations of its specific, sugar ligand, Hoiejii and colleague~ extended ~ ~ ~ ~ their procedure to enable a determination of the dissociation constant of the lectin-sugar complex to be made. Affinity electrophoresis has also been used to study sugar-binding heterogeneity of lectins and their chemically modified derivative^.'^'^

2. Isolation and Purification of Lectins It is only within the past decade that lectins have been purified to a homogeneity demonstrable by physicochemical and immunological criteria. Although Sumner and Howell crystallized the jack-bean lectin (concanavalin A or con A) over forty years ago, they were unable to free it completely from carbohydrate contaminants.lo2 Isolation of lectins generally begins with a saline (or buffer) extraction of the finely ground, seed meal. Pre-extraction with organic solvents (for example, methanol or diethyl ether) is often employed to remove lipid or other interfering s ~ b s t a n c e s . ' ~ ~Ammonium ~ ' ~ ~ - ' ~ ~ sulfate fractionation, centrifugation, and dissolution of the precipitate yields a supernatant liquor containing the lectin(s). Plant agglutinins may be isolated from saline extracts by conventional, protein-purification techniques, affinity chromatography, or a combination thereof. Virtually all contemporary, lectin-purification schemes employ affinity chromatography that exploits the specific, sugar-binding capacity of the l e ~ t i n . ' ~Simply , ' ~ ~ stated, a carbohydrate ligand with which the lectin interacts is insolubilized, the lectin is adsorbed as the extract is percolated slowly over the adsorbent, and displacement of bound lectin is accomplished by elution, either with a sugar that competes for lectin sites with the specific adsorbent, or by altering the nature of the eluant (by lowering the pH, increasing the ionic strength, or adding denatur(127a) V. HoiejXi, M. Ticha, and J. Kocourek, Biochim. Biophys. Acta, 499, 290-300 ( 1977). (127b) V. Hoiejs'i, M. Ticha, and J. Kocourek, Biochim. Biophys. Acta, 499,301-308 (1977). (128) A. K. Allen, A. Neuberger, and N. Sharon, Biochem. J., 131, 155-162 (1973). (129) Y. Nagata and M. M. Burger,J. Biol. Chem., 249,3116-3122 (1974). (130) J. H. Shaper, R. Barker, and R. L. Hil1,Anal. Biochem., 53, 564-570 (1973). (131) C. E. Hayes and 1. J. Goldstein,J. Biol. Chem., 249, 1904-1914 (1974). (132) H. Lis, R. Lotan, and N. Sharon, Ann. N.Y. Acad. Sci., 234,232-238 (1974).

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ants). Such insoluble, naturally occurring, or chemically modified substances as ~ h i t i n , ' ~S~e Jp ~h ~a d e ~ , ' ~ and ~-'~ agarose ~ or S e p h a r ~ s e ' ~ ~ - ' ~ ~ have also been employed as affinity matrices. Applications of affinity chromatography to lectin purification have been s u m m a r i ~ e d . ~ *AJ ~few ~ examples of the diverse approaches utilized for affinity-column synthesis and used in the isolation of lectins follow; these are discussed in detail in subsequent Sections. HoPejSi and K o c ~ u r e k copolymerized '~~ a series of alkenyl glycosides with acrylamide, whereas Matsumoto and O s a ~ aincorporated '~~ a variety of sugar residues into starch; both materials have been used149as affinity matrices in lectin isolation. Reaction of epoxy-activated Sepharose 6B with 2-acetamido-2-deoxy-~-glucoseand -D-galactose afforded affinity columns used for the purification of wheat-germ agglutinin and soybean lectin, r e ~ p e c t i v e l y . Affinity ' ~ ~ ~ chromatography on aminoethyl poly(acrylamide) gels containing reductively aminated disaccharide residues (lactose, melibiose, maltose, andN,N'-diacetylchitobiose) was used by Baues and Gray149b to isolate lectins from the B . simplicifolia seeds, castor beans, jack beans, lentils, and wheat germ. Several purification schemes employed Sephadex affinity-chromatography; a - ~ glucopyranosyl- and a-D-mannopyranosyl-binding lectins from the R. Bloch and M. M. Burger, Biochem. Biophys. Res. Commun., 58,13-19 (1974). B. B. L. Agrawal and I. J. Goldstein, Biochem. ]., 96,23C-25C (1965). B. B. L. Agrawal and I. J. Goldstein, Biochim. Biophys. Acta, 147,262-271 (1967). M. 0. J. Olson and I. E. Liener, Biochemistry, 6, 105-111 (1967). K. Aspberg, H. Holmen, and J. Porath, Biochim. Biophys. Acta, 160, 116-117 (1968). I. K. Howard and H. J. Sage, Biochemistry, 8,2436-2441 (1969). M. Tomita, T. Osawa, Y. Sakurai, and T. Ukita, Znt.]. Cancer, 6,283-289 (1970). J. L. Wang, J. W. Becker, G. N. Reeke, Jr., and G. M. Edelman,]. Mol. B i d , 88, 259-262 (1974). G. Entlicher, J. V. KodtiF, and J. Kocourek, Biochim. Biophys. Acta, 221,272-281 (1970). M. Tichi, G. Entlicher, J. V. KoBtii, and J. Kocourek,Biochim. Biophys. Actu, 221, 282-289 (1970). S. Toyoshima, T. Osawa, and A. Tonomura, Biochim. Biophys. Acta, 221,514-521 (1970). M. Tomita, T. Kurokawa, K. Onozaki, N. Ichiki, T. Osawa, and T. Ukita, Experientia, 28,84-85 (1972). B. Ersson, K. Aspberg, and J. Porath, Biochim. Biophys. Acta, 310,446-452 (1973). G. L. Nicolson, J. Blaustein, and M. E. Etzler, Biochemistry, 13,196-204 (1974). S . Olsnes, E. Saltvedt, and A. Pihl,]. BioZ. Chem., 249,803-810 (1974). V. Ho'rejSi and J. Kocourek, Biochim. Biophys. Acta, 297, 346-351 (1973). I. Matsumoto and T. Osawa, Biochem. Biophys. Res. Commun., 46, 1810-1815 (1972). (149a) P. Vretblad, Biochim. Biophys. Acta, 434, 169-176 (1976). (149b) R. J. Baues and G. R. Gray,]. B i d . Chem., 252, 57-62 (1977).

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seeds of Canavalia ensiformis (con A),134-136 Vicia c r a ~ c a , ~Vicia ~' faba,139*140 Pisum sativus (pea),14' and Lens culinaris (lenti1)'38,142~L43 were isolated in this way. Likewise, such P-D-galaCtOpyranOSylbinding proteins as ricin (Ricinus c ~ m m u n i s ) ' ~and ~ . ' abrin ~ ~ (Abrus p r e c a t o r i ~ s ) have ' ~ ~ been purified on agarose. LecMns from soybean, Wistaria jloribunda, Bauhinia purpurea alba, and Sophora japonica could be isolated by affinitychromatography on acid-treated Sepharose 6B, but not on untreated Sepharose.LsoaCross-linked guaran was used for isolating the lectins from Helix pomatia, Glycine max, Ricinus communis, and Echinocystis lobata (wild cucumber), and the a-Dgalactopyranosyl-binding protein from Bandeiraea simplicifolia; however, the lectins from Dolichos bijlorus, Phaseolus lunatus, and Sophora japonica failed to bind to this ab~orbent.'~"A further procedure involves entrapment of polysaccharide in poly(acry1amide) gels. The lectins from the peanut (Arachis hypogaea) and Bandeiraea simplicifolia seeds were isolated by using guaran-entrapped bead^.^^^^,^^^^ The use of 1,4-butanediol diglycidyl ether to couple carbohydrate ligands to Sepharose has also afforded affinity matrices for lectin p u r i f i ~ a t i o n . ' ~ ~ ~ Adsorption to insolubilized, hog-mucin type A + H substance, folafforded purlowed by elution with 2-acetamido-2-deoxy-~-galactose ified lectins from the seeds of Dolichos bijlorus,lo8Phaseolus lunatus (lima bean),lS1and Helix pomatia (the edible snail).63Similarly, chitin has been used for purifying the 2-acetamido-2-deoxy-~-glucosebinding lectins from Triticum vulgaris (wheat germ),133Solanum tuberosum (potato),152and Bandeiraea simplicifolia. 125 A soluble adsorbent, the tris(p-azophenyl a-L-fucopyranoside) conjugate of phloroglucinol, was employed by Yariv and to isolate the L-fucose-binding protein from Lotus tetragonolobus. An alternative approach involves insolubilized glycoproteins. Thus, an affinity system for the isolation of the lectin of red kidney-bean (Phaseolus v ~ l g a r i s ) ' ~involved ~ * ' ~ ~ thyroglobulin-Sepharose, and, for (150) S. Olsnes and A. Pihl, Eur. J . Biochem., 35, 179-185 (1973). (150a) H. J. Allen and E. A. Z. Johnson, Carbohydr. Res., 50,121-131 (1976). (150b) J. Lonngren, I. J. Goldstein, and R. Bywater, FEBS Lett., 68,31-34 (1976). (150c) M. Horisberger, Carbohydr. Res., 53,231-237 (1977). (150d) K. Sutoh, L. Rosenfeld, and Y. C. Lee, Anal. Biochem., 79,329-337 (1977). (150e) R. Uy and F. Wold, Anal. Biochem., 81,98-107 (1977). (151) W. Galbraith and I. J. Goldstein, FEBS Lett., 9, 197-201 (1970). (152) M. A. Leon, personal communication. (153) J. Yariv, A. J. Kalb, and E. Katchalski, Nature, 215,890-891 (1967). (154) R. L. Felsted, R. D. Leavitt, and N. R. Bachur, Biochim. Biophys. Acta, 405, 72-81 (1975).

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the isolation of Limulus polyphemus, bovine submaxillary mucinSepharose.’” Fetuin-Sepharose was employed for the isolation of the agglutinins from wheat germ (Triticum vulgaris), horse-shoe crab (Limulus polyphemus), jack bean (Canavalia ensiformis), and several other sources.’56Wheat-germ agglutinin was also isolated by using insolubilized ovomucoid.ls’ Finally, erythrocytes treated with formaldehyde and glutaraldehyde have been used as adsorbents for lectin is~lation.’~~-’~~ Although seeds are the common source of lectin activity, there are reports (some contradictory) of agglutinins occurring in leaves, stems, and root^.^^,^^ For example, black-locust (Robinia pseudoaccacia) lectin’60was isolated from its bark, potato (Solanum tuberosum) lectin16’ from its tubers, and poke-weed (Phytolacca americanum) mitogen162 from its roots. A new class of plant cell-wall and membrane-bound lectins (“p-lectins”) has been d e ~ c r i b e d . ~A~treatise ~ , ’ ~ collected ~~~~~ several schemes for the purification of l e c t i n ~ . ’ ~ ~ J ~ ~ ~

3. Carbohydrate-binding Specificity of Lectins The carbohydrate-binding specificity of lectins varies greatly with respect to the binding of simple sugars, the precipitation of carbohydrate-containing macromolecules, and the agglutination of plant and animal cells. In order that a lectin may be a truly useful tool in carbohydrate chemistry and other areas (for example, biology), it is necessary that its carbohydrate-binding specificity be established; this is generally accomplished by the Landsteiner hapten-inhibition technique.166Sugar-lectin complementarity is established by comparing (155) J. D. Oppenheim, M. S. Nachbar, M. R. J. Salton, and F. Aull, Biochem. Biophys. Res. Commun., 58, 1127-1134 (1974). (156) B.-A. Sela, J. L. Wang, and G. M. EdelmanJ. B i d Chern., 250,7535-7538 (1975). (157) S. Avrameas and B. Guilbert, Biochimie, 53,603-614 (1971). (158) R. W. Reitherman, S. D. Rosen, and S. H. Barondes,Nature, 248,599-600 (1974). (159) T. P. Now& and S. H. Barondes, Biochim. Biophys. Acta, 393,115-123 (1975). (160) R. Bourrillon and J. Font, Biochim. Biophys. Acta, 154, 28-39 (1968). (161) E. Gellhom, in “Handbuch der Biochemie des Menschen und Tiere von Oppenheimer,” F. Schiff, ed., Gustav Fischer, Jena, 1925, Vol. 2, pp. 346-377. (162) J. Borjeson, R. Reisfeld, L. N. Chessin, P. D. Welsch, and S. D. Douglas,J. E x p . Med., 124,859-872 (1966). (163) H. Kauss and C. Glaser, FEBS Lett., 45,304-307 (1974). (164) D. J. Bowles and H. Kauss, Plant Sci. Lett., 4,411-418 (1975). (165) Methods Enzymol., 38 (Part B), 313-388 (1972). (165a) Methods Enzymol., 34 (Part B), 317-331 (1974). (166) K. Landsteiner, “The Specificity of Serological Reactions,” Dover Publications, New York, Revised Edition, 1962, pp. 1-330.

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sugars on the basis of the minimal concentration required to inhibit

(I) the precipitin reaction between the lectin and a reactive macromolecule,167-170 or (2) the hemagglutination reaction.10~19~21~22~77.7s.”71 Other procedures, including equilibrium dialysis,172ultraviolet spect r o ~ c o p y ,elution ~ ~ ~ ~from ’ ~ ~solid-phase ads or bent^,^^".'^^ and precipitation with na~Ura~63,102,104.106~117,120,121,124,170,176--178 and model carbohydrate-protein conjugates113J26J79 have also been informative. The use of alkyl a-and P-glycosides as hapten inhibitors, in addition to free sugars, yields vital information on the anomeric specificity of the lectin, but it is also necessary to test di- and higher oligo-saccharides, in order to interpret studies on the structural features of polysaccharides and glycoproteins correctly. (Sugars purchased commercially may contain hemagglutinin substances, and must be purified prior to Glycosides of aromatic aglycons provide information about the nature of the protein site adjacent to the anomeric carbon atom ofthe sugar, but can be misleading insofar as lectin specificity for oligosaccharides is concerned.113J67J80-184 In effect, the interaction of the protein with an aromatic moiety is explored, not the carbohydrate-binding specificity of the lectin; this situation dictates caution in interpreting such experiments. Although the interaction of lectins with polysaccharides, glycoproteins, and glycolipids is, without dispute, more complex than with simple sugars, the results of hapten-inhibition studies employing (167) G. F. Springer and P. Williams, Biochem. J., 85,282-291 (1962). (168) I. J. Goldstein, C. E. Hollerman, and E. E. Smith, Biochemistry, 4,876-883( 1965). (169) L. L. So and I. J . Goldstein,J. lnzmunol., 99, 158-163 (1967). (170) K. 0. Lloyd, E. A. Kabat, and S. Beychok,J. Immunol., 102, 1354-1362 (1969). (171) A. H. Rule and W. C. Boyd, Transfusion (Philadelphia), 4,449-456 (1964). (172) L. L. So and I. J. Goldstein, Biochim. Biophys. Acta, 165,398-404 (1968). (173) G. S. Hassing and I. J. Goldstein, Eur. J. Biochem., 16, 545-556 (1970). (174) W. Bessler, J. A. Shafer, and I. J. GoldsteinJ. B i d . Chem., 249,2819-2822 (1974). (175) S. M. Chien, S. Singla, and R. D. Poretz,]. Immunol. Methods, 8,169-174 (1975). (176) G. W. G. Bird, Vox Sang., 4, 307-313 (1959). (177) H. Markowitz,J. lmmunol., 103, 308-318 (1969). (178) S. Hammarstrom, A. A. Lindberg, and E. S. Robertsson, Eur.J.Biochem., 25,274282 (1972). (179) I. J. Goldstein and R. N. Iyer, Biochim. Biophys. Acta, 121, 197-200 (1966). (179a) M. D. Marquardt and J. A. Gordon, Nature, 252, 175-176 (1974). (180) R. D. Poretz and I. J. Goldstein, Biochem. Pharmacol., 20,2727-2739 (1971). (181) F. G. Loontiens, J. P. Van Wauwe, R. De Gussem, and C. K. D e Bruyne, Carbohydr. Res., 30, 51-62 (1973). (182) J.-P. Van Wauwe, F. G. Loontiens, H. A. Carchon, and C. K. De Bruyne, Carbohydr. Res., 30,249-256 (1973). (183) T. Irimura, T. Kawaguchi, T. Terao, and T. Osawa, Carbohydr. Res., 39,317-327 (1975). (184) R. D. Poretz, H. Riss, J. W. Timberlake, and S. M. Chien, Biochemistry, 13,250256 (1974).

141

LECTINS: CARBOHYDRATE-BINDING PROTEINS

mono- and oligo-saccharides and their derivatives have, in all cases, been applicable to studies with complex saccharides. (A single example, the unexpected reactivity of the pea lectin with a glycopeptide, has yet to be explained.I6') The higher affinity-constants observed for binding of lectins to glycoproteins and cell-surface carbohydrates than to simple sugars can be rationalized on the basis of multivalent interactions between the lectin (almost all lectins contain two or more binding sites; see, for example, Ref. 186) and the complex sa~charide.'~'-'~~ Furthermore, nonspecific interactions between the lectin and the macromolecule, and the influence of steric factors, must be considered, in addition to the specific interaction between the carbohydrate and the le~tin.'~~*'~3 In early studies, Morgan and WatkinsZ2observed that sugars (for example, L-fucose, 2,6-dideoxy-~-lyxo-hexose, L-galactose, and 6-deoxy-~-talose)that inhibit the L-fucose-binding lectin from Lotus tetragonolobus all have the same configuration at C-3 and C-4. Similarly, K r i i ~ enoted ~ ~ that sugars (2-acetamido-2-deoxy-~-galactose, D-galactose, lactose, and melibiose) that inhibit the agglutinin from Sophora japonica seeds all have the same configuration at C-3 and C-4. Generalizing from these and his own studies, Make1a78suggested that lectin-reactive monosaccharides may be divided into four classes, based on their configuration at C-3 and C-4 of the pyranose forms (see Fig. 1).

c)

no no I

noo

0 OH

noo

no HO

I II 111 IV FIG. 1.-Classification of Pyranoses into Four Croups, Based on the Configurations of the 3- and 4-Hydroxyl Groups (after Makela7a).

(185) J. PospiSilovh, G. Entlicher, and J. Kocourek, Biochim. Biophys. Acta, 362,593597 (1974). (186) M . D . Stein, I. K. Howard, and H. J. Sage,Arch. Biochem. Biophys., 146,353-355 (1971). (187) M. D . Stein, H. J. Sage, and M. A. Leon, Arch. Biochem. Biophys., 150,412-420 (1972). (188) P. W. Majerus and G. N. Brodie,J. B i d . Chem., 247,4253-4257 (1972). (189) S. Hammarstrom, Scand. J. Immunol., 2, 53-66 (1973). (190) C. L. Homick and F . b r u s h , Immunochemistry, 9,325-340 (1972). (191) C. E. Hayes and I. J . Coldstein,J. B i d . Chem., 250, 6837-6840 (1975). (192) M . W. Davey, J. W. Huang, E. Sulkowski, and W. A. Carter,J. Biol. Chem., 249, 6354-6355 (1974). (193) S. K. Podder,A. Surolia,and B. K. Bachhawat,Eur.J. Biochem., 44,151-160(1974).

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For a time, before extensive and systematic studies on pure lectins were conducted, this classification served a useful purpose. Thus, agglutinins derived from the pea(Pisurn sativum)20and the lentil (Lens bound Makela's group I11 sugars, being ~ u l i n a r i s ) ' ~preferentially ~*'~ inhibited by D-glucose and D-mannose, whereas lectins from the seeds of Sophora japonica,17 Ricinus c o r n m ~ n i s , ~and ~ * ~Bandeiraea ~~ ~ i m p l i c i f o l i a ~were ~ ~ * inhibited '~~ by group I1 carbohydrates. Sugars of group I inhibit the lectins from Lotus tetragonolobusZ2 and the L-fucose-binding lectin of Ulex e u r o p e u ~ .As '~~ yet no lectin has been isolated that interacts with Makela's group IV sugars (D-idose, D-gulose, L-glucose, and L-xylose). The limitations of this classification have already been mentioned. Unlike human agglutinins, which generally require a disaccharide hapten as the smallest fragment that will inhibit precipitation, the binding site of most lectins accommodates a single glycosyl residue. In a few cases, however, the nature of the glycosidic linkage plays a role in determining the binding specificity. Thus, isomaltose and other oligosaccharides containing nonterminal, a-D-( 1+6)-glucosidic bonds have a higher affinity for con A than oligosaccharides having nonreduclinkages.'68J97 A moreing a-D-( 1+2, -+3, or -4)-glucosidic pronounced linkage-specificity was observed by O ~ a w a 'for ~ the Laburnum alpinum and Cytisus sessilifolius lectins. Only disaccharides containing a glycosidic bond to a secondary hydroxyl group were bound by these lectins; for example, laminarabiose and cellobiose are good inhibitors, whereas gentiobiose is a noninhibitor. A similar specificity is exhibited by the B . simplicifolia 2-acetamido-2deoxy-D-glucose-binding 1 e ~ t i n . l ~ ~ The fact that several lectins bind di- and tri-saccharides'2J more strongly than alkyl glycosides indicates that the reducing sugar may contribute to the binding energy of the lectin-carbohydrate complex. For example, a trisaccharide inhibits soybean lectin better than the corresponding disaccharide lacking its "reducing" sugar residue.lg8 and The same phenomenon was observed for the Dolichos bi.orus108 lunatus) lectins. The precise position of a del i m a - b e a ~(Phaseolus ~'~~ terminant sugar in an oligo- or poly-saccharide is also important to lectin reactivity. Pereira and KabatZo0observed that the location of (194) G. L. Nicolson and J. Blaustein, Biochim. Biophys. Acta, 266, 543-547 (1972). (195) 0.Makela, P. Makela, and M. Kriipe, Z. Immunitaetsforsch.Exp. Ther., 117,220229 (1959). (196) I. Matsumoto and T. Osawa, Vox Sang., 21, 548-557 (1971). (197) E. E. Smith and I. J. Goldstein, Arch. Biochem. Biophys., 121, 88-95 (1967). (198) M. E. A. Pereira, E. A. Kabat, and N. Sharon, Carbohydr. Res., 37,89-102 (1974). (199) W. Galbraith and I. J. Goldstein, Biochemistry, 11, 3976-3984 (1972). (200) M. E. A. Pereira and E. A. Kabat, Biochemistry, 13,3184-3192 (1974).

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143

a-L-fucopyranosyl end-groups in various oligosaccharides is of paramount importance in determining the reactivity with the Lotus tetragonolobus lectin (compare the L-fucose-binding lectin of Euonymus europeus; see Section VII,3). Examples in which the reducing sugar residue does not contribute to the affinity of a lectin for a disaccharide include the Bandeiraea simplicifolia lectin, for which, methyl a-D-galactopyranoside, melibiose, and 2-0-a-D-ga~actopyranosyl-Dglucose all bind to the same extent.l3I On the other hand, the peanutlectin combining-site interacts in a highly specific way with the disaccharide 2-acetamido-2-deoxy-3-O-~-D-ga~actopyranosy~-D-ga~actose201-203 Only for con A [which binds (1+2)-a-D-manno-oligosaccharide^^^^*^^*^^^] and a series of lectins (wheat-germ agglutinin,128potato and Ulex europeus lectinZo8)that bind N-peracetylated chito-oligosaccharides has it been shown that the carbohydratebinding site is complementary to three or more sugar units (see later). With but few exceptions, lectins interact with the nonreducing, terminal glycosyl groups of polysaccharide and glycoprotein chain-ends. Con A, on the other hand, in addition to its interaction with a - ~ mannopyranosyl and a-D-glucopyranosyl terminal groups, binds internal 2-0-a-D-mannopyranosyl residues.209Similarly, three other D-mannose-specificlectins (those of the pea, the lentil, and broad bean) reportedly interact with the (reducing) D-mannose residue of 2-0-(2acetamido-2-deoxy-~-~-g~ucopyranosy~)-~-mannose.~'~ Wheat-germ agglutinin (and, most probably, the 2-acetamido-2-deoxy-~-glucosebinding lectins of the potato and Ulex europeus) interacts with internal, 4-O-substituted, 2-acetamido-2-deoxy-&glucopyranosyl residues.128 Lectins differ markedly with respect to their anomeric specificity. Some, such as con A (Refs. 168, 169, and 197), the lectins from Ban(201) G. Uhlenbruck, G. I. Pardoe, and G. W. G. Bird, Z . Immunitaetsforsch. Allerg. Klin. Immunol., 138, 423-433 (1969). (202) R. Lotan, E. Skutelsky, D. Danon, and N. Sharon,J. Biol. Chem., 250,8518-8523 ( 1975). (203) M. E. A. Pereira, E. A. Kabat, R. Lotan, and N. Sharon, Cnrbohydr. Res., 51, 107118 (1976). (204) L. L. So and I. J. Goldstein,J. Biol. Chem., 243, 2003-2007 (1968). (205) I. J. Goldstein, Ado. E x p . Med. Biol., 55, 35-53 (1974). (206) G. I. Pardoe, G. W. G. Bird, and G. Uhlenbruck, Z. Immunitaetsforsch. Allerg. Klin. Immunol., 137, 442-457 (1969). (207) A. K. Allen and A. Neuberger, Biochem. J,, 135,307-314 (1973). (208) I. Matsumoto and T. Osawa, Arch. Biochem. Biophys., 140,484-491 (1970). (209) I. J. Goldstein, C. M. Reichert, A. Misaki, and P. A. J. Gorin, Biochim. Biophys. Acta, 317, 500-504 (1973). (210) R. Kaifu, T. Osawa, and R. W. Jeanloz, Carbohydr. Res., 40, 111-117 (1975).

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deiraea simpli~ifolia,'~~ the pea,211and Lotus tetragonolobus,22.200 exhibit pronounced, anomeric specificity, whereas other lectins, such as those from soybean1g8~212 and castor bean'46(RCA,), appear to be almost indifferent. Many lectins tolerate some variation at C-2 of the sugars which they bind. Thus, con A (Refs. 168 and 169) and the lectins from the pea (Pisum sativum),211lentil (Lens ~ u l i n a r i s ) , ' ~and ~ , ' fava ~ ~ bean (Vicia f a b ~ ) all ~ ' exhibit ~ a primary specificity for D-mannose, but will also bind D-glucose and, to a lesser extent, 2-acetamido-2-deoxy-~-glucose. A considerable number of lectins display a preferential affinity for 2-acetamido-2-deoxy-~-galactose, but also react, to different extents, with D-galactose (for example, the lectins from Dolichos bi.orus,'08 * ~ ' ~ ) , there is a Phaseolus l ~ n a t u s , 'and ~ ~ Helix p ~ m a t i a , ' ~ ~Conversely, series of lectins that display a primary specificity for D-galactose, and cross-react to a varying degree with 2-acetamido-2-deoxy-D-galactose. These include the a-D-galactopyranosyl-binding proteins from Banand Ricinus c ~ m m u n i s . ' ~ ~ deiraea ~implicifolia'~' In general, lectins tolerate very little variation at C-3 of the sugars they bind, although a report213indicates that the Viciafaba lectin has a greater affinity for 3-O-methyl-D-glucosethan for D-glucose (compare, ~ ~ *pea ~ ~lectin211). ~ the eel a g g l ~ t i n i n ' and The 4-hydroxyl group of carbohydrates is also critically involved in lectin binding. In general, D-glucose(D-mannose)-binding lectins20,'23,'43,'68,213 do not interact with D-galactose, and vice versa.131~146 Similarly, 2-acetamido-2-deoxy-~-glucose-binding lectins do not interLectins that bind act with 2-acetamido-2-deoxy-~-galactose.'~~~~~~-~~~ 2-acetamido-2-deoxy-~-galactose do not generally interact with 2-acetamido-2-deoxy-~-glucose,although the Helix pomatia agglutinin interacts with 2-acetamido-2-deoxy-~-glucose,albeit with only 10% of the affinity of 2-acetamido-2-deoxy-~-galactose.~~ Certain sugars in their furanose form unequivocally bind to con A (for example, D-fructose and ~ - a r a b i n o s e ) .There ~ ~ ~ ~is ' ~reason ~ ~ ~ ~to~expect that the pea, lentil, and Vicia fuba lectins will also bind D-fmctofuranosyl and D-arabinofuranosyl end-groups. A single report in its that wheat-germ agglutinin binds 2-acetamido-2-deoxy-~-glucose furanose form needs confirmation.lZ8

-

J. P. Van Wauwe, F. G. Loontiens, and C. K. D e Bruyne, Biochim. Biophys. Acta, 379,456-461 (1975). H. Lis, B.-A. Sela, L. Sachs, and N. Sharon, Biochim. Biophys. Acta, 211,582-585 (1970). A.K. Allen, N. N . Desai, and A. Neuberger, Biochem. J . , 155, 127-135 (1976). G. F. Springer and P. Williamson, Vox Sang., 8, 177-195 (1963). L. L. So and I. J. Goldstein, Carbohydr. Res., 10,231-244 (1969).

LECTINS: CARBOHYDRATE-BINDING PROTEINS

145

Finally, a group of lectins having a complex, sugar specificity that has yet to be established includes agglutinins from the red kidney-bean (Phaseolus vulgaris),216 the spindle tree(Evonymus europaea L.),217 the Vicia graminea,219,220 scarlet runner-bean (Phaseolus coccineus L.),218 and the meadow mushroom (Agaricus bisporus).221

4. Nomenclature of Lectins In any rapidly developing area of science, it is necessary to establish a systematic nomenclature, a problem not yet confronted in the already extensive field oflectin biochemistry. The names of a few lectins derive from their genus of origin (for example, ricin, abrin, concanavalin A, and favin for the lectins from Ricinus communis, Abrus precatorius, Canavalia ensiformis, and Viciafaba, respectively); others bear common names (for example, pea, soybean, and lima-bean lectins from the seeds of Pisum sativum, Glycine max, and Phaseolus lunatus, respectively). Particularly inappropriate is the accepted designation PHA for the phytohemagglutinin of the red kidney-bean (Phaseolus vulgaris). Employing the nomenclature ofblood-group ~ e r o l o g y ,Prokop ~ ~ ~ ,and ~~~ his coworker^^^^,^^^ proposed a nomenclature for plant and animal agglutinins. As an example, the lectins from Dolichos bi$orus, Helix hortensis, and Helix pomatia, all of which agglutinate type A erythrocytes were termed ADb,AHH, and AHP-the A standing for antigen, and Db, HH, and HP, for the initial letters of the Latin name. At present, most new lectins are cited by their genus and species names; for example, the lectin from Wistaria Poribunda and the lectin from Sophora japonica. (In a few cases, the sugar-binding specificity of the lectin has also been included in the designation.) At least two serious deficiencies are associated with all of these designations. First, it is theoretically possible, and indeed known, that several lectins having different sugar-binding specificities are present in the same plant-seed (for example, Ulex europeus contains a-Lfucopyranosyl-binding and 2-acetamido-2-deoxy-/3-~-glucopyranosyl-binding lectins208,22s,,226; Bandeiraea simplicifolia con(216) (217) (218) (219) (220) (221) (222) (223) (224) (225) (226)

R. Kornfeld and S. Kornfeld, J . Biol. Chern., 245,2536-2545 (1970). F. Pacak and J. Kocourek, Biochirn. Biophys. Acta, 400, 374-386 (1975). N. Nowakova and J. Kocourek, Biochirn. Biophys. Acta, 359,320-333 (1974). G. F. Springer, H. Tegtmeyer, and S. V. Huprikar, Vox Sang., 22,325-343 (1972). M. J. Prigent and R. Bourrillon, Biochirn. Biophys. Acta, 420, 112-121 (1976). H. J. Sage and J. J. Vasquez, J . Biol. Chern., 242, 120-125 (1967). A. S. Wiener, Transfusion (Philadelphia), 1, 308-320 (1961). A. S. Wiener, Blood, 27, 110-125 (1966). 0. Prokop, G. Uhlenbruck, and W. Kohler, Vox Sang., 14,321-333 (1968). L. L. Flory, Vox Sang., 11, 137-156 (1966). I. Matsumoto and T. Osawa, Biochirn. Biophys. Acta, 194, 180-189 (1969).

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tains a-D-galaCtOpyranOSyl-’3’ and 2-acetamido-2-deoxy-D-glucosylbinding lectinslZ5).Second, these designations are generally quite uninformative with regard to the sugar-binding specificity of the lectin. There is certainly a need for an informative nomenclature based on a distinguishing characteristic or feature, similar to that formulated for enzymes.227 (Were a physiological hnction for lectins to be discovered, however, it might be necessary to revise such a system; see Section 1,5.) Makela classified7*lectins into four groups, based on the configuration of C-3 and C-4 of the most complementary pyranose (see Fig. 1). K r i i ~ elisted ~ ~ three groups. The rationale for this system was that lectins can usually be assigned to one of the four groups. However, this framework is severely limited, because it fails to stipulate the configuration of C-2 or of the anomeric carbon atom. Thus, concanavalin A, wheat-germ agglutinin, and the 2-acetamido-2-deoxy-~glucopyranosyl-binding protein from the seeds of Bandeiraeu simplicifolia all conform to Makela’s group I11 lectins and yet, they preferentially bind such disparate sugars as a-D-mannopyranose, N,N’,N’’-triacetylchitotriose,and 2-acetamido-2-deoxy-~-glucopyranose, respectively. Inasmuch as new lectins are being isolated, characterized, and utilized in a variety of systems, we now suggest a nomenclature that cites the genus and species, followed by the sugar-binding specificity in parentheses. If a lectin is not completely specific for a single sugar and anomeric form, it would be necessary to cite two or three structures. If, on the other hand, the lectin exhibits no anomeric preference, the sugar would be cited without the a or /3 designation [for example, the lectins from Canaualia ensiformis (a-D-Manp > a - ~ - G l c p> a-DGlcNAcp), Triticum vulgaris (N,N’,N”-triacetylchitotriose > N,N’diacetylchitobiose > > p-~-GlcNAcp),and Lotus tetrugono2obus (aL - F u ~].~Use ) of this nomenclature would obviously require adequate characterization of the sugar-binding specificity of each lectin so described.

5. Function of Lectins Although numerous physiological roles have been speculated for plant lectins, there is a paucity of supportive data for any of them. The interaction of lectins with animal cells and carbohydrates is almost certainly the result of coincidental complementarity between the lectin (227) “Enzyme Nomenclature,” Recommendations (1972) of the International Union of Pure and Applied Chemistry and the International Union of Biochemistry; Revision of the Recommendations of 1964. American Elsevier Publishing Co., New York, 1972, pp. 1-443.

LECTINS: CARBOHYDRATE-BINDING PROTEINS

147

combining-site and the sugar c ~ n f i g u r a t i o nOn . ~ the ~ ~ other ~ ~ ~ hand, it is probably no accident that these carbohydrate-binding proteins are ~ ~suggested that adapted to bind certain carbohydrate^.^^ K r i i ~ efirst lectins might function as “Kohlenhydratfixieren” (carbohydrate catchers), with roles in such physiological functions as sugar transport, stor~ , ~ ~a ~distinct * ~ ~ ~ possibility. age, or i m m o b i l i z a t i ~ n ~ Jbeing An antibody-like role for the lectins, intended to counteract soil bacteria, was postulated by P ~ n i n ~and ~ OSaint-PaulZ3* (compare, Ref. 232). Indeed, the lectin of Vicia cracca inhibits the growth of a soil micro-organism known to metabolize the seed coats of this s p e ~ i e s . 2 ~ ~ Wheat-germ agglutinin inhibits synthesis of chitin, hyphal growth, and spore germination by binding to the hyphal tips of Trichoderma viride and Fusarium ~ o l a n iIn. ~ all~these ~ cases, a mechanism must be established whereby the plant lectin is transported from the seeds to the roots, and then secreted into the soil. A more-interesting hypothesis predicted a relationship between the agglutinin of a leguminous species and the bacteria of the genus Rhizobium found in the root nodules of the and theorized that the lectin might bind the bacteria to the roots. Although such a relationship is not established by the demonstration that a lectin agglutinates the rhizobial micro-organisms that inhibit that species of legume, there have been several such reports, none of them experimentally convincAs evidence against such a relationship, Makela78found that ing.2366-237e extracts from the seeds of Trifolium pratense, Pisum sativum, Vicia faba, and Phaseolus vulgaris agglutinate neither their own Rhizobium leguminosarum strains nor the strains of one another. Similarly, Dazzo and H ~ b b e 1 demonstrated 1~~~~ that con A combined strongly with vari(228) A. Ensgraber, Ber. Dtsch. Bat. Ges., 29, 349-361 (1958). (229) W. C. Boyd, D. L. Everhart, and M. H. McMaster,J. Immunol., 81, 414-418 (1958). (230) W. Punin, Z . Naturforsch., Teil B , 7,48-50 (1952). (231) M. Saint-Paul, Transfusion (Philadelphia), 4, 3-37 (1961). (232) P. Albersheim and A. J. Anderson, Proc. Natl. Acad. Sci, U.S.A., 68, 1815-1819 (1971). (233) D . A. Jones, Heredity, 19,459-469 (1964). (234) D. Mirelman, E. Galun, N. Sharon, and R. Lotan, Nature, 256,414-416 (1975). (235) M. Krupe, Z . Immunitaetsforsch. E x p . Ther., 107,450-464 (1950). (236) J. Hanlblin and S. P. Kent, Nature (London) New Biol., 245,28-30 (1973). (237) B. B. Bohlool and E. L. Schmidt, Science, 185, 269-271 (1973). (237a) F. Dazzo and D . Hubbell, Appl. Microbiol., 30,1017-1033 (1975). (237b) J. S. Wolpert and P. Albersheim, Biocliem. Biophys. Res. Commun., 70,729-737 (1976). (237c) F. Dazzo, C. Napoli, and D . Hubbell, Appl. Microbiol., 32, 166-171 (1976). (237d) F. Dazzo and D . Hubbell, Plant Soil, 43,717-722 (1975).

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ous nodulating and non-nodulating strains of Rhizobium regardless of their respective hosts. O t t e n s ~ o s e P speculated ~~ that the production of agglutinins may be stimulated by infection with rhizobial microorganisms. More-convincing evidence for the participation of lectins in the recognition processes between leguminous plants and rhizobial bacteria comes from the studies of Dazzo and his colleagues. These workers isolated, from clover seeds and roots, a lectin that binds Rhizobium trifolii organisms to clover root-hairs. Of the sugars (“2-deoxy-D-glucose”) inhibtested, only 2-deoxy-~-arubino-hexose ited this binding interaction; this 2-deoxy sugar was also shown to be a constituent of the capsular heteropolysaccharide S unique to R. trifolii. The striking parallel between the simple sugars that interact with jack-bean lectin (concanavalin A) and the monosaccharides that serve as substrates for yeast hexokinase (see Fig. VI.3 in Ref. 239) suggested a possible enzymic function for the l e ~ t i nHowever, . ~ ~ ~ investigation failed to detect any hexokinase, phosphatase, or phosphorylase activity. The more obvious possibility of a glycosidase activity for lectins must be examined critically, in view of the abundance of glycosidases in most p l a n t - s e e d ~Unlike . ~ ~ ~ glycosidases, the activity of lectins does not increase after germination, and several purified-lectin preparations do not hydrolyze the disaccharides that inhibit their agglutination of animal cells.z0~z28 The observation that UDP-Dgalactose binds to the a-D-galactopyranosyl-binding lectin from Bandeiruea simplicifolia seeds signaled a possible D-galactosyltransferase role for this lectin, although there is actually no evidence to support this view.131 A few investigators have studied the appearance and disappearance oflectins during the life cycle of the plant.z4z,243 J. M. Jones and coworke r reported ~ ~ that ~ Maclura ~ pomiferu agglutinin begins to accumulate (238) F. Ottensooser, Arch. Biol., 39,76-85 (1955). (238a) F. B. Dazzo, C. Napoli, and D. Hubbel1,Appl. Enuiron. Microbiol., 32,166-171 (1976). (238b) F. B. Dazzo and W. J. Bril1,Appl. Enuiron. Microbiol., 33, 132-136 (1977). (239) M. Dixon and E. C. Webb, “Enzymes,” Academic Press, New York, 2nd Edition, 1964, p. 217. (240) I. J. Goldstein, C. M. Reichert, and A. Misaki, Ann. N.Y. Acad. Sci., 234,283-296 ( 1974). (241) K. M. L. Agrawal and 0. P. Bahl,J. Biol. Chem., 243, 103-111 (1968). (242) J. M. Jones, L. P. Cawley, and G. W. Teresa, J. Immunol., 98,364-367 (1967). (243) I. K. Howard, H. J . Sage, and C. B. Horton,Arch. Biochem. Biophys., 149,323-326 (1972).

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during the early development of the seed, reaches a maximum as the seed achieves maturity, and decreases slowly during germination, but it could still be detected in low concentrations at a seedling age of six months. All lectin activity was localized within the seed, none being found in either the seed coat or the leaves of the mature tree. On studying the same phenomenon in the lentil (Lens culinaris), Howard and noted the appearance of lectin during early development and differentiation ofthe seed embryo; a possible involvement in the germination and maturation process was thus indicated. Con A and the lectin from Phnseolus vulgaris were shown to stimulate the germination of The castor-bean agglutinin, present only in the matrix region of protein bodies of the seed e n d o ~ p e r m , 2 ~ ~ ~ . ~ undergoes slow degradation during germination, in contrast to storage proteins .243b Talbot and Etzler isolated a possible “prolectin” from the stems and leaves of the Dolichos biflorus plant.243dPresent in microgram quantities, the prolectin cross-reacts with antibodies to the seed lectin. The cross-reacting material, which neither agglutinates human blood-group A erythrocytes, nor binds to insolubilized, hog bloodgroup (A + H ) substance (as does the seed lectin), has one subunit identical to subunit I A of the seed lectin, and a unique subunit of higher molecular weight. In common with the seed lectin, both “prolectin” subunits have an N-terminal alanine residue, suggesting an extension at the carboxyl end of one of the polypeptide chains. Mialonier and also discovered the presence of a substance in the leaves of Phaseolus vulgaris L. (var. red) that cross-reacted with antiserum to the seed lectin. Finally, it has been shownz4 that addition of black-bean (Phaseolus vulgaris) phytohemagglutinin to the normal diet of the bruchid beetle that can eat phytohemagglutinin-free cowpeas (Vigna unguiculata), but not P . vulgaris seeds, kills the bruchid larvae. It was concluded2” that “a major part of the adaptive significance of phytohemagglutinins in black bean and other legume seeds is to protect tham from attack b y insect seed predators.” (243a) D. Southworth, Nature, 258, 600-602 (1975). (24311) R. J . Youle and A. H. C. Huang, Plant Physiol., 58,703-709 (1976). (243c) R . E. Tully and H. Beevers, Plant Physiol., 58,710-716 (1976). (243d) C. F. Talbot and M. E. Etzler, Fed. Proc., 36,795 (1977). (243e) G . Mialonier, J.-P. Privat, M . Monsigny, G . Kahlem, and R. Durand, Physiol. Veg., 11,519-537 (1973). (244) D. H. Janzen, H. B. Juster, and I. E. Liener, Science, 192, 795-796 (1976).

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11. D-MANNOSE(D-GLUCOSE)-BINDING LECTINS 1. Concanavalin A of the Jack Bean (Canavalia ensiformis) (jack bean; a-D-Manp > a - ~ - G l c p> a-~-GlcNAcp) a. Introduction; Physical and Chemical Characterization.Concanavalin A (con A) is without doubt the most notable of the plant agglutinins. First isolated and crystallized by Sumner and H 0 ~ e 1 1 , ~ ~ ~ , ~ who identified it as the phytohemagglutinin of the jack bean this lectin exhibits a series of remarkable (Canuvulia properties. It has been employed as a structural probe for carbohydrates in s ~ l u t i o nas ’ ~well as at cell surfaces,25as a lymphocyte mitogen,248,249as a reagent for differentiating normal from malignant ce11s,31~2so~251 as an anticancer agent,33,252 and, in its interaction with carbohydrates, as a model for the antibody-antigen reaction.72*102,’21~168~253 Currently, its interaction with hormone-like receptors (for example, insulin) is being studied i n t e n ~ i v e l y . ~Sepharose~-~~ coupled con A (con A-Sepharose) is being used to isolate membrane glycoproteins, enzymes, cell-surface receptors, and viral glycoproteins, all of which possess con A-reactive oligosac~harides.~~ A novel immuno-electrode composed of con A covalently attached to a poly(viny1 chloride) membrane has been constructed, and shown to be capable of sensing yeast D - ~ ~ l a n n a nTwo . ’ ~ ~volumes devoted entirely to con A have been p u b l i ~ h e d , ~ ~ ~ ~ ~ ~ Sumner and HowellLo2 prepared con A by extracting jack-bean meal (pre-extracted with aqueous acetone and ethanol) with 5% sodium chloride solution, and dialyzing the salt extract against distilled water containing toluene. From 100 g of meal, these investigators obtained 2.5-3.0 g of crystalline con A. The lectin, which could not be entirely freed of carbohydrate,lo2agglutinated cat, dog, guinea-pig, horse, and rabbit erythrocytes, but not the red blood-cells ofthe cow, goat, human, pig, or sheep.10224462447 In all respects, con A exhibited the properties of a protein: it was (245) J. B. Sumner,]. B i d . Chem., 37, 137-142 (1919). (246) J. B. Sumner, S. J. Howell, and A. Zeissig, Science, 82, 65-66 (1935). (247) J. B. Sumner and S. I?. Howell,]. Ztnmunol., 29, 133-134 (1935). (248) M . Wecksler, A. Levy, arid W. G. Jaffh, Acta Cient. Venez., 19, 154-156 (1968). (249) A. E. Powell and M. A. Leon, E x p . Cell Res., 62,315-325 (1970). (230) M. Inbar and L. Sachs, Nature, 223, 710-712 (1969). (251) H. Ben-Bassat, M. Inbar, and L. Sachs,]. Membr. Biol., 6, 183-194 (1971). (252) J. Shoham, M. Inbar, and L. Sachs, Nature, 227, 1244-1246 (1970). (253) E. J. Hehre, Bull. Soc. Chim. Biol., 42, 1581-1585 (1960). (254) J. Janata,J. Am. Chem. Soc., 97, 2914-2916 (1975). (255) H. Bittiger and H. P. Schnebli, “Concanavalin A as a Tool,” Wiley, New York, 1976, pp. 1-639.

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denatured102by 0.1 M hydrochloric acid and 0.1 M sodium hydroxide, readily digested by pepsin102(but it resisted tryptic digestionlo2),contained 3.36% of tryptophan, 5.2% of tyrosine, and 0.4% of c y ~ t e i n e , ~ ~ ~ and had an isoelectric pointlo2of 5.5and a molecular weight of 96,000in the jack-bean lectin neutral, phosphate b ~ f f e r . ~A~metalloprotein, '=~~~ contains Mn2+ and Ca2+; removal of the metal ions abolished the hemagglutinating In its ability to precipitate glycogen and starch from solution, agglutinate erythrocytes from many animal species, and clump certain bacteria (such as Mycobacterium and Actinomyces),con A was likened to an antibody.'02Sumner and Howell102also suggested "that the chemical component in stromata [of rabbit erythrocytes] with which con A unites may be glycoprotein. . ." These same investigators observed102 that cane sugar "prevents the agglutination of starch as well as the agglutination of erythrocytes," thus demonstrating the first hapten inhibition of the con A system by sugars of low molecular weight. Inhibition of the turbidimetric reaction between con A and polysaccharide has been used for the quantitative analysis of D - m a n n o ~ e . ' ~ ~ ~ In 1965,the isolation of con A in pure form was reported by Agrawal and C o l d ~ t e i n . ' Exploiting ~ ~ ~ ' ~ ~ the observation that con A precipitates dextrans,120,121~253,260 these investigators adsorbed the jack-bean lectin on cross-linked dextran gel (Sephadex),and eluted it with D-glucose, a competitive inhibitor of this interaction.120,168 Some time later, Olson and L i e n e F also purified con A to homogeneity by adsorption to Sephadex and elution with M acetic acid. These reports constituted the first instance of lectin purification by affinity chromatography. Physical characterization of con A revealed monodispersity in the ultracentrifuge at pH 2-5, and a two-peak pattern at pH 7, suggesting pH-dependent association of s ~ b ~ n i t s It . ~is ~ now ~ *known ~ ~ ~ * ~ ~ ~ (256) J. B. Sumner and V. A. Graham,]. B i d . Chem., 64,257-261 (1925). (257) J, B. Sumner, N. Gralkn, and I.-B. Eriksson-Quensel, Science, 87,395-396 (1938). (258) J. B. Sumner, N. GralBn, and 1.-B. Eriksson-Quensel,]. Biol. Chem., 125,45-48 (1938). (259) J. B. Sumner and S. F. Howell,]. Biol. Chem., 115,583-588 (1936). (259a) R. D. Poretz and I. J. Goldstein, Carbohydr. Res., 4,471-477 (1967). (260) J. A. Cifonelli, B. A. Lewis, R. Montgomery, and F. Smith,ABstr. Pap. Am. Chena. soc. Meet., 129,313 (1956). (261) B. B. L. Agrawal and I. J. Goldstein, Biochim. Biophys. Acta, 133,376-379 (1967). (262) B. B. L. Agrawal and I. J. Goldstein,Arch. Biophys. Biochem., 124,218-229 (1968). (262a) K. D. Hardman and C. F. Ainsworth, Biochemistry, 11,4910-4919 (1972). (263) J. L. Wang, B. A. Cunningham, and G. M. Edelman, Proc. N a t l . Acad. Sci. U.S.A., 68, 1130-1134 (1971). (264) A. B. Edmundson, K. R. Ely, D. A. Sly, F. A. Westholm, D. A. Powers, and I. E. Liener, Biochemistry, 10, 3554-3559 (1971).

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that con A consists of polypeptide subunits, ofmolecular weight 26,000. At pH 5.6 and below, two protomers are associated in a dimer of molecular weight 52,000; above pH 5.6, the dimers aggregate, forming tetramers. As aggregation is pH- and temperature-dependent,2s4a molecular weight determinations must be scrutinized in terms of the conditions of measurement. The original determination of 96,000 (in neutral, phosphate buffer) by Sumner and coworker^^^^*^^^ is in excellent agreement with current estimates based on amino acid analyses (dimer, 52,000; tetramer, 104,000). Molecular-weight determinations from analytical, ultracentrifuge data136,261,262,265,266 vary between 50,000 and 120,000. Con A holds the distinction of being the first lectin to have its amino acid sequence determined,264.267-271 and the first for which the threeThe dimensional crystal-structure has been solved. multiple isomorphous-replacement method, using a series of heavymetal derivatives (sodium mersalyl, Pb(NO,),, K,PtCl,, K2PtC16,and mercuric phenylacetate), revealed the overall molecular architecture of crystalline con A by low-resolution, X-ray d i f f r a ~ t i o n , 2 ~and ~ - ~later, ~~3~~ by high-resolution studies at267-269375*276 200 and262a*274 240 pm to be a composite of four identical protomers (molecular weight 26,000).The pseudotetrahedral cluster has an aggregate molecular weight of (264a) M. Huet, Eur. J . Biochem., 59,627-632 (1975). (265) A. J. Kalb and A. Lustig, Biochim. Biophys. Acta, 168,366-367 (1968). (266) G. H. McKenzie, W. H. Sawyer, and L. W. Nichol, Biochim. Biophys. Acta, 263, 283-293 (1972). (267) G. M. Edelman, B. A. Cunningham, G. N. Reeke, Jr., J. W. Becker, M. J. Waxdal, and J. L. Wang, Proc. Natl. Acad. Sci. U.S.A., 69, 2580-2585 (1972). (268) G. N. Reeke, Jr., J. W. Becker, B. A. Cunningham, G. R.Gunther, J. L. Wang, and G. M. Edelman, Ann. N.Y. Acad. Sci., 234, 369-382 (1974). (269) G. N. Reeke, Jr., J. W. Becker, B. A. Cunningham, J. L. Wang, I. Yahara, and G. M. Edelman, Ado. E x p Med. Biol., 55, 13-33 (1975). (270) J. L. Wang, B. A. Cunningham, M. J. Waxdal, and G. M. Edelman, J . Biol. Chem., 250, 1490-1502 (1975). (271) B. A. Cunningham, J. L. Wang, M. J. Waxdal, and G. M. Edelman, J . Biol. Chem., 250, 1503-1512 (1975). (272) K. D. Hardman, M. K. Wood, M. Schiffer, A. B. Edmundson, and C. F. Ainsworth, Proc. Natl. Acad. Sci. U.S.A., 68, 1393-1397 (1971). (273) K. D. Hardman, M. K. Wood, M. Schiffer, M. Edmundson, and C. F. Ainsworth, Cold Spring Harbor Symp. Quant. Biol., 36,271-275 (1971). (274) K. D. Hardman, Adu. E x p . Med. Biol., 40, 103-123 (1973). (274a) J. Greer, H. W. Kauhan, and A. J. Kalb,J. Mol. Biol., 48, 365-366 (1970). (275) J. W. Becker, G. N. Reeke, Jr., J. L. Wang, B. A. Cunningham, and G. M. Edelman, J . Biol. Chem., 250, 1513-1524 (1975). (276) G. N. Reeke, Jr., J. W. Becker, and G. M. EdelmanJ. Biol. Chem., 250,1525-1547 (1975). (277) F. A. Quiocho, G.N. Reeke, Jr., J. W. Becker, W. N. Lipscomb, and G. M. Edelman, Proc. Natl. Acad. Sci. U.S.A., 68, 1853-1857 (1971).

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104,000. The single polypeptide-chain protomers (however, see later) are compactly folded, forming dome-shaped structures approximately 4.2 nm high x 4.0 nm wide x 3.9 nm thick. Each subunit contains one Mn2+,one Ca2+,and one carbohydrate binding-~ite,'~~,~~~-~~~ believed, until recently, to be situated in a prominent cavity in the molecular Two such subunits are joined with their flattened bases proximal, to form roughly ellipsoidal domes. The dimers pair across one another, to form tetramers as depicted in Fig. 2.

FIG.2.-Schematic Representation of Con A T e t ~ a r n e r[Ca, .~~~ Mn, and S indicate the position of the Ca2+,Mn2+,and carbohydrate-specific binding-sites, respectively. I localizes the position of the hydrophobic binding-site present in crystals of the I222 space group (reproduced by permission of Nature).]

The most striking feature of the con A secondary structure is its high content (>50%) ofp-pleated sheet, a finding supported by c.d. and 0.r.d. studies.281-285a A theoretical study of 0-sheet-breaking residues was (278) J. Yariv, A. J. Kalb, and A. Levitzki, Biochim. Biophys. Acta, 165,303-305 (1968). (279) G. H. McKenzie and W. H. Sawyer, J . Biol. Chem., 248,549-556 (1973). (280) J. W. Becker, G. N. Reeke, Jr., and G. M. EdelmanJ. Biol. Chem., 246,6123-6125 (1971). (281) R. Zand, B. B. L. Agrawal, and I. J. Goldstein, Abstr. Pap. Am. Chern. SOC.Meet., 156, BIOL-132 (1968). (282) C. Kay, FEBS Lett., 9,78-80 (1970). (283) M. N. Pflumm, J. L. Wang, and G. M. Edelman,J. Biol. Chem., 246,4369-4370 (1971). (284) R. Zand, B. B. L. Agrawal, and I. J . Goldstein, Proc. Natl. Acad. Sci. U.S.A., 68, 2173-2176 (1971). (285) W. D. McCubbin, K. Oikawa, and C. M. Kay, Biochem. Biophys. Res. Commun., 43,666-674 (1971).

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conductedzE6 on con A. One extensive, @-pleatedsheet forms almost the entire back surface of the protomer, and is responsible for most of the noncovalent interactions bonding two protomers to form dimers, and two dimers to form tetramers. There are two additional regions of @-structure;the rest of the protein exists as random coil. The interested reader is referred to extensive discussionsz6za~26E~~6g~z74~z76~zE7 on the molecular structure of con A. A second remarkable structural feature is the presence in native con A of several molecular species: an intact subunit of molecular weight 25,500 containing 237 amino acids, and three fragments of the subunit termed Al, A‘,, and A2 (Ref. 263), B1, B2, and B2a (Ref. 288),and I, 11, and I11 (Ref. 264). Disc gel-electrophoresis in dodecyl sodium sul8 fate,263,2EE gel filtration in guanidiniuni chloride44 propanoic M urea,zEEa or 40% acetic acid,zE6and ion-exchange chromatography on Amberlite CG-50 (Ref.264) or D E A E - c e l l u l ~ sallowed e ~ ~ ~ separation of these species (compare, Ref. 28813). By end-group and amino acid analyses of the fragments, it was found that the fragments are all derived from the intact, polypeptide subunit; Al and A2 result from cleavage ofthe polypeptide chain between residues 118 (Asn)and 119 (Ser), and At1 (present in low proportion) is derivedz63from A,. Tryptic, peptide maps confirmed these Interestingly, there are no obvious differences between the three-dimensional structures or biological activities of con A tetramers made up entirely of intact subunits and of tetramers containing fragment^,^^^,"^ However, there are some differences in the physical and chemical properties of the two species. For example, incubation of native con A in 1% ammonium hydrogencarbonate at 37” results in the formation of a precipitate that contains the intact subunit and the naturally occurring fragments of the molecule, whereas the solution phase contains only the intact subA similar fractionation may be achieved by gel filtration on Bio-Gel P-100 (Refs. 279 and 290) and by gradient elution from Sephadex with D-g~ucosezE9; the fragmented molecules are eluted at (285a) W. D. McCubbin, K. Oikawa, and C. M.Kay, F E B S Lett., 23, 100-104 (1972). (286) E. A. Kabat and T. T. Wu, Proc. Natl. Acad. Sci. U.S.A., 70, 1473-1477 (1973). (287) K. D. Hardman and I. J. Goldstein, in “Immunochemistry of Proteins,” M. Z. Atassi, ed., Plenum Press, New York, 1977, Vol. 2, pp. 373-416. (288) Y. Abe, M. Iwabuchi, and S.-I. Ishii, Biochem. Biophys. Res. Commun., 45, 12711278 (1971). (288a) M. 0. J. Olson and I. E. Liener, Biochemistry, 6, 3801-3808 (1967). (289) B. A. Cunningham, J. L. Wang, M. N. Pflumm, and G. M. Edelman, Biochemistry, 11,3233-3239 (1972). (290) W. H. Sawyer, S. Hammarstrom, G. Moller, and I. J. Goldstein,Eur.J.Immunol., 5, 507-510 (1975).

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lower concentrations of D-glucose than the intact molecules. The origin of the fragments is unknown, but they could result from proteolytic cleavage prior to, or during, the isolation.z63Moving-boundary electrophoresisZ6' and isoelectric-focusing studies290aalso revealed the heterogeneity of purified con A. An isoelectric point of 4.5-5.5 was electrophoresis ~~a on cellulose aceobtained by isoelectric f o c u ~ i n g , ~ tate,290acataphoresis,lo2and the minimum-solubility method,z90b in contrast to pZ 7.1 k O . 1 obtained by free-boundary electrophoresisz6zand electrophoresis on cellulose acetate.z62 The availability of the intact subunits was a prerequisite to sequence determination. Tryptic and chymotryptic digestion ofthe intact subunit produce both fragments of high molecular weight and smaller peptides,zE9neither of which are the same as the naturally occurring con A fragments. Three fragments (FlyFZ, and F3)were generated by degradation with cyanogen b r ~ m i d eas, was ~ ~ to ~ be ~ expected ~ ~ ~ from a polypeptide containing two methionyl residues. The order ofthe three peptides was established by end-group analysis, and confirmed by sequence analysis of the two methionine-containing, overlapping peptides isolated from tryptic digests.z91Sequence analysis of the F, and Fz pepgave the complete sequence of 237 amino tidesZ7O and the F3peptideZ7* acids. As already indicated, each con A subunit contains one Caz+and one Mnz+ ion. Both metals are needed for carbohydrate-binding, and, hence, for all of the biological activities of the protein (compare Ref. 292), a dependence first established by Sumner and and The metals may be later verified by several investigators.'73~z7E~z93~z94 removed from the protein by acidification with 0.1M hydrochloric acid and dialysis against distilled water,zsgor by dialysis against 1.0M acetic acidszs3Binding of Mnz+ to demetallized con A must precede Caz+ binding,173,z94 the Mnz+being bound to a specific site, S1. It appears that the binding of the transition-metal ion induceszg4the formation of a showed specific Ca2+ion-binding site (S2). Shoham and (290a) G. Entlicher, J. V. KoStii, and J. Kocourek,Biochim. Biophys. Acta, 236,795-797 (1971). (290b) F. A. Czonka, J. C. Murphy, and D. B. Jones,]. Am. Chem. Soc., 48, 763-768 (1926). (291) M . J. Waxdal, J. L. Wang, M. N . Pflumm, and G. M. Edelman, Biochemistry, 10, 3343-3347 (1971). (292) C. F. Brewer, D. M. Marcus,A. P. Grollman, and H. Sternlicht,]. Biol. Chem., 249, 4614-4616 (1974). (293) B. B. L. Agrawal and I. J. Goldstein, Can. J. Biochem., 46, 1147-1150 (1968). (294) A. J. Kalb and A. Levitzki, Biochem. J., 109,669-672 (1968). (295) M . Shoham, A. J. Kalb, and I. Pecht, Biochemistry, 12, 1914-1917 (1973).

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that the Ca2+site is specific; it binds Caz+or Cd2+,but not Ba2+,transition metals, or Sm3+.Ni2+ may be substituted for Mn2+without any substantial change in carbohydrate-binding activity of the l e ~ t i n . ~ ~ ~ Mn2+and Ca2+appear to stabilize the conformation of the subunit; in their absence, the four carboxyl groups clustered at the metal bindingsites would strongly repel one another.274For this reason, it is, perhaps, not surprising that the demetallized apoprotein cannot be crystallized in the same space-group.2g6A U.V. difference-spectrum resulted when Mn2+and Ca2+were added to the metal-free protein.2g7Potentiometric titration, and chemical modification, of the demetallized protein indicated that two histidine residues, pK, 6.8, are ligands for the transition-metal ion.2g8Titration of Caz+ion indicated involvement of two carboxyl groups (pK, 4.2).2g9 The X-ray diffraction data revealed that the two metal ions are -450 pm apart.262a*27s Edelman and coworker^^^^.^^^ identified Glu 8, Asp 10, Asp 19, His 24, and 2 water molecules as coordinate ligands for Mn2+;and Asp 10, two water molecules, and the backbone carbonyl groups of Tyr 12, Asn 14, and Asp 19 as ligands for Ca2+.Hardman and AinsworthZBza and HardmaP4 obtained the same results for Mn2+,but reported only one water molecule and Asp 208 in addition to the four amino acid side-chains reported by Edelman’s group. Con A prepared by Sephadex affinity-chromatography may not contain its full complement of metal ions, giving rise to forms that differ in their carbohydrate-binding ability.300,301 This deficiency may be alby addition of Mn2+and Ca2+.Gadolinium (Gd3+)binds to a site (S3) distinct from the Mn2+( S l ) and Ca2+(S2)binding sites. Alter and his showed that binding of Mn2+to con A is cooperative in the presence, and noncooperative in the absence, of Ca2+;the degree of cooperativity increases with pH. A kinetic study of the binding of Mn2+and Ca2+to con A over the pH range of 5.3 to 6.4 has been conducted.304b (296) A. Jack, J. Weinzierl, and A. J. Kalb,J. Mol. Biol., 58, 389-395 (1971). (297) R. J. Doyle, D . L. Thomasson, R. D. Gray, and R. H. Clew, FEBS Lett., 52,185-187 (1975). (298) G. Gachelin and L. Goldstein, FEBS Lett., 26,264-266 (1972). (299) G. Gachelin, L. Goldstein, D. Hofnung, and A. J. Kalb, Eur. J . Biochem., 30, 155-162 (1972). (300) T. Uchida and T. Matsumoto, Biochim. Biophys.Acta, 257,230-234 (1972). (301) B. Karlstrom, Biochim. Biophys. Acta, 329,295-304 (1973). (302) A. D. Sherry and G. L. Cottom, Arch. Biochem. Biophys., 156,665-672 (1973). (303) A. D. Sherry, A. D. Newman, and C. G. Gutz, Biochemistry, 14,2191-2196 (1975). (304) B. H. Barber, B. J. Fuhr, and J. P. Carver, Biochemistry, 14,4075-4082 (1975). (304a) G. M. Alter, E. R. Pandolfino, D. J. Christie, and J. A. Magnuson, Biochemistry, 16,4034-4038 (1977). (304b) R. D. Brown 111, C. F. Brewer, and S. H. Koenig, Biochemistry, 16,3883-3896 (1977).

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Koenig and coworkers30sreported the dependence on magnetic field and temperature of solvent proton spin-lattice relaxation-rates for zinc and manganese derivatives of con A in solution at pH 5.6; they concluded that there is one rapidly exchanging water molecule ligand on the Mn2+ion (residence lifetime, 2.5 psec at 25") and, further, that monosaccharide binding to manganese-con A has little effect on the relaxation rates of solvent protons. This suggests that sugars do not bind directly to the transition metal. Essentially similar results were obtained by Villafranca and Viola.306 Con A has also been isolated by Sephadex affinity-chromatography and Canavalia maritima30ecbeans. Both from Canavalia gladiata306a,b*c proteins were immunologically indistinguishable306C from C. ensiformis con A. Interestingly, whereas C. ensiformis and C . gladiata con A contain two metliionine residues per protein subunit, only one .~~~~ methionine residue is found in the C. maritima l e ~ t i n Analysis indicated that the methionine residue at position 129 in C. ensiformis from C. maritima con A. con A is

b. The Concanavalin A-Saccharide Binding-site.-In

1971, Becker

and coworkersza0reported locating the con A saccharide-binding site. They infused o-iodophenyl P-D-glucopyranoside, a glycoside inhibitor of con A-levan precipitation,lEO into lectin crystals (1222)and analyzed the crystalline, lectin-glycoside complex by X-ray crystallography. Difference electron-density projections at a resolution of 280 pm positioned the iodine atoms directly within the prominent surface cavities of the protein (see Fig. 2). The site identified was 350-600 pin wide, -750 pm high, and 1.8 nm deep. The iodine atom was deeply buried in the cavity; the glycopyranoside ring was, assumedly, situated between the iodine atom and the surface, with the anomeric carbon atom toBecker and coworkers also wards the interior of the protein.267*268~280 speculated that, inasmuch as con A binds nonreducing termini of polysaccharides, in order to bind at the same site as o-iodophenyl P-D-ghcopyranoside, the polysaccharide terminus would have to be oriented in the reverse direction, with the anomeric carbon atom towards the surface of the protein, and the polysaccharide extending out into the solution.268,280 In the case of D-glucose, having equatorial (305) S. H. Koenig, R. D. Brown, 111, and C. F. Brewer, Proc. Natl. Acad. Sci. U.S.A., 70, 475-479 (1973). (306) J. J. Villafranca and R. E. Viola, Arch. Biochem. Biophys., 165, 51-59 (1974). (306a) S. Nakaniura and R. Suzuno, Arch. Biocheni. Biophys., 111,499-505 (1965). (306b) H . Akedo, Y. Mori, Y. Tanigaki, K. Shinkai, and K. Morita, Biochim. Biophys. Act(/., 271, 378-387 (1972). (306c) D. R. Hague, Platit Physiol., 55, 636-642 (1975).

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groups at C-2, C-3, and C-4, reversal would leave these three hydroxyl groups in the same relative position. Each particular saccharide would bind in one or the other orientation, depending on the restrictions imposed by the site and the configurational features of the specific sugar.z68*280 Brewer and coworkers studied the binding of I3C-enriched methyl aand /3-D-glucopyranoside to con A by carbon-13 nuclear magnetic A or Mn-con A to the resonance s p e ~ t r o s c o p yAddition . ~ ~ ~ ~ of ~~ Zn-con ~ D-glucoside resulted in line broadening in the I3C-n.m.r. spectrum of the sugar, without detectable changes in the chemical shifts of the carbon resonances. [13C]Sugarcarbon-13 spin-lattice relaxation-times (TJ, measured in the presence and absence of the two proteintransition metal derivatives, was used to calculate the distance between Mnz+and each bound, sugar carbon atom. The results indicated that methyl a-D-ghcopyranoside binds (to the protein) in the 4C, conformation, with C-3 and C-4 closest to the Mnz+,at a mean distance of 1 nm. This value contrasts with 2 nm proposed from the results of X-ray crystallographic s t ~ d i e s Different . ~ ~ ~ binding-orientations ~ ~ ~ ~ ~ ~ ~ ~for methyl a-and /I-D-glucopyranosidewere postulated, in which the 2-, 3-, and 4-hydroxyl groups of the p anomer bind at positions occupied by the 6-, 4-, and 3-hydroxyl groups of the a anomer, respectively.30s Two further studies, one by Villafranca and Viola,309who carried out I3C spin-lattice relaxation measurements on the interaction of methyl a-D-glucopyranoside with con A, and the other by Alter and Magn u ~ o n , ~ ~who O characterized the binding of 2-deoxy-2(trifluoroacetamido)-D-glucose to the lectin, placed the saccharide at 1.0 to 1.4 nm from the Mnz+ion, in agreement with the results of Brewer and coworker^.^^^^^^^ Villafranca and Viola’s mode1309placed C-1, C-2, and C-6 closest to the Mnz+(see Fig. 3). On the other hand, support for the cavity proposed by Becker and coworkerszs0comes from p.m.r.spectral measurements of the longitudinal relaxation-times of the methyl proton of methyl a-D-mannopyranoside with Znz+-and Gdz+substituted con A.311*31z (307) C. F. Brewer, H. Sternlicht, D. M. Marcus, and A. P. Grollman, Proc. Natl. Acad. Sci. U.S.A.,70, 1007-1011 (1973). (308) C. F. Brewer, H. Sternlicht, D. M. Marcus, and A. P. Grollman, Biochemistry, 12, 4448-4457 (1973). (309) J. J. Villafranca and R. E. Viola, Arch. Biochem. Biophys., 160, 465-468 (1974). (310) G. M. Alter and J. A. Magnuson, Biochemistry, 13,4038-4045 (1974). (311) B. H. Barber, A. Quirt, and J, P. Carver, Adu. E r p . Med. B i d , 55, 325 (1975). (312) B. J. Fuhr, B. H. Barber, and J. P. Carver,Proc. N a t l . Acad. Sci. U.S.A.,73,322-326 (1976).

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FIG.3.-Distances from the Con A Mn2+Site to the Seven Carbon Atoms of Methyl cu-DGlucopyranoside.811(Reproduced by permission of Arch. Biochem. Biophys.)

In an important paper, Hardman and Ainsworth313showed that con A crystals contain a cavity that binds a number of relatively nonpolar molecules (methyl p-hydroxybenzoate, phenyl phosphate, o-iodobenzoic acid, o-iodoaniline, and dimethylmercury) and, most important, o-iodophenyl P-D-galactopyranoside. As a noninhibitor of the con A system, o-iodophenyl P-D-galactopyranoside serves as a control substance for o-iodophenyl P-D-glucopyranoside binding. Binding of the galacto analog and several unrelated nonpolar substances at a position identical with the purported D-glucoside binding-site strongly suggested that this cavity is not the specific carbohydrate-binding site of the jack-bean lectin. Rather, it appears to be a nonspecific binding site for hydrophobic molecules (perhaps lipid).314Interestingly, native crystals of con A cracked and dissolved in methyl a-D-mannopyranoside of 21 mM concentration, and in 5 mM o-iodophenyl P-D-glucopyranoside, but not in 5 mM o-iodophenyl P-D-galactopyranoside (which does not bind to the protein in soluti~n).~ These l~ results suggested that molecules binding at the carbohydrate-specific site either sterically interfere with the crystallattice packing, or produce in the protein conformational changes which do so. Conformational changes may occur as a result of substrate binding to con A, as monitored by U.V. difference spectroscopy, (313) K. D. Hardman and C. F. Ainsworth, Biochemistry, 12,4442-4448 (1973). (314) W. G. Jaffk and A. Palozzo, Actn Cient. Venez., 22,102-105 (1971). (315) R. J. Doyle, E. P. Pittz, and E. E. Woodside, Cnrbohydr. Res., 8,89-100 (1968).

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circular dichroism studies,283*285 n.m.r.-spectral measurements,316electrophoretic mobility,317 and the use of a “reporter” group [2-(bromomethyl)-4-nitrophenol].318 Alkali- and urea-induced conformational changes in con A have also been studied.319The demonstration that ligands which bind to con A in solution [for example, methyl a - ~ - m a n n o p y r a n o s i d e , 2MnZ+ ~ ~ ~ (Refs. ~ ~ ~ * ~173,285,297, ~~ and 321),and Ca2+ (Refs. 173, 285, 297, 316, and 321)] stabilize and protect the protein against aggregation,320heat denaturation,318enzymic digestion (pronase),318precipitation of fragments during incubation289at 3 T , pH 7.9,and the time-dependent change in molecular weight observed in the absence of ligand,266s318 also supports the possibility of ligandinduced, conformational change. Further evidence that there may be fundamental differences between the carbohydrate-binding properties of con A in solution and in the crystalline state was provided by Bessler and coworkers174using U.V. difference displacement spectroscopy. Certain ligands, such as p-nitrophenyl a-D-mannopyranoside, bind to con A, thereby inducing pronounced ultraviolet spectral changes. A second ligand binding to the same site can displace the chromogenic ligand from con A, quenching the difference spectrum. Bessler and coworkers found that all saccharides inhibiting con A-polysaccharide interaction are able to displace p-nitrophenyl a-D-mannopyranoside from con A, whereas no quenching of the difference spectrum was noted with myo-inositol (compare Ref. 322), o-iodophenyl P-D-galactopyranoside, and p-nitrophenol, all of which are noninhibitors of con A-polysaccharide i n t e r a ~ t i 0 n .The l ~ ~ data were interpreted in terms of a single carbohydrate binding-site per monomeric unit for the protein in solution. Inhibition indices of a number of glycosides were linearly related to their association constants, and provided further support for a single, carbohydrate-specific, b i n d i n g - ~ i t e . ~ ~ ~ (316)B. H. Barber and J. P. Carver, Can. J . Chem., 53,371-379 (1975). (317)H. Akedo, Y. Mori, M. Kobayashi, and M. Okada, Biochem. Biophys. Res. Commun., 49, 107-113 (1972). (318)R. J. Doyle, S. K. Nicholson, R. D. Gray, and R. H. Glew, Carbohydr. Res., 29, 265-270 (1973). (319)M. N. Pflumm and S. Beychok, Biochemistry, 13,4982-4987(1974). (3u)) L. L. So and I. J. Goldstein,]. Biol. Chem., 242, 1617-1622 (1967). (321)R. J. Doyle, D. L. Thomasson, and S. K. Nicholson, Carbohydr. Res., 46,111-118 (1976). (322)K.D.HardmanandC. F.Ainsworth,Nature(London)New Blol., 237,54-55(1972). (323)F.G. Loontiens, J. P. Van Wauwe, and C. K. De Bruyne, Carbohydr. Res., 44, 150-153 (1975).

LECTINS: CARBOHYDRATE-BINDING PROTEINS

161

The carbohydrate-specific binding-site of con A has been identified in crystals of the con A-methyl a-D-mannopyranoside (or its 2-deoxy-2iodo derivative) ~ o m p l e ~ It . ~was ~ ~necessary * ~ ~ ~to* obtain ~ ~ ~a new crystalline form of the carbohydrate-con A complex, as protein crystals of the I222 space group cracked and dissolved upon addition of such sugar derivatives as methyl a-D-mannopyranoside. Growth of the new crystalline form was obtained by incubating con A with a large excess of methyl a-D-mannopyranoside, followed by replacement of the D-mannoside with o-iodophenyl P-D-glucopyranoside or methyl 2-deoxy-2-(2-iodoacetamido)-c~-~-glucopyranoside.32~ The gross conformations of the monomers, dimers, and tetramers in the new space group C222, were found very similar to those of the carbohydrate-free protein. The carbohydrate binding-site at 600-pm resolution was 700 pm and 1.1nm from the Ca2+and Mn2+,respect i ~ e l yThese . ~ ~ ~results are in good agreement with those obtained by n.m.r. s p e ~ t r o s c o p y . The ~ ~ ~amino - ~ ~ ~acyl side-chains in closest proximity to the bound carbohydrate appear to be324Tyr 12 and 100, Asp 16 and 208, Asn 14, Leu 99, Ser 168, and Arg 228. The finding of Asp 16 and 208 corroborates evidence from chemical modification, and titration (see later).326Tyrosyl groups have similarly been implicated.327This carbohydrate-specific site is 3.5nm from the iodophenyl (hydrophobic) site originally proposed as the carbohydrate binding-site.280Essentially similar results were obtained by Becker and coworkers32gin structural studies on demetallized, inactive con A, and on glutaraldehyde crosslinked crystals (space group 1222) complexed with methyl 2-deoxy-2iodo-a-D-mannopyranoside.Removal of bound Mn2+ and Caz+ from native con A resulted in a loss of carbohydrate-binding activity, suggesting an association between the metal-binding and saccharidebinding sites.325Indeed, interpretation of electron-density maps localized significant structural differences between native con A and metal-free con A (compare Ref. 296) in the vicinity of the carbohydrate-binding site.325Studies at higher resolution will undoubtedly provide a more definitive structural solution to con A-saccharide interaction. Efforts to identify the amino acyl residues involved in (324) K. D. Hardman and C . F. Ainsworth, Biochemistry, 15, 1120-1128 (1976). (325) J. W. Becker, G. N. Reeke, Jr., B. A. Cunningham, and G. M. Edelman,Nature, 259, 406-409 (1976). (326) G. S . Hassing, I. J. Goldstein, and M. Marini, Biochim. Biophys. Acta, 243,90-97 (1971). (327) R. J. Doyle and 0. A. Roholt, Life Sci., 7 , 841-846 (1968).

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IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

carbohydrate-con A interaction have been r e p ~ r t e d . ~ These ~ ~ - in~~~ clude chemical-modification s t ~ d i e s , 3 ~ “affinity ~ ~ ~ labelling attempts,326,332*333 and hydrogen-ion titration in the presence and absence of methyl a-~-mannopyranoside.~~~ Approximately eight carboxyl groups (pK, 3.8) per 100 kg of protein (2 carboxyl groups/subunit) are not titratable when methyl a-D-mannopyranoside is present.326Furthermore, the same glycoside protected con A carboxyl groups from amidation with g l y ~ i n and e~~ n i~t r o t y r ~ s i n emethyl ~ ~ ~ esters. Dean and Homer334 measured the quenching of the fluorescence of 4-methylumbelliferyl a-D-mannopyranoside by con A as a function of pH, thereby implicating a carboxyl group (pK, 3.5)in the binding phenomenon. Carboxyl groups have also been implicated in the binding of saccharides to wheat-germ agglutinin.335 Treatment of con A with sodium acetate and acetic anhydride acetyThe lated 84% of the amino groups and 31% of the phenolic modified protein was immunochemically indistinguishable from native con A, but could not be absorbed to S e p h a d e ~ even , ~ ~ ~in the presence of 1 mM MnZf.Although retaining only-50% of its activity,32s*330 acetylated con A exhibited unaltered saccharide-binding specificity. Acetylation and succinylation reportedly converted tetrameric into dimeric con A, thereby altering its biological properties.336These results suggested that free amino groups and many tyrosyl residues are neither directly involved in carbohydrate binding nor in maintaining the structural integrity of the protein. Like acetylation, derivatization of con A with maleic anhydride gave a stable, dimeric species having an essentially unchanged binding-~ite.~~’ Maleylated con A required the presence of high concentrations of salt to precipitate d e ~ t r a n .Doyle ~~] and RoholPz7observed that reaction of con A with tyrosyl-modifying reagents (tetranitromethane, N-acetylimidazole, and iodine) decreased, but did not abolish, glycogen-precipitating ability. Hassing and G o l d ~ t e i obtained n ~ ~ ~ similar results with N-acetylimidazole. The combined evidence suggested that “accessible” tyrosyl residues prob(328) B. B. L. Agrawal, I. J. Goldstein, G. S . Hassing, and L. L. So, Biochemistry, 7, 4211-4218 (1968). (329) G. S. Hassing and I. J. Goldstein, Eiochim. Biophys. Acta, 271,388-399 (1972). (330) R. J. Doyle and D. C. Birdsel1,J. Bocteriol., 109, 652-658 (1972). (331) N. M. Young, Biochim. Biophys. Acta, 336, 46-52 (1974). (332) M. Beppu, T. Terao, and T. Osawa,]. Eiochem. (Tokyo), 78, 1013-1019 (1975). (333) A. R. Frstser, J. J. Hemperly, J. L. Wang, and G. M. Edelman, Proc. Natl. Acad. Sci. U.S.A.,73, 790-794 (1976). (334) B. R. Dean and B. R. Homer, Biochim. Biophys. Acta, 322, 141-144 (1973). (335) R. H. Rice and M. E. Etzler, Biochemistry, 14,4093-4099 (1975). (336) G. R. Gunther, J. L. Wang, I. Yahara, B. A. Cunningham, and G. M. Edelman, Proc. Natl. Acad. Sci. U.S.A., 70, 1012-1016 (1973).

LECTINS: CARBOHYDRATE-BINDING PROTEINS

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ably do not contribute significantly to stabilization of the carbohydrate-protein complex. Other amino acid modifications gave ambiguous results, or denatured the protein.329The effect of chemical modification on the circular dichroism spectrum has been reported.285a Fluorescence studies indicated that neither the specific binding of methyl a-D-mannopyranoside nor alternation of the con A quaternary structure changed the number or accessibility of the solvent-exposed tryptophan residues in the l e ~ t i n . ~ ~ ‘ Affinity-labelling experiments employing methyl 2-deoxy-2-(pdiazobenzamid0)-a-D-glucopyranosideand p-diazophenyl glycosides of D-glucose, D-mannose, and maltose failed to label con A carbohydrate-binding sites specifically.329However, partial success was achieved by using a photoaffinity-labelling ligand, p-azidophenyl a - ~ - m a n n o p y r a n o s i d eThe . ~ ~lack ~ ~ ~ of ~ ~success in these experiments can probably be attributed to the low association-constants of the affinity-labelling reagents. Kinetic parameters for the binding of p-nitrophenyl a-D-mannomethyl a-D-mannopyrano~ide,~~~ and the anomers pyranoside, of methyl D-gl~copyranoside~~~ to con A have been reported. Con A apparently binds its ligands of low molecular weight more slowly than do other proteins. Specifically, most second-order rate-constants for protein-ligand complexing are 107-108 sec-I M - l , whereas the constants for the binding of con A to p-nitrophenyl a-D-mannopyranoside or methyl a- and P-D-glucopyranosides are less than lo5 sec-’ M - l . postulated that complex-formation with con Brewer and A is not a simple, one-step, reversible reaction, but involves a ligandinduced, conformational change such as that described by equation 1 , wherein PD, and PD, represent different conformers of the complex. 3383339

kl

k2

P+D$PD,ePD, k-2

Whereas a simple, one-step pathway for complex-formation requires that, when one reactant is in large excess, the observed pseudo-first(337) R. Pelly and P. Horowitz, Biochim. Biophys. Acta, 427, 359-363 (1976). (338) D. Gray and R. H. Glew,J. Biol. Chem., 248,7547-7551 (1973). (339) S. D. Lewis, J. A. Shafer, and I. J . Goldstein, Arch. Biochem. Biophys., 172, 689-695 (1976). (340) J. J. Grimaldi and B. D . Sykes,J. Biol. Chem., 250,1618-1624 (1975). (341) C. F. Brewer, D. M . Marcus, A. P. Grollman, and H. Sternlicht, in “Lysozyme Proceedings of the Lysozyme Conference,” E. Osserman, S. Beychok, and R. Canfield, eds., Academic Press, New York, 1974, pp. 225-239.

164

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

order rate-constant (kobs) shall increase linearly with increasing excess of reactant, the two-step pathway requires that kobs shall become independent of [PI when k,[P] >> 1. Attempts by Lewis and coworkers339to obtain such kinetic evidence for a two-step pathway were unsuccessful; their results indicated that, if the two-step pathway obtains, k, <360 M-'. Gimaldi and S y k e ~ ~observed ~O a substrate-induced, conformational change in the binding of methyl a-wmannopyranoside to con A by using stopped-flow n.m.r. spectroscopy. These workers340reported avalue for k, of 6200 M-l. The data340also indicated that the rate of the conformational change is at least 100times lower with methyl a-D-mannopyranoside than the lower limit calculated for the corresponding confonnationa1change were the binding ofp-nitrophenyl a-D-mannopyranoside also a two-step process. Unfortunately, these studies340were complicated by the fact that metal binding and ligand binding were studied simultaneously. It is to be hoped that further studies of the kinetics of binding of ligand to con A will reveal the nature of the interactions or conformational changes that accompany ligand binding, or both. c. Preparation and Biological Activity of Mono-, Di-, and Tetravalent Concanavalin A.-Multivalence apparently accounts for many biological properties of lectins, for example, cell agglutination, patch and cap formation, mitogenesis, and polymer precipitation. The influence ofvalence on biological activity may be investigated by using lectins of various valences. For example, tetravalent, lima-bean lectin is a more potent hemagglutinin, mitogen, and glycoprotein precipitant than the divalent s p e ~ i e s . ' Glutaraldehyde ~~,~~~ cross-linked, soybean agglutinin343and polymeric tetra- and multi-valent l e ~ t i are, n ~ simi~ ~ ~ larly, more potent than the native (divalent) agglutinin.343 Con A has also been manipulated in order to probe the biological effects of multivalence. The equilibrium between naturally occurring, dimeric (bivalent) and tetrameric (tetravalent) con A is affected by pH, temperature, and chemical derivatization. The dimeric form at pH s5 retains specific, carbohydrate-binding ~apacity."~ However, equilibrium dialysis experiments suggested firstly, that dimer and tetramer differ slightly in binding affinity, and secondly, that dimers containing a fragmented protomer possess only one b i n d i n g - ~ i t e ?(C.d. ~ ~ studies (342) R. W. Ruddon, L. M. Weisenthal, D. E. Lundeen, W. Bessler, and I. J. Goldstein, P ~ o cNatl. . Acad. S C ~ U.S.A., . 71, 1848-1851 (1974). (343) R. Lotan, H. Lis, A. Rosenwasser, A. Novogrodsky, and N. Sharon, Biochem. Biophys. Res. Commun., 55, 1347-1355 (1973). (343a) B. Schechter, H. Lis, R. Lotan, A. Novogrodsky, and N. Sharon,Eur.J.Immunol., 6,145-149 (1976).

LECTINS: CARBOHYDRATE-BINDING PROTEINS

165

also revealed small, conformational differences between intact and fragmented species.344)Below pH 5, predominantly dimeric con A does Moreover, dimers isolated not precipitate polysaccharides.173~204~290~309 by gel filtration at physiological pH failed to precipitate glycogen, although the mitogenic activity was equivalent to that of the t e t ~ a m e r . ~ ~ ~ (It is important to distinguish between the properties of lectin in solution and at a cell surface.29o)Dimers induced by lowered temperatures did not agglutinate erythrocytes, although electron microscopy showed they could bind soluble g l y ~ o p r o t e i n s . ~ ~ ~ The mitogenic activities of native (tetravalent) and succinylated or acetylated (bivalent) con A were Whereas the protein concentration-dependent, DNA synthetic response of mouse splenocytes to native con A exhibited a sharp peak, succinyl con A stimulated DNA synthesis over a broad range ofprotein c o n ~ e n t r a t i o nMixing . ~ ~ ~ of native con A with its dimeric, succinylated derivative at pH 4.5 resulted in the formation of hybrid molecules. A dimer species consisting of equimolar amounts of native con A protomer and its succinyl derivative was isolated, and s h o ~ n to ~ have ~ ~ a molecular weight of50,OOO at both pH 5 and pH 7. Treatment of con A with trypsin reportedly yielded nonagglutinating, monovalent con A, which restored normal growth to tumor (Py 3T3) Monovalent con A (operationally defined as protease-treated, nonagglutinating con A) has been prepared, and used indiscriminately by cell biologists without extensive characterization.347,348 Preparation of monovalent con A fragments from demetallized lectin has been reported.349Alternatively, partial blocking of the carbohydrate-binding sites was achieved by using a combination of succinylation and photoaffinity labelling (with p-azidophenyl a - ~ m a n n o p y r a n o ~ i d e ) . ~Affinity ~ ~ , ~ ~ chromatography ~,~~~ of the derivatized protein yielded a fraction consisting of dimers having, at pH 5, one free and one blocked saccharide-binding site. Aggregation and protomer7. A~ peptide exchange ~ ~ ~ ~ r at r pH e d ~ ~ reportedly , ~ ~ ~capable , ~ of ~ ~ (344) W.H. Sawyer,G. H. McKenzie, and L. W. Nicho1,Aust.J. Biol. Sci., 27,l-6 (1974). (345) C. Huet, M.Lonchampt, M. Huet, and A. Bernadac,Biochim. Biophys. Acta, 365, 28-39 (1974). (345a) A. R. Fraser, J. L. Wang, and G. M. Edelman,J. Biol. Chem., 251,4622-4628 (1976). (346) M. M. Burger and K. D. Noonan, Nature, 228,512-515 (1970). (347) M. S. Steinberg and I. A. Gepner, Nature, 241, 249-251 (1973). (348) P. M.Evans and B. M. Jones, Exp. Cell Res., 88,56-62 (1974). (349) D. L.Thomasson and R. J. Doyle, Biochem. Biophys. Res. Commun., 67, 15451552 (1975). (350) M. Beppu, T. Terao, and T. Osawa,J. Biochem. (Tokyo), 79, 1113-1117 (1976).

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inhibiting the hemagglutinating and mitogenic activities of native con A was obtained by limited digestion of the lectin with t r y p ~ i n . ~ ~ ~ d. Interaction of Concanavalin A with Po1ysaccharides.-Sumner and Howell noted that con A precipitated glycogen and yeast mannan, and agglutinated starch granule^.'^^*^^^ F. Smith and his colleagues later measured glycogen and yeast mannan by a turbidimetric a . ~ s a y ~ ~1~ * ~ " ; mg of a given polysaccharide incubated with an aliquot of con A proVarious polysaccharides duced a turbid solution quantitated by A,. were differentiated on the basis of their "glycogen value," the turbidity ratio comparing one mg of the given polysaccharide to one mg of rabbit-liver glycogen in the standard assay."7~121~3s44-359 Turbidimetry indicated that precipitate formation depended on the concentration of the protein as well as on that of the polysaccharide, the time-period of incubation, and the ratio of protein to polysachar ride.'^^,^"^^^^ Precipitin curves determined turbidimetrically differed from those determined by analysis of precipitated nitrogen.360For these reasons, early turbidimetric studies conducted on crude (rather than purified) con A are difficult to interpret. The quantitative precipitin method, wherein increasing amounts of polysaccharide or glycoprotein are added to standard aliquots of con A, and the resulting precipitate is washed, and analyzed for nitrogen, is now considered the method of choice for studying protein-polysaccharide i n t e r a c t i ~ n . ' ~ ~ * ~ ~ ~ A typical precipitin-curve for the reaction320between con A and dextran B-1355-Sis presented in Fig. 4. Analogous to antibody-antigen precipitin curves, there are three zones: lectin excess (all added dextran is precipitated), equivalence (virtually all dextran and lectin are precipitated), and polysaccharide excess (soluble complexes are formed). Optimal precipitation of con A by dextran B-1355-Soccurred in 24 h at 2 9 , between pH 6.1 and 7.2, and was unaffected320by (buffered) concentrations of sodium chloride of up to 4.2 M . From precipitin studies on a large number of polysaccharides, Gold(351) D . S. Seidl, A. Palozzo, A. Levy, V. Azavache, M. Jaff6, and W. G. Jaff6,Experientia, 31,37-38 (1975). (352) J. B. Sumner and D. J. O'Kane, Enzymologia, 12,251-253 (1948). (353) J. A. Cifonelli and F. Smith, Anal. Chem., 27, 1639-1641 (1955). (354) J. A. Cifonelli, R. Montgomery, and F. Smith,J. Am. Chem. Soc., 78,2488-2489 ( 1956). (355) J. A. Cifonelli and F. Smith,]. Am. Chem. Soc., 79,5055-5057 (1957). (356) R. Montgomery, Arch. Biochem. Biophys., 67, 378-386 (1957). (357) D. J. Manners and A. Wright,J. Chem. Soc., 4592-4595 (1962). (358) 0. Kjolberg, D. J. Manners, and A. Wright, Comp. Biochem. Physiol., 8,353-365 (1963). (359) E. E. Smith, Z. H. Gunja Smith, and I. J. Goldstein, Biochem. J . , 107, 715-724 (1968). (360) R. D . Poretz and I. J. Goldstein, Immunology, 14, 165-174 (1968).

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- 500

E

Dextran Img)

FIG. 4.-Quantitative, Precipitin Curve of Dextran B-13553 with Con A.309 [The total amount of dextran in the precipitate is also illustrated. Con A, 42 pg of nitrogen. (Reproduced by permission of the Journal of Biological Chemistry.)]

stein and coworkers120,121 suggested that all branched polysaccharides having multiple, terminal (nonreducing) a-D-glucopyranosyl, a-Dmannopyranosyl, or D-fmctofuranosyl groups would precipitate with in con A. The “chain-end mechanism” postulated,120~121~16s~1s7*zo4~359 which tetravalent con A interacts with specific glycosyl residues of polysaccharide or glycoprotein chain-ends, is diagrammed in Fig. 5.

Mullivalent polysaccharide

#

Ea!er

FIG. 5.-Diagranimatic Representation of the Chain-end Mechanism of Con APolysaccharide (Glycoprotein) Interaction.

168

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

This chain-end mechanism has been modified209*240 to account for the observation that 2-0-substituted a-D-mannopyranosyl (and, probably, 2-0-substituted a-D-ghcopyranosyl) residues in internal regions of polysaccharides and glycoproteins also interact with con A. Branched a~~~g~ucans,102,120,121.2S3,320.3S3-35918d61.362

a-~~mannans,102,120,121,204,3S2-3S4,363

and precipitate with con A, whereas linear polymers may bind, but will not precipitate the protein.'21*204*3s9*362 These polysaccharides are discussed in detail in the following Sections. Con A-polysaccharide complex-formation is probably stabilized largely by hydrogen bonding and charge-dipole interaction.16g~310*320*365 As charged sugars are not bound, it is unlikely that charge-charge interactions are involved. Nonpolar forces have, however, not been excluded. Nonspecific precipitation between con A and various polyelectrolytes (such as fucoidan, RNA, heparin and phosphorylated dextran, starch, and glycogen) has been ~ b ~ e r v e d High . ~ ~concentrations ~ , ~ ~ ~ * ~ ~ ~ ~ of salt (1.0 M NaC1) inhibited the con A-heparin reaction, suggesting that nonspecific, charge-charge interaction had been involved. Complete inhibition of specific precipitation would be expected to result from addition of competing sugar hapten, but not by high concentrations of salt.320However, the results of inhibition studies are not always decisive; sugar haptens occasionally produce partial inhibition of con A-polyelectrolyte precipitation, but higher concentrations of saccharides are generally necessary than those required for complete inhibition of con A-neutral glycan interaction^.^^^ Perhaps, specific saccharide interaction with con A modifies the surface charge-distribution on the protein as a result of conformational changes (see earlier). Nonspecific, hydrophobic forces might also be involved in lectin~~J~~ polysaccharide or, more likely, l e c t i n - g l y c o p r ~ t e i n ~interaction (see Section II,l,j). Preliminary information regarding certain structural features of (361) M. E. Preobrazhenskayaand E. L. Rosenfel'd, Biokhimiya, 33,784-791 (1968). (362) I. J. Goldstein, R. D. Poretz, L. L. So, and Y. Yang, Arch. Biochem. Biophys., 127, 787-794 (1968). (363) M. E. Slodki, R. M. Ward, and J. A. Boundy, Biochim. Biophys. Acta, 304,449-456 (1973). (364) B. A. Lewis, M. J. S. Cyr, and F. Smith, Carbohydr. Res., 5,194-201 (1967). (365) R. D. Poretz and I. J. Goldstein, Biochemistry, 9,2890-2896 (1970). (366) R. J. Doyle, E. E. Woodside, and C. W. Fishel, Biochem. I., 106,35-40 (1968). (366a) V. Buonassisi and P. Colburn, Arch. Biochem. Biophys., 183,399-407 (1977). (367) N. DiFerrante and R. Hrgovcic, FEES Lett., 9,281-283 (1970). (368) R. J. Doyle and T.-J.Kan, FEES Lett., 20,22-24 (1972).

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polysaccharides can sometimes be obtained by determining the binding strength between the two components of a con A precipitate. To a first approximation, this is proportional to (1) the “solubility” of the precipitate,320and (2) the proportion of a sugar hapten that is needed in order to inhibit precipitation320 by 50%. These two operationally defined parameters have proved extremely useful in comparative studies of a series of related polysaccharides (for example, dextrans) permitting some tentative conclusions regarding the extent and nature of branching, and the number of glycosyl residues in exterior, oligosaccharide side-chains (see later). e. Interaction of Concanavalin A with Glycogen and Amy1opectin.-As already mentioned, Sumner and Howell’s discovery of con A-polysaccharide precipitation102eventually led to the definition of “glycogen value” (g.v.), determined t u r b i d i m e t r i ~ a l l y .F. ~~~*~~~ Smith and coworker^,^^^^^^^ noting that the extent of reaction depended on the origin of the glycogen and that amylopectin did not react, suggested that con A could be used to distinguish between these two classes of branched a-D-glucans. Manners and his ~ o l l e a g u e s reported ~ ~ ~ ~ ~ a~gross * relationship between the g.v. and the degree of branching for many glycogen specimens; they also found no reaction with amylopectin and amylopectin @limit dextrin. On the other hand, sweet corn (Zea mays) phytoglycogen gave a g.v. similar to those of animal glycogens, affirming the structural relationship between these two groups of p o l y s a c ~ h a r i d e s . ~ ~ ~ Inasmuch as periodate abolished, whereas beta-amylase enhanced, the reactivity of glycogen with con A, Smith and coworkers suggested117 that the protein probably interacted with “intact inner branches of the glycogen molecule,” in addition3s7 to “hydroxyl groups of the molecule.” Goldstein and coworkerslZ1determined the optimal concentration of lectin for precipitation, and showed that potato amylopectin did precipitate with the jack-bean lectin. In a turbidimetric study of the interaction of con A with amylopectin, glycogen, and their enzymically and chemically degraded products, E. E. Smith and coworkers3s9 confirmed that the extent of interaction of polysaccharides with con A is highly dependent on the concentration of protein. For example, floridean starch gave no turbidity with con A at 1 mg/ml, whereas yeast mannan, dextran B-13554,and rabbit-liver glycogen all formed prec i p i t a t e ~ .However, ~~~ with con A at 5 mg/ml, floridean starch exhibited3sgthe same reactivity as dextran B-1355s. The molecular weight of the polysaccharide also affects con A-

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

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polysaccharide i n t e r a ~ t i o n .Chemically ~ ~ ~ . ~ ~ ~ or enzymically degraded polysaccharides of decreased molecular weight generally reacted more extensively with jack-bean lectin than their undegraded counterp a r t ~ .Assuming ~ ~ ~ * that ~ ~ con ~ A binds nonreducing termini, increased polysaccharide reactivity could be a consequence of increasing, by degradation,3ssthe number of chain ends per unit weight. Glycogens extracted from rabbit liver by three different procedures were investigated by quantitative precipitation (see Fig. 6): glycogen having the lowest molecular-weight distribution, obtained by extraction with potassium hydroxide solution, attained maximal precipitation of nitrogen at the lowest concentration of glycogen, followed by the trichloroacetic acid-extracted and the water-extracted glycogens.369 Glycogen extracted with potassium hydroxide solution also inhibited precipitation more effectively than the trichloroacetic acid-extracted glycogen (the glycogen-excess region of the precipitin curve).36sThe same pattern was also observed for preparations of levan and dextran of differing molecular weight.369A particularly interesting feature of the con A-glycogen precipitation reaction is the very broad range of maximal precipitation (compare the dextran B-1355-S precipitin curve in Fig. 4); this distinctive type of curve was also given by corn (Zea mays) phytoglycogen, but not by corn amylopectin.

1 0

I

I

0.4

1

I

0.8

I

I

1.2

1

I

I

I

I

I

I

1 . 1

20 2.4 2 8 Glycogen (mg 1

1.6

1

4.0

I

I

6.0

I

I

8.0

I

1

10.0

FIG. 6.-Quantitative, Precipitin Curves of Rat-liver Glycogen with Con A. (0, Potassium hydroxide-extracted glycogen; A, trichloroacetic acid-extracted glycogen; U, water-extracted glycogen. Con A, 48 pg of nitrogen. After Ref. 369.) (369) L. L. So and I. J. Goldstein,J. Immunol., 102,53-57 (1969).

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The influence of the molecular weight and the polydispersity of the polysaccharide preparation on the con A-polysaccharide interaction was also studied by two-dimensional, agar-gel diffusion. Homogeneous polysaccharides generally gave single, sharp, precipitin bands with con A, whereas polydisperse preparations gave diffuse bands of variable breadth. Polysaccharides and glycoproteins of low molecular weight usually have high diffusion coefficients, and form bands close to the con A well. On the other hand, species having high molecular weight form precipitin bands close to the polysaccharide we11.120,359~369 Quantitative studies with glycogens and amylopectins of known structure and molecular weight are needed in order that a full understanding of these interactions may be achieved.

f. Interaction of Concanavalin A with De~trans~~~.-Dextrancon A interaction is of special significance, because the isolation of this lectin by affinity chromatography on cross-linked dextran gels ( S e p h a d e ~ ) ' ~ *resulted -'~~ from the discovery that con A precipitated with dextrans. The availability of a large number of dextrans containing a variety of different a-D-glucosidic linkages370also makes this an ideal system for investigating structure-activity relationships. Cifonelli and Smith3s5reported that, although dextran B-512 did not form a precipitate with con A, a "dextrin"-derived dextran synthesized by Acetobacter capsulatum did precipitate. Furthermore, con A displayed a specificity similar to that of type XI1 pneumococcus rabbit antibody in its differential reactivity with dextrans, especially those ( purportedly containing a high proportion of a - ~ -1+=2)-glucosidic bond^.^^^,^^^*^^' Validating this relationship, Suzuki and Hehre372 showed that the proportion of kojibiose isolated following acetolysis of dextrans paralleled both the extent of type-XI1 cross reactivity and, with con A. broadly speaking, the degree of precipitation253*,260*371~372 Each of 23 dextrans studied by Goldstein and coworkerslZ1formed precipitates with purified con A, although seven of them [all containing >90% of a - ~ 1+6)-like -( D-glucosidic linkages]370required high concentrations of lectin for precipitate formation. Similar results were reported by Preobrazhenskaya and R ~ z e n f e l ' d ~these ~ ' ; findings were also corroborated by gel-diffusion studies.120Formation of precipitate with con A was approximately related to the content of non a - ~ -1(4 6 ) like linkages, determined from periodate-oxidation data.I2' The dextran from Streptococcus bovis (previously considered to be (370) R. L. Sidebotham, Adu. Carbohydr. Chem. Biochem., 30,371-444 (1974). (371) E. J. Hehre, K a g a k u No Ryoiki, 9,454-455 (1965). (372) H. Suzuki and E. J . Hehre, Arch. Biochem Biophys., 104, 305-313 (1964).

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linear)373afforded a typical precipitin curve when levels of con A higher than those required to precipitate dextran B-13554 were employed.362 Nigerose was isolated from this dextran, indicating the possible origin of branch points.362Dextran B-512(F) and the dextran from Betacoccus arabinosaceous (L. rnesenteroides), Birmingham strain, likewise gave precipitin curves with high levels of con A; a chemically synthesized, linear (1+6)-a-D-gl~can~~~ did not precipitate the protein.362From these studies, it was suggested that all native dextrans are branched.362 The solubility of the dextran-con A precipitates at equivalence is a revealing parameter. Compared to the dextran B-1355-con A complex, which had320a solubility of 1.5 p g of N/ml, the solubility of S. bovis-, dextran B-512(F)-, and B . arabinosaceous dextran-con A precipitates were 28, 19, and 11pg of N/ml, respectively.362Similarly, 0.024, 0.03, and 0.22 pmoles of methyl a-D-glucopyranoside were needed in order to inhibit the precipitation reaction between con A and dextrans S. bovis, B-512(F), and B . arabinosaceous, respectively.362These two parallel parameters indicate the affinity of the dextran for con A. Methylation studies showed that B . arabinosaceous dextran had one branch point per 6 to 7 D-gh2osyl residues375;18% of all branches consisted of a single D-glucosyl Thus, this dextran is approximately three times as ramified as B-512(F), that has one branch per 23 Not surprisingly, the con A-B-512(F) dextran precipitate is more soluble than, and -7 times as readily inhibited by methyl a - ~ glucopyranoside as, the con A-B. arabinosaceous dextran precipitate. By these criteria, S . bovis dextran is more closely related to B-512(F) dextran than to B . arubinosaceous d e ~ t r a nOne . ~ ~additional ~ parameter that correlates well with structure is the maximum amount of con A nitrogen precipitated by each of these dextrans: 28,39, and 83% of con A added to the dextrans from S. bovis, B-512(F),and B . arabinosaceous, respectively.362Torii and his colleagues378employed fractional precipitation with con A to demonstrate the microheterogeneity of dextran B-1397. Dextran B-1355-S was adsorbed to three different forms of immobilized con A (con A-agaro~e,~’~ poly(L-leucy1)-con A,379and (373) R. W. Bailey, Biochem. J., 71, 23-26 (1959). (374) E. R. Ruckel and C. Schuerch,J. Am. Chem. Soc., 88,2605-2606 (1966). (375) S. A. Barker, E. J. Bourne, G. T. Bruce, W. B. Neely, and M. StaceyJ. Chem. Soc., 2395-2399 (1954). (376) E. J. Bourne, D. H. Hutson, and H. Weigel, Biochem. J., 86,555-562 (1963). (377) J. W. Van Cleve, J. W. Schaffer, and C. E. Rist,J. Am. Chena. Soc., 78,4435-4438 (1956). (378) M. Torii, K. Sakakibara, B. P. Alberto, and A. Misaki, Biochem. Biophys. Res. Commun., 72,236-242 (1976). (379) K. 0. Lloyd, Arch. Biochem. Biophys., 137, 460-468 (1970).

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glutaraldehyde-insolubilized con A),380whereas dextran B-512 was not adsorbed. These results were explained in terms of dextran structure. Corroboration for the chain-end mechanism of con A-polysaccharide interaction was obtained when Duke and coworkers381showed that dextran N-4, similar in structure to dextran B-512, lost its con A precipitating capacity after nonreducing (terminal) a-D-glucopyranosyl groups had been converted into glucopyranosyluronic acid groups by catalytic oxidation. Although it is probable that 2-O-substituted a-Dglucopyranosyl residues (such as those found in the internal regions of dextrans) would also interact with con A, this has not been demon~trated.~~~ Until complete, structural information is available for each of these dextrans, it is not possible to relate dextran structure to con A reactivity. However, such factors as polydispersity, molecular frequency and nature of branching, type of a-D-glucosidic linkages, and, very important, the number of glycosyl residues in the exterior chains, all contribute to the con A reactivity of the polymer.362The capacity for precipitate formation with con A is now virtually a standard procedure in studies on dextran s t r u c t ~ r e . ~ ~ ~ . ~ ~ ~ g. Interaction

of Concanavalin A with Mannans.-Yeast

mannan (gum) was among the first polysaccharides reported by Sumner and his colleague^^^^^^^^ to precipitate with con A. Sumner’s results were confirmed by Cifonelli and F. Smith; the interaction of con A with yeast mannan was distinguished by the very high “glycogen values” obtained in a turbidimetric a s ~ a y . ’ ~ This ’ ” ~ ~behavior was attributed to the extensive branching in the molecule (34%), and, hence, to the large number of chain ends.lZ1 The interaction between con A and a-mannans from a variety of .~~~ mannans micro-organisms was studied by So and G o l d ~ t e i n The from Saccharomyces cerevisiae, Saccharomyces rouxii, and Sarcina sp., and the phosphomannan from Saccharomyces pini Y-2579, all gave classical precipitin curves similar to those obtained for d e x t r a n ~ , 3 ~ ~ , ~ ~ ~ A synthetic, linear but unlike the extended curves given by glyc0gen.3~~ (1+3)-a-D-mannopyranan did not precipitatezo4with con A. The greater reactivity of a-mannans compared to other con A-reactive polysaccharides was indicated by several observations: (1)the con A(380) E. H. Donnelly and I. J. Goldstein, Biochem.], 118, 679-680 (1970). (381) J. Duke, I. J. Goldstein, and A. Misaki, Biochim. Biophys. Actu, 271, 237-241 (1972). (382) E. E. Smith, FEBS Lett., 12,33-37 (1970). (383) M. Kobayoshi, K. Shishido, T. Kikuchi, and K. Matsuda, Agric. B i d . Chem., 37, 357-365 (1973).

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IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

excess region of the precipitin curve rose more steeply than for any the other polysaccharide or g l y ~ o p r o t e i n(2) ,~~ ~ con A-mannan precipitates exhibited the lowest “solubility” of any carbohydrate-containing (3) the con A-mannan premacromolecule (approximately 1pg/ml),204 cipitation reaction was more difficult to inhibit with carbohydrate hapten than with any other con A-reactive p ~ l y m e r , ’and(4) ~ ~ * ~D-mannose ~~ derivatives were the most potent inhibitors of the con A These observations are corroborated by evidence that con A contains a specific-binding locus for the 2-hydroxyl group of D-mannose, whereas the 2-hydroxyl group of D-ghCOSe actually constitutes a destabilizing By utilizing the Ouchterlony technique of two-dimensional, agar-gel diffusion, Slodki and his colleagues investigated the interaction of con A with O-pho~phonomannans.~~~ They suggested a possible correlation between the extent of precipitate formation with con A and the content of a-D-( 1+2)-mannosidic linkages. Chemically synthesized D-mannans have also been investigated.384 The D-mannan prepared by polymerization of 1,6-anhydro-P-~mannopyranose reacted vigorously with the jack-bean lectin, indicating that it was most probably a highly branched polymer having multiple a-D-mannopyranosyl chain-ends. A mannodextran, prepared by grafting D-mannose units onto dextran B-512, precipitated a higher proportion of con A than the parent d e ~ t r a n . ~ ~ ~ Hapten-inhibition studies employing D-mannose-containing oli-( gosaccharides led to the important discovery that a - ~ 1-+2)-linked D-mannopyranosyl residues were exceptionally reactive with con A, the di- and tri-saccharides [a-D-Manp-(1+2)-D-Man and a-D-Manp(1+2)-a-D-Manp-( 1-*2)-~-Man]being 4 and 20 times as potent as .~~~~ findings ~~~ suggest that the methyl a - D - m a n n o p y r a n o ~ i d eThese con A binding-site could be complementary to a sequence of three, or four, a-D-( 1-*2)-mannopyranosyl r e s i d ~ e s .On ~ ~the , ~other ~ ~ hand, -( or a-D-( 1+6)-linked D-mannosyl residues do not internal a - ~ 1+3)interact with con A. The implication of a-D-( 1+2)-mannopyranosyl residues as con A binding-sites in animal glycoproteins adds further significance to these studies.240 Fluorescein- and mercury-labelled con A386-388have been used to demonstrate the presence of D-mannan in yeast c e l l - ~ a l l s and ~ ~ ~in~ ~ ~ ‘ (384) R. Robinson and I. J . Goldstein, Carhohydr. Res., 13,425-431 (1970). (385) B. Lindberg and S. Svensson, Actu C h m . Scand., 24, 711-713 (1970). (386) J. S. Tkacz, E. B. Cybulska, and J. 0. Lampen,j. Bacteriol., 105, 1-5 (1971). (387) M. Horisberger, H. Bauer, and D. A. Bush, FEBS Lett., 18,311-314 (1971). (388) H. Bauer, M. Horisberger, D . A. Bush, and E. Sigarlakie, Arch. Mikrobiol., 85, 202-208 (1972).

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bud scars388 of Saccharomyces cerevisiae. By employing a con A-Sepharose column, Lloyd379demonstrated adsorption of yeast (Saccharomyces cerevisiae) mannan, and fractionation of a galactomannan from Cladosporium werneckii.

h. Interaction of Concanavalin A with D-Fructans.-Precipitate formation between p-D-fructans (levans) and con A was first demonstrated by Goldstein and So'zo~3sg; levans from NRRL B-512 and B-1662, and from Erwinia ananas, Bacillus megatherium, and Aerobacter levanicum, formed precipitin bands with con A by Ouchterlony, two-dimensional, agar-gel diffusion. A levan from Leuconostoc mesteroides, strain C , likewise364precipitated con A. In a comprehensive study of D-fructans, So and Goldstein obtained typical precipitin curves between levans from Aerobacter levanicum No. 15552, Bacillus ~ contrast ~'~~~~ megatherium KM, and Bacillus subtilis and the l e ~ t i n . In to dextran B-1355-S and a variety of a-D-mannans that precipitated 95-98% of the total con A added, these levans maximally pre~ipitated"~ only 70-78% of added con A. A relatively weak precipitin reaction was generated between con A and the D-gluco-D-fructanfrom the Hawaiian ti plant; at equivalence, only 41% of added con A was precipitated."' The con A-levan (A. levanicum) precipitate, moreover, was more s o h ble than the con A-dextran B-1355-S (Ref. 320) or con A-S. cerevisiae a-D-mannanZo4 precipitates. A further indication of the lowered reactivity of D-fructans compared to those of D-mannans, dextrans, and glycogens was the relative ease with which sugar haptens inhibited precipitation. Only 16% of the sugar hapten was needed to inhibit, by 50%, the A . levanicum levan-con A precipitation reaction that was necessary for the con A-dextran B-135543 system.215 Hapten inhibition studies showedz1' that sugars containing the D-arabinofuranoid ring system bind to con A. Thus, methyl p-Dfructofuranoside, methyl a-D-arabinofuranoside, 2,5-anhydro-~mannitol, and 2,5-anhydro-~-glucitolall inhibited the con A-polysaccharide system. Interestingly, although inulobiose and inulotriose both inhibited the dextran-con A precipitation reaction, inulin itself inhibited the con A-dextran system but did not form a precipitate with the lectin."' Further discussion of the binding of furanoid sugars to con A is included in Section II,l,l. i. Interaction of Concanavalin A with Teichoic Acids.-Con A specifically precipitates with teichoic acids containing a-D-g~ucopyranosy~ end-groups, but not the and 2-acetamido-2-deoxy-a-~-glucopyranosyl corresponding p-glycosyl groups. Furthermore, both the organisms

176

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

and the isolated cell-walls derived therefrom are agglutinated by the lectin. Reeder and E ~ k s t e dstudied t ~ ~ ~ the interaction of con A with teichoic acids from Staphylococcus aureus and Staphylococcus epidermidis by gel diffusion,and precipitation in a fluid system. The teichoic acid from strain T, of S . epidermidis contains a-D-glucopyranosyl residues, and it precipitated with con A, whereas strain Tz, which is p-D-glucosylated, did not. Classical precipitin curves resulted when con A interacted with strains T, and 412 (also a-D-ghcosylated); the precipitation was specifically inhibited by D-glucose, 2-acetamido-2-deoxy-~-glucose, and methyl a-and /3-D-glucopyrano~ides.~~~ Con A also precipitated, in agar gel, with teichoic acids from strains Copenhagen and 416 of S. aureus, but not with strains Foggie and Smith.389From the work of Sanderson and coworkers, it is known that S . aureus Copenhagen teichoic acids contain both a- and p-linked 2-acetamido-2-deoxy-~-glucosyl resid~es.~ Interaction ~ ~ ~ ~ ~ 'of these teichoic acids with con A resulted in the precipitation of only the a-D-linked species.389Thus, not only was a fractionation achieved, but Reeder and Eckstedt also demonstrated that the a-and p-linked amino sugars occurred on separate teichoic acid molecules, instead of the anomers being present in a single chain.389Failure ofcon A to react with teichoic acid from the Foggie and Smith strains of S . aureus confirmed the presence of p-D-linked 2-acetamido-2-deoxy-~-glucosyl residues. Previous determinations of a- or P-specificity involved either hapten inhibition of precipitation with specific a n t i b ~ d y ,enzymic ~ ~ ~ , ~degra~~ d a t i ~ n , ~or~ O optical rotatory dispersion Archibald and Coapes observed that both the intracellular and membraneous teichoic acids isolated from Streptococcus fecalis 8191 gave, with con A in agar gel, sharp precipitin lines that dissolved when methyl a-D-glucopyranoside was added.3g5Moreover, Lactobacillus plantarum wall teichoic acid, which also contains a-Dglucopyranosyl substituents, precipitated with con A, whereas the Staphylococcus epidermidis 12 teichoic acid, containing P-Dglucopyranosyl substituents, did not.395Additionally, these authors demonstrated395cell-wall agglutination by con A. (389) W. J. Reeder and R. D. Ekstedt,]. ImmunoZ., 106,334-340 (1971). (390) A. R. Sanderson, J. L. Strominger, and S. G. Nathenson,J. B i d . Chem., 237, 3603-3613 (1962). (391) A. R. Sanderson, W. G. Juergens, and J. L. Strominger, Biochem. Biophys. Res. Commun., 5, 472-476 (1961). (392) S. I. Morse,J. Exp. Med., 117, 19-26 (1963). (393) M. Torii, E. A. Kabat, and A. E. Bezer,]. E x p . Med., 120, 13-29 (1964). (394) E. A. Kabat, K. 0. Lloyd, and S. Beychok, Biochemistry, 8,747-756 (1969). (395) A. R. Archibald and H. E. Coapes, Biochem. J., 123,665-667 (1971).

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Interaction between con A and cell-wall digests of Bacillus subtilis 168 resulted in insoluble complexes.330Interestingly, the precipitation reaction exhibited two pH optima, at pH 3.1 and 7.4. Similar to specific, neutral polysaccharide-con A interaction, the precipitation reaction that was optimal at pH 7.4 was completely inhibited by methyl a - ~ mannopyranoside, whereas the precipitation reaction that peaked at pH 3.1 was only partially inhibited. By using a purified, teichoic acid preparation, virtually no inhibition with sugar hapten could be demo n ~ t r a t e d Only . ~ ~ ~the precipitation reaction optimal at pH 3.1 was sensitive to increase in salt concentration, indicating that a chargecharge interaction had probably occurred between positively charged con A and negatively charged teichoic a ~ i d .Doyle ~ ~ and ~ *coworkers ~ ~ ~ utilized a con A-Sepharose column for the large-scale isolation of B . subtilis 168 teichoic from 45 mg of autolyzate added to a con A-Sepharose column, they recovered 21 mg of pure teichoic acid by elution with methyl a-D-glucopyranoside solution. Bacteriophage $125 infects B. subtilis 168 by binding to CY-Dglucopyranosyl residues of the cell-wall teichoic acids. Prior treatment of B . subtilis 168 with con A completely inhibited phage The inhibition depended on the concentration of con A, and was reversible in the presence of methyl ~~-D-glucopyranoside.~~~ The organization of the surface teichoic acid was investigated by electron microscopy after complexing cells with con A.399Binding studies with 14Clabelled con A revealed teichoic acids on the external, but not internal, face of the cell wall.400The number of con A-reactive sites increased significantly following limited enzymic digestion of the cell, indicating that some teichoic acids are probably embedded within the peptidoglycan matrix.400 j. Interaction of Concanavalin A with Glyc~proteins.~~~-For many years, the reaction of jack-bean extract with the components of various animal secretions and body fluids, notably plasma102*10"107~402,403 and gastric juice,404has been recognized. Kabat and coworkers dem(396)T.-J. Kan, R. J. Doyle, and D. C. Birdsell, Carbohydr. Res., 31,401-404 (1973). (397)R. J. Doyle, D. C. Birdsell, and F. E. Young, Prep. Biochem., 3, 13-18 (1973). (398)D. C.Birdsell and R. J. Doyle,]. Bacteriol., 113, 198-202 (1973). (399)D. C.Birdsell, R. J. Doyle, and M. Morgenstem,]. Bacteriol., 121,726-734(1975). (400)R. J. Doyle, M. L. McDannel, J. R. Helman, and U. N. Streips,]. Bacteriol., 122, 152-158 (1975). (401)A. Surolia, S.Bishayee, A. Ahmad, K. A. Balasubramanian, D. Thambi-Dorai, S. K. Podder, and B. K. Bachhawat, Ado. E x p . Med. Biol., 55,95115 (1975). (402) S. Murakawa and S. Nakamura, Bull. Yamaguchi Med. Sch., 10,11-29 (1963). (403) S. Nakamura, S. Tominaga, A. Katsuno, and S. Murakawa, Cornp. Biochem. Physiol., 15,435-444 (1965). (404) A. E. Clark and M. A. Denborough, Biochem.]., 121,811-816 (1971).

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IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

onstrated the interaction of con A with blood-group substances isolated from hog-stomach linings and from a specimen of human-stomach A product formed by alkaline, borohydride degradation of hog lining.170 gastric-mucin, namely, 4-0-(2-acetamido-2-deoxy-a-~-glucopyranosy1)-D-galactitol, inhibited con A-mucin precipitation, suggesting a basis for this interaction.405Human ovarian-cyst mucins having B, I, and i activities reacted similarly with jack-bean lectin.406-408 Complex-formation between con A and various glycoproteins has now been elucidated. For example, con A specifically binds several providing a basis for isolation purified, glycoprotein lectins,10s*,240,409-411 ~~~ of lectins by affinity chromatography on con A - S e p h a r ~ s e .Certain myeloma proteins and immunoglobulins,409~412 ribonuclease B (Ref. 409),o v a l b ~ r n i nand , ~ ~carcinoembryonic ~ antigen4I3also precipitated with con A. Additional con A-reactive glycoproteins and glycopeptides, identified by inhibition of hemagglutination, mitogenesis, or polysaccharide precipitation, include serum proteins ( t r a n ~ f e r r i n ) ,im~~~ munogl~bulins,~ and ~ ~thyr~globulin.~~' ~ ~ ~ ~ - ~ ~ ~ Con A also precipitates mannan-protein complexes such as occur in extracellular /3-Dglucanases from Saccharomyces c e r e v i ~ i a eLectin-reactive .~~~~ glycoproteins from cell membranes are discussed in Section VIII. Occasionally, con A-glycoprotein complexes are stabilized by nonspecific, hydrophobic interaction, rendering them insoluble even in the presence of known, carbohydrate inhibitors (for example, methyl a-D-mannopyranoside). In these instances, organic solvents (for examThe experiple, ethylene glycol) may dissociate the complex. (405) C. Moreno and E. A. Kabat,J. lmmunol., 102, 1363-1367 (1969). (406) F. Maisonrouge-McAuliffe and E. A. Kabat, Arch. Biochcm. Biophys., 175,71-80 (1976). (407) F. Maisonrouge-McAuliffe and E. A. Kabat, Arch. Biochem. Biophys., 175, 8189 (1976). (408) F. Maisonrouge-McAuliffe and E. A. Kabat, Arch. Biochem. Biophys., 175, 90113 (1976). (409) I. J. Goldstein, L. L. So, Y. Yang, and Q. C. Callies,J. Immunol., 103, 695-698 (1969). (410) W. Bessler and I. J . Goldstein, F E B S Lett., 34, 58-62 (1973). (411) W. G. Jaffb, A. Levy, and D . I. Gonzilez, Phytochemistry, 13,2685-2693 (1974). (412) M. A. Leon, Science, 158, 1325-1326 (1967). (413) S. Hammarstrom, E. Engvall, B. G. Johansson, S. Svensson, G. Sundblad, and I. J. Goldstein, Proc. Natl. Acad. Sci. U.S.A.,72, 1528-1532 (1975). (414) B. R. Andersen, lmmunochemistry, 6, 739-749 (1969). (415) P. S. Chase and F. Miller, Cell. lmmunol., 6, 132-139 (1973). (416) R. Kornfeld and C. Ferris,J. Biol. Chem., 250,2614-2619 (1975). (417) S. Toyoshima, M. Fukuda: and T. Osawa, Biochemistry, 11,4000-4005 (1972). (417a) P. Biely, Z. Kritkjr, and S. Bauer, Eur. J. Biochem., 70, 75-81 (1976). (418) M. W. Davey, E. Sulkowski, and W. A. Carter, Biochemistry, 15,704-713 (1976). (419) E. F. Plow and H . Resnick, Biochim. Biophys. Acta, 221,657-661 (1970).

LECTINS: CARBOHYDRATE-BINDING PROTEINS

179

mental systems studied concerned con A and i n t e r f e r ~ n ' and ~ ~ ,ri~~~ cin.lg3Thus, superimposed upon primary, specific interaction between con A and glycoproteins (by carbohydrate binding) may be secondary, nonspecific, nonpolar interactions; the latter may be of pronounced importance in considering the reactions between con A and cellular surfaces.

k. Interaction of Convanavalin A with Miscellaneous Polysaccharides.-In addition to branched a-D-glucans, a-D-mannans, and p-D-fructans, con A also interacts with numerous other polysaccharides that contain the proper determinant sugars. These polysaccharides include lipopolysaccharides (for example, those isolated from Salmonella typhimurium, S . bredeney, and S. senftenberg) which contain terminal a-D-glucopyranosyl certain pneumococcal, capsular polysaccharides (for example, S XII, which contains kojibiosyl e n d - g r o u p ~ ) ' ~ arabinogalactans ~ , ~ ~ ~ * ~ ~ ~ ; (which possess nonreducing, terminal D-arabinofuranosyl g r o ~ p s ) ~ arabinoman~~-~~~; nans42ss427; and polysaccharides from Histoplasma capsulatum (all of which reportedly contain D-mannose, D-galactose, and traces of D-gh~CoSe'~~). The precipitin reaction between con A and certain Klebsiella polysaccharides possessing 2-0-substituted a-D-mannopyranosyl residues has already been noted.209*240 1. Carbohydrate-binding Specificity of Concanavalin A.-The carbohydrate-binding specificity of con A has been studied in great detail by a variety of approaches: ( a ) con A precipitation by naturally occurring, and model, carbohydrate-containing macromolecu1es,I02,113,117,120,121,126,170,177,253,3j5,420,423 ( b ) quantitative, hapten inhibi( c ) hemagglutination and tion of precipitation,168-170*197*204,215*365 lymphocyte stimulation,249( d ) displacement of the lectin from ' ~ U.V. ~ ~ ~spectroscopy, ~~ S e p h a d e ~ ,(' e~)~equilibrium d i a l y s i ~ ,(f) .~~~ results ~ ~ ~permit ~ ~ ~ a~ mapping ~ of and (g) n.m.r. ~ p e c t r o s c o p yThese (420) I. J. Goldstein and A. M. Staub, Immumchemistry, 7, 315-319 (1970). (421) J. A. Cifonelli, P. Rebers, M. B. Perry, and J. K. N. Jones, Biochemistry, 5,30663072 (1966). (422) I. J. Goldstein, J . A. Cifonelli, and J. Duke, Biochemistry, 13, 867-870 (1974). (423) I. J. Goldstein and A . Misaki,]. Bacteriol., 103, 422-425 (1970). (424) T. M. Daniel and J. J . Wisnieski, Am. Reo. Respir. Dis., 101, 762-764 (1970). (425) T. M. Daniel, Am. Rezj. Respir. Dis., 110, 634-640 (1974). (426) T. M . Daniel and L. S. Todd, Am. Reu. Respir. Dis., 112,361-364 (1975). (427) T. M. Daniel and A. Misaki, A m . Rev. Respir. Dis., 113,705-706 (1976). (428) G. Betail, L. Genaud, and M. Coulet, Ann. Inst. Pasteur, 123, 731-740 (1972). (429) R. D . Brown, 111, C. F. Brewer, and S. H. Koenig,Ado. E x p . Med. Biol., 55,323-324 (1975).

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IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

the con A binding-site in terms of the various saccharide hydroxyl groups with which the protein interacts. The simplest known sugar derivatives that interact with the jackand 1,4bean lectin are 1,5-anhydro-2-deoxy-~-arabino-hexitol anhydro-~-arabinitol.l~~~~~ These alditols contain the minimal structural features required for binding to con A, namely, unmodified hydroxyl groups of the D - W U ~ ~ configuration ~ O at C-3, C-4, and C-5 of the 1,5-anhydrohexitol, or C-2, C-3, and C-4 of the 1,4-anhydropentitol ring systems. Introduction of an axial 2-hydroxyl group of the L-gZycero configuration into 1,5-anhydro-2-deoxy-~-arabino-hexitol enhances binding affinity to the lectin, whereas an equatorial 2-hydroxyl group of the D-gly Cero configuration diminishes its complementarity.168,169,'97,204*215,365 Configurationally related sugars will also interact with the protein. For example, a-L-sorbopyranose and p-Dfructopyranose can formally be related to 1,5-anhydro-D-glucitol and 1,5-anhydro-~-mannitol, respectively, and a-and p-D-arabinofuranose, a- and p-D-fructofuranose, 2,5-anhydro-~-mannitol,and 2,5-anhydroFig. 7 outlines these D-glucitol to 1,4-anhydro-~-arabinitol.~~~~~~~ configurational relationships. CH,OH

FIG. 7.-Configurational Relationships Among Furanoid and Pyranoid Sugars that Interact with Con A. (Hydroxyl groups critical for binding to con A are underlined. Note the relationship between the hydroxyl groups in the furanoid and pyranoid R,, R3, systems.) (a) R,, R,, R,, R4, R, = H : 1,5-anhydro-2-deoxy-~-arabino-hexitol; €&, R, = H; R, = OH : 2-deoxy-a-~-arabino-hexopyranose; R,, R,, R3. R, = H; R4 = OH : 1,5-anhydro-~-glucitol;R,, R,, R4,R, = H; R3 = O H : 1,5-anhydro-~-mannitol; R,, R3, & = H; Rz = R4 =OH : a-D-glucopyranose; R,, €&, €& = H; R, = R, = OH : a - ~ mannopyranose; R,, R,, RI = H; R4 = R5 = OH : a - ~sorbopyranose; R,, R,, R, = H; R3 = R5 = OH : P-D-fructopyranose; (b) R,, R, = H : 1,4-anhydro-~-arabinitol; R, = H; R2 = OH : a-D-arabinofuranose; R, = H; R, = OH : p-Darabinofuranose; R, = CH,OH; R2 = OH : a:D-fructofuranose; R, = CH,OH; R, = OH : p-Dfructofuranose.

Con A exhibits a pronounced preference for the a anomer of D-manno- and D-gluco-pyranose. In fact, a-D-mannopyranosyl residues are most complementary to the carbohydrate-binding site168-170,197,204,21.5,365 of con A; there are loci in the carbohydratespecific, binding site of con A that interact with each hydroxyl group, as well as with the anomeric oxygen atom of this residue.365Epimeric

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181

TABLEI Inhibition of Concanavalin A-Dextran 13554 Precipitation by Mono- and Oligo-saccharides” and Some of Their Derivatives Saccharide D-Glucose DMannose D-Fructose D-Galactose D-Allose Methyl a-D-glucopyranoside Methyl P-Dglucopyranoside Methyl a-Dmannopyranoside Methyl 2-acetamido-2-deoxy-a-~-glucopyranoside

20

4.4 8.8 104 (8%)b 97 (4%) 2.5 67 0.6

5.0

Methyl P-D-fructopyranoside Methyl a-D-hctopyranoside Methyl a-D-fructofuranoside Methyl p-D-fructofuranoside Methyl a-L-sorbopyranoside

0.85 107 (40%) 16 5.7 3.1

Maltose Isomaltose Cellobiose Laminarabiose Gentiobiose Sucrose

4.3 2.2 100 (2%) 102 (0%) 100 23

Sophorose

7

2-O-~-~Galactopyranosy~-D-g~ucose

7

Methyl a-sophoroside Methyl P-sophoroside ~

Micromoles giving 50% inhibition

1.2 25

~~

“Data taken from Refs. 168,169,197,215,365, and 430.*Numbers in parentheses refer to percentage inhibition given by the pmoles of saccharide noted.

a-D-glucopyranosy~residues have %th to V5th of the affinity of methyl a-D-mannopyranosyl residues, whereas a-D-galactopyranosy1residues Hapten-inhibition studies with a exhibit no affinity.16B-170,197.2”.215,s65 series of deoxy, deoxyfluoro, and 0-methyl derivatives of D-glucose and D-mannose permit a more-definitive analysis of the binding mechanism The observation that 6-deoxy-~-glucose, (see Table I).168,169~197”40~36s (430)I. J. Goldstein, R. N. Iyer, E. E. Smith, and L. L. So, Biochemistry, 6,2373-2377 (1967).

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IRWIN J. GOLDSTEIN A N D COLLEEN E. HAYES

6-O-methyl-~glucose, and methyl 6-deoxy-6-fluoro-a-D-g~ucopyranoside all poorly inhibit the con A-dextran precipitation reaction suggests that it may be the hydrogen atom of the 6-hydroxyl group that interacts1ss~240*365 with con A. A report that D-glucuronic acid is an inhibitor of the con A system could not be s u b ~ t a n t i a t e d . ~ ~ ' * ~ ~ ~ Similarly, the failure of methyl 4-deoxy-a-~-xylo-hexopyranoside, methyl 4-O-methyl-a-~-glucopyranoside, and 4-deoxy-4-fluoro-~glucose to bind to con A implicates the hydrogen atom ofthe 4-hydroxyl group of D-gIucose and D-mannose in the binding phenomenon.240In and contrast, although methyl 3-deoxy-a-~-arubino-hexopyranoside methyl 3-O-methyl-a-~-glucopyranosideare poor inhibitors, 3-deoxy3-fluoro-D-glucose binds to the protein almost as well as D-glucose, s ~ g g e s t i n ga~role ~ ~ for * ~the ~ ~oxygen atom of the 3-hydroxyl group in binding to con A. Finally, inasmuch as the 2-methyl ether of D-mannose and 2-deoxy-2-fluoro-D-mannose inhibit con A-polysaccharide interaction to the same extent, we suggest that the oxygen atom on C-2 of D-mannose participates in hydrogen bonding to the [It is is a of some interest that, whereas 2-acetamido-2-deoxy-~-mannose noninhibitor, the ortho ester 1,2-0-(1-methoxyethy1idene)-fi-D-mannopyranose, as well as the disaccharide 2-O-fi-D-glucopyranosyl-Dmannose, binds to con A approximately as well as D-mannose Methyl a-D-glycopyranosides of D-mannose and D-glucose are six to seven times as inhibitory as their corresponding 1,8anhydrohexitol derivatives, prompting the suggestion that the a-D-glycosidic oxygen atom contributes to the binding energy of the con A-carbohydrate ~ o m p l e ~In. substantiation ~ ~ ~ * ~ ~ of~ this view, a - ~ glucopyranosyl fluoride inhibits the con A system to the same extent as D - g l u c o ~ eThese . ~ ~ ~ data are summarized in Tables I and I1 and Fig. 8.

H

H

FIG.8.-Carbohydrate-binding Specificity of Con A. (The hydroxyl groups of the a-D-mannopyranosyl group that are most critically involved in binding to con A are italicized. Hydrogen and oxygen atoms believed to participate in hydrogen bonding are overscored.)

Con A will tolerate considerable modification at C-2 of D-glucose. In fact, 2-deoxy-~-arabino-hexoseis bound more strongly than ~ - g l u c o s e .Whereas ~ ~ ~ * ~the ~ ~2-methyl and 2-ethyl ethers of D-glucose

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TABLEI1 Inhibiting Power of Various Monosaccharide Derivatives" ~

Quantity needed for 5w0inhibition0 (pmoles)

Compound ~

Methyl 2-deoxy-a-~-urubino-hexopyranoside Methyl a-D-mannopyranoside Methyl a-D-glucopyranoside

0.95 0.34 1.30

D-Mannose 2-0-Methyl-D-mannose p-Nitrophenyl a-D-mannopyranoside p-Nitrophenyl2-O-methyl-a-~-mannopyranoside

2.0 1.9 0.17 0.19 1.5

2-Deoxy-2-fluoro-D-mannose

DGlucose 3-Deoxy-~-ribohexopyranose 3-Deoxy-3-fluoro-D-glucose Methyl a-Dglucopyranoside Methyl a-D-galactopyranoside Methyl 4-deoxy-a-~xyZo-hexopyranoside Methyl 4O-methyl-a-~-glucopyranoside D-Glucose 4-Deoxy-4-fluoro-~-g~ucose Methyl a-Dglucopyranoside Methyl a-D-xylopyranoside Methyl 6-deoxy-a-~-glucopyranoside Methyl 6-deoxy-6-fluoro-a-~-glucopyranoside 1,5-Anhydro-~-glucitol Methyl a-D-ghcopyranoside Methyl P-Dglucopyranoside

7.2 1.3 37.0

DGlucose a-DGlucopyranosyl fluoride

25 25

"Because these data were collected in a series of experiments over a long period of time, only the numerical values within each set may be directly compared. a m e r e numbers are given in parentheses, 50% inhibition was not attained; the numbers in parentheses indicate the percent inhibition given by the pmoles of saccharide noted. Reprinted, with permission, from Ann. N.Y. Acud. Sci.140

are equivalent to D-glUCOSe in inhibitory potency, 2-acetamido-2deoxy-D-glucose is only 50% as active, and the 2-chloro-2-deoxy and 2-deoxy-2-iodo derivatives are '/loth and +oth as potent as ~ - g l u c o s e . ~ ~ ~ itself does not bindlg7to con Interestingly, 2-amino-2-deoxy-D-glucose A, probably owing to the positively charged amino group (pK 7.8).

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IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

A comprehensive examination of furanoid sugars allows the generalization that 1,4-anhydro-~-arabinitol(see Fig. 7) possesses2" the minimal structural requirements for interaction with con A. The disposition of the hydroxyl groups in this sugar derivative is similar to the orientation of the hydroxyl groups at C-3, (2-4, and C-5 of the D-mannopyranosyl residue.21sOf the pentoses, only D-arabinofuranose contains the required binding loci2l5;con A interacts with Mycobacterium bovis cell-wall arabinogalactan (containing nonreducing, terminal, a-Darabinofuranosyl (compare, Ref. 424). Both 2,5-anhydro-~-mannitoland 2,5-anhydro-~-glucitolcontain the requisite 1,4-anhydro-~-arabinitol ring system.21s*240 The D-mannitOl derivative is about 3.5 times as potent an inhibitor as the D-glUCitOl derivative, perhaps by virtue of the fact that it may bind to con A either through the hydroxyl groups at C-1, (2-3, and C-4, or (2-3, C-4, and C-6 (compare Ref. 429). Finally, the important observation that methyl aand @-D-fructofuranosideare inhibitors (the @ anomer being approximately three times as active as the a anomer) provides an explanation for the interaction of con A with plant and bacterial levans (see Section II,l,h). Hapten-inhibition studies with a group of disaccharides indicated that, with but one exception, namely, s o p h o r ~ s (see e ~ ~ later), ~ only those disaccharides containing nonreducing, terminal a - ~ glucopyranosyl or a-D-mannopyranosyl groups i n t e r a ~ t e d ~ ~with ~*"~~'~~ con A. Thus, isomaltose, kojibiose, maltose, and nigerose (in order of potency) inhibited the con A-dextran precipitation reaction.lsa In contrast, laminarabiose and cellobiose were noninhibitors, gentiobiose was a very poor inhibitor, and, unexpectedly, sophorose was a strong i n h i b i t ~ r ' ~ ~(see * ' ~Table ~ * ~ ~I).~ Although a more limited number of D-mannose-containing disaccharides was investigated, the same pattern emerged: a d i n k e d D-mannobioses were good inhibitors, whereas 4-0-@-D-mannopyranosyl-D-mannose was a n0ninhibit0r.l~~ Later, it was found that, like sophorose, 2-O-P-D-mannopyranosyl-Dmannose also binds209to con A. Investigation of higher oligosaccharide series revealed several important feature^.'^' On a molar basis, all members of the homologous "maltodextrin" series of oligosaccharides (maltose to maltodecaose, inclusive) inhibited the con A-dextran precipitation reaction to the same extent.lg7The same was true of isomalto-oligosaccharides (isomaltose to isomaltoheptaose, inclusive), and the methyl isomaltoside series (methyl a-isomaltoside to methyl a-isomalto-octaoside, i n c l ~ s i v e ' ~ ~ ) . These data indicated that the con A combining-site was complementary to a single, nonreducing, terminal a - ~ 1+4)-( or a - ~ 1+6)-(

LECTINS: CARBOHYDRATE-BINDING PROTEINS

185

glucopyranosyl group.Is7 The failure of the cyclo-hexa- and -heptaamyloses to inhibit is consistent with the absence of nonreducing, terminal D-glucosyl groups from their ring-like molecules.1s7 In sharp contrast to the malto- and isomalto-oligosaccharide series, the inhibiting power of a series of manno-oligosaccharides containing (1+2)-linked a-D-mannopyranosyl residues increased as the number of units was i n ~ r e a s e d .Thus, ~ ~ ~ mannobiose ~~~' was five, and mannotriose and mannotetraose were twenty, times as potent, respectively, as methyl a-D-mannopyranoside.2".43lThese results raised the possibility that the con A combining-site may be composed of a series of subsites similar to that found for wheat-germ agglutinin'28 (see Section 111,4); in this case, the subsite specificity would be complementary to seFurther support quences of a-D-( 1-*2)-mannopyranosyl residues for an extended binding-site derives from spectrophotometric studies that indicated that con A can interact simultaneously with hydroxyl groups on both a-D-mannopyranosyl units of the chromogenic ligand p-nitrophenyl 2-O-a-D-mannopyranosy~-a-D-rnannopyran0side.~~~~ Examination of a series of branched oligosaccharides and of those containing several different kinds of glycosidic linkages was informative.lS7All gluco-oligosaccharides containing terminal, nonreducing isomaltosyl groups (for example, panose) inhibited to the same extent as isomaltose; the same phenomenon was observed for oligosaccharides containing maltosyl e n d - g r o u p ~ .These ' ~ ~ data lend support to the hypothesis that it is mainly the terminal, nonreducing, a - ~ glycopyranosyl groups of simple and complex a-D-glycans with which con A interactsls7(however, see later). The surprisingly low inhibitory alactivity of the trisaccharide 4,6-di-O-a-~-g~ucopyranosy~-D-g~ucose, though it possesses two nonreducing a-D-glucosyl groups, was attributed to steric hindrance to the close approach of the protein.1s7 The discovery that sophorose (2-O-@-D-g~ucopyranosy~-D-g~ucose) was a good inhibitor of the con A system430forced a re-examination of the concept of an exclusive "chain-end mechanism" for con @-D-Ghcopyranosyl residues A-carbohydrate interaction.121~1e7~zo4~z40~365 as they occur in oligo- and poly-saccharides do not i n t e r a ~ t ' ~with ~*'~~ con A. Therefore, the 3-, 4-, and 6-hydroxyl groups of the reducing D-glUCOSe residue of sophorose were implicated as jack-bean lectin binding-loci (see Fig. 9). A comprehensive study of the interaction of con A with sophorose and its derivatives supported this view430(see Table I). .2043431

(431) I. J. Goldstein, Adu. E n p . Med. Biol., 55, 35-53 (1975). (431a) T. J. Williams, J. A. Shafer, and I. J. Goldstein,Abstr. Pap. Am. Chem. SOC. Meet., 174, CARB-64 (1977).

186

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

Sophorose ~2-O-~-o-glucopyronosyl-o-glucose)

FIG. 9.-Structure

of Sophorose (2-0-p-DGlucopyranosyl-D-glucose). (The site of interaction with con A is outlined. From Ref. 240. Printed by permission of Ann. N.Y. Acad. Sci.)

Over ten years ago, Hehre and his pointed out that 2-0-substituted a-D-glucopyranosyl residues, which occur in many dextrans, possess the configurational features necessary for interaction with con A (see earlier). Glycosylated a-D-mannopyranosyl residues should also interact with con A; this has been shown conclusively by inhibition and precipitation s t ~ d i e s . ~Thus, ~ ~ although , ~ ~ ~ ,the ~ ~trisac~ 142)charides 0-a-D-galactopyranosyl-( 1+2)-0-a-~mannopyranosy~-( D-mannose and 0-a-D-galactopyranos yl-( 1+6)-O-a-D-mannopyranosyl-( 1+2)-D-mannose each possesses a terminal, a-D-galactopyranosyl group, they nevertheless are more potent inhibitors of the con Adextran system than methyl a - D - m a n n o p y r a n o ~ i d e .Nonspecific 2~~~~~~~~~~ interaction of a-D-galactopyranosyl residues with the .protein could contribute to enhanced activity. Reduction of the first-mentioned trisaccharide with sodium borohydride yielded the corresponding alditol, which still possessed considerable inhibitory potency owing to its internal, (1+2)-substituted, a-D-mannopyranosyl residue.209 2-0-P-D-mannoSimilarly, 2-0-~-D-g~ucopyranosy~-D-mannose~09 pyranosy~-D-mannose,209and 2-0-(2-acetamido-2-deoxy-~-~-glucopyranosy~)-D-mannose210 all inhibited the con A system. The lastnamed disaccharide is of particular interest, because it occurs commonly in erythrocyte and lymphocyte surface-glycoproteins, as well as in plasma proteins (for example, immunoglobulin^).^^^^^^^^^^^ BY hemagglutination inhibition, this disaccharide and its methyl a-glycopyranoside were shown to be as effectively bound as methyl a-D-mannopyranoside."O On the other hand, the same investigators (432) R. Kornfeld,J. Keller, J. Baenziger, and S. Kornfeld,]. B i d . Chem.,246,3259-3268 (1971). (433) F. Miller, Immunochemistry, 9,217-228 (1972).

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observed that the disaccharide methyl furanoside was inactive, supporting the hypothesis that it is the reducing 2-0-substituted a - ~ mannopyranose residue that is the locus21ofor interaction with con A. Kornfeld and Ferris416studied intact, and partially degraded, immunoglobulin glycopeptides for their ability to interact with con A. The most potent inhibitor was the branched glycopeptide 1containing two terminal @-~-GlcNAcp-( 1+2)-a-D-Manp units. Kornfeld and Ferris4I6 R

t

p-o-GlcNA~p-(I-- 2)- a - ~ - M a n p (1- 3)- p-o-Manp 6

t

a-~-Manp 2

t

1 ~-D-G~cNAcP 1

conjectured that “in glycopeptides, such terminal P - N acetylglucosamine residues [may] assume a more favorable conformation for interaction with the saccharide binding site of concanavalin A than the same residue can assume in smaller glycosides.” Although the results of inhibition studie~’~’make it highly unlikely that 2-acetamido-2-deoxy-~-~-glucopyranosyl residues interact in a speci$c way with con A, nonspecific interaction with the protein is possible. Alternatively, glycosyl substitution at 0-2 may enhance the interaction of a-D-mannopyranosyl residues with con A; this possibility is supported by the observation that the methyl 2-O-a-(and @)-D-glucopyranosyl-a-D-glucopyranosideshave a greater affinity for con A than has methyl a - ~ - g ~ u c o p y r a n o s i d e . ~ ~ ~ ~ ~ ~ ~ Further support for the reactivity of internal, 2-0-linked7 a-Dmannopyranosyl residues with con A comes from precipitation studies with macromolecules of known structure. A lipopolysaccharide from Klebsiella 0 group 5 and the capsular polysaccharide from Klebsiella K-24 (after deacetylation) generatedzo9classical precipitin curves in their reaction with con A. Several investigators have also reported that glycopeptides and glycoproteins lacking con A-reactive sugars at glycosyl chain-ends have the capacity to interact with the lectin.416,433 In all these cases, there are internal, 2-0-substituted a-D-mannopyranosyl with con A. residues that may act as loci for (434)S. Komfeld, J. Rogers, and W. Gregory,]. B i d . Chem., 246,6581-6586 (1971). (435)C. A. Presant and S. Kornfeld,]. Biol. Chem., 247,6937-6945 (1972).

188

IRWIN J. GOLDSTEIN A N D COLLEEN E. HAYES

The influence of the aglycon on the inhibitory potency of a large number of alkyl /3-D-glucopyranosides and aryl a- and p-Dglucopyranosides and -mannopyranosides has been investigated.'80*181*323,365*436 The inhibiting power of all of the P-D-glucopyranosides of primary alcohols that were examined was virtually identical, regardless of the degree of substitution at the p-carbon atom of the a g l y c ~ n On .~~~ the other hand, those /3-D-glucopyranosides that possess aglycons branched at the a-carbon atom displayed inhibiting powers inversely related365to the degree of substitution at this carbon atom (see Table 111).The lack of any linear correlation between the Taft TABLEI11 Inhibition of Concanavalin A Precipitation by Aliphatic and Aromatic P-D-Glucopyranosides" Inhibitor (aglycon) Methyl Ethyl Butyl Neopentyl Isobutyl Benzyl Isopropyl Cyclohexyl Cyclopentyl tert-Butyl Phenyl 3-Methylphenyl 3-Eth ylphenyl 3-Isopropylphenyl 3-tert-Butylphenyl 3,5-Dimethylphenyl 3,5-Di-tert-butylphenyl

Micromoles required for 50% inhibition 37 37 36 37 35 37 100 65 46 165

7.0 3.9 3.0 2.4 1.8

2.4 0.70

"Data abstracted from Refs. 180 and 365.

substituent constant of the aglycon and the inhibiting power of the saccharide indicated that polar effects are not involved in the binding of the aglycon to the protein, and is consistent with the concept that the P-D-glucopyranosidic oxygen atom is not involved365in the binding of the glycoside to con A. In contrast, data have accumulated that suggest the presence of a (436) R. D. Poretz and I. J. Goldstein,Arch. Biochem. Biophys., 125,1034-1035 (1968).

LECTINS: CARBOHYDRATE-BINDING PROTEINS

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region on the con A molecule, adjacent to the specific, saccharidebinding site, which is capable of interacting specifically with the On investiaromatic ring of phenyl ~-D-glucopyranosides.160~161~3z3*436 gating the inhibition potency of a variety of ortho-, metu-, and parusubstituted-phenyl P-D-glucopyranosides, Poretz and Goldstein observed no electronic substituent effect (m)for p-substituents and only moderately greater effects foro- a n d m - s u b s t i t u e n t ~ .Nor ' ~ ~was ~ ~ ~there ~ any correlation with the Van der Waals radii or the surface area of the substituents. However, 2,6-disubstitution of the phenyl group (for example, 2,6-dimethylphenyl and pentafluorophenyl p-Dglucopyranoside) drastically lessened the inhibiting power of the saccharide.lsO These results suggest that a specific orientation of the phenyl ring with respect to the pyranose ring is required in order that the saccharide may interact maximally with a corresponding region on the protein molecule.1s0 In contrast to the relative insensitivity ofthe con A system to substitution of the aromatic ring of phenyl p-D-glucopyranosides by polar groups, there was a very good correlation between the Hammett substituent-constants and the inhibiting activity of p-substitutedphenyl a-D-glucopyranosides and a-D-mannopyranosides. The influence of hydrophobic substituents on the inhibition potency of aryl a- and p-D-gluco- and a-D-manno-pyranosides was also investigated. A linear correlation was demonstrated between the hydrophobicity of the substituent and the inhibiting power of the glycoside for 0-,m-, and p-substituted-phenyl p-D-glucopyranosides. In his studies on the interaction of organic compounds with proteins, Hansch and coworkers introduced the concept ofrr, a parameter indica' , ~been ~~ tive of the hydrophobicity of an atom or group of a t o r n ~ . ~11~has defined as log P J P , where P , is the partition coefficient ofa substituted solute in a water-lipophile binary system, and PH is the partition coefficient of the parent compound. In this case, the partition of a substituted-aromatic glycoside in 1-octanol-water compared to that of the parent compound'60*436 was determined. As indicated, there was an excellent correlation between the hydrophobicity (rr) of m-substituted-aryl P-D-glucosides and their inhibition potency. In fact, the most potent inhibitor tested was 3,5-di-tert-butylphenyl p-Dg l u c o p y r a n ~ s i d e (see ' ~ ~ ~Table ~ ~ ~ 111).Loontiens and colleagueslsl extended this relationship by demonstrating a good correlation between of p-alkyl-substituted-phenyl p-Dthe hydrophobicity glucopyranosides and their binding to con A. (437)T. Fujita, J. Iwasa, and C. Hansch,J. Am. Chem. Soc., 86,5175-5180 (1964). (438)K. Kiehs, C.Hansch, and L. Moore, Biochemistry, 5,2602-2605 (1966).

190

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

In summary, on the con A molecule (in solution), there is a hydrophobic region adjacent to the carbohydrate-binding site that interacts specifically with the phenyl groups of P-D-glucopyranosides. The phenyl group must be linked directly to the glycosidic oxygen atom. (Benzyl P-D-glucopyranoside has only 20%of the inhibition potency of Hydrophobic substituents in the phenyl P-~-glucopyranoside~~"). meta- and para-positions are highly effective in enhancing the inhibition potency of the glycosides. 2. Lens culinaris syn. esculenta (Lentil) (lentil; a-D-Manp > CY-D-GIC~, CY-D-G~CNAC~) Hemagglutinating activity in the common lentil (Lens culinaris or Lens esculenta) was first reported by Landsteiner and R a ~ b i t s c h e k . ~ ~ ~ Several laboratories have since isolated the lentil lectin in pure form, and studied its physical-chemical properties and interaction with carbohydrates.138,142,143,440-442

The lentil lectin consists of two proteins that differ in their electrophoretic mobility. These proteins, termed LcH-A and LcH-B by and I and I1 by TichC and colleague^,'^^ may Howard and be isolated as their natural mixture by iso-ionic precipitation and DEAE-cellulose chr~matography,'~~ or by specific adsorption to Sephadex G-50, G-100, or G-150 [but not G-25 (compare Refs. 134 and 440)], followed by elution with 0.1 M D - ~ ~ u c o s ~ ' ~ ~ or ~'~~~'~ glycine-hydrochloric acid buffefl4Oof pH 2. Both proteins were found in all individual seeds examined; however, their relative proportions varied with the lentil source.441 (Toyoshima and coworkers143 reported a single protein; nevertheless, two components are evident in their elution pattern of the lectin from Sephadex by 0.1 M D-glucose.) The two lectins differ slightly in their affinity for Sephadex G-150, and can be separated by careful pooling of peak fractions eluted by 0.1 M D - g l u ~ o s e . 'Alternatively, ~~ they can be separated by vertical, starch-gel e l e c t r o p h o r e ~ i sor, ~ ~ most ~ conveniently, by 0-(carboxymethy1)cellulose c h r ~ m a t o g r a p h y . ~The ~ ' , ~ ~isolectins have identical molecular weights, values reported ranging from 42,000 to 63,000, with 52,000 being the best estimate.'42,'43,440*441 The proteins (439) K. Landsteiner and H. Raubitshek, Zentrulbl. Bukt. Parusitenk. Infektionskr. Hyg., Abt. 1. Orig., 45, 660-667 (1907). G. Entlicher, M. Tichi, J. V. KoStiF, and J. Kocourek,Experientiu, 25,17-I9 (1969). I. K. Howard, H. J. Sage, M . D. Stein, N . M. Young, M. A. Leon, and D. F. Dyckes, J. Biol. Chem., 246, 1590-1595 (1971). (442) M. Paulovi, M. Tichi, G. Entlicher, J. V. Koztif, and J. Kocourek, Biochim. Biophys. Actu, 252, 388-395 (1971).

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were immunochemically identical, as measured by double immunodiffusion using an antiserum prepared against the natural mixture,441and displayed identical hemagglutinating properties.441Each protein gave a single, symmetrical peak in the analytical ultracentrifuge, and a single, sharp, protein band by disc-gel electrophoresis. The lectins have almost identical amino acid cornpositi~ns,~~' and contain a preponderance of aspartic acid (and/or asparagine), 1.8 residues of cysteineM1per threonine, serine, and valine142*143,441; ~ ~ ? ~ ~ ~ and molecular weight of 49,000; but no m e t h i ~ n i n e . ' (Toyoshima coworkers'43reported 4 residues of methionine per 63 kg of protein; this was perhaps, due to examination ofa different variety of lentil.) The difference in electrophoretic behavior and adherence to O-(carboxymethy1)cellulose has been attributed441to the higher conand tent of lysine in LcH-B. End-group analysis gave t h r e ~ n i n eas ' ~the ~ N-terminal amino acids, and serine as the C-terminal amino The lectins contain -1.5-3% of neutral carbohydrate'43.441(principally D-glucose, as well as 2-amino-2-deoxy-Dglucose) Tryptic peptide mapping of each lectin revealed half of the expected number ofpeptides (15-16 peptides ofthe expected 32-37). The lectins share 15 identical, or closely similar, tryptic peptides"'; this suggests that each protein is a molecule consisting of identical halves.441However, a unique peptide was identified for LcH-A which stained gray with ninhydrin. The amino terminal sequence of the first 25 amino acids of the a- and p-subunits of the pea and the lentil lectins has been the determined. It was found that, of the 25 residues analyzed,"za*b~c N-terminal a-chain of the lentil and pea lectins differed only at three positions, and the p-chains at two positions. Metal analysis revealed one atom of Mn2+ and six atoms of Ca2+ associated with each 66.5 kg of ~ r 0 t e i n .Addition l~~ of 5 mM Ca2+and mM Mn2+to the native lectins enhanced both hemagglutination and mannan precipitation. This indicates that either some metal loss occurred during purification, or that the metal binding-sites are never completely occupied442(compare Refs. 300 and 301). Mn2+stimulated the hemagglutinating activity of all lentil-lectin preparations more than did Ca2+,whereas Caz+promoted mannan precipitation more effectively.442 Demetallization, by dialysis against 0.1 M EDTA followed by M acetic (442a) E. Van Driessche, A. Foriers, A. D. Strosberg, and L. Kanarek, FEBS Lett., 71, 220-222 (1976). (442b) A. Foriers, E. Van Driessche, R. De Neve, L. Kanarek, and A. D. Strosberg,FEBS Lett., 75,237-240 (1977). (442c) A. Foriers, C. Wuilmart, N. Sharon, and A. D. Strosberg, Biochem. Biophys. Res. CO~WZU 75,~ 980-986 ., (1977).

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IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

acid, decreased the Mn2+content and hemagglutination activity by -95%, without altering the Ca2+content.442The demetallization treatment may have caused partial denaturation, as Mn2+addition failed to reactivate the protein.442The electron paramagnetic resonance specto be similar to that trum of the lentil phytohemagglutinin was of con A. A detailed study of the subunit structure of the lentil lectin (Lens culinaris Moench; var. Hrotovicka) was conducted by Fliegerovh and coworkers.444Both isolectins dissociated readily in acidic solution (0.1 M glycine-hydrochloric acid buffer, pH 2.2, or M acetic acid) or in 8 M urea, and were resolved into their component polypeptide chains by gel filtration on columns of Bio-Gel P-100 or Sephadex G-200. There are two distinct species of subunits: heavy chains (H)and light chains (L) of approximate molecular weights 18,000 and 8,000, respectively (determined by gel filtration). Howard and confirmed dissociation of an LcH mixture in 6 M guanidinium chloride-0.1 M 2-mercaptoethanol by sedimentation-equilibrium ultracentrifugation and by disc-gel electrophoresis in dodecyl sodium sulfate. The molecular weights obtained by dodecyl sodium sulfate disc-gel electrophoresis were identical with the H and L chains isolated as already described. The H subunits tended to aggregate in dilute buffer solut i o n ~ . ~ "Because dissociation into subunits did not require 2-mercaptoethanol, the possibility that subunits are linked by disulfide bonds was excluded.444 Dissociation of the phytohemagglutinin subunits was accompanied by a complete release of Mn2+from the protein.444Carbohydrate and Ca2+remained bound to both subunits. Very low hemagglutinating activity was associated with the heavy subunit; the light subunit was inactive. Addition of Mn2+and Ca2+to a mixture of H and L chains failed to restore hemagglutinating Amino acid analysis of the isolated subunits showed that only the H chains contained cysteine (one residue per molecule).444Unfortunately, this amino acid analysis did not include methionine, reported absent by one groupM1and present by a second group.143End-group analysis showed N-terminal valine and threonine for the H and L chains, respectively. Treatment with carboxypeptidase gave serine as the C-terminal amino acid for both subunits. On the basis of these data, it appears that both lentil isolectins consist of 2H and 2L subunits giving a noncovalently linked aggregate of (443) M. Tichjr, M. Tichi, and J. Kocourek, Biochim. Biophys. Acta, 229,63-67 (1971). (444) 0. Fliegerovi, A. Salvetovi, M. Tichl, and J. Kocourek, Biochim. Biophys. Acta, 351,416-426 (1974).

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rnolecular weight 52,000-in good agreement with the determinations of Howard and and of Hayman and C r ~ m p t o nAs . ~ the ~~ isolated subunits differed both in amino acid composition and N-terminal amino acid, the L subunit is probably not a fragment of the H chains. The precise relationship between the two lentil lectins remains unclear, but it would appear from the available data that there is considerable homology between them, and that one could have arisen from the gene coding for the other b y minor m ~ t a t i o n ( s ) . ~ ~ l The lentil lectins are nonspecific with regard to hemagglutination of human erythrocyte^,'^^,'^^ but they do exhibit a distinct species specificity. They agglutinate erythrocytes of some species of rabbit,138,446 and horse,138but not sheep,138goat,138,446 or The importance of accurately identifying the seed variety is illustrated by the report of FialovL and coworkers447They isolated a new type of hemagglutinin from Lens esculenta Moench, subspecies microsperma (Baumg.)Barulina, termed “small-seed” lentil as opposed to the “large-seed” lentil (Lens esculenta Moench. Lens culinaris Med.). In contrast to the purified, large-seed lentil isolectins, the small-seed lentil hemagglutinin was retarded but not adsorbed to Sephadex. [Addition of Mn2+and Ca2+did not change this property (compare Refs. 300 and 301)]. The purified, small-seed lectin consisted of a single homogeneous protein which corresponded in electrophoretic mobility to phytohemagglutinin I1 from the large-seed lentil [poly(acrylamide) gel, pH 8.91. The small-seed lectin was likewise composed of two subunits (molecular weight 8,000 and 18,000), and had an aggregate molecular weight of 53,300 *2,500. Although similar in its amino acid and carbohydrate composition to the large-seed lectins, the small-seed protein contained only one-fourth of the 2-amino-2-deoxy-~-g~ucose residues. Analysis revealed the same two N-terminal amino acids (valine and threonine) as in the two large-seed lentil i ~ o l e c t i n s . ~ ~ Hemagglutination inhibition with D-glucose, D-mannose, and their methyl a-D-glycosides showed no difference between the two varieties of lentil seeds.447However, the small-seed lectin showed a lower agglutinating titer against human group B erythrocytes compared to Al, Az,and 0 cells, and a higher electrophoretic mobility on starch gel than the large-seed l e ~ t i n s . ~ ~ ~ (445) M. J. Hayman and M . J. Crumpton, Biochem. Biophys. Res. Commun., 47,923-930 (1972). (446) L. Bures’, G. Entlicher, M. TichP, and J. Kocourek, Experientia, 29, 1546-1547 (1973). (447) D. Fialovi, M. Tichi, and J. Kocourek, Biochim. Biophys. Acta, 393, 170-181 (1975).

194

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

Using equilibrium dialysis, Stein and coworkers186found that there are approximately two saccharide binding-sites per LcH-A molecule with K, = 230+30 M-' for D-mannose and K, = 100+24 M-' for methyl a-Dglucopyranoside. These are extremely low binding-constants (see Section IX, Table XXV). The interaction of the lentil lectins with p ~ l y s a c c h a r i d e s ' ~ ~ ~ ~ ~ ~ and g l y c o p r ~ t e i n s ~has ~ ~ been ~ ' ~ ~ studied by many investigators. Precipitation of muscle glycogen and yeast(Sacchar0myces cereuisiae) D-mannan by the lentil lectins was reported by TichL and coworke r ~ . On ~ the ~ ~other * ~hand, ~ ~ sixteen dextrans failed to precipitate with the leQtil lectins, although interaction with two of these (B-1299-L and B-1299-S) was demonstrated by their inhibition of lectin-human erythrocyte agglutinati~n.'~~ In view of their very low D-glUCOSe and D-mannose binding-constants,'86 it is, perhaps, not surprising that the lentil lectins do not precipitate dextrans as readily as does con A.'23*'38 The 0-phosphonomannan from Pichia pinus effectively inhibited hemagglutination and also precipitated with the lectin both in agar-gel double-diffusion and in solution.123The flocculation profile was very similar to an antibody-antigen precipitin curve, and proceeded best at 20°,pH 6-8,and an ionic strength >0.1. The lentil lectin p r e ~ i p i t a t e d ' ~several ~ , ' ~ ~ glycoprotein components of human serum: a,-macroglobulin, IgM, P,-glycoprotein, as well as traces of IgA and IgG. Transferrin, ceruloplasmin, and haptoglobin were unreactive. Glycopeptides from transferrin and IgM, but not ovalbumin, inhibited the Lens culinaris-glycoprotein precipitation system.'0g Young and coworkers stated that, whereas the lentil lectin discriminated less well between simple sugars than does con A, it was superior in distinguishing the aforementioned glycopeptide~.'~~ Classified with lectins inhibited by Makela's group I11 sugars, the lentil lectin is primarily specific for a-D-mannopyranosyl r e s i d u e ~ . ' ~ ~ J ~ ~ Inhibition studies are summarized in Table IV. D-Glucose and 2-acetamido-2-deoxy-~-glucoseinhibited to the same extent (displaying about twice the potency of D-fructose),whereas D-galactose was a n o n i n h i b i t ~ r . ' ~ ~ ~ ' ~D-Glucose ~ . ' ~ ~ . ' ~ protected ~ the lentil lectins from heat denaturation, whereas D-galactose did a-D-Linked D-glUCOSe disaccharides interacted with the lentil lectin, whereas gentiobiose and cellobiose were, re~pectively,'~~ a poor inhibitor and a noninhibitor. Aromatic aglycons (phenyl, p-nitrophenyl, and benzyl groups) enhanced binding of the a-D-linked glycosides of D-glucose and 2-acetamido-2-deoxy-~-glucose.'~~ Replacement of the hydroxyl groups at C-3, C-4, or C-6 of methyl a-D-glucopyranoside by hydrogen atoms abolished the inhibiting capacity, thereby indicating the impor-

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195

TABLEIV Comparative Inhibitory Data on the Lentil Lectina Inhibitor D-Mannose DGlucose D-Fructose 2-Acetamido-2-deoxy-~-glucose Methyl a-D-glucopyranoside Methyl P-D-glucopyranoside Methyl a-D-mannopyranoside Methyl 2-acetamido-2-deoxy-a-~-glucopyranoside Methyl 2-acetamido-2-deoxy-~-~-glucopyranoside 3-0-Methyl-D-glucose Maltose Isomaltose Cellobiose Gentiobiose

B'

C"

1.0 3.8 5.4 2.1 1.1

1 4 8 4 8

1.0 1.0

2.1

X

A"

0.7

0.5 8' 1.4

8 4 X

16

1.0 >4.0 0.25 1.o 0.4 >4.0 4.0

"All data normalized to D-mannose = 1.0."From Ref. 123;molarity required to produce 50% inhibition of precipitation of lentil lectin with phosphonomannan or glycoprotein. "From Ref. 143;minimum amounts (mg/ml) completely inhibiting 4 hemagglutinating doses of lentil lectin. x, no inhibition at 20 mg/ml. dFrom Ref. 213;millimolarity needed to produce 50% inhibition of agglutination.

tance of these groups for binding to the 1 e ~ t i n .That l ~ ~ the configuration of the 3- and 4-hydroxyl groups is of critical importance was also indicated by the fact that D-allOSe and D-galactose, the C-3 and C-4 epimers O f D-glUCOSe, were noninhibitor~.'~~ On the other hand, there is some latitude in substitution at C-2; D-mannose, 2-deoxy-arabino-hexose, and D-glucose (in order of potency) all inhibited lentil hemagglutination. In a comparative study of D-mannose(D-glucose)-binding lectins, Allen and coworkers213discovered that 3-0-methyl- and 3-0-benzylD-glucose were better inhibitors ofthe lentil lectin than D-glUCOSe itself (see Table IV). Like the jack-bean and pea lectins, the lentil agglutinin interacted with 2-0-(2-acetamido-2-deoxy-~-~-g~ucopyranosy~)-D-mannose~~~ providing evidence that the lentil lectin binds to internal 2-0substituted D-mannopyranosyl residues that occur in animal glycoproteins. Van Wauwe and coworkers182studied the effect of various parasubstituents on the binding of phenyl a-D-mannopyranosides to the lentil lectin. As with con A and the pea lectin, binding of p-substituted-phenyl a-D-mannopyranosides correlated fairly well with the Hammett substituent constant uH'in which electron-releasing

196

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

substituents favored binding. The hydrophobicity of the substituents contributed little or nothing to the binding. Inasmuch as extensive maleylation of free amino groups did not abolish the hemagglutinating and poly saccharide-precipitating activity of the lentil lectin, it is probable that amino groups are not involved in the carbohydrate-binding site of the l e ~ t i nTreatment . ~ ~ ~ ~ of the lentil lectin with 1-acetylimidazole resulted in the modification of 3-5 tyrosyl and 7-17 amino The modified proteins had lost their hemagglutinating activity, but retained their capacity to interact with polysaccharides and to bind to In summary, the lentil lectins exhibit a carbohydrate-binding specificity similar to those of the pea lectin and con A; primary specificity is towards a-Dmannopyranosyl residues (2), and secondary is to a - ~ -

2

glucopyranosyl and 2-acetamido-2-deoxy-a-~-glucopyranosy~ residues, Furthermore, 2-0-substituted a-D-mannopyranosyl residues interact with the protein.210 Many unanswered questions remain with respect to the lentil lectins. These include the chemical relationship between the two lectins, a comparison of their carbohydrate-binding specificity, a study of the binding of furanoid sugars (for example, a- and p-arabinofuranosides) to the lectins, and a determination of the H or 0 atoms of each hydroxyl group which may be involved in the binding phenomenon. There is also the question as to the size of the binding site; will the lentil-lectin site be more complementary to sequences of a-D-(1+2)mannopyranosyl residues than is con A (compare Ref. 204)?

3. Pburn sativurn (Pea) ~, (garden pea; a-D-Manp > u - D - G ~ c a-~-GlcNAcp) Although there are several reports on the extraction, partial purification, and properties of a blood-group ABO nonspecific agglutinin from the pea,141.448 only recently have the lectins of the garden pea (Pisum (4474 D. VanEurovi, M. Tichi, and J. Kocourek, Biochim. Biophys. Acta, 453,301-310 ( 1976). (448) S . V. Huprikar and K. Sohonie, Enzymologia, 28, 333-345 (1965).

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sativum) been isolated in pure form. Conventional purificationtechnique^,^^^,^^^ or affinity chromatography on followed by elution with D-glucose solution or acidic b ~ f f e r ~ yield , ~ ~ ~ , ~ ~ pure preparations. The hemagglutinating activity of the pea resides in two closely related agglutinins. The isolectins [termed I and I1 (Ref. 141), and A and B (Ref. 453)], separable by DEAE-cellulose ion-exchange chromatogidentical molecular weights of raphy at pH 8.4 and 8.8, have141*453 approximately 50,000, and remarkably similar amino acid compositions. Aspartic acid and threonine preponderate, whereas methionine and half-cystine are a b ~ e n t . ~Only ~ ~ *traces ~ ~ ~of* carbohydrate ~ ~ ~ (<0.5%)are present.141*453*454 The two pea-agglutinins differ in electrophoretic m ~ b i l i t y .The ~ ~acidic ~,~~ (PI ~ 4.1) component A and neutral (PI 6.5) component B occur as a mixture in the ratio453of 1:2. The isoelectric points of A and B were later found to vary with buffer composition; Entlicher and K o c o ~ r e k reported ~ ~ ~ PI 5.9 and 7.0 for the two pea-agglutinins; a hybrid form had PI 6.35 (see Ref. 455). Approximately one atom of Mn2+and 2.5 atoms of Ca2+are bound per lectin molecule.141 (Ethylenedinitri1o)tetraacetate (EDTA) inhibits both hemagglutination and precipitation of yeast m a n n a r ~ . ~Succes’~ sive dialysis against EDTA andM acetic acid removed most ofthe Ca2+, but did not change the Mn2+content. The apoprotein failed to precipitate D-mannan, and the hemagglutination titer was decreased456by 75%. Subunit structural studies demonstrated two types of polypeptide chains. The smaller subunit (a),molecular weight 7,000-10,500, has N-terminal valine, whereas the larger subunit (p), molecular weight 12,000-18,000, has threonine in the N-terminal Kocourek and coworkers4s4separated the subunits on Biogel P-100 in 5 M guanidinium hydrochloride buffer containing EDTA [monitoring by dodecyl sodium sulfate-poly(acry1amide) electrophoresis]. The subunits a and p occurred in the same relative proportion in each purified (449) T. Shinohara, Proc. J p n . Acad., 47,331-336 (1971). (450) G. Betail, J. Guillot, and M. Coulet, C. R. Soc. Biol., 163, 150-152 (1969). (451) J. Guillot, M. Mustier, G. Betail, A. M. Chabanier, and M. Coulet, C . R. Soc. Biol., 163, 152-154 (1969). (452) K. Onodera and T. Shinohara, Agric. Biol. Chem., 37, 1661-1666 (1973). (453) I. S. lrowbridge,J. Biol. Chem., 249, 6004-6012 (1974). (454) T. Maiik, G. Entlicher, and J. Kocourek, Biochim. Biophys. Acta, 336, 53-61 ( 1974). (455) G. Entlicher and J. Kocourek, Biochim. Biophys. Acta, 393, 165-169 (1975). (456) M. Paulova, G. Entlicher, M. Tichi, J. V. KoStif, and J. Kocourek, Biochim. Biophys. A c ~ u237, , 513-518 (1971). (457) G. Betail, M. Coulet, and J. Guillot, C. R. Soc. B i d , 163, 1771-1775 (1969).

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IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

component, as well as in the native mixture. N-Terminal sequences of the a- and @-subunitsof the pea lectin were determined442aand shown to exhibit a remarkable degree of homology with the analogous subunits of the lentil lectin."2b*c Trowbridge's results using isoelectric focusing in 8 M urea differed slightly.453 Components A and B of affinity-purifiedpea-lectin gave two major protein-bands. The more-basic band (p) was common to both proteins, but there were two types ofa subunits: an acidic species came from the A component, and a more-basic species from the B component. Entlicher and K o c ~ u r e kconfirmed ~~~ these results, adding that the The amino common subunit (p)was larger than the unique subunits (a). acid compositions of the a and /? subunits show significant differe n c e ~ .The ~ ~possibility ~ * ~ ~ ~that one is generated from the other by proteolytic cleavage was excluded on the basis of tryptic peptide mapping.4s3The two pea-lectins are evidently tetrameric molecules consisting of two light (a)and two heavy ( p ) polypeptide chains united by noncovalent forces. They may be represented as a2p2and az'p2,as their a subunits are unique. A hybrid molecular species aa'p2(PI6.35) might be generated in alkaline media.454 Equilibrium-dialysis studies showed two equivalent, sugar-binding sites on each isolectin: intrinsic association-constants (Ka') were 1450 f 2 3 0 M-' for binding to D-mannose and 773 f126 M-' to methyl a - ~ glucopyrano~ide.~~~ Photo-oxidation of the pea lectin at pH 8.2 led to stepwise inactivation of the protein.458Tryptophanyl, histidyl, arginyl, and tyrosyl residues were progressively decomposed, along with a loss of proteinbound Mn2+.The conformation of the protein, namely, a mixture of p-structure and random coil, remained unchanged during photooxidation (as measured by circular dichroism). Kocourek and coworke r suggested ~ ~ that ~ certain ~ tryptophan residues might be of importance in the carbohydrate-binding mechanism of the pea lectin. Under defined, mild conditions, the reaction of the pea lectin with (2-nitropheny1)sulfenylchloride results in sulfenylation of only 2 of the 10 tryptophan residues of the lectin molecule, with simultaneous loss of biological activity. Both sulfenylated tryptophan residues belong to the two heavy subunits of the lectin. Enzymic hydrolysis, and separation of the tryptic peptides, gave one homogeneous, yellow, octapeptide containing the modified tryptophan residue. The octapeptide either is directly a part of the pea-lectin binding-site, or plays an important role in maintaining the tertiary structure of the binding site. According to the amino acid composition and amino acid sequence, the (458) L. BureH, G. Entlicher, and J. Kocourek, Biochim. Biophys. Acta, 285,235-242 (1972).

LECTINS: CARBOHYDRATE-BINDING PROTEINS

199

octapeptide isolated from the pea lectin is almost identical4sgwith a corresponding peptide in con A. The carbohydrate-binding specificity of the pea lectins has been precipitastudied by inhibition of hemagglutination,20~1zz*213~4z8~440~44g~452 tion,122,21f and equilibrium d i a l y s i ~ . Makela ~ ~ ~ , ~demonstrated ~~ that sugars conformingto his group I11 sugars were the best inhibitors ofthe hemagglutination of human erythrocytes .20 In decreasing order of effectiveness they were: D-mannose, trehalose, and turanose > 2amino-2-deoxy-D-glucose, maltose > 2-acetamido-2-deoxy-~-glucose, D-fiXCtOSe, and sucrose. He suggested that the pea lectin could distinguish between the a-and P-D-glycosides of D-glUCOSe, D-mannose, and 2-acetamido-2-deoxy-~-glucose.~~ Other investigators obtained ~ ~ ~by ~ ~pre~~ similar results by inhibition of h e r n a g g l ~ t i n a t i o n ,and cipitation studies.211 Inhibiting sugars enhanced the electrophoretic mobility of the lectin on starch gels in 0.03 M acetate buffer at pH 5.0 in proportion to the sugar concentration and the inhibitory power.440 A thorough study of pea-lectin, carbohydrate-binding specificity was conducted by Van Wauwe and coworkers211(see Table V). By haptenTABLEV Inhibition of the P. satiuum Lectin-P. pinus Phosphonomannan Precipitation Reaction by Various Carbohydrates‘ Inhibitor

Concentration giving inhibition (mM)

D-Mannose D-Glucose D- Fructo se L-Sorbose Methyl a-D-mannopyranoside Methyl a-D-glucopyranoside Methyl P-D-glucopyranoside Phenyl a-D-glucopyranoside Phenyl P-D-glucopyranoside Maltose Isomaltose Cellobiose Gentiobiose Melibiose

0.7 1.7 3.0 6.2 0.3 0.84 7.4 0.44 3.5 0.75 0.9 15.0 (O%)* 11.0 15.0 (0%)

“Taken from Ref. 21 1. *Numbers in parentheses represent the percentage inhibition given by the concentration of carbohydrate noted.

(459) M. Cennakovi, G. Entlicher, and J. Kocourek, Biochim. Biophys. Acta, 420,236245 (1976).

200

IRWIN J. GOLDSTEIN A N D COLLEEN

E. HAYES

inhibition measurements on the precipitin reaction between the Pisum sativum lectin and Pichia pinus 0-phosphonomannan, these investigators showed that configurationally related monosaccharides, namely, D-mannose,D-glucose, D-fructose, and L-sorbose, bound to the lectin. Unmodified hydroxyl groups at C-4 and C-6 of the D-glucopyranose ring were essential for protein binding. Methyl a-Dmannopyranoside was 2.8 times as potent as the corresponding a-Dglucoside, indicating a positive contribution of the axial 2-hydroxyl group of D-mannose to the binding energy of the protein-carbohydrate complex (compare Ref. 452). Pea lectin, unlike con A, appears to be relatively insensitive to variations at c - 2 of D-ghOSe; 2-deoxy-Darabino-hexose, D-glucose, 2-deoxy-2-fluoro-D-glucose, and 2-acetamido-2-deoxy-~-g~ucose all inhibit to the same extent. Although 2-deoxy-2-fluoro-D-mannose and D-mannose are equally effective, 2-acetamido-2-deoxy-D-mannose failed to inhibit hemagglutination211 (compare, Ref. 197). In contrast to changes at C-2, modification at the 3-hydroxyl group notably affects pea-lectin binding. Although 3-deoxy-3-fluoro-~glucose has about one-third the activity of D-glUCOSe, the 3-0-methyl and 3-0-benzyl derivatives of D-glucose are -10 times as potent as D-glUCOSe.211.213 Likewise, methyl 2,3-di-O-methyl-a-Dglucopyranoside is 18 times as inhibitory as methyl a - ~ glucopyranoside. In this respect, the pea lectin is dissimilar to con A, which does not tolerate substitution at C-3 of D-glucose, except for the 3-deoxy-3-fluoro derivative.240 The pea lectin binds a-D-linked D-glucobioses, but not p-D-linked disaccharides (except for gentiobiose, which has one-eleventh the inhibitory effect of isomaltose). Like con A, it interacts with 2-0(2-acetamido-2-deoxy-~-D-g~ucopyranosyl)-~-mannose, suggesting that it may be capable of binding to glycoproteins containing internal 2-0-substituted a-D-mannopyranosyl residuesS2l0 Binding of several fluoro sugars by the pea lectin allowed elucidation of the specific atom of the hydroxyl groups that is recognized by the lectin.211 Inasmuch as 4-deoxy-4-fluoro- and 6-deoxy-6-fluoro-~glucose do not bind, it is probable that the hydrogen atoms of the 4- and 6-hydroxyl groups are hydrogen-bonded to the pea lectin. By the same reasoning, the oxygen atoms of the 2- and 3-hydroxyl groups of D-mannose are implicated in lectin interaction; this is precisely the pattern observed for con A (see Fig. 8).169,240-365 Significant differences were found for the binding of methyl a- and P-D-xylopyranoside to the pea lectin.211These glycosides, lacking hydroxymethyl groups on (2-5,are less effective, by factors Of V240 and 6/20,

LECTINS: CARBOHYDRATE-BINDING PROTEINS

201

than the corresponding methyl a- and P-D-glucopyranosides; methyl P-D-xylopyranoside is -4-6 times as potent as the corresponding a anomer. These results conform to a hypothesis advanced by Brewer and colleagues for the con A binding-site308:the 5-(hydroxymethyl) group of methyl P-D-glucopyranoside binds in the same position occupied by the 2-hydroxyl group of methyl a-D-glucopyranoside. The effect of para-substitution on the affinity of phenyl a - ~ mannopyranoside (and phenyl a-D-glucopyranoside) for the pea lectin was investigated by Van Wauwe and coworkers.'82 Affinities were related to the electronic properties of the substituents, expressed as the Hammett substituent-constants. Thus, electron-releasing substituents favored the binding of p-substituted-phenyl a-D-mannopyranoside (and a-D-glucopyranoside) by an increase in electron density either at the anomeric oxygen atom or in the phenyl ring. The same phenomenon applied to con A (Refs. 180 and 181) and the lentil lectin.182The contribution of the hydrophobic character of para-substituents was small. On the other hand, the corresponding aryl P-D-glucosides bound to the pea lectin independent of both the polar and hydrophobic nature of the substituent.182 The pea lectin precipitated with muscle glycogen, yeast mannan from Saccharom yces cereuisiae, and 0-phosphonomannan from Pichia pinus. 122 All of these reactions were inhibited by specific, sugar haptens (D-mannose, D-glucose, and D-fructose).'22 A comparative study of the hemagglutinating activities of the pea (Pisum satiuum), the lentil (Lens culinaris), and the jack-bean (Canaualia ensiformis) lectins towards the erythrocytes of fourteen different species that the pea and lentil lectins were, in all instances, more active than con A. Of great interest was the finding that, among these three lectins, con A alone was completely inactive against human erythrocytes (blood group, unspecified).446 The pea lectin, like con A, agglutinated normal, embryonic fibroblasts of human and rat origin at high lectin concentrations (1.500 mg/ ml), whereas, various, rat tumor-cells transformed in vitro (spontaneously, and by Rous sarcoma virus) were agglutinated at very low concentrations (5 Fg/ml) of l e ~ t i n . ~ ~ ~

4. Viciu fuba (Fava Bean) (fava or broad bean; a-D-Manp > a - ~ - G l c p>> a-~-GlcNAcp) Apart from its capacity to agglutinate human erythrocytes nonspecifically,the fava bean(Vicia faba) has been known for many years as the (460) P. Vesel?, G . Entlicher, and J. Kocourek, Erperientia, 28, 1085-1086 (1972).

202

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

cause of favism among certain population groups, notably those from countries bordering the eastern Mediterranean.461Favism is an acquired, hemolytic anemia associated with metabolic abnormalities of the erythrocyte (for example, instability to glutathione, and low D-glucose 6-phosphate dehydrogenase activity.) Although aggravated by ingestion of the raw or partially cooked fava bean, the disease does not appear to be due to the presence of the fava-bean agglutinin. In their pioneering paper on the blood-group specific hemagglutinins, Boyd and Reguera5 observed that a saline extract of the fava bean ~~ agglutinated all types of human erythrocytes. Both K r i i ~ eand M&ela78included the fava bean in their compendia of plant agglutinins. Cregar and GiffordM2confirmed that fava-bean extracts agglutinated human, red blood-cells without regard to blood group, and also agglutinated guinea-pig, rabbit, and albino-rat erythrocytes. Erythrocyte aggregation was greatly enhanced by incorporating gum acacia (but not gum ghatti or gum tragacanth) in the agglutination medium, but was inhibited by human serum (apparently the y-globulin f r a ~ t i o n ) . ~ ~ , ~ ~ ~ Treatment of erythrocytes with proteolytic enzymes464(for example, ficin, and an exopolypeptidase from Streptomyces griseus) greatly increased agglutinability by fava-bean extracts, whereas treatment of the cells with tannic acid inhibited a g g l u t i n a t i ~ nVarious . ~ ~ ~ murine and rat tumor-cells were also agglutinated by fava-bean extracts.13QThe agglutination and toxicity of a purified preparation of the lectin were tested against Yoshida sarcoma cells.466Hashem and K a b a r i t ~re~~~ ported that fava-bean extracts were mitogenic for human, peripheral lymphocytes. The Viciafuba lectin was first purified by Tomita and by affinity chromatography of crude extracts on a column of Sephadex G-50; however, no physical-chemical properties were reported. Also employing Sephadex affinity-chromatography (elution with 0.1 M D-glucose), Wang and coworkers140isolated the lectin in pure form. Gel electrophoresis in dodecyl sodium sulfate gave a major protein-band (mol. wt. 18,000), constituting 80-85% of the total material, and two minor bands with molecular weights of -16,000, and 9,000 (Ref. 140). As (461) A. Luisada, Medicine, 20,229-250 (1941). (462) W. P. Creger and H. Gifford, Blood, 7,721-728 (1952). (463) K. L. Roth and A. M. Frumin,J. Lab. Clin. Med., 56, 695-700 (1960). (464) E. Suescun and A. M. Frumin, Am. J . Clin. Pathol., 49,602-605 (1968). (465) E. Suescun, E. A. Pachtman, and A. M. Frumin, Vox Sang., 18,77-80 (1970). (466) M. Tomita, T. Kurokawa, K. Onozaki, T. Osawa, Y. Sakurai, and T. Ukita, 1nt.J. Cancer, 10,602-606 (1972). (467) N. Hashem and A. Kabarity, Lancet, (1) 1428-1429 (1966).

LECTINS: CARBOHYDRATE-BINDING PROTEINS

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identical patterns were obtained in the presence and absence of 2-mercaptoethanol, it appeared that there were no interchain disulfide bonds in the 1 e ~ t i n .The l ~ ~components of molecular weight 18,000and 9,000 stained with the periodic acid-Schiff reagent, indicating bound carbohydrate. Molecular-weight determinations by sedimentation equilibrium and X-ray diffraction gave values of 51,000 and 53,000, respectively. These data are consistent with several possible subunit structures, which include a tetramer (consisting of two chains of 18,000 and two of 9,000 molecular weight) and a more-unusual structure composed of three polypeptide chains140of molecular weight 18,000. The possibilities of other molecular fragments (for example, polypeptide of molecular weight 16,000) cannot be excluded (compare Ref. 263). A tetrameric structure composed of two polypeptide chains of molecular weight 17,300 and two of molecular weight 14,300 was also proposed by Allen and Rechromatography on Sephadex removed a polypeptide (mol. wt. 9,000), believed to be a contaminant (compare Ref. 140). Notable differences in the proportions of aspartic and glutamic acids, and isoleucine and lysine were observed in the two subunits.467aPreliminary, X-ray crystallographic data140indicated that the fava lectin crystallized in the orthorhombic space group P212121. Proceeding from the observation that 2-acetamido-2-deoxy-3-0methyl-D-glucose was a potent inhibitor of the fava-bean lectin, Allen and purified the Vicia fubu phytohemagglutinin by affinity chromatography, using 2-amino-2-deoxy-3-O-methyl-~glucose covalently attached through the amino group to CH-Sepharose (an w-hexanoic acid derivative of agarose). The lectin, whose molecular weight was determined by gel filtration to be -47,500, is believed to be composed of two apparently identical subunits of molecular weight 24,000. Fragmented subunits, similar to those in con A and soybean agglutinin, were found in active preparations of this lectin. A glycoprotein, the agglutinin contains 2-acetamido-2-deoxy-~-glucose and mannose. The carbohydrate moiety is presumably bound by way of an N-asparaginyl linkage, as treatment with alkali failed to release carbohydrate. Studies on the carbohydrate-binding specificity of the fava-bean lectin, as determined by hapten inhibition of hemagglutination,140.2’3.46*~469 showed that the lectin is inhibited by Makela’s group 111 sugars (see Table VI). Hemagglutination by the lectin was inhibited by (467a) H . J. Allen and E. A. Z. Johnson, Biochim. Biophys. Acta, 444,374-385 (1976). (468) C. B. Perera and A . M. Frumin, Science, 151,821 (1966). (469) J . K. N. Lee, E. A. Pachtman, and A. M. Frumin, Proc. Natl. Acad. Sci. U.S.A.,234, 161-169 (1974).

204

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

TABLEVI Comparison of the Inhibitory Effect of Various Saccharides on the Agglutinating Lectinsa Activity of Four D-Mannose/D-Glucose-specific Concentration (mM) needed to produce 50% inhibition of agglutination Lectin of Inhibitor

Concanavalin A

D-Mannose D-Glucose 3-O-Methyl-~-glucose 3-O-Benzyl-D-glucose Methyl a-D-glucopyranoside Methyl 2,3-di-O-methyl-aD-glucopyranoside Methyl 2-acetamido-2deoxy-a-D-glucopyranoside Methyl 2-acetamido-2deoxy-3-O-methyl-a-~glucopyranoside Methyl 2-acetamido-2deoxy-4-O-methyl-a-~glucopyranoside Methyl 2-acetamido-2deoxy-6-O-methyl-a-~glucop yranoside Methyl 2-acetamido-2deoxy-P-D-glucopyranoside Methyl 2-acetamido-2deoxy-3-O-methyl-P-~glucopyranoside Trehalose Kojibiose Nigerose Maltose Isomaltose Sophorose Laminarabiose Cellobiose Gentiobiose

9 25 >200 67 6.3 16 6.3

200

L. culinaris

P. sativum

V .faba

50 50 12.5 33

25 25 1.6 4.2

6.3 25 1.6 2.1

33

12.5

25

2.1 50

4.6

0.5 25

1.6

0.5 25

2.3

100

100

>200

>200

200

200

>200

>200

>200

>200

200

>200

>200 3.1 2.1 8.3 3.1 1.2 4.2 50 >200 100

50 50 8.3 100 50 19.5 50 67 >200 200

25 12.5 2.1 67 12.5 4.9 17 67 200 100

“From Ref. 213. Published by permission of the Biochemical ]oumaZ.

50 12.5 4.2 33 12.5 9.8 17 33 200 100

LECTINS: CARBOHYDRATE-BINDING PROTEINS

205

D-mannose, D-glucose, 2-acetamido-2-deoxy-~-glucose, D-fructose, L-sorbose, maltose, and sucrose, but not by L-arabinose, D-XylOSe, D-ribose, D-galactose, L-fucose, 2-amino-2-deoxy-D-galactose, 2-acetamido-2-deoxy-~-galactose, 2-amino-2-deoxy-~-g~ucose, lactose, D-mannitol, or D-glucit01.~~**~~~ Methyl a-D-mannopyranoside, a,atrehalose, and melezitose were also strong inhibitors of agglutination.140A rough titration indicated that D-mannose is approximately four times as inhibitory as D-glucose or maltose.'39 These data establish a specificity for a-D-mannopyranosyl residues (2) similar to that of con A, and place the lectin in the same class as those of the pea (Pisum sativum) and the lentil (Lens culinaris). Sugar-inhibition of yeast-cell agglutination gave similar results.469a It has been shown that substitution of 0-3 of D-glucose and 2-acetamido-2-deoxy-~-glucoseby methyl or benzyl groups greatly enhances the binding of these sugars to the V. f a b a lectin213;this is also true of the lentil and pea lectins, but not of con A. The best inhibitor of all of the monosaccharide derivatives for the pea, the lentil, and the V. f a b a lectins (but not con A) is methyl 2,3-di-O-methyl-a-~glucopyranoside, which is 25-50 times as inhibitory for the V. f a b a and P . sativum lectins as methyl a-~-glucopyranoside.~~~ These results suggest that there is a hydrophobic area in the lectin binding-site that interacts with the methyl group of the 3-0-methyl derivatives and the The 3-0methylene (or benzyl) group of 3-0-benzyl-~-glucose.~'~ substituted D-mannose derivatives have not yet been tested. Table VI presents comparative inhibitory data on four D-mannose(D-glucose)-binding l e c t i n ~Such . ~ ~ ~studies are exceptionally useful in defining fine differences in the specificity of a group of lectins having similar, but not identical, sugar-binding specificity. A comparative study of the binding of four D-mannose(D-glucose)binding lectins (pea, lentil, con A, and V. f a b a ) to 6C3HED murine ascites tumor-cells indicated that the pea, lentil, and V. f a b a lectins bind to a group of cell-surface glycoproteins different from those to which con A binds. Cell-surface glycoprotein heterogeneity was demonstrated for con A and the pea l e ~ t i n . ~ ~ ~ , ~ ~ ~ ~ From the interest expressed in the fava-bean lectin, we may expect to receive more-complete information on both its structure and carbohydrate-binding specificity in the near future.

(469a) P. Ziska, Acta B i d . Med. Germ., 35, 1575-1576 (1976). (46913) H. J. Allen and E. A. Z. Johnson, Biochim. Biophys. Acta, 436,557-566 (1976).

206

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

111. 2-ACETAMIDO-2-DEOXY-D-GLUCOSE-BINDING LECTINS 1. Bandeiraea simplicqolia I1 (p-~-GlcNAcp= a-~-GlcNAcp)

A lectin having high affinity for nonreducing 2-acetamido-2-deoxya- and -P-D-ghcopyranosyl groups was isolated from Bandeiruea

sirnplicifolia seed-extracts by affinity chromatography on chitin.125Elution of the chitin column with 2-acetamido-2-deoxy-~-glucose gave virtually pure lectin, designated BS 11. Traces of the a-Dgalactopyranosyl lectin, also present in these seeds and designated BS I (see Section V), were removed by passage of purified BS I1 through a The 2-acetamido-2-deoxy-~melibionate-Biogel P-300 c01urnn.l~~ glucose-binding lectin is a tetrameric structure composed of four apparently identical subunits of molecular weight -30,000; an aggregate molecular weight of 113,000was determined by gel filtration. A glycoprotein (4% of carbohydrate), BS I1 contains a high proportion of hydroxylic and acidic amino acids, and two methionine and three cysteine residues per subunit (a disulfide bridge links two subunits). Each subunit contains one carbohydrate-binding The BS I1 lectin does not agglutinate A, B, or 0 erythrocytes, but will agglutinate acquired-B, T-activated, and Tk polyagglutinable ce11s.125,470 BS I1 gave precipitin-like curves with p-azophenyl 2-acetamido-2-deoxy-a- and -p-D-glucopyranoside-bovineserum albumin conjugates-the model substrates first employed in its detection, Shier's antigen A [N,N'-diacetylchitobiosyl-poly(L-aspartate) p~lymer"~] also precipitated BS I1 1 e ~ t i n . Glycogen l~~ and dextran formed precipitates at high concentrations of 1 e ~ t i n . l ~ ~ Carbohydrate-binding specificity-studies were performed by sugar inhibition of the lectin-p-azophenyl 2-acetamido-2-deoxy-~-~-glucopyranoside-bovine serum albumin precipitating system. The most effective monosaccharide inhibitor found (see Table VII) was 2acetamido-2-deoxy-~-glucose; it was over 400 times as inhibitory as D-glucose. Of the common amino sugars examined, only the aforemenexhibited lectin reactivity, estioned 2-acetamido-2-deoxy-~-glucose tablishing the necessity for an equatorial acetamido group at C-2 of the D-hexopyranosyl ring. The failure of D-galactose and 2-acetamido-2deoxy-Dgalactose to inhibit the precipitin reaction also established a (469c) S. Ebisu, P. N. Shankar Iyer, and I. J. Goldstein, Carbohyd. Res., 61, 129-138 (1978). (470) W. J. Judd, M . L. Beck, B. L. Hicklin, P. N. Shankar Iyer, and I. J. Goldstein, Vox Sang., 33,246-251 (1977).

LECTINS: CARBOHYDRATE-BINDING PROTEINS

207

TABLEVII Carbohydrate-binding Specificity of Bandeiraea simplicifolia 2-Acetamido-2-deoxy-~-glucose-binding Lectin"

Sugar inhibitors ~

Micromoles of inhibitor required for 50% inhibition

~~

2-Acetamido-2-deoxy-~-glucopyranose Methyl 2-acetamido-2-deoxy-cr-~-glucopyranoside Methyl 2-acetamido-2-deoxy-fl-~-glucopyranoside p - Nitrophenyl 2-acetamido-2-deoxy-a-D-glucopyranoside p - Nitrophenyl2-acetamido-2-deoxy-fl-~-glucopyranoside 2-Acetamido-2-deoxy-D-mannose 2-Acetamido-2-deoxy-D-galactose D-Glucose Methyl a-Dglucopyranoside Methyl 0-D-glucopyranoside Methyl a-D-galactopyranoside 3-0-( 2-Acetamido-2-deoxy-a-~-glucopyranosy~)-~-g~ucopyranose Maltose Cellobiose Gentiobiose N, N'-Diacetylchitobiose N, N ', N"-Triacetylchitotriose

0.017 0.010 0.080 0.005 0.03 17.0 0% at 50 pmol 7.4 1.3 16.0 0% at 100 pmol 0.01 1.8 1.6 16.0 0.0045 0.0062

~~

"Data taken from Ref. 125.

requirement for an equatorial 4-hydroxyl group. Comparison of the inhibiting capacity of methyl or phenyl 2-acetamido-2-deoxy-a-Dglucopyranoside with those of their respective anomers indicated that the a anomer is bound six to eight times as avidly as the corresponding /3 anomer. The situation becomes complex on considering disaccharides. Maltose and cellobiose are equivalent inhibitors (gentiobiose is a poor inhibitor) and N,N'-diacetylchitobiose is about twice as potent as the disaccharide having a nonreducing a-D-linked 2-acetamido-2-deoxyD-glucosyl group [a-~-GlcNAcp-( 1+3)-~-Glc]. The data suggest that the BS I1 sugar-binding site is complementary to a nonreducing group (3). BS I1 is the 2-acetamido-2-deoxy-a- or -~-D-g~ucopyranosyl first lectin described as having a primary specificity for both a- and p-D-linked 2-acetamido-2-deoxy-~-glucose.Furthermore, a p-D(1+6)-glycosidic linkage seemingly destabilizes the lectin-carbohydrate complex; in this regard, BS I1 resembles the lectins from Laburnum alpinum and Cytisus sessifoZius.'9~471 (471)W. M. Watkins and W. T. J. Morgan, Vox Sang., 7, 129-150 (1962).

208

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

NHAc

3

It has now been established that the BS I1 lectin interacts with the same determinant in blood-group substances derived from hog and human stomach linings as con A (see Section II,1, j), namely, nonreducing, terminal 2-acetamido-2-deoxy-a-~-glucopyranosyl groups ,471a 2. Cytisus sessilifolius [P-D-GlcNAcp-( 1+4)-P-~-GlcNAc]

Renkonen originally discovered the strong anti-H(0) and anti-A, serological activity of Cytisus sessilifolius extrack6 K o u l ~ m i e s , ~ ~ ~ , ~ ~ Kriipe7', and Make1a78later confirmed his findings. Saliva from secretors (but not nonsecretors) was shown to inhibit the hemagglutination a finding substantiated by precipitin band-formation in Ouchterlony plates.474Although the agglutination of 0 cells by C. sessilijolius seed-extracts was neutralized by high dilutions of human and hog H ( 0 ) substances,22~77~78~196~473~475 the agglutinin cannot be classified with eel serum, Lotus tetragonolobus, and Ulex europeus I, because it is not inhibited by L-fucose or its derivatives. Rather, it resembles Ulex europeus I1 (Refs. 77, 208, and 225)and Laburnum alpinums*22*77~471; N,N'-diacetylchitobiose best inhibits 0 erythrocyte hemagglutination induced by these l e ~ t i n s . *The ~ ~erythrocyte ' ~ ~ ~ ~ ~structure ~ with which "Cytisus-type" (Ref. 208) anti-H(0) agglutinins react is not clear. The complementary 2-acetamido-2-deoxy-P-D-glucosyl group may not occur in a terminal, nonreducing position on the blood-group determinant; in fact, internal 2-acetamido-2-deoxy-/3-~-glucopyranosyl residues were demonstrated in soluble blood-group substance.24 Moreover, purified Bacillus fulminans a-L-fucosidase destroyed 0-erythrocyte agglutinability by Cytisus-type anti-H(0) lectins concomitantly with blood-group 0 reactivity.,08 (471a) C. Wood, E. Kabat, S. Ebisu, and I. J. Goldstein, manuscript submitted. (472) R. Koulumies, Ann. Med. E x p . Bid. Fenn., 27, 185-188 (1949). (473) R. Koulumies, Ann. Med. E x p . BioZ. Fenn., 28, 160-167 (1949). (474) F. J. Grundbacher, Science, 81,461-463 (1973). (475) V. P. Rege, T. J. Painter,W. M. Watkins,and W. T. J. Morgan,Nature, 203,360-363 (1964).

LECTINS: CARBOHYDRATE-BINDING PROTEINS

209

Matsumoto and Osawa synthesized an affinity adsorbent for Cytisus sessilifolius lectin by cross-linking N,N',N''-triacetylchitotriose to insoluble starch.'49Gel filtration of a fraction precipitated with ammonium sulfate was followed by adsorption of the hemagglutinin to the affinity r n a t r i ~ . ' ~Glycine-hydrochloric ~,~~~ acid buffer (pH 3.0) eluted a homogeneous protein, as judged by ultracentrifugation and poly(acrylamide) disc-gel electrophoresis (pH 8.9).The chemical composition S) was not deterof this protein (molecular weight 110,000;~,~,,=6.8 mined, nor was its subunit structure investigated. Carbohydrate-binding specificity studies have been conducted on crude C.sessilifolius extracts by hemagglutination inhibition. K r i i ~ e ~ ~ first reported inhibition by salicin [o-(hydroxymethy1)phenyl p-Dglucopyranoside], a finding confirmed by other^.'^^^^^'^^ The p-D-linked D-glucobioses cellobiose and laminarabiose were also good inh i b i t o r ~ , ' ~ *whereas ~ ~ * ~ ~gentiobiose ' and s o p h ~ r o s e were ~ ~ ' poorly inhibitory. N,N'-Diacetylchitobiose conformed most closely to the lectin saccharide binding-site (4) as judged by hemagglutination inhibi-

I

NHAc

NHAc 4

tion.'g*471,475,477 All a-D-linked disaccharides of D-glucose tested were A trisaccharide, believed to be 0-a-L-fucopyranosylnoninhibitor~.'~ ( 1+2)-0-~-D-galactopyranosyl-(1-*4)-2-acetamido-2-deoxy-D-g1ucose, was also a good inhibitor, suggesting that an internal (1-*4)-2acetamido-2-deoxy-~-glucosylresidue could interact with the C.sessilifolius b i n d i n g - ~ i t ePhenyl . ~ ~ ~ and p-nitrophenyl (but not m e t h ~ l ) ' ~ , ~ ~ ~ P-D-glycosides of D-glucose and 2-acetamido-2-deoxy-~-glucose were comparable to (or somewhat better than) salicin as inhibitor^.'^^'^^ (476) I. Matsumoto and T. Osawa, Biochemistry, 13,582-588 (1974). (477) T. J. Painter, V. P. Rege, and W. T. J. Morgan, Nature, 199,569-570 (1963).

2 10

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

Phenyl and p-nitrophenyl P-D-galactopyranoside, as well as lactose, were poorly i n h i b i t ~ r y . ' ~ The , ~ ~high ~ lectin-reactivity reported for 2'0-L-fucosyllactose is interesting, but inconsistent with the observed carbohydrate-binding specificity of the lectin (the authors might well have tested the very active trisaccharide just noted).ls6 Makelazoand Osawal9 suggested the C. sessilifolius lectin as a reagent for preliminary examination of a,p-anomerism in disaccharides of It should, however, be D-glucose and 2-acetamido-2-deoxy-~-glucose. noted that p-D-linked oligosaccharides must possess a P-D-(143 or 4)-glycosidic linkage in order to bind to the lectin ( ~ o p h o r o s eand ~~l g e n t i o b i o ~ e lare ~*~ noninhibitors). ~~ In summary, both the physical-chemical and carbohydrate bindingspecificity studies on this interesting and important lectin are incomplete; additional characterization is required before the lectin can be employed as a meaningful, structural probe.

3. Solanum tuberosum (Potato) [potato; p-~-GlcNAcp -( 1+4)-[P-~-GlcNAcp-( 1+4)1,-/3-~-GlcNAc > P-D-G~cNA -( 1+4)-P-~-GlcNAcp ~~ -( 1+4)-D-GkNAc] The presence of a nonspecific, erythrocyte agglutinin in potato tubers (Solanum tuberosum) was first recognized by Gelhorn16' in 1925. Using a crude preparation from potatoes, M a r c u s s ~ n - B e g u ninvesti~~~ gated the agglutinability of a variety of animal red-cells and the inhibitory effect of several different sera on the hemagglutination reaction. Later, Kriipe77*480 reported that the potato lectin agglutinated erythrocytes of several mammalian species, including guinea pig, human (irrespective of blood-group type), mouse, rabbit, and sheep. Furthermore, he found that blood-group substances, but not simple sugars, inhibited a g g l u t i n a t i ~ n . ~ ~ ~ Although Singh and coworkers481 obtained an active, agglutinin preparation following acetone precipitation, M a r i n k o v i ~ hfirst ~~~ seriously attempted to purifjl the potato lectin by employing acetone fractionation and cellulose ion-exchange chromatography. Homogeneity, by electrophoresis in starch gel and by ultracentrifugation, was demonstrated. He determined the glycoprotein nature of the lectin; chemical analysis showed arabinose, a preponderance of acidic amino acids, and a high molar content of cysteine residues.48zThe agglutinin (478) G. BBtail and M. Coulet, C. R. Acad. Sci., 168,295-299 (1974). (479) H. Marcusson-Begun, 2. Immunitaetsforsch. Exp. Ther., 45,49-73 (1926). (480) M. Krupe, Hoppe-Seyler's Z . Physiol. Chem., 299,277-282 (1955). (481) G. Singh, S. D. Verma, and G. W. G. Bird, Z. Immunitaetsforsch.Exp. Ther., 121, 181-184 (1961). (482) V. A, Marinkovich,]. Immunol., 93, 732-741 (1964).

LECTINS: CARBOHYDRATE-BINDING PROTEINS

211

was inactivated when treated with 0.2M 2-mercaptoethanol for 90 min at 37". No specificity for A, B, 0, MN, or Rh antigens was exhibited. Inhibition of hemagglutination could not be demonstrated, using a variety of mono- and di-saccharides and saliva specimens.482Despite an earlier reporP on the heat stability of potato extract, Marinkovich that his preparation rapidly lost activity on heating to 80". Pardoe and coworkerszo6observed that treatment of a series of ten different, animal erythrocytes with pronase or neuraminidase had virtually no effect on the agglutination titer of the potato lectin. By employing conventional, protein-purification techniques (ammonium sulfate fractionation, and DEAE-cellulose, CM-cellulose, and Sephadex chromatography),Allen and NeubergerZo7 purified the potato lectin to homogeneity (yield: 38 mg from 4.5 kg of potato tubers), and showed it to be a glycoprotein containing -50% (by weight) of carbohydrate. The authors suggested that the lectin was composed of two (or possibly more) identical subunits (molecular weight -46,000), bound noncovalently, giving a molecular weight of 85,000to 100,000. Attempts to purify the potato lectin on ovomucoid- and N,N'diacetylchitobiose-substituted Sepharose failed; the lectin was so strongly bound that it could not be removed from the adsorbent by displacement with urea.483To circumvent this difficulty, Delmotte and employed p-aminobenzyl2-acetamido-2-deoxy-l-thio-~D-glucoside-substituted Sepharose, and eluted the lectin with 0.1 M acetic acid (yield: 58 mg from 128 g of the protein in potato-tuber extract). Arabinose was the preponderant sugar in the glycosyl part of the potato lectin, along with smaller proportions of galactose, glucose, and, Because the carbohydrate possibly, 2-acetamido-2-deoxy-~-glucose.~~~ residue survived dialysis against 0.5M sodium hydroxide solution, the presence of alkali-labile,484glycosidic linkages involving the hydroxyl groups of serine and threonine was excluded. Allen and Neuberger207 suggested that arabinosyloxy-L-proline constituted the major carbohydrate-protein linkage; this linkage occurs in the cell wall of many plants, including the potato.485,486 The amino acid composition of the potato lectin is unusual in a number ofrespects: the most abundant amino acid is hydroxy-L-proline (the first lectin reported to contain this amino acidzo7),11.5% of the residues are half-cystine (compare wheat-germ agglutinin, with 20%of (483) F. Delmotte, C. Kieda, and M. Monsigny, FEBS Lett., 53, 324-330 (1975). (484) R. D . Marshall and A. Neuberger, Adv. Carbohydr. Chem. Biochem., 25,407-478 (1970). (485) D. T. A. Lamport, Biochemistry, 8, 1155-1163 (1969). (486) M. F. Heath and D. H. Northcote, Biochem. J., 125, 953-961 (1971).

212

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

h a l f - c y ~ t i n e ' ~ ~L-phenylalanine ~'~~), is absent, and ornithine is a possible, although disputed, constituent of the lectin.204s483 The carbohydrate-binding specificity of the potato lectin has been studied by examining the extent to which carbohydrates of known structure inhibit hemagglutination of rabbit or human erythrocytes (see Table VIII). The Solanum tuberosurn agglutinin is specifically inhibited by oligosaccharides containing 2-acetamido-2-deoxy-~-glucose,20G*207,483 but not by 2-acetamido-2-deoxy-~-glucose itself77*207*480 (see, however, Ref. 483), or its methyl a- and p-glycopyrano~ides.~~~ Several earlier investigators were similarly unable to demonstrate inhibition of hemagglutination by monosaccharides .77*480 A series of disaccharides containing a 2-acetamido-2-deoxy-~-glucoseresidue in the reducing position, namely, /3-D-Galp-(1+3)-~-GlcNAc, a-D-Galp(1+3)-~-GlcNAc,and P-D-Galp-(1+4)-~-GlcNAc,also failed to inhibit the hemagglutinin reaction between potato lectin and animal erythrocytes treated with pronase and neuraminidase.20G Interestingly, Matsumoto and OsawaIg6reported that p-nitrophenyl at a concen(and phenyl) 2-acetamido-2-deoxy-~-~-glucopyranoside, tration of 10 mg/ml, did not inhibit the agglutination reaction between the potato lectin and human erythrocytes, whereas both p-nitrophenyl 2-acetamido-2,6-dideoxy-~-~-glucopyranoside and phenyl 2-acetat a level amido-3-0-(D-l-carboxyethyl)-2-deoxy-~-~-glucopyranoside, of 5 mg/ml, completely inhibited the hemagglutinin reaction. The TABLEVIII Inhibition, by Various Sugars, of the Hemagglutinating Activity of the Potato (Solanum tuberosum) Lectin" Concentration required for 50% inhibition (mM)

Compound ~~

~~

2-Acetamido-2-deoxy-~-glucose Methyl 2-acetamido-2-deoxy-a-~-glucopyranoside Methyl 2-acetamido-2-deoxy-~-~-glucopyranoside Benzyl 2-acetamido-2-deoxy-ar-~-glucopyranoside N, N'-Diacetylchitobiose N, N',N"-Triacetylchitotriose N,N', N", N"'-Tetraacetylchitotetraose N,N',N",N"', N""-Pentaacetylchitopentaose p-~-GlcNAcp-(1+4)-MurNAcb p-~-GlcNAcp-(1+4)-p-MurNAcp-( 1+4)-P-~-GlcNAcp( 1+4)-MurNAc P-D-Galp-(1+4)-~-GkNAc p-~-Glcp-( 1-+4)-D-Glc

"Data taken from Ref. 207. "MurNAc

=

N-acetylmuramic acid.

0% at 200 mM 0% at 200 mM 0% at 200 mM

40 0.1 0.05 0.005 0.002 8 0.1 O%at3mM 0% at 200 mM

LECTINS: CARBOHYDRATE-BINDING PROTEINS

2 13

interpretation of these data requires further clarification, especially in view of the observation that benzyl 2-acetamido-2-deoxy-a-~-g1ucopyranoside was reported by Allen and NeubergerZo7to be a weak inhibitor. The inhibitory power of the N-peracetylated chito-oligosaccharides increases with increase in chain length up to the tetraose; this suggests an extended b i n d i n g - ~ i t e ~similar ~ ~ ~ ~to~ that ~ * ~proposed *~ for wheatgerm agglutinin.lZ8However, the combining site of the potato lectin may be somewhat larger, as it is complementary to a tetrasaccharide rather than a trisaccharide. Delmotte and coworkers483observed that the aromatic ring in p-nitrophenyl glycosides of N-peracetylated chitobiose and chitotriose enhanced the binding affinity of these ligands to the lectin. Like wheat-germ agglutinin, cellobiose and N-acetyllactosamine206~zo7 [P-D-Galp-(1+4)-~-GlcNAc]were not inhibitors of the potato lectin. It is noteworthy that the bacterial cell-wall oligosaccharides, [p-~-GlcNAcp-( 1+4)-P-~-MurNAc], (n = 1,or 2) (see Table VIII), are weaker inhibitors than the corresponding chito-oligosaccharide h o m o l ~ g s . ~ ~ ~ In summary, the lectin from potato tubers (Solanurn tuberosum) is a

h;' CH,OH

I

NHAc

NHAC

5

2 14

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

nonspecific, blood-group agglutinin that exhibits a carbohydratebinding specificity similar to that of wheat-germ agglutinin, in that it binds to /3-~-(1+4)-linked oligomers of 2-acetamido-2-deoxy-Dglucose. Its binding site can accommodate a tetrasaccharide (5) (possibly, a pentasaccharide). Finer details of this interesting glycoprotein are still lacking, and it is hoped that further studies will be forthcoming so that its potential as a structural probe may be realized. 4. Triticum uulgaris (Wheat Germ) [wheat germ; j3-~-GlcNAcp-( 1+4)-/3-~-GlcNAcp-(1+4)-GlcNAc > p-D-GlcNAcp-( 1+4)-/3-D-GlcNA~]

One of the first reports of the interaction between carbohydratebinding proteins and tumor cells was made by Aub-andcolleaguesz8in 1963; a wheat-germ, lipase preparation agglutinated malignant cells. Subsequently, Burger and Goldberg30reported that virally transformed cells were agglutinated to a greater extent than their respective, parent cell-lines. Wheat-germ agglutinin (WGA) is not a blood-group-specific hemagglutinin; it will agglutinate all types of human erythrocytes, as well as a variety of normal and neoplastic animal cells. Some years later, the agglutinating principle of wheat germ was isolated, and crystallized, by Nagata and BurgeP' and LeVine and coworkers48s;both studies reported a glycoprotein having a molecular weight of 23,500. Others purified WGA b y conventional techniques,'z8~'z9~48g*4go and by affinity chromatography on synthetic adsorbents: [6-aminohexyl 2-deoxy-/3-~-urubino-hexopyranoside-Sepharose 4B (Ref. 130), p-aminobenzyl 2-acetamido-2-deoxy-l-thio-/3-~g l u c o p y r a n 0 s i d e , 4 ~2-acetamido-N-(6-aminohexanoyl)-2-deoxy-/3~~~~~~~ D-glucopyranosylamine-Sepharose 4B (Ref. 491)], immobilized o v o m ~ ~ o iand d chitin.'= , ~ ~ ~ Careful ~ ~ ~analysis ~ ~ ~ revealed ~ ~ the protein (487) Y. Nagata and M. M. Burger,]. Biol. Chem., 247,2248-2250 (1972). (488) D. LeVine, M. J. Kaplan, and P. J. Greenaway, Biochem. J . , 129,847-856 (1972). (489) B. Ozanne and J. Sambrook, Nature (London) New Biol., 232,156-160 (1971). (490) Y. Nagata, A. R. Goldberg, and M. M. Burger, Methods Enzymol., 32, 611-615 (1974). (490a) M. E. Rafestin, A. Obrenovitch, A. Oblin, andM. Monsigny,FEBS Lett., 40,62-66 (1974). (490b) P. Bouchard, Y. Moroux, R. Tixier, J.-P. Privat, and M. Monsigny, Biochimie, 58, 1247-1253 (1976). (491) R. Lotan, A. E. S.Gussin, H. Lis, and N. Sharon, Biochem. Biophys. Res. Commun., 52,656662 (1973). (492) M. M. Burger, Proc. Natl. Acad. Sci. U.S.A., 62, 994-1001 (1969). (493) V. T. Marchesi, Methods Enzymol., 28, Part B, 354-356 (1972).

LECTINS: CARBOHYDRATE-BINDING PROTEINS

215

to be devoid of covalently bound c a r b ~ h y d r a t e ' ~ ~ -hence, ~ ~ ~ . ~WGA ~~~; is not a glycoprotein. Hemagglutinating activity was associated with several fractions eluted from columns of SE-Sephadex and SPS e p h a d e ~ . ' ~Rice ~ . ~and ~ ~ E t ~ l e 1 3isolated ~~ 200-250 mg of purified WGNkg of raw wheat-germ (Bouchard and coworker^^^^^ reported 500 mg of WGNkg of wheat germ); this was distributed among four active fractionP5 as follows: 35% of I, 50% of 11,, 5% of IIb, and 10% of 111. WGA fractions I, 11,, and I11 were indistinguishable, focusing in a sharp band at pH 8.7k0.3 in isoelectric focusing experiments; WGA IIb focused at pH 7.7 k0.3. The amino acid compositions of the four, chromatographically distinct, forms indicated considerable similarity (although fraction I contained no histidine), and were comparable to related results of others.128,12s,133,4s1 There was a very high content of half-cystine (but no free sulfhydryl groups) and glycine. A genetic basis for the occurrence of multiple forms of WGA has been advanced.494a There is now general concurrence that WGA is a dimeric protein, of molecular weight 36,000, composed of two identical subunits (molecular weight 18,000).129,335*495 Denaturants (8 M urea), pH extremes (pH 1-11), or high concentrations of salt promoted subunit exchange among WGA I, 11,, and 111, or with chemically modified (N-acetyl and N-methyl derivatives), electrophoretic variants to give hybrid agglutinins. These experiments provided further evidence for the structural similarity and dimeric nature of the three WGA forms.335All four isolectins of wheat-germ agglutinin exhibited similar c.d. Analysis of the c.d. spectrum in the ultraviolet region indicated -12% The of p-structure, and no evidence of an a-helical X-ray crystallographic analysis of the agglutinin has been solved at 200-pm r e ~ o l u t i o n . Wheat-germ ~ ~ * ~ ~ ~ ~agglutinin dimers (4 x 4 x 7 nm) are composed of two closely associated protomers, each consisting of 164 amino acids. Each protomer consists of four structurally homologous and spatially distinct domains.49saCrystals of the agglutinin containing the 4-methylumbelliferyl P-glycoside of N,N'-diacetylchitobiose have been prepared. (494) J.-P. Privat, F. Delmotte, G. Mialonier, P. Bouchard, and M. Monsigny, Eur. J . Biochem., 47,5-14 (1974). (494a) R. H. Rice, Biochim. Biophys. Acta, 444, 175-180 (1976). (495) R. H. Rice and M. E. Etzler,Biochem. Biophys. Res. Commun.,59,414-419(1974). (495a) M. W. Thomas, E. F. Walborg, Jr., and B. Jirgensons,Arch. Biochem. Biophys., 178,625-630 (1977). (496) C. S. Wright, C. Keith, R. Langridge, Y. Nagata, and M. M. Burger,J.Mol. Biol., 87, 843-846 (1974). (496a) C. S. Wright,J. Mol. Biol., 111,439-457 (1976).

216

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

Equilibrium-dialysis studies on the number of carbohydratebinding sites per subunit have been conducted, but the results are somewhat discordant. Thus, LeVine and coworkers,488 using 2-acetamido-2-deoxy-~-glucose, reported one binding site per subunit of molecular weight 23,000, whereas Nagata and BurgerlZ9found two binding sites per molecular equivalent of subunit. In order to exclude the possibility that two molecules of 2-acetamido-2-deoxy-~-glucose might bind to one subunit within the same binding site (see later), Privat and coworker^"^ studied the binding of NaBT4-reduced N,N”N”,N’”-tetraacetylchitotetraose to WGA. In agreement with the results of Nagata and Burger,lz9 two moles of tetrasaccharide were bound per mole of polypeptide chain. By means of a fluorescencequenching technique, the same investigators demonstrated that two moles of the 4-methylumbelliferyl P-glycosides of 2-acetamido-2deoxy-D-glucose, N,N’-diacetylchitobiose, and N , N ’ ,N ’ ’-triacetylchitotriose were similarly bound to a WGA monomeric unit? (compare Ref. 497a). Inasmuch as the fluorescence of all of these glycosides was quenched to the same extent (as opposed to the findings of Van Landschoot and it was proposed that the aglycon occupied the same subsite in the three cases, and that the lectin binding-site contains only three subsites. If it is confirmed that there are two identical and noninteracting subsites per subunit, it would be expected that there is an element of symmetry in each polypeptide chain; for example, the subunit might consist of two (identical) halves resulting from gene duplication. Indeed, X-ray crystallographic data suggest the possibility of gene quadruplication followed by divergent e v o l ~ t i o n . ~ ~ ~ ~ Studies on the specific precipitation of WGA by glycoproteins, glycolipids, bacterial cell-wall peptidoglycan, and model substrates have been reported. Shier1I4chemically linked N,N ’-diacetyl-p-chitobiosylamine to poly(L-aspartic acid), to afford a model substrate termed antigen A [poly(N,N’-diacetyl-p-chitobiosylaspartate)] which formed a precipitin band1I4 with WGA. A saturated solution of N,N’-diacetylchitobiose completely inhibited the reaction. Antigen A gave4s8a precipitin curve with WGA. Similarly, Watanabe and Hakomori demo n ~ t r a t e da ~precipitin ~~ reaction by agar-gel diffusion between WGA and purified glycosphingolipids from human adenocarcinoma and blood-group A erythrocytes. The latter, blood-group A sphingolipid (497) J.-P. Privat, F. Delmotte, and M. Monsigny, FEBS Lett., 46,229-232 (1974). (497a) A. Van Landschoot, F. G . Loontiens, R. M. Clegg, N. Sharon, and C. K. De Bruyne, Eur. J . Bfochem., 79,275-283 (1977). (498) I. J. Goldstein, S. Hammarstrom, and G . Sundblad, Biochim. Biophys. Acta, 405, 53-61 (1975). (499) K. Watanabe and S. Hakomori, FEBS Lett., 37,317-320 (1973).

LECTINS: CARBOHYDRATE-BINDING PROTEINS

217

was composed of 1.0 fucose, 0.9 glucose, 2.1 galactose, 0.92-amino-2deoxyglucose, and 1.7 2-amino-2-deoxygalactoseunits. Smith degradation caused decomposition of fucose and 2-amino-2-deoxygalactose, whereas the reactivity with WGA intensified, suggesting that the site recognized b y WGA may be in the interior chain.499A ceramide trisaccharide [O-2-acetamido-2-deoxy-~-~-glucopyranosyl-( 1+3)-0-@D-galactopyranosyl-(1+4)-p-~-glucopyranosylceramide]isolated from degraded, blood-group glycolipid was also strongly WGA-reactive, suggesting that WGA can bind nonreducing (terminal) 2-acetamido-2deoxy-D-glucosyl groups as In order to form a lattice with the lectin, the glycolipid is most probably present in the micellar form, in which case, multiple 2-acetamido-2-deoxy-D-glucosyl groups would be available for interaction with the protein. Privat and coworker^"^ diazotized p-aminophenyl N,N'-diacetylp-chitobioside to bovine serum albumin, and demonstrated that it precipitated with WGA in agar gel; N,N'-diacetylchitobiose inhibited this interaction. Carcinogenic embryonic antigen (CEA) precipitated with WGA in agar gels4B9and in solution.498CEA that had undergone three Smithdegradation cycles also gave a precipitin curve.498As may be seen from Fig. 10, each subsequent degraded product precipitated with WGA, including the products after periodate oxidation and reduction, but prior to mild, acid hydrolysis.

-

6

m

3.5

'EI

.-.-'04 a c

g 3 g 2

e

L

21 10

20

30 40 5 0 60 CEA antigen ( p g )

.,

70

00

FIG. 10.-Precipitation of Carcinoembryonic Antigen (CEA) and its Smith-degraded Products by Wheat-germ Agglutinin.*8(0,Native CEA; 0 ,periodate-oxidized, borohydride-reduced CEA; V, Smith-degraded CEA, stage I; V, periodate-oxidized, borotwo cycles of Smith-degraded CEA, hydride-reduced, Smith-degraded CEA I; stage 11; 0 , periodate-oxidized, borohydride-reduced, stage 11; A, three cycles of Smith-degraded CEA. Conditions: 30 pg of wheat-germ agglutinin per tube; total volume, 200 pl. Reproduced by permission of Biochim. Biophys. Acta.)

218

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

p-Azophenyl 2-acetamido-2-deoxy-~-~-glucopyranoside-bovine serum albumin conjugate precipitated with WGA, and this system was used to investigate the sugar-binding specificity of the agglutinin.498 Interaction of WGA with a soluble, linear peptidoglycan secreted by Micrococcus luteus, and with the teichoic acid of Staphyloccus aureus H , was demonstrated by agar-gel diffusion, quantitative precipitation, and inhibition of trypsinized rabbit-erythrocyte h e m a g g l u t i n a t i ~ n . ~ ~ ~ No interaction was observed with the teichoic acid from a phageresistant mutant (S. aureus 52A2) that lacked 2-acetamido-2-deoxy-~glucose residues. Burger and Goldberg30 first reported that, of the sugars found in glycoproteins, only 2-acetamido-2-deoxy-~-glucoseand N,N’-diacetylchitobiose inhibited the agglutination of polyoma virus-transformed BHK cells and chemically-induced L1210 leukemia cells (compare Refs. 501-504). Sugar inhibition of rabbit erythrocyte agglutination by WGA demonstrated that, of the common amino sugars, 2-acetamido-2-deoxy-~glucose alone inhibited the agglutination reaction.lZ8Neither 2-amino2-deoxy-D-glucose,2-acetamido-2-deoxy-~-galactose, nor S-acetamido2-deoxy-~-mannosewas inhibitory, demonstrating the necessity for an equatorial hydroxyl group at C-4 and the D-glyCerO configuration at C-2 (compare Ref. 498).WGA exhibited little or no anomeric preference for methyl glycopyranosides of 2-acetamido-2-deoxy-~-glucose.~~~~~~~ Employing a precipitation reaction between WGA and p-azophenyl 2-acetamido-2-deoxy-~-~-glucopyranoside-bovine serum albumin conjugate, Goldstein and coworkers showed that p-nitrophenyl 2-acetamido-2-deoxy-~-~-glucopyranoside was a poor inhibitor, and the methyl a- and P-glycopyranosides of D-glUCOSe, D-mannose, and D-galactose were noninhibit01-s.~~~ (Earlier, the same p-azophenyl 2-acetamido-2-deoxy-~-~-glucopyranoside-bovine serum albumin conjugate was shown to inhibit WGA-induced h e m a g g l u t i n a t i ~ n . ~ ~ ) Like 2-amino-2-deoxy-~-glucose, 2-acetamido-l-(~-aspart-4-oyl)-2deoxy-P-D-glucopyranosylaminewas a noninhibitor of both hemagglutination128and p r e c i p i t a t i ~ n .These ~ ~ ~ data suggest that an uncharged amide group (for example, N-acetyl or N-propionyl group) (500)R. Lotan, N. Sharon, and D. Mirelman, Eur. J . Biochem., 55,257-262 (1975). (501) R. Liske and D. Franks, Nature (London), 217,860-861 (1968). (502)G.Uhlenbruck, G.I. Pardoe, and G . W. G.Bird, Nuturwissenschuften, 55,347 (1968). (503)G.Uhlenbruck,W.Gielen, and G . I. Pardoe,Z. Krebsforsch., 74,171-178(1970). (504)R.Lotan and N. Sharon, Biochem. Biophys. Res. Commun., 55,1340-1346(1973).

LECTINS: CARBOHYDRATE-BINDING PROTEINS

219

must be present at C-2 in the D-glycero configuration; neighboring, free carboxyl and amino groups interfere with the binding phenomenon. On the other hand, uncharged 2-acetarnido-N-acetyl-2-deoxy-~-~glucopyranosylamine inhibited to the same extent as the a- or pglycopyranosides of 2-acetamido-2-deoxy-~-glucose,indicating that nitrogen may replace the glycosidic oxygen atom without any sacrifice in inhibitory potency.128Nonspecific binding of N-acetylneuraminic acid, possibly to the same site as that to which 2-acetamido-2-deoxy-~glucose binds, was also reported505(compare Refs. 505a-d). Methylation at 0 - 4 or 0 - 6 (for example, 2-acetamido-2-deoxy-4- or -6-O-methyl-~-glucose)had no effect on inhibitory activity, whereas similar substitution at 0 - 3 gave an inactive derivative.128These results suggest that oligosaccharides containing 4- and 6-0-substituted (perhaps, also, 4,6-di-0-substituted) 2-acetamido-2-deoxy-~-glucose residues may bind to WGA; this has been substantiated for certain 4-0substituted 2-acetamido-2-deoxy-~-glucoseoligosaccharides,'28*4Qsbut has yet to be demonstrated for the 6-0-substituted derivative (see later). Of the oligosaccharides examined for their capacity to inhibit WGA, only those containing 2-acetamido-2-deoxy-~-glucoseresidues proved effective. Lactose, cellobiose, cellotriose, and N-acetyllactosamine (2acetamido-2-deoxy-4-O-~-D-ga~actopyranosyl-~-g~ucose) were all inactive as inhibitors of the p-azophenyl 2-acetamido-2-deoxy-P-~glucopyranoside-bovine serum albumin-WGA reaction.4Q8Table IX presents representative inhibition data. By far the most interesting series of WGA sugar inhibitors are the N-peracetylated chito-oligosaccharides. Confirming Burger and Goldberg's results,3OWGA showed a much higher affinity forN,N'-diacetylchitobiose than for 2-acetamido-2-deoxy-~-glucose. In fact, this affinity increased as the homologous series was ascended, such that the biose was several hundred and the triose several thousand times more tightly bound than 2-acetamido-2-deoxy-D-glucose. Comparative data, from studies in five different systems, on WGA binding of N-peracetylated chito-oligosaccharides have been tabulated.4g8Although, in some systems, N-peracetylated chitotetraose and chito(505) P. J. Greenaway and D. LeVine, Nature (London) New Biol., 241,191-192 (1973). (505a) P. Cuatrecasas, Biochemistry, 12, 1312-1323 (1973). (505b) W. R. Redwood and T. G. Polefka, Biochim. Biophys. Acta, 455,631-643 (1976). (505c) D. H. Boldt, S. F. Speckart, R. L. Richards, and C. R. Alving, Biochem. Biophys. Res. Commun., 74,208-214 (1977). (505d) V. P. Bhavanandan, J. Umemoto, J. R. Banks, and E. A. Davidson, Biochemistry, 16,4426-4437 (1977).

220

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES TABLEIX Carbohydrate-binding Specificity of Wheat-germ Agglutinin Concentration required for 50% inhibition Inhibitor

nmoV2OO pla

N,N', N", N"', N""-Pentaacetylchitopentaose 1.0 N, N ',N", N"'-Tetraacetylchitotetraose 1.3 N,N',N"-Triacetylchitotriose 1.6 N , N'-Diacetylchitobiose 45 N,N'-Diacetylchitobiitol 500 Chitobiose >4,500 >700 a-D-Manp-(1+3)-,9-D-Manp-( 1+4)-~-GlcNAc P-DManp-( 1+4)-P-~-GlcNAcp-(1 + 4 ) - p ~ 33 GlcNAcp-(l+N)-Asn 39 p-~-ClcNAcp-(1+4)-/3-~GlcNAcp-(l+N)-Asn 2-Acetamido-N-(4-~-aspa1toyl)-2-deoxy-/3-~-gluco350 pyranos ylamine p-Nitrophenyl 2-acetamido-2-deoxy-/3-~-gluco1,100 pyranoside Methyl 2-acetamido-2-deoxy-/3-~-glucopyranoside 3,300 Methyl 2-acetamido-2-deoxy-a-~-glucopyranoside 5,000 2-Acetamido-2-deoxy-~-glucose 5,900 2-Amino-2-deoxy-~glucose 2-Acetamido-2-deoxy-~-galactose Methyl 2-acetamido-2-deoxy-3-O-methyl-a-~-

glucopyranoside Methyl 2-acetamido-2-deoxy-4-O-methyl-a-~glucopyranoside Methyl 2-acetamido-2-deoxy-6-O-methyl-a-~glucopyranoside Cellobiose Cellotriose

>63,000

31,000

mM*

0.005 0.01 0.01 0.5

0% at 35 mM

10 10 30 0% at 200 mM 200 200 10

10 >2,000 >3,000

"Inhibition of wheat-germ agglutinin-p-azophenyl 2-acetamido-2-deoxy-/3-~-glucopyranoside-bovine serum albumin precipitation by various saccharides. Data taken from Ref. 498. bInhibitory effect of various sugars on the agglutinating activity of wheatgerm agglutinin against rabbit erythrocytes. Data from Ref. 128.

pentaose showed a somewhat higher affinity for WGA than did chitotriose, the WGA combining-site appears to be complementary to a sequence of three @-D-( l-+4)-linked 2-acetamido-2-deoxy-~-glucose units (6), with additional glycosyl residues adding little to the free energy of binding.

221

LECTINS: CARBOHYDRATE-BINDING PROTEINS

0 CH,OH

‘

I NHAc

0

NHAC 6

Studies on the homologous, N-peracetylated chito-oligosaccharides led Allen and coworkerslZ8to postulate that the WGA binding-site consists of a system of adjacent subsites similar to that assumed for hen’s egg-white lysozyme506(see Fig. 11).They observedlZ8that the bacterial cell-wall disaccharide [P-DGlcNAcp-(1+4)-MurNAc] was a weaker inhibitor than N,N’-diacetylchitobiose, whereas the tetrasaccharides (N-peracetylated chitotetraose) and [p-D-GlcNAcp-(1-4)MurNAcplz were about equally as active as inhibitors. Subsites A, B,

Subs it e :

A

B

C D-G~cNAcp - 0 - R 8- D - GlCNAC p - (1- 4) - D -GlCNAC

D

6-D - GlCNACp - (1- 4 ) -@- D - GlCNACp - (1- 4)- D - GlCNAC 8- D - GlcNAc p - (1-4LMurNAc 4-D - GlcNAc p - (l+ 4)-p-MurNAcp-(l-c 4)-p-D -GlcNAc p- (l--l)-MurNAc FIG. 11.-System of Subsites Proposed for the Binding Site of Wheat-germ Agglutinin.lZ8 (506) C. C. F. Blake, L. N. Johnson, G. A. Mair, A. C. T. North, D. C. Phillips, andV. R. Sarma, Proc. R. Soc. London Ser. B , 167,378-388 (1967).

222

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

and C were envisaged as binding 2-acetamido-2-deoxy-~-glucose residues, and subsite D as accommodating aglycons of the glycosides. They also suggestedlZ8that 2-acetamido-2-deoxy-~-glucose itself is bound only to subsite C, and that its 3-hydroxyl group must be unsubstituted. On the other hand, oligosaccharides composed of 2acetamido-2-deoxy-~-glucoseresidues may bind to WGA even if the residue that occupies subsite B contains a substituent at (2-3, as in N acetylmuramic acid. Thus, as indicated previously, bacterial cell-wall tetrasaccharide binds as well as N-peracetylated chitotetraose.lZ8 The relative affinity of WGA for the mono-, di-, and tri-saccharide differs according to the system employed. Although the reason for these differences is not yet clear, it seems probable that they are related to the ability of the “substrate” used in the inhibition assays to occupy one or more subsites in WGA. Whereas fluorescence enhancement and equilibrium dialysis are direct measurements of binding strengths, inhibition assays depend on the ability of the hapten to displace the “substrate” from the site. In the case of inhibition ofprecipitation using p-azophenyl P-D-GlcNAc-bovine serum albumin, it may be assumed that only one subsite is When 2-acetamido-2-deoxy-~glucose is added as an inhibitor, both competitive and noncompetitive binding of 2-acetamido-2-deoxy-~-glucose to the agglutinin may occur, thus increasing the concentration needed for 50% inhibition. In contrast, only competitive binding will occur with N , N ’ , N ’ ’ triacetylchitotriose, assuming that WGA has three subsites. On the other hand, N,N’-diacetylchitobiose may bind both noncompetitively and competitively. However, as compared to 2-acetamido-2-deoxy-~glucose, the relative contribution of noncompetitive binding to total binding will be smaller with the b i o ~ e . ~ O ~ Like 2-amino-2-deoxy-~-glucose,the chito-oligosaccharides having unsubstituted 2-amino groups do not bind to WGA, due, presumably, to repulsion of their positively charged -NH3+groups.128*408 The ability of WGA to interact with P-D-Manp-(1+4)-p-~-ClcNAcp(1+4)-P-~-GlcNAcp-(l+N)-Asn and p - ~ - G l c N A c p -1-+4)-P-~( GlcNAcp-(l+N)-Asn with approximately the same affinity as N , N ’ d i a c e t y l c h i t o b i ~ s efurther ~ ~ ~ supports the observations of Privat and coworker^"^ and Shier114;macromolecules containing multiple N,N’diacetylchitobiosyl residues precipitate with the agglutinin. The carbohydrate-binding sites of WGA are probably situated at the surface of the protein molecule. The pH dependences of association constants for lectin-chitotriose binding indicate that an ionizable group, pK = 3.9, is probably involved in complex-formation.404*504 This observation is especially noteworthy, inasmuch as protein carboxyl

LECTINS: CARBOHYDRATE-BINDING PROTEINS

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groups have also been implicated in the carbohydrate-binding sites of con A (see Ref. 326). Reductive methylation (treatment with HCHO in the presence of NaBH4)had no detectable effect on the carbohydratebinding ability of the protein.33sSubsequent 0-acetylation with acetic anhydride prevented binding of the protein to ovomucoid-Sepharose and greatly lowered its erythrocyte-agglutinating ability; inhibition was reversible by treatment with h y d r o ~ y l a m i n eSimilarly, .~~~ acetylation of native WGA, modifying available amino and phenolic groups, greatly lowered the agglutinating ability.335Regeneration of free tyrosine by brief treatment with hydroxylamine restored the ability of WGA to agglutinate erythrocytes. These experiments suggest that free amino groups (for example, the 6-amino groups of lysine residues) are not involved in carbohydrate binding, whereas the hydroxyl groups of tyrosine residues may make contact with bound ~ a r b o h y d r a t eMod.~~~ ification ofcarboxyl groups with glycine methyl ester or glycinamide in the presence of a water-soluble carbodiimide abolished the erythroagglutinating activity of the protein, supporting the role of carboxyl groups of the lectin in the carbohydrate-binding m e c h a n i ~ m . ~ ~ ~ . ~ ~ ~ Fluorescence studies led to the conclusion that at least two of the three tryptophan residues in WGA are highly accessible to solvent molecules.494Inasmuch as the fluorescent, tryptophan residues were not fully protected by N,N',N''-triacetylchitotriose from quenching by iodide, it was postulated that they were not directly in the WGA binding-site."' Treatment of WGA with N-bromosuccinimide in 0.1 M acetic acid-8 M urea modified all of the tryptophan residues, whereas only two of three tryptophan residues were modified when the reaction was conducted in 0.1 M citrate (pH 6.0) buffer.508Oxidation (in acetic acid-urea) of one tryptophan residue per subunit led to almost complete (97%)loss of hemagglutination activity and a 3.5-fold decrease in the affinity constant for N,N',N"-triacetylchitotriose, and rendered the subunits unable to form the native dimer.s08No significant changes in the circular dichroism spectrum of WGA were observed after oxidation of three tryptophan residues, suggesting that no gross conformational changes had occurred.508 The presence of 2-acetamido-2-deoxy-~-glucose, -D-galactose, -D-mannose, or the N-acetylated chito-di-, -tri-, and -tetra-saccharides induced conformational changes in the l e ~ t i n . @ The ~ ~changes at 270290 nm of the c.d. spectrum were attributed to perturbations of tyrosine and, possibly, tryptophan and disulfide c h r o m o p h ~ r e s . ~ ~ ~ ~ (507)J.-P, Privat and M. Monsigny, Eur. J . Biochem., 60,555-567 (1975). (508)J.-P.Privat, R. Lotan, P. Bouchard,N. Sharon, and M. Monsigny,Eur.]. Biochem., 68,563-572 (1976).

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IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

It has now been found that WGA will form a specific precipitate with keratan from cornea and nasal cartilage50ea;these glycosaminoglycans contain alternate, (1+4)-linked 2-acetamido-2-deoxy-~-D-glucopyranosyl residues. Similarly, pneumococcus S 14 capsular polysaccharide (containing 2-acetamido-2-deoxy-~-~-glucosyl residues substituted by glycosyl groups at both 0-4 and 0-6) gave a precipitin CUrve508a.508b with WGA (compare Ref. 502). S 14 polysaccharide that had been subjected to controlled, Smith degradation also precipitated with WGA; this derived polysaccharide contains 2-acetamido-2-deoxy-~-~-glucosyl residues linked solely at 0-6. These results demonstrate unequivocally that 2-acetamido-2-deoxy-p-Dglucosyl residues linked ( 1 4 6 ) or (1+4, 1 4 6 ) are capable of interacting50sa,50sb with WGA. Elution of a chitin column, charged with wheat-germ extract, with 0.01 M acetic acid (as opposed to 0.05 M hydrochloric gave protein(s) mitogenic for purified, human peripheral-lympho~ytes.~~~~ This discovery is contrary to several past reports (see, for example, Ref. 156)that showed that WGA had no mitogenic activity. The relationship between WGA and the newly described protein(s),termed wheat-germ mitogen, has yet to be established.

5. Ulex europeus I1 (Gorse or Furze Seed) [gorse or furze seed; ~-~-GlcNAcp-(1+4)-D-GlcNAcl The extract of Ulex europeus seeds has long been used in serological 1aboratorie~'~J~ as a reagent for typing blood-group 0 erythrocytes, determining subgroups of blood types A and AB, and assessing secretor status (the occurrence of H-active substance in saliva). This use was based upon the initial observation of Cazal and Lalauriesl that three species of Ulex seeds (Ulex provincialis Lois, Ulex jussiaei Webb, and Ulex europeus L.) contained strong hemagglutinating activity against blood-group 0 erythrocytes, with weaker activity against Az cells, and occasional reactivity with B cells. These results were subsequently confirmed by Boyd and Shapleigh,12*13 F l ~ r yand , ~ K~ ~~+ p e Subgroup .~~ AzB reacted with the extract, whereas AIB did not.lZSaliva of secretor individuals inhibited 0 erythrocyte agglutination by Ulex europeus extract, regardless of the individual's blood type.13 (508a) H. E. Carlsson, J. Lonngren, I. J. Goldstein, J. E. Christner, and G. W. Jourdain, FEBS Lett., 62, 38-40 (1976). (508b) S.Ebisu, J. Lonngren, and I. J. Goldstein, Carbohydr. Res., 58,187-191 (1977). (508c) J. M. Brown, M. A. Leon, and J. J. LightbodyJ. Immunol., 117,1976-1980 (1976). (508d) G. W. G. Bird and J. Wingham, Vox Sung., 19,132-139 (1970).

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Flory fractionated U. europeus extracts with ethan01.2~~ The 30%-and 50%-alcohol precipitates agglutinated type 0 erythrocytes, a reaction that was readily inhibited by P-D-glycosides (cellobiose, lactose, or salicin), but unaffected by L - ~ U C O S On ~ . ~the ~ ~ other hand, 0 erythrocyte agglutination by the 70%-alcohol precipitate was inhibited by L-fucose (slightly by D-arabinose, D-ribose, and D-lyxose), but not by any of the aforementioned glycosides. Furthermore, the 30%- and 50%-alcohol precipitates reacted with all human, buccal (epithelial cheek) cells, whereas the 70%-alcohol precipitate agglutinated only the buccal cells of secretors. On the basis of these observations, Flory suggestedzz5that Ulex extract contained at least two agglutinins having different carbohydrate-binding specificity. Apparently, lectins of two alternative specificities interact with type 0 erythrocytes. Eel serum-type anti-H(0) agglutinins (Anguilla anguilla, Lotus tetragonolobus, and Ulex europeus I) are best inhibited by L-fucosides; they are considered in Section VI. Cytisus-type antiH ( 0 ) agglutinins (Cytisus sessilifolius, Laburnum alpinum, and Ulex Purified, europeus 11) are inhibited by N,N’-diacetylchitobi~se.~~~ H-decomposing enzyme from Bacillus fulminans, an L-fucosidase, destroyed the agglutinability of 0 erythrocytes not only by L-fucosebinding anti-H(0) lectins but also by N,N‘-diacetylchitobiose-binding anti-H(0) lectins as Although L-fucose does not inhibit the N,N’-diacetylchitobiose-binding lectins directly, it evidently affects the complementarity of complex oligosaccharides to the binding sites of these lectins. A structure with 2-acetamido-2-deoxy-~-~-glucosyl residues occurs in an internal position of blood-group substance oligosaccharide chains.z4 Proceeding from Flory’s observations, Matsumoto and Osawa purified two lectins of distinctly different specificities from Ulex europeus extracts196~08~2z6~50s; an L-fucose-binding protein, Ulex I (Ref. 226), and a 2-acetamido-2-deoxy-~-glucose-binding protein, Ulex I1 (Ref. 208). Ulex I1 was obtained in pure form from the 40-70%-saturated ammonium sulfate precipitate by cellulose ion-exchange, poly(viny1 chloride) block electrophoresis, and Biogel P-200 gel-filtration.z08The lectin was homogeneous in the analytical ultracentrifuge, and by poly(acry1amide) disc-gel electrophoresis. An Szo,, value of 6.5 S, but no value for the molecular weight, was reported. Amino acid analysis revealed Ulex I1 to be rich in acidic and hydroxy amino acids, and low in those containing sulfur. Carbohydrate analysis gave 21.7% (by weight) of carbohydrate (mannose, galactose, arabinose, and 2-amino-2deoxyglucose). Ulex lectin I1 has also been purified14sby adsorption to (509) T. Osawa and I. Matsumoto, Methods Enzymol., 28, Part B, 323-327 (1972).

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IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

N,N"N"-triacetylchitotriose-substituted starch, followed by elution with 0.05 M glycine hydrochloride buffer, pH 3.0. Characterization of the carbohydrate-binding specificity of Ulex europeus I1 is still very rudimentary. Studies by inhibition of hemagglutination have been completed, using only a few of the appropriate sugars necessary to define specificity of the binding. Makela found7*that crude extracts from Ulex seeds were inhibited by L-fucose and salicin, whereas Kriipe7' did not include L-fucose among the sugars which inhibited Ulex-0 erythrocyte agglutination (compare Ref. 508d). Oligosaccharides isolated from blood-group substances, and containing (nonreducing) p-D-linked 2-acetamido-2-deoxy-~glucosyl groups inhibited such Cytisus-type anti-H(0) lectins as U. europeus I1 (see Ref. 475). The investigation of Matsumoto and Osawa10s~500 is the most complete to date; it reveals a binding specificity for (1+4)-linked 2-acetamido2-deoxy-~-~-glucosyl derivatives. The binding site appears to be an extended one, capable of accommodating N,N'-diacetylchitobiose (4) or N,N',N"-triacetylchitotriose (6). Salicin, phenyl p-D-glucopyranoside, and cellobiose were poor inhibitors; maltose, lactose,N-acetyllactosamine [@D-Galp-(1-*4)-D-GlcNAc], 2-acetamido-2-deoxy-~-galactose, and 2-acetamido-2-deoxy-~-glucosewere noninhibitors.

Iv. 2-ACETAMIDO-2-DEOXU-D-GALACTOSE-BINDING LECTINS 1. Dolichos biflorus (Horse Gram) (horse gram; a-D-GalNAcp >> a-D-Galp)

The phytohemagglutinin of Dolichos bi$orus is one of three wellcharacterized, blood-group A-specific, plant lectins (see also Phaseolus lunatus and Helix pomatia), and one of two phytohemagglutinins to find application in routine serology (see also Ulex e u r ~ p e u s ) . The '~ agglutination of human A, eyrthrocytes by Dolichos extracts was first reported by Bird.510-512 When the lectin was compared to human anti-A sera with respect to agglutination of erythrocytes from representative blood-group types, complete correspondence was observed.512A, cells were agglutinated by the lectin at titers between 8,192 and 32,768, and A2 cells at titers of 4-16. Furthermore, Dolichos lectin specificity was retained when tested with papain-treated r e d - ~ e l l s . ~ Subsequently, '~ Boyd and Shapleigh2observed that saliva of type A, secretor individu(510) G. W. G. Bird, Curt-. Scf.,20,298-299 (1951). (511) G. W. G. Bird, Indian]. Med. Res., 40,289-293 (1952). (512) G.W. G . Bird, Nature (London), 170,674 (1952). (513) G. W. G. Bird, Nature (London), 174, 1015 (1954).

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als formed a precipitate with Dolichos bijlorus extracts, and also inhibited A, erythrocyte agglutination. Saliva of Az secretors gave very weak reactivity in both assays; B and 0 secretor saliva were inactive. Similar observations were made by From agar-gel diffusion and precipitin studies of ovarian cyst A, and Az substances with Dolichos extract, Bird'O~'~~ concluded that the lectin made no qualitative distinction between the two subgroups of A. Hog gastric-mucin A substance reacted in a manner identical to the cyst substances. In addition to its reactivity with A, erythrocytes, secretor saliva, and ovarian-cyst substances, Dolichos bijorus lectin agglutinated streptococci of the serological group C, but failed to react with a group C variant whose cell-wall polysaccharide, unlike normal group C organisms, lacked terminal 2-acetamido-2-deoxy-cu-~-galactosyl sidechains.s14A precipitin band with peptone A substance was also demonstrated in agar plates.51sStepwise fractionation of Dolichos seedextracts with ammonium sulfate and Sephadex G-200 or CM-cellulose chromatography was conducted by Kiihnemund and coworkers.516 They reported that the streptococcus-agglutinating activity was associated with a glycoprotein of molecular weight -130,000, and they conducted preliminary amino acid and carbohydrate analyses. Etzler and Kabat purified the agglutinating principle from Dolichos seed extracts by adsorption to insoluble polyleucyl hog A H sub~tance.'~~ Specific * ~ ' ~ elution of the adsorbent with 2-acetamido-2deoxy-D-galactose gave a protein that appeared homogeneous by immunodiffusion, immunoelectrophoresis, disc-gel electrophoresis under acid and alkaline conditions, and sedimentation analysis. The amino acid composition of the protein of molecular weight 141,000 reflected a high content of aspartic acid and serine, little methionine, and no cysteine. Carbohydrate analysis showed a sugar content of 2.4% of hexose, 1.6% of hexosamine, and 1.5% of 2-acetamido-2-deoxyhexose. An alternative purification of Dolichos bijorus lectin was published by Font and coworkers.S18Fractional precipitation with ammonium

+

(514)W.Kohler and 0. Prokop, Z. Immunitaetsforsch. Allerg. Klin. Immunol., 133, 171-179 (1967). (515) G . Uhlenbruck, I. Sprenger, A. J. Leseney, J. Fontand, and R. Bourrillon, Vox Sung., 19,488-495 (1975). (516)V. 0. Kuhnemund, W. Kohler, and 0. Prokop, Hoppe-Seyler's Z. Physiol. Chem., 349,1434-1436 (1968). (517)M.E. Etzler, Methods Enzymol., 28 (Part B), 340-344 (1972). (518) J. Font, A. M. Leseney, and R. Bourrillon, Biochim. Biophys. Acta, 243,434-446 (1971).

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IRWIN J. GOLDSTEIN AND COLEEN E. HAYES

sulfate was followed by gel filtration and ion-exchange chromatography, yielding a homogeneous preparation [by starch-gel and poly(acry1amide)-gel electrophoresis, as well as by ultracentrifugal analysis]. They reportedSl8a sedimentation coefficient of 6.3 S, an isoelectric point of pH 5.5, and a carbohydrate content of 3.75% by weight. The amino acid analysis was in good agreement with that of Etzler and Kabat.lo8The glycosyl portion of the lectin was composed largely of mannose residues, with small proportions of glucose, fucose, xylose, and arabinose residues. Periodic acid oxidation resulted in rapid loss of agglutinating activity, accompanied by only minor decomposition of neutral sugars, and no loss of hexosamine. Subsequent studies by PBre and coworkerss19 demonstrated a molecular weight of 30,000 for a subunit. Gel filtration in 6 M guanidinium chloride, dodecyl sodium sulfate electrophoresis, and peptide mapping of the tryptic digest confirmedSl9that four noncovalently associated subunits comprise the native lectin of molecular weight 120,000. Only N-terminal alanine was found, suggesting that the four subunits are identical. Carter and E t ~ l e r ~confirmed ~O the presence of one methionyl residue per subunit by cyanogen bromide cleavage ofDolichos lectin into two fragments. The peptide fragments, separated by anion exchange and gel filtration, were distinguished, in that the amino terminal segment, of molecular weight 15,000, contained carbohydrate, whereas the carboxyl terminal segment, of molecular weight 12,000, did not. Acid hydrolysis, and analysis of the pronase-digested glycopeptide, gaveszoa mixture of serine, aspartic acid, mannose, and 2-acetamido-2-deoxy-Dglucose in the ratios of 1:5:20-25:5-10. 2-Acetamido-N-(~-aspart-4oyl)-2-deoxy-~-D-glucopyranosy~amine was also identified in a partial, acid hydrolyzate.”O What had, in earlier ~ t u d i e sappeared , ~ ~ ~to ~be~a homogeneous, ~ ~ ~ ~ ~ ~ protein preparation was further fractionated into electrophoretically distinguishable isolectins A and B by chromatography on concanavalin A-Sepharo~e.~ The ~ ~B* form, ~ ~ ~ which comprised less than 12% of the original lectin sample, was not bound, whereas the A form was bound and was specifically elutedsZ1as a biphasic peak with a gradient of methyl a-D-glucopyranoside solution. Analysis, for carbohydrate content, of fractions obtained from different portions of the elution profile revealed considerable heterogeneity in the relative proportions of D-mannose and 2-acetamido-2-deoxy-~-glucose per molecule of pro(519)M.PBre, J. Font, and R. Bourrillon, Biochim. Biophys. Acta, 365,40-46 (1974). (520)W. G.Carter and M. E. Etzler, Biochemistry, 14,5118-5122(1975). (521)W. G. Carter and M. E. Etzler,J. Biol. Chem., 250,2756-2762 (1975).

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tein. Forms A and B, with respective apparent molecular weights of 113,000 and 109,000, were present as native proteins in the seed, instead of arising from modification or degradation during the course of the purification.521The two species showed considerable similarity in amino acid composition, were indistinguishable in immunodiffusion against antisera to the seed extract, and exhibited identical amino(alanine) and carboxyl-terminal (leucine and valine) amino acids. Studies on the carbohydrate specificity of forms A and B showed essentially no difference. Dodecyl sodium sulfate-urea gel-electrophoresis ofthe purified isolectins revealed four distinguishable types of subunit, IA and IIA in form A, IB and IIB in form B, whereas dodecyl sodium sulfate gel-electrophoresis revealed only two types of subunit, a band of material of molecular weight 26,500 from form A, and a band of molecular weight 26,000 from the B form.521A so-called “prolectin” has been isolated from the leaves and stems of 6-weeks-old Dolichos bijlorus plants243a(see Section 1,5). Subunits IA and IIA were isolated by ion-exchange chromatography on DEAE-cellulose in 8 M urea.s22Sedimentation-equilibrium analysis in 8 M urea gave subunit molecular weights of 27,700 and 27,300 for IA and IIA, respectively. The isolated subunits did not differ substantially in amino acid composition, or antigenicity. Although they both contained N-terminal alanine, digestion of IA with carboxypeptidase A released leucine and valine simultaneously, whereas IIA was not hydrolyzed by this enzyme under identical conditions. On the basis of their results, Carter and EtzleP2 proposed a stoichiometry of IA211A2 for the principal form of Dolichos bijlorus lectin. The first 30 aminoterminal amino acids of the IA and IIA subunits of the D . bijlorus lectin were shown to be supporting the suggestion that the two subunits may differ from one another only at their carboxyl-terminal ends .520,522 The carbohydrate-binding specificity of purified Dolichos bijlorus lectin has been studied in detail by Etzler and Kabat.’08 In precipitin studies conducted on the Dolichos lectin, 87-99% was precipitated by ovarian cyst A substance, whereas only 60-70% was precipitated by hog A substance; the latter result was attributed to the formation of soluble, lectin-oligosaccharide complexes. A, substances were more reactive than A2 substances; however, no reaction was observed with B or 0 substances, even following mild hydrolysis with acid or two stepwise, Smith degradations. N-Deacetylation of A substance with (522) W. G. Carter and M. E. Etzler, Biochemistry, 14,2685-2689 (1975). (522a) M. E. Etzler, C. F. Talbot, and P. R. Ziaya, FEBS Lett., 8 2 , 3 9 4 1 (1977).

230

IRWIN J. GOLDSTEIN AND COLEEN E. HAYES

Clostridium tertium N-deacetylating enzyme abolished Dolichos lectin reactivity; restoration was achieved by re-N-acetylation. Sugar inhibition of human A substance-lectin precipitation suggested that the lectin combining-site was more complementary to 2-acetamido-2-deoxy-a-~-galactopyranosyl end-groups. The A-active disaccharide, a-~-GalNAcp-(1+3)-~-Gal,and trisaccharide, a - D GalNAcp-(1+3)-@-D-Galp-(1+3)-D-GlcNAc7 were equivalent in inhibitory capacity to methyl 2-acetamido-2-deoxy-a-~-galactopyranoside (0.55 pmole for 50% inhibition). The methyl a-glycoside was 2.5 times as effective as 2-acetamido-2-deoxy-~-galactose,whereas the ethyl @-glycosidewas less effective than the latter. The following compounds were noninhibitory: 2-acetamido-2-deoxy-~-glucose, 2-acetamido-2-deoxy-~mannose,2-amino-2-deoxy-~galactose,2-amino-2deoxy-D-glucose, 2-acetamido-2-deoxy-~-galactitol,Dgalactose, L-fucose, D-mannose, and D-glucose. Two disaccharides in which 0-3 of 2-acetamido-2-deoxy-~-galactose was substituted by a @-Dgalactosyl or a 2-acetamido-2-deoxy-a-~-glucosylgroup were also inactive. The best inhibitor tested, needing only 0.32 pmole for 50% inhibition, was the A-active, reduced pentasaccharide shown in formula 7. The authors U-D

-

-GalNAc p- (l+ 3)- 0-0 -Calp- (1- 4)- 8-D-GlcNAcp ( l - d ) - R 2

t1

U-L

-Fuc~ 7

suggested that L-fucosyl substitution either confers a more favorable conformation upon the nonreducing 2-acetamido-2-deoxy-a-~galactosyl group, as compared to the di- and tri-saccharides mentioned previously, or itself contributes to stabilization of lectin-oligosaccharide binding, implying an extended carbohydrate-binding site on the Dolichos lectin molecule. PBre and coworkers investigated the circular dichroic spectrum of Dolichos lectin in the presence and absence of 2-acetamido-2-deoxyD-gala~tose.~'~ The far-ultraviolet, c.d. spectrum of Dolichos lectin displayed weak, negative bands at 217 and 230 nm, with a positive band at 197 nm. By analogy to the spectrum of con A, whose complete, threedimensional structure is known, the authors concluded that the Dolichos lectin has a preponderance of the aperiodic, bent structure stabilized by hydrophobic interactions, and a significant content of (523) M.PBre, R. Bourrillon, and B. Jirgensons, Biochim. Biophys. Acta, 393, 31-36 (1975).

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231

p-pleated-sheet conformation. The addition of 2-acetamido-2-deoxyDgalactose caused a significant diminution in the amplitude of the c.d. spectrum at 280-286 nm and at 290 nm, at pH 6.8-7.5. No alteration was observed at pH 8.25. As the 280-300-nm spectral zone is related to the tryptophanyl and tyrosyl chromophores, Pkre and coworkers suggested523that these residues may be perturbed upon sugar binding. Addition of dodecyl sodium sulfate to the lectin disrupted its tertiary structure, and induced some a-helix formation. Purified Dolichos bijlorus lectin has been applied to the study of glycoproteins and glycolipids from mammalian cell^.^^^-^^^ Fluorescein isothiocyanate-conjugated lectin was employed in a study of the rat stomach and d ~ o d e n u m . " From ~ both organs could be isolated A-active material by adsorption to Dolichos lectin-agarose columns, and elution with 2-acetamido-2-deoxy-~-ga~actose.Fluoresceinlabelled lectin was also used to examine sections from various regions of rat s m a l l - i n t e ~ t i n eDifferential .~~~ fluorescent staining of epithelial cells lining the crypts and villi of the intestine was observed from the proximal to the distal end, suggesting differential localization of cellsurface and secretory components in these regions. Furthermore, a Dolichos lectin-Sepharose column, in tandem with a Lotus tetrugonolobus lectin-Sepharose column, proved effective in separating hog gastric-mucin into A-substance and H-substance devoid of crossc o n t a r n i n a t i ~ nFinally, . ~ ~ ~ the interaction between Dolichos lectin and chick embryonic fibroblasts as a function of development was studied by Roguet and B o u r r i l l ~ n The . ~ ~ ~number of lectin-binding sites per cell remained constant, but the apparent association-constant decreased from day 8 to day 16. The effect of lectin on [3H]thymidine incorporation was age-dependent in the chick fibroblast. 2. Glycine max (Soybean) (soybean; a-D-GalNAcp 5 0-D-GalNAcp >> a-D-Gab)

The nutritional superiority of heated soybean meal in contrast to raw was initially thought to reflect the presence of a growthrepressive, heat-labile s u b ~ t a n c e . ~ Thus, ~ ~ -Liener ~ ~ ~ and P a l l a n s ~ h ~ ~ ~ (524) M.E. Etzler, Ann. N.Y. Acad. Sci., 234,260-275 (1974). (525)M.E. Etzler and M. L. Branstrator,J.Cell B i d , 62,329-343 (1974). (526)M.E. A. Pereira and E. A. Kabat,J. E x p . Med., 143,422-436 (1976). (527)R. Roguet and R. Bourrillon, Biochim. Biophys. Acta, 389,380-388(1975). (528)T. B. Osborne and L. B. Mende1,J. Biol. Chem., 32,369-387 (1917). (529) I. E. Liener and M. J. Pallansch,J. Biol. Chem., 197,29-36 (1952). (530)I. E. Liener,J. Nutr., 49,527-539 (1953). (531) I. E. Liener and T. A. Seto, Cancer Res., 15,407-409 (1955).

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and LienePO fractionated a toxin, from defatted soybean flour, that exhibited some growth-repressive activity in feeding studies, strong hemagglutinating activity, and lability to heat and to pepsin digestion.532However, they observed no specific effect of the substance on tumor growth, or on the urease, lipoxidase, or anti-tryptic activities known to be present in soybean extracts. Although Liener and Pall a n s ~ found h ~ ~ no ~ statistically significant correlation between toxicity and hemagglutinating activity, they implied that the two activities were due to a single entity. These early studies were conducted on a preparation partially purified by fractional precipitation with ammonium sulfate and Several purification schemes have since been reported to yield homogeneous preparations. Wada and coworkers534used the technique of recycling, moving-boundary electrophoresis to obtain a hemagglutinating protein that manifested homogeneity in ultracentrifugation, starch-gel electrophoresis, and anion-exchange chromatography. Stead and applied an ammonium sulfate- and acetoneprecipitated fraction to a DEAE-cellulose column, and obtained six peaks with a stepwise gradient of sodium chloride. They reported no correlation between the toxicity, hemagglutinating activity, and trypsin inhibition of the six peaks, confirming the earlier work of Rackis and An alternative purification, in which ammonium sulfate fractionation was followed by chromatography on calcium phosphate, was reported by Lis537and Lis and to give a protein identical to a sample obtained from Liener.530Hemagglutinating activity was confined to a single peak of the calcium phosphate, gel-elution profile. Rechromatography of this fraction on calcium phosphate, CMcellulose, or Sephadex G-50 yielded a single, protein peak. Further analysis by chromatography on Sephadex G-50 in 4 M guanidinium chloride, ultracentrifugation, and gel electrophoresis at both acidic and alkaline pH did not reveal significant contamination. However, when the purified agglutinin was chromatographed on DEAE-cellulose, four distinct peaks of hemagglutinating activity were f o ~ n d .Each ~ ~ * ~ ~ ~ (532)I. E. Liener,J. Bid. Chem., 233,401-405 (1958). (533)M.J. Pallansch and I. E. Liener, Arch. Biochem. Biophys., 45,366374(1953). (534) S. Wada, M.J. Pallansch, and I. E. Liener,J. B i d . Chem., 233,395-400 (1958). (535)R. H.Stead, H. J. H. de Muelenaere, and G. V. Quicke, Arch. Biochem. Biophys., 113,703-708 (1966). (536)J. J. Rackis, H. A. Sasame, R. L. Anderson, and A. K. Smith,]. Am. Chem. Soc., 81, 6265-6270 (1959). (537)H. Lis, N. Sharon,and E. Katchalski,Biochim. Biophys. Acta, 83,376-378(1964). (538)H. Lis, N. Sharon, and E. Katchalski,]. Biol. Chem., 241,684-689 (1966). (539)H. Lis, C. Fridman, N. Sharon, and E. Katchalski,Arch. Biochem. Biophys., 117, 301-309 (1966).

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pooled peak was rechromatographed on DEAE-cellulose, followed by calcium phosphate, and, finally, again on DEAE-cellulose. The four hemagglutinins were not separable on calcium phosphate or CMcellulose at pH 4.0; they migrated identically in gel electrophoresis at pH 4.5, and had almost identical amino acid compositions, but varied slightly in their content of mannose and 2-amino-2-deoxyglucose. In verification of these results, Catsimpoolas and Me~e15~O demonstrated the presence of four, immunochemically indistinguishable, hemagglutinating proteins by isoelectric focusing of soybean extract. Thus, it appears that soybean agglutinin exists as multiple, highly similar forms. Two affinity systems for the purification of soybean agglutinin have been developed. Gordon and coworker^^^^,^^^ coupled N-(6-aminohexanoy1)-P-D-galactopyranosylamineto cyanogen bromide-activated Sepharose, to afford a specific adsorbent for the agglutinin. Elution with D-galactose gave, in 90% yield, a major hemagglutinin from which minor agglutinins could be removed by DEAE-cellulose, according to Lis and Sharon.g7Soybean agglutinin so prepared was homogeneous by gel electrophoresis, and identical, with respect to electrophoretic mobility, and amino acid and carbohydrate analyses,538to agglutinin prepared by previous methods. A second, simpler, affinity adsorbent was prepared by Allen and N e ~ b e r g e Pby ~ ~reaction of 2-amino-2deoxy-D-galactose with CH-Sepharose 4B in the presence of a carbodiimide. The adsorbent bound 12 mg of agglutinin per ml, which was 1% of the total protein applied. Elution with Dgalactose solution gave one major and several minor agglutinins which were separated by anion-exchange chromatography. The electrophoretic mobility and chemical composition were in accord with those for soybean agglutinin prepared by other method^.^^,^^^ The biophysical characteristics of soybean agglutinin were first investigated by Pallansch and Liener533with a protein preparation known to contain minor contaminants. They determined a sedimentation coefficient of 6.4 S , a diffusion coefficient of 5.72 X cm2 sec-', an extinction coefficient E,l& = 15.7, a molecular weight (by sedimentation) of 105,000, and an isoelectric point at pH 6.1. Catsimpoolas and MeyeP40 confirmed that the isoelectric point is pH 6.0, and reported that dissociation of the lectin in phenol-acetic acid in the presence of (540) N. Catsimpoolas and E. W. Meyer, Arch. Biochem. Biophys., 132,279-288 (1969). (541) J. A. Gordon, S. Blumberg, H. Lis, and N. Sharon, FEBS Lett., 24,193-196 (1972). (542) J. A. Gordon, S. Blumberg, H. Lis, and N. Sharon, Methods Enzymol., 28, Part B, 365-368 (1972). (543) A. K. Allen and A. Neuberger, FEBS Lett., 50,362-364 (1975).

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2-mercaptoethanol and urea gave two subunits. Lis and coworkers538 analyzed a highly purified, lectin sample, and estimated a sedimentation coefficient of 6.0 S, a diffusion coefficient of 5.0 x lo-' cm2.sec-1, and calculated the molecular weight of the protein to be 110,000. Without question, the most complete, biophysical characterization of soybean agglutinin is that of Lotan and The lectin, affinity-purified according to Gordon and was homogeneous by poly(acry1amide) gel-electrophoresis at pH 4.5, 8.9, and 7.2 in the presence of a detergent. Furthermore, electrophoresis in an acrylamide gradient of 4 to 8% with 8 M urea yielded a single, protein band, as did isoelectric focusing. Sedimentation-velocity studies in buffers of pH 2.2 to pH 10.8 at protein concentrations of 3 to 10 mg per ml resulted in a single, symmetrical peak of s&,w = 6.0L0.125 S. The molecular weight calculated was 122,000 k1,300, using an experimentally determined, partial specific volume of 0.745mug. This value was in good agreement with the molecular weight of 120,000 L 10,000 determined by gel filtration. The protein formed aggregates having high molecular weight if stored for a long time in the lyophilized state."5 A subunit molecular weight of 30,000 was obtained by poly(acrylamide) gel-electrophoresis in dodecyl sodium sulfate, by gel filtration in detergent, and, finally, by sedimentation equilibrium in 6 M guanidinium the authors reported an extinction coefficient of = 12.8 cm-'. Although, on the basis of end-group analysis (1mole of N-terminal alanine per 30,000 g of protein in 8 M urea) and electrophoretic s t ~ d i e s , 5the ~ ~soybean agglutinin appeared to be a tetrameric protein composed of identical subunits, Lotan and coworkers546later reported resolution of two types of subunit in the ratio of 1:1,either by electrophoresis at alkaline pH in the presence of urea or detergents, or by chromatography on DEAE-cellulose in Tris buffer, pH 7.3, with 8 M urea. Several groups have investigated the chemical composition of soybean agglutinin. The early amino acid analysis of Wada and coworke r ' differs ~ ~ ~considerably from the later analyses by Lis and cow o r k e r ~ .They ~ ~ ,found ~ ~ almost twice the content of serine, leucine, and lysine, and substantially increased proline than those reported by Wada and whereas the contents of methionine, (544)R. Lotan, H.W. Siegelman, H. Lis, and N. Sharon,]. Biol. Chem., 249,1219-1224 (1974). (545)R. Lotan, H.Lis, and N. Sharon, Biochem. Biophys. Res. Commun., 62,144-150 (1975). (546) R.Lotan, R. Cacan, M. Cacan, H. Debray, W. G . Carter, and N. Sharon,FEES Lett., 57, 100-103 (1975).

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isoleucine, and arginine were comparatively diminished. The results of Lis and c o ~ o r k e r sconcur ~ ~ ~ with , ~ ~ each ~ other. Wada and coworke r calculated ~ ~ six ~ residues ~ of cysteine per molecule of lectin on the basis of elemental analysis, although no free sulfhydryl groups were found. Both of the later amino acid analyses suggested that soybean agglutinin is devoid of c y ~ t e i n e . " ~Like , ~ ~ several ~ other lectins, soybean lectin is comparatively rich in acidic and hydroxylic amino acids. Of the nonidentical subunits separated by Lotan and coworkers,s46 subunit I1 contains two more aspartic acid residues, one additional glutamic acid residue, and one lysyl residue fewer, as compared to subunit I. End-group analysis revealed only N-terminal alanine.534*544*546 The amino-terminal residues of the soybean agglutinin have been s e q ~ e n c e d . ~ ~ Eleven ~~,"~ of the first amino-terminal residues are identical with that of the peanut lectin and of the /+chain of the lentil l e ~ t i nAmong . ~ ~ ~the 14 nonidentical residues, 9 could have resulted from a single nucleotide substitution for the lentil lectin, and 8 for the peanut lectin.442c These data indicate a rather high level of sequence homology among the three lectins, and suggest a common ancestry for the genes coding for these three plant-lectins. Chemical modification of soybean agglutinin by acetylation of its amino groups resulted in little loss of agglutinating activity, whereas the protein was quite sensitive to modification of its tyrosyl Failure of the protein to react with 2-iodoacetamide or p-(chloromercuri)benzoate in 6 M urea confirmed that it was devoid of sulfhydryl groups. A metalloprotein containing151Ca2+and Mn2+,the soybean lectin is inactivated by A13+, Fe3+,and Pb2+,whereas MnZ+, Ba2+,Mf+, Ag+, Li+, and K+ are without The soybean lectin is a glycoprotein containing -7% (by weight) of carbohydrate comprised of mannose and 2-amino-2deoxyglucose.534=538~539~s44~546 Each subunit of the agglutinin carries an oligosaccharide chain composed of nine mannosyl and two 2-amino-2deoxyglucosyl residues.s48A glycopeptide of molecular weight 4,600 was isolated from pronase-digested, native agglutinin. Digestion with glycosidase revealed that mannose occurs in three, distinct regions of the oligosaccharide, separated by 2-acetamido-2deoxy-D-glucosyl residues, Exhaustive digestion with a-Dmannosidase and 2-acetamido-2-deoxy-~-~-glucosidase allowed Lis and coworkerssmto isolate the carbohydrate-protein linkage region of the glycopeptide ,and to characterize it as 2-acetamido-(~-aspart-4-oyl)(547) I. E. Liener and S. Wada,J. B i d . Chem., 222,695-704 (1956). (548) N. Sharon, H. Lis, and R. Lotan, CoZZoq. Int. C. N . R. S., 221,693-710 (1974). (1968). (549)H.Lis, Isr. J . Chem., 6, 114~ (550)H.Lis, N.Sharon,andE. Katchalski,Biochim.Biophys. Acta, 192,364-366(1969).

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IRWIN j. GOLDSTEIN AND COLLEEN E. HAYES

2-deoxy-P-D-glucosylamine. Oxidation of five of the nine mannosyl units per subunit of soybean agglutinin with periodic acid caused no diminution of hemagglutinating activity.ss1Reduction of the oxidized lectin with sodium borotritide afforded a radioactive product indistinguishable from the native protein by several criteria.551 The carbohydrate-binding specificity of soybean agglutinin appears to be directed towards both anomers of 2-acetamido-2-deoxy-~galactose.z1z~ss52 By inhibition of hemagglutination, Lis and coworkers21z found that four disaccharides, in which 2-acetamido-2-deoxy-~-( P-D-(1+3), and P-D-(1+4), galactose was linked P-D-(1+6), a - ~ 1+3), respectively, to D-galactose, were approximately equivalent to 2-acetamido-2-deoxy-~-galactose inhibition. In a comprehensive, immunochemical study, Pereira and coworkersszzextended these observations by measuring mono- and oligo-saccharide inhibition of soybean agglutinin-human blood-group precursor substance precipitation (see Table X).Several conclusions emerge from their results. Firstly, the lectin exhibited greatest affinity for 2-acetamido-2-deoxy-~-galactose, its glycosides, and oligosaccharides in which this was the nonreducing, terminal, sugar group; the reaction was inhibited to a lesser extent by D-galaCtOSe and its derivatives. Secondly, a slight preference for a-over P-glycosidically linked sugars was evident, as was a preference for aromatic over alkyl aglycons. Thirdly, substitution of blood-group A-active oligosaccharides by L-fucosyl residues greatly diminished their binding capacity, although L-fucose was linked to the penultimate D-galactosyl residue, leaving nonreducing, terminal 2-acetamido-2deoxy-D-galactosyl groups unsubstituted. An analogous "blocking effect" occurred upon L-fucosyl substitution of lactose derivatives and blood-group B-active oligosaccharides. Fourthly, on comparing P-Dgalactosyl disaccharides, it was evident that 6-0-linked sugars were more reactive than their 4-0- or 3-0-substituted counterparts. Finally D-glucose, D-mannose, L-fucose, L-rhamnose, 2-acetamido-2-deoxywere ineffective. In D-glucose, and 2-acetamido-2-deoxy-~-mannose summary, soybean agglutinin is most complementary to 2-acetamido2-deoxy-a-~-galactopyranosylend-groups (8).

NHAC

8

(551)€7. Lotan, H.Debray, M. Cacan, R. Cacan, and N. Sharon,J. B i d . Chem., 250, 1955-1957 (1975).

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TABLEX Inhibition of Soybean Agglutinin-Human Blood-group Precursor Substance Precipitation by Mono- and Oligo-saccharidesSs2 sugar

Concentration giving 50% inhibition ( p M )

Phenyl 2-acetamido-2-deoxy-a-~-galactopyranoside 2-Acetamid0-3-0-(2-acetamido-2-deoxy-a-~-galactopyranosyl)2-deoxy-6-O-~~ga~actopyranosy~-D-g~ucose (A-active trisaccharide) 3-0-(2-Acetamido-2-deoxy-a-~-galactopyranosy~)-~-ga~actose (A-active disaccharide) Methyl 2-acetamido-2-deoxy-cY-Dgalactopyranoside Ethyl 2-acetamido-2-deoxy-~-~-galectopyranoside 2-Acetamido-2-deoxy-~-galactose p-Nitrophenyl a-D-galactopy-ranoside Mono-L-fucosyl A-active pentasaccharide 6~-P-D-Gdactopyranosyl-D-glucose 2-Acetamido-2-deoxy-6-O-~-~galactopyranosyl-~-glucose Methyl a-Dgalactopyranoside p-Nitrophenyl P-D-galactopyranoside Mnose Stachyose 3-O-a-D-Gdactopyranosy~-Dga~actose Methyl P-Dgalactopyranoside 2-Acetamido-2-deoxy-4-O-~-~g~actopyranosyl-~-glucose 2-Acetamido-2-deoxy-3-O-~-~galactopyranosy~-~-glucose Lacto-N-tetraose Di-L-fucosyl A-active pentasaccharide Lactose D-Galactose 2'-O-~-Fucosyllactose(2-O-~-fucopyranosyl-4-O-~-t1-gdactopyranosyl-D-glucose)

0.014 0.019

0.025 0.025 0.037 0.10 0.48 0.60 0.70 0.70 0.71 0.71 0.71 0.71 0.71 1.40 1.40 1.40 1.40 1.40 1.60 2.40 2.40

Noninhibiting sugars DGlucose 2-Acetamido-2-deoxy-~-glucose (and its methyl a-and P-glycosides) D-Mannose %Acetamido-2-deoxy-~-mannose Methyl a-L-fucopyranoside L-Rhamnose &Acetamido-2-deoxy-3-O-~-~-glucopyranosyl-~g~lactose 3-0-(2-Acetamido-2-deoxy-~-~glucopyranosyl)-~-gdactose 4-O-~-~Glucopyranosyl-~galactose

Lactodifucotetraose Difucosyl B-active oligosaccharides ~~

(552) M. E. A. Pereira, E. A. Kabat, and N. Sharon, Carbohydr.Res., 37,89-102 (1974).

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IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

Equilibrium-dialysis experiments revealed two 2-acetamido-2deoxy-D-galactose binding-sites per molecule of soybean agglutinin.544 This value was confirmed by gel filtration of the lectin on a column equilibrated with a radioactive ligand. The noninteracting, identical sites exhibited an association constant of K, = 3.0 x lo41iter.mole-' for this 2-acetamido-2-deoxyhexose. Soybean agglutinin precipitated several, purified, blood-group subs t a n c e ~ Maximal .~~~ precipitation was achieved with type A, substances; on a weight basis, Lea substances were -60-70% as active as A, substances, whereas Az substances were considerably less active. I-active blood-group precursor substances gave good precipitin reactions. However, B-active substances reacted poorly, despite their conattributed this tent of terminal a-D-galactosyl groups. The finding to the L-fucosyl blocking-effect already noted. H-substances were unreactive. Two streptococcal polysaccharides differed in their reaction with the soybean agglutinin. A group C polysaccharide (nongroups) formed a reducing, terminal 2-acetamido-2-deoxy-~-galactosyl precipitate, whereas a group A polysaccharide (terminal, nonreducing 2-acetamido-2-deoxy-~-glucosyl groups) did not. In another study, Irimura and observed inhibitory activity of neuraminidase-treated, porcine thyroglobulin, porcine submaxillary mucin, and bovine submaxillary mucin in a hemagglutination assay. Based on the structures proposed for these glycopeptides, their results The reactivity are in agreement with those of Pereira and of porcine-thyroglobulin glycopeptide Byfrom which sialic acid and galactose have been enzymically removed, thereby exposing nongroups, remains reducing (terminal)2-acetamido-2-deoxy-~-~-glucosyl ~nexp1ained.l~~ Soybean agglutinin exhibits several striking, biological activities. The lectin agglutinates both rabbit and human erythrocytes (type A > type 0 > type B),212rabbit red-cells binding five to six times as much iodinated agglutinin as do human cells.QQTrypsinized erythrocytes exhibited dramatically increased agglutinability, and the amount of lectin bound increased only slightly.QQ Binding is reversible by addition of 2-acetamido-2-deoxy-~-galactose.The lectin also agglutinates neuraminidase-treated, murine splenocytes, causing them to undergo blast transformation and to exhibit an accelerated rate of DNA synthesis.553Stimulation is inhibitable by 2-acetamido-2-deoxy-D-galactose. Chemical cross-linking of native, soybean agglutinin into dimers and higher oligomers greatly enhanced its hemagglutinating and (553) A. Novogrodsky and E. Katchalski, Proc. Natl. Acad. Sci. U.S.A., 70,2515-2518 (1973).

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lymphocyte-transforming activities.343Lyophilization of soybean agglutinin gives rise to polymeric forms of the lectin that are two orders of magnitude more mitogenic towards pig lymph-node cells than dimeric le~tin.~~~a In further biological studies, Sela and coworkersss4found that, although normal hamster, human, mouse, and rat cell-lines were not agglutinated by soybean lectin, transformed counterparts of the cell lines from mouse, human, and rat were agglutinable. Furthermore, mild, proteolytic digestion rendered normal cells agglutinable. The change in agglutinability could not be explained in terms of altered receptor-density.sssAgglutination was again reversed by the competing sugar. The major lectin from a soybean cultivar (Glycine max cv D68-127) was purified by chromatography on hydroxylapatite and DEAE-cellulose. A tetrameric glycoprotein (molecular weight 92,000), the lectin is composed of four subunits, molecular weight 23,000, and is specific for 2-acetamido-2-deoxy-~-galactose.~~~~

3. Helix pomutiu (Edible Snail) (edible snail; a-D-GalNAcp > a-~-GlcNAcp>> a-~-G:alp) The edible snail, Helix pomatia, contains a lectin that specifically agglutinates human type A, but not types B or 0, erythrocytes.s8~60~61~63~100~ss6~s63 The albumin gland, a part of the sexual apparatus, contains rather large amounts of the agglutinin (8% of the soluble protein).562Several other species of snails (for example, Helix and Euphadra periomphala56s)also conhortensisYs8 Otala lactea,6z*ss4 tain specific agglutinins. (See Refs. 76 and 566 for a discussion of snail lectins.) (554)B.-A. Sela, H. Lis, N. Sharon, and L. Sachs,]. Membr. Biol., 3,267-279 (1970). (555)B.-A. Sela, H.Lis, N. Sharon, and L. Sachs, Biochim. Biophys. Acta, 249,564-568 (1971). (555a) D. W. Fountain and W.-K. Yang, Biochim. Biophys. Acta, 492, 176-185 (1977). (556) Z.Kim, G.Uhlenbruck, 0. Prokop, and D. Schlesinger, Z. Immunitaetsforsch. Allerg. Klin. Immunol., 130,290-295(1966). (557)I. Ishiyama and T. Yomaguichi,Jpn. J . Leg. Med., 20,285-288 (1966). (558)0. Kiihnemund and W. Kohler, Experientia, 25,1137-1138 (1969). (559)T. Takatsu, M.Mukaida, and I. Ishiyama,Jpn. J. E x p . Med., 41,411-421 (1971). (560)I. Ishiyama, M. Mukaida, and A. Takatsu, Ann. N.Y. Acad. Sci., 234,7594(1974). (561) S. Hammarstrom, Ann. N.Y. Acad. Sci., 234, 183-197 (1974). (562)S. Hammarstrom, Methods Enzymol., 28, Part B, 368-383 (1972). (563)W. Knobloch, I. Knobloch, W.-E. Vogt, S. Schnitzler, and M. Bottger, Z. Immunitaetsforsch. Allerg. Klin. Immunol., 139, 119-128 (1970). (564)H. M.Bhatia, W. C. Boyd, and R. Brown, Transfusion (Philadelphia), 7, 53-59 (1967). (565)I. Ishiyama and A. Takatsu, Vox Sang., 19,522-526 (1970).

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IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

Purification of the snail hemagglutinin from Helix pomatia has been accomplished by adsorption to insolubilized human, or hog, bloodgroup A s u b s t a n ~ [also, e ~ to ~ poly(acry1amide) ~ ~ ~ ~ ~ ~ ~ gel polymerized in the presence of blood-group substance from human A m e c ~ n i u m ~ ~ ~ ] followed by elution with 2-acetamido-2-deoxy-~-galactose (5-15 mM). Purification by DEAE- and CM-cellulose chromatography has also been Despite its specificity for a-D-linked 2-acetamido-2deoxy-D-galactose, the snail agglutinin can also be purified by adsorption to Sephadex G-100 and G-200, followed by elution with 2-acetamido-2-deoxy-~-galactose, D-galactose, or D-glucose, or at an acid pH.558,559,567,568 Although the agglutinin has a very low affinity for D-glucose, the large number of (nonreducing) a-D-glucosyl end-groups in Sephadex must provide for the hexavalent protein enough binding loci to cause it to be adsorbed to the matrix. The purified hemagglutinin was homogeneous by gel filtration and A immuno-electrophoresis, and in the analytical ultracentrifuge. molecular weight of 79,000 was determined by the sedimentation equilibrium method561*56g (100,000by sedimentation and velocity meas u r e m e n t ~and , ~ ~ 53,000 by gel filtration563). Analysis showed that the snail agglutinin contained a preponderance of acidic and hydroxylic amino acids and a large proportion of proline r e s i d ~ e s . ~ Uncharacteristic ~ * ~ ~ ~ - ~ ~ ~of lectins from leguminous-plant seeds, the hemagglutinin contained 18 half-cystine residues and 10 molecular proportions of methionine per molecule of protein. About 8%(by weight) ofcovalently bound carbohydrate was found; this was principally D-galactose and D-mann~se.'~ Free sulfhydryl groups were shown by Hammarstrom and coworkers to be absent.56gReduction of the protein in 6 M guanidinium chloride with an excess of l,Pdithiothreitol, followed by alkylation with iodoacetate, led to the introduction of 18 moles of acetate per mole of agglutinin. The mean molecular weight of the reduced, alkylated subunit was determined to be 13,000 by gel filtration on a calibrated column.569Digestion with trypsin, followed by peptide mapping, was When the consistent with the presence of a single type of snail hemagglutinin was treated with 6 M guanidinium chloride, either alone, or at pH 4.0 (for 0.25-48 h), it gave, upon gel filtration on a calibrated column, a single species, of molecular weight 26,000(566) R. T.Pemberton, Ann. N.Y.Acad. Sci., 234,95-121 (1974). (567)I. Ishiyama and G . Uhlenbruck, Comp. Biochem. Physiol. A, 42,269-276 (1972). (568)I. Ishiyama and G . Uhlenbruck, 2. Immunitaetsforsch. Allerg. Klin. Immunol., 143, 147-155 (1972). (569) S. HammarsWm, A.Westoo, and I. Bjork, Scand.J. Immunol., 1,295-309(1972).

LECTINS : CARBOHYDRATE-BINDING PROTEINS

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31,000. Equilibrium dialysis against a blood-group A-active, reduced pentasaccharide revealed the presence of one carbohydrate bindingsite per molecular weight of 17,000, with K&= 5 x lo31iter.mole-' (at 25"), and570AGO - 21.1 kJ.mole-' (-5.04 kcal.mole-'). Scatchard plots were linear, indicating homogenous, noninteracting sites.s70 On the basis of these data, the following model of the subunit structure is proposed: the snail hemagglutinin consists of 6 identical, polypeptide chains (subunits), each containing one intrachain disulfide bond and a carbohydrate binding-site. Furthermore, two subunits are linked by an intrachain, disulfide bond to form subunit dimers of molecular weight 26,000, and three dimers (mol. wt. 26,000) are held together by noncovalent interactions.569 The utility of the Helix pomatia lectin as a probe for the detection of terminal (nonreducing) 2-acetamido-2-deoxy-a-~-galactosyl groups in biopolymers and cell surfaces (blood cells, tumor cells, and microorganisms) has been noted.60,61.100,s62,567 Studies of precipitation between the Helix pomatia agglutinin and a wide range of polysaccharides and glycoproteins have been conducted by many investigators, including Uhlenbruck and Prokop,61 Prokop and coworkers,100and Hammarstrom and his colleagues.63J78*s61~s62~570 Human blood-group A substance (and, to a lesser extent, B, H, and Lea blood-group substances), desialized ovine submaxillary mucin, group C streptococcal polysaccharide (group C and H streptococci are specifically a g g l ~ t i n a t e d ~ ~ ' ) and hog group A + H substance, all form precipitates with the agglutinin by virtue of their content of nonreducing, a-D-linked 2-acetamido-2-deoxy-~-ga~actosyl terminal groups.61,63,'00~'78~560-562,570 A synthetic, carbohydrate-protein conjugate, p-azophenyl2-acetamido2-deoxy-~-~-galactopyranoside-bovine serum albumin, was also shown to precipitate the H. pomatia l e ~ t i n Tay-Sachs .~~~ ganglioside was reported to precipitate with the snail lectin.61It was also observed that guaran and Staphylococcus aureus teichoic acid, containing nonreducing (terminal) a-D-galactopyranosyl and 2-acetamido-2-deoxy-a-~galactopyranosyl groups, respectively, also interacted with the agglutinin, whereas teichoic acids containing p-D-linked 2-acetamido-2deoxy-D-galactosyl end-groups, or macromolecules having p-D-linked D-galactopyranosyl end-groups, were ina~tive.~~'"O Hammarstrom and c o ~ o r k e r s ' ~ also ~ investigated a series of (570) S . Hammarstrom and E. A. Kabat, Biochemistry, 10,1684-1692 (1971). (571) W. Kohler and 0. Prokop, Z . Immunitaetsforsch. Allerg. Klin. Immunol., 133, 50-53 (1967). (572) G. Uhlenbruck and W. Gielen, Hoppe-Seyler's 2. Physiol. Chem., 348,1693-1696 (1967).

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lipopolysaccharides for their capacity to precipitate the snail agglutinin. Lipopolysaccharides isolated from Salmonella typhimurium rough mutants of chemotype Ra and Rb precipitated the Helix pomatia lectin, whereas no precipitation was obtained with lipopolysaccharides of the parent, smooth strain, or of Rc, Rd, and Re mutants. It was suggested that nonreducing 6-O-a-D-galactopyranosy~-D-glucosyl groups in the lipopolysaccharides provided the binding sites for the agg1~tinin.l~~ Extensive information on the carbohydrate-binding specificity of the Helix pomatia lectin was obtained by examining the extent to which a large number of sugars inhibited the precipitation reaction between agglutinin and human blood-group A substance or Salmonella ~ J ~Table ~ ~ ~ ~ ~XI). ~~~~ typhinurium SH 180 1 i p o p o l y s a c ~ h a r i d e ~(see TABLEXI Inhibition of HeZix pomatia Hemagglutinin-Blood-group A Substance Precipitation by Various Saccharidesss2 Saccharide

2-Acetamido-2-deoxy-~-galactose 2-Acetamido-2-deoxy-~-glucose DGlucose DGalactose D-Mannose 2-Amino-2-deoxy-~galactose 2-Amino-2-deoxy-~-glucose Methyl 2-acetamido-2-deoxy-a-~-galactopyranoside Ethyl 2-acetamido-2-deoxy-/3-~-galactopyranoside Methyl 2-acetamido-2-deoxy-a-~-glucopyranoside Ethyl 2-acetamido-2-deoxy-/3-~-glucopyranoside Methyl a-Dgalactopyranoside Phenyl 2-acetarnido-2-deoxy-a-~-galactopyranoside cY-D-GalNAcp-(1+3)-/3-D-Galp-( 1+3)-~-GlcNAc

Micromoles required for 50% inhibition 1.65 9.0 >18.4 >15.8 >22.3 >9.7 >18.8 0.76 >3.60 >4.0 10.0 >10.9 1.65 0.96

Carbohydrate-specificity studies involving hemagglutination inh i b i t i ~ n ~ ~ J (and " ' * ~sugar ~ ' displacement from S e p h a d e ~ , 5 and ~ ~an immunosorbent of human blood-group A substance,567gave essentially the same results. Methyl 2-acetamido-2-deoxy-a-~-galactopyranoside was the best inhibitor tested.63~556*56',562~567 The observation that a blood-group Type A pentasaccharide inhibited to approximately the same extent as this glycoside led to the conclusion that the combining site accommodates a

LECTINS : CARBOHYDRATE-BINDING PROTEINS

243

single, a-D-linked glycosyl unit.63 Preference for the a anomer was indicated by the 4-fold higher affinity of the methyl a-glycoside of 2-acetamido-2-deoxy-~-galactose over the parent amino sugar, and the fact that the ethyl P-D-glycoside was noninhibitory, even at very high concentration^.^^ 2-Amino-2-deoxy-D-galactosewas a noninhibitor; apparently, a positively charged 2-amino group destabilizes the carbohydrate-protein complex.63*s6',s62 Methyl 2-acetamido-2-deoxya-D-galactopyranoside is bound four times as avidly as the C-4 epimer (methyl 2-acetamido-2-deoxy-a-~-glucopyranoside), indicating a preference for an axial 4-hydroxyl group.63J00~s61*562 On the other hand, 2-acetamido-2-deoxy-~-mannose, the C-2 epimer, does not bind to the snail agglutinin. The presence of an equatorially oriented N-acetyl or 2-0-acetyl group is essential for strong binding to the hemagglutinin. D-Galactose is a poor inhibitor, showing less than one percent of the activity of 2-acetamido-2-deoxy-~-galactose. Melibiose, although a poor inhibitor, displaced the snail agglutinin from S e p h a d e ~ . ~ ~ ~ The data indicate that H. pomatia lectin exhibits a specificity for 2-acetamido-2-deoxy-cr-~-galactopyranosyl end groups (8),but will also interact with 2-acetamido-2-deoxy-c~-~-glucopyranosyl and, to a more limited extent, a-D-galactopyranosy1groups. The hexavalent nature of the snail lectin must also be considered a factor in its Such multivalence enhances the affinity of the lectin for multivalent, configurationally related structures, for example, Sephadex and g ~ a r a n(See . ~ ~p. 249 for further binding-studies.) Cell-binding studies on human erythrocytes, several human, urinary-bladder, carcinoma cell-lines, and an osteogenic-sarcoma cellline have been conducted.189After treatment with neuraminidase, -80% of human lymphocytes will bind the H . pomatia lectin. Neuraminidase-treated lymphocytes can also be fractionated on Helix pomatia hemagglutinin coupled to Sepharose beads.s72b 4. Phaseolus lunatus syn. limensis (Lima Bean) (lima bean; a-D-GalNAcp > a-D-Gab)

The lima-bean agglutinin holds the distinction of being the first lectin shown to exhibit blood-group specificity. Although Boyd initially observed, in 1945, that the lima-bean lectin specifically agglutinated type A erythrocyte^,^,^ he did not publish his observation until 1949, (572a) S. Hammarstrom, U. Hellstrom, P. Perlmann, and M.-L. Dillner,J.Exp. Med., 138, 1270-1275 (1975). (572b) U. Hellstrom, S. Hammarstrom, M.-L. Dillner, H. Perlmann, and P. Perlmann, Scand. J . lmmunol., 5, Suppl. 5, 45-55 (1976).

244

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

when it appeared together with a report on the agglutinins from a large variety of plants5 The lima-bean lectin specifically agglutinated type A erythrocytes in the following order of decreasing activity: A, > AIB > A2 > A2B. Some varieties of lima bean showed a slight agglutination of type B ce11s.2*5*22*573 The proportion of lectin activity in lima beans differed from variety to variety, and genetic studies suggested the presence of a locus for lectin Between 1945 and 1970, investigators studying the lima-bean lectin employed crude, saline extracts or, at most, partially purified preparation^.^^^.^^^ By fractional precipitation with alcohol, Boyd and coworkers103achieved partial purification of the Sieva lima-bean lectin. One-third of the nitrogen present in this preparation was precipitated by hog-mucin, type A substance; ultracentrifugal and electrophoretic studies revealed the presence of two, and four, components, respectively. Lima-bean lectin precipitated blood-group A and B secretor saliva, but not 0; it did not precipitate the saliva of any n o n ~ e c r e t o r s . ~ , ~ ~ ' ~ ~ K r i i ~ e substantiated ?~ Boyd's results, using secretor saliva as an inhibitor of lima-bean lectin-erythrocyte agglutination. Types AIB, A1A2, A1O, and A 2 0 saliva all inhibited the lima-bean lectin, whereas type 00 saliva and type 00 ovarian-cyst material were noninhibitory. In one of the first applications of the Landsteiner hapten-inhibition technique to lectin studies, Morgan and Watkins22demonstrated that purified, group A substance, at a dilution of 1:500,000, inhibited limabean-extract agglutination of Al cells, as did 2-acetamido-2-deoxy-~galactose, whereas preparations of human ByH, and Lea substances, and of material extracted from 0 stroma, were without activity at a dilution of 1:lOO. K r i i ~ verified e ~ ~ inhibition of the lima-bean lectin by 2-acetamido-2-deoxy-~-galactose,and reported that the lectin did not react with chicken, guinea pig, mouse, rabbit, or sheep erythrocytes. Make1a78showed that the lima-bean lectin reacted equally well in saline, serum, or poly(vinylpyrro1idinone) media with normal, or papain-treated, human, type A erythrocytes, and he added bovine erythrocytes to the list of unreactive, red blood-cells. Scheinberg and c o l l e a g ~ e sstudied ~ ~ ~ *the ~ ~binding ~ of lima-bean lectin to types A, By and AB cells, and observed that the lectin could be partially purified by adsorption to A2 cells, followed by elution at 60". (573)A. Chattoraj,]. Irnrnunol., 98, 757-763 (1967). (574)K. F. Schertz, W. Jurgelsky, and W. C. Boyd, Proc. Natl. Acad. Sci. U.S.A., 46, 529-532 (1960). (575)H.M.Bhatia, Y. C. Kim, and W. C. Boyd, Vox Sang., 15,278-286 (1968). (576)K. C. Atwood and S. L. Scheinberg, Science,l29,963-964(1959). (577)S. L.Scheinberg and D. T. 0. Wong, J . Immunol., 92,520-528 (1964).

LECTINS: CARBOHYDRATE-BINDING PROTEINS

245

Bhatia and further purified the lectin preparation of Boyd and coworkers103by using Bio Gel P-200 molecular sieve. The protein peak first eluted contained the “specific,” anti-A hemagglutinin. However, this preparation gave 3 peaks in the ultracentrifuge, and ~ hog A substance precipitated only 35% of the protein p r e ~ e n t . “Although treatment of the “specific” lectin with 2-mercaptoethanol and 2-iodoacetamide had no effect on its activity, the reduced, alkylated protein gave two peaks in gel chromatography, with the activity residing in the peak of material of low molecular weight. This experiment suggested breakdown of the lectin into smaller subunits by reduction of disulfide bonds.s7sRetention of hemagglutinating activity after reduction and alkylation is surprising, in view of subsequent results indicating the necessity of free thiol groups for lectin activity (see later). Investigating lima-bean extracts for cytoagglutinin activity, found that sarcoma 180 cells were agglutinated. Inasmuch as adsorption with type A, red blood-cells removed both the hemagglutinin and the cytoagglutinin activity of the lima-bean extract, the two activities appeared to reside in the same molecule; this conclusion is in contrast to that for the Phaseolus vulgaris lectin (see later). Tunis further noted579that lima-bean, navy-bean, and kidney-bean activities were inhibited by (ethylenedinitrilo)tetraacetate,an observation later attributed to the presence of metal ions in these lectin~.’~’ Affinity labelling of the lima-bean lectin was attempted by Matsubara and Boyd.580*s81 Diazotized p-aminophenyl a-glycosides of D-glucose, D-galactose, 2-amino-2-deoxy-D-glucose, 2-amino-2-deoxy-Dand 2-acetamido-2galactose, 2-acetamido-2-deoxy-D-g~ucose, deoxy-D-galactose were used as the affinity labelling compounds. The diazotization reaction failed to decrease the anti-A activity of the lectin, but actually increased the ability of the lectin to agglutinate type B erythrocytes and, in some cases, even promoted the agglutination of 0 erythrocytes. Although these results are interesting, they must be scrutinized in terms of the experimental conditions. The combination of using an impure lectin preparation (of 30% purity), glycosides having unreported properties (some not even synthesized at the time), and a prolonged reaction-time (22 h), together with the lack of adequate controls (for example, protection with 2-acetamido-2-deoxy-~galactose and other sugars), militates heavily against specific, activesite labelling; more probably, nonspecific labelling occurred. The (578) (579) (580) (581)

M . Tunis,]. Zmmzcnol., 92, 864 (1964). M. Tunis,J. Zmmunol., 95,876-879 (1965). S. Matsubara and W. C. Boyd,J. Zmmunol., 91, 641-643 (1963). S. Matsubara and W. C. Boyd,J. Zmmunol., 96,25-28 (1966).

246

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

same authorssE2reported that (p-phenylazobenzoy1)ation of the limabean and Sophora japonica lectins enhanced their respective specific activities. Boyd and coworkers5E3 also labelled lima-bean lectin with 13'1(indirectly, by coupling of ['311]-p-iodoanilineto protein by d i a z o t i ~ a t i o n ) ~ ~ ~ and visible dyes,s84~sEs and quantitated the binding to type A erythrocytes. The isolation of the lima-bean lectin(PhaseoZus Zunatus) in pure form was r e p ~ r t e d ' in ~ ~ 1970. ~ ' On ~ ~employing ~ ~ ~ ~ salt ~ ~ fractionation ~ ~ in conjunction with pH adjustment, followed by gel filtration on Bio-Gel A-0.5, Gould and ScheinbergsE6isolated two active components from the lima bean (Phaseolus Zunatus, var. thorogreen). Components I1 and I11 (designated in order of elution from the gel-filtration column) were essentially pure by poly(acry1amide) gel-electrophoresis and by ultracentrifbgation; the molecular weights computed were 269,000 and 138,000, respectively. Galbraith and G ~ l d s t e i n ' ~ used ' ~ ' ~specific ~~~~~ adsorption to insolubilized, type A blood-group substance, followed by elution with 2-acetamido-2-deoxy-~-galactose and recycling, Sephadex G-200 chromatography, to obtain the same two proteins. Components I1 and I11 were pure, as shown by poly(acry1amide) gel-electrophoresis, and their molecular weights were 247,100 and 124,400, respectively. (Pole lima-beans, PhaseoZus Zunatus, var. Carolina or Sieva, were used for these s t ~ d i e s . ' ~Although ' ~ ~ ~ ~ ) an inactive precipitate slowly formed during prolonged storage at 4", there was no indication that either component was transformed into the other. A new, affinity-chromatographic procedure has been employed to purify the lima-bean lectin~.~lO On treatment with 1,4-dithiothreitol or 2-mercaptoethanol in dodecyl sodium sulfate, components I1 and I11 both yielded'g9,5E6 subunits of molecular weight 31,000. Poly(acry1amide) gelelectrophoresis, in the presence of 1%of dodecyl sodium sulfate alone, gavesE6a component in the range of 60,000. Amino acid analysis (see later) showed two half-cystine residues per subunit. Direct titration of components I1 and I11 with 5,5'-dithiobis(2-nitrobenzoic acid), in the absence or presence of 8 M urea or dodecyl sodium sulfate, gave'99*sE6 (582) S. Matsubara and W. C. Boyd, J . Zmmunol., 96,829-831 (1966). (583) W. C. Boyd, H. M. Bhatia, M. A. Diamond, and S. Matsubara,]. Zmmunol., 89, 463-470 (1962). (584) J. T. Miller, W. C. Boyd, and M. A. Diamond, Vox Sang., 13,449-460 (1967). (585) H. M. Bhatia, C. K. Yang, J. Jaumatte, and W. C. Boyd, ZndianJ. Med. Res., 56, 1525-1530 (1968). (586) N. R. Gould and S. L. Scheinberg, Arch. Biochem. Biophys., 137, 1-11 (1970). (587) W. Galbraith and I. J. Goldstein, Methods Enzymol., 28, Part B, 318-323 (1972).

LECTINS : CARBOHYDRATE-BINDING PROTEINS

247

one sulfhydryl group per subunit of molecular weight 31,000. However, after reduction with 1,4-dithiothreitol, two sulfhydryl groups could be titrated.1Bs~s86 These data indicated that both components consist of apparently identical subunits of molecular weight 31,000; two of these polypeptides are linked by an interchain, disulfide bridge, to form a subunit of molecular weight 62,000, with two thiol groups remaining free. Component I1 contains four dimers (or eight polypeptide chains), and component 111,two dimers (or four chains); the dimers are held together by strong, noncovalent forces that require strong detergents or dissociating agents to disaggregate them. The hemagglutinating activity of the lima-bean lectins is strongly dependent on the integrity of the free sulfhydryl groups. N-Ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic acid), and p-(chloromercuri)benzoate inhibited the activity of the proteins.1BB,s88 2-Acetamido-2-deoxy-~-galactose,a specific inhibitor of the hemagglutination of type A erythrocytes by lima-bean lectin, offered protection against inactivation by the aforementioned sulfhydryl reagents.s88 Complete, immunological cross-reactivity was demonstrated between components I1 and I11 with rabbit anti-component I11 antiserum, thereby providing strong evidence that the two molecular species are closely related.lBB Analysis similar amino acid distributions for the limabean lectin components I1 and 111. Neither contained methionine, but each contained two half-cystine residues per subunit. Both components were rich in aspartic acid, serine, and leucine.1s1J0B~s86 The limabean lectins, which are glycoproteins,1s1~1gB~s86 contain 3 4 % of carbohydrate consisting of mannose, fucose, and 2-amino-2-deoxyglucose, and traces of arabinose and x y l o ~ e . ' ~Carbohydrate ~*~~~ analysis of the glycosyl moiety of the lima-bean lectin gave a structure consisting of four residues of mannose, two of 2-amino-2-deoxyglucose, and 0.5 molecule of f ~ c o s e Three . ~ ~ ~ of the D-mannosyl residues have a-Dglycosidic bonds, which accounts for the precipitation reaction1s1*'gs,410*588a between the lima-bean lectin and con A. Ions of Mn2+and Ca2+were bound to the purified, lima-bean lectins.lS1*lBB Removal of Mn2+lowered the hemagglutination titer by 75%. (Ethylenedinitri1o)tetraacetate completely inhibited the precipitin reaction between lima-bean lectin component I11 and type A bloodgroup substance (compare Ref. 579). Several divalent-metal cations restored activity to the demetallized protein or (ethylenedinitri1o)tetraacetate-treatedlectin; the addition of Ca2+, (588) N. R. Could and S. L. Scheinberg,Arch. Biochern. Biophys., 141,607-613 (1970). (588a) A. Misaki and I . J. Goldstein,]. Biol. Chem., 262,6995-6999 (1977).

248

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

Co2+,Fez+,Mg2+,Mn2+,Ni2+, Sr2+,or Zn2+gave equivalent results.'s9 The specific-titer activity of lima-bean lectin components I1 and I11 towards type A human erythrocytes was 5,100 and 1,300, respectively, ~~~*~~~ and, towards type B erythrocytes, 20 and 5.1, r e s p e c t i ~ e l y .The hemagglutinating activity of component I1 is, thus, four times the act i ~ i t y ' of ~~ component , ~ ~ ~ 111. Neither component reacted with type 0 human, red blood-cells, or native or trypsinized, rabbit erythro~ytes.'~~~'~~ Equilibrium-dialysis studiessss using I4C-labelled methyl 2-acetamido-2-deoxy-a-~-galactopyranoside gave straight-line, Scatchard plots for both components, thereby suggesting homogeneous, noninteracting binding-sites. Component I11 has two binding sites per mole of protein, and component 11, four binding sites per mole of protein, with association constants of 1.01 x lo31iter.mole-' and 0.93 x 103 liter.mole-', respectively. Interestingly, tetravalent component I1 was much more m i t o g e n i ~than ~~~ divalent component 11. The reaction of partially purified, lima-bean lectin with hog gastricmucin type A substance exemplified the precipitin-like curve obtained for lectin-polysaccharide or -glycoprotein reactions.103 Classical precipitin-curves between purified components I1 and I11 and type A blood-group substance were also Maximal precipitation of component I1 (equivalence) occurred at a lower ratio of A substance per mole of protein than for component 111. Under conditions where type A substance precipitated 90% of the lectin, types A2 and B pre~ i p i t a t e d ' ~66 ~ *and ' ~ ~13%, respectively, of component 11, and 21 and 0% of component 111.Neither ofthe lima-bean lectins precipitated with type 0 blood-group substance. The specificity of the binding site of the lima-bean lectin has been probedlS9by hapten inhibition of the precipitation reaction between components I1 and I11 and blood-group A substance, and by inhibition 2-Acetamido-2-deoxy-~-galactose was the of h e m a g g l u t i n a t i ~ n . ~ ~*~~~ best monosaccharide inhibitor tested, being respectively 20 and 4 times as potent as 2-acetamido-2-deoxy-~-glucoseand D-galactose. The preference of the lectin for the a-anomeric linkage shown in formula 8 was established by the three- to four-fold greater inhibitory potency of and methyl a - ~ methyl 2-acetamido-2-deoxy-a-~-galactopyranoside galactopyranoside over the respective Yosizawa and Miki"" similarly observed that 2-acetamido-2-deoxy-~1-3)galactose and 0-(2-acetamido-2-deoxy-a-D-galactopyranosyl)-( (589) W. Bessler and I. J. Goldstein, Arch. Biochern. Biophys., 165, 444-445 (1974). (590) Z. Yosizawa and T. Miki, Proc. Jpn. Acad., 39, 187-192 (1963). (591) L. A. Murphy and I. J. Goldstein, unpublished results.

LECTINS : CARBOHYDRATE-BINDING PROTEINS

249

D-galactose were good inhibitors of the hemagglutination reaction, 1+3)-~-gawhereas 0-(2-acetamido-2-deoxy-~-D-galactopyranosy1)-( lactose was a poor inhibitor. Moreover, melibiose was approximately twice as good an inhibitor as lactose. Methyl 4-deoxy-Pfluoro-a-Dgalactopyranoside had one-third of the effect of methyl a-D-galaCtOpyranoside, whereas D-fucose was very similar to D-galactose in inhibitory potency."' Interestingly, methyl 2-deoxy-2-(p-nitrobenzamido)-and -(paminobenzamido)-a-D-galactopyranosidewere the most potent inhibitors tested.lSsThese data suggest that, on the protein, there may be a region that can interact specifically with an aromatic moiety at C-2 of 2-amino2-deoxy-~-galactose.Table XI1 presents some representative, inhibition data. TABLEXI1 Inhibition of Lima-bean Lectin by Saccharides"

Inhibitor

Micromoles of inhibitor for 50% inhibition

Methyl 2-deoxy-2-(p-nitrobenzamido)-cr-~-galactopyranoside Methyl 2-acetamido-2-deoxy-a-~-galactopyranoside Methyl 2-acetamido-2-deoxy-~-~-galactopyranoside 2-Acetamido-2-deoxy-~-galactose 2-Acetamido-2-deoxy-~-glucose Methyl a-Dgalactopyranoside Methyl P-D-galactopyranoside Melibiose Lactose D-Galactose D-Fucose Methyl 4-deoxy-4-fluoro-c~-D-galactopyranoside

0.5 2.6 17.0 8.0 158 34 200 28 51 38 45 107

"Inhibition of the precipitation reaction between lima-bean lectin component I11 and human blood-group A s u b s t a n ~ e . ~ ~ ~ . ~ ~ '

A comparative study of the carbohydrate-binding specificity of four 2-acetamido-2-deoxy-~-galactose-binding lectins (from Dolichos bi$orus, Glycine max, Helix pomatia, and Phaseolus lunatus) has been conducted by using a series of model macromolecules for direct precipitation and a variety of mono- and oligo-saccharides as hapten inh i b i t o r ~The . ~ ~data ~ indicated that the combining site of all four lectins (592)S . Hammarstrom, L. A. Murphy, I. J. Goldstein, and M. E. Etzler, Biochemistry, 16,2750-2755 (1977).

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IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

corresponds to the size of a monosaccharide unit. The contact groups in 2-acetamido-2-deoxy-~-galactose that are probably involved in hydrogen-bond formation with the four lectins are: for Helix pomatia, the carbonyl oxygen atom of the 2-acetamido group and the oxygen atom of the 4-hydroxyl group; for soybean agglutinin (Glycinemax), the carbonyl oxygen atom of the 2-acetamido group and the hydrogen atoms of the 4- and 6-hydroxyl groups; for the Dolichos biflorus and lima bean (PhaseoZus Zunatus) lectins, only the 2-acetamido group was identified as a probable binding-locus. In view of its discovery as the first blood-group-specific lectin, it is surprising that the lima-bean lectin is still one ofthe least studied, plant agglutinins. Undoubtedly, this lectin merits closer scrutiny.

5. Sophora japonica (Japanese Pagoda Tree) (Japanese pagoda tree; P-D-GalNAcp > P-D-Galp) The agglutinin of Sophora japonica seeds was first described by Kriipe and B r a ~ n . "Shortly ~ thereafter, Morgan and Watkins22obtained preliminary evidence that the lectin in the seed extract, which agglutinated A and B cells more strongly than 0 erythrocytes, interacted with both type A and type B blood-group substance. Absorption of the extract with type A erythrocytes left neither A nor B agglutinating activity in the supernatant liquor; absorption with B erythrocytes gave the same result. Furthermore, type A substance inhibited lectin agglutination of B cells, as well as of A cells, as did type B substance. Sugar inhibition ofhemagglutination demonstrated the reactivity of the lectin towards 2-acetamido-2-deoxy-D-galactose, lactose, D-galaCtOSe, and melibiose (in order of decreasing reactivity), whereas L-arabinose and D-hcose showed weak inhibiting activity. Later studies of hemagglutination inhibition employing crude extract^'^ or partially purified preparation^^^^-^^^ confirmed the work of Morgan and Watkins.22 Osawa and Akiya partially purified S . japonica extract by precipitation with an organic solvent and ammonium sulfate f r a c t i o n a t i ~ n . ~ ~ ~ Ultracentrifugation and starch gel-electrophoresis revealed multiple (593) (594) (595) (596) (597) (598) (599)

M. Kriipe and C. Braun, Naturwissenschaften, 39,284-285 (1952). T. Osawa and S. Akiya, Bull. Tokyo Med. Dent. Unio., 8,299-305 (1961). T. Osawa and S. Akiya, Bull. Tokyo Med. Dent. Univ., 8,287-298 (1961). Z. Yosizawa and T. Miki, Proc. Jpn. Acad., 39, 182-186 (1971). T. Terao and T. Osawa,J. Biochem. (Tokyo), 74, 199-201 (1973). R. D. Poretz, Methods Enzymol., 28, Part B, 349-354 (1972). P. Balding and E. R. Gold, Z. Immunitaetsforsch. Allerg. Klin. Immunol., 145, 156-165 (1973).

LECTINS : CARBOHYDRATE-BINDING PROTEINS

251

components. The protein was heat-labile, agglutinated both A and B erythrocytes, and contained 17.3% (by weight) of carbohydrate (Dgalactose, D-arabinose, and D-xylose). The lectin of Sophora japonica has now been purified to hom~geneity.'*~ Poretz * ~ ~ ~and coworkers184specifically adsorbed the saline-extracted protein onto insolubilized A plus H-active hog gastric-mucin, and eluted the bound lectin with D-galactose. The physicochemical purity of the D-galactose-eluted protein was assessed by electrophoretic, immunochemical, and centrifugal methods. Amino acid analysis revealed a relatively high content of acidic amino acids, no methionine, and 5 half-cystinyl residues per molecule of molecular weight 132,800 (determined by gel filtration). The presence of carbohydrate was established by chemical analysis [7.8%(by weight) of and by reaction mannose, xylose, and 2-acetamido-2-deoxy-glucose], with con A. An unusual, pH profile was obtained by the hemagglutination assay: activity rose rapidly with increasing pH, to a maximum at pH

8.5. Early sugar-specificity studies on partially purified S . japonica lectin was complementary suggested that 2-acetamido-2-deoxy-~-galactose .~~~ D-fUCOSe, D-gUlOSe, D-talOSe, to the lectin b i n d i n g - ~ i t eD-Galactose, 2-amino-2-deoxy-D-galactose, and 2-amino-2-deoxy-D-glucose also inhibited lectin-B erythrocyte a g g l ~ t i n a t i o nP-D-Galactopyranosides .~~~ were better inhibitors than a-D-galactopyranosides. Whereas methyl groups at 0-2,-3,and -6of D-galactose did not diminish the inhibitory capacity, 4-0-methyl-D-galactose was a noninhibitorVSg4 On a weight basis, blood-group A and B substances were far more reactive than any of the simple sugars tested.594 Sugar inhibition of highly purified, lectin-type B blood-group substance precipitation was achieved by Poretz and coworker^.^^^,^^ Hemagglutination inhibition studies were conducted by Irimura and coworkers.lm This work is summarized in Table XIII. A comparison of 2-acetamido-2-deoxy-~-galactose(and its glycosides) with D-galaCtOSe (and its glycosides) confirmed a preferential binding of the lectin to the 2-acetamido-2-deoxy-~-galactosyl structure. Moreover, on contrasting the P-glycoside of either sugar with the a-linked anomer, it is evident that S.japonica exhibits a stronger affinity for the p-anomeric configuration. Among p-D-linked D-galactopyranosides, the aromatic aglycon contributed importantly to stabilization of the lectin-saccharide complex in comparison to the aglycon of alkyl glycosides. N-Acetyllactosamine is of special interest, in that it inhibits hemagglutination to the same extent as phenyl 2-acetamido-2-deoxy-P-Dgalactopyranoside, regardless of the apparent preference of the lectin over D-galaCtOSe, and aromatic for 2-acetamido-2-deoxy-~-galactose

252

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES TABLEXI11

Sugar Inhibition of Sophora japonica Lectin

Sugar

Micromoles required Concentration required for 50% inhibition for B erythrocyte of B-substance hemagglutination p r e c i p i t a t i ~ n ' ~ ~ ~ ' ~inhibitionla ~ (mM) 4.6

2-Acetamido-2-deoxy-D-galactose p-Nitrophenyl2-acetamido-2deoxy-P-D-galactopyranoside Phenyl2-acetamido-2-deoxy-P-~galactopyranoside Phenyl2-acetamido-2-deoxy-a-~galactopyranoside Methyl 2-acetamido-2-deoxy-P-Dgalactopyranoside Methyl 2-acetamido-2-deoxy-a-~ galactopyranoside D-Galactose p-Nitrophenyl P-D-gdactopyranoside Phenyl P-Dgalactopyranoside Phenyl a-D-galactopyranoside Methyl P-D-galactopyranoside Methyl a-Dgalactopyranoside 2-Acetamido-2-deoxy-4-O-P-Dgalactopyranos yl-D-glucose (N-acetyllactosamine) Lactose Melibiose

0.21

0.8 1.6

2.7 3.5 25 1.2 3.9

2.7

6.3 13

11 18 0.81 6.6 20

over alkyl glycosides.'@ This result suggests the possibility of an extended binding-site complementary to a unit larger than a monosaccharide unit. Indeed, Balding and Gold599found that the strongest hemagglutination inhibitors were sugars in which a D-galactopyranosyl group was linked 3-0-p- or 4-0-p- to the penultimate 2-acetamido-2-deoxy-~-glucopyranosylresidue: N-acetyllactosamine, lacto-N-biose I, and lacto-N-tetraose. The data suggest a groups (9). specificity for 2-acetamido-2-deoxy-~-~-galactopyranosyl

NHAC

9

LECTINS : CARBOHYDRATE-BINDING PROTEINS

253

The observation that S. japonica lectin agglutinates B erythrocytes slightly more strongly than A cells, whereas 0 cells are agglutinated only at high concentrations of l e ~ t i n , together ~ ~ ~with ~ ~ sugar J ~ ~ ~ ~ inhibition data, suggests that the lectin may react with non-ABO blood-group structures. Some investigators have reported agglutination of erythrocytes regardless of blood type594*596; others have found variable results, depending on the source of the seeds.5g9 In order to clarify the nature of the erythrocyte structure responsible for S.japonica lectin binding, Chien and coworkersa0'investigated the relationship between cell agglutination and the I antigenic determinant. By comparison of the reactivity of purified lectin with human, red blood-cells of various I phenotypes (adult I, adult i, cord blood i) and that of human anti-B or anti-A sera with the same cells, theya0' demonstrated that lectin agglutination was closely related to the presence of the I antigen. Furthermore, the hemagglutination inhibiting capacity of Smith-degraded, type B blood-group substance having decreased nonreducing (terminal) a-D-galactosyl and a-L-fucosyl groups, but increased P-D-galactosyl termini, in comparison to that of native B substance was not diminished, nor was its capacity to precipitate the lectin from solution substantially lessened. However, this chemical degradation completely destroyed the B determinant, as evidenced by the low inhibitory activity of degraded B substance in the B erythrocyte-anti-B serum agglutination reaction. In addition, digestion of B substance with a-D-galactosidase, which diminished the nonreducing (terminal) a-D-galactosylgroups, without concomitantly increasing the number of P-D-galactosyl termini, increased the concentration of oligosaccharide required for hemagglutination inhibition without altering that needed for inhibition of lectin agglutination. Enzymic digestion substantially lessened the capacity of B-active blood-group substance to precipitate the lectin from solution. Chien and coworkersao1concluded that S. japonica lectin reacts with P-D-galactosyl residues (believed to represent the I antigenic determinant)602common to both A and B erythrocytes of I phenotype. Balding and Gold599studied the agglutination of various, red bloodcell types by extracts ofSophora japonica seeds from varied geographical sources. Whereas a purified lectin sample from R. D. Poretz (seeds from a supplier in the United States) and lectin from Japanese seeds did not agglutinate 0 cells, a sample from Portugal did. All three lectin (600) J. T. Miller and W. C. Boyd, Vox. Sang., 13,209-217 (1967). (601) S. M. Chien, T. Lemanski, and R. D. Poretz, Immunochemistq, 11, 501-506 (1974). (602) T. Feizi, E. A. Kabat, G. Vicari, B. Anderson, and W. L. Marsh,]. E x p . Med., 133, 39-52 (1971).

254

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

samples reacted strongly with OEn(a-) cells, which contain a lowered proportion of surface sialic acid due to a genetic abnormality. If it is assumed that the lectin can react with a- or P-D-galactosides or 2-acetamido-2-deoxy-~-galactosides, the lectin may interact not only with blood-group A and B substances, but also with nonreducing (terminal) P-D-galactosyl groups on (a) I-precursor substance (incompletely biosynthesized A or B substance), (b)incompletely biosynthesized, or partially degraded, M or N substances, (c) desialized, myxoviral receptors, and (d) OEn(a-) Irimura and coworkerslmemployed a series of native and degraded glycopeptides as inhibitors of S. japonica lectin hemagglutination. Desialized, porcine thyroglobulin gave good inhibition by virtue of its O-P-D-galactopyranosyl-(l-*4)-2-acetamido-2-deoxy-~-~-glucopyranosyl nonreducing termini. On the other hand, desialized bovine submaxillary-mucin did not react, despite the presence of O-(e-acetamido2-deoxy-a-D-galactopyranosyl)-serine (or -threonine), which may have been sterically unavailable to the lectin. Finally, acid hydrolysis of nonreducing (terminal) a-L-fucosyl groups from degraded, porcine submaxillary-mucin exposed unsubstituted 0-0-D-galactopyranosyl(1-*4)-2)-acetamido-2-deoxy-a-~-galactopyranosyl-serine (or -threonine), resulting in a two-fold diminution of inhibitory capacity. This result implies that a-L-fucose either represented a binding locus, or conferred a more favorable conformation on the disaccharide to which it was attached.

V. BGALACTOSE-BINDING LECTINS 1. Abrus precatorius (Jequirity Bean) (jequirity bean; P-D-Gab > cw-D-Gab)

In his very early study of Ricinus communis and Abms precatorius seed-extracts, Stillmark observed toxic and hemagglutinating activities in both.603The strongly toxic effects of the extracts were originally attributed to the hemagglutinating a ~ t i v i t y , 6but ~ ~ it, ~has ~ ~since been demonstrated that these activities are associated with distinct proteins present in Abrus precatorius e x t r a ~ t s . ' ~Interest ~ ' ~ ~ *in~purifying ~~ the toxic protein, abrin, was greatly enhanced by reports of its anti-tumor activity.32Olsnes and coworker^^^'*^^^ purified abrin by a combination of ion-exchange and affinity chromatography, and studied the mechanism (603)H.Stillmark, Arb. Phannakol. Inst. Dorpat, 3,59-151 (1889). (604)H. Hellin, Ph.D. Thesis, Universitkt zu Dorpat (1891). (605)A. H.Kahn, B. Gul, and M. A. Rahman,]. Immunol.,96,554-557(1966).

LECTINS : CARBOHYDRATE-BINDING PROTEINS

255

of its toxic action. The protein gave a single band in disc-gel electrophoresis, and exhibited a molecular weight of 65,000 by gel filtration. Abrin was split into two polypeptide chains of molecular weights 35,000 and 30,000 on reduction with 2-mercaptoethan01.~~~ The larger nontoxic subunit, designated the B chain, bound to carbohydrate, as evidenced by its ability to agglutinate erythrocytes in the presence of anti-abrin serum.147,150 The smaller chain, designated the A chain, was incapable of agglutinating erythrocytes in the presence of anti-abrin serum, but manifested considerable activity as an inhibitor of protein synthesis in a cell-free system. Olsnes and coworkersao6concluded that the mechanism of action of the toxins abrin and ricin was virtually identical: the toxin binds to the cell surface by virtue of the B chain; and the A chain is pinocytosed, whereupon it inhibits the chain-elongation step of protein synthesis. An alternative purification of abrin, involving chromatography in three separate, ion-exchange systems, gave two toxic proteins designated607abrin A and abrin C. The two toxins were homogeneous by sedimentation and electrophoretic analysis, and similar in molecular weight (abrin A, 60,100; abrin C, 63,800), but differed slightly in amino acid composition. However, the proteins differed markedly in their affinity for Sepharose 4B:abrin A was not bound, whereas abrin C was bound, and was eluted specifically with D-galactose. Wei and coworkers8O7suggested that abrin C may be identical to the toxin studied by Olsnes and Pihl,lS0and abrin A to that reported by Lin and coworkers."* The effect of utilizing widely different seed-sources in the three investigations is difficult to assess. On reduction with 2-mercaptoethanol, the abrin A of Wei and coworkers607gave subunits of molecular weights 32,000,29,500, and 28,000 in the ratios of 2: 1:1, whereas abrin C gave equal proportions of subunits of molecular weight of 33,000 and 28,000. Abrin C was crystallized by Wei and Einstein,609and preliminary, X-ray crystallographic studies were made. Limited data are available with respect to the carbohydrate-binding specificity of abrin. Olsnes and coworkers147demonstrated that the agglutination of erythrocytes by abrin in the presence of anti-abrin antiserum was inhibited best by D-galactose. Lactose, melibiose, and D-fucose (in the order of decreasing potency) gave weaker inhibition, whereas D-glucose, D-mannose, D- and L-arabinose, D-xylose, (606) S. Olsnes, K. Refsnes, and A. Pihl, Nature, 249,627-631 (1974). (607) C. H. Wei, F. C. Hartman, P. Pfuderer, and W.-K. Yang,J. Biol. Chern., 249, 3061-3067 (1974). (608) J.-Y. Lin, Y . 4 . Shaw, and T.-C. Tung, Toxicon, 9,97-101 (1971). (609) C. H. Wei and J. R. Einstein,J. B i d . Chem., 249,2985-2986 (1974).

256

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

D-fructose, D-ribose, and maltose were without effect. Equilibriumdialysis analysis147revealed one binding site for lactose on the abrin molecule, with an apparent association constant of 8 x lo3 it4-l. The hemagglutinin of A. precatorius was also purified and characterized, by both Olsnes and coworkers'47and Wei and coworkers.610 The latter authors reported that a lectin preparation, homogeneous by electrophoresis, isoelectric focusing, and sedimentation analysis, gave a molecular weight of 130,000&5,000 by gel filtration."O A molecular weight of 125,400 to 126,000 was obtained by equilibrium sedimentation and calculation from the chemical composition. The protein exhibited an isoelectric point at pH 5.0, a carbohydrate content (mannose) of5% by weight, and subunits ofmolecular weight 33,800 and 32,200 in the presence of 2-mer~aptoethanol.~~~ Olsnes and c o ~ o r k e r s ' ~concluded ' that their lectin preparation probably contained two highly similar proteins that differed in their subunit structure. After fractionation on DEAE-cellulose, the active fraction was specifically adsorbed to Sepharose 4B, and was eluted with D-gahCtOSe. The resultant preparation, after chromatography on CMcellulose, had a very high hemagglutinating activity and very little toxicity, and migrated as a single, symmetrical peak when sedimented in a sucrose density-gradient. The protein of molecular weight 134,000 split into two components having molecular weights of 68,000 and 69,000 in the presence of dodecyl sodium sulfate, and further into subunits of molecular weight 36,000, 35,000, and 33,000 in the presence of 2-mercaptoethanol and a detergent. The relative amounts of the two larger polypeptide chains was variable, but that of the small chain was constant. The authors suggested that the two agglutinins differ in their heavy chain but share the subunit of molecular weight 33,000. Again, a disparity in the seed source used by Olsnes and cow o r k e r ~and ~ ~by ~ Wei and coworkers610may account, in part, for the differences noted by the two groups. Roy and coworkers6'l purified both the toxin and the lectin ofAbrus precatorius Linn. by using a combination of ammonium sulfate fractionation, CM-cellulose ion-exchange, and affinity chromatography on Sepharose 4B. The two proteins were obtained in crystalline form, exhibiting homogeneity by immunochemical and ultracentrifugal criteria. The molecular weight reported for the lectin, namely, 132,000, agreed with previous values. 147m0 Biophysical characterization gave s Z O ~ , =~ 6.7 S, DZOo,w= 4.7 x 10-7cm2.sec-1,andF = 0.73for the protein.611 (610) C. H. Wei, C. Koh, P. Pfuderer, and J. R. Einstein,J. B i d . Chern., 250,4790-4795 ( 1975). (611) J. Roy, S. Som, and A. Sen, Arch. Biochern. Biophys., 174,359-361 (1976).

LECTINS : CARBOHYDRATE-BINDING PROTEINS

257

A pH-dependent dissociation of the tetramer to dimers of molecular weight 64,000 occurred between pH 2 and 7 .Analysis ofthe C-terminal amino acid showed both alanine and leucine, and the N-terminus was valine. The possibility of multiple isolectins was indicated by multiple band-formation of isoelectric-focused hemagglutinin.611 A. precatorius lectin agglutinates human type B and 0 erythrocytes more effectively than type A cells.'47The agglutination reaction was inhibited by D-galactose, lactose, melibiose (in order of decreasing activity), and, to a lesser extent, by L-arabinose and D-fUCOSe. Although limited, the data suggest a specificity for P-Dgalactopyranosyl groups (10). Two binding-sites for lactose were observed by Olsnes and coCH,OH

OH 10

workers14' in equilibrium-dialysis experiments; the calculated association constant was 8 x 103M - l , which was identical to that of abrin. Wei and coworkers obtained crystals ofA. precatorius lectin, and conducted preliminary X-ray crystallographic studies.610Whereas fresh preparations of abrin were extremely toxic and nonmitogenic, storage of the glycoprotein for several months at 4"rendered it relatively nontoxic and highly mitogenic.612The chemical relationship between the subunits of the toxin and the agglutinin has not yet been established, nor have the stereochemical features involved in carbohydrate binding to either protein been studied in detail.

2. Arachis hypogaea (Peanut) [peanut;P-D-Gab-(1+3)-~-GalNAc > D - G ~ N H = , a-D-Galp] Polyagglutinability, the agglutination of erythrocytes irrespective of blood type by a high percentage of adult, human sera, is a problem encountered in routine serology.613 T-Polyagglutinability usually arises as a result of in vitro contamination of blood specimens (although authentic cases of in vivo T-polyagglutinability have been reported) through the degradative action of bacterial neuraminidase. Until re(612) S. J. Kaufman and A. McPherson, Cell, 4,263-268 (1975). (613) G. W. G. Bird and J. Wingham, Scand. J. Haematol., 8,307-308 (1971).

258

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

cently, complex and time-consuming absorption studies were needed in order to establish the presence of the T-antigen. With the discovery of the T-antigen-reactive peanut lectin, determination of T-polyagglutinability was greatly ~ i m p l i f i e d . ~Agglutination '~,~'~ of A, B, or 0 erythrocytes by peanut lectin occurs only after digestion of these cells with neuraminidase, which exposes the T determinant.20',202 Two affinity-purification schemes for the lectin in peanuts have been reported.202*61s Lotan and coworkers chromatographed a solubilized, ammonium sulfate fraction of the seed extract on Sepharose-N-(6aminohexanoyl)-~-D-galactopyranosylamine.202 The bound lectin was eluted with D-galactose as a single peak in 87%yield. Homogeneous by disc-gel electrophoresis (pH 4.3, 7.2, and 8.9), gel filtration, and sedimentation analysis, the protein exhibited a molecular weight of 110,000by gel filtration. A molecular weight of 111,000 was calculated from biophysical data ( S Z " ~ , ~= 5.7 S, D&,w= 5.0 x lo-' cm2.sec-', ij= 0.73). In the presence of detergent, the protein dissociated into subunits of molecular weight 27,000-28,000, The amino acid composition reported by Lotan and coworkers showed a high content of acidic and hydroxylic amino acids, relatively little methionine, tryptophan, and histidine, and the complete absence of cysteine. The finding of a unique sequence for the five NH2-terminal amino acids of the peanut lectin suggested four identical subunits.202Sequence homology with the p-chain of the lentil (see Section II,2) and pea (see Section II,3) lectins and the soybean lectin (see Section IV,2) has been S ~ O W I I . ~ Peanut lectin has also been isolated on poly(acry1amide)-entrapped guaran beads and on poly(acry1amide) copolymerized with ally1 a - ~ galactopyrano~ide.'~" Terao and coworkers fractionated the seed extract on Sepharose 6B; peanut lectin was retarded with respect to contaminating proteins.816 The homogeneous protein (disc gel-electrophoresis at pH 4.3 and 7.1, and by ultracentrifugation) had a molecular weight of 106,500, calculated from sedimentation data ( S L = , ~6.05 S). Detergent-dissociated subunits had molecular weight 27,000. The chemical composition reported was at variance with the results of Lotan and coworkers.202Terao and coworkersrn3reported considerably less serine, threonine (due, perhaps, to their using unextrapolated values), tryptophan, and arginine; moreover, they found no methionine, but reported 16.6 moles (614)W. C. Boyd, D. M. Green, D. M. Fujinaga, J. S. Drabik, and E. WaszczenkoZacharczenko, Vox Sung., 4,456-467 (1959). (615)G. W. G. Bird, Vor Sung., 9,748-749 (1964). (616)T.Terao, T.Irimura, and T. Osawa,Hoppe-Seyler's Z. Physiol. Chem., 356,16851692 (1975).

~ ~

LECTINS : CARBOHYDRATE-BINDING PROTEINS

259

of cysteine per mole of lectinS6l6 Neither group found covalently bound carbohydrate.202*616 Early studies on the carbohydrate-binding specificity of peanut lectin suggested that P-glycosidically linked D-galactosyl residues might be an important part of the T-antigen to which the lectin bound. Uhlenbruck and coworkerszo1found particularly strong inhibition of hemagglutination by 2-acetamido-2-deoxy-3-O-~-~-galactopyranosyl D-galactose, and by glycoproteins and gangliosides carrying this disaccharide in a nonreducing (terminal) position. Later studies confirmed these results.202,203,604 The investigations of Dahr and coworker^^'^,^*^ suggested that the peanut-lectin receptor was part of the base-labile oligosaccharide 11 that is responsible for MN blooda-AcNeu - (2-3)-p-o-Galp

- (1-

3)-GalNAc- (l+O)-Ser, Thr 6

t

2 a-AcNeu 11

group activity. Desialization by neuraminidase or acid hydrolysis revealed the peanut-reactive structure, which was labile to digestion with periodate, D-galactose oxidase, and P-D-galactosidase. An extensive, immunochemical investigation of the specificity of peanut agglutinin was conducted by Pereira and coworkers.z03Corroborating previous work, only derivatives of D-galactose gave significant inhibition of peanut agglutinin-blood-group precursorsubstance precipitation (see Table XIV). Of the monosaccharides tested, 2-amino-2-deoxy-~-galactosewas the most effective inhibitor, whereas 2-acetamido-2-deoxy-~-galactoseand S-deoxy-~-lyxohexoseZo2were inactive, suggesting the importance of a hydrogenbonding substituent on C-2. By comparison of methyl a-Dgalactopyranoside with its 6-0-methyl-substituted counterpart, and of D-galactose with D-fucose, it is evident that the 5-(hydroxymethyl) group is also implicated in lectin-saccharide interaction. Lotan and coworkers further reported that D-galaCtOSe 6-sulfate7 D-galacturonic acid, and L-arabinose are noninhibitory, confirming the importance of an unsubstituted, 5-(hydroxymethyl) group.z02Monosaccharides lacking inhibitory activity were D-glucose,2-acetamido-2-deoxy-~-glucose, D-mannose, 2-acetamido-2-deoxy-D-mannose, L-fucose, L-rhamnose, and maltose.z0z~203 (617) W. Dahr, G. Uhlenbruck, and G. W. G . Bird, Vox Sung.,27,29-42 (1974). (618) W. Dahr, G. Uhlenbruck, and G. W. G. Bird, Vox Sung., 28, 133-148 (1975).

260

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

TABLEXIV Inhibition of Peanut Agglutinin-Blood-group Precursor Substance Precipitation by saccharide^'^^ Micromoles required for 50% inhibition

Sugar

D-Galactose 2-Amino-2-deoxy-D-ga1actose Methyl a-D-galactopyranoside p-Nitrophenyla-D-galactopyranoside Methyl P-D-galactopyranoside p-NitrophenylP-Dgalactopyranoside Methyl 6-O-methyl-a-~-galactopyranoside DFucose 2-Acetamido-2-deoxy-~-galactose 2-Acetamido-2-deoxy-3~-~-D-galactopyranosy~-~-ga~actose 2-Acetamido-2-deoxy-3-O-~-galactopyranosyl-~-galactito~

6.0

2.7 2.7 2.7 4.0 4.0 6.0 10.0 >60 0.11 2.7 3-0-a-D-Galactopyranos yl-D-galactose 7.0 6-O-a-~-Galactopyranosy~-6-0-c~-~galactopyranosyl-P-D-g~ucopyranosyl D-fmctoside 7.0 3-0(2-Acetamido-2-deoxy-c~-D-ga~actopyranosy~)-D-ga~actoseinactive at 4.0 4-O-P-D-Galactopyranosyl-D-g~ucose 2.7 6-O-~-D-Galactopyranosyl-D-glucose 2.7 6-O-a-~-Galactopyranosyl-D-glucose 10.0 2-Acetamido-2-deoxy-3-O-~-~-ga~actopyranosyl-D-glucose 10.0 2-Acetamido-2-deoxy-4-O-~-~-galactopyranosyl-~-glucose 1.5

The disaccharide 2-acetamido-2-deoxy-3-O-~-D-ga~actopyranosy~-Dgalactose (12)shows the corresponding group was, without question,

OH 12

the most complementary to the lectin binding-site. Reduction of this disaccharide resulted in a 25-fold decrease in reactivity. Substitution at 0 - 2 or -4of the nonreducing (terminal) D-galactosyl group with an Lfucosyl or a 2-acetamido-2-deoxy-~-glucosyl group, respectively, greatly diminished the inhibiting capacity. This finding suggests that

LECTINS: CARBOHYDRATE-BINDING PROTEINS

26 1

peanut lectin does not recognize (internal) D-galaCtOSyl residues. The results of Irimura and coworkers183are at variance with these findings, in that substitution of an L-fucosyl group on 0-2 of the nonreducing (terminal) D-galaCtOSyl group of 2-acetamido-2-deoxy-O-~-~-galactopyranosyl-D-galactopyranosylserine/threonine(the oligosaccharide core of porcine, submaxillary mucin) increased the binding to peanut lectin by a factor of 2.5. Further substitution by a 2-acetamido-2-deoxyD-galaCtOSyl group at 0-3of the same D-galactosyl residue did not alter the binding characteristics. These discrepancies remain unexplained. With respect to the anomeric carbon atom of nonreducing (terminal) D-galactosyl groups, a discrepancy exists between monosaccharides and disaccharides. Whereas, methyl a-D-galactopyranoside was 1.5 times as effective as methyl P-D-gdactopyranoside, 6-0-P-D-galactopyranosyl-D-glucose was 3 times as potent as its a-D-linked counterpart; lactose gave better inhibition than melibiose,zOzand other oligosaccharides having nonreducing (terminal) a-D-galactosyl groups A preference for 4-0-P-D-linkage were relatively poor inhibit01-s.~~~ over 3-0-p-D-linkage was suggested by comparison of the activity of 2-acetamido-2-deoxy-4-0-~-~-ga~actopyranosyl-~-g~ucose with that of 2-acetamido-2-deoxy-3-O-~-~-galactopyranosyl-~-glucose. The precipitin reaction with peanut agglutinin revealed considerable heterogeneity among blood-group substances having the same Thus, some samples of A-, B-, and H-active substances precipitated almost 100% of the lectin at equivalence, whereas others failed to react, or gave an intermediate result. In no case did A, substances give precipitation. Strikingly, all A-, B-, and H-active substances gave very strong precipitin reactions after mild, acid hydrolysis, or one-step Smith degradation. Moreover, all precursor blood-group substances (I-active) gave strong precipitin reactions. The authorszo3suggested that the peanut agglutinin reacts with a determinant other than that which accounts for A, Byor H specificity, which although normally concealed, can be exposed by chemical degradation. Incomplete biosynthesis of oligosaccharide chains, and variable, steric hindrance of the access of lectin to reactive, short chains by unreactive, large oligosaccharide chains were explanations invoked by Pereira and coworkers to account for the blood-group substance heterogeneity observed with peanut l e ~ t i n . ~ ~ ~ A major disagreement exists with respect to the biological activity of peanut lectin.fi16*619 Terao and coworkers616could not demonstrate mitogenicity of peanut lectin towards either normal, or neuraminidase(619) A. Novogrodsky, R. Lotan, A. Ravid, and N. Sharon,J. Immunol., 115,1243-1248 (1975).

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IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

treated, human, peripheral-blood lymphocytes. On the other hand, Novogrodsky and coworkers observed stimulation of DNA synthesis in neuraminidase-treated, rat or human lymphocytes by this l e ~ t i n . ~ l ~ Untreated lymphocytes did not respond, nor did normal or neuraminidase-digested lymphocytes of the mouse or guinea pig.61gThe question of the mitogenicity of peanut lectin, therefore, remains unresolved. 3. Bandeiraea simplicifolia I (cY-D-Gab > a-D-GalNAcp)

A human blood-group B-specific phytohemagglutinin, first observed in Bandeiraea simplicifolia seeds by the Makelas,620has now been purified by Hayes and G~ldstein.'~' An ammonium sulfate fraction of the seed extract was chromatographed on a melibionyl-Bio-gel P-300 column (compare Refs. 148,149b, and 150c). Elution ofthe specifically adsorbed protein was effected with D-galactose. The protein appeared homogeneous by disc gel-electrophoresis (pH 4.3), immunoelectrophoresis, gel filtration, and sedimentation analysis. Electrophoresis at pH 9.5, and isoelectric focusing, revealed multiple bands. Later studies revealed the presence of multiple, lectin specificities in Bandeiraea simplicifolia ~ e e d ~The . a-D-galactos ~ ~ ~ * ~yl-binding ~ ~ lectin, designated BS I, was completely precipitated from solution by the galactomannan guaran. A molecular weight of 114,000 was calculated from sedimentation velocity centrifugation ( S O Z ~ , ~ = 7.52 x S, Dio,w= 5.2 x lo-' cm2.sec-', ij= 0.69 cm3.g-'), and was corroborated by gel filtration and calculation based on the chemical composition. The glycoprotein lectin (9.0%, by weight, of carbohydrate, namely, mannose, fucose, xylose, and 2-amino-2-deoxyglucose) dissociated in the presence of detergent into subunits having molecular weight 28,500. Amino acid analysis demonstrated the abundance of hydroxylic and acidic amino acids, -0.5 molecule of methionine, and one free-thiol cysteine residue per subunit. Chemical modification, with 5,5'-dithiobis(2-nitrobenzoic acid), of 2-3 molar proportions of cysteine residues per mole of protein destroyed the hemagglutinating activity.I3l However, both native and demetallized lectin failed to react with methyl methanethiosulfonate (MeSS02Me), indicating that the thiol groups were most probably "buried," and not directly involved in carbohydrate- or metalbinding.622Amidation of 9 carboxyl groups per subunit with glycine methyl ester hydrochloride lowered the precipitating capacity of the (620) 0. Makela and P. Makela, Ann. Med. E x p . Biol. Fenn., 34,402-404 (1956). (621) I. J. Goldstein, L. A. Murphy, and T. Ebisu, Pure Appl. Chem., 49, 1095-1103 (1977). (622) J. Lonngren and I. J. Goldstein, Biochim. Biophys. Acta, 439, 160-166 (1976).

LECTINS : CARBOHYDRATE-BINDING PROTEINS

263

polysaccharide622by 80%. Inclusion of methyl a-D-galactopyranoside during amidation afforded some protection; 8 residues per subunit were modified, giving a protein that had 80% of the activity of native lectin. On the other hand, acetylation of 8 lysyl side-chains and 2 hydroxyl or thiol groups per subunit caused virtually no perturbation of the carbohydrate-binding activity.622These studies suggest that carboxyl groups participate in carbohydrate binding, either directly, or indirectly by way of stabilization of the conformation or metal binding, whereas free amino and sulfhydryl groups do not.622 The BS I lectin requires bound calcium for activity.131Two moles of calcium and 1.25moles of magnesium per mok of protein were found by atomic absorption spectroscopy. Inactive, metal-free lectin, obtained by exhaustive dialysis, could be reconstituted by addition of calcium, cadmium, or strontium (magnesium restored 80% of the activity). Although bound-calcium was not removed by dialysis against EDTA, inclusion of this chelating agent in the precipitin reaction resulted in complete inhibition. Conformational analysis of BS I by c.d. spectroscopy indicated that the protein contained 30-40% of structure in its native conformat i ~ nThe . ~c.d. ~ ~spectrum was relatively insensitive to alteration in pH, removal of bound metal, and addition of methyl a-D-galactopyranoside. However, addition of dodecyl sodium sulfate or 2,2,2-trifluoroethanol resulted in the formation of some a-helical structure, and was accompanied by the loss of polysaccharide-precipitating capacity. Urea (8 M ) irreversibly denatured the lectin. BS I agglutinates human, type B and AB erythrocytes strongly, and Al cells weakly, and does not agglutinate Az or 0 ce11s.131,195-624 (Old seed samples were reported to be more specific for B erythrocytes than fresh seeds, which contained some anti-A activity.6zo)Polysaccharides and glycoproteins having terminal (nonreducing) a-D-galactopyranosy1 groups gave131*6zs a precipitin reaction with BS I. Thus, type B bloodgroup substance and a-D-galactopyranosyl-substituted, branched polysaccharides precipitated all of the lectin from solution under optimal conditions, whereas type A blood-group substance gave a much diminished precipitate. Type Az and H ( 0 ) substances did not react, nor A series of did fetuin or orosomucoid (either native or de~ialized).'~~ polysaccharides was s t ~ d i e d 'by ~ ~Ouchterlony-diffusion ~ ~ ~ ~ and quantitative-precipitin analysis with BS I. The shape of precipitin curves with six leguminous-seed galactomannans correlated with their (623) J. Lonngren, I. J. Goldstein, and R. Zand, Biochemistry, 15,436-440 (1976). (624) W. J. Judd, E. A. Steiner, B. A. Friedman, C. E. Hayes, and I. J. Goldstein, Vox Sang., 30,261-267 (1976). (625) C. E. Hayes, L. A. Murphy, and I. J. Goldstein, to be published.

264

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

galactose to mannose ratio (C yamopsis tetragonolobus, Cassia alata, Ceratonia siliqua L., Caesalpinia spinosa, Crotalaria mucronata, and Leucaena glauca). Weaker precipitin-reactions were obtained with Tsuga canadensis galactoglucomannan and Torulopsis gropengiesseri galactomannan. Larch egalactan and sugar-beet L-arabino-P-Dgalactan did not react.131A synthetic melbionate-bovine serum albumin conjugate also precipitated with the lectin.lz6Cell-surface galactomannan from Schixosaccharomyces pombe cells was localized by goldlabelled BS I lectin,BZsaand a-D-galaCtOSyl-COntining components on mouse neuroblastoma cells by 1311-labelledlectin.62sbThe BS I lectin was also shown to give rise to an immediate-type skin-reaction in a sensitized, laboratory worker.62sc Makela and colleagues investigated the specificity ofB. simplicifolia The , ' ~ ~a - ~ seed-extracts by hemagglutination i n h i b i t i ~ n . ~ ~ galactosides melibiose and raffinose gave good inhibition, as did D-Fucose and D-galactose and 2-acetamido-2-deoxy-~-galactose. L-arabinose were weakly inhibitory. The carbohydrate-binding specificity of BS I has been studied in detail by sugar inhibition of lectin-galactomannan p r e c i p i t a t i ~ n . ' ~ ~ , ~ ~ ~ Representative data are summarized in Table XV. That BS I preferentially binds a-D-linked D-galactopyranosides was readily established by comparison of glycosides having nonreducing (terminal) a - ~ galactopyranosyl groups with those which are P - ~ - l i n k e d . ~Fur~"~~ thermore, a-glycosides of D-galactose were bound with equivalent affinity, regardless of the nature of the aglycon, whereas P-glycosides having aromatic aglycons were more avidly bound than those having A relatively important bindingsaccharide or hydrocarbon agly~ons.'~' locus resides in the 2-hydroxyl group of the D-ga~actopyranosy~ configuration, as deduced by a comparison of methyl 2-acetamido-2deoxy-D-galactopyranoside, 2-deoxy-~-lyxo-hexose,and D-talose, all of which inhibited poorly. Likewise, the importance of the 3-hydrogen atom in stabilizing the lectin-saccharide complex was suggested by comparing D-galactose with D-gulose, and p-nitrophenyl P-Dgalactopyranoside with its 3-deoxy-3-fluoro d e r i v a t i ~ e . ' ~Examina'*~~~ tion of analogs of D-galactose altered at C-5 (D-fUCOSe, L-arabinose, methyl 6-0-methyl-a-D-galactopyranoside,and 6-deoxy-6-fluoro-~galactose) led to the conclusion that the oxygen atom of the (625a) M. Horisberger and J. Rosset, Arch. Microbiol., 112, 123-126 (1977). (625b) S . Basu, J. R. Moskal, and D. A. Gardner, in "Ganglioside Function: Biochemical and Pharmacological Implications," G. Porcellati, B. Ceccarelli, and G . Tettamanti, eds., Plenum, New York, 1976. PP.45-63. (625c) T. M. Kanellakes and K. P. Mathews:j. Allergy Clin. lmmunol., 56,407-410 (1975).

265

LECTINS: CARBOHYDRATE-BINDING PROTEINS

TABLEXV Inhibition of Bandeiraea simplicifolia I Lectin-Galactomannan Precipitation'zss1gs

Sugar

Concentration required for 50% inhibition (mM)

Methyl a-D-galactopyranoside, melibiose, p-nitrophenyl a-D-gdactopyranoside, 2-O-a-D-galactopyranosyl-D-g1LIcose, O - ~ - D - g ~ a C t O p y r ~ O S y l ~ y O - i n O S i t o l p-Nitrophenyl P-D-galactopyranoside Raffinose D-Galactose Methyl P-D-galactopyranoside Methyl 6-O-methyl-a-~-galactopyranoside Methyl 2-acetamido-2-deoxy-a-D-galactopyranoside 6-Chloro-6-deoxy-D-galactose 6-Deoxy-6-fluoro-D-galactose DTalose

0.62 0.84 1.20 1.90 5.40 5.50 9.20 10.4 10.4 20.0 Inhibition at 25 mM

40" 18 17b 15 15 14 8

2-Deoxy-D-lyxo-hexose DFucose 4-Deoxy-~-xylo-hexose 3-O-P-~-Galactopyranosyl-D-arabinose Methyl a-wglucopyranoside Lactose L-Arabinose 4-Deoxy-4-fluoro-D-ga~actose Galactitol D-LyXOSe Methyl a-D-lyxofuranoside Methyl a-D-xylofuranoside Methyl a-D-mannopyranoside DGulose L-Fihamnose

" Inhibition at 20 mM.

0' 1

1 0 0 0 0 0

Inhibition at 4.2 mM. Inhibition at 10.4 mM.

5-(hydroxymethyl) group was most probably involved in hydrogen bonding to the lectin. The important binding-loci for BS I-saccharide interaction are italicized in formula 13.

OH 13

266

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

Equilibrium-dialysis studies on the binding of methyl a-D-galactopyranoside to BS I revealed the existence of one carbohydrate-binding site per subunit for the tetrameric protein, withlgl an intrinsic association constant of 8.6 x lo4M-' at 2", and 3.3 x lo4M-' at 20". These values correspond to a free energy of binding, AGO' (pH 7.2), of -26 kJ.mole-' and -25.36 kJ.mole-' at 2" and 20", respectively. The sites appeared to be homogeneous and n~ninteracting.'~' The BS I that had been lZ5I-labeled by a diazonium coupling technique626was employed in a study of lectin-receptor density on the B erythro~yte.'~' Scatchard analysis of the binding data demonstrated a variation in receptor density among type B individuals from 7.2 x lo4to 13.4 x lo4 sites per erythr~cyte.'~' Likewise, apparent associationconstants for lectin-cell interaction varied from 1.1 x lo' to 2.9 x lo7 M-1. A specific adsorbent has been prepared by covalent coupling of BS I to cyanogen bromide-activated Sepharose 4B, and its interaction with model, carbohydrate-protein conjugates has been studied.627The carbohydrate-binding specificity of the lectin was retained. The authorssZ7further demonstrated the utility of the specific adsorbent by effecting a single-step purification of the Cassia alata seedgalactomannan. Further investigation revealed that BS I consists of five isolectins: these are tetrameric structures composed of two, unique subunits (A and B) in various proportions.621*628 They are designated BS I(A4),BS I(&B), BS I(AZ&),BS I(A&), and BS I(B4). All are glycoproteins, as revealed by a fluorescent, glycoprotein reagent.s2gThe subunits are indistinguishable on the basis of size and immunochemical reactivity, but differ in isoelectric point.621,s28 Subunit A is devoid of methionine, whereas B contains one methionyl residue per polypeptide chain. The single cysteinyl residue of subunit B was titrated with 5,5'-dithiobis(2nitrobenzoic acid).621,628 On the other hand, the cysteinyl residue of subunit A could only be titrated in 6 M guanidinium chloride. Perhaps the most interesting difference between the subunits is their carbohydrate-binding specificity.621*62s BS I(&)agglutinated type A erythrocytes (specific titer, 170) but not type B cells, and precipitated A substance, indicating an affinity for a-D-linked 2-acetamido-2deoxy-D-galactosyl residues. Cross-reactivity with a-D-galactosyl res(626) C. E. Hayes and I. J. Coldstein, And. Biochem., 67,580-584 (1975). (627) T. T. Ross, C. E. Hayes, and I. J. Goldstein, Carbohydr. Res., 47,91-97 (1976). (628) L. A. Murphy and I. J. Goldstein,J. Biol. Chem., 252,47394742 (1977). (629) A. E. Eckhardt, C. E. Hayes, and I. J. Goldstein, And. Biochem., 73, 192-197 (1976).

LECTINS : CARBOHYDRATE-BINDING PROTEINS

267

idues was evidenced by precipitation of BS I(&) with type B substance and guaran, but not larch arabinogalactan. Sugar inhibition of BS I(A4)-guaran precipitation confirmed this conclusion; methyl 2-acetamido-2-deoxy-a-~-galactopyranoside was 20 times as inhibitory as methyl a-D-galactopyranoside. In contrast to BS I(A4), BS I(AB,,B,) is highly blood-group B specific (specific titer, 120); these forms did not agglutinate A erythrocytes, precipitated only B substance and guaran, and showed no reactivity towards A substance or larch arabinogalactan. Moreover, methyl a-D-galactopyranoside inhibited BS I(AB3,B4)guaran precipitation, whereas methyl 2-acetamido-2-deoxy-a-~galactopyranoside was one-hundredth as potent. In many respects, the finding of related isolectins (of BS I) closely parallels structural studiess30 on Phaseolus vulgaris agglutinin (see Section V11,l). It will be interesting to determine whether there is sequence homology between BS I subunits A and B, like that between and furthermore, to discover a structural the two P . vulgaris basis for the observed differences in specificity. 4. MacZura pomgera syn. aurantica (Osage Orange) (Osage orange; D-Gab, D-GalNAcp) Osage-orange seeds (Maclura pornifera) contain a nonspecific, blood-group h e m a g g l ~ t i n i n . " ~Ulevitch .~~~ and coworkerss33reported the isolation of Maclura pornifera lectin by affinity chromatography of seed extracts on a column prepared by condensing 2-amino-2-deoxyD-galaCtOSe with an aminosuccinyl-Sepharose. The bound-protein fraction, eluted with melibiose, gave a single peak when chromatographed on Sephadex G-200, and a single band on disc-gel electrophoresis, and appeared homogeneous in the analytical ultracentrifuge. A molecular weight of 80,000 to 100,000 was determined by gel filtration. Amino acid analysis revealed a high content of glycine and aspartic acid, with relatively little cysteine, methionine, and histidine. Bausch and Poretz purified M.pornifera agglutinin on insolubilized immunochemically homogenepolyleucyl, hog-gastric m ~ c i nThe . ~ ~ ous protein migrated as a single component at acidic or alkaline pH in poly(acry1amide) gel electrophoresis, and focused as a single band (630) J. B. Miller, R. Hsu, R. Heinrikson,andS.Yachnin,Proc.Natl.Acad.Sci. U.S.A.,72, 1388-1391 (1975). (631) J. M. Jones, L. P. Cawley, and G. W. Teresa, Vox Sang., 12,211-214 (1967). (632) G. I. Pardoe, G. W. G. Bird, G. Uhlenbruck, I. Sprenger, and M. Heggen, Z. Immunitaetsforsch. Allerg. Klin. Zmmunol., 140,374-394 (1970). (633) R. J. Ulevitch, J. M. Jones,and J. D. Feldman, Prep. Biochem., 4,273-281 (1974). (634) J. N. Bausch and R. D. Poretz, Fed. Proc., 35, 1530 (1976).

268

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

having an isoelectric point PI 4.75.In sharp contrast to the results of Ulevitch and Bausch and Poretz determined a molecular weight of 43,000 b y gel filtration.634Subunits of molecular weight 11,000 were observed by gel filtration in dodecyl sodium sulfate or guanidinium Regrettably, the only studies on the sugar-binding characteristics of the MacZura pornifera lectin were conducted by hemagglutination inhibition of crude seed-extracts rather than on a purified, protein Consequently, disparate results were obtained. Representative hemagglutination inhibition are presented in Table XVI, notwithstanding our serious reservations concerning the TABLEXVI Hemagglutination Inhibition of Crude Macluru pornifera Lectin by Simple Sugars83s

Sugar

Melibiose Stachyose 2-Acetamido-2-deoxy-~-galactose 2-Deoxy-D-lyxo-hexose D-Galactose 2-Amino-2-deoxy-~-galactose Raffi nos e

L-Rhamnose D-Ribose Maltose

Concentration required for 100% inhibition (mM)

4.0 5.0 5.0 10 150 300 370 400 400 400

significance of studies conducted on crude seed-extracts. Melibiose and stachyose, having terminal (nonreducing) a-D-galactopyranosyl groups, were effective inhibitors, whereas raffinose, having the same terminal (nonreducing) group, was a very poor inhibitor. Equivalent to melibiose and stachyose, 2-acetamido-2-deoxy-~-galactosewas 60 times as inhibitory as the related 2-amino-2-deoxy-~-galactose,and twice as effective as 2-deoxy-~-Zyxo-hexose.Noninhibitory sugars included D-arabinose, L-arabinose, 2-acetamido-2-deoxy-~-glucose, D-glucose, 2-amino-2-deoxy-~-glucose,D-fUCOSe, L-fucose, cellobiose, lactose, D-lyxose, D-mannose, melezitose, L-sorbose, sucrose, a,atrehalose, D-XylOSe, and ~ - x y 1 o s e . These ~ ~ ~ findings ~ ~ ~ ~ must - ~ ~be~ considered preliminary, until they are confirmed with purified lectin. (635) L. P. Cawley, J. M. Jones, and G. W. Teresa, Transfusion (PhiludeZphia), 7,343346 (1967).

LECTINS : CARBOHYDRATE-BINDING PROTEINS

269

In disagreement with the data presented in Table XVI, Pardoe and coworkers632reported that D-galactose is a better inhibitor than melibiose, whereas 2-acetamido-2-deoxy-D-galactose does not inhibit this lectin. Sprenger and coworkers636found that stachyose inhibits rather poorly, its activity being of the same order of magnitude as that of 2-amino-2-deoxy-~-galactose. Chuba and coworkers637 stated that methyl a-D-mannopyranoside inhibited hemagglutination, although no data were presented; this result is difficult to reconcile with previous reports. It may tentatively be concluded that the lectin binds sugars related to D-galactose. No clear difference has been established between the inhibitory potency of 2-acetamido-2-deoxy-~-galactoseand D-galactose. Although lactose was found noninhibitory in two s t ~ d i e s ,several ~ ~ ~ ,reports ~ ~ ~ of lectin interaction with terminal, nonreducing, p-D-linked D-galactopyranosyl groups of glycopeptides e ~ i s t . ~Thus, ~ ~ Pardoe * ~ ~ and ~ * coworkers632 ~ ~ ~ found neuraminidasetreated, horse-erythrocyte glycopeptide (having terminal p-Dgalactopyranosyl groups) the most potent hemagglutination-inhibitor of an extensive series of polysaccharides and glycoproteins tested. Similarly, Sprenger and coworkers636demonstrated inhibition by a number of mucins having the same, nonreducing terminus. Finally, Chuba and coworkers637and Ahmend638studied the interaction of the lectin with antarctic-fish, antifreeze glycoproteins. The glycopeptides are comprised of repeating glycotripeptide units; the disaccharide side-chains of 2-acetamido-2-deoxy-3(or 4)-O-P-D-ga~actopyranosyl-Dgalactose interact strongly with Mucluru pornifera lectin. On the other hand, melibiose and a-D-galactopyranosyl-substitutedglycoproteins ~ ~ * ~ ~using ~,~~~ were also shown to inhibit h e m a g g l u t i n a t i ~ n . ~Results 2-acetamido-2-deoxy-~-galactosyl-substitted glycoproteins are somewhat puzzling in view of the sugar inhibition studies (see Table XVI). Nonreducing (terminal) 2-acetamido-2-deoxy-a-~-galactopyranosyl groups occur in peptone A substance and in horse-erythrocyte glycoprotein digested sequentially with neuraminidase and p-Dgalactosidase; both were good inhibitor^.^^,^^^ Globoside I (terminal 2-acetamido-2-deoxy-~-~-galactosyl groups) and p-aminophenyl 2acetamido-2-deoxy-p-~-galactoside, diazotized and coupled to ovalbumin, were without inhibitory ~apacity."~ In summary, despite a considerable amount of investigation, the ~

(636) I. Sprenger, G. Uhlenbruck, and G. I. Pardoe, Haematologia, 4,373-378 (1970). (637) J. V. Chuba, W. J. Kuhns, R. F. Nigrelli, J. R. Vandenheede, D. T. Osuga, and R. F. Feeney, Nature, 242, 342-343 (1973). (638) A. I. Ahmed, D. T. Osuga, and €4. F. Feeney,]. B i d . Chem., 248,8524-8527 (1973).

270

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

sugar-binding requirements of purified M . pomifera lectin have not yet been delineated. Furthermore, there are few biophysical data concerning the molecular weight of this lectin, and no data regarding its subunit structure, metal requirements, or presence of a glycosyl moiety; it may be hoped that further studies will soon become available.

5. Ricinus communis (Castor Bean) (castor bean; P-D-Gab > cu-D-Galp) The (toxic)castor bean has been associated with folk medicine since antiquity; the Ricinus communis bean was first described in the sixthcentury Sanskrit work on medicine, Susruta A y u r u e d ~Dixons40 . ~ ~ and, later, Stillmark603attributed the toxicity of these seeds to an extractable protein. As seed extracts agglutinated erythrocytesYso3 it was assumed that toxicity was the result of agglutination. It is now recognized that two, chemically distinct, carbohydrate-binding proteins are present in castor beans: a toxin and a h e m a g g l ~ t i n i n . Extensive ~ ~ ~ * ~ ~ bibliog~ raphies on the lectins of R. communis have been compiled by Olsnes and PihP9 and Balint.643 Multiple purification-schemes have been applied to the separation of the toxin and the hemagglutinin.144J46~147~150*194*641-s“1 Fractionation using salt and ethanol precipitation led to c r y s t a l l i ~ a t i o nof~the ~~~~~~ toxin known as ricin or ricin D. The hemagglutinin was isolated, free from toxic activity, by ion-exchange chromatography and gel filtration.642*s46-s4* With the introduction of affinity chromatography on Sepharose 4B, to which both proteins bind, purification of the two R. (639)S. Olsnes and A. Pihl, in “Receptors and Recognition Series: The Specificity and Action of Animal, Bacterial and Plant Toxins,” Chapman and Hall, London, 1976. (640)T. Dixon, Aust. Med. Gaz., 6, 137-155 (1887). (641)T.Takahashi, G. Funatsu, and M. Funatsu,]. Biochem. (Tokyo), 52,50-53 (1962). (642)M. Ishiguro, T. Takahashi, G. Funatsu, K. Hayashi, and M. Funatsu,]. Biochem. (Tokyo), 55,587-592 (1964). (643)G. A. Balint, Toxicology, 2, 77-102 (1974). (644)E.A. Kabat, M. Heidelberger, and A. E. Bezer,]. B i d . Chem., 168,629-639(1947). (645)M. Kunitz and M. R. McDonald,]. Gen. Physiol., 32,25-31 (1948). (646)E.Waldschmidt-Leitz and L. Keller, Hoppe-Seyler’s Z. Physiol. Chem., 350,503509 (1969). (647)E.Waldschmidt-Leitzand L. Keller, Hoppe-Seyler’s Z. Physiol. Chem., 351,990994 (1970). (648) L. G. Gurtler and H. J. Horstmann,Biochim. Biophys. Acta, 295,582-594 (1973). (649)S. Olsnes and A. Pihl, Biochemistry, 12,3121-3125 (1973). (650)M. Lhermitte, G.Lamblin, P. Degand, and P. Roussel, Biochimie, 57,1293-1299 (1975). (651) S.Olsnes, K. Refsnes, T. B. Christensen, and A. Pihl, Biochim. Biophys. Acta, 405, 1-10 (1975).

LECTINS: CARBOHYDRATE-BINDING PROTEINS

271

communis lectins was greatly simplified.144~'46~'47Jso~1g4~649 The D-galactose eluate of Sepharose columns was further separated into toxic and agglutinating components by gel filtration146*194,650 or ionexchange chromatography.147~1s0~649~650 Alternatively, Nicolson and coworkers reported selective elution of ricin from Sepharose with 2-acetamido-2-deoxy-D-galactose.146 Subsequent D-galactose elution of the hemagglutinin effected a single-step p u r i f i ~ a t i o n . ' ~ ~ Ricin, which has a molecular weight of 60,000, consists of two nonidentical, disulfide-bonded, polypeptide chains.'46J50Jg4~64E~64g The subunits A and B have14s~64E~651 respective molecular weights of -28,000 and 32,000. The amino acid composition of the isolated subunits has been determined, and they were found to be remarkably similar.B4E~6s1 The B subunit has relatively more aspartic acid and cysteine, but relatively less glutamic acid and phenylalanine than the A subunit. Both subunits contain low proportions of basic amino acids and methionine. No reduced cystinyl residues occur in the native toxin."' The A and B subunits gave almost identical, tryptic peptide maps, but differed in their N-terminal (isoleucine-A, alanine-B) and C-terminal amino acids (serine-A, phenylalanine-B).642,64E In contrast, Lhermitte and coworkers observed650 N-terminal glycine and serine for the intact toxin. Sequencing studies were conducted on ricin subunits by Li and coworkers.652 Circular dichroism studiesss3showed that the ricin A chain contained 0.3%of a-helix, and the B chain, -25%. Both subunits contain covalently bound carbohydrate: the A chain, mannose, and the B chain, mannose and 2-amin0-2-deoxyglucose.~~~ Ricin has been crystallized,844*645*654*s55 and its crystal structure studied.656 Ricin is extremely toxic to eukaryotic cells. The experiments of Olsnes and Pih1150*639*s49 and Pappenheimer and coworker~ demonstrated ~~~ that one of the two subunits binds to the cell membrane, presumably by way of a carbohydrate structure, whereas the second subunit inhibits protein synthesis by a catalytic mechanism in a cell-free system. This suggests that toxicity may result from the (652)S. S.-L. Li, C. H. Wei, J.-Y.Lin, andT.-C.Tung,Biochem. Biophys. Res. Commun., 65,1191-1195(1975). (653)M. Funatsu, G.Funatsu, M. Ishiguro, and K. Hara,Jpn.J. Med. Sci. Biol., 30-32 (1973). (654)1.-Y. Lin, Y . 4 . Shaw, and T.-C. Tung, Toxicon, 9,97-101 (1971). (655) M.Funatsu, G.Funatsu, S. Ischiguro, S. Nanno, and K. Hara, Proc.J p n . Acad., 47, 718-723 (1971). (656)C. H. Wei, J . Biol. Chem., 248,3745-3747 (1973). (657)J.-Y. Lin, K.Lin, C.-C. Chen., and T . C . Tung, Cancer Res., 31,921-924 (1971). (658)A. M. Pappenheimer, S. Olsnes, and A. A. Harper,J. Immunol., 113, 835-841 (1974).

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IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

binding of the toxin to mammalian cells by way of its B subunit, followed by ingestion of toxin (or A chain), and, finally, by A chain inhibition of protein synthesis.639 R . communis agglutinin, like the toxin, is composed of two types of subunits bound by disulfide bonds. In the presence of 2-mercaptoethanol and a detergent, the protein of molecular weight 120,000 breaks down into subunits; Olsnes and coworker^'^' reported subunits of molecular weight 31,000 and 34,000; Nicolson and coworke r ~ 29,500 , ~ ~and ~ 37,000; and Giirtler and Horstmann,M827,500, 30,000, and 33,000. The smaller subunit is reportedly an inhibitor of protein synthesis.639The amino acid composition revealed a polypeptide rich in aspartic and glutamic acids, serine, threonine, leucine, and isoleucine, with low proportions of cysteine, methionine, or phenylalanine.M8*6so No reactive sulfhydryl groups were detected in the presence or absence of urea.639Threonine and alanine were reported648 as theN-termini of the subunits; serine, lysine, and phenylalanine were found in the C-terminal position.646On the other hand, Lhermitte and reported N-terminal alanine and glycine. The agglutinin contains about 12%(by weight) of carbohydrate (mannose, glucose, and 2-amin0-2-deoxyglucose)~~~ not required for its carbohydrate-binding a ~ t i v i t y .The ~ ~ failure ~ , ~ ~ of ~ EDTA to inhibit R. communis agglutinin suggested its lack of dependence on metal cations for its activity.I8The lectin is relatively stable to freeze-thawing, alteration of pH, detergents, and Immunochemical studies, tryptic peptide mapping, and end-group analysis suggested that the toxin and agglutinin may have one common ~ a compari~ ~ ~ and one unique type of subunit e a ~ h . ~Thus, son of tryptic peptide maps gave146a ratio of identical to unique peptides of 1:l. Furthermore, antisera to either the toxin or the agglutinin cross-reacted with the other protein.146,147,6s8,660,662 Shimazaki and coworkers663analyzed the c.d. spectrum of both toxin and agglutinin in the presence and absence of lactose. The spectra of the two proteins were similar, but there were differences in band strength between them. The toxin contains 15%of a-helix and 52%of @pleated sheet; the agglutinin, 13%of a-helix and 51% of P-pleated

-

(659) A. Surolia, A. Ahmad, and B. K. Bachhawat,Biochin~Biophys. Acta, 371,491-500 (1974). (660) M. Jacoby, Beitr. Chern. Physiol. Pathol., 1, 51-77 (1902). (661) M. Jacoby, Beitr. Chern. Physiol. Pathol., 2, 535-544 (1902). (662) S . Olsnes and E. Saltvedt,J. Imrnunol., 114, 1743-1748 (1975). (663) K. Shimazaki, E. F. Walborg, Jr., G. Neri, and B. Jirgensons, Arch. Biochern. Biophys., 169,731-736 (1975).

~

~

LECTINS: CARBOHYDRATE-BINDING PROTEINS

273

sheet. Conformational transitions occurred in both spectra upon the addition of lactose. The hemagglutinin agglutinates human erythrocytes without regard to type. The toxin, on the other hand, agglutinates cells only after addition of antiricin antiserum, or at high concentrations, where dimer formation 0 c c ~ r r e d . l ~ ~ The carbohydrate-binding specificity of purified agglutinin has been ~ ~ ~ * ~ J ~ ~of studied by sugar inhibition of h e m a g g l u t i n a t i ~ n , ~ inhibition ' ~ ~ inhibition of lectinlectin-hog A + H substance p r e ~ i p i t a t i o n ,and alfalfa galactomannan precipitation. lZ4The results obtained by sugar inhibition studies in precipitating systems are in very close agreement. Table XVII presents representative data from the work of van Wauwe TABLEXVII Sugar Inhibition of R. communis Hemagglutinin-Alfalfa Galactomannan PrecipitationlZ4

Sugar p-Nitrophenyl P-D-galactopyranoside Lactose Methyl P-D-galactopyranoside p-Nitrophenyl 2-acetamido-2-deoxy-~-~-galactopyranoside p-Nitrophenyl a-D-galactopyranoside Methyl a-D-galactopyranoside Melibiose Raffinose D-Galactose D-Fucose L-Arabinose

Inhibitor required for 50% inhibition (mM)

0.026 0.05 0.16 0.225 0.26 0.29 0.32 0.35 0.39 0.51 1.42

and c o w ~ r k e r s . ' The ~ ~ following sugars were reported to be noninhibitors of lectin-alfalfa galactomannan interaction: D-mannOSe, D-ghOSe, L-fucose, and D - r i b o ~ e .Nicolson '~~ and found L-rhamnose comparable to methyl P-D-galactopyranoside as an inhibitor of lectin-hog A H blood-group substance precipitation, and extended the list of noninhibiting sugars to include galactitol, 2-acetamido-2-deoxy-~-galactose,2-amino-2-deoxy-D-galactose, D-lyxose, 2-acetamido-2-deoxy-~-glucose,and L-glucose. These results are, with some exceptions, in accord with those of hemagglutination inhibition studies. Olsnes and coworkers'47 found methyl a - ~ galactopyranoside to be a noninhibitor of lectin-human type B erythrocyte agglutination. Furthermore, Irimura and reported

+

274

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

that the phenyl and methyl glycosides, both a-and p-, of 2-acetamido2-deoxy-D-galactopyranose were noninhibitors of hemagglutination, whereas 2-acetamido-2-deoxy-4-O-~-D-ga~actopyranosy~-D-g~ucose (N-acetyllactosamine) was as effective as phenyl p-Dgalactopyranoside. Finally, Nicolson and B l a ~ s t e i n 'reported ~~ that L-arabinose does not inhibit hemagglutination, whereas it does inhibit H substance pre~ipitati0n.I~~ Hemagglutination studies are hog A complicated, in that the receptor structure(s) is not known, quantitation is imprecise, and nonspecific interaction between lectin and cell cannot be excluded. These considerations may, in part, explain the discrepancies noted. It may be concluded from these studies that sugars of the D-galactopyranose configuration are bound most effectively by R . communis agglutinin. The lectin exhibits some preference for p-Dgalactosides (lo),although the a anomer is also bound. The binding site of the lectin may recognize more than a simple monosaccharide determinant. Thus, lactose is three times as effective as methyl p-Dgalactopyranoside and, according to Irimura and coworkers,lS N-acetyllactosamine, but neither phenyl nor methyl 2-acetamido-2deoxy-P-D-galactopyranoside,inhibits hemagglutination. Equilibrium-dialysis experiments indicated that R. communis agglutinin binds two moles of lactose per mole of protein, with an association constant of 1.5 x lo4M - l , whereas ricin binds one mole of lactose per mole of protein with an identical association ~0nstant.I~' Two binding sites for p-nitrophenyl P-D-galactopyranoside,association constant 1.65 x lo4M-I, were observed by van Wauwe and coworkerstZ4 on studying the agglutinin. The equilibrium-dialysis experiments of Podder and coworkers193differed slightly: an association constant of 2.2 x lo3 M-' was reported for the two lactose-binding sites of the hemagglutinin. The carbohydrate-binding specificity of the toxin is very similar to that of the agglutinin, with one important difference; the toxin was inhibited by 2-acetamido-2-deoxy-D-galactose, whereas the agglutinin was The order of decreasing inhibitory capacity by sugars in the toxin-hog A H substance precipitating system is lactose > methyl P-D-galactopyranoside, methyl a-D-galactopyranoside > D-galactose, > melibiose > raffinose, D-fUCOSe, 2-acetamido-2-deoxy-~-galactose L-rhamnose, ~ - a r a b i n o s e . ' ~ ~ The interaction of R. communis agglutinin with polysaccharides has been i n v e ~ t i g a t e d . ' The ~ ~ *lectin ~ ~ ~ precipitates galactomannans of al-

+

+

(664) J. P. Van Wauwe, F. G . Loontiens, and C. K. de Bruyne, Biochim. Biophys. Actu, 354, 148-151 (1974).

LECTINS : CARBOHYDRATE-BINDING PROTEINS

275

falfa, fenugreek, and guar in a very similar way (compare Ref. 659). The reaction between the agglutinin and locust-bean galactomannan or larch arabinogalactan, as compared to that of the galactomannans, is considerably weaker, reflecting a lower ga1actose:mannose ratio in the former polysaccharide, and the presence of nonreducing (terminal) L-arabinosyl groups (which bind poorly) in the latter. The linear, partially sulfated carrageenans did not precipitate with the lectin, nor did beef-lung galactan. A series of amyloids (tamarind, balsamine, and capucine) gave precipitin reactions proportional to their galactose:glucose:xylose ratios; increased substitution of the main chain side-chains was with reactive 2~-~-D-galactopyranosyl-a-D-xylosy~ correlated with more-complete precipitation of the lectin at lower concentrations of the polysaccharide. Prior treatment of capucine amyloid with P-D-galactosidase resulted in the destruction of precipitating capacity, confirming the presence of nonreducing (terminal) p-I>galactopyranosyl groups. studied the interaction of fragments Codington and obtained from epiglycanin, the major membrane-glycoprotein of a murine, mammary carcinoma, with a series of lectins, including Ricinus communis. The fragments only became inhibitors of hemagglutination after neuraminidase treatment. This finding together with chemical analysis suggested the presence of penultimate p-Dgalactopyranosyl residues. A comparative study of D-galactose-binding lectins was made by Irimura and who reported that porcine thyroglobulin glycopeptide B, both untreated and neuraminidase-digested, was a good inhibitor of hemagglutination, whereas porcine and bovine submaxillary-mucin glycopeptides were not inhibitory, even after removal of sialic acid, 2-acetamido-2-deoxy-~-galactose,and L-fucose residues. The latter result is difficult to explain, in that removal of these sugar residues would be expected to leave the core structure pGal+aGalNac+ Ser/Thr, which would be complementary to the specificity of the lectin. Adair and Kornfeld666studied the binding of '251-labelledR . communis lectins to human erythrocytes, isolated a lectin-reactive, erythrocyte glycoprotein by affinity chromatography on a lectin-Sepharose column, and compared several glycoproteins, glycopeptides, and simple sugars in a standard inhibition-assay (see Table XVIII). The lectinreactive, erythrocyte glycoproteins were 1,200 times as inhibitory as (665) J. F. Codington, K. B. Linsley, R. W. Jeanloz, T. Irimura, and T. Osawa, Carbohydr. Res., 40, 171-182 (1975). (666) W. L. Adair and S. Kornfeld,]. Biol. Chem., 249,4696-4704 (1974).

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IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

TABLEXVIII Inhibitory Activity of Various Carbohydrates, Glycopeptides, and Glycoproteins in a Standard, r251-Lectin-Erythrocyte Ghost-binding Assayess Concentration required for 50% inhibition" Substance Affinity-purified erythrocyte receptor for ricin Affinity-purified erythrocyte receptor for R . communis hemagglutinin Erythrocyte sialoglycoprotein Erythrocyte sialoglycoprotein digested with neuraminidase IgG glycopeptide Fetuin glycopeptide Transferrin glycopeptide Lactose D-Galactose 2-Acetamido-2-deoxy-~-galactose

Rich (pM)

R. communis Hemagglutinin ( p M )

0.5

1.4

0.5 18

0.6 15

4.5 50 108 185 108 650 280

8 67 25 180 100 830 3000

"Normalized to content of D-galactose.

simple sugars in this assay. Multivalent interaction and a possibly extended, carbohydrate-binding site may account for this finding. Clear differences in specificity between ricin and the agglutinin were observed with fetuin glycopeptide, affinity-purified erythrocyte receptor for ricin, and 2-acetamido-2-deoxy-~-galactose. Extracts of R. communis seeds formed p r e c i p i t a t e ~ ' ~ ~ with ~''~~~~~ salivary mucins, ovarian-cyst, blood-group substances, and pneumococcal polysaccharide XIV. Furthermore, the Sepharosecoupled agglutinin reacted with each of fifty monoclonal, IgM immunoglobulins by way of a site on the Fc fragment,667and with IgG, immunoglobulins.66sAs IgG3does not differ from IgG,, IgG2,and IgG, in its carbohydrate content, this result suggests that IgG3 differs conformationally from the other immunoglobulins.668 Surolia and also coupled the R. communis lectin to Sepharose, and demonstrated its utility by separating guaran from (667)M.Harboe, E. Saltvedt, 0. Closs, and S. Olsnes, Scnnd. J. lmmunol., 4,Suppl. 2, 125-134 (1974). (668)E.Saltvedt, M.Harboe, I. Folling, and S. Olsnes, Scand. J. Immunol., 4,287-294 (1975). (669)A. Surolia, A. Ahmad, and B. K. Bachhawat, Biochim. Biophys. Acta, 404, 83-92 (1975).

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glycogen (the guaran was specifically bound, and eluted with 0.1 M lactose). Desialated ceruloplasmin and fetuin were readily bound by the immobilized agglutinin.66sSmall fractions of “native fetuin” and native ceruloplasmin” were retarded, and, upon analysis, it was found that the retarded material had a lower content of sialic acid compared to the native g l y c o p r o t e i n ~It . ~was ~ ~ suggested that columns of R . comrnunis lectin might be used as an analytical tool for the separation of partially sialated glycoproteins. “

VI. L-FUCOSE-BINDING LECTINS 1. Anguilla anguilla (Eel Serum) (eel; CX-L-FU~~, 3-0-Me-~-Fucp,3-0-Me-o-Gab) An extremely potent source of natural anti-blood-group H ( 0 )activity ~~,~”~~~ was discovered in the serum of the eel, Anguilla ~ n g u i l l a . ~ ’ .Eel anti-H(0) agglutinins, which some investigators consider to be primitive antibodies, are found in approximately 50% of eels.21However, important differences exist between eel-serum lectins and conventional immunoglobulins; the antigenic stimulus (if any) responsible for anti-H(0) activity in eel serum has yet to be identified.671Eel sera agglutinate human 0 erythrocytes strongly, and A2 cells to some extent, The agglutination of 0 but do not react with types A, or B ce11s.21,64,65~670 cells by eel sera was readily neutralized by saliva from secretor indiv i d u a l ~ , ’ by ~ , ~soluble ~ blood-group H ( 0 ) substance, and by sugars Early related to the type 0 oligosaccharide structure. hemagglutination inhibition studies revealed that the carbohydratebinding site of the eel agglutinin was most complementary to methyl a-~-fucopyranoside.~’*”~~~’*~~~ Specifically, studies showed that L-fucose was strongly inhibitory, whereas D-fucose was not, that the pyranose form was essential, and that an a-L-glycosidic linkage, in contrast to the p-L-linkage, contributed positively to the binding of L-fucose. On comparing difucosides with milk oligosaccharides, Watkins and Morgan concluded4” that 2-O-a-~-linkedL-fucosyl residues were bound more avidly than 3- or 4-O-a-~-fucosylresidues. This fairly straightforward concept of the stereochemical features defining the specificity of eel serum was, however, complicated by an unexpected finding: extremely low concentrations (for example, 0.3 puglml) of an L-fucose-free polysaccharide from Taxus cuspidata com(670) S. Sugishita, juzenkwai-Zasshi, 40(5), 1938 (1935). (671) G. F. Springer and P. R. Desai, Vox Sang., 18, 551-554 (1970). (672) R. Kuhn and H. G. Osman, Hoppe-Seyler’s Z . Physiol. Chem., 303, 1-8 (1956).

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IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

TABLEXIX Eel Serum-0 Erythrocyte Agglutination and Eel Serum-H(O) Substance Precipitation: Sugar Inhibition Studies167*674*676

Sugar

Minimum amount Amount giving completely inhibiting 50% inhibition 4 hemagglutinating of precipitation doses (mg/ml) (pmoles)

L-Fucose and related sugars L-Fucose Methyl a-L-fucopyranoside p-Aminophenyl a-L-fucopyranoside Methyl P-L-fucopyranoside 2-0- Meth yl-~-fucose Methyl 2-O-methyl-a-~-fucopyranoside Methyl 2-O-methyl-P-~-fucopyranoside 3-O-Methyl-~-fucose 2,3-Di-O-methyl-~-fucose Methyl 2,3-di-O-methyl-a-~-fucopyranoside Methyl 2,3-di-O-methyI-P-~-fucopyranoside 2,3,4-Tri-O-methyl-~-fucose 2,3,5-Tri-O-methyl-~-fucose 2-Acetamido-2-deoxy-~-fucose

0.1-0.2 0.02 0.02 0.3 0.05 0.02 0.15 0.05-0.1 0.05 0.02 0.3 5.0 >5

1.2-1.5 0.3-0.5 0.25 13% at 2.0 0.6 0.6 30% at 2.0 0.6 0.4 0.25 2.7 1.75

D-Fucose and related sugars D-Fucose 2-O-Methyl-D-fucose 3-O-Methyl-D-fucose Methyl 3-0-methyl-a-D-fucopyranoside Methyl 3-0-methyl-/3-D-fucopyranoside 2,3-Di-O-methyl-D-fucose Methyl 2,3-di-O-methyl-a-D-fucopyranoside Methyl 2,3-di-O-methyl-~-~-fucopyranoside 2,3,4-Tri-O-methyl-D-fucose 2,3,5-Tri-0-methyl-D-fucose

Methyl a-Bfucopyranoside Methyl P-D-fucopyranoside Methyl 2-O-methyl-a-(or P-)D-fucopyranoside

inactive 2.5 0.05 0.02-0.05 0.02 0.05 0.05 0.15 1.2-2.5 >5 inactive inactive inactive

Galactose and related sugars D-Galactose L-Galactose 2-O- Methyl-D-galactose 3-O- Methyl-D-galactose 2,3-Di-O-methyl-~galactose 3,4-Di-O-rnethyl-D-galactose

2,3,4-Tri-O-methyl-~-galactose 3-O-a-D-Galactopyranosyl-D-galacl :ose

inactive inactive 2.5-5 0.1 0.1 >5 0.3 >5

inactive 0.5 0.5 0.3 0.5 0.4 0.7 1.2-2.5 inactive inactive inactive

LECTINS : CARBOHYDRATE-BINDING PROTEINS

279

pletely inhibited the agglutination of O-erythrocytes by eel serum.'18 The serological activity of Taxus cuspidata twig polysaccharide was shown to be due to its content of 2-O-methyl-~-fucose.~~~ As a consequence of this observation, Springer and his associates systematically s t ~ d i e d ' the ~ ~ interaction ,~~~ of D- and L-fucose derivatives with eel serum (see Table XIX). Unexpectedly, 2- or 3-O-methylation conferred inhibitory activity on the otherwise totally inactive, parent sugar, D-fucose. High inhibitory activity was also exhibited by 2,3-di-0methyl-D-fucose. However, further methylation at 0-4 or 0-5 greatly lessened the binding capacity. Interestingly, 2-0-, 3-0-, or 2,3-di-0methyl-L-fucose derivatives were slightly more inhibitory than the free sugar. As in the D series, further substitution at 0-4or 0-5 diminished the lectin binding. Whereas 3-O-methyl-D- and -L-fucose displayed equivalent activity, the 2-O-methyl enantiomorphs differed substantially. These findings were difficult to reconcile in terms of the concept of stereospecificity in protein-ligand interaction. Based on his observation of space-filling models, Kabat675suggested that rotation of 3-O-methyl-D-fucoseby 180"about its major axis would align this sugar with methyl a-L-fucopyranoside in such a way that centers of electronegativity, hydrogen-bonding capability, and hydrophobicity would become virtually superposable. Another feature ofthe eel-serum specificity that is unlike that ofother lectins is the existence of anomeric preference only within the L-hcose ~ e r i e s . ' ~ Thus, ' , ~ ~ ~a-L-hcopyranosides are two to five times as efficient as inhibitors as their parent compounds, whereas their p-Lglycosidically linked counterparts have half to one-third the effect. On the other hand, the anomers of methyl 3-0-methyl-~-fucopyranoside are of equivalent activity, being approximately twice as active as the parent saccharide. In the D series, over 90% of the binding energy is evidently accounted for by parts of the sugar molecule other than the anomeric carbon atom and the aglycon. A second, plant polysaccharide devoid of L-fucose, that from Sassafras albidum, also displayed strong H ( 0 ) activity when tested with eel serum (2.0 puglml gave complete inhibition of h e m a g g l ~ t i n a t i o n ~Of ~~). its constituent sugars, 3-O-methyl-~-galactoseis apparently responsible for blood-group H ( 0 ) activity. An ensuing study of methylated D-galactoses revealed that 3-0-methyl-D-galactose and 2,3-di-0(673) G . F. Springer, N. Ansell, and H. W. Ruelius, Naturwissenschuften, 43,256-257 (1956). (674) G. F. Springer, P. R. Desai, and B. J. Kolecki, Biochemistry, 3,1076-1085 (1964). (675) E. A. Kabat, Biochem. I., 85,291-293 (1962). (676) G. F. Springer, T. Takahashi, P. R. Desai and B. J. Kolecki, Biochemistry, 4, 2099-2113 (1965).

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IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

methyl-D-galactose were as active as L-fucose, although D-galactose itself was n ~ n i n h i b i t o r y . ~ ? ~ reported an extensive list of saccharides Springer and that were weakly inhibiting or noninhibiting. These included the 2-acetamido-2-deoxy derivatives of D-galactose, D-glUCOSe, D-ribose, D-talose, and D-arabinose, 3,6-dideoxy-~-and -D-xylo-hexose, L-lyxohexosulose, L-arabinose, D-fructose, and 2-deoxy-D-erythro-pentose. Three cardiac glycosides, namely, panstrosid, strospesid, and chartreusin, were effective inhibitors of eel-serum agglutinin by virtue of A p-Dthe content of p-D-linked 3-O-methyl-~-fucose(D-digitalo~e).'~~ glycoside of 2,3-di-O-methyl-~-fucose,namely, streblosid, also gave good i n h i b i t i ~ n . ' ~ ~ , ~ ~ ~ Perhaps the most unusual aspect of eel serum-carbohydrate interaction, discovered during the course of precipitin inhibition studies, was the demonstration that 3-0-methyl-D-fucose could itself function as a specific precipitinogen of eel-serum a g g l ~ t i n i n . ~Over ~ ' . ~90% ~ ~ of the eel serum anti-H(0) activity was precipitated by addition of 4 to 8 pmoles of this sugar per ml of serum. This precipitin reaction, which was also obtained with 3-O-methyl-D-galactose,was specifically inhibited by L-fucose and other known inhibitor^.^^^,^^^ The precipitinogen, D-digitalose, could be converted into an inhibitor of precipitation by methylation at 0-1or 0-2, or reduction at C-2. Other common, antiH ( 0 ) reagents, including the lectins ofLotus tetragonolobus, were not precipitated by D-digitalose. By assuming a lattice theory of precipitation, Springer and Desai6??suggested the novel idea that the complementary grouping for binding to eel-serum agglutinin might be smaller than a monosaccharide. The minimal requirements for inhibiting sugars (see Fig. 12A) were stated66to be "a methyl substituent

1

HO

1

HO

(A) (B) FIG. 1 2 . 4 A ) 3-O-Methyl-~-fucose,an Inhibitory Hapten for the Eel Agglutinin, and (B) 3-O-Methyl-~fucose,a Precipitinogen of Eel Agglutinin. [The inhibitor (A) is inverted by 180". Areas reactive with the agglutinin are marked by an arrow (t).Note the similarity between the two molecules. (From Springer and De~ai.~'?)]

(677) G. F. Springer and P. R. Desai, Biochemistry, 10, 3749-3761 (1971).

LECTINS : CARBOHYDRATE-BINDING PROTEINS

28 1

attached equatorially to a pyranose ring, an ether oxygen adjoining this methyl group, and an axial, oxygen-carrying substituent cis to the methyl group on a contiguous C atom.” Additional specific requirements for precipitinogens includess (see Fig. 12B) “three vicinal oxygens protruding from a C1 pyranose ring. The oxygen at C-3 must carry an apolar group, and the two oxygens flanking this group must be capable of hydrogen bonding. One of these latter oxygens must be equatorial and trans to the oxygen at C-3 and the other axial and cis.” Digitalose-precipitated, eel-serum agglutinin was quantitatively recovered by dialysis of the precipitate, thereby affording a novel, affinity purification of the protein.66*671*677*678 The homogeneity of the isolated agglutinin was assessed by disc gel-electrophoresis (pH 5 to lo), moving-boundary electrophoresis (pH 8.6), zone electrophoresis on paper and on cellulose acetate, immunodiffusion, and sedimentation in From the biophysical constants measured the ultra~entrifuge.~~**~~~~~~~ ( ~ 2 0 0 ,= ~ 7.2 S, DZ”o,w = 5.0 X lo7 cm2.sec-’, ij = 0.705), Springer and coworkers calculated a molecular weight of 123,000 for this almost spherical, globular protein.679Dissociation by detergent, or succinylation, gave material having a molecular weight of 40,000, and treatment with 2-mercaptoethanol yielded subunits of molecular weight 10,000. From these data, the authors67gproposed, for eel-serum agglutinin, a tertiary structure in which three subunits, each composed of four disulfide-bonded, polypeptide chains of molecular weight 10,000, are associated noncovalently. Chemical analysis of the protein revealed substantial proportions of aspartic and glutamic acid, alanine, glycine, serine, and threonine, with small proportions of methionine, tryptophan, and phenylalanine. Very little carbohydrate was found: 0.39% (by weight) comprised of 2-amin0-2-deoxyglucose.~~~ End-group analysis demonstrated equimolar amounts of N-terminal serine and alanine, and C-terminal serine and g l y ~ i n eThe . ~ ~molar ~ ratios77of monosaccharide precipitinogen to protein at equivalence was 5.73: 1. A comparative, c.d.-spectral analysis of individual, eel-serum antiH ( 0 ) specific proteins showed virtually identical patterns.680A strong, positive band centered at 197 nm suggested the /3-conformation. Furthermore, a negative band at 213 nm, weak positive bands at 235 nm, (678) G. F. Springer, P. R. Desai, and J. C. Adye, Ann. N.Y. Acad. Sci., 234, 312331 (1974). (679) A. Bezkorovainy, G. F. Springer, and P. R. Desai, Biochemistry, 10, 37613764 (1971). (680) B. Jirgensons, G. F. Springer, and P. R. Desai, C o m p . Biochem. Physiol., 34, 721-725 (1970).

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IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

-290 nm, and 295 nm, and a strong, positive band at 270 nm gave no indication of a-helical structure. The c.d. spectra were markedly different from those of human 7 s immunoglobulin.680 An alternative purification of eel-serum agglutinin was reported by Matsumoto and 0 ~ a w a . L-Fucose l~~ was coupled to starch by use of epichlorohydrin. Eel serum was chromatographed on the resulting adsorbent, and bound protein was in two peaks with glycine hydrochloride buffer, pH 3.0. Peak B (60% of the eluate) was homogeneous by gel electrophoresis at pH 8.9, and by analytical ultracentrifugation. In agreement with Springer and coworkers,679Matsumoto and O ~ a w a reported '~~ a sedimentation coefficient of 7.2 S. However, they estimated a molecular weight of 140,000 by gel filtration, a value considerably higher than that reported earlier.679 Cell-binding studies conducted with [1251]-eel-serumagglutinin 1.7 x 106receptorsites per 0 erythrocyte, with K, = 2.7 x lo6 A4-l. Binding of eel-serum agglutinin to red cells of types A, and B gave a biphasic curve, suggesting two types of receptor. Linear curves were obtained in the presence either of human anti-A, or of anti-B serum. Competitive inhibition in cell-binding studies on 0 erythrocytes was observed between eel serum, Ulex europeus I, Ulex europeus 11, and Cytisus sessil$olius lectins, suggesting that they interact with the same, or closely associated, cell-surface structures.476 In addition to H ( 0 ) substances, Matsumoto and OsawalS6reported hemagglutination inhibition by B and A substances and neuraminidase-digested, porcine submaxillary-mucin. Although Lea substance did not inhibit, a closely related milk oligosaccharide, lacto-N-fucopentaose 11, did exhibit activity in this assay. On screening 22 invertebrate extracts, Baldo and coworkers found eel-serumreactive material in 15 species.681 A fucoxylomannan from the fruiting bodies of Fomes annosus (Fr.) Cook was isolated by precipitation with purified, eel-serum agglutinin.682 2. Lotus tetragonolobus (Asparagus Pea) 2-O-Me-~-Fucp) (asparagus pea; (Y-L-Fuc~, The blood-group H ( 0 ) specific hemagglutinating activity of Lotus tetragonolobus extracts was originally documented in the 1948 report of Renkonen6, and this was confirmed by other.^.^^,^','^ In a landmark (681)B. A. Baldo, G. Uhlenbruck, and G. Steinhausen, Vor Sang., 25,398-410 (1973). (682)K.Axelsson, H.Bjomdal, S. Svensson and S. Hammarstrom, Acta Chem. Scand., 25,3645-3650(1971).

LECTINS: CARBOHYDRATE-BINDING PROTEINS

283

study, Morgan and Watkins22attempted to establish the nature of the serological specificity of seed extracts by investigating the ability of blood-group substances and simple sugars to inhibit hemagglutination. Included in this investigation were extracts prepared from Lotus tetrugonolobus. In agreement with the results of Renkonen,6they22found that the extract agglutinated type 0 cells considerably better than A2 cells, whereas types A1, B, and AB were not agglutinated. Lotus-0 cell agglutination was inhibited by purified, human, or porcine, H(0)active blood-group substance, and by L-fucose, alone of the four constituent monosaccharides of the ABO blood-group substances.22Furthermore, methyl a-L-fucopyranoside was twice as active as the parent sugar, whereas the /3 anomer and 2-deoxy-~-Zyxo-hexosewere half as active. These early inhibition-studies were instrumental in establishing that a-L-fucosyl residues are important, immunodominant structures of H-active blood-group substances. Kuhn and 0sma1-1~'~ confirmed, and extended, the work of Morgan and Watkins,22 and suggested that serologically active fucosides must be ofthe L configuration and in the a-1;-linked, pyranose form. The hemagglutinin of Lotus seeds has been purified by three, different, affinity techniques based on its binding capacity for a - ~ fucopyranosides. Yariv and specifically precipitated the lectin with the trifunctional fucosyl dye, 1,3,5-tri-(p-a-~-fucosyloxyphenylazo)-2,4,6-trihydroxybenzene. Following dissolution of the precipitate in 2% L-fucose, an ion-exchange resin removed the dye, leaving a protein preparation that appeared homogeneous by sedimentation analysis. A molecular weight of -107,000 was determined by sedimentation equilibrium. Kalbs= subsequently separated the aforementioned protein preparation into three components by ion-exchange chromatography on DEAE-cellulose. Sedimentation equilibrium established the following molecular weights: component A, 120,000; component B, 58,000; and component C, 117,000 (designated in order of salt elution from DEAE-cellulose at pH 7.6). A refined, affinity purification for L-fucose-binding Lotus proteins was reported by Blumberg and ~ o w o r k e r s . N-(6-Amino-l-oxo~~~,~~~ hexy1)-/3-L-fucopyranosylamine was coupled to cyanogen bromideactivated Sepharose 4B. The three proteins, A, B, and C, were bound by (683) A. J. Kalb, Biochim. Biophys. Acta, 168, 532-536 (1968). (684) M. Blumberg, J. Hildesheim, J. Yariv, and K. Wilson, Biochim. Biophys. Acta, 264,171-176 (1972). (685) J. Yariv, A. J. Kalb, and M. Blumberg, Methods Enzymol., 28 (Part B), 356-360 (1972).

284

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

the adsorbent at 4", whereas only A and C were bound at 25". Proteins A and C were further separated by ion-exchange chromatography. Each of the three gave a single band in disc gel-electrophoresis. Pereira and KabatZo0adsorbed the L-fucose-binding proteins on polyleucyl hog A H substance. The dialyzed, L-fucose eluate gave a single band against antiserum to the crude seed-extract, both by double diffusion in agar and by immunoelectrophoresis. However, disc gelelectrophoresis, at pH 9.3 and 2.7, revealed three components; these were subsequently separated by preparative, isoelectric focusing. Fraction I, isoelectric point pH 5.4, was identified as the component B of Kalbsm;fraction 11, isoelectric point pH 6.2, as component C; and fraction 111, isoelectric point pH 7.1, as component A. Pereira and KabaPooinvestigated the subunit structure of the three components by dodecyl sodium sulfate disc-gel electrophoresis. With or without 2-mercaptoethanol, proteins A (111)and B (I) each exhibited a single band, ofmolecular weight 27,800, whereas protein C (11)gave a band of material having molecular weight 27,000. Equivalent fucosebinding weights of 31,000,31,000, and 32,000 for A, B, and C, respectively, were reported by Blumberg and coworkers.6s4KalbsS3determined equivalent binding-weights of 28,0OO,29,000, and 30,000 for A, B, and C. It appears likely that A and C are tetrameric, whereas B is a dimeric protein. Chemical analysis of the three, related, L-fucose-binding proteins revealed certain similarities, but sufficient differences to establish their unique character. Thus, component A contains 8%of hexose and 1.4% of 2-amino-2-deoxyglucose (by weight); component B, 4% of hexose and 0.8% of 2-amino-2-deoxyglucose; and component C, 8% of hexose and 1.2% of 2-amino-2-deoxyglucose.683Each protein is devoid of cysteine, extremely low in methionine content, and relatively rich in aspartic acid (asparagine), threonine, and serine. However, they differ noticeably in the relative content of the dibasic amino acids.683 The capacity of simple sugars to inhibit Lotus extract-0 erythrocyte agglutination was an important observation in early studies concerning . ~ ~initial ~ ~ ~re* ~ ~ ~ ~ ~ the chemical basis of serological s p e ~ i f i ~ i t yThese ports prompted Springer and c o w o r k e r ~ ' ~ to ' , ~synthesize, ~~ and test, methyl ethers of fucose and their methyl glycosides, as well as an extensive list of unrelated sugars, as inhibitors of Lotus extract-0 cell agglutination. In accordance with earlier work, methyl a - ~ fucopyranoside elicited the greatest inhibition. The following sugars were of approximately equivalent potency (half as active as methyl a-L-fucopyranoside): p-aminophenyl a-L-fucopyranoside, L-fucose, 3-02-O-methyl-~-fucose, methyl 2-O-methyl-a-~-fucopyranoside, methyl-L-fucose, 2,3-di-O-methyl-~-fucose, and methyl 2,3-di-0-

+

LECTINS: CARBOHYDRATE-BINDING PROTEINS

285

methyl-a-L-fucopyranoside. The p-L-linked methyl glycosides of L-fucose and 2-O-methyl-~-fucosehad one-tenth the effect of methyl a-L-fucopyranoside. Fucoses methylated at 0-4, D-fUCOSe, S-deoxy-~xylo-hexose (colitose), and fucofuranosides were inactive. Although these experiments were conducted on a crude, lectin preparation, they illuminate important features of Lotus lectin-carbohydrate interaction, namely, the common requirement of the three constituent components for the pyranoid (as opposed to the furanoid) form, the 0-2 and 0-3 atoms, and the hydrogen atom of the 4-hydroxyl group of the L-fucose configuration. During the course of their work, Springer and W i l l i a r n ~ o nmade '~~ the startling observation that, although D-fucose was totally inactive, 2-0-methyl-D-fucose possessed considerable inhibiting capacity. Methyl glycosidation eliminated the binding of this D-fucose derivative. Kabat675explained this apparent anomaly introduced by 2- or 3-0-methylation on the basis of structural similarities between D- and L-fucose derivatives as noted in three-dimensional models. Thus, a similarity in profile and in regions of low hydrogen-bonding capability is evident between %O-methyl-~-fucoseand L-fucose, if the latter is rotated 180" about its horizontal axis. This phenomenon is even more apparent in the binding of D-fucose derivatives to the L-fucose-binding lectin of the eel167(see Section V1,l). Pereira and KabatZo0undertook a careful study of Lotus lectin carbohydrate-binding specificity employing blood-group-active substances, related oligosaccharides, and derivatives of L-fucose as inhibitors of precipitation of affinity-purified lectin with blood-group H substance. The data (see Table XX) indicated that the sugar-binding sites of components A, B, and C are essentially homogeneous with respect to specificity, although they differ in affinity. The ratios of relative inhibitory capacity for three L-fucose derivatives were constant for the three proteins and the unfractionated mixture, thereby demonstrating their complementarity. However, the published association constants for methyl a-L-fucopyranoside binding are different: 1.2 x lo4 M - l , 0.6 x lo4M - * , and 3.7 x lo4M-' for A, B, and C, respecti~ely.~~~ The immunochemical studies of Kabat and coworkers200,686 revealed some striking subtleties in Lotus tetragonolobus sugar-specificity. Precipitin reactions between the lectin and several blood-group H substances demonstrated strong affinity of the lectin for this determinant. H substances precipitated 83438% of the purified Lotus lectin at (686) L. Rovis, E . A. Kabat, M. E.A. Pereira, and T. Feizi, Biochemistry, 12, 5355-

5360 (1973).

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

286

TABLEXX Sugar Inhibition2O0of the Precipitin Reaction Between Human H Substance and Lotus tetragonolobus Lectins A, B, and C ~~

~

Lectin fraction

Sugar inhibitor

Nanomoles for

50% inhibition

Relative inhibiting power compared to that of methyl a-Lfucopyranoside

A (111)

Methyl a-L-fucopyranoside 2'-O-~-Fucosyllactose L-Fucose

30 90 110

100 33 27

B (1)

Methyl a-L-fucopyranoside 2'-O-~-Fucosyllactose L-Fucose

13 31 41

100 39 29

c (11)

Methyl a-L-fucopyranoside 2'-O-~-Fucosyllactose L-Fucose

90 240 330

100 38 27

Unfractionated

Methyl a-L-fucopyranoside 2'-O-~-Fucosyllactose L-Fucose

50 150 180

100 33 28

equivalence. Interestingly, precipitin studies of human blood-group A2 substances showed them to be -33% to 70%as active as H substances, but human Al blood-group substances showed no reaction at all. Lea substances precipitated the lectin, whereas B substances gave relatively poor precipitation, and I-active precursor (as well as i-active) substances did not react. Of all oligosaccharides investigated by Pereira and Kabat,20° an H-active difucosyl oligosaccharide, H JS RIMS2.5, exhibited the strongest inhibition (see Table XXI). It was, in fact, superior to methyl a-L-fucopyranoside and two monofucosyl oligosaccharides tested ( H JS RL 0.75, 2-fucosyllactose). All of these oligosaccharides have a main chain of 4-O-@-D-galactopyranosyl-D-g~ucoseor 2-acetamido-2-deoxyD-glUCOSY1 residues, which is representative of the type 2 chains found in blood-group substances.ss7The L-fucosyl residues are substituted at the 2-hydroxyl group of galactose and, in difucosyl derivatives, the 3-hydroxyl of glucose (or 2-acetamido-2-deoxy-~-glucose).However, if the fucosyl residue(s) is attached to a type l chain (~-O-@-Dgalactopyranosyl-D-glucose or 2-acetamido-2-deoxyglucose),the in(687) E. A. Kabat, Ado. Chem. Ser., 117,334-340 (1973).

287

LECTINS: CARBOHYDRATE-BINDING PROTEINS

TABLEXXI Inhibition of Human Blood-group H Substance-Lotus tetragonolobus Lectin Precipitation by Sugars and Oligosaccharides200of Low Molecular Weight"

Sugar

LX-L-FUC

(Y-L-FUC

1

1

.1

.1

Nanomoles for 50% inhibition

2 3 P-D-Gal-( 1+4)-P-~-GlcNAc-(1-+6)-R (H JS RIMS2.5)

40

Methyl mL-hcopyranoside Lacto-difucotetraose*

50 80

(Y-L-FUC 1

.1 2 P-D-GaI-(1+4)-P-~-GlcNAc-(1+6)-R (H JS RL0.75) 2'-0-~-Fucosyllactose L-Fucose Lacto-N-fucopentaose 111 3-0-L-Fucosyllactose Lacto-N-fucopentaose 11 Lacto-N-fucopentaose I Lacto-N-difucohexaose I 3-O- Methyl-L-fucose Fucitol Urine-B oligosaccharide Urine-A oligosaccharide

105 150 180 450 575 700 inactive inactive inactive inactive inactive inactive ~~

"Data of Ref. 200, using affinity-purified, unfractionated lectin. bFor chemical structures of oligosaccharides, see Ref. 200.

hibiting capacity is abolished (for example, lacto-N-fucopentaose I, lacto-N-difucohexaose I). Likewise, substitution of the type 2 chain galactose at C-3 with 2-acetamido-2-deoxy a-D-galaCtOSe (A-active urine oligosaccharide), or at 0 - 3 with a-D-galactose (B-active urine oligosaccharide) negates the inhibitory activity of the oligosaccharide. The authorszo0concluded that Lotus lectin manifests a high degree of specificity for unsubstituted, type 2 chains of bloodgroup H-active substances. The structural constraints demonstrated for Lotus lectin-oligosaccharide interaction were cited by Pereira and KabaPoO in their re-examination of A, and Az blood-group specificity. As

288

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

Lotus lectin does not react with A,, they suggested that all type 2 chains of A, substance must be substituted by 2-acetamido-2-deoxy-a-~galactosyl groups, thereby blocking their reactivity. Similarly, reaction of Lotus lectin with type A2 material indicated the existence of some unsubstituted, type 2 chains on A2 substances. The chemical studies of Kabat and coworkersasaestablished two types of type 2 determinant in H substance. If a difference in 2-acetamido-2-deoxy-~galactosyltransferase substrate-specificity between A, and A2 individuals is postulated, the authors suggested that simultaneous reactivity of A2 substances with anti-A antisera and Lotus lectin may be explained by substitution of one type 2 chain-structure with the blood-group A determinant sugar, whereas the other unsubstituted, type 2 chainstructure would remain available to react with the lectin. A qualitative difference between Al and A2 substances would then exist, together with a quantitative difference in A-determinants. A fraction enriched in type 2 chains was obtained by fractionating A2 substance (cyst 14) on a Lotus-Sepharose column.526Fractionation of pooled, hog gastricH blood-group glycoproteins on the same immobilized mucin A Lotus column yielded fractions showing only A, only H, or AH activity.526 In studies of secretor and nonsecretor saliva antigens, Grundbacher ~ ~ S ~ a ~strong, ~ * ~but ~ diffuse, ~ precipitin band and C O W O ~ ~ observed between nonsecretor saliva (A, By or 0 donors) and the Lotus lectin, whereas secretor saliva of all types formed a strong, diffuse band, as well as a weaker band, by Ouchterlony gel-diffusion. The two bands were poorly separated, complicating interpretation of the patterns. The weaker band appeared identical to the antigen reactive with Ulex europeus, and was presumed to be the H antigen. However, the stronger band appeared, by Ouchterlony diffusion, to differ from the H, Lea, and Leb antigens. The antigen described was not present in saliva, or on red cells of the Oh (Bombay) type. Inbar and coworkersas9determined that r3H]-acetylatedlectin did not bind to the surface of normal cell-lines (hamster and rat embryo, and mouse 3T3 cells) or to the transformed counterparts of these cells. Trypsinization did not alter these results. The authors concludedssgthat L-fucosyl residues were not exposed on the cell surface. In view of the specificity studies of Kabat and coworkers,200*686 this interpretation may be incorrect; L-fucosyl residues may be rendered sterically inaccessible by glycosylation of neighboring sugar residues. (688) P. W. Napier, D. L. Everhart, and F. J. Gmndbacher, Vox Sang., 27, 447-458

+

(1974). (689) M. Inbar, I. Vlodavsky, and L. Sachs, Biochim. Biophys. Acta, 255, 703-708 ( 1972).

LECTINS : CARBOHYDRATE-BINDING PROTEINS

289

3. Ulex europeus I (Gorse or Furze Seed) (gorse or furze seed; C X - L - F U ~ ~ ) Since Cazal and Lalaurie's discoverys1of anti-H(0) hemagglutinating activity in extracts of three Ulex species (Ulex provincialis L., Ulex jussiaei Webb, and Ulex europeus L.), U . europeus extract has become a standard, serological reagent.14 It is used in typing 0 blood, in distinguishing Az from Al, and in the assessment of secretory status (the occurrence of H-active substance in s a l i ~ a ) . ' ~ -0' ~cells are strongly agglutinated, Az and AzB react more weakly, whereas A, and AIB are not aggl~tinated.'~-'~~~~,~~*~~~ Secretor saliva, irrespective of type, inhibits O-erythrocyte agglutination by U . europeus e ~ t r a c t . ' ~ There are two lectins of distinct, sugar-binding specificity in Ulex extract^.'^^^^^^ Using ethanol fractionation, FloryZz5initially discriminated between Ulex I, which is readily inhibited by L-fucose derivatives, and Ulex 11, which binds P-D-glucosides. (A comprehensive discussion of Ulex I1 purification and properties is presented in Section 111,5.)Flory's 50-70% ethanol precipitate enriched in Ulex I, was slightly inhibited by D-arabinose, D-ribose, and D-lyxose, in addition to ~ - f u c o s eThis . ~ ~ ~preparation agglutinated human, epithelial, cheekcells of secretory individuals Matsumoto and Osawa196~208~226~509 isolated and studied both Ulex I and I1 (see Section 111,5).Ulex europeus hemagglutinin I, which precipitated between 0 and 40% saturation with ammonium sulfate, was purified by CM-cellulose chromatography followed by gel filtration.226 (The same authors were unable to adsorb U . europeus I to insoluble L-fucose-substituted starch.149)Homogeneity of the preparation was assessed by ultracentrifugation and gel filtration. Hemagglutination of 0 erythrocytes by hemagglutinin I was completely inhibited by L-fucose and by secretor saliva. These authors149reported an szo,w value of 6.5S,a molecular weight of 170,000,and an amino acid content rich in aspartic acid and serine, but relatively poor in methionine and cysteine. The protein had 3.8%(by weight) of neutral sugar and 1.4% of a hexosamine. By using an a-L-fucosyl-poly(acry1amide) adsorbent, with L-fucose elution, Hoiejii and Kocourek obtainedag0a pure preparation of U. europeus I, as judged by cellulose acetate electrophoresis and sedimentation analysis. A major and a minor protein component were observed on poly(acry1amide)disc gel-electrophoresis, pH 8.9. In contrast to the protein reported by Matsumoto and Osawa,226Hor'ejii and K o ~ o u r e determined k~~~ a molecular weight of 46,000by sedimentation (690) V. HolejG and J. Kocourek, Biochim. Biophys. Acta, 336,329-337 (1974).

290

IRWIN J, GOLDSTEIN AND COLLEEN E. HAYES

equilibrium centrifugation, 40,000 by poly(acrylamide) gel electrophoresis in dodecyl sodium sulfate, and 60,000-65,000 by gel filtration. No observable dissociation of the protein occurred upon reduction, or dissolution in 4 M urea. Although the amino acid analysis was in good agreement with earlier results, the neutral sugar determined by Ho?ejs’i and Kocourekago(7.2%) was twice that determined previously.226A single polypeptide chain was implicated by the finding of only N-terminal serine. Interestingly, EDTA had no effect on the hemagglutinating activity, although the lectin contained bound metals (two calcium ions and one zinc or manganese ion per 46 kg of protein).690 A second, affinity-purification scheme was developed by Frost and coworkers691; they synthesized the mixed 6-aminohexyl ~ , P - L fucopyranosides, and coupled these to CNBr-activated Sepharose 4B. Elution, by L-fucose solution, ofthe resin-adsorbed lectin gave 13mgof purified protein from 100 g of seeds. The sample was homogeneous by gel electrophoresis at pH 4.5 and pH 8.1, and by immunodiffusion against specific, rabbit antiserum.691As evidence of its biological purity, 0 erythrocyte agglutination was 97% inhibitable by 10 mM L-fucose. In contrast to the results of Matsumoto and OsawazZaand HoZejBi and K o c o ~ r e kFrost , ~ ~ ~and coworkersss1reproducibly observed, in dodecyl sodium sulfate gel-electrophoresis, two closely similar bands that had molecular weights of 31,000 and 32,000. (U. europeus lectin prepared by elution of formalized erythrocytes with L-fucose also gave two bands in dodecyl sodium sulfate e l e c t r o p h ~ r e s i s . ~ ~ ~ ) Characterization of the carbohydrate-binding specificity of Ulex lectin I is still very rudimentary. All reported studies applied the hemagglutination inhibition technique, but used only a few of the appropriate sugars necessary to the defining of binding specificity. Make1a78found that a crude extract from Ulex seeds was inhibited by L-fucose and salicin, whereas Kriipe7’did not include L-fucose among the sugars that inhibited Ulex-0 erythrocyte agglutination. Inhibition analysis of purified U . europeus I revealed a binding site complementary to a - ~ - f u c o s i d e s .Ally1 ~ ~ ~a-L-fucopyranoside ~~~~-~~~ and 2’-O-~-fucosyllactosegave strong inhibition of hemagglutination, whereas L-fucose, lacto-N-fucopentaose I, and lacto-N-fucopentaose I1 were slightly less e f f e c t i ~ e . ’The ~ ~ *monosaccharides ~~~ L-arabinose, D-ribose, D-galactose, D-glucose, D-mannose, 2-acetamido-2-deoxy-~and the disaccharides glucose, and 2-acetamido-2-deoxy-~-galactose, (691) R. G. Frost, R. W. Reitherman, A. L. Miller, and J. S. O’Brien, A n d . Biochem., 69, 170-179 (1975). (692) T. Osawa and I. Matsumoto, Methods Enzymol., 28, Part B, 323-327 (1972).

LECTINS : CARBOHYDRATE-BINDING PROTEINS

29 1

lactose, maltose, cellobiose, and gentiobiose were noninhibitory.1g63690-692 In addition to 1mM L-fucose, Frost and coworkers noted6** that 50 mM D-mannose and 10 mM methyl a-D-mannopyranoside gave 50% inhibition of 0 cell agglutination. We note a possible explanation in the stereochemical relationship between L-fucose and D-mannose: rotation of L-fucopyranose by 180"about its horizontal axis results in its 4-, 3-, and 2-hydroxyl groups becoming superposable on the 2-, 3-, and 4-hydroxyl groups of D-mannopyranose. Complex, blood-group-active substances have been employed as ~~~~~ and ~ Matsumoto found6s2 inhibitors of h e m a g g l u t i n a t i ~ n . 'Osawa inhibition of both Ulex lectins by human A-, B-, and H-active substances, whereas Lea substance and porcine, submaxillary mucin bound only to Ulex 11.Chuba and coworkers693reported that substances from porcine stomach (A-active), equine stomach (B-active), and baboon stomach (A-, B-, and O-active substances), as well as human A-, B-, and O-active substances, bound equally well to both U . europeus lectins. However, porcine, submaxillary mucin and nonsecretor saliva inhibited only Ulex I1 activity. VII. OTHERLECTINS

1. Phaseolus vulgaris (Red Kidney-bean) Lymphocyte mitogenicity, an intriguing and distinctive biological activity of several lectins, was first discovered in red kidney-bean (Phaseolus vulgaris) extract^.^^^^^^ (For reviews of lectin-induced mitogenesis, see Refs. 35-37a.) Biochemical studies on this phytohemagglutinin (regrettably termed PHA, see Section I,4) have been complicated by the large number of varieties of P . vulgaris investigated; red kidney-bean, black kidney-bean, wax bean, pinto bean, and navy bean are all members of this genus and species. Further complexity is introduced by the multifarious biological properties that various species exhibit. They possess leuco- and erythro-agglutinating activity and the ability to induce lymphocyte blast transformation with accompanying stimulation of DNA, RNA, and protein synthesis that culminates in cell division. ~ and Raubitschek discovered the In the early 1 9 0 0 ' ~Landsteiner hemagglutinating properties of kidney-bean Dorset and HenleyI6 later used navy-bean extracts to separate cellular elements (693) J. V. Chuba, W. J. Kuhns, J. D. Oppenheim, M . S. Nachbar, and R. F. Nigrelli, Immunology, 29, 17-30 (1975). (694) D. A. Hungerford, A. J. Donnelly, P. C. Nowell, and S. Beck, Am. J . Hum. Genet., 11, 215-236 (1959).

292

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

from blood, in preparing an antiserum against hog cholera. Goddard and MendelGg5partially purified the navy-bean hemagglutinin, and characterized it as a protein that clumped the erythrocytes of chicken, dog, duck, man, mouse, rabbit, and rat. The demonstration by Li and Osgood’s that Phaseolus vulgaris and Phaseolus communis beanextracts could be used to separate leucocytes from erythrocytes proobmoted isolation of the proteins in pure form. Rigas and OsgoodGg6 tained from P . vulgaris a purified “mucoprotein” which they freed of polysaccharide by dissociation at pH 1.0. The protein was homogeneous in electrophoresis (pH 2.0 to 8.0),had PI 6.5, and contained 3.4% of reducing substance.696 It agglutinated human erythrocytes irrespective of the blood-group type.6g6 Further purification of the red kidney-bean lectin was conducted by Rigas and Johnson by using ammonium sulfate fractionation, anion exchange, and gel-permeation c h r o m a t ~ g r a p h y .The ~ ~ ~homogene,~~~ ous glycoprotein, molecular weight 128,000, contained alanine as the only detectable N-terminal amino acid.697*6gs The lectin was rich in aspartic acid, leucine, serine, and threonine, but low in sulfurcontaining amino acids .697*698 Prolonged incubation of the agglutinin in 8.0 M urea, followed by starch gel-electrophoresis in 8.0 M urea, gave eight discernible components which were interpreted as eight unique subunits. The hemagglutinating and mitogenic activities of the lectin were associated with a single molecule, as shown by adsorption to erythrocyte ~ t r o m a . ” ~Other * ~ ~ ~investigators speculated that the lymphocyte-stimulating and agglutinating activities of the kidney bean might reside on separate r n ~ l e c u l e s . ~ Repeated ~ ~ - ~ ~adsorption ~ of partially purified, Phaseolus vulgaris extracts with erythrocytes gave a supernatant solution that exhibited mitogenic activity and was solely leucoagglutinating; both activities were removed by leucocyte adsorp(695)V. R. Goddard and L. B. Mende1,J. Biol. Chem., 82,447-463 (1929). (696)D . A. Rigas and E. E. Osgood,J. Biol. Chem., 212,607-615 (1955). (697)D. A. Rigas and E. A. Johnson, Ann. N.Y. Acad. Sci., 113,800-818(1964). (698)D.A. Rigas, E. A. Johnson, R. T. Jones, J. D. McDermed, and V. V. Tisdale, in “Chromatographie et Methodes de Separation Immediate,” G. Parissakes, ed., Association of Greek Chemists, Athens, Greece, 1966,Vol. 11, pp. 151-223. (699)J . Bojeson, R. Bouveng, S . Gardell, b. Nordbn, and S. Thunell, Biochim. Biophys. Acta, 82,158-161 (1964). (700)T.Punnett and H. H. Punnett, Nature (London), 198,1173-1175(1963). (701) P. Barkhan and A. Ballas, Nature (London), 200, 141-142 (1963). (702)A. Rivera and G. W. Mueller, Nature (London), 212, 1207-1210 (1966). (703)C. T.Mordman, A. de la Chapelle and R. Grasbeck, Acta Med. Scand. Suppl.,

412,49-58(1964). (704)T. Weber, C. T. Nordman, and R. Grasbeck, Scand. J . Haematol., 4, 77-80 (1967).

LECTINS : CARBOHYDRATE-BINDING PROTEINS

293

t i ~ n . ~By Ousing ~ chromatography on SE-Sephadex C-50, Weber and his colleagues704separated P . vulgaris preparations into two lymphocytestimulating fractions: one that was solely leucoagglutinating and lymphocyte-stimulating, and a second that was both strongly erythroagglutinating and capable of agglutinating mixtures of leucocytes and erythrocytes. The discovery that erythroagglutinating activity was inactivated by 8.0 M urea at a moderately high rate, whereas mitogenic activity was relatively stable, reinforced the suggestion that these two activities might reside on different proteins (or Indeed, Rigas and Head705resolved the P . vulgaris lectin into eight components by poly(acry1amide) gel-electrophoresis in 8.0 M urea-borate buffer, pH 8.7. The eight bands (numbered in reverse order of migration towards the anode) stained for both protein and carbohydrate. Band 2, and, to a somewhat lesser extent, band 3, were the most potent erythroagglutinins, displaying no detectable mitogenicity. Bands 5 and 6 exhibited the highest mitogenic activity towards human peripheral lymphocytes, with only marginal erythroagglutinating It was hypothesized that different ratios of mitogenic to erythroagglutinating activities might reflect combinations of a mitogenic and an erythroagglutinating subunit in various ratios .697*705 Analysis of tryptic glycopeptides from a highly purified, lectin sample suggested that at least eight heteropolysaccharide chains of two or more different types were p r e ~ e n t . ~ ~ ~ . ~ ~ ~ Commercially available phytohemagglutinin (PHA-P) prepared from red kidney-beans (Phaseolus vulgaris) gave 17 different protein bands when analyzed by poly(acry1amide) gel-electrophore~is.~~~ The same sample, chromatographed on CM-Sephadex and Sephadex G-150, yielded several, distinct, mitogenic proteins having differing hemagglutinating capacity.708The most potent mitogen isolated (LPHA) was homogeneous by several criteria; it also had considerable leucoagglutinating activity.?08A mixture of (at least two) closely related proteins (H-PHA) possessing 250 times the hemagglutinating activity of L-PHA was also isolated. Slightly less mitogenic than L-PHA, this material possessed some leucoagglutinating activity.708The amino acid and carbohydrate compositions of the two molecular species were (705) D. A. Rigas and C. Head, Biochem. Biophys. Res. Commun., 34, 633-639 ( 1969). (706) D. A. Rigas, C. Head, and C. Eginitis-Rigas, Physiol. Chem. Phys., 4, 153-165 (1972). (707) E. A. Johnson and D. A. Rigas, Physiol. Chem. Phys., 4,245-256 (1972). (708) L. W. Allen, R. H. Svenson, and S. Yachnin, Proc. Natl. Acad. Sci. U.S.A.,63, 334-341 (1969).

294

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

similar, but H-PHA contained about twice as much carbohydrate as L-PHA (both contained 2-amino-2-deoxyglucose and mannose, with lower proportions of xylose and arabinose or fucose), and had a slightly higher molecular weight, as determined by molecular sieving.708 Two distinct subunits in the P . vulgaris lectins were reported b y Allan and C r u m p t ~ nand ,~~ confirmed ~ by Oh and Conrad,710Weber and coworkers,711and Yachnin and his c o l l e a g ~ e s . ~In~ ~fact, - ~ ~red ~ kidney-bean lectins comprise a family of five mitogenic glycoprot e i n ~ ~ each ' ~ ; contains four subunits held together by noncovalent The individual isolectins contain various proportions of L and R s u b ~ n i t s , "in~a manner reminiscent oflactic acid dehydrogenase i s ~ z y m e sThe . ~ ~subunits ~ can be distinguished by N-terminal amino acid sequence, isoelectric point, and biological properties.712 Leucoagglutinin (L-PHA) consists of four identical L-subunits, with serine as the N-terminal amino acid and having PI 5.25; it exhibits strong affinity for lymphocyte receptors, but little for those of red (H-PHA) contains four identical, b l o o d - ~ e l l sThe . ~ ~hemagglutinin ~~~~~ R subunits having an N-terminal amino acid sequence beginning with alanine, and712 PI 5.95; this form exhibits a strong affinity for erythrocyte-membrane receptors.714The erythroagglutinin (H-PHA), which is probably the molecular species isolated by Rigas and coworke r ~ ,was ~ reported711 ~ ~ * ~to ~have ~ a somewhat higher molecular weight (150,000)than the leucoagglutinin (L-PHA). The three intermediate, mitogenic glycoproteins are tetramers that contain various proportions of L and R subunits (LR,, LzRz,L3R).A schematic representation of the five P . vulgaris isolectins is depicted in Fig. 13. The mixed,

FIG. 13.-Schematic Representation of the Tetrameric Structure of the Five Isolectins from Phaseolus vulgaris. [Each form consists of various proportions of L (N-terminal Ser) and R (N-terminal Ala) subunits. From Ref. 630. (Published by permission of Proc. Natl. Acad. Sci. U.S.A.)] (709) D. Allan and M. J. Crumpton, Biochem. Biophys. Res. Commun., 44, 1143-1148 ( 1971). (710) Y. H. Oh and R. A. Conard, Arch. Biochem. Biophys., 152,631-637 (1972). (711) T. W. Weber, H. Aro, and C. T. Nordman, Biochim. Biophys. Acta, 263,94-105 (1972). (712) J. B. Miller, C. Noyes, R. Heinrikson, H. S. Kingdon, and S. Yachnin,J. E x p . Med., 138,939-951 (1973). (713) S. Yachnin and R. H. Svenson, Immunology, 22, 871-883 (1972).

LECTINS: CARBOHYDRATE-BINDING PROTEINS

295

erythrocyte-lymphocyte agglutination activity (mixed agglutination) displayed by these isolectins reflects their hybrid structures. These hybrid species also evoke a lymphocyte mitogenic response in proportion to their content of L subunits. Interestingly, treatment of the agglutinins with sodium metaperiodate affected only slightly their agglutinating activity while abolishing their lymphocyte-stimulating activity.?' The L and R subunits have been isolated in homogeneous form by isaelectric focusing in 8 M urea.630,712 They have identical molecular weights, and both lack methionine and cysteine. The subunits differ in amino acid sequence from residues 1to 7 (from theN-terminus), but are identical in positions 8-24 and in their three C-terminal residues.630 The twelfth residue in each subunit is a glycosylated asparagine residue; the carbohydrate composition of the R and L subunits is identical.630Thus, there are striking similarities between the subunits, and it would appear that the differences in biological properties between the two species are the result of relatively restricted differences in their primary structure.630It is, indeed, interesting that a similar set of five tetrameric isolectins composed of two different subunits (A and B) has also been discovered628for the Bandeiraea simplifdia I lectin (Section V73). L e ~ c o a g g l u t i n i n ~ ~ ~ ~(L4) ~ ~ was ' , ~p~ ~ .r ~i ~f "i ~e ~don ~~ a~preparative ~,~~~ scale by fractional precipitation with ethanol, followed by ionexchange chromatography on DEAE-cellulose and SP-Sephadex, and exclusion chromatography on Sephadex G-150. The crystalline glycoprotein was homogeneous by numerous physicochemical and immunochemical riter ria.^^^,^'^ It is composed of four L-subunits, molecular weight 31,000 (compare 34,000 given in Ref. 712), and has an aggregate molecular weight7I8of126,000( s & =~6.87 S). This glycoprotein lacks sulfur-containing amino acids, but contains high proportions of aspartic acid, leucine, serine, threonine, and valine; it also contains 2-amino-2-deoxyglucose and mannose as the only carbohydrates718 (compare Ref. 629). In addition, leucoagglutinin also contains Mn2+and Ca2+, which are essential both for lymphocyte-stimulating and leucoagglutinating activities718(compare Ref. 151). (714) S. Yachnin, L. W. Allen, J. M. Baron, and R. H. Svenson, Cell. Immunol., 3, 569-589 (1972). (715) T. H. Weber, Scand. J . Clin. Lab. Inoest., 24, S u p p l . 111, 1-80 (1969). (716) T. Weber and R. Grasbeck, Scand. /. Clin. Lab. Inoest., 21, S u p p l . 101, 14 ( 1968). (717) N. 0. Kaplan, Brookhawen Symp. Biol., 17, 131-153 (1964). (718) V. Risanen, T. H. Weber, and P. Grisbeck, Eur. J . Biochem., 38, 193-200 (1973).

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IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

Resolution of affinity-purified, red kidney-bean lectin by ionexchange chromatography afforded71Bafive distinct, homogeneous glycoproteins, each of molecular weight 115,000 (&4,130).The tetrameric isolectins (termed E4, LIE3,L2E2,L3E1, and L4)differed in properties in the expected way: E4 was the most potent erythroagglutinin (active at nanogram concentrations), L4was the most potent lymphocyte mitogen, and intermediate forms displayed both properties.71sa Dissociation in vitro of the native isolectins in 6 M guanidinium chloride, followed by removal of dissociation agents, led to reconstitution of the original, isolectin The P . vulgaris lectins have now been isolated by affinity chromatography on columns of Sepharose conjugated with porcine thyroglobulin14BJs4 and fetuin,lS6both of which glycoproteins bind the lectin. In light of the evidence, it is difficult to reconcile it with a report that the RNA- and DNA-stimulating activity of Phaseolus vulgaris extracts is not due to proteins.71B A hemagglutinin from the wax bean (Phaseolus vulgaris; var. Sure Crop Stringless Wax) was purified by Takahashi and coworkers720;its amino acid distribution was similar to that of the red kidney-bean lectins (no sulfur-containing amino acids, and high proportions of aspartic acid, serine, and thre~nine).~*O The oligosaccharide chain(s) reportedly contained (in addition to mannose and 2-amino-2deoxyglucose) glucose, arabinose, galactose, fucose, and xylose. In view of the difficulty in removing polysaccharides from the lectin, some of the carbohydrate may result from an impurity. Supporting this suggestion, the same investigators isolated, from the lectin, a glycopeptide of carbohydrate composition grossly different from that of the original g l y c o p r ~ t e i nOn . ~ ~the ~ other hand, Sela and coworkers722isolated from the wax bean (Phaseolus uulgaris; var. Brittle-Wax) two hemagglutinins that also contained arabinose, glucose, galactose, and fucose, in addition to mannose. Moreover, the lectins were tetramers (subunit molecular weight 30,000)of molecular weight 125,000+5,000. (718a) R. D. Leavitt, R. L. Felsted, and N. R. Bachur,]. Biol. Chem., 252,2961-2966 (1977). (718b) R. L. Felsted, M. J. Egorin, R. D. Leavitt, and N. R. Bachur,]. Biol. Chem., 252, 2967-2971 (1977). (719) M. L. Goldberg, W. Rosenau, and G. C. Burke, Proc. Natl. Acad. Sci. U.S.A., 64,283-289 (1969). (720) T. Takahashi, P. Ramachandramurthy, and I. E. Liener, Biochim. Biophys. Acta, 133, 123-133 (1967). (721) T. Takahashi and I. E. Liener, Biochim. Biophys. Acta, 154,560-564 (1968). (722) B.-A. Sela, H. Lis, N. Sharon, and L. Sachs, Biochim. Biophys. Acta, 310, 273-277 (1973).

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297

Isolectins from the “haricot” kidney-bean (Phaseolus vulgaris) were fra~tionated.~’~ They displayed723two types of glycoprotein subunit (molecular weight 30,000 +1,000 and 35,000 +1,000). Although these glycoproteins were shown to be agglutinins of red and white bloodcells, they had negligible effects on lymphocyte transformati~n.’~~ Two lectins from Phaseolus vulgaris (Blue Lake) were isolated by Dahlgren and his colleagues.724One of the lectins (PHA-a”)was purified to homogeneity. It had a molecular weight of 83,000, which is considerably lower than that of other P . vulgaris lectins, although it was supported by electron-microscope data.725PHA-a” exhibited erythroagglutinating, leucoagglutinating, and mitogenic properties; attempts to separate these activities failed.724 The lectin from Phaseolus vulgaris L. (var. red), isolated by conventional protein-purification procedures, was shown to be localized in A glycoprothe cytoplasm of the cotyledon and embryo of the tein (6.6%carbohydrate), the lectin had molecular weight 128,000, and contained high proportions of aspartic acid, serine, and tryptophan, but no c y ~ t e i n e . ~ ~ ~ ~ Proteins from the black kidney-bean were fractionated.726Analysis showed species that had both hemagglutinating and mitogenic properties; these agglutinins proved to be g l y c o p r o t e i n ~Rabbit . ~ ~ ~ antiserum, prepared against proteins from black kidney-beans, cross-reacted with the water-soluble proteins from white and red kidney-beans (although each variety of bean gave very different immunoelectrophoretic pattern~).~~~,~~~ The lectins from Phaseolus vulgaris are not readily inhibited by simple gar^,^^^^^^^,^^^.^^^ despite reports that 2-acetamido-2-deoxy-Dgalactose selectively inhibited both agglutinating and mitogenic acIt is now tivities of a partially purified, P . vulgaris fairly well accepted that a complex, as yet ill-defined, saccharide structure is required for binding to the P . vulgaris lectin. To elucidate the carbohydrate-binding specificity of the lectin, glycoproteins and glycopeptides have been used. Specifically, lectin-reactive structures (723) A. Pusztai and W. B. Watt, Biochim. Blophys. Acta, 365, 57-71 (1974). (724) K. Dahlgren, J. Porath, and K. Lindahl-Kiessling, Arch. Biochem. Biophys., 137, 306-314 (1970). (725) S. Hoglund and K. Dahlgren, Eur. J . Biochem., 17,23-26 (1970). (726) W. G. Jaffi, and K. Hannig, Arch. Biochem. Biophys., 109,BO-91 (1965). (727) W. G. Jaffi,, 0. Briicher, and A. Palozzo, Z. Immunitaetsforsch. Allerg. Klin. Immunol., 142,439-447 (1972). (728) H . Borberg, J. Woodruff, R. Hirschhorn, B. Gesner, P. Miescher, and R. Silber, Science, 154, 1019-1020 (1966). (729) H. Borberg, I. Yesner, B. Gesner, and R. Silber, Blood, 31, 747-757 (1968).

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(assayed by inhibition of hemagglutination, or precipitate formation) are subjected to sequential, chemical modification (for example, Smith degradation), or treatment with purified, specific glycosidases, or both; the degraded product is re-assayed. The validity of these studies depends on careful monitoring of the chemically or enzymically released sugars, and on a rigorous characterization of the product; unfortunately, these imperatives have not always been appreciated. Treatment of human erythrocytes with trypsin released a soluble glycopeptide (molecular weight 10,000)that bound to purified P . vulgaris lectin and thereby abolished its erythroagglutinating and lymphocyte-stimulating properties.730Pronase-digested glycopeptide was chromatographed on D E A E - c e l l u l ~ s e . Carbohydrate ~ ~ ~ * ~ ~ ~ and amino acid analyses, followed by sequential cleavage ofsugars from the nonreducing end(s) with specific glycosidases, to the branched structure proposed in formula 14. The derived glycopeptide a-AcNeu 2

1

6

t 334

p- D - GlcNAc p

p- D -GlcNAc p

?t

a

a - D- M a p - (1-

?t

?

a)-@-D-Manp - (l--?)-D-G1cNAcP-c Asn 14

was an excellent inhibitor of P . vulgaris lectin-induced cellagglutination. Neuraminidase cleavage of the sialic acid residue did not affect the activity, whereas treatment of the desialized product with P-D-galactosidase essentially abolished its ability to inhibit h e m a g g l u t i n a t i ~ n ~ ' ~(see ~~~ Table ~~~~ XXII). ~ - ~ ~Interestingly, ~ removal ofthe single, free P-D-galactosylgroup from glycopeptide I resulted in a A second erythroloss of about 50% of its inhibitory potency.2'6,432*730-732 cyte glycopeptide has been described in which both P-D-galactosyl residues were penultimate to sialic acid.730Neuraminidase treatment (730) S. Komfeld and R. Kornfeld, Proc. Natl. Acad. Sci. U.S.A., 63, 1439-1446 (1969). (731) A. M. Leseney, R. Bourrillon, and S. Kornfeld, Arch. Biochem. Biophys., 153, 831-836 (1972). (732) R. Kornfeld and S. Kornfeld, Ann. N.Y. Acad. Sci., 234,276-282 (1974). (733) R. Kornfeld, W. T. Gregory, and S. A. Komfeld, Methods Enzymol., 28, Part B, 344-349 (1972).

LECTINS: CARBOHYDRATE-BINDING PROTEINS

299

TABLEXXII Inhibition of PhaseoEus oulgaris Lectin-Erythrocyte Agglutination by Simple and Complex C a r b ~ b y d r a t e s ~ ' ~ * ' ~ ~ Compound Erythrocyte trypsin fragmenP Erythrocyte glycopeptide I" Immunoglobulin G glycopeptide I b Immunoglobulin G glycopeptide 11* Fetuin glycopeptideb Transferrin glycopeptide Lacto-N-tetraose [P-D-Galp-(1+3)-P-~-GlcNAcp-(1+3)-p-~Galp-(1+4)-~-Gk] Lacto-N-neotetraose p-D-Galp-( 1+4)-P-~-GlcNAcp-(1+3)-PD-Galp-(1-+4)-~-Glc] N- Acetyllactosamine [P-D-Galp-(1+4)-~-GlcNAc] Lactose 2-Acetamido-2-deoxy-~-galactose 2-Acetamido-2-deoxy-~-glucose D-Galactose

Nanomoles needed to give 1 inhibitory unir" 0.8 0.3 0.35 1.6 5.0 15.0 >500 >500 >500 >500

22,000 >20,000 >20,000

"Amount of material necessary to inhibit erythrocyte agglutination completely in the standard system for three minutes. *All contain the sequence A c N e u + p - G a l p + P-D-GlcNAcp+D-Manp in their structure.

also did not affect its activity as an inhibitor.730Porcine erythrocyte glycoproteins isolated by phenol-saline extraction gave precipitin bands (immunodiffusion in agar gel) against commercial (Difco) P. vulgaris preparation^.^^**^^^ Only neuraminic acid-containing fractions, resolved by isoelectric focusing, inhibited P. vulgal-is-type 0 erythrocyte h e m a g g l ~ t i n a t i o n . ~ ~ " ~ ~ ~ A series of linear, model oligosaccharides having the terminal sequence P-D-galaCtOpyranOSyl-(1-+3,4)-2-acetamido-2-deoxy-~-glucose (see Table XXII) had less than 0.1%of the inhibitory capacity of glycopeptide I, whereas D-galactose, lactose, 2-acetamido-2-deoxy-~glucose and 2-acetamido-2-deoxy-~-galactosewere n o n i n h i b i t ~ r y . ~ ~ ~ ~ ~ ~ ' (734) G. Uhlenbruck, U. Reifenberg, and R. Oyen, 2. Naturforsch., Teil B , 24, 147 (1969). (735) G. Uhlenbruck, G. Wintzer, K. Schumacher, H. Oerkermann, G. I. Pardoe, W. D. Hirschmann, G. Alzer, and R. Gross, in "The Role of Lymphocytes and Macrophages in the Immunological Response," D. C. Dumonde, ed., SpringerVerlag, Berlin, Heidelberg, New York, 1971, pp. 87-90. (736) G . Uhlenbruck, G. Wintzer, B. Salfner, K. Schumacher, H. Oerkermann, W. D. Hirschmann, and G. Alzer, K2in. Wochenschr., 48, 1369-1370 (1970).

300

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

On the other hand, two glycopeptides isolated from432 immunoglobulin G, and glycopeptides derived from f e t ~ i and n ~ t~r a~n ~ f e r r i nwhich ,~~~ have mannose residues in their cores as well as GaldGlcNAc sequences in their outer chains, proved to be good inhibitors.732From these studies, it would appear that a branched structure containing at least two p-D-galactopyranosylgroups that are terminal or residues that are penultimate to sialic acid are required for efficient binding to PHA. The core region of the oligosaccharide chain is probably involved in maintaining the proper spatial orientation. Multiple p-D-galaCtOSyl groups were also implicated as P. vulgaris lectin-reactive sites by Pardoe and her colleagues, who found that orosomucoid and erythrocyte glycopeptides from the hog, the horse, and the human were potent inhibitors of P. vulgaris-induced h e m a g g l u t i n a t i ~ n . ~ ~ ~ A glycoprotein that reacted with P. vulgaris lectin is secreted in all human saliva and ovarian-cyst fluid.739 Binding to P . vulgaris lectin was abolished by proteolysis (with trypsin or pronase), or mild, alkaline hydrolysis, but was unaffected by sequential digestion with neuraminidase (all detectable sialic acid was liberated) and p-Dgalactosidase (less than 1%of the galactose was released).739 Osawa and his colleagues examined the binding of porcine~ . ~ ' ~ bindthyroglobulin glycopeptides to P . vulgaris l e ~ t i n . ' ~Although ing of the lectin to thyroglobulin glycopeptide B can be rationalized on the basis of the presence of three p-D-galactopyranosyl units (one terminal group and two residues penultimate to sialic acid), binding to glycopeptide A [(Man),+( GlcNAc),+Asn] is difficult to e ~ p l a i n . ' ~ ~ , ~ ' ' Sequential treatment of glycopeptide B with neuraminidase and p-Dgalactosidase diminished its inhibitory capacity to approximately onet ~ e l f t h . ' ~Residual ~ , ~ ' ~ activity could be explained by incomplete release of galactose. Additional studies on the isolation and inhibitory activity of glycopeptides from human erythrocytes have been made.740,741 Red kidney-bean extracts formed precipitates with several, normal, animal sera, as well as with individual ~ e r u m - c o m p o n e n t s . ' ~ ~ * ~ ~ ~ ~ ~ (737) R. G . Spiro,J. Biol. Chem., 239,567-573 (1964). (738) G. A. Jamieson, in "Protides of the Biological Fluids," H. Peefers, ed., American Elsevier, New York, 1966, pp. 14 and 71. (739) J. A. Strauchen, C. F. Moldow, and R. Silber, J. Zmmunol., 104, 766-768 (1970). (740) Y. Akiyama and T. Osawa, Proc. J p n . Acad., 47, 104-109 (1971). (741) Y. Akiyama and T. Osawa, Hoppe-Setller's Z. Physiol. Chem., 353, 323-331 (1972). (742) L. Beckman, Nature, 195,582-583 (1962). (743) W. H. Marshall and L. C. Norins, Aust. J. E x p . Biol. Med., 43, 213-228 (1965). (744) N. H. Holland and P. Holland, Nature, 207, 1307-1308 (1963).

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301

The interaction of partially purified, commercial preparations of P . vulgaris with a large number of serum glycoproteins was studied by Morse.'osThe lectin precipitated a,-macroglobulin, @lipoproteins, and immunoglobulin M. Preparations of a,-glycoprotein, orosomucoid, and several immunoglobulin A myeloma proteins also reacted.lo5 Chondroitin 4-sulfate, dermatan sulfate, and heparin also precipitated a partially purified preparation of red kidney-bean; the precipitation reaction was completely inhibited by 0.5 M sodium chloride solution.366a Yachnin demonstrated"O that it was the Phaseolus vulgaris hemagglutinin (H-PHA), not leucoagglutinin (L-PHA), that formed a precipitate in agar gel with both normal serum and the a-globulin derived therefrom. He also showed'll that fetuin, which possesses oligosaccharide units similar in structure to those of red blood-cells, bound and precipitated H-PHA, but not L-PHA. Gel-filtration studies revealed that H-PHA formed soluble complexes with fetuin, whereas the affinity of L-PHA for fetuin was much lower, and any molecular complexes that formed dissociated rapidly. Despite these differences, fetuin is able to inhibit both H- and L-PHA-induced, lymphocyte transformation."' Pusztai and Watt723similarly observed that fetuin would inhibit hemagglutination of red cells by PHA. According to their increasing content of R (hemagglutinating) subunits, affinity-purified and resolved P . vulgaris isolectins exhibited an increasing capacity to precipitate with 14 different animal sera.74s Purified, carcinoembryonic antigen (CEA), which has sugar sequences similar to those in erythrocyte glycoprotein and fetuin, precipitated with purified l e u ~ o a g g l u t i n i n .Periodate ~~~ oxidation destroyed the capacity of CEA to interact with l e u ~ o a g g l u t i n i n . ~ ~ ~ An early study implicated bound-sialic acid as a site of reaction with wax-bean h e m a g g l ~ t i n i n . ~ ~ ~ In summary, although the carbohydrate-binding specificity of the P . vulgaris lectins has still not been defined, it would appear that a complex structure consisting of at least two fi-D-ga~actopyranosy~ groups that are terminal, or residues that are subterminal to sialic acid, is required. The D-mannose residues in the core region of P . vulgaris lectin-reactive glycoproteins appear to be involved in binding to the lectin.216*417-733*73s,747 In fact, it has been proposed that a trisaccharide (745) R. L. Felsted, R. D. Leavitt, and N. R. Bachur, Comp. Biochem. Physiol., B , 55, 417-421 (1976). (746) R. L. Northrup and I. E. Liener, Proc. Soc. E x p . Biol. Med., 100, 105-108 (1959). (747) S. Kornfeld and R. Kornfeld, in "Glycoproteins of Blood Cells and Plasma," G. A. Jamieson and T. J. Greenwalt, eds., Lippincott, Philadelphia, 1971, pp. 50-67.

302

IRWIN J, GOLDSTEIN AND COLLEEN E. HAYES

unit [P-D-Galp-(1+3,4)-P-~-GlcNAcp-(1+2)-D-Man] is an essential component of glycoproteins reactive with the red kidney-bean l e ~ t i nKaifu . ~ ~ and ~ Osawa have748synthesized the trisaccharide cited [containing a P-D-( 1+4)-galactosidic linkage], and reported that it inhibited 0-erythrocyte-P. vulgaris lectin hemagglutination. If confirmed, this will rank as an important discovery in lectin chemistry. 2. Vicia graminea Historical interest in the Vicia graminea lectin has closely paralleled interest in the chemical relationship between M and N blood-group specificity. This blood-group N-specific hemagglutinin was discovered by Ottensooser and S i l b e r s ~ h m i d twho , ~ ~ ~noted a striking correspondence between human anti-N sera (which is rare) and Vicia graminea extracts. Although it agglutinated only NN and M N cells, the lectin could be adsorbed with MM erythrocytes,750and it agglutinated751MM cells stored in citrate buffers ofhigh pH. It had not yet been established whether M and N substances were cross-reactive products of two alleles utilizing a common precursor substance, or were characterized by a precursor-product relationship such that incomplete biosynthesis would lead to the expression of both antigens. Nagai and Springer isolated752blood-group M substance from erythrocyte stroma, and demonstrated that this unreactive glycoprotein became a potent inhibitor of Vicia graminea hemagglutination following acid-catalyzed hydrolysis of sialic acid, a result confirmed by Lisk o w ~ k a Neuraminidase .~~~ digestion likewise converted M-substance into a product indistinguishable, by Vicia graminea reactivity, from N - s ~ b s t a n c e , whereas ~ ~ ~ * ~ N-active ~~ substances were unaffected by neuraminidase d i g e ~ t i o n ~and ~ ~mild - ~ ~ hydrolysis ~ with a ~ i d . ' ~ ~ , ~ ~ ' Once sialic acid had been removed from M-substance, Vicia graminea (748) R. Kaifu and T. Osawa, Carbohydr. Res., 52, 179-185 (1976). (749) F. Ottensooser and K. Silberschmidt, Nature, 172,914 (1953). (750) P. Levine, F. Ottensooser, M. J. Celano, and W. Pollitzer, Am.]. Phys. Anthropol., 13,29-36 (1955). (751) T. van Wageningen and L. E. Nijenhuis, Vox Sang., 5,572-573 (1960). (752) Y. Nagai and G. F. Springer, Fed. Proc., 21,67(d) (1962). (753) E. Lisowska, Nature, 198,865-866 (1963). (754) M. Kriipe and G . Uhlenbruck, Z . Immun. Allergieforsch., 126,408-414 (1964). (755) G. Uhlenbruck and M. Kriipe, Vox Sang., 10,326-332 (1965). (756) G . Uhlenbruck and M. Kriipe, 2. Immunitaetsforsch. E x p . Ther., 124, 342-345 (1962). (757) G. F. Springer and K. Hotta, Fed. Proc., 22,2261 Abs. (1963). (758) G. F. Springer, Y. Nagai, and H. Tegtmeyer, Biochemistry, 5,3254-3272 (1966). (759) E. Lisowska, Eur. J . Biochem., 10,574-579 (1969).

LECTINS : CARBOHYDRATE-BINDING PROTEINS

303

reactivity could be abolished by treatment with D-galactose oxidase or P-D-galactosidase,as was also true of native N-substance.219~678~754~7s5*7s8~76 Deacetylation of erythrocyte N-substance also resulted in the loss of lectin rea~tivity."~ The oligosaccharide with which Vicia graminea lectin reacts was labile to alkaline borohydride reduction-hydrolysis after desialization, suggesting that it was attached by an 0-glycosyl linkage to the polypeptide chain.7s92-Acetamido-2-deoxy-O-~-galactosylgalactitol was released by this reaction. These studies suggested that M-substance resulted from sialic acid substitution on the nonreducing (terminal)P-D-galactosyl group of N-substance by the product of the M-blood-group gene, a sialyltransfera~e.~~~~~~~ Vicia graminea lectin has been purified to homogeneity by Prigent and Bourrillon.220An ammonium sulfate fraction of the extract of the ground seed was chromatographed on DEAE-cellulose, and then procThe eluted protein (yield: 50 essed by gel filtration on Sephadex G-150. mg/kg of seeds) was homogeneous by sedimentation analysis, electrophoresis, immuno-electrophoresis, and isoelectric focusing. Sedimentation equilibrium studies gave a calculated molecular weight of 105,000 ( s ; ~ ,=~ 5.3 S). Subunits of molecular weight 25,000were obtained by electrophoresis in the presence of a detergent. The chemical composition of Vicia graminea lectin was similar to that of many other lectins, being high in its content of serine, and aspartic and glutamic acids, and relatively deficient in cysteine and methionine. In addition, three amino acids having hydrophobic sidechains, namely, glycine, isoleucine, and leucine, were found in abundance. The protein contained 7.3% of carbohydrate, principally manalong with lesser proportions of nose and 2-acetamido-2-deoxyglucose, fucose, galactose, and glucose. Most investigators have reported that Vicia graminea extracts do not bind any known mono- or oligo-saccharide, as assayed by hemagglutiAmong those tested were sialic acid, nation inhibition.21S.220*678*7s8~761,762 D-galactose, methyl a-D-galactopyranoside, methyl P-D-galactopyranoside, o-nitrophenyl P-D-galactopyranoside, 2-acetamido-2-deoxy-Dgalactose, lactose, melibiose, 3-O-a-D-galactopyranosy~-D-galactose, 6-0-P-D-ga~actopyranosyl-D-gdactose, 2-acetamido-2-deoxy-3-0-P-Dgalactopyranosyl-D-galactose (the disaccharide determinant of desialyzed N-substance), 2-acetamido-2-deoxy-3-(and4-)O-P-D-galactopyranosyl-D-glucose, lacto-l\r-tetraose, ~acto-l\r-neotetraose,D-glUCOSe, (760) E. Romanowska, Vox Sang., 9,578-588 (1964). (761) G . Uhlenbruck and W. Dahr. Vox Sang., 21,338-351 (1971). (762) J. F. Codington, A. G. Cooper, M. C. Brown, and R. W. Jeanloz, Biochemistry, 14,855-859 (1975).

304

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

2-acetamido-2-deoxy-~-glucose,L-fucose, D-fUCOSe, and D-mannose. On the other hand, Springer and his colleagues678noted inhibition of Vicia graminea-induced agglutination of N N erythrocyte by 1acto-Ntetraose, lacto-N-neotetraose, 2-acetamido-2-deoxy-3-(and4 - ) 0 - p - ~ galactopyranosyl-D-glucose, and D-galactose (in decreasing order of potency). This discrepancy awaits resolution through continued analysis. It is conceivable that Vicia graminea lectin has an extended binding-site complementary to an oligosaccharide more complex than those tested (compare Refs. 183, 216, 276, and 417). None of thirty amino acids were inhibit01-y.~~~ The lectin reacts with bovine subma~illary-mucin,~~~ as well as with a large glycoprotein released by proteolysis from the surface of TA3Ha cells of a mouse rnamrnary-~arcinorna.~~~

3. Miscellaneous Lectins Numerous other lectins have been isolated; however, information regarding their physical and chemical properties and carbohydratebinding specificity is incomplete. We present here, short descriptions of several lectins that have interesting biological and carbohydratebinding properties. Although many of these substances are already being used in a variety of studies, it is probable that more-complete characterization will enhance their value considerably. Extracts of Vicia cracca seeds were among the first plant-materials shown to exhibit anti-A hemagglutination a ~ t i v i t y . ~ Gel , ~ ~filtration ,~~,~~~ of a seed extract on Sephadex G-100 led to the discovery of two lectins in V. cracca seeds.137The column eluate exhibited an increased, anti-A ~pecificity'~~; elution of the Sephadex column with 0.1 M D-glUCOSe solution displaced a second, blood-group, nonspecific lectin with carbohydrate-binding properties similar137to those of con A. Some mitogenic activity was demonstrated in the latter, nonspecific 1e~tin.I~' The anti-A lectin was isolated on an affinity adsorbent prepared by conjugating ovarian-cyst A substance to agarose beads.764The anti-A lectin was displaced with 0.1 M acetate buffer, pH 4.0. A substance having blood-group A activity was isolated from hog gastric-mucin by .~~~ ovarian-cyst precipitation with V. cracca anti-A l e ~ t i nSubsequently, A-substance was isolated on Vicia cracca lectin immobilized on a g a r o ~ e Some . ~ ~ ~ subfractionation of the A-substance was achieved. (763) M. Kriipe, Z. lmmunitaetsforsch. E x p . Ther., 111, 22-31 (1954). (764) T. Kristiansen, L. Sundberg, and J. Porath, Biochim. Biophys. Acta, 184, 93-98 (1969). (765) T. Kristiansen and J. Porath, Biochirn. Biophys. Acta, 158, 351-357 (1968). (766) T. Kristiansen, Biochim. Biophys. Acta, 388, 246-253 (1974).

LECTINS : CARBOHYDRATE-BINDING PROTEINS

305

2-Acetamido-2-deoxy-~-galactose was the best inhibitor ofVicia cracca agglutination of A, erythrocyte^^^,^^,^^; melibiose and raffinose were weak inhibitor^.^^ (A, cells are more subject to agglutination than Az ~ e l l s . ~Interestingly, ~,~~) D-mannose, D-glucose, and maltose were the best inhibitors of rabbit and pig cells, reflecting reaction of the second, D-mannose-binding lectin with these animal cells.78Two blood-group A lectins were isolated from Vicia cracca seed-extracts by affinity chromatography on an adsorbent containing matrix-bound 2-acylamido-2-deoxy-~-galactose.~~~~ Continuous, pH-gradient elution gave two fractions, each of which consisted of several agglutinating species. Both lectin fractions had a molecular weight of 125,000and a subunit weight of 33,000. D-Galactose was only 1% as potent as 2-acetamido-2-deoxy-~-galactose in inhibiting hemagglutination of A, erythrocytes by the V. cracca l e c t i n ~ At . ~ pH ~ ~ 8, ~ 2-acetamido-2deoxy-D-galactose binds to the lectins with K,,,, = 6 x lo3A 4 - l . Extracts from the seeds of Laburnum alpinum were first shown to possess anti-H(0) activity by Renkonen,6 and this was confirmed by Morgan and Watkins22and other^.^^,^^, Although the L. alpinum lectin(s) has not been purified, hemagglutination-inhibition studies conducted on seed extracts reveal a specificity towards N,N'diacetylchitobiosyl residues.19*196~471 Human A, H, and neuraminidasetreated human Lea blood-group substances were also extremely good inhibitors of the Laburnum l e ~ t i n . ' ~ ~ ~ ~ ' ~ The hemagglutinin from Bauhinia purpurea alba seeds was purified by specific adsorption on Sepharose 4B, and subsequent displacement with 0.1 M The purified lectin, homogeneous in the ultracentrifuge and by electrophoresis on poly(acry1amide)gel had a molecular weight of 195,000,and contained 11% of carbohydrate (principally mannose and 2-amino-2-deoxyglucose, with smaller proportions of xylose, glucose, galactose, and f u c o ~ e ) . The ' ~ ~ most notable feature of the amino acid analysis was the absence of methionine, and the high .~~~ proportions of aspartic acid, serine, and t h r e ~ n i n e Carbohydratebinding, specificity studies, performed by hemagglutination inhibition analysis, revealed the lectin to be most reactive towards 2-acetamido-2-deoxy-~-galactose.~~'~~~~ D-Galactose, lactose, ~~,'~~ melibiose, and N-acetyllactosamine were all -50% as a ~ t i v e . ~Desialized, ovine submaxillary-mucin [which contains terminal (nongroups] was exreducing) 2-acetamido-2-deoxy-a-~-galactopyranosyl ceptionally The purified lectin agglutinated erythrocytes, independent of their ABO and MN blood-group types767;this nonspecific (766a) H. Riidiger, Eur. J . Biochem., 72,317-322 (1977). (767) T. Irimura and T. Osawa, Arch. Biochem. Biophys., 151, 475-482 (1972).

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IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

activity is in contrast to the anti-N reactivity described by others for ~ , ~ ~ ' a second crude extracts of Bauhinia purpurea alba s e e d ~ . ~Perhaps, lectin exhibiting anti-N specificity is present in B . purpurea extracts, or anti-N specificity is present in only certain varieties of B . purpurea. Ersson and isolated a D-galactose-binding lectin from ) ~affinity ~ chromatography on sunn-hemp seeds (Crotalariaj ~ n c e aby acid-treated, galactan gel. The lectin, eluted with 0.1 M lactose solution, had molecular weight 120,000 by gel filtration, contained 5% of covalently bound carbohydrate, and was inhibited best by lactose'45 (compare Ref. 78). D-Galactose and 2-amino-2-deoxy-~-galactosehad 12%, and melibiose, -50%, of the activity of 1 a ~ t o s e .The l ~ ~ C. juncea lectin was especially mentioned by Makelazofor its ability to distinguish between a-and /3-D-galactosides. C. juncea lectin, immobilized by coupling to agarose-gel beads, was employed as an adsorbent for serum proteins.7s8The C. juncea lectin was also purified on a matrix prepared by coupling D-galactose to Sepharose 6B activated with divinyl ~ u l f o n eHomogeneous .~~~~ by several physical criteria, the lectin was shown to be a tetrameric protein, subunit molecular weight -31,000. It is a glycoprotein (9.8%of carbohydrate: mannose, 2-amino2-deoxyglucose, fucose, and xylose), contains bound metallic ions ( Ca2+and Mgz+),and has a high content of aspartic acid and serine, but no methionine or ~ y s t i n e . ' ~ ~ ~ A sialic acid-binding lectin has been isolated from horse-shoe crab (Limulus polyphemus) hemolymph by both c o n ~ e n t i o n a 1 and ~~~*~~~ affinity chromatographic (insolubilized, bovine subma~illary-mucin,'~~ and formalinized, horse erythrocyte^'^^) procedures. The purified lecis composed of 18 to 20, nontin (molecular weight, -400,000)1sg*769-771 covalently bound subunits (molecular weight, -20,000)15B~76s9-771 in the form of a ring-shaped structure,772contains covalently bound carbohydrate (-4%, by weight, of 2-amino-2-deoxyglucose and neutral of acidic amino acids, as well as high s ~ g a r ) , 'and ~ ~ a. preponderance ~~~ proportions of glycine and leucine.17B~76s*770 Three moles of cysteine and 2 to 3 moles of methionine per mole of subunit were also reported to be present in the Limulus a g g l ~ t i n i n . ~The ~ ~ ,sequences ~~~ of the (768) B. Ersson and J. Porath, FEBS Lett., 48, 126-129 (1974). (768a) B. Ersson, Biochim. Biophys. Acta, 494,51-60 (1977). (769) J. J. Marchalonis and G . M. Edelman,J. MoZ. Biol., 32,453-465 (1968). (770) A.-C. Roche and M. Monsigny, Biochim. Biophys. Acta, 371, 242-254 (1974). (771) C. L. Finstad, R. A. Good, and G . W. Litman, Ann. N.Y. Acad. Sci., 234, 170182 (1974). (772) H. Femindez-Moran, J. J. Marchelonis, and G. M. Edelman, J . MoZ. Biol., 32,467-469 (1968). (772a) R. Kaplan, S . S.-L. Li, and J. M. Kehoe, Biochemistry, 16,4297-4303 (1977).

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N-terminal-24 (Ref. 771) and -50 amino acids (Ref. 772a) have been determined. The Limulus lectin agglutinates e r y t h r o ~ y t e s (. ~irre~~-~~~ spective of blood type), human l e u c ~ c y t e splatelets,776 ,~~~ and tumor Neuraminidase-treated cells could no longer be agglutinated by Limulus serum.777Calcium ions are required for, and sometimes and they induce a enhance, the agglutination reaction,155J59,769*770J75 change in the c.d. spectrum of the l e ~ t i n . ~ N-Acetylneuraminic ~l a ~ i d and ~ ~ D-glucur~nic'~~ ~ J ~ ~ (but not D-galacturonic) acid specifically inhibited agglutination of horse erythrocytes b y the LimuZus lectin. 2-Acetamido-2-deoxy-~-glucosewas also reported to be an inhibitor by some investigator^,^^^,^^^ but not by others.159Human orosomucoid770 and carcinoembryonic antigen775inhibited agglutination b y the Limulus lectin. A precipitin reaction in immunodiffusion gels was observed with purified, bovine submaxillary-mucin, but not against the desialated m ~ c i n . These ' ~ ~ studies indicated that the Limulus polyphemus lectin possesses the capacity to react with biopolymers and cells containing terminal sialic acid residues. As such, it should prove to be an exceptionally usefbl reagent for identification and isolation purposes. Labelled with ferritin and fluorescein, the lectin should be capable of localizing neuraminic acid residues on cell surfaces. Limulin has also been shown to be mitogenic toward human, peripheral lymphocytes .777a An L-rhamnose-binding protein has been isolated from the culture filtrate of Streptomyces 27S5 by Fujita and his c o l l e a g ~ e s ~they ~*~~~; used conventional procedures, and affinity chromatography on insolubilized gum arabic. (It was necessary to use M D-galaCtOSe in order to displace the lectin.") The purified lectin, a protein of molecular weight 11,000, is d i s t i n g u i ~ h e dby ~ ~its high content of alanine, glycine, and valine (corresponding to 47% of the total amino acid residues); low content of carbohydrate (1.8%,corresponding to one residue ofhexose, which could represent an impurity); and an unusual, c.d. spectrum (large positive peak at 226 nm). The lectin yielded a typical (773) H. Noguchi, Zentralbl. Bakteriol. Parasitenkd. Infektionskr. H y g . Abt. 1. Orig., 34,286-288 (1903). (774) E. Cohen, A. W. Rose, and F. C. Wider, Life Sci., 4,2009-2016 (1965). (775) E. Cohen, M. Rozenberg, and E. J. Massaro, Ann. N.Y. Acad. Sci., 234, 28-33 ( 1974). (776) G. I. Pardoe, G. Uhlenbruck, and G . W. G. Bird, Immunology, 18, 73-83 (1970). (777) E. Cohen, Truns. N.Y. Acad. Sci., 30,427-443 (1968). (777a) A.-C. Roche, Y. Perrodon, B. Halpern, and M. Monsigny, Eur. /. lmmzcnol., 7 , 263-267 (1977). (778) Y. Fujita, K. Oishi, and K. Aida, Biochem. Biophys. Res. Commun., 53,495-501 (1973).

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precipitation-curve with gum arabic, showed a high, rather specific, titer against type B erythrocytes (256 times that for A or 0 cells), and was strongly inhibited in its agglutination of B erythrocytes by bloodgroup B substance from human saliva. Hemagglutination inhibition analysis indicated a specificity for L-rhamnose. D-Galactose, methyl a-D-galactopyranoside, melibiose, and L-arabinose had lo%, D-fUCOSe 2.5%, and phenyl a-and P-D-galactopyranoside -40% of the potency of ~ - r h a m n o s e . ~Galactitol, ~ , ~ ~ ~ D-ga~actono-1,4-~actone, 2-amino-2deoxy-D-galactose, and 2-acetamido-2-deoxy-~-galactose were essentially n ~ n i n h i b i t o r s . ” *On ~ ~the ~ other hand, guar gum, locust-bean gum, and gum arabic, all of which contain terminal (nonreducing) a-D-galactopyranosyl groups, were very good inhibitors. Once it is more completely characterized, this interesting lectin should prove a useful structural probe for biopolymers containing L-rhamnose residues. A nonspecific lectin was isolated from the meadow mushroom (Agaricus campestris) by chromatography on DEAE-cellulose.si~221 The lectin, pure by ultracentrifugation, electrophoresis on cellulose acetate, and immunoelectrophoresis, had molecular weight 64,000, and contained 4% of ~ a r b o h y d r a t eA. ~four-chain ~ structure (AzB,) was proposed for the hemagglutinin, based on gel-filtration studies in dissociating solvents, and tryptic peptide-analysis.sl Four “buried” sulfhydryl groups were detectedSs1Erythrocytes from all of the major blood-types gave equal titers against the A. compestris lectin; most of the animal erythrocytes were also agglutinated. Of the many sugars tested, none inhibited human, red blood-cell agglutination. However, a sonic suspension of red-cell ghosts did produce inhibition of lectininduced hemagglutination.221 Presant and K ~ r n f e l disolated ~ ~ ~ two lectins (PHA-A and PHA-B) from the commercial mushroom (Agaricus bisporus) by DEAEcellulose and 0-phosphonocellulose chromatography, and studied their cell-binding properties. Glycopeptides from erythrocytemembranes, fetuin, and immunoglobulin A were potent inhibitors of the hemagglutination reaction. A complex, carbohydrate-binding specificity similar to that for the Phaseolus vulgaris isolectins was indicated. Isolation of a 2-acetamido-2-deoxy-~-galactose-binding protein from a slime mold, Dictyostelium discoideum, was reported by Rosen and Affinity chromatography on Sepharose 4B, followed by (779) S. D. Rosen, J. A. Kafka, D. L. Simpson, and S. H. Barondes, Proc. Natl. Acad. Sci. U.S.A., 70,2554-2557 (1973). (780) D. L. Simpson, S. D. Rosen, and S . H. Barondes, Biochemistry, 13, 3487-3493 (1974).

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elution with D-galactose solution, gave the pure lectin, called disA tetramer having molecular weight 100,000, the lectin consists of four, apparently identical, subunits (molecular weight 25,000). A pure protein, discoidin is rich in aspartic acid, glutamic acid, and 3-hydroxy amino acids, and it also contains a considerable proportion of half-cysteine residues.780It is believed that discoidin may mediate intercellular adhesion and aggregation during starvation of the organism. The lectin is a powerful agglutinin of sheep erythroc y t e ~ . Carbohydrate-binding ~ ~ ~ * ~ ~ ~ specificity studies, monitored by hemagglutination inhibition of formalinized sheep-erythrocytes, revealed a primary specificity for 2-acetamido-2-deoxy-~-galactose.~~~ Lactose and D-galactose were approximately %th and l/icth as active. Interestingly, 3-O-methyl-D-gluCOSe was equivalent to S-acetamido2-deoxy-~-galactosein its inhibitory potency (compare Ref. 213). A similar, carbohydrate-binding protein was isolated from another cellular slime-mold, PoZysphondyZium pallidurn, by adsorption to formalinized, human erythrocytes followed by elution with D-galactose The purified lectin, subunit molecular weight 25,000, has a primary specificity for D-galactose; lactose is 4 times as effective as an inhibitor, and 2-acetamido-2-deoxy-D-g~ucosehas one quarter of the potency O f D-galaCtOSe.78'This lectin is also believed to be involved in cellular a g g r e g a t i ~ n . ~ ~ ' Potent, mitogenic activity was reported by Farnes and his coll e a g u e ~to~be ~ ~present in the extracts of pokeweed (pigeon berry; PhytoZncca nmericnna). Extracts of whole (ripe and unripe) berries, seed, pulp, and stem produced slight, and variable, erythrocyte agglutination, and were mitogenic. High erythrocyte-agglutinating activity and mitosis ofleucocytes were found with root and leaf extracts.771 Subsequently, Borjeson and coworker^'^^,^^^ reported the isolation from pokeweed-root extracts of a homogeneous substance (a single band in disc gel-electrophoresis, and a single line in immunoelectrophoresis) that possessed three biological properties: hemagglutination, leukoagglutination, and mitogenesis. Adsorption studies with red cells or stroma removed the hemagglutinating activity, without altering the mitogenic activity, whereas adsorption with leukocytes resulted in loss of both the mitogenic and the leukoagglutinating activities (compare (781) S. D . Rosen, D. L. Sinipson, J. E. Rose, and S. H. Barondes, Nature, 252, 128, 149-151 (1974). (782) P. Farnes, B. E. Barker, L. E. Brownhill, and H . Fanger, Loncet, (2) 1100-1101 ( 1964). (783) L. N. Chessin, J . Borjeson, P. D. Welsh, S. D. Douglas, and H. L. Cooper, J. E x p . Mcd., 124, 873-884 (1966).

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Ref. 703). Reisfeld and colleagues784 employed preparative, acrylamide-gel electrophoresis to isolate pokeweed mitogen as a homogeneous glycoprotein. It had molecular weight 32,000, an unusually high content of half-cysteine residues [but no free sulfhydryl groups, as determined by titration with 5,5-dithiobis(2-nitrobenzoic acid)], and contained 3.2% of carbohydrate (4residues of mannose, 1-3 each of fucose and glucose, and 2.8 of hexosamine). W a ~ d a fractionated 1~~~ a saline extract of pokeweed roots on hydroxylapatite, followed by Sephadex-gel filtration. Five glycoproteins were obtained, designated Pa-1 through Pa-5. Each glycoprotein had distinctive, physical and chemical properties, and biological activities, and each appeared in the plant at different times of the year.78sAll components had molecular weights in the range of 19,000-31,000, contained 1.8-12.5% of covalently bound carbohydrate, and 8 to 50 residues of half-cystine. Glycoprotein Pa-1 was the most potent hemagglutinin (no significant difference in hemagglutination titer for ABO cells was noted). All of these glycoproteins were mitogenic over an unusually wide range of concentration of protein compared to other mitogenic l e ~ t i n s .However, ~ ~ ~ , ~only ~ ~ Pa-1 was mitogenic for both the B and the T classes of murine lymphocytes.786This difference in mitogenic specificity is directly referable to the physicochemical properties of the glycoproteins. Only Pa-1 behaves as a multimeric protein in gel filtration in nondissociating solvents. Furthermore, of the mitogenic P . americana glycoproteins, Pa-1 has the fewest disulfide bonds.785,788 Yokoyama and coworkers787performed a similar fractionation of P . americana extracts by employing DEAE-cellulose and affinity chromatography on immobilized, desialized, humanerythrocyte glycopeptides. Five mitogens (Pa-1 through Pa-5) were isolated, and characterized. In agreement with the results of Waxdal and B a ~ h a monly , ~ ~Pa-1 ~ was found to be mitogenic both for murine B-cells and T-cells, whereas the other glycoproteins were T-cell mitogens.787Two phytomitogens were isolated from saline extracts of Phytolacca esculenta (shoriku) roots by salting out with ammonium sulfate and chromatographing on DEAE-cellulose and Sephadex G-100 columns.788Fraction E-2 had properties (molecular weight 32,000; 18 residues of half-cystine; 5.3% of carbohydrate) similar to (784) R. A. Reisfeld, J. Borjeson, L. N. Chessin, and P. A. Small, Jr., Proc. Natl. Acad. Sci. U.S.A., 58, 2020-2027 (1967). (785) M. J. Waxdal, Biochemistry, 13,3671-3677 (1974). (786) M. J. Waxdal and T. Y. Basham, Nature, 251, 163-164 (1974). (787) K. Yokoyama, 0. Yano, T. Terao, and T. Osawa, Biochim. Biophys. Acta, 427, 443-452 (1976). (788) H. Tokuyama, Biochim. Biophys. Acta, 317,338-350 (1973).

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those of the pokeweed mi tog en^.^^^ The carbohydrate-binding specificity of the lectins from the genus Phytolacca is still unknown. This system is of outstanding interest and importance to cellular immunologists; it could materially assist in unravelling the differential requirements for B- and T-cell activation. A phytohemagglutinin (termed Robin) from Robinia pseudoacacia (black locust) seed-extracts was isolated by conventional techniques.l6O The lectin had a molecular weight of -100,000 ( s ; ~ ,=~ 4.39 S), contained 17% of covalently bound carbohydrate (mannose, 2-amino-2deoxyglucose, fucose, and xylose, and a trace of glucose), and was rich in acidic amino acids, hydroxyl amino acids, and leucine, but lacked cysteine.160The R. pseudoacacia lectin possesses a significant content of @pleated, sheet conformation, but little, if any, a - h e l i ~ . "Treatment ~ of the glycoprotein with pronase and trypsin released a glycopeptide which, after purification on Sephadex, was found to contain essentially ~ , ' ~purified ~ glycopeptide all of the activity of the h e m a g g l ~ t i n i n . ' ~The contained 65% of neutral sugar (mainly mannose) and 2-amino-2d e o x y g l u c ~ s e Fifteen .~~ minutes after treatment with 0.05 M periodate,'89 half of the hemagglutinating activity was lost. Simple sugars do not inhibit the R. pseudoacacia lectin, but a sialoglycoprotein isolated from the urine of a pregnant woman was found to be a potent inhibitor of lectin h e m a g g l u t i n a t i ~ nThe . ~ ~ ~R . pseudoacacia lectin has been used as a membrane probe.731 A D-mannose-Sepharose 6B column791was used to isolate a bloodgroup nonspecific, D-mannose-binding lectin from Vicia ervilia extract^.^^*^'^ Gel-filtration studies in dissociating solvents, and ultracentrifugation analysis, led to the suggestion that the molecule of the V . ervilia lectin is composed of four subunits, two of type A (molecular weight 4,700) and two of type B (molecular weight 2l,OOO), to give an aggregate molecular weight of 53,000,in agreement with the amino acid composition and the ultracentrifuge data. The agglutination reaction was inhibited by D-glucose, D-mannose, D-fructose, methyl aD-mannopyranoside, maltose, melezitose, and a,a-trehalose. This carbohydrate-binding specificity places the V .ervilia lectin in the same class as con A and the lectins from the lentil, the pea, and the broad bean (V.faba). Two lectins, one a potent m i t ~ g e n the , ~ ~other ~ displaying strong (789) J. Font and R. Bourrillon, Biochim. Biophys. Acta, 243, 111-116 (1971). (790) M. Lemonnier, Y. Goussault, and R. Bourrillon, Carbohydr. Res., 24, 323-331 (1972). (791) N. Fornstedt and J. Porath, FEBS Lett., 57, 187-191 (1975). (792) B. E. Barker and P. Farnes, Nature, 215,659-660 (1967).

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IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

hemagglutinating and leucoagglutinating activity, were separated from seed extracts of Wistaria floribunda by SE-Sephadex chromatography.793,794 The mitogen was a glycoprotein of molecular weight -70,000. It contained 11.4% of carbohydrate (5.8% of mannose, 3.5% of 2-amino-2-deoxyglucose,and smaller proportions of arabinose, fucose, and xylose), and had an amino acid composition rich in acidic and hydroxylic amino acids and low in sulfur-containing amino Both the purified, mitogenic lectin and the strongly hemagglutinating fraction A (compare Ref. 794) were nonspecific with regard to bloodgroup types ABO, and were strongly inhibited in their agglutination of erythrocytes by 2-acetamido-2-deoxy-~-galactose(the hemagglutinin, (The mitogen was also reported to be inhibited more by N,N'-diacetylchit~biose.~~~) The hemagglutinin was inhibited somewhat more strongly by lactose than by melibiose, whereas these disaccharides were equally potent with respect to the m i t ~ g e n . ~ ~ ~ Mitogenic and cell-binding studies were reported for the mitogen and ~ ~ , hemagglutinin, ~~~*'~~ purified by adsorption the h e m a g g l ~ t i n i n . ~ The onto insolubilized, hog (A + H active) gastric-mucin, followed by elution with D-galaCtOSe solution, appeared to be homogeneous by isoelectric focusing (PI, 5.5), but actually consisted of a mixture of di-, . ~ ~ ~ chromatography tetra-, and octa-meric forms of the l e ~ t i nRecycling on Sephadex G-200 showed the tetramer (mol. wt. 125,000) to constitute 85% of the mixture. The lectin agglutinated human and murine lymphocytes, but was nonmitogenic towards these cells.794Kurokawa and his colleagues796also isolated a hemagglutinin from W. floribunda seeds. A homogeneous glycoprotein of molecular weight 68,000, the lectin contained 3.2% of carbohydrate (mannose, galactose, and 2-amino-2-deoxyglucose)in the molar ratios of 1:2:1.The lectin, shown to contain two equivalent binding-sites for 2-acetamido-2-deoxy-~galactose (K, = 1.28 x lo4M-'),dissociated into two identical, monovalent subunits upon reduction with 2-mercaptoethanol, indicating the presence of two subunits of mol. wt. 32,000 linked through a disulfide bridges7% Hemagglutination-inhibition studies showed the W . floribunda lectin to be complementary to 2-acetamido-2-deoxy-p-~galactopyranosyl units. A more complete investigation of the W. (793)S. Toyoshima, Y. Akiyama, K. Nakano, A. Tonomura, and T.Osawa, Biochemistry, 10, 4457-4463 (1971). (794)G. Cheung, A. Haratz, M. Katar, and R. D. Poretz, Abstr. P a p . Chem. Congr. North Am. Continent I , BMPC 19 (1975). (795)T. Osawa and S. Toyoshima, Methods Enzymol., 28, Part B, 328-332 (1972). (796)T. Kurokawa, M. Tsuda, and Y. Sugino, /. Biol. Chem., 251, 5686-5693 (1976). (797)S. Toyoshima and T. Osawa,J. Biol. Chem., 250, 1655-1660 (1975).

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Joribunda lectins should be rewarding, in that these substances will be useful probes for both the chemist and the molecular biologist. Two lectins were also isolated from Maackia amurensis seeds, one a potent mitogen (MAM), the other a strong hemagglutinin (MAH).798 Boyd and coworkersssfirst described a hemagglutinin in M . amurensis seeds. Purification involved affinity chromatography on procinethyroglobulin glycopeptides-Sepharose, and gel filtration.798Both lectins were glycoproteins (mannose and 2-amino-2-deoxyglucose) of molecular weight 130,000. Complex, carbohydrate-binding patterns are apparent. Among the simple sugars, only 2-acetamido-2-deoxy-~ galactose and maltose had moderate inhibitory activity against both MAH and MAM. Glycopeptide B from porcine thyroglobulin was a potent inhibitor of MAM hemagglutination, but had no effect against MAH. On the other hand, glycopeptide B and its sequential, enzymicdegradation products inhibited the mitogenic activity of both M . amurensis lectins (MAH also has a weak mitogenic Multiple agglutinins were isolated from the hemolymph of the male . ~ ~ of ~ the agglutinins (LAg-2) was lobster (Homarus a m e r i c a n u ~ )One reported to possess a site complementary to 2-acetamido-2-deoxy-ogalactose; a second (LAg-1) was complementary to N-acetylneuraminic acid.s00 Two lectins were purified by Bloch and his colleaguesso1from the . ~ ~of the blood-group seeds of the pea tree (Caragana a b o r e s c e n ~ )One nonspecific glycoproteins (I) was purified by affinity chromatography column; on a 2-acetamido-2-deoxy-~-galactose-substituted-Sepharose the lectin is composed of two types of polypeptide chain (molecular weight 30,000), cross-linked by disulfide bonds to form dimers that appear to be in equilibrium with tetramers.sO1A strong agglutinin for ABO erythrocytes, the lectin also agglutinates Ehrlich ascites cells, Lectin 11, and has a specificity for 2-acetamido-2-deoxy-~-galactose. a minor component, binds to underivatized Sepharose, and exhibits low hemagglutinating activity.s01 Seeds from Euonymus europeus contain a mixture of lectins that exhibit anti-(B + H) and A2 activity but no anti-Al activity,77~80~s0*~803 a

-

(798) T. Kawaguchi, I. Matsumoto, and T. Osawa, J . B i d . Chem., 249, 2780-2792 (1974). (799) J. L. Hall and D. T. Rowlands, Jr., Biochemistry, 13,821-827 (1974). (800) J. L. Hall and D. T. Rowlands, Jr., Biochemistry, 13, 828-832 (1974). (801) R. Bloch, J. Jenkins, J. Roth, and M. M . Burger,J. Biol. Chem., 251,5929-5935 (1976). (802) F. Pacik and J. Kocourek, Biochim. Biophys. Acta, 400,374-386 (1975). (803) J. Petxyniak, M. E. A. Pereira, and E. A. Kabat, Arch. Biochem. Biophys., 178, 118-134 (1977).

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finding that prompted the suggestion that these seed extracts could be employed for distinguishing between subgroups A, and Az. Pacik and KocourekEo2purified one of the E . europeus lectins (termed phytohemagglutinin I) by preparative electrophoresis, and showed it to be a homogeneous glycoprotein (1.9% of neutral sugar) of molecular weight 127,000; the lectin contained bound Caz+(and traces of Mg2+, Zn2+,and Cu2+),and aspartic acid as the sole, N-terminal amino acid. None of the simple sugars tested inhibited lectin-induced hemagglutination. Petryniak and his colleaguesso3purified the E . europeus lectin by adsorption onto insoluble polyleucyl hog A + H blood-group substance, followed by elution with lactose. The lectin, heterogeneous by several criteria, had a molecular weight of 166,000 (compare Ref. 802) and gave subunits of molecular weight 17,000 and 35,000 by electrophoresis in dodecyl sodium sulfate on poly(acry1amide) gel. The purified product, a glycoprotein (4.8% of D-galaCtOSe, 2.9% of D-glucose, and 2.8% of 2-acetamido-2-deoxy-~-glucose),precipitated B and H, but not Al, blood-group substances. Quantitative, hapteninhibition studies revealed theE. europeus lectin to be most specific for an a-D-Galp-[cx-DFucp-(1+2)]-( 1+3)-p-D-Galp-( 1+3 or 4)-&~-GlcNAc structure.803 A 2-amino-2-deoxy-~-hexose-binding lectin has been isolated from barley.804A homogeneous protein of molecular weight 31,000, the barley lectin is devoid of carbohydrate and half-cysteine residues. The lectin binds to the coat glycoprotein of barley stripe-mosaic virus by way of 2-amino-2-deoxy-~-glucoseand -D-galactose units.E04Only the free amino sugars, namely, 2-amino-2-deoxy-~-glucose,-D-galactose, and -D-mannose inhibit barley-lectin-induced aggregation of barley stripe-mosaic virus.804 The hemolymph from the elongate clam Tridacna maxima (Roding) contains a lectin that agglutinated human type O - e r y t h r o c y t e ~ . ~ ~ ~ - ~ ~ ~ Purification of the agglutinin was accomplished by adsorption onto polyleucyl-arabinogalactan followed by elution with a solution of 2-acetamido-2-deoxy-~-galactose.*~~ The isolated lectin (termed Tridacnin) gave a single band in gel diffusion, but several bands in disc-gel electrophoresis and isoelectric focusing.*05P-D-Galactans from (804) J. Partridge, L. Shannon, and D. Gumpf, Biochim. Biophys. Acta, 541,470-483 (1976). (805) B. A. Baldo and G . Uhlenbruck, FEBS Lett., 55,25-29 (1975). (806) G. Uhlenbruck, B. A. Baldo, and G . Steinhausen, Z . Immunitaetsforsch. Allerg. Klin. Immunol., 150,354-363 (1975). (807) B. A. Baldo and G. Uhlenbruck, Carbohydr. Res., 40, 143-151 (1975). (808) K. Eichmann, G. Uhlenbruck, and B. A. Baldo, Immunochemistry, 13, 1-6 (1976).

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Helix pomatia (L-galactose is also present), bovine lung, Larix occidentalis, and Lymnaea stagnalis L., as well as Pneumococcus type XIV polysaccharide and hog H-substance, all precipitate the T . maxima agglutinin.E06-E0E Hapten-inhibition studies revealed a primary specunits, although ificity for p-linked 2-acetamido-2-deoxy-~-galactosyl the lectin also interacted strongly with nonreducing (terminal) p-Dgalactopyranosyl groups; oligosaccharides containing a - ~ galactopyranosyl residues were poor inhibitors or n o n i n h i b i t o r ~A. ~ ~ ~ ~ ~ ~ ~ comparative study of the interaction of the T . maxima lectin, Axinella polypoides (sponge)agglutinin, and mouse-myeloma protein 5539 with (1+6)-P-D-galactans has also been reported.E0E A blood-group A lectin was isolated from frog(Rana catesbiana) eggs by gel filtration and poly(acry1amide)-gel e l e c t r o p h o r e ~ i sA. ~glyco~ protein containing 1.8%of carbohydrate, the lectin had a molecular weight of 210,000 and a carbohydrate-binding specificity that appears to be directed towards nonreducing trisaccharide units [a-D-GalNAcp(1+3,4)-~-~-Galp-(1+4,3)-~-D-GlcNAcp-(l+R)], as well as internal p-~-Galp-( 1+4)-P-D-GlcNAc residues.809The possibility exists that the lectin preparation, although showing a single band in electrophoresis, may be a mixture of two proteins having different s p e c i f i c i t i e ~ . ~ ~ A P-D-galactopyranosyl-binding protein was isolated from extracts of electric-organ tissue of the electric eel (Electrophorus electricus) by affinity chromatography on desulfated agarose (ECD-Sepharose).810 Termed “electrolectin,” the protein has a molecular weight of 33,000, agglutinates trypsinized rabbit erythrocytes, and is specifically inhibited by P-Dgalactopyranosides (lactose or o-nitrophenyl p-Dgalactopyranoside), but not by a-D-galactopyranosides (melibiose or raffinose).E1O Commencing with the discovery by Dodd and his colleaguesE” that sponges also contain hemagglutinins, several sponge lectins have been studied.E12-E14 Most sponge lectins are considered to be relatively nonspecific, in that they agglutinate A, By0,and AB erythrocytes to the same extent. Differential agglutination of animal erythrocytes has, however, been observed.E11 (809) F. Sakakibara, G. Takayanagi, H. Kawauchi, K. Watanabe, and S. Hakomori, Biochim. Biophys. Actu, 444, 386-395 (1976). (810) V. I. Teichberg, I. Silman, D. D. Beitsch, and G. Resheff, Proc. Nutl. Acad. Sci. U.S.A., 72, 1383-1387 (1975). (811) R. Y. Dodd, A. P. MacLennan, and D. C. Hawkins, Vox Sung., 15, 386-391 ( 1968). (812) S. Khalap, T. E. Thompson, and E. R. Cold, Vox Sung., 18,501-526 (1970). (813) S. Khalap, T. E. Thompson, and E. R. Cold, Vox Sang., 20,150-173 (1971). (814) H. Bretting, Z. Immunitaetsforsch. Allerg. Klin. lmmunol., 146,239-259 (1973).

3 16

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

Brettingsl*adsorbed to human A, B, and 0 erythrocytes two proteins from crude extracts of the sponge Aaptos papillata. One of the proteins exhibited hemagglutinating activity, and was isolated by gel filtration.81sOn using a column of polyleucyl blood-group A + H substance followed by a column of DEAE-cellulose, and preparative, disc-gel electrophoresis, Bretting and coworkersaI6isolated three Aaptos lectins (I, 11, and 111). Poly(acry1amide)gel electrophoresis of lectin I in the presence of dodecyl sodium sulfate gave two bands, of molecular weight 12,000 and 21,000 respectively; Aaptos lectins I1 and I11 each gave only one band, of molecular weight 16,000. The last two lectins had similar amino acid compositions, whereas Aaptos lectin I had a distinctive amino acid composition.816Lectin I precipitated bloodgroup substances having terminal (nonreducing) 2-acetamido-2deoxy-D-glucosyl groups; N , N', N", N" '-tetraacetylchitotetraose was the best inhibitor ofAaptos lectin I, being 2,000 times as active as 2-acetamido-2-deoxy-~-glucose. Lectins I1 and I11 precipitated blood-group substances containing nonreducing (terminal) 2-acetamido-2-deoxy-~-glucosyl groups, 2-acetamido-2-deoxy-~galactosyl groups, or sialic acid residues, and were best inhibited by N,N',N"-triacetylchitotriose. Aaptos lectin I was also precipitated by the monovalent hapten p-nitrophenyl2-acetamido-2-deoxy-a-~-galactopyranoside.s'6 A lectin from the sponge Axinella spec. was purified by Gold and colleague^,^'^ and was found to be an acidic protein having a molecular weight of 15,000-18,000. It contained a negligible proportion of carbohydrate, had an isoelectric point of 3.9, and contained bound Ca2+and Fe3+. Lacto-N-tetraose was the best inhibitor of Axinella-induced hemagglutination. Bretting and KabaP' isolated, and resolved, three hemagglutinins from Axinella polypoides. The two main lectins (I and 11) were studied. A. polypoides lectins I and I1 had molecular weights 21,000 and 15,000, Both agglutinins contained -0.5% of carbohydrate, and they were shown to be unrelated immunochemically and by amino acid composition.818Both lectins precipitated Al, Az, B, and Lea blood-group substances, and were inhibited best by terminal (nonreducing) P-D-galactopyranosyl groups.81a Finally, although they are not classified as lectins, the interesting and (815) H. Bretting and L. Renwrantz, Z. Immunitaetsforsch. Allerg. Klin. Immunol., 147,250-261 (1974). (816) H. Bretting, E. A. Kabat, J. Liao, and M. E. A. Pereira, Biochemistry, 15, 5029-5038 (1976). (817) E. R. Gold, C. F. Phelps, S. Khalap, and P. Balding, Ann. N . Y . Acad. Sci., 234, 122-128 (1974). (818) H. Bretting and E. A. Kabat, Biochemistry, 15,3228-3236 (1976).

LECTINS: CARBOHYDRATE-BINDING PROTEINS

317

useful interactions between myeloma immunoglobulins and polysaccharides should be mentioned (see, for example, G l a u d e m a n ~ ~ ' ~ ) . VIII. CELL-SURFACE, LECTIN-REACTIVE GLYCOPROTEINS The nature of those cellular structures with which lectins interact has been probed by (a) light- and electron-microscope analysis of tissue sections following reaction with appropriately derivatized lectins, ( b ) mono- and oligo-saccharide inhibition of radiolabelled-lectin binding, (c) competitive binding between two lectins of known specificity, (d) the effect of glycosidase digestion on lectin reactivity, and, most conclusively, ( e ) isolation of the reactive structures themselves. Although extensive use has been made of lectins (derivatized with ferritin, fluorescein, and radioisotopes) as histological stains, this topic is beyond the scope of the present article; a limited bibliography is presented for the convenience of interested readers.92a,820-833 Several interesting discoveries have been made as a result of the investigation of lectin-cell interaction. Many normal and transformed cells exhibit differential agglutinability when tested with con A (Refs. 31, 250, 834, and 835), wheat-germ agglutinin (Refs. 28-30, 492, and 834-837), R. cornrnunis a g g l ~ t i n i n ,soybean ~ ~ ~ , ~agg1utiniqss4 ~~ and lentil l e ~ t i n .In~ ~many ~ cases, proteolytic digestion increases the agglutinability of normal ce11s.492~836,837 The binding of a lectin to a cell is C. P. J. Glaudemans, Adu. Carbohydr. Chem. Biochem., 31, 313-346 (19735). W. Bernhard and S. Avranieas, E x p . Cell Res., 64, 232-236 (1971). N. K. Gonatas and S. Avrameas,j. Cell Biol., 59,436-443 (1973). C. Huet and J. Garrido, E x p . Cell Res., 75, 523-527 (1972). G. L. Nicolson and S. J. Singer, Proc. Natl. Acad. Sci. U.S.A., 68, 942-945 (1971). (824) R. M. Pratt, Jr., and W. A. Gibson, J. Histochem. Cytochem., 21,229-232 (1973). (825) J . Roth and K. Thoss, Experientia, 30,414 (1974). (826) J . Roth and K. Thoss, E x p . Pathol., 10, 258-267 (1975). (827) J . Roth, K. Thoss, M. Wagner, and H . W. Meyer, Histochemistry, 43, 275-282 (1975). (828) J. Roth, M. Wagner, and K. Thoss, E x p . Pathol., 11, 67-72 (1975). (829) S. B. Smith and J.-P. Revel, Deu. Biol., 27, 434-441 (1972). (830) J . D. Stobo and A. S. Rosenthal, E x p . Cell Res., 70,443-447 (1972). (831) R. W. Stoddart and J. A. Kienian, Histochemie, 33, 87-94 (1973). (832) K. Thoss and J. Roth, E x p . Pathol., 11, 155-161 (1975). (833) M . J . Cline and D. C. Livingston, Nature (London) New Biol., 232, 155-156 (1971). (834) A. A. Moscona, Science, 171,905-907 (1971). (835) J . Roth, G. Neupert, and K. Thoss, Exp. Pathol., 10, 309-317 (1975). (836) M. M. Burger, Fed. Proc., 32, 91-101 (1973). (837) R. R. Gantt, J . R. Martin, and V. J. Evans, J. Natl. Cancer Iizst., 42, 369-374 ( 1969).

(819) (820) (821) (822) (823)

318

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

a necessary, but not sufficient, condition for cell agglutination. For example, certain normal cells bind con A and wheat-germ agglutinin, but are not agglutinated, whereas their highly agglutinable, transformed counterparts bind equivalent amounts of these lectins .489*833 (These findings have been c o n t e ~ t e d . ~Extended, ~ ~ * ~ ~ or ~ )complex, lectin saccharide-binding sites were demonstrated for the P. vulgaris agglutinin’83,216~730*747 and the Agaricus bisporus l e ~ t i nby ~ ~using j purified, membrane-derived glycopeptides as inhibiting structures in place of simple oligosaccharides.216~435~730~747 The investigation of lectin-cell interaction by means of binding studies with labelled lectins is informative, but limited, in that the carbohydrate-binding sites of most lectins are still incompletely characterized. A comprehensive understanding of lectin-cell interaction requires, in addition to pure lectins of known properties, highly purified, lectin-reactive molecules that are also well characterized. In very few (if, indeed, any) cases has this ideal been achieved. It is to be hoped that such more-complete analyses will be forthcoming. In this Section, we consider in four sections, based on their cellular origin, lectin-reactive, membrane glycoproteins and glycopeptides that have been at least partially purified and characterized: 1 , erythrocytes and platelets; 2, lymphocytes; 3, neuronal tissue; and4, tumor cells. In reviewing lectin-reactive, cell-membrane glycoproteins, we have avoided the term “lectin receptor.” The term “receptor” should, we believe, be reserved for those unique, membrane structures that bind external molecules in a highly specific way, thereby transmitting signals from the environment to the interior of the cell. Hormones and drug receptors are examples of such unique structures. The interaction between lectins and cell surfaces is more general, in that lectins will react with a n y cell-surface, glycosyl moiety that is complementary to its binding site. It is, of course, possible for a lectin to interact with a unique “receptor” as defined; but this is only one of the cell-surface, carbohydrate-containing structures available. Several reviews deal A ~valuable ~~~~~~~~~~~ with lectin-reactive, membrane g l y c o p r o t e i n ~ . study on the activities of lectins and their immobilized derivatives in detergent solutions has been 1. Erythrocytes and Platelets Lectin-reactive glycoproteins of the erythrocyte membrane have been studied extensively. Of these, the major sialoglycoprotein has (838) K. D. Noonan and M. M. Burger,J. Biol. Chem., 248,4286-4292 (1973). (838a) R. Lotan, G . Beattie, W. Hubbell, and G. L. Nicolson, Biochemistry, 16, 17871794 (1977).

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319

been most completely characterized. This glycoprotein has a molecular ~~~-~~~ weight of 53,000-55,000,and contains 55-60% of ~ a r b o h y d r a t e . It carries the immunodeterminant structure for blood-group MN, but not ABO, specificity.842 The carbohydrate part of the major sialoglycoprotein occurs in two, distinct, oligosaccharide chains, an 0-glycosylically linked t e t r a s a c ~ h a r i d e and ~ ~ ~ an N-glycosylically of -6:1. Thomas and linked octa~accharide,~~"."~ in the ratio435,841,843 W i n ~ l e rdetermined ~~~ the structure of the 0-glycosylically linked chains to be those depicted in formula 15. 2-Acetamido-20-AcNeu 2

1

6 0-AcNeu- (2- J)-p-D-Galp- (I-- 3 ) - -GalNAcp~ (l+O)-Ser ,Thr 15

deoxygalactose was found only in 0-linked chains; 2-acetamido-2deoxyglucose and mannose were constituents of the N-linked oligosac.216,730,842

glycopepThe K ~ r n f e l disolated ~ ~ ~a P~. vulgnris ~ ~ ~ ~lectin-reactive * ~ ~ ~ tide from human erythrocytes; it derived horn the major sialoglycoprotein. The trypsin-released glycopeptides were reduced with alkaline borohydride (without loss of lectin reactivity), digested with pronase, and chromatographed repeatedly on Sephadex gels and DEAEcellulose. The purified glycopeptide represented a 3.7% yield of the initial activity. The single, N-glycosylically bound chain was composed of sialic acid, D-galactose, D-mannose, and 2-acetamido-2-deoxy-~glucose residues linked to asparagine. Digestion with glycosidase revealed a branched structure; one of two nonreducing (terminal) p - ~ galactopyranosyl groups was terminally substituted by sialic did not a ~ i d ~ as ~ shown ~ , ~ in formula ~ ~ * 14. ~ Although ~ ~ , desialization ~ ~ ~ alter kidney-bean lectin reactivity, removal of D-galaCtOSe abolished the inhibitory capacity. The chloroform-methanol-extracted, major sialoglycoprotein was 90 times as effective as its glycopeptide fragment in the kidney-bean lectin-erythrocyte hemagglutination inhibition assay, an effect ascribed to its m u l t i ~ a l e n c e . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ (839) J. P. Segrest, R. L. Jackson, E. P. Andrews, and V. T. Marchesi, Biochem. Biophys. Res. Commun., 44, 390-395 (1971). (840) J. P. Segrest, 1. Kahane, R. L. Jackson, and V. T. Marchesi, Arch. Biochem. Biophys., 155, 167-183 (1973). (841) M. Fukuda and T. Osawa,J. Biol. Chem., 248,5100-5105 (1973). (842) D. B. Thomas and R. J. Winzler,J. Biol. Chem., 244,5943-5946 (1969). (843) R. L. Jackson, J. P. Segrest, I. Kahane, and V. T. Marchesi, Biochemistry, 12, 3131-3138 (1973).

320

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

~ ~ ~ to and Robinia p ~ e u d o a c a c i aappear The lectins of the interact with the N-linked oligosaccharide of the major, erythrocyte sialoglycoprotein. The trypsin-released glycopeptide already mentioned inhibited hemagglutination by these two lectins. Alkaline borohydride removal of serine- and threonine-bound oligosaccharides had no effect on lectin reactivity. Furthermore, P . vulgaris lectin com~~ petitively inhibited binding of radiolabelled R. p ~ e u d o a c a c i aor~lentil l e ~ t i n ~ to3erythrocytes, ~ Conversely, R. pseudoacacia and lentil lectins partially blocked kidney-bean lectin binding. These lectins may interact with unique portions of a single oligosaccharide. The lectin of commercial mushroom (Agaricus bisporus), on the other hand, apparently binds to 0-glycosylically linked chains.435 Whereas trypsin-released glycopeptide I (with both types of chains) inhibited lectin-induced hemagglutination, neither fragment of the alkaline borohydride-reduced sialoglycoprotein exhibited activity. However, glycopeptide B, a pronase-digested fragment of glycopeptide I possessing only 0-linked oligosaccharide chains, was strongly inhibitory. Presant and K ~ r n f e l dsuggested ~~~ that the mushroom (A. bisporus) lectin displays an extended binding-site, part of which recognizes the 0-glycosylic linkage to serine or threonine. Chemical or enzymic removal of D-galactose from desialized glycopeptide B abolished the mushroom-lectin reactivity. Jackson and coworkerss43also studied glycopeptides derived from the major sialoglycoprotein of the erythrocyte membrane. Purified glycoprotein was degraded with trypsin; a combination of ionexchange chromatography and gel filtration then separated glycopeptides a-1, a-2, a-3, and p. Only glycopeptide a-1 contained 2-acetamido-2-deoxy-~-glucose residues, in which it accounted for 40% of the total hexosamine and was found only in N-glycosylic linkand age, presumably to asparagine. 2-Acetamido-2-deoxy-~-galactose sialic acid were constituents of each glycopeptide. Reduction with alkaline borohydride destroyed virtually all of the 2-acetamido-2deoxy-D-galactose, suggesting that it occurred only in 0-glycosylic linkage. Approximately half of the serine and threonine units of each glycopeptide were substituted by 2-acetamido-2-deoxy-~-galactose residues.843 Only the a-1 glycopeptide reacted with wheat-germ agglutinin, as determined by a hemagglutination inhibition assay. Inasmuch as the a-1 glycopeptide alone contains N-linked oligosaccharide chains, these chains must account for the wheat-germ reactivity. It is more difficult to explain the finding that the P. vulgaris lectin was inhibited by both the a-1and the p glycopeptide, but not by the a-2 or a-3 glycopeptide, as all have 0-linked carbohydrate chains. This observation is at variance with the Kornfelds’ demonstration216that P .

LECTINS : CARBOHYDRATE-BINDING PROTEINS

32 1

vulgaris lectin reacted only with the N-linked chains. It is possible that the reactive, 0-linked oligosaccharide of the P-glycopeptide differs from the tetrasaccharide structure elucidated by Thomas and Winzler.842In fact, Jackson and coworkers843demonstrated, by trypsin treatment of red cells, that the a-3 and P-glycopeptides are derived from buried portions (of the major sialoglycoprotein) which are unavailable to trypsin and, therefore, would not have been among the glycopeptides studied by Thomas and W i n ~ l e r . * ~ ~ KubLnek and colleagues844isolated a glycopeptide reactive with pea agglutinin from human, B erythrocyte ghosts. The purification scheme involved delipidation of ghosts with chloroform-methanol, followed by pronase digestion. Released glycopeptides were precipitated with ethanol, dissolved in 0.1 M acetic acid, and separated into two neutral, sugar-containing fractions (I and 11) by Sephadex G-25 gel-filtration. Fraction I, reactive with pea lectin, was further separated by verticalpaper electrophoresis in pysidine-acetate buffer, pH 5.6, into 5 peptides. Peptide 1.3 exhibited 79% of the pea-lectin reactivity present in unseparated fraction I, 14.3%of the total, neutral sugar of the erythrocyte ghost, and considerable blood-group B activity. Moreover, it inhibited both pea and lentil lectin-induced hemagglutination 32 times as effectively as D-glucose; the reactivity of the glycopeptide with con A was 5 times that with D-glucose. Glycopeptide 1.3 was comprised of 1 sialic acid, 4 D-galactose, 2 D-mannose, 1 L-fucose, 8 2-acetamido-2deoxy-D-glucose, 1 aspartic acid, 1 serine, and traces of threonine, glutamic acid, glycine, and alanine residues, giving a calculated molecular weight of -4,000. By virtue of its content of 2-acetamido-2deoxy-D-glucose, this glycopeptide may contain N-glycosylically linked chains derived from the major sialoglycoprotein.216~43s~730~747 PospiSilovL and coworkers'8s investigated the chemical structure of the pea lectin-reactive glycopeptide isolated by KubLnek and coworke r ~After . ~ refining ~ the chemical analysis, and applying glycosidase digestion, they proposed the structure depicted in formula 16. Despite Gal

Gal

-

GlcNAc

GlcNAc

\ Man

/

- GlcNAc

Asn

16

the fact that pea hemagglutinin is not inhibited by D-galactose, 90% of the pea reactivity of this glycopeptide is reportedly due to the two (844) J. KubBnek, G. Entlicher, and J. Kocourek, Biochim. Biophys. Acta, 304, 93102 (1973).

322

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

nonreducing (terminal) D-galactosyl groups.1ssThis result is difficult to reconcile with those from the sugar inhibition analysis of pea agglutinin specificity (see Section 11,3), The possible presence of two D-mannose units, one being a 2-0-a-D-mannopyranosyl residue, would account for the reactivity of this glycopeptide with the pea lectin (compare Refs. 209 and 210). Adair and Komfeld investigated the behavior of detergent-extracted, erythrocyte glycoproteins with a series of l e ~ t i n s .Triton ~ ~ ~ X-100*~~~ sodium borate buffer solubilized 40-50% of erythrocyte ghost protein and 75-85% of sialic acid, leaving undissolved between 10 and 30% of the binding capacity for the following lectins: Abrus precatorius, Agaricus bisporus, Lens culinaris, Phaseolus vulgaris, Ricinus communis, and Triticum vulgaris. Inhibition by cell extracts was assayed by reduction of [1251]-lectinbinding to erythrocyte ghosts. Affinitycolumn chromatography employing Sepharose-coupled R . communis agglutinin (RCAIzo)or wheat-germ agglutinin resulted in further purification of the detergent extract.aaaBoth columns were deliberately overloaded in order to provide maximal yields of reactive glycoproteins and to lessen nonspecific adsorption; this procedure should result in the selective adsorption of the glycoprotein of highest affinity in the event that several lectin-reactive species should be present. The lactose eluate of RCAlz0-Sepharose columns gave three protein bands (plus minor bands), but no clear bands staining with periodic acidSchiff reagent.aaaOne broad band, encompassing the major, sialoglycoprotein region, and probably representing several species, resulted when gel slices were assayed for hexosamine. The glycoproteins reactive with the lectins from A. bisporus, P. vulgaris, and L. culinaris all failed to bind to RCA120-Sepharose.66a The combined evidence suggests that the R. communis agglutinin does not bind the major, erythrocyte sialoglycoproteinaaawith which the mushroom, kidneybean, and lentil lectins r e a ~ t . ~ The ~ ~ R. , ~communis, ~ ~ * ~ affinity~ ~ * ~ ~ ~ isolated glycoprotein was 1,200 times as active as D-galactose in the standard inhibition assay.6a6Moreover, it inhibited A . precatorius lectin, but not the mushroom, wheat-germ, or kidney-bean agglutinins. In contrast, when erythrocyte-ghost extract was applied to a wheatgerm agglutinin-Sepharose column, the 2-acetamido-2-deoxy-~glucose eluate contained only the major sialoglycoprotein, as judged by electrophoretic mobility and chemical composition.66aThis preparation inhibited wheat-germ agglutinin 15,000 times as effectively as 2-acetamido-2-deoxy-~-glucose,and blocked A . bisporus and P . vulgaris lectin binding to erythrocyte ghosts as well. Pronase degradation of the native glycoprotein decreased its inhibitory activity by 98%; the authorsaa6ascribed this observation to the conversion of a multivalent

LECTINS : CARBOHYDRATE-BINDING PROTEINS

323

into a monovalent hapten. Neuraminidase treatment decreased wheat-germ agglutinin reactivity to one-ninth of its previous level. In agreement with Adair and Kornfeld,666Kahane and coworkersw5 reported a single-step purification of the major, erythrocyte sialoglycoprotein from detergent-extracted ghosts by means of wheat-germ agglutinin-Sepharose affinity chromatography. Furthermore, R. communis and P . vulgaris hemagglutinin-Sepharose columns also bound the major sialoglycoprotein, whereas con A and P . vulgaris leukoagglutinin-Sepharose columns did A slightly different, membrane-extraction procedure was employed by find la^^^^ in a study of erythrocyte glycoproteins. (Ethylenedinitri1o)tetraacetate- and Triton X-100-extracted, membrane glycoprotein was chromatographed on columns of Sepharose-coupled con A or lentil lectin. Eight percent of the applied material bound to con A-Sepharose, and was specifically eluted with methyl a - ~ mannopyranoside solution, whereas the corresponding figure for the lentil column was 30%.There was no apparent difference between the two elution-profiles, or between the protein-stained, electrophoreticgel patterns. However, staining with the periodic acid-Schiff reagent showed that lentil-Sepharose had retained both the major sialoglycoprotein and a minor glycoprotein [PAS 2, component a (Ref. 847); component I11 (Ref. 848)],whereas con A-Sepharose retained only PAS 2. Of the total, minor glycoprotein(s), 20-30%bound to con A-Sepharose, 60-80% bound to lentil lectin-Sepharose, and a fraction did not bind to either column. Moreover, a portion of the glycoprotein unreactive with con A-Sepharose did adsorb to a lentil lectin-Sepharose column. On the basis of these findings, find la^^^^ postulated heterogeneity in the oligosaccharide of PAS 2. In accordance with the results of Kahane and coworker^,^^ there was no evidence of interaction between con A and the major sialoglycoprotein. Fukuda and Osawawl utilized detergent extraction, ion-exchange chromatography, and gel filtration to isolate the major sialoglycoprotein of human, 0-erythrocyte membrane in pure form. Purity was demonstrated in ultracentrifugal studies and b y dodecyl sodium sulfate acrylamide-gel electrophoresis. The glycoprotein was devoid of tryptophan and cysteine, and contained very little methionine, phenylalanine, or tyrosine. Sialic acid and D-galactose were abundant; (845) I . Kahane, H. Furthmayr, and V. T. Marchesi, Biochim. Biophys. Acta, 426, 464-476 (1976). (846) J. B. C. Findlay,J. B i d . Chem., 249,4398-4403 (1974). (847) M. S. Bretscher,J. Mol. Biol., 59, 351-357 (1971). (848) G. Fairbanks, T. L. Steck, and D. F. H. Wallach, Biochemistry, 10, 2606-2617 (1971).

324

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

2-acetamido-2-deoxy-D-galactose, 2-acetamido-2-deoxy-~-glucose, D-mannose, and L-fucose were also identified. By hemagglutination inhibition assay, kidney-bean lectin and wheat-germ agglutinin interacted strongly with the glycoprotein, and C . sessilifolius lectin and the Ulex europeus lectins [both blood-group H ( 0 ) specific] interacted with moderate affinity. Inhibition by the glycoproteins of the lectins from S . japonicu, eel serum, B . purpureu, and V. gruminea (bloodgroup N specific) was weak; R . communis lectin, con A, and lima-bean lectin were not inhibited. Alkaline borohydride cleavage of O-glycosylically linked chains severely lowered the reactivity with the blood-group N and H ( 0 ) specific lectins, but increased the reactivity towards several blood-group, nonspecific lectins (con A, R . communis, lentil, pea, V. fuba).This suggested that, in some way, O-linked chains mask the reactivity of N-linked chains. In contrast to Kahane and cow o r k e r ~and ~ ~FindlayYM6 ~ Fukuda and 0sawas4l demonstrated interaction between the major sialoglycoprotein and con A. Furthermore, theys4*reported interaction between R. communis agglutinin and the major sialoglycoprotein, as did Kahane and coworkerss45;however, this The resolufinding is contested by the results of Adair and Kornfeld.666 tion of these discrepancies awaits further investigation. Human A erythrocyte stroma, pre-extracted with 26% potassium chloride solution, yielded soluble glycopeptides upon repeated digestion with a - c h y m ~ t r y p s i n . ~After ~ ~ J ~precipitation l by 90%-saturated ammonium sulfate, and extraction with phenol, this preparation contained inhibitory activity toward several lectins. Fractionation was performed with DEAE-cellulose, Sephadex G-100, and Sepharose 6B. Numerous fractions were obtained, none of which were characterized. Several fractions exhibited slight preferential reactivity towards lentil, lima-bean, pea, wheat-germ, and V. faba lectins. The use of digestion with a-chymotrypsin during the course of extraction precludes direct comparison of these glycopeptides with the tryptic peptide fragments derived from the major, erythrocyte glycoprotein studied previous~y,216.730,747,S43

Akedo and purified two rat-erythrocyte glycoproteins which interacted with con A of the nata bean, Canavalia gladiata. Rat-erythrocyte stroma were solubilized with dodecyl sodium sulfate and 2-mercaptoethanol. The acetone precipitate of the supernatant liquor was redissolved in detergent, and separated by preparative, acrylamide-gel e l e c t r o p h o r e ~ i s . ~ Two ~ ~ electrophoretically homogeneous, con A-reactive glycoproteins, BPI and BP2 (molecular weight 200,000 and 300,000, respectively), were eluted. They were comprised of protein, neutral sugar (25% by weight, BPI; 19% by weight, BPJ, and small proportions of sialic acid; neither had sig-

LECTINS : CARBOHYDRATE-BINDING PROTEINS

325

nificant hexosamine contents. P. vulgaris agglutinin did not react with either glycoprotein. Human-platelet membranes have been extracted with lithium 3,5diiodosalicylate, and the soluble glycoproteins separated on O-phosphonocellulose.a4pThe purified, membrane glycoprotein, molecular weight 100,000, was immunochemically identical to a sample purified from platelet-membrane extract by means of con A-Sepharose affinity chromatography. No further characterization was reported. 2. Lymphocytes

A mixture of lentil lectin-reactive glycoproteins from pig lymphocyte-plasma membrane was isolated by lentil lectin-Sepharose chromatography of sodium deoxycholate-solubilized membrane^."^ Eighty-seven percent of the protein applied (17% of hexose) passed through unretarded, and 13% of the applied protein (83% of hexose) was bound, and eluted with methyl a-D-glucopyranoside solution. Recovery was 95% of the material applied, in contrast to the recovery in similar experiments conducted on con A-Sepharose columns (80% recovery).850The eluate from the lentil column, which contained at least ten glycoproteins, blocked lymphocyte transformation induced by lentil or kidney-bean l e ~ t i n s . " ~ A highly purified, con A-reactive glycopeptide was obtained from calf thymocytes by PospiiilovL and colleaguess5' by using essentially the same protocol developed by KubLnek and coworkersa44to isolate the pea lectin-reactive glycoprotein from erythrocytes. Pronase diges.~~~ tion of delipidated membranes yielded soluble g l y c ~ p e p t i d e sThese were separated by gel filtration, and repeated, vertical, descending paper-electrophoresis in pyridine-acetate buffer, pH 5.6, yielding 12 mg of con A-active glycopeptide from 16 g of membranous material,8s1 The glycopeptide represented 0.15% of the membrane hexose, and contained 10.4% of carbohydrate. Analysis revealed the presence of D-mannose, D-galactose, D-glucose, L-fucose, 2-amino-2-deoxy-Dglucose, and sialic acid, as well as many amino acids (glycine and alanine preponderated). A minimum molecular weight of 12,000 was calculated. The glycopeptide inhibited [1311]conA binding to calf thymocytes 200 times more effectively than methyl CX-D(849) R. L. Nachman, A. Hubbard, and B. Ferris, J . Biol. Chem., 248, 2928-2936 (1973). (850) D. Allan, J. Auger, and M. J. Crumpton, Nature (London) New Biol., 236, 2325 (1972). (851) J. PospiHilovi, C. HaSkovec, G . Entlicher, and J. Kocourek, Biochim. Biophys. A c ~ u373,444-452 , (1974).

326

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

mannopyranoside (compared on a molar basis), and, in addition, blocked con A-induced, DNA synthesis by calf thymocytes.8s1 3. Neuronal Cells

Lectin affinity columns have been used to isolate, and purify, glycoproteins and glycopeptides from neuronal tissue, especially brain. Gombos and coworkersa52employed glutaraldehyde-insolubilized con A to isolate a series of glycopeptides from the pronase-treated, microsoma1 fraction of rat brain. These glycopeptides were rich in mannose and 2-acetamido-2-deoxyglucose,and stimulated neurite growth in tissue culture.8s2 Similar results were obtained by Susz and coll e a g u e ~ who , ~ ~ ~chromatographed whole-brain extracts, solubilized with deoxycholate and dodecyl sodium sulfate, on con A-Sepharose. In a continuation of these studies, it was shown that 25-30% of the total glycopeptides, obtained by papain treatment of delipidated, whole brain, bound to a con A-Sepharose column.8s4Elution with 5% methyl a-D-glucopyranoside solution gave a series of glycopeptides that contained mainly mannose and 2-acetamido-2-deoxyglucose,as well as small proportions of galactose and f ~ c o s eTreatment .~~~ of a purified, glycopeptide fraction with a-D-mannosidase drastically lowered its affinity for con A, suggesting the presence of terminal (nonreducing) a-D-mannopyranosyl groups,8s4 A water-soluble, 50%-methanol-soluble, acidic glycoprotein was isolated from rat brain by affinity chromatography on con A-Sepharose.8ss The glycoprotein, pure by poly(acry1amide)gel-electrophoresis at pH 8.8, had an apparent molecular weight of 14,500 +1,400 by dodecyl sodium sulfate gel-electrophoresis, No analysis for carbohydrate was reported.8ss Employing columns of immobilized, lentil lectin and wheat-germ agglutinin, Gurd and MahlersS6isolated a series of glycopeptides from deoxycholate-extracted, synaptic-plasma membranes; no analytical data were provided. In a similar study, Zanetta and coworkersss7 chromatographed the dodecyl sodium sulfate-solubilized extract from

(852) G. Gombos, J. C. Hermetet, A. Reeber, J.-P. Zanetta, and J. Treska-Ciesielski, FEBS Lett., 24, 247-250 (1972). (853) J. P. Susz, H. I. Hof, and E. G. Brunngraber, FEBS Lett., 32, 289-292 (1973). (854) J. I. Javaid, H. I. Hof, and E. G. Brunngraber, Biochim. Biophys. Acta, 404, 74-82 (1975). (855) G. Ramirez, K. G. Haglid, B. Karlsson, and L. Ronnback, FEBS Lett., 38, 143-146 (1974). (856) J. W. Gurd and H. R. Mahler, Biochemistry, 13,5193-5198 (1974). (857) J,-P. Zanetta, I. G. Morgan, and G. Gombos, Bruin Res., 8 3 , 3 3 7 4 4 8 (1975).

LECTINS: CARBOHYDRATE-BINDING PROTEINS

327

delipidated rat-brains on a con A-Sepharose column. A portion of the extract (fraction C1) bound to the affinity column, and was eluted with methyl a-D-glucopyranoside solution.857Fraction C 1 was markedly enriched with respect to D-mannose and 2-acetamido-2-deoxy-D-glucose; its electrophoretic profile was complex, showing multiple 4. Tumor Cells

The experiments of Walborg and his colleagues have focused on two rat-hepatoma lines that grow in ascitic form and exhibit different agglutinability patterns with con A and wheat-germ a g g l ~ t i n i n . ~ ~ ~ - ~ ~ Whereas Novikoff hepatoma cells are readily agglutinated by both lectins, AS-SOD hepatoma cells are much more s u ~ c e p t i b l e ~ to ~~~~~’-~ agglutination by wheat-germ agglutinin than by con A. Papain digestion of either tumor line not only rendered the cells agglutinable by low concentrations of either wheat-germ agglutinin or con A, but also released sialoglycopeptides capable of inhibiting con A- or wheat-germagglutinin-induced, guinea-pig-erythrocyte h e m a g g l ~ t i n a t i o n . ~ ~ ~ . ~ ~ ~ A crude, sialoglycopeptide fraction (representing 65-80% of neuraminidase-labile sialic acidEs8)was obtained by Sephadex G-50 chromatography of the papain By employing Sephadex chromatography of the pronase digest, Wray and W a l b ~ r g partially ~ ~ ~ resolved the crude mixture of glycopeptides from Novikoff cells into a fraction of molecular weight >3,300 that inhibited both lectins, and a fraction of molecular weight 2,000-3,300 that inhibited only con A. Each fraction was further resolved by ionexchange c h r o m a t ~ g r a p h yAlternatively, .~~~ the crude papain-digest of either tumor line was separated into sialic acid-containing fractions A, B, and C by chromatography on Sephadex G-50 equilibrated with 0.1 M acetic a ~ i d . AS30D ~ ~ ~ fraction - ~ ~ A ~ demonstrated wheat-germ agglutinin and con A reactivity, fraction C contained only con A reactivity, and fraction B inhibited neither lectin.861Fraction A from AS30D cells was degraded with pronase, and resolved861into A1 and A11 on Sephadex G-200. A1 (excluded from Sephadex G-200)appeared to be resistant to (858) E. F. Walborg, Jr., R. S. Lantz, and V. P. Wray, Cancer Res., 29, 2034-2038 (1969). (859) V. P. Wray and E. F. Walborg, Jr., Cancer Res., 31,2072-2079 (1971). (860) D. F. Smith and E. F. Walborg, Jr., Cancer Res., 32,543-549 (1972). (861) D. F. Smith, G. Neri, and E. F. Walborg, Jr., Biochemistry, 12, 2111-2118 (1973). (862) G. Neri. D. F. Smith, E. B. Gilliam, and E. F. Walborg, Jr., Arch. Biochem. Biophys., 165,323-330 (1974). (863) G. Neri, D. F. Smith, E. B. Gilliam, and E. F. Walborg, Jr., in “Comparative Biochemistry and Physiology of Transport,” L. Bolis, K. Bloch, S. E. Luria, and F. Lynen, eds., North-Holland Publishing Co., Amsterdam, 1974.

328

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

proteolysis, and exhibited both wheat-germ agglutinin- and con A-inhibiting activity, whereas A11 was nonreactive by hemagglutination inhibition.861A1 was separated into three components by ion exchange on DEAE-cellulose in pyridine-acetic acid buffer. Component DAI-2, representing 12% (by weight) of the crude sialoglycopeptide mixture, strongly inhibited wheat-germ agglutinin; it was composed of relatively high proportions of aspartic and glutamic acids, and serine and threonine, with no methionine or arginine. Furthermore, this component was comprised of 18.8% of 2-amino-2-deoxy-D-glucose and 12.7% of D-gahCtOSe, with lesser proportions of 2-amino-2-deoxy-~galactose, D-mannose,L-fucose, D-glucose, and sialic acid. On the other hand, the fraction most reactive with con A was DC-2, obtained by ionexchange chromatography of fraction C. DC-2 represented 7% (by weight) of the original sialoglycopeptide mixture, but was not chemically analyzed.861 A similar resolution of Novikoff cell fraction A was achieved by using862*sm Sephadex G-200. Fraction A1 had a high molecular weight (100,000 to 200,000) and possessed lectin reactivity, whereas A11 did not. DEAE-cellulose ion-exchange in pyridine-acetic acid buffer distinguished eight component^.^^^*^^^ Con A reactivity was associated with DAI-2 (which was ten times as active as the crude mixture); peak, wheat-germ-agglutinin reactivity was associated with DAI-4 (four times as active as the crude mixture). Chemical analysis of a DAI-1,2 mixture gave results very similar to those of AS-SOD fraction DAI-2; glutamic and aspartic acids, serine, and threonine were abundant, and 30% of the carbohydrate was hexosamine; D-mannose, D-glucose, D-galactose, L-fucose, and sialic acid were also present.862 Inhibitory activity of fraction A1 towards wheat-germ agglutinin and con A paralleled the agglutinability of the tumor cell from which it derived: Novikoff A1 exhibited potent reactivity towards both lectins; AS-SOD A1 reacted strongly with wheat-germ agglutinin, and minimally863with con A. The lectin-reactive component of A1 appeared to be a peptide highly substituted with oligosaccharide side-chains, as indicated by its exclusion from Sephadex G-200, its high content of amino acids known to be involved in protein-carbohydrate linkages, and its resistance to proteo1ysksa The structure of the reactive oligosaccharide was not determined. Mouse L1210 leukemia cells were the source of a fraction containing wheat-germ-agglutinin-reactive g l y c o p r o t e i n ~ . ~Insoluble ~~-~~~ (864)M. M. Burger, Nature (London),219,499-500 (1968). (865) V. K. Jansons and M. M. Burger, Biochim. Biophys. Acta, 291, 127-135 (1973). (866) V. K. Jansons, C. K. Sakamoto, and M. M. Burger, Biochim. Biophys. Acta, 291, 136-143 (1973).

LECTINS : CARBOHYDRATE-BINDING PROTEINS

329

material obtained by hypotonic incubation of L1210 cells, when extracted with either phenol, guanidinium chloride, pyridine, or lithium 3,5-diiodosalicylate, yielded a supernatant solution containing at least four components (by gel electrophoresis), each of The mixture which contained 2-acetamido-2-deoxy-~-glucose.~~~ inhibited wheat-germ lectin agglutination of L1210, Py3T3, and PyBHK transformed cells,x6s but not agglutination by con A or Phaseolus vulgaris hemagglutinin.866Antiserum to the mixture of wheatgerm-agglutinin-reactive glycoproteins showed specificity towards L1210 cells, but it did not react with normal-mouse lymphocytes or erythrocytes, with which wheat-germ agglutinin does react.x66The antiserum blocked wheat-germ-agglutinin, but not con A agglutination of Py3T3 cells.866Resolution of the supernatant solution on Sephadex G-200 in 33% pyridine with 2-mercaptoethanol gave two broad peaks; activity was associateds6' primarily with a peak having a molecular weight of 40,000 to 60,000. A lithium 3,5-diiodosalicylate extract of mouse L929 cells, and amino prelabelled in vitro with 2-amino-2-deoxy-~-~H]glucose [14Clacids,was separated by chromatography on Sephadex G-200 into two fractions.s67Fraction A was excluded from the gel, and contained fraction B, one third of the applied 2-amino-2-deoxy-~-[~H]g~ucose; retarded by Sephadex G-200, contained two-thirds of the applied 2-amino-2-deoxy-~-[~H]g~ucose. Fraction A was applied to a column of con A-Sepharose from which a glycoprotein of molecular weight -100,000 was eluted with methyl a-D-mannopyranoside solution. The glycoprotein migrated as a single component in dodecyl sodium sulfate gel-electrophoresis and represented 51% of the 2-amino-2-deoxy-~[3H]glucoseapplied. Similar results were obtained when plasma membrane, labelled by means of D-galactose oxidase and potassium borotritide, was extracted and separated in an analogous manner.867Amino acid analysis showed a relative abundance of serine, glycine, glutamic acid, and alanine. Valine was found in the N-terminal position. Cleavage of the intact glycoprotein with cyanogen bromide resulted in five fragments; each contained bound 2-amino-2-deoxy-~-[~H]glucose, suggesting that the L929 membrane glycoprotein was multi-substituted by oligosaccharide chains.s67 Nachbar and coworkers investigated the composition of Ehrlich ascites, tumor-cell plasma membrane by using a series of lectin-Sepharose adsorbents ( R . communis hemagglutinin, con A, wheat-germ agglutinin, and soybean agglutinin).s68The Ehrlich cells (867) R. C. Hunt, C. M. Bullis, and J. C. Brown, Biochemistry, 14, 109-115 (1975). (868) M. S. Nachbar, J. D. Oppenheim, and F. Aull, Biochim. Biophys. Acta, 419, 512-529 (1976).

330

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

were agglutinated by RCAIzo,wheat-germ agglutinin, con A, and soybean agglutinin in decreasing order of agglutinability; Ulex europeus I did not agglutinate the cells. For membrane protein from Ehrlich cells labelled in vivo with ~-[~H]2-amino-2-deoxyglucose, 80-90% was extracted by 10% deoxycholate in Tris [2-amino-2-(hydroxymethyl)-1,3-propanediol] buffer. When the extract was separated by means of one of the four lectin-Sepharose columns, electrophoretic profiles of the specifically bound, sugar-eluted fractions showed both common and unique peaks for each lectin. The fractions were neither purified, nor characterized with regard to chemical composition.868 Kim and coworkers examined glycopeptides from normal, and cancerous, colonic m ~ c o s a .Pronase ~~~ digestion of disrupted cell-membranes gave a soluble glycopeptide fraction. The fractions obtained from normal tissues inhibited Dolichos biflorus, but not Ricinus communis, hemagglutination, whereas the reverse was true of fractions obtained from malignant, colonic mucosa. No further analysis was carried O U ~ . ~ ~ ~ Carcinoembryonic antigen (CEA), a glycoprotein (molecular weight 200,000) purified to homogeneity from malignant, human, gastrointestinal-tract tumors,870 contains 50% (by weight) of carbohydrate.413Hammarstrom and coworkers413 listed a series of lectins that precipitated CEA, including con A, and P. vulgaris, R . communis, and wheat-germ agglutinins. Helix pomatia, Dolichos biflorus, and B . simplicifolia I did not precipitate CEA. One-cycle, Smith-degraded CEA, in which all L-fucose, sialic acid, 70% of the D-galaCtOSe, and 15-30% of the D-mannose had been destroyed, showed strong reactivity with H . pomatia lectin, but the reactivity with R. communis and con A was abolished. After two cycles of Smith degradation, the H . pomatia reactivity was lost. Wheat-germ-agglutinin-reactivity was unimpaired throughout four cycles of Smith degradation (see Fig. 10).The authors postulated413that most of the wheat-germ-agglutinin-reactive 2-acetamido-2-deoxy-~-glucoseis situated in the interior of the carbohydrate chain, in N-glycosylic linkage to asparagine. Two sublines of the TA3 mouse mammary carcinoma, St and Ha, were studied by Codington and coworkers762with respect to their reactivity with the Vicia graminea lectin. The Ha subline, which grows in allogeneic, as well as syngeneic, mice, adsorbed 100 to 400 times as much V. graminea lectin as subline St, which grows only in syngeneic (869) Y. S. Kim, R. Isaacs, and J. M. Perdomo, Proc. Natl. Acad. Sci. U S A . , 71,48694873 (1974). (870) P. Gold and S. 0.Freedman,J. E x p . Med., 121, 439-462 (1965).

LECTINS : CARBOHYDRATE-BINDING PROTEINS

331

mice. Furthermore, proteolysis of Ha cells lowered the lectin adsorption by 90%, and released glycopeptides that completely inhibited V. gruminea-N erythrocyte agglutination at levels of 5-10 puglml. Proteolysis of subline St cells did not release lectin-reactive material. Carbohydrate analysis of the papain-solubilized glycopeptides revealed the presence of D-galactose, 2-acetamido-2-deoxy-~-galactose, 2-acetamido-2-deoxy-~-glucose, and sialic In summary, many investigations of lectin-reactive, membrane glycoproteins and glycopeptides have been initiated. The molecules have been obtained both by proteolysis of whole cells and by detergent extraction of plasma membranes. In many instances, affinity chromatography on lectin-Sepharose adsorbents has proved extremely useful in separating and purifying lectin-reactive glycoproteins. Unfortunately, analyses of purified material have most often included compositional, but not structural, characterization (with the exception of the work of Osawa and c o ~ o r k e r s , ' ~Koriifeld ~ , ~ ~ ~ and colleagues,216*434*435*666,730*731,747 and PospiBilovii and coworker^'^^) and

. . H(0)-Determmant

~ - ( 1 - 3 ) - ~ - ~ - G l c N A ~ p - (- l- --- - - - - - - - Ser,Thr. \

J I

FIG. 14.-Hypothetical, Composite "Megalo-oligosaccharide" Structure for Bloodgroup Substances, Showing the Carbohydrate-binding Loci for Various Lectins. [a, Dolichos bijlorus; b, Phaseolus lunatus; c, Helix pomatia; d, Glycine max; e , Bandeiraea simplic$olia I (A4); f, Bandeiraea simplicifolia I (BJ; g, Abrus precatorius; h, Sophora japonica; i, Triticum vulgaris; j, Cytisus sessilifolius; k, Canuvalia ensiformis; 1, Bandeiraea simplicifolia 11; m, Ricinus communis; n, Ulex europeus; 0, Arachis hypogaea; p, Lotus tetragonolobus; q, Anguilla anguilla; and r, Vicia graminea.]

332

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

must therefore be considered incomplete. It will be both interesting and valuable to compare lectin-reactive, membrane glycoproteins from normal cells and transformed cells, erythrocytes and lymphocytes, platelets, and other sources when information regarding their structures becomes available. Figs. 14 to 16 present the structures of a series of naturally occurring glycopeptides, and summarize the carbohydrate-binding specificity of a number of lectins in terms of the carbohydrate units with which they interact.

+

4,6) - P - D - Manp 1

I

4

p - D - Glc NAcp 1

I

4 p-D-GlcNAcp

b, f , m,n

1 1

N Asn

FIG. 15.-Human IgA, Glycopeptides7' Showing the Carbohydrate-binding Loci for Various Lectins. [a. Limulus polyphemus; b, Triticum vulgaris; c, Ricinus communis; d, Sophora japonica; e, Abrus precatorius; f, Cytisus sessilifolircs; g , Phuseolus vulgaris; h, Canavalia ensiformis; i, Lens culinaris; j, Pisum sativus; k, Vicia faba; 1, Bandeiraea simplicifolia 11; m, Solanum tuberosum; and n, Ulex europeus 11.1 (871) J. Baeiiziger and S. Kornfeld,J. Biol. Chem., 249, 7260-7269 (1974).

LECTINS: CARBOHYDRATE-BINDING PROTEINS

333

I

1

-

8- D- XJrlp (1

---t

4

1

6)-p -D- GlcNACp

h, i , j , k

i

N Asn

FIG. 16.-Glycopeptide from Pineapple-stem Bromelins7z~873 Showing the Carbohydrate-binding Loci for Various Lectins. [a, Canavalia ensijormis; b, Lens culinaris; c, Pisum sativum; d, Vicia faba; e, Lotus tetragonolobus; f, Anguilla anguilla; g, Ulex europeus I; h, Triticum vulgaris; i, Solanum tuberosum; j, Cytisus sessilifolius; and k, Ulez europeus 11.1

A lectin from the seeds of Duturu strumonium L. Cjimson weed) was isolated by affinity chromatography on the insoluble polysaccharides from Aspergillus r ~ i g e r . ~A’ ~glycoprotein (28% neutral sugar, preponderantly arabinose), the lectin is blood-group ABO-nonspecific, contains large proportions of cystine and glycine, and 6.3%of hydroxyproline, and has a molecular weight of 120,000. Chito-oligosaccharides bind to the Daturu l e ~ t i n . ~ ~ , ~ ~ ~ Bausch and P ~ r e t purified z ~ ~ ~ the Muclura pomifera seed-lectin (see Section V,4) to homogeneity on insolubilized polyleucyl hog-gastric mucin. The lectin, a glycoprotein containing 0.8% of neutral sugar, has a molecular weight of 40,000, and is “composed of two pairs of dissimilar polypeptide chains stabilized by noncovalent interactions” (Ref. 875).Relatively rich in acidic and hydroxy amino acids, the lectin contains a very small proportion of methionine, but is devoid of cysteine. Studies on sugar-inhibition of hemaggl~itination~~~ indicated that the Macluru lectin has a high specificity for a-D-galactopyranosyl end-groups and for 2-acetamido-2-deoxy-~-galactose. (872) Y. Yasuda, N. Takahashi, and T. Murachi, Biochemistry, 9, 25-32 (1970). (873) Y . 4 . Lee and J. R. Scocca,J. Biol. Chem., 247,5753-5758 (1972). (874) V. HoiejGi and J. Kocourek, Biochim. Biophys. Acta, 532, 92-97 (1978). (875) J. N. Bausch and R. D. Poretz, Biochemistry, 16,5790-5794 (1978).

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

334

IX.TABLES TABLEXXIII Physical and Chemical Properties of Purified Lectins" Subunit structure Latin name (common name) Abrus precatorius hemagglutinin (jequirity bean)

toxin (abrin)

Anguilla anguilla (eel) Aruchis hypogaea (peanut)

Molecular weight

Molecular weight

132,000(Ref. 611) 134,000(Ref. 147) 36,000,35,000,and 33,000 125,000-132,000 33,800and 32,200 (Ref. 610) (Ref. 610) 63,800(Ref. 607) 33,000 and 28,000 (Ref. 607) 65,000(Ref. 150) 35,000and 30,000 (Refs. 147,150) 123,000 10,000 110,000 27,000-28,000 106,500

Number

2

1 4

1

l2

12 4

Bandeiraea simplicifolia

I

114,000

I1

113,000 104,000(pH 7) 52,000 (pH 5)

Canaualia ensiformis (jack bean)

Cytisus sessilifoliolizrs Dolichos bijlorus (horse

28,500 (5isolectins: &, A3B, AdL BJ 30,000 26,000

110,000

4 4 4 (PH 7) 2 (PH 5) n.d.

gram)

A

113,000 109,000 Glycine max (soybean) 120,000-122,000 Helix pomatia (edible snail) 79,000 Lens culinaris (lentil) 42,000-63,000 335,000 400,000

26,500 26,000 30,000 13,000 18,000 8,000 19,000 20,000

120,000 58,000 117,000

27,800-28,000 27,800-29,000 27,000-30,000

4 2 4

269,000,247,000 138,000,124,000

31,000 31,000

8 4

B

Limulus polyphemus (horse-shoe crab)

4 4 4 6

2 1 4 18-20

Lotus tetragonolobus (asparaguspea)

A B

C Phaseolus lunatus limensis (lima bean)

I1

I11

LECTINS : CARBOHYDRATE-BINDING PROTEINS

Glycopmtein Carbohydrate (per cent by weight)

Amino acid composition’’

Sugars

Cysteine

Methionine

8

Man (Ref. 610) Man, Glc, GlcNAc

21.6

18.3

5

Man, Glc, GlcNAc

3

Man, Glc, GlcNAc

4.2 10

0.39 -

GlcNAc

n.d. 16.6

6.7

Man, Fuc, Xyl, GlcNAc

4.0 -

Man, Fuc, GlcNAc -

147,611,651

6.7

610,651 147,150,607 612,651

11-13

+

References

3 0

n.d. n.d. n.d.

679 202 616

1

0.4

Ca2+,Mgz+

131

1

3 2

Ca2+,Mg2+ Ca2+,Mn2+

n.d.

n.d.

125 136,258,261, 262,262a,265, 267,271,324 149,476

4

-

n.d. -

2

4 1 2 - (4)

neutral sugars, GlcNAc

3

2-3

n.d. 108,519-521 n.d. 108,520,521 Ca2+,MnZ+ 151,538,544,546 561,569,570 Ca2+,Mn2+ 138,141-143, 441,442,444 n.d. 159,769-771

Gal, GlcNAc Gal, GlcNAc Gal, GlcNAc

0

trace trace trace

n.d. n.d. n.d.

Man, GlcNAc, Fuc Man, GlcNAc, Fuc Man, GlcNAc

2 2

3.8-4.7 1.3 7 8 1.5-3

Man, GlcNAc Man, GlcNAc Man, GlcNAc Gal, Man Glc, GlcNAc

>4

3-5 3-5 10-11

Metals

n.d.

Man, Glc, GlcNAc

9.4 4.8 9.2

335

-

3

0 0

0

0 0 0

200,683,684 200,683,684 200,683,684

Ca2+,Mn2+ 151,199,586,588 Ca2+,Mn2+ Ca2+,MnZ+ 630,697,698, 705-716,718 (Continued)

336

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES TABLEXXIII (Continued)

Subunit structure

Latin name (common name)

Molecular weight

Phaseolus vulgaris (red kidney-bean)

126,000-136,000

Pisum sativum (pea)

49,000-53,000

Ricinus comniunis hemagglutinin (castor bean)

120,000

toxin (ricin) Solanum tuberosum (potato) Sophora japonica (pagoda tree) Triticum vulgaris (wheat germ) Ulex europeus (gorse, h n e , whin seed) I

I1 Vicia faba (horse, broad, or fava bean)

Vicia graminea

60,000 100,000

Molecular weight

29,000-34,000 (5 isolectins: L,, L& LR,, L% R J CY,7,000-10,500 p, 12,000-18,000 29,500 and 37,000 (Ref. 146) 31,000 and 34,000 (Ref. 147) 27,500 and 30,000 (Refs. 648, 651) 28,000 and 32,000 (Refs. 146, 147, 150, 194, 648, 649) 46,000

132,800 36,000

Number

4

2

1 4

4

2 2

n.d. 18,000

43,000, 45,000 (Ref. 158) 31,000, 32,000 (Ref. 691) 40,000-65,000 (Ref. 690) 170,000 (Ref. 226) n.d.

2

n.d.

n.d.

47,500-53,000

24,000

105,000

18,000 9,000 17,300 13,300 25,000

2

(Ref. 213) R

2 4

LECTINS : CARBOHYDRATE-BINDING PROTEINS

Glycoprotein Carbohydrate (per cent by weight) trace (<0.5)

Sugars

337

Amino acid compositionL

Cysteine

Methionine

Metals

141,453-455

Glc, Xyl

0

0

4.7

Man, Glc, GlcNAc

1.69, 1.77

1.05, 1.23

146,147,648, 650,651

5.5-6

Man, Glc, GlcNAc Ara, Gal, Glc, GlcNAc Man, Xyl, GlcNAc

0.78 0.71, 0.95 1 n.d.

146,147,150, 194,648-650

50

9.81 1.32, 1.82 25

7.8 0

5.2-7.2

21.7 3

7.3

5 17-18

Man, Fuc, Glc, Xyl, Ara, Glc, GlcNAc

Man, Gal, Ara, GlcNAc GlcNAc, Man

Man, Glc, Fuc, Gal, GlcNAc

2

Ca2+,MnZ+

References

207

0

n.d.

184,598

2

n.d.

128-130,335, 495

2-3

Ca2+,Mn2+, Zn2+

158,196,225, 226,509,690, 691

+

+

n.d.

0

0

n.d.

194,208,225, 226,509 140,2 13,467a

8

8

n.d.

220

“+,present; -,absent; n.d., notdetermined. *Molesofamino acid permole ofsubunit.

0 0

00

TABLEXXIV

Blood-group Specificity and Carbohydrate-binding Specificity of Purified Lectins Latin name

Blood-group specificity

147,603 21,64 65,670

Abrus precatorius Anguilla anguilla Arachis hypogaea Bandeiraea simplicifolia BS I BS I1 Canaoalia ensiformis

References

neuraminidase-digested A, B, 0, or T antigen B >> A, Tk

nonspecific

Cytisus sessilifolius

0 > A2 > A,

Dolichos bijlorus Glycine mar Helix pomatia

Al >> As A>O>B A

Lens culinaris

nonspecific

Lotus tetragonolobus

0 >> A2

202,710 131,195,622, 625 125,470 102,246,247 6,77,78,472, 473,476 108,510-512 212 60,63,100, 189 138,142 6,22,77,78

Carbohydrate specificity

References

P-D-Galp > a-DGalp (Y-L-FUCP 3-0-Me-DFucp 3-O-Me-~Galp P - ~ G a l p -1+3)-~-GalNAcp (

147 21,22 167,471 672,674,676 20 1,617-6 19

a - ~ - G a l p> a-DGalNAcp

20,131,195, 626 125 168-170, 204,215,365 19,20,471, 475,477 108 212,552 61,63,100, 178,561,562 109,123,138, 143,213 22,77,78, 167,672,674

P-D-GICNACP= a-DGlcNAc a-DManp > a - ~ - G l c p> a-DGlcNAcp P-wGlcNAcp-( 1+4)-P-~GlcNAc > cellobiose a-DGalNAcp a-D-GalNAcp > P-DGalNAcp a-DGalNAcp >> a-DGlcNAcp a-DManp > a-DGlcp, a-DGlcNAcp

~Y-L-Fuc~, 2-O- Me-nFucp

1z Y Q 0

r

U

M

4

z

5

5r r

M

M

z

M

5*

!2

Maclura pomifera

Phaseolus lunatus syn.

nonspecific A, > A2 >> B

631,632 2,5,22,199

D-Galp, DGalNAcp a-DGalNAcp > a-DGalp

limensis

Phaseolus vulgaris Pisum sativum

nonspecific nonspecific

696 141,448

Ricinus communis

nonspecific

Solantcni tuberosum

nonspecific

147,603,641, 642 77,480,482

Sophora japonica

Triticum vulgaris

A>B>>O I-antigen nonspecific

22,78,184, 593,599,601 501

Vicia faba

nonspecific

Vicia graminea Ulex europeus

N )"

750,752-758

Ulex I

0 >> A2

Ulex I1

0 >> A2

12-14,77,81, 225 12-14,77,81, 225

5,462

a-D-Manp > a - ~ G l c p> a-D-GlcNAc P-DGalp > a - ~ G a l p P-D-GlcNAcp-( 1+4)-M-D-GlcNAcp-(1+4)&P-DGlcNAc P-D-GalNAcp > P - ~ G a l p> a-D-Galp P-D-GlcNAcp-( 1+4)-P-D-GIcNAcp-( 1+4)-P-DGlcNAcp > P-DGlcNAcp-(1+4)-GlcNAc a-DManp > a-~-GlcNAcp

632,635-637 22,151,199, 591 730,731 122,211,213, 428,440,448, 449,452 124,146,147, 183,194 206,207,483 175,183 184 30,128,498

0

3

..5 n 0

140,213,468, 469

CY-L-FLIC~ P-DGlcNAcp-(1+4)-P-~-GlcNAc

n P ~ G a l p 1+4)-P~GlcNAcp-( -( 1+2)-a-~Manp P ~ - G a l p -1+4)-P-~-GlcNAcp-( ( 1+2)-w~-Manp

0 0 (D

IRWIN J. GOLDSTEIN AND COLLEEN E. HAYES

340

TABLEXXV Lectin-Carbohydrate Association as Studied by Equilibrium Dialysis

Latin source

Abrus precotorius (jequirity bean) Bandeiraea simplicifoliu I Cunavalia ensiforrnis (concanavalin A from jack bean) Glycine mas (soybean)

Helix pomatia (snail) Lens culinnris (lentil)

Carbohydrate ligand lactose methyl a-mgalactopyranoside methyl a-D-mannopyranoside 2-acetamido-2-deoxy-~galactose A substance pentasaccharide D-mannose methyl a-mglucopyranosicle

& ("C) (M-')

na

References

lo3(----)*

2/4

147

8.6 x lo4 (2") 3.3 x 104 (207 2.06 x 104 (20)

4/4

626

44

172,278

(4")

2/4

544

lo3 (25")

6/6

570

2.3 x lo2 (4") 1.0 x 102 (4")

2/2 2/2

186

1.2 x 104 (37 0.6 x 104 (37 3.7 x 104 (3")

44 2/2 414

683

1.01 x 103 (20) 0.93 x lo3 (2')

2/4 4/8

589

1.5 x 104(----)b 2/4 1.65 X lo4 (25") 2/4

147 124

8x

3.0 x

5x

lo4

Lotus tetragonolobus (asparagus pea) A B C

Phaseolus lunatus (lima bean) I11 I1 Ricinus conzniunis (castor bean)

Pisum satiuum (pea) Triticum vulguris (wheat-germ agglutinin)

Wistaria floribundn

L-fucose

methyl 2-acetamido-2deoxy-a-D-galactopyranoside lactose p-nitrophenyl p-Dgalactopyranoside D-mannose methyl C X - D - ~ ~ L W O pyranoside 2-acetamido-2-deoxy-~glucose

1.4 X lo2 (4") 8 x lo2 (4")

214 2/4

453

1.3 x 10" (4")

4/2

129

N,N',N",N"'-tetraacetyl- 5.3 x 104 (20")

4/2

115

chitotetraitol 2-acetamido-2-deoxy-~galactose

2/2

796

1.28 x 104 (----)I'

"Binding sites per subunit = n. !'(----), temperature not reported.

BIOCHEMISTRY OF PLANT GALACTOMANNANS

BY PRAKASH M. DEY* Department of Biochemistry, Royal Holloway College, University of London, Egharn Hill, Egham, Surrey, TW20 OEX, England I. Introduction ... .. ... ........ . . .. . . .. . . .. . .............. . .. .. ..... ... .. . 1. Occurrence .... .. .................. .......... .... ... .. .... .... ...... 2. Location in uiuo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , , , . . . 3. Isolation . . . .... .. ...... ... .. .... ... .. .. . . . . , .. . .... . .......,. .. ... .. 4. Structure.. . . . . . . . . . . . . . . . . . . . . . . . . . , . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Biosynthesis ......... .. .. .... ........... ..... . ... .... .. ...... ... .. ..... 111. Biochemical Degradation . . . . . . . . . . . . . . . . . . . , . . , . . . . . . . . . . . . . . . . . . . . . . . . 1. General Considerations . . . . . . , , . . , , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . , . 2. Enzymes Involved.. . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . .. IV. Function ...............................................................

341 343 345 345 347 352 356 356 361 375

I. INTRODUCTION Plant galactomannans are reserve polysaccharides composed of linear chains of (1*4)-linked P-D-mannopyranosyl residues having single stubs of a-D-galactopyranosyl groups joined by (1+6)-linkages along the chain. These polysaccharides are also known as gums; their use as substances for mummification can be traced back to 3000 B.C. in ancient Egypt, and, hence, they are humorously called “Pharaoh‘s” polysaccharides. The importance of these polysaccharides can be seen in their wide use in industry, notably, food,*-” pharmaceutical^,'^-^^ *The author is indebted to Professors J. B. Pridham and J. E. Courtois, and Dr. D. R. Davies, for their valuable suggestions.

(1) D. A. Rees, Biochem. J., 126,257-273 (1972). (2) M. Glicksman and E. H. Farkas, Fr. Pat. 2,119,365 (1972);Chern. Abstr., 78,96,287 (1973). (3) M. H. Yueh and E. D. Schilling, Gel. Pat. 2,104,743 (1971);Chem. Abstr., 76,35,4339 (1972). (4) P. Kovacs, Food Technol. (Chicago),27, 26-30 (1973). (5) H. R. Schuppner, Can. Pat. 824,635 (1969); Dairy Sci. Abstr., 32, 1488 (1970). (6) A. J. Leo and E. Bielskis, U.S. Pat. 3,396,039 (1968); Chem. Abstr., 69, 66,286 (1968). 34 1

342

PRAKASH M. DEY

cosmetics,20--21 paper p r o d ~ c t s , ~paints ~ - ~ ~and plasters,26-28welldrilling and mining,2e-33and explosives and fire-fighting.34-37 (7) B. Weinstein, U.S. Pat. 2,856,289 (1958);Chem. Abstr., 53, 1583e (1959). (8) L. L. Little, U.S. Pat. 3,370,955 (1968);Chem. Abstr., 68, 94,742 (1968). (9) H. Burton, H. R. Chapman, and D. J. Jayne-Williams, Proc. Int. Dairy Congr. 16th, Copenhagen, 3,82 (1962). (10) M. Glicksman, “Gum Technology in the Food Industry,” Academic Press, New York, 1969, p. 130. (11)W. A. Carlson, E. M. Ziegenfuss, and J. D. Overton, Food Technol. (Chicago), 16,5044 (1962). (12) E. Nuemberg and E. Rettig, Pharm. Ind., 36, 194-198 (1974). (13) E. Nuernberg and E. Rettig, Drugs Made Ger., 17,26-28,28-31 (1974). (14) E. Nuernberg, Ger. Pat. 1,290,661 (1969);Chem. Abstr., 70, 118,096 (1969). (15) Laboratories Dausse S. A,, Fr. Pharm. Pat. M. 7794 (1970);Chem Abstr., 76,131,509 (1972). (16) E. Nuernberg, E. Rettig, and H. Mueller, Ger. Pat. 2,130,545 (1972); Chem. Abstr., 78, 62,171~(1973). (17) E. Nuernberg, H. Mueller, H. Nowak, and P. Luecker, Ger. Pat. 2,017,495 (1971);Chem. Abstr., 76, 17,808 (1972). (18) Synthelabo S. A., Fr. Pat. 2,073,254 (1971);Chem. Abstr., 77,39,247 (1972). (19) E. Merck A. G., Neth. Pat. 6,504,974 (1965);Chem. Abstr., 64, 14,042g (1966). (20) R. J. Chudzikowski,J. Soc. Cosmet. Chem., 22,43-60 (1971). (21) J. I. Gonzales, Fr. Pat. 2,067,649 (1971);Chem. Abstr., 78,20,125 (1973). (22) R. Nordgren, U.S. Pat. 3,225,028 (1965);Chem. Abstr., 64, 6891c (1966). (23) J. W. Opie and J. L. Keen, U.S. Pat. 3,228,928 (1966); Chem. Abstr., 64, 11,430~ (1966). (24) D. J. Chrisp, U.S. Pat. 3,301,723 (1967);Chem. Abstr., 67, 92,485~(1967). (25) M. H. Yueh and E. D. Schilling, Fr. Pat. 2,080,462 (1971); Chem. Abstr., 77, 103,610 (1972). (26) J. Fath and M. Rosen, U.S. Pat. 3,700,612 (1972); Chem. Abstr., 78, 59,856 (1973). (27) D. J. Pettitt, U.S. Pat. 3,658,734 (1972); Chem. Abstr., 77, 35,846 (1972). (28) G. Benz, Ger. Pat. 1,206,777 (1965); Chem. Abstr., 64, 629Oc (1966). (29) R. E. Walker, U.S. Pat. 3,215,634 (1965);Chem. Abstr., 64, 382Oc (1966). (30) N. H. Black and L. L. Melton, U.S. Pat. 3,227,212 (1966); Chem. Abstr., 64, 7941b (1966). (31) V. V. Horner and R. E. Walker, U.S. Pat. 3,208,524 (1965); Chem. Abstr., 64, 503b (1966). (32) W. C. Browning, A. C. Perricone, and K. A. Eking, U.S. Pat. 3,677,961 (1972); Chem. Abstr., 77, 128,458 (1972). (33) F. B. b o p , S. Afr. Pat. 69,06946 (1970);Chem. Abstr., 73, 111,681 (1970). (34) J. J. Yancik, R. E. Schulze, and P. H. Rydlund, U.S. Pat. 3,640,784 (1972); Chem. Abstr., 76, 101,828 (1972). (35) P. R. Goffart, Fr. Pat. 1,533,471(1968); Chem. Abstr., 71, 23,404 (1969). (36) E. I. du Pont de Nemours and Co., Inc., Fr. Pat. 1,537,625(1968); Chem. Abstr., 72,45,652 (1970). (37) W. W. Morgenthaler, US. Pat. 3,634,234 (1972); Chem. Abstr., 76, 143,009 (1972).

BIOCHEMISTRY OF PLANT GALACTOMANNANS

343

Earlier reviews on g a l a c t o m a n n a n ~ ~mainly ~ - ~ ~ described their general chemistry. Dea and Morrison43discussed the latest investigations on plant galactomannans with respect to their intermolecular interactions and their interactions with other polysaccharides.

1. Occurrence The distribution of galactomannans in the plant kingdom is limited; the rich sources are the members of the family Leguminoseae. They have also been found in the species of Annonaceae (custard apple Ebenaceue family),44 Convolvulaceue (morning glory family),44*4s (ebony family),46Loganiaceae (Buddleia family),46and PaZmae (palm Amongst the leguminous plants, galactomannans are located in the endospemic part of the seeds; Dea and Morrison43 summarized the sources in which these polysaccharides have been detected. Baileys7 suggested that legume-seed galactomannans may (38) R. L. Whistler and C. L. Smart, “Polysaccharide Chemistry,” Academic Press, New York, 1953, p. 291. (39) B. N. Stepanenko, Bull. Soc. Chim. Biol., 42, 1519-1536 (1960). (40) F. Smith and R. Montgomery, “Chemistry of Plant Gums and Mucilages,” Reinhold, New York, 1959. (41) P. A. J. G r i n and J. F. T. Spencer, Adu. Carbohydr. Chem., 23,367-417 (1968). (42) See “Carbohydrate Chemistry,” R. D. Guthrie, ed., Specialist Report, The Chemical Society, London, 1970, Vol. 3, p. 236 (43) I. C. M. Dea and A. Morrison, Ado. Carbohydr. Chem. Biochem., 31, 241-312 (1975). (44) P. Kooiman, Carbohydr. Res., 20,329-337 (1971). (45) S. N. Khanna and P. C. Gupta, Phytochemistry, 6,605-609 (1967). (46) P. Kooiman and D. R. Kreger, K. Ned. Akad. Wet. Proc. Ser. C, 63, 634-645 (1960). (a7) G. 0. Aspinall, E. L. Hirst, E. G . V. Percival, and J. R. Williamson, J . Chem. SOC.,3184-3188 (1953). (48) F. Klages, Ann., 509, 159-181 (1934). (49) F. Klages, Ann., 512, 185-194 (1935). (50) A. K. Mukherjee, D. Choudhury, and P. Bagchi, Can. J . Chem., 39, 1408-1418 (1961). (51) V. Subrahamnyan, G. Bains, C. Natarajan, and D. Bhatia,Arch. Biochem. Biophys., 60,27-34 (1956). (52) C. V. N. Rao and A. K. Mukherjee,J. Indian Chem. Soc., 39,711-716 (1962). (53) C. V. N. Rao, D. Choudhury, and P. Bagchi, Can. J . Chem., 39,375-381 (1961). (54) H. S. El Khadem and M. A. E. Sallam, Carbohydr. Res., 4,387-391 (1967). (55) V. K. Jindal and S. Mukherjee, Cum. Sci., 38,459-460 (1969). (56) V. K. Jindal and S. Mukherjee, IndianJ. Chem., 8,417-419 (1970). (57) R. W. Bailey, in “Chemotaxonomy of the Leguminoseae,” J. B. Harborne, D. Boulter, and B. L. Turner, eds., Academic Press, London, 1971, p. 503.

344

PRAKASH M. DEY

have taxonomic importance, and put forward two main reasons for this: firstly, that different species have various levels of the polymer, and secondly, that the ratio of D-mannose to D-galaCtOSe (expressed as M/G in this text) also varies. However, the yield of galactomannans may vary with the method of extraction. It has been generally experienced that the solubility of the polymer in water increases as the ratio M/G decreases. The efficiency of aqueous extraction therefore depends upon the in vivo composition of the galactomannan. The total time involved in the extraction procedure may also alter the yield, because of enzymic degradation (for example, by a-D-galactosidases), which may alter the composition of the polysaccharide and hence affect its solubility. a-D-Galactosidases are generally present in leguminous seeds.s8 Other galactomannan-degrading enzymes may have similar effects on the solubility of the polysaccharide. Therefore, the taxonomic importance of these polysaccharides should be considered with caution. The levels of galactomannan are very high (15-38% of the dry weight of seed) in some members of the sub-families Caesalp i n i ~ i d e a e ~ ~and - ~ *Indiofereae.59,61m Some members of the subfamilies Astragaleae,6' C r ~ t a l a r i e a e , ~ ~M* i~r' n- ~o ~s e ~ e , ~ ~ * ~ ~ * ~ ~ * ~15-25% ~ of the polysacchaS o p h o r e ~ e ,and ~ ~ T r i f ~ l e a e , have Genisteae,72*73 G l y ~ i n e a e , ~ ~ ride, whereas those of De~rnoieae,~',~' and Loteae6'*7sa76 have only 1-15%. On the other hand, the seeds of several kinds of legumes do not contain any g a l a c t ~ m a n n a n . ~ ~ (58) D. Barham, P. M. Dey, D. Griffiths, and J. B. Pridham, Phytochemistry, 10, 1759-1763 (1971). (59) E. Anderson, Ind. Eng. Chem., 41,2887-2890 (1949). (60) J. Y. Morimoto, I. C. J. Unrau, and A. M. Unrau, J . Agric. Food Chem., 10, 134-137 (1962). (61) H. L. Tookey, R. L. Lohmar, I. A. Wolff, andQ. JonesJ. Agric. FoodChem., 10,131133 (1962). (62) H. L. Tookey, V. F. Pfeiffer, and C. R. Martin, J. Agric. Food Chem., 11, 317-321 (1963). (63) J. Y. Morimoto and A. M. Unrau, Hawaii F a r m Sci., 11, 6-8 (1962). (64) S. A. I. Rizvi, P. C. Gupta, and R. K. Kaul, Planta Me& 20,24-32 (1971). (65) A. M. Unrau and Y. M. Choy, Carbohydr. Res., 14, 151-158 (1970). (66) V. P. Kapoor, Phytochemistry, 11, 1129-1132 (1972). (67) A. S. Cerezo,J. Org. Chem., 30,924-927 (1965). (68) C. Leschziner and A. S. Cerezo, Carbohydr. Res., 15,291-299 (1970). (69) A. M. Unrau,J. Org. Chem., 26,3097-3101 (1961). (70) J. S. G. Reid and H. Meir, Z. Pfanaenphysiol,, 62,89-92 (1970). (71) M. P. Sinha and R. D. Tiwari, Phytochemistry, 9, 1881-1883 (1970). (72) J . E. Courtois and P. Le Dizet, Bull. Soc. Chim. Biol., 45, 731-741 (1963). (73) Z. F. Ahmed and A. M. Rizk,J. Chem. U.A.R., 6,217-226 (1963). (74) R. L. Whistler and J. Saamio,J. Am. Chem. Soc., 79, 6055-6057 (1957). (75) R. Somme, Acta Chem. Scand., 20, 589-590 (1966). (76) R. Somme, Acta Chem. Scand., 21,685-690 (1967).

BIOCHEMISTRY OF PLANT GALACTOMANNANS

345

In addition to the seed galactomannans, the leaf and stem tissues of red clover (Trifolium pratense) have been shown to contain a galactoglucomannan; this has a main chain of (1+4)-linked p-D-glucopyranosyl and P-D-mannopyranosyl residues, to which are attached single stubs of (1+6)-linked a-D-galactopyranosyl g r o ~ p s . ~ ~ , ’ ~ Such polysaccharides have also been isolated from the stem of the tropical legume Stylosanthes h ~ m i l i sand , ~ ~from some softwoods.80*81

2. Location in vivo In general, galactomannans do not exist together with the starch granules, but are present in seeds that are rich in oligosacfamily. In such seeds, they are usually charides of the &nose located in the endosperm tissues (which lie outside the embryo and are surrounded by the testa). They serve as reserve carbohyd r a t e ~ . By ~ ~light-microscopy, , ~ ~ ~ ~ ~ Reid and Meiers3 examined the location of galactomannan in fenugreek seeds (Trigonella foenum-graecum); Fig. 1 shows that the endosperm cells are completely filled with galactomannan. There are two known exceptions to the finding of a galactomannan in the endosperm: (a) Gymnocladus dioica,@ in which the polysaccharide lies in the inner side of the seed coat, and (b) Glycine max,8sin which it occurs in the hull.

3. Isolation Isolation43 involves pulverizing the seeds into a flour which is then extracted with cold or hot water,65s76*8688 alkali,89 or dilute The solubility of galactomannans has been discussed in detail by Dea and Morrison.43The galactomannan from the crude extract may be purified by various methods, including the use of (77) B. D. E. Gaillard and R. W. Bailey, Phytochemistry, 7,2037-2044 (1968). (78) A. J. Buchala and H. Meier, Carbohydr. Res., 31,87-92 (1973). (79) M. Alam and G. N. Richards, Aust. J . Chem., 24,2411-2416 (1971). (80) T. E. Timell, Ado. Carbohydr. Chem., 19,247-302 (1964);20,409-483 (1965). (81) J. E. Courtois, Bull. Soc. Bot. Fr., 115, 309-344 (1968). (82) A. Tschirch, “Angwandte Pflanzenanatomie,” Urgan and Schwarzenburg, Vienna, 1889. (83) J. S. G. Reid and H. Meier, Caryologin, 25,219-222 (1973). (84) E. B. Larson and F. Smith,J. Am. Chem. Soc., 77,429-432 (1955). (85) G. 0. Aspinall and J. N. C. Whyte, J. Chem. Soc., 5058-5063 (1964). (86) K. F. Horvei, and A. Wickstrflm, Acta Chem. Scand., 18,833-835 (1964). (87) N. R. Krishnaswami, T. R. Seshadri, and B. R. Sanna, Cum. Sci., 35, 11 (1966). (88) R. Somme, Acto Chern. Scand., 22,870-876 (1968). (89) P. V. Subbarao and M. V. L. Rao, IndianJ. Chern., 3,361-363 (1965).

346

PRAKASH M. DEY

FIG. 1.-A Cross-section of the Outer Part of a Seed of Trigonella foenum-graecum Before Mobilization of the Galactomannan, Showing the Three-layered Seed-coat (S) and a Small Part of the Cotyledon (C), with the Endosperm in Between. [The aleurone layer (A) is the outer cell-layer of the endosperm, and the rest of the endosperm is composed of large cells that have thin, primary walls and are completely filled with the dark-stained galactomannan (G). Stained with the periodic acid-Schiff reagent; x300 (reproduced, by permission, from Ref. 199).]

primary alcohols,so copper ~ o m p l e ~ barium , ~ hy~ ~ d r o ~ i d e , a~ c~e*t ~y ~l a t i ~ n , and ~ ~ *acetylpyridinium ~~~~ bromide comp l e ~ The . ~ ~ method of copper complexing, however, has been shown to cause chain cleavageag7 As with most polysaccharides, galactomannans isolated chemically may be heterogeneous and polydisperse with respect to both (90) C. M. Rafique and F. Smith,]. Am. Chem. SOC., 72,4634-4636 (1950). (91) A. M. Unrau and Y. M. Choy, Can.]. Chem., 48,1123-1128 (1970). (92) P. Andrews, L. Hough, and J. K. N. Jones,/. Am. Chem. Soc., 74, 4029-4032 (1952). (93) P. Andrews, L. Hough, and J. K. N. Jones,]. Chem. SOC., 2744-2750 (1952). (94) A. J. Erskine and J. K. N. Jones, Can.J . Chem., 34,821-826 (1956). (95) H. Meier, Methods Carbohydr. Chem., 5,45-46 (1965). (96) R. G. Morley, Ph.D. Thesis, University of Salford (1972). (97) N. Sugiyama, H. Shimahara, T. Andoh, M. Takemoto, and T. Kamata, Agric. B i d . Chem., 36, 1381-1387 (1972).

~

~

BIOCHEMISTRY OF PLANT GALACTOMANNANS

347

molecular weight and composition. In such cases, the yield and nature of the polysaccharide depend upon the method of extraction employed.8s 4.

Structure

The fundamental structure of the plant galactomannan is the following. [P-D- Manp-( 1+4)-In-P-D- M anp-( 1 4 ) -

6

t1

a-D-Galp This structure has been elucidated by extensive work over the past four decades. The main techniques used were r n e t h y l a t i ~ n , ~ ~par*~~-'~~ tial h y d r ~ l y s i s , ' ~ ~periodate -'~~ o x i d a t i ~ n ,and ~ ~ Jspecific, ~ enzymic hyd r ~ l y s i s . ' ~ ~A- "more ~ detailed account of the structure is given in a review43(see also, Refs. 46 and 113-115). The frequency ofsubstitution by D-gdactosyl groups along the main chain of the D-mannan varies according to the source of the p o l y ~ a c c h a r i d e ~ ~some * ' ~ ~examples ; are shown in Table I. It is evident that, in galactomannans having M/G ratios of 1.0, all of the D-mannosyl residues carry a D-galaCtOSyl group; this is shown by the inability of P-D-mannanases to hydrolyze the D-mannan backbone (98)V. P. Kapoor, Indian J . Chem., 11, 13-16 (1973). (99)F.Smith,J. Am. Chem. Soc., 70,3249-3253 (1948). (100)E.L. Hirst and J. K. N. Jones,]. Chem. Soc., 1278-1282 (1948). (101)D. S. Gupta and S. Mukherjee, Indian]. Chem., 11,505-506(1973). (102)Z.F.Ahmed and R. L. Whistler,J. Am. Chem. Soc., 72,2524-2525 (1950). (103)D. S. Gupta and S. Mukherjee, IndianJ. Chem., 13,1152-1154(1975). (104)R. L. Whistler and J. Z. Stein,]. Am. Chem. Soc., 73,4187-4188 (1951). (105)R. L. Whistler and D. F. Durso,]. Am. Chem. SOC.,73,4189-4190 (1951). (106)0. E. Moe, S. E. Miller, and M. H. Iwen,]. Am. Chem. SOC., 69, 2621-2625 (1947). (107)J. E.Courtois and P. Le Dizet, Carbohydr. Res., 3, 141-151 (1966). (108)J. E.Courtois and F. Petek, Methods Enzymol., 28,565 (1966). (109)P. Hui, Ph.D. Thesis, Juris, Zurich (1962). (110)J. E. Courtois and P. Le Dizet, Bull. SOC. Chim. B i d , 52, 15-22 (1970). (111) I. C. M. Dea, C. Hitchcock, S. Hall, and A. Morrison, unpublished results. (112)J. E. Courtois and P. Le Dizet, Bull. SOC. Chim. B i d . , 50, 1695-1710 (1968). (113)P. S. Kelkar and S. Mukherjee, Indian J . Chem., 9, 1085-1087 (1971). (114)D.S. Gupta and S. Mukherjee, Indian ]. Chem., 11, 1134-1137 (1973). (115)E. L.Richards, R. J. Beveridge, and M. R. Grimmett, Aust. J . Chem., 21, 21072113 (1968). (116)B. V. McCleary, N. K. Matheson, and D. M. Small, Phytochemistry, 15, 11111117 (1976).

PRAKASH M. DEY

348

TABLEI

M/G Ratios" of Some Plant Galactomannans Source

M/Ga

Medicago satiua L. (alfalfa, lucerne) Trifolium repens L. (white clover) Trigonella foenum-graecum (fenugreek) Ceratonia siliqua (carob, locust bean) Cyamopsis tetragonoloba bar) COCOS nucifera (coconut) Gleditsia amorphoides Cassia absus Cleditsia triacanthos (honey locust)

1.0- 1.25

"Ratio

References

70,92,117,118

1.0-1.3

72,86

1.1-1.2

70,93,119

1.2-5.25 1.3-7.0 2.0 2.5 3.0 3.2-3.8

59,99,100,107,120,121 59,61,62,120-122 53 67 98,123- 125 59,68,107

of D-mannopyran0se:D-galactopyranose.

(see Section 111,2,b).However, this does not hold if the galactomannan (M/G = 1.O) possesses 6-O-a-D-ga~actopyranosy~-a-D-ga~actopyranosy~ groups instead of single D-galactosyl groups. It has been suggested that galactomannans having an M/G ratio of >1.0 have a regular distribution of D-galaCtOSyl groups along the main chain of the D-mannan.126p127 On the other hand, Courtois and Le Dizet110,112 demonstrated the formation of D-manno-oligosaccharides, and a galactomannan having an M/G ratio of almost 1.0, following the hydrolysis of the polysaccharide (M/G = 4)from GZeditsia ferox and carob (M/G = 4) by a P-D-mannanase. This observation supports the view that, in these polymers, there is an alternation of zones in which (117) J. E. Courtois, C. Anagnostopoulos, and F. Petek, Bull. SOC. Chim. Biol., 40, 1277-1285 (1958). (118) R. J. McCredie, Diss. Abstr., 19, 432 (1958). (119) K. M. Daoud, Biochem. J . , 26,255-263 (1932). (120) P. A. Hui and H. Neukom, Tappi, 47, 39-42 (1964). (121) R. D. Jones and A. Morrison, unpublished results. (122) E. Heyne and R. L. Whistler,J. Am. Chem. SOC.,70,2249-2252 (1958). (123) V. P. Kapoor and S. Mukherjee, Curr. Sci., 38, 38 (1969). (124) V. P. Kapoor and S. Mukherjee, Phytochemistry, 10,655-659 (1971). (125) V. P. Kapoor and S. Mukherjee, IndianJ. Chem., 10,155-158 (1972). (126) H. Deuel, R. Leunberger, and G. Huber, Helw. Chlm. Acta, 33,942-946 (1950). (127) K. J. Palmer and M. J. Ballantyne, J . Am. Chem. SOC.,72, 736-741 (1950).

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349

each D-mannosyl residue carries a D-galaCtOSyl residue and zones devoid of these residues. Dea and Morrison43favored the block (or zone) structure, and discussed this aspect in relation to X-ray analysis and the interaction of galactomannans with other polysaccharides. To study the consecutive or the block structure of galactomannans, C. W. Baker and W h i ~ t l e r ' ~applied ~ , ' ~ ~ a technique whose principle was based on the fact that 6-deoxy-6-C-p-tolylsulfonylhexopyranosides are susceptible to alkali-catalyzed, glycosidic hydrolysis. 130-132 They followed the sequence of p - e l i m i n a t i ~ n ,m ' ~e~t h y l a t i ~ n ,acid ' ~ ~ hydroly s i ~ ,r'e~d~~ c t i o n , and ' ~ ~ acetylation. This resulted in the formation of 1,5-di-0-acetyl-2,3,4,6-tetra-O-methyl-~-mannitol when the Dmannosyl residues were substituted at 0-6 with a-D-galactosyl groups and at 0 - 4 by a D-mannosyl group which was itself not substituted at 0-6.But, when the latter D-mannosyl group was substituted at 0 - 6 with an a-D-galactosyl group, 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl-~mannitol was formed. By comparing the ratios of the two products, the author^'^^^^^^ suggested that guar galactomannan (M/G = 2) has a regular structure, with alternate D-mannosyl residues substituted with D-galaCtOSyl groups, whereas carob galactomannan (M/G = 4) has blocks of 25 D-mannosyl residues which are substituted with D-galaca molecular tosyl groups. They further s ~ g g e s t e d ' ~that, ~ * 'assuming ~~ weight of 210,000 for this galactomannan, the average length of each unsubstituted block is 85 linearly ( 1+4)-linked, p-D-mannopyranosyl residues. This unsubstituted chain-length is in agreement with the requirement observed for the association of the polysaccharide with carrageenan. 136*137 Lindberg and performed methylation analyses on the products resulting when guar and carob galactomannans were oxidized with periodate and then reduced with borohydride. The results indicated that a simple, alternating structure for guaran, and a simple, block structure for carob gum, are not possible. The authors suggested an (128) C. W. Baker and R. L. Whistler, Carbohydr. Res., 45,237-243 (1975). (129) C. W. Baker and R. L. Whistler, Methods Carbohydr. Chem., 7 , 152-156 (1976). (130) B. Lindberg and H. Lundstrem, Actu Chem. Scand., 20,2423-2426 (1966). (131) H. Bjorndal and B. W&ngstr@m,ActaChem. Scand., 23,3313-3320 (1969). (132) 0. Lann, B. Lindberg, and S. Svensson, Carbohydr. Res., 20, 39-48 (1971). (133) H. E. Conrad, Methods Carbohydr. Chem., 6,361-364 (1972). (134) G. A. Adams, Methods Carbohydr. Chem., 5, 269-276 (1965). (135) P. D. Bragg and L. Hough,J. Chem. SOC.,4347-4352 (1957). (136) I . C. M. Dea, A. A. Mekinnon, and D. A. Rees,J. Mol. Biol., 68, 153-172 (1972). (137) T. F. Child and N. G. Pryce, Biopolymers, 11,409-429 (1972). (138) J. Hoffman, B. Lindberg, and T. Painter, Actu Chem. Scand., 30, 365-366 (1976).

350

PRAKASH M. DEY

almost random distribution of the D-galaCtOSyl groups. A closer examination of Baker and Whistler's results'28 reveals that they do not, in fact, exclude the possibility of an almost random structure for both polysaccharides. It has been generally presumed that the results of structural studies on polysaccharides may be interpreted as indicating the presence of regular, repeating units. This view should, however, be accepted with c a u t i ~ n . ~Galactomannans '*~~ having similar M/G ratios may have markedly different patterns of distribution of D-galactosyl groups along the D-mannan main-chain. This is often reflected in the ''anomalous'' pattern of the action of a-D-galactosidase on the polymers'07~'09~110 (see also, Section 111,2,a). Interpretation of the results of structural investigations is difficult, because of variable extractability of the polymer from the same source, and because each extract may have a different M/G r a t i ~ , leading ~ ~ ~ to ~ a~ batch-to-batch ~ . ~ ~ ~ variation in the fine structure.43 The products formed by the action of the endo-acting, white-clover p-D-mannana~e'~'on the galactomannans of guar (M/G = 2), carob (M/G = 4),and Gleditsia ferox (M/G = 4) have been examined'39 by t.1.c. (see Fig. 2). For guar gum, none of the products of the enzymic hydrolysis migrated from the base line (see Fig. 2C), despite a linear increase, during the first 50 min, in the reducing power. This could mean that the polysaccharide had been cleaved, presumably at the points where a sufficient number of D-mannosyl residues were unsubstituted, to produce a mixture of fairly large, oligomeric fragments. Thus, the distribution of D-galactosyl side-chain groups is unlikely to follow a regular, alternating pattern in guaran. The Gleditsia digest contained four D-manno-oligosaccharides of the same homologous series (see Fig. 2B,c,d,e,f) and three that contained both D-mannose and D-galactose (indicated as * in Fig. 2,B). On the other hand, the carob digest had five members of the D-manno-oligosaccharide series (Fig. 2A,c,d,e,f,g) and only two sugars consisting of both monosaccharides (indicated as * in Fig. 2A). These results tended to suggest that neither of the two polysaccharides possesses uniform, block structures, and that the D-galactosyl groups are distributed more randomly in Gleditsia gum than in carob gum. The action of a-Dgalactosidase on these polysa~charides~~ also shows that fewer, isolated a-D-galactosyl groups are interspersed in the unsubstituted regions of carob gum than in Gleditsia galactomannan; this is in agreement with the general structural scheme proposed by Courtois and Le Dizet."O (139) P. M.Dey, unpublished results. (140) J. Williams, H.Villarroya, P. M. Dey, and F. Petek, Int. Congr. Biochern., IOth, Hamburg, 623 (1976).

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FIG. e.-Thin-layer Chromatographic Separation of the Products of the Action of White-clover P-D-Mannanase on Various substrate^.'^^ [A, Carob galactomannan; B, Gleditsia ferox galactomannan; C, guar galactomannan; D, ivory-nut mannan; and E, salep glucomannan. a, DMannose; b, ~ g l u c o s e ;c, mannobiose; d, mannotriose; e, mannotetraose; f, mannopentaose; and g, mannohexaose. The homologous series of Dmanno-oligosaccharides consist of (1+4)-linked P-D-mannopyranosyl residues. In A and B, the symbol * represents the unidentified, D-galactose-containing oligosaccharides, and, in E, the symbol t represents the unidentified, D-glucose-containing oligosac charides.]

Deviations from the main structure with respect to “anomalous” glycosidic linkages have also been reported in some galactomannans. These are P-D-(1+3)- and P-D-( 1+2)- linkages in the D-mannan backbone,91.100*141 with substitution by a-D-galactosyl groups at 0 - 2 of some of the D-mannosyl residues.lz4The presence O f D-galaCtOSe stubs made -( up of more than one D-galactose unit, linked together by a - ~ 1+6)linkages, has also been ~ h o ~ n . ~ ~ , ~ ~ * ~ ~ ~ (141) V. P. Kapoor and S. Mukherjee, Can.J . Chem., 47,2883-2887 (1969).

PRAKASH M. DEY

352

11. BIOSYNTHESIS The biosynthesis of plant galactomannans has attracted relatively little attention and, as yet, no general pathway has been defined. It has been generally concluded that D-galactose is stored in the seeds in the form of galactomannan, by random attachment of D-galaCtOSyl groups to a main-chain of D-mannan.92*'00 Hough and coworkers'42 pointed out that a plausible way whereby a galactomannan may be synthesized is by trans-D-galactosylation as shown. G

Raffinose

+ .-..M-M-M.-.-.

D-Mannan polymer

I + sucrose * .....M-M-M.... galactomannan

where M = D-mannosyl residue and G = D-galactosyl group. In 1970, Reid and Meier'43 studied the biosynthesis of galactomannan in fenugreek seeds at different stages of maturation. The polysaccharide is present at all stages, except in the very young seeds; the maximum yield was obtained from 9-10-week-old seeds (at almost the fully mature stage). The most interesting feature of the polysaccharide is that the M/G ratio (1.2) remains the same during various stages of development until maturation. In addition, both free Dmannose and D-manno-oligosaccharides, which might act as primers for the synthesis, are absent. It therefore seems unlikely that the D-galactosyl groups are attached randomly to a D-mannan polymer. Instead, the results suggest that D-galactose and D-mannose units are deposited simultaneously, to form the galactomannan. Courtois and Le D i ~ e also t ~ ~found unchanged M/G ratios for galactomannans in maturing seeds of Gleditsia ferox and white clover. The immature seeds of fenugreek also contain sucrose, myo-inositol, and D-glucose, galactinol (1L-~-O-a-D-galactopyranosy~myo-inositol), stachyose, in addition to ga1a~tornannan.I~~ In the mature seeds, when the deposition of the polysaccharide is complete, stachyose is still found in abundance. This oligosaccharide is known to be formed through the transfer of a-D-galaCtOSyl groups from galactinol to raff i n 0 ~ e . Courtois l~~ and proposed the following pathway for formation of stachyose.

(142) M. E. Henderson, L. Hough, and T. Painter,J. Chem. Soc., 3519-3522 (1958). (143) J. S. G . Reid and H. Meier, Phytochemistry, 9, 513-520 (1970). (144) W. Tanner and 0. Kandler, Plant Physiol., 41, 1540-1542 (1966). (145) A. Sioufi, F. Percheron, and J. E. Courtois, Phytochemistry, 9, 991-999 (1970).

BIOCHEMISTRY OF PLANT GALACTOMANNANS

353

+

UDP-Dgalactose + myo-inositol --* galactinol UDP UDP-D-galactose sucrose + raffinose UDP Galactinol raffinose + stachyose myo-inositol

+

+

+

+

It has been suggested that a similar mechanism of D-galactosyl transfer may be involved in the biosynthesis of galactomannan, with the participation of G D P - ~ - m a n n o s e . ' ~ ~ Courtois *'~~ and coworkers 14s have, in fact, detected UDP-D-galactose and GDP-D-mannose in fenugreek seeds. In the absence of a well-worked-out, biosynthetic pathway for plant galactomannans, a useful comparison may be made with that of a related polysaccharide, namely, glucomannan. This plant polysaccharide mostly consists of D-mannosyl and D-glucosyl residues joined, ~ ~ - ' ~ ~and in the form of a linear chain, by P - D - ( 1 ~ 4 ) - l i n k a g e ~ . 'Hassid coworkers153isolated from mung-bean (Phaseolus aureus) seedlings a particulate-enzyme fraction that catalyzes the transfer of D-['4c]glucose from UDP-D-['~]glucose into cellulose. This incorporation was stimulated in the presence of GDP-D-mannose, but the product formed under these conditions differed from cellulose.'s4 It was further found that the D-mannosyl group of GDP-~-['~C]mannoseis, in fact, incorporated into a polymer characterized as g l u ~ o m a n n a n . ' ~ ~ E l b e i ~ ~ ' studied ~ ~ , ' ~ the ~ properties of the enzyme system responsible for the biosynthesis of glucomannan, and showed that GDP-D['Cjglucose is utilized only in the presence of GDP-Dmannose. However, incorporation of radioactivity from GDP-~-['~C]mannose was inhibited by GDP-D-glucose. Structural analysis showed that the polymer contains 3 to 4 D-mannose units per D-glucose molecule. This is evidence for the existence of a biosynthetic pathway dependent on a nucleoside 5'-(glycosyl diphosphate). (146)J. E. Courtois, in "Plant Carbohydrate Biochemistry," J. B. Pridham, ed., Academic Press, London, 1974,p. 1. (147)C. T. Bishop and F. P. Cooper, Can. J. Chem., 38,793-804 (1960). (148)A. Tyminski and T. E. Timel1,J. Am. Chem. Soc., 82,2823-2827 (1960). (149)M. 0. Gyaw and T. E. Timell, Can. J. Chem., 38,1957-1966 (1960). (150)T. E. Timell, Methods Carbohydr. Chem., 5, 137-138 (1965). (151)H.J. Rogers and H. R. Perkins, "Cell Walls and Membranes," Spon, London, 1968. (152)0.Perila and C. T. Bishop, Can. J. Chem., 39, 815-826 (1961). (153)A. D.Elbein, G . A. Barber, and W. Z . Hassid,]. Am. Chem. Soc., 86, 309-310 (1964). (154) G.A. Barber, A. D. Elbein, and W. Z . Hassid,J. Biol. Chem., 239, 4056-4061 (1964). (155)A. D. Elbein and W. Z. Hassid, Biochem. Biophys. Res. Commun., 23, 311318 (1966). (156)A. D.Elbein,]. Biol. Chem., 244, 1608-1616 (1969). (157)A. D.Elbein, Methods Enzymol., 28,560 (1972).

354

PRAKASH M. DEY

Some micro-organisms also contain galactomannans, O-phosphonogalactomannans, O-phosphonomannans, and peptidoOphosphonogalactomannans .158-162 The galactomannans from Trichophyton and Microsporum species have been ~ h a r a c t e r i z e d .Gander ~~ and coworkers183-166 characterized the peptido-O-phosphonogalactomannan from Penicillium charlesii, and studied its biosynthesis (see also, Ref. 167).The polysaccharide molecule contained 90 Dmannopyranosyl residues linked through a - ~1+-2)( and a - ~1+6)-linkages, ( and to 0-3 of D-mannose units of this backbone were attached 8 to 10 poly(Dgalactofuranosyl) chains of variable length. The D-galaCtOfUranOSyl residues in these chains were linked together through @D-( b 5 ) linkages.ls3It was shown that P . charlesii produced -20% of the total peptido-O-phosphonogalactomannan before the depletion of NH4+ from the growth medium, and the rest was formed during NH4+ starvation. Incorporation studies with exogenous precursors, namely, D-['~]ghCOSe,~-['~C]threonine, and NaH32P04,showed that the product was the result of a biosynthetic process, and was not derived by the autolysis of cell wa11s.164*166 The presence of UDP-D-galactofuranose has also been demonstrated in P . charlesii168;Fobes and G a n d e F detected an enzyme [uridine 5'-(a-~-galactopyranosyl diphosphate): NAD 2-~-hexosyloxidoreductase] that oxidized UDP-Dgalactopyranose to a D-lyxo-hexos-2-dose derivative. The authors suggested that a possible role of this enzyme might be the conversion of the D-hexosyl group of UDP-D-galactose from the pyranose into the furanose form. The complete, biosynthetic situation is not yet clear. In addition to GDP-D-mannose as the D-mannosyl d o n ~ r , * ~ ~ * ' ~ ' D -

-

(158) S. A. Barker, 0. Basarab, and C. N. D. Cruickshank, Carbohydr. Res., 3, 325332 (1967). (159) A. Jeanes and P. R. Watson, Can.]. Chem., 40, 1318-1325 (1962). (160) M. E. Slodki, Biochirn. Biophys. Acta, 57, 525-533 (1962). (161) K. 0. Lloyd, Biochemistw, 9,3446-3453 (1970). (162) T. R. Thieme and C. E. Ballou, Biochemistry, 10,4131-4129 (1971). (163) J. E. Gander, N. H. Jentoft, L. R. Drewes, and P. D. Rick, J . Biol. Chem., 249, 2063-2072 (1974). (164) J. E. Gander, Annu. Reu. Microbiol., 28, 103-119 (1974). (165) L. R. Drewes and J. E. Gander,]. Bacteriol., 121,675-681 (1975). (166) L. R. Drewes, P. D. Rick, and J. E. Gander, Arch. Microbiol., 104, 101-104 (1975). (167) F. A. Troy, F. E. Frerman, and E. C. Heath, Methods Enzymol., 28,602 (1972). (168) G. Trejo, J. W. Haddock, G. J. F. Chittenden, and J. Baddiley, Biochem. ]., 122, 49-57 (1971). (169) W. S. Fobes and J. E. Gander, Biochem. Biophys. Res. Commun., 49, 76-83 (1972). (170) N. H. Behrens and E. Cabib,]. Biol. Chem., 243,502-509 (1968). (171) L. P. Kozak and R. K. Bretthauer, Biochemistry, 9, 1115-1122 (1970).

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355

mannosyl-lipid intermediate^'^^-^^^ have also been shown to participate in the biosynthesis of D-mannose-containing oligo- and polysaccharides in plants, animals, and microbial systems. However, further research is needed in order to establish the biosynthetic pathway of plant galactomannans (see also, Section 111,2,d). Reid and Meier- performed a histological examination of the process of deposition of galactomannan in the endosperms of maturing fenugreek-seeds. There was no galactomannan in the very young seeds (2 weeks after anthesis), and the endosperm was not (172) I. M. Weiner, T. Higuchi, L. Rothfield, M. Saltmarsh-Andrew, M. J. Osborn, and B. L. Horecker, Proc. Natl. Acad. Sci. U.S.A., 54,228-235 (1965). (173) A. Wright, M. Dankert, and P. Robbins, Proc. Natl. Acad. Sci. U.S.A., 54, 235241 (1965). A. Wright, M. Dankert, P. Fennessey, and P. Robbins, Proc. Natl. Acad. Sci. U.S.A., 57, 1798-1803 (1967). M. Scher, W. J. Lennarz, and C. C. Sweeley, Proc. Natl. Acad. Sci. U.S.A.,59, 1313-1320 (1968). M. Scher and W. J. Lennarz,]. Biol. Chem., 244,2777-2789 (1969). M. Lehar, T. H. Chiu, and W. J. Lennarz, J . Biol. Chem., 244, 5890-5898 (1969). A. Wright,J. Bacteriol., 105,927-936 (1971). K. Nikaido and H. Nikaido,]. Biol. Chem., 246, 3912-3919 (1971). F. A. Troy, F. E. Frerman, and E. C. Heath, J . Biol. Chem., 246, 118-133 (1971). K. Takayama and D. S. Goldman,J. Biol. Chem., 245,6251-6257 (1970). P. Babczinski and W. Tanner, Biochem. Biophys. Res. Commun., 54, 11191124 (1973). R. M. Barr and F. W. Hemming, Biochem. J., 126, 1193-1202, 1203-1208 ( 1972). J. F. Caccam, J. J. Jackson, and E. H. Eylar, Biochem. Biophys. Res. Commun., 35,505-511 (1969). W. Tanner, P. Jung, and N. H. Behrens, FEBS Lett., 16,245-248 (1971). C . J. Waechter, J. J. Lucas, and W. J. Lennarz, J . Biol. Chem., 248, 75707579 (1973). J. L. Strominger, Y. Higashi, H. Sandermann, K. J. Stone, and E. Willoughby, in “Biochemistry of the Glycosidic Linkage,” R. Piras and H. G. Pontis, eds., Academic Press, London, 1972, p. 135. J. B. Richards, P. J. Evans, and F. W. Hemming, In “Biochemistry of the Glycosidic Linkage,” R. Piras and H. G. Pontis, eds., Academic Press, London, 1972, p. 207. H. Kauss, in “Biochemistry of the Glycosidic Linkage,” R. Piras and H. G. Pontis, eds., Academic Press, London, 1972, p. 221. W. Tanner, P. Jung, and J. C. Linden, in “Biochemistry of the Glycosidic Linkage,” R. Piras and H. G. Pontis, eds., Academic Press, London, 1972, p. 227. W. Z. Hassid, in “Biochemistry of the Glycosidic Linkage,” R. Piras and H. G. Pontis, eds., Academic Press, London, 1972, p. 315. M. Scher and W. J. Lennarz, Methods Enzymol., 28,563 (1972).

356

PFtAKASH M . DEY

cellular at this stage. In the fifth week after anthesis, while the seeds were green and immature, deposition of galactomannan started in the cells next to the embryo. The polysaccharide was seen at the periphery of the cells and encroaching inwards into the cytoplasm. In the mature, green seeds (8 weeks after anthesis), endosperm cells were completely filled with galactomannan that had spread up to the border of the aleurone layer. Meier and Reidlg3conducted a detailed, electron-microscope study of the deposition of galactomannan in the endospermic cells of fenugreek. They observed that the cells had stacked rough, endoplasmic reticulum (ER) at the initial stage of deposition of galactomannan; this was followed by the appearance of vacuoles in the intracisternal space of the ER, and their membranes entrapped cytoplasmic pockets. It was suggested that the rough E R makes contact with the plasmalemma, and discharges the galactomannan-containing enchylema, outside the cell, with local disruption of the cell membrane. At the advanced stage of development, a larger deposition of galactomannan was seen within the protoplast, inside the ER vacuoles. This mode of secretion, although not very common, has been shown to take place in a few instance^.^^^^^^^ The general mode of secretion is, however, through the Golgi v e s i ~ l e s . ' ~ ~ ~ ' ~ ~

111. BIOCHEMICALDEGRADATION

1. General Considerations It has been known for some time that the albuminous seeds of the family Leguminosae contain, in the endosperm, galactomannan as the reserve polysaccharide. The exalbuminous seeds, on the other hand, have the main reserve in the cotyledon in the form of starch, oil, and p r ~ t e i n s . ' The ~ ~ ~seed ' ~ ~ galactomannan-reserves are generally mobilized during the process of germination.43~57~8'*82*1sg~zos Detailed study (193) H. Meier and J. S. G . Reid, Planta, 133,243-248 (1977). (194) T. Rochmilevitz and A. Fahn,Ann. Bot. (London),37, 1-9 (1973). (195) J. M. Unzelman and P. L. Healey, Protoplasma, 80, 285-303 (1974). (196) J.-C. Roland and D. Sandoz,]. Microsc. (Paris), 8,263-268 (1969). (197) M. Rougier,]. Microsc. (Paris), 10, 67-82 (1971). (198) F. N. Howes, "The Nature and Uses of Gums," Chronica Botanica, Waltham, Massachusetts, 1949. (199) J. S. G . Reid, Planta, 100, 131-142 (1971). (200) K. Sehgal, H. S. Nainawatee, and B. M. Lal, Biochenz. Physiol. Pflanz., 164, 423-428 (1973). (201) J. S. G . Reid and H. Meier, Planta, 106,44-60 (1972). (202) J. S. G . Reid and H. Meier, Planta, 112,301-308 (1973).

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of the metabolism of galactomannan during seed germination has been limited to only a few species. Working with fenugreek seeds soaked in water for 24 h at 2426", Reidlg9 defined the start of germination as the penetration of the seed coat by the radicle; this required a further 6 h. A histological study revealed that there was no morphological change in the endosperm or the cotyledon during the first phase of germination (18 h after the appearance of the radicle; compare Fig. l).The level of galactomannan remained constant in the endosperm, and that of stachyose and verbascose decreased, but that of sucrose increased; the levels of sucrose and free D-galactose were approximately equal. On the other hand, cotyledons were free from starch and D-galactose, and there was a decrease in the total carbohydrates of low molecular weight, except sucrose, which increased. The dissolution of galactomannan occurred (see Fig. 3) in the second phase (lasting for 24 h,

FIG.3.-A Cross-section Similar to Fig. 1, Showing the Seed During Mobilization of Galactoinannan (S, Three-layered Seed-coat). [The galactomannan (G) in the endosperm is in the process of being dissolved, and the dissolution zone (D) begins at the aleurone layer (A) and spreads inwards towards the cotyledons (C). Stained with the periodic 0 by permission, from Ref. 199).] acid-Schiff reagent; ~ 4 0 (reproduced, (203) A. Seiler, Planta, 134,209-221 (1977). (204) J. S. G. Reid, C. Davies, and H. Meier, Planta, 133,219-222 (1977). (205) B. V. McCleary and N. K. Matheson, Phytochemistry, 13,1747-1757 (1974).

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following the first phase). The decrease in the galactomannan level (there was no change in the M/G ratio) was due to secretion of hydrolytic enzymes from the active, aleurone layer into the endosperm. This is comparable to starch hydrolysis in cereal grains during germination.20sThe galactomannan reserve and the raffinose family of sugars completely disappeared from the endosperm at the end of this phase. The hydrolysis products, such as sucrose, D-galaCtOSe, D-mannose, and P-D-(1+4)-linked mannobiose, reached their highest levels during 6-12 h of the second phase, and were lowered to traces by the end of this phase. These sugars appear to be translocated to the cotyledons, as evidenced by the concomitant synthesis of starch (see Fig. 4)and the presence of a high level of sugars of low molecular weight. A transitory synthesis of starch in the cotyledons of fenugreek as the endosperm dissolved had been indicated by NadelmannZo7in 1889. The sugars taken up by the cotyledons are likely to have been converted Interestingly, ~~,~~~ into starch through the established p a t h ~ a y s . ~ although the cotyledons are capable of starch synthesis, such synthesis

0

--phase 1

phase 2

phase 3

Germinotion time ( h )

FIG. 4.-Disappearance of Galactomannan from the Endosperm, and Appearance of Starch in the Cotyledon, During Germination of Seeds of Trigonella foenum-gruecum that had Already Been Soaked for 24 Hours at 24-26”. (Adapted from Figs. 5 and 6 of Ref. 199.) (206) T. A. Villiers, “Dormancy and Survival of Plants,” Arnold, London, 1975. (207) H. Nadelmann, Ber. Dtsch. Bot. Ges., 7,248-255 (1889). (208) D. R. Davies, in “Plant Carbohydrate Biochemistry,” J. B. Pridham, ed., Academic Press, London, 1974, p. 61. (209) M. A. R. De Fekete and H. G. Vieweg, in “Plant Carbohydrate Biochemistry,” J. B. Pridham, ed., Academic Press, London, 1974, p, 127.

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FIG.5.-A Cross-section Comparable to Figs. 1 and 3. [Here, no galactomannan is left in the endosperm (E), only a remnant of which remains between the seed coat (S) and the cotyledon (C). PAS stained; ~ 4 1 (reproduced, 5 by permission, from Ref. 199).]

does not occur until the seeds germinate. On the other hand, in exalbuminous legume seeds in which the endosperm disappears during maturation, the reserve starch is stored in the cotyledons; this may be due to environmental adaptation undergone by the galactomannancontaining seeds which enables them to absorb and retain water. During the third, and final, phase (24 h following the second phase), galactomannan is absent (see Fig. 5 ) , and the endospermic carbohydrates of low molecular weight also disappear completely and rapidly. Cotyledons, however, have a high level of starch (see Fig. 4). The pattern of disappearance of galactomannan was also studied in dry-isolated, fenugreek endosperms, which were aseptically incubated under “germination” conditions. The process, and the results, were identical to those found in whole-seed experiments, except that the levels of free D-galactose and D-mannose were higher, probably because these sugars could not be translocated and metabolized in the absence of the cotyledons. In addition, the decomposition of galactomannan began slightly earlier in the isolated endosperms; this may be due to (a) the ease of diffusion of oxygen into the endosperm, causing aerobic respiration to start, and, possibly, (b) the ease of removal of

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any metabolic inhibitor from the tissues. ShiroyaZ1Ohas also shown that oxygen is required for the disappearance of the raffinose family of sugars in germinating cotton-seeds. As the absence of embryo in the isolated-endosperm experiment had no effect on the mobilization of galactomannan, this would indicate that, unlike the process in cereals,211-214 the embryo does not participate in the catabolic process. It was also confirmedm1that the composition of the galactomannan of the isolated endosperms of fenugreek, crimson clover (Trifolium incarnatum), and lucerne were unchanged (the M/G ratios were -1) throughout the “germination” period. On the other hand, Courtois and coworkers145showed a change of M/G ratio from 1.05 to 7.45 in the galactomannan of 24hour-germinated7 fenugreek seeds. However, the latter experiment was conducted with whole seeds (see also Section 111,2,b). It was also suggested that, during germination of the seeds of Gleditsia ferox and G . triacanthos, there was an initial removal of Dgalactosyl groups from the polysaccharides, followed by the cleavage of the D-mannan backbones, giving rise to D-mannooligosaccharides (see also, Section 111,2,a). Reid and MeierZo1 suggested that the presence of free D-galactose in the endosperm during the early stages of germination might be due to the non-living nature of the storage tissues. Cotyledons, on the other hand, consist of living cells where Dgalactose is rapidly metabol i ~ e dPridham . ~ ~ ~ and coworkers216have actually detected a D-gdaCt0kinase in Viciafuba seeds, and related this enzyme to the metabolism Of D-galaCtOSe through phosphorylation (see also, Refs. 145, 217, and 218). A similar observation was made by Shiroya,210who examined the breakdown of raffinose and stachyose in germinating cotton-seeds; by infiltration of D-galactose into the seeds, he showed that an effective mechanism for the utilization of D-galactose exists. Reid and MeierZo1 found that the breakdown of galactomannan in isolated, fenugreek endosperms is strongly inhibited by cycloheximide (210)T. Shiroya, Phytochemistry, 2, 33-46 (1963). (211)M. Black, “Control Processes in Germination and Dormancy,” Clarendon Press, Oxford, 1972. (212)H. Yomo, Hukko Kyokaishi, 16,444-448 (1958). (213)D.Cohen and L. G . Paleg, Plant Physiol., 42, 1288-1296 (1967). (214)J. E. Varner and G . R. Chandra, Proc. Nutl. Acad. Sci. U.S.A., 52, 100-106 (1964). (215)H. Coring, E.Reckin, and R. Kaiser, Flora (Jena),Abt. A, 159,82-103 (1968). (216)J. B. Pridham, M. W. Walter, and H. G. J. Worth,J. E r p . Bot., 20, 317-324 (1969). (217)S. Clermont, M. J. Foglietti, and F. Percheron, Compt. Rend., 276, 843-847 (1973). (218) M.J. Foglietti and F. Percheron, Biochimie, 56,473-475 (1974).

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{3-[2-(3,5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyllglutarimide} and by 2,4-dinitrophenol. They related this to inhibition of the synthesis of protein, and oxidative phosphorylation, respectively, in the aleurone layer. An electron-microscope examinationzo1showed the presence of a large number of ribosomes in the aleurone cells, even at the beginning of germination. There were large numbers of polyribosomes in the first phase of germination, and these were membranebound to small vesicles and to flat cisternae which were probably newly formed and derived from ER. As the germination progressed, the content of aleurone cells vanished, and vacuoles appeared. It was, therefore, concluded that the galactomannan-mobilizing enzymes are synthesized in the aleurone layer, and that the dissolution of the polysaccharide is not controlled by the embryo. 2. Enzymes Involved Hylin and Sawai2I9 isolated, and crystallized, an enzyme from Leucaena leucocephala seeds which they termed “galactomannan depolymerase.” This enzyme catalyzed a rapid fall in the viscosity of solutions of galactomannan (obtained from the same source), at pH 5.3, to a low value, after which, no further change in the viscosity or increase in the reducing power occurred. The maximum depolymerizing activity was isolated from the 7-day-germinated seeds. It was suggested that, in the initial stages of germination, the polysaccharide is depolymerized into fragments of considerable size, which are then further degraded into D-galactose and D-mannose units. The galactomannan of L. leucocephala seeds consists of 57% of D-mannose and 43% of D-galactose, and the degree of polymerization (d,p.) is 150. The main backbone of the D-mannan has some @-D-(1+3)-linkages, in addition to the usual @-D-(1+4)-linkages, and some D-galaCtOSyl residues occupy intra-chain positions.69If an almost random, general structure of the polysaccharide is considered (see also, Ref. 112), a few unsubstituted areas along the main chain would be expected. These areas would, undoubtedly, be the points of attack by an endo-@-D-mannanase; this enzyme would, however, be unable to by-pass the points where the intra-chain D-galactose units occur. It would be of interest to examine the substrate specificity of the depolymerase, in order to establish its identity. A similar galactomannanase has also been isolated from germinated guar seeds.z00

(219) J. W. Hylin and K. Sawai,J. Biol. Chem., 239, 990-992 (1964).

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Whistler and coworkers220showed that, on incubation with guaran solution, enzyme preparations from guar seeds increase the reducing power and decrease the viscosity, but the ratio of these two effects differed for different enzyme preparations. Thus, they predicted that two or more guaran-hydrolyzing enzymes must be present in guar seeds. These may be (a) an enzyme that would hydrolyze the /3-Dmannosidic bonds in the main chain (for example, an endo-P-Dmannanase), and (b)an enzyme that would hydrolyze a-D-galactosidic bonds (for example, a-D-galactosidase). According to Reese and Shibata,22‘the biodegradative hydrolysis of galactomannan requires at least three different enzymes, namely, a-D-galactosidase, endo-p-D-mannanase, and p-D-mannosidase, In addition to these enzymes, there is also a report222of a phosphorylase, identified as oligo-/3-~-mannosyl-(1+4)-phosphorylase, which may take part in the degradation. These enzymes will now be discussed in detail. a. a-D-Ga1actosidase.-This enzyme (melibiase, a-D-galactoside galactohydrolase, EC 3.2.1.22)catalyzes the following reaction.

“0 CH,OH

OR

+ q0

CH20H

OH An ff-D-galactoside

+

“>..H

ROH

OH D

- galactose

where R is an alkyl or aryl group, or a glycosyl (mono or oligo) residue or group. Under suitable conditions, the enzyme can also catalyze de nouo syntheses of oligosaccharides and transfer reaction^.^^^,^^^ a-D-Galactosidases have been isolated from animal, plant, and microbial sources; a review223includes details of the isolation, properties, and characterization of these enzymes. Amongst plants, they have (220) R. L. Whistler, W. E. Eoff, and D. M. Doty, J . Am. Chem. SOC.,72, 4938-4949 (1950). (221) E. T. Reese and Y. Shibata, Can. J . Microbial., 11, 167-183 (1965). (222) M. J. Foglietti and F. Percheron, Compt. Rend., 274, 130-132 (1972). (223) P. M. Dey and J. B. Pridham,Ado. Enzymol., 36,91-130 (1972). (224) J. B. Pridham and P. M. Dey, in “Plant Carbohydrate Biochemistry,” J. B. Pridham, ed., Academic Press, London, 1974, p. 83.

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been detected in leguminous and other species, especially those which contain or utilize a-D-galactosidic substrates.43~s7~81~z0s~,223~zzs~ It is well established that, in plants, one of the functions of WDgalactosidases is to cleave a-D-galactosyl groups from a-D-galactose-containing oligo- and poly-saccharides. The degradation products thus formed serve as a ready source of energy and of cell metabolites. These enzymes have also been associated with the metabolism of galactolipids in plantszz8andwith chloroplast-membrane f ~ n c t i o n . ~ ~ ~ ~ The specificities of a-D-galactosidases vary widelyzz3;the enzymes a~~ Mortierella ~ v i n ~ c e ahydrolyze ~~~ substrates from Vicia s a t i ~ and of small molecular weight, such as phenyl a-D-galactopyranoside, but do not act on larger substrates, such as galactomannans. However, the s ~ coffee ~ ~ beans108hydrolyze both enzymes from Phaseolus ~ u l g a r iand groups of substrates. A more detailed account of substrate specificity is given in a review.z23It seems apparent that the specificity and function of an a-D-galactosidase are related to the nature of the a-D-galactosidic compounds that may occur in a given source. Thus, a galactomannan from Gleditsia ferox (M/G = 4) was attacked by the a - ~ galactosidase from that source, but was unaffected by the enzyme from Penicillium p a x i l l u ~Courtois . ~ ~ ~ and Le D i ~ eshowed t ~ ~ ~ that the mode of action of a-D-galactosidases on galactomannans may vary with the source of the enzyme, even though these may come from galactomannan-containing species. However, the enzymes were able to liberate D-galactose more readily from a galactomannan having a low content of D-galactose than from one having a high content thereof. Hui and Neukom120reported that, despite lengthy incubation, a - ~ galactosidase from coffee beans liberated only 20% of the total Dgalactose residues from guar galactomannan (M/G = 2), and 70% from that of carob (M/G = 4),and that complete removal of =galactose by this enzyme was not possible; this conclusion was in agreement with similar, earlier findings."' Agrawal and Bahl, using enzyme prepara,~~~ tions from both Aspergillus nigerZ3"and Phaseolus v ~ l g a r i s also (225) D. French, Adu. Carbohydr. Chem., 9, 149-184 (1954). (226) J. E. Courtois and F. Percheron, Mem. SOC. Bot. Fr., 29-39 (1965). (227) P. M.' Dey and K. Wallenfels, Eur. J . Biochem., 50, 107-112 (1974). (228) P. S. Sastry and M. Kates, Biochemistry, 3, 1271-1280 (1964). (229) E. S. Bamberger and R. B. Park, Plant Physiol., 41, 1591-1600 (1966). (230) S. Gatt and E. A. Baker, Biochim. Biophys. Acta, 206, 125-135 (1970). (231) F. Petek, E. Villarroya, and J. E. Courtois, Eur. J . Biochem., 8, 395-402 (1969). (232) H. Suzuki, S . C. Li, and Y. T. Li,J. Biol. Chem., 245, 781-786 (1970). (233) K. M. L. Agrawal and 0. P. Bah1,J. Biol. Chem., 243, 103-111 (1968). (234) J. E. Courtois and P. Le Dizet, Bull. SOC. Chim. Biol., 45,743-747 (1963). (235) 0. P. Bahl and K. M. L. Agrawa1,J. Biol. Chem., 244,2970-2978 (1969).

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obtained only a 30-40% removal of the total D-galaCtOSe from these two polysaccharides. Interestingly, the a-D-galactosidase isolated from guar seeds removed all ofthe D-galactose residues from guaran, leaving a residue of water-insoluble D-mannan.236 Coffee-bean a-D-galactosidase has been extensively studied with respect to its specificity for various galactomannans. The e n ~ y m e ~ ~ . ' ~ ~ liberated D-galactose at comparable rates during the initial stages of reaction with the galactomannans from white clover, lucerne, fenugreek (all with M/G = l),Genista scoparia (M/G = L6), Caesalpinia spinosa (M/G = 3),Gleditsia ferox, and honey locust (both having M/G = 4).The results of prolonged action were, however, different. The hydrolysis of the polysaccharide from white clover gave a product having an M/G ratioIo7 of 1.7, whereas hydrolysis of lucerne galactomannan yielded a product with an M/G ratiol10 of 3.0. The products from those of G. ferox and carob (M/G = 4)had M/G ratios ~ ~ ~ 'indicates ~~ a difference in the of -30 and 5.3,r e s p e ~ t i v e l y . This fine structures of the two groups of polysa~charides.~~ Courtois and Le Dizet"O suggested that the a-D-galactosidase initially removes the randomly distributed D-galactosyl groups which are situated next to the unsubstituted D-mannosyl residues in the polymer. This is followed by a slower action on the extremities of the uniform blocks of D-galactosyl groups along the D-mannan backbone. The enzyme is, however, unable to remove all the D-galactosyl groups from the polymer. The authors further suggested1I0that the action of a-D-galactosidase would thus produce longer blocks of unsubstituted D-mannosyl residues which would be relatively hydrophobic in nature. These parts must then fold back, more or less over, the blocks carrying D-gdactosyl groups, thereby making the latter increasingly inaccessible to further enzymic cleavage. It is also tempting to envisage a form of secondary structure having hydrophobic interactions between inter- or intra-chain, unsubstituted D-mannose blocks; this may also make the D-galactosyl groups inaccessible to the enzyme. This situation can be compared to that of "buried" sulfhydryl groups in some proteins. a-DGalactosidase activity has been shown to increase during the germination of seeds of various species, notably, fenugreek, 145*202 g ~ a r , 'Gleditsia ~~ f e r o ~ , ~lucerne,205 ~' carob,20s soybean,20s runner

(236) S. R. Lee, Ph.D. Thesis, Univ. of Minnesota, Minneapolis (1965); Chem. Abstr., 68, 111,694~(1968). (237) J. E. Courtois and P. Le Dizet, Bul. SOC. Chim. Biol., 48, 190-191 (1966).

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bean,233,238*239 cotton,210coffee,240and various other^.^^^^^^' In most of these, a concomitant depletion of a-D-galactosidic, reserve carbohydrates takes place. Dry fenugreek seedszo2have a high level of a-D-galactosidase in the embryo, but only negligible amounts in the endosperm. However, during germination, whereas the enzyme level in the embryo remains constant, it increases sharply in the endosperm; this process parallels the degradation of galactomannan. Similar observations have been made on the incubation of dry-isolated embryos, and of endosperms under “germination” conditions. In the in vivo experiments, the increase in enzyme level was strongly diminished by cycloheximide. The inhibitor acted by preventing protein synthesis in the aleurone tissues. SeilerZo3has actually shown the synthesis of a-D-galactosidase by demonstrating the in vivo incorporation of ~-[‘~C]serine into the enzyme protein during the germination of carob beans. Reid and MeierzoZsuggested that galactomannan is not the natural substrate for the embryo a-D-galactosidase. Rather, the enzyme may be responsible for the hydrolysis of the raffinose family of sugars. It has been found139that a-D-galactosidase from germinated fenugreek seeds could be resolved into two active peaks by chromatography on CM-cellulose, whereas the separation is not possible by gel filtration. It will be of interest to examine the substrate specificities of the enzymes from the embryo and the endosperm. McCleary and MathesonZosshowed the presence of multimolecular forms of a-D-galactosidase (designated A, B, and C) in germinated seeds of carob, guar, lucerne, and soybean. They achieved the separation of the isoenzymes by DEAE-cellulose chromatography. Forms A and C were common to all species examined; carob and soybean had a third form, B. By column chromatography on Sephadex G-200, enzyme C from soybean was further resolved into two peaks of activity, I, and 11. Form A was also present in the dry seeds, and its level did not increase significantly on germination. It was suggested that A and B, which are confined to the cotyledon-embryo part of the seeds, are mainly responsible for the hydrolysis of a-Dgalactosidic oligosaccharides. On the other hand, form C is an endospermic enzyme, and it increases rapidly on seed germination, (238) (239) (240) (241)

D. Lechevallier, Compt. Rend., 258,5519-5522 (1964). D. Lechevallier, Compt. Rend., 250,2825-2827 (1960). M. Sadaksharaswami and G. Ramachandra, Phytochemistry, 7, 715-719 (1968). D. Lechevallier, Compt. Rend., 255, 3211-3213 (1962).

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PRAKASH M . DEY

except in soybean, which has a very low level of galactomannan. The enzyme C was shown to be highly specific for the hydrolysis of galactomannan. Two a-D-galactosidases from Viciu fubu have been resolved by gel filtration.223During germination, form I of high molecular weight, (preponderant in the dry seeds) declined, with a slow increase in the level of form I1 (of low molecular weight). The changes in the enzyme levels that occur during maturation and germination have been described in There are indications242that some compartmentation of a-D-galactosidase activity in organelles, in the form of spherosomes, occurs in V. fuba seeds; this may explain the simultaneous increase in the levels of both a-D-galactosidase and its substrates during seed maturation.

b. P-D-Mannanase.-This enzyme [endo-p-D-mannanase, (1+4)-pD-Inannan mannohydrolase, EC 3.2.1.781 catalyzes the hydrolysis of p-D-( 1+4)-mannopyranosyl linkages of mannans, galactomannans, glucomannans, galactoglucomannans, and D-manno-oligosaccharides.243In addition, some p-D-mannanases have also been to hydrolyze such linkages as those in p-D-Manp-(1+4)-p-D-Glcpand p-~-Glcp-( 1+4)-P-D-Manp-. P-D-Mannanases have been detected in r n i c r ~ - o r g a n i s m s , ~ ~ ~ - ~ ~ ' plants,220~204~243~2"2-255 and animals.243~256-25s Microbial P-D-mannanases (242) P. M. Dey, M. T. Fordom, and J. B. Pridham, unpublished results. (243) R. F. H. Dekker and G. N. Richards, Ado. Carbohydr. Chem. Biochem., 32, 277-352 (1976). (244) S. Innawi, Agric. Biol. Chem., 25, 155-163 (1961). (245) H. Lyr and E. Novak, 2. Allg. Mikrobiol., 2,86-98 (1962). (246) E. Ahlgren, K. E. Eriksson, and 0. Vesterberg, Acta Chem. Scand., 21, 937944 (1967). (247) Y. Misawa, M. Matsubara, M. Hatano, M. Hara, and T. Inuzuka, Nippon Shokuhin Kogyo Gakkai Shi, 14,286-291 (1967); Chem. Abstr., 68,86,259e (1968). (248) K. E . Eriksson and W. Rzedowski, Arch. Biochem. Biophys., 129, 683-688 (1969). (249) Y. Hashimoto, Nippon Nogei Kagaku Kaishi, 44,287-292 (1970); Chem. Abstr., 73, 108,395d (1970). (250) G. Keilich, P. J. Bailey, and W. Liese, Wood Sci. Technol., 4,273-283 (1970). (251) S. Emi, J. Fukumoto, and T. Yamamoto, Agric. Biol. Chem., 36, 991-1001 (1972). (252) B. V. McCleary and N. K. Matheson, Phytochemistry, 14, 1187-1194 (1975). (253) H. Villarroya and F. Petek, Biochim. Biophys. Acta, 438, 200-211 (1976). (254) P. Halmer, J. D. Bewley, and T. A. Thorpe, Planta, 130, 189-196 (1976). (255) S. Clermont-Beaugiraud and F. Percheron, Bull. SOC. Chim. Biol., 50, 633-639 (1968). (256) H. Bierry and J. Giaja, Biochem. Z., 40,370-378 (1912).

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are known to be both inductive and constitutive, occurring as extracellular or intracellular enzymes.243It has been suspected that the p-D-mannanases detected in the digestive tracts of various animals . ~ ~ ~ and may be due to symbiotic r n i c r o - ~ r g a n i s m s Dekker characterized various p-D-mannanases. Studies on the properties of plant p-D-mannanases have been hampered by the difficulty in obtaining the enzyme free from interfering glycosidases, notably, a-D-galactosidase, P-D-glucosidase, and p-D-mannosidase, Beaugiraud and PercheronZ6O had only limited success in obtaining, from germinated, fenugreek seeds, a p-D-mannanase free from a-D-galactosidase. They found that continuous-flow electrophoresis gave an a-D-galactosidase preparation lacking detectable @-D-mannanase activity, but that the reverse was not possible. A similar result was obtained when separation by CMcellulose chromatography was attempted.139By using ion-exchange chromatography and gel-filtration, S. R. Lee236 resolved multiple forms of p-D-mannanase from germinated guar-seeds; one of the forms was free from a-D-galactosidase activity. Extensive purifications ofp-Dmannanases have been achieved, the sources being lucerne,253white clover,'40 and konjaP1; the enzyme from Bacillus subtilisZs1has been crystallized. Most 6-D-mannanases are endo-acting enzymes, as evidenced by a rapid fall in viscosity when they act on polymeric substrates, producing a series of Dmanno-oligosaccharides of different d.p. values, 110.1 12,243,251,285,262-265 Amongst the D-manno-oligosaccharides, those having a d.p. > 2 are hydrolyzed, but the minimum d.p. requirement of a readily hydrolyzable substrate is14032433253 -4. In a detailed study, Villarroya and PetekZs3found that the lucerne p-D-mannanase is unable to hydro(257) P. J. Kooiman,J. Cell. Comp. Physiol., 63, 197-201 (1964). (258) F. L. Meyers and D. H. Northcote, Biochem. J . , 6 9 , 5 4 ~(1958). (259) J. E. Courtois, F. Petek, and T. Dong, Bull. SOC. Chim. Biol., 44, 11-21 (1962). (260) S. Beaugiraud and F. Percheron, Compt. Rend., 259,3879-3881 (1964). (261) H. Shimihara, H. Suzuki, N. Sugiyama, and K. Nisizawa, Agric. Biol. Chem., 39, 301-312 (1975). (262) Y. Hashimoto and J. Fukumoto, Nippon Nogei Kagaku Kaishi, 43, 317-322 (1969); Chem. Abstr., 72,53,856b (1970). (263) S. Clermont-Beaugiraud, M. Charpentier, and F. Percheron, Bull. SOC. Chim. Biol., 52, 1481-1495 (1970). (264) Y. Tsujisaka, K. Hiyama, S. Takenishi, and J. Fukumoto, Nippon Nogei Kagaku Kaishi, 46, 155-161 (1972); Chem. Abstr., 77, 44,667s (1972). (265) N. Sugiyama, H. Shimahara, T. Andoh, and M. Takemoto, Agric. Biol. Chem., 37, 9-17 (1973).

368

PRAKASH M. DEY

lyze p a - (1+4)-linked mannobiose (M,) and mannotriose ( M3). The hydrolysis of M4 is slow, and results in M3 (lo%), M, (80%), and D-mannose (10%). On the other hand, M5 is rapidly hydrolyzed, producing equal amounts of M2 and M3, and only -2% of D-mannose. The enzymic action on Me liberated M3 (50%), M4 (25%), and M2 (25%);on further incubation, M4 was degraded to a mixture of M3 (55%), M2 (41%), and D-mannose (4%). Based on these results, Villarroya and Petek2= suggested the following mode of action.

(MA M-M (M3) M-M-M (M4) M-M-&M-&M (M,) M-M-M-bM-M (Me) M-M-M+M-$M-M where M = P-D-(1+4)-linked D-InannOpyranOSyl residues, 4 = preferential points of attack, J, = secondary points of attack, and f = slow attack. Beaugiraud and Percheron2ss~z60 showed that the enzyme from germinated fenugreek seeds separately hydrolyze both M4 and Ms, yielding M3, M,, and traces of D-mannose from each. To explain the hydrolytic products from M4, the authors suggested that some of the initially formed M, units are linked to M4 molecules, presumably by transglycosylation, producing Me; this could then be cleaved into two M3 units. The action of white-clover p-D-mannanase" on ivory-nut mannan produced139 a mixture of D-manno-oligosaccharides (Mz-M6), as shown in Fig. 2,D. On the other hand, the enzyme from fenugreek seeds mainly produced M,, M3, and traces of D-manno~e.'~~ The enzyme from white clover also hydrolyzes salep (Orchis militaris) glucomannan (D-mannose/D-glucose = 3), yielding a mixture of ohgosaccharides, some of which contain D-glucose, and also traces of free D-glUCOSe (see Fig. 2,E). The presence of free D-glucose and a disaccharide containing D-mannose and D-glUCOSe units may indicate the ability of the enzyme to act upon /3-~-(1+4)-linkages between these two sugar residues. It will be of interest to investigate whether the D-glUCOSe units of the oligosaccharides are situated terminally or in the middle of the chain. However, the enzyme from lucerne2s3does not produce any D-glucose-containing oligosaccharides, even following a long incubation with the glucomannan; M, and M3 are the

BIOCHEMISTRY OF PLANT GALACTOMANNANS

369

only products. On the other hand, a purified enzyme from konjac rapidly decreased the viscosity of konjac glucomannan, but the reducing power liberated was very This shows the presence of only a few points of attack on the polysaccharide. The ability of p-D-mannanases to hydrolyze galactomannans is generally related to the M/G ratio of the polysaccharide; a higher value facilitates the degradation.l16 It has been found that, during the initial period of reaction (20 min),140the white-clover enzyme produces almost equal amounts of reducing power from the galactomannans of carob (M/G = 4) and Gleditsia ferox (M/G = 4), but the reducing power was almost half with that of guar gum (M/G = 2),and one-tenth with that of white clover (M/G = 1.7); the K , values for the polysaccharides were, respectively, 0.16, 0.08, 0.83, and 1.1%(the concentrations are expressed as w/v). Despite a similar, overall M/G ratio, Gleditsia galactomannan was more strongly bound to the enzyme than that of carob. Analysis of the hydrolysis products showed that the former yielded a greater number of D-galactose-containing oligosaccharides than the latter; the Gleditsia polymer also yielded some free D-mannose (see Fig. 2,A and B). This is further evidence for a more random structure for Gleditsia galactomannan (see also Section 1,4),thus providing a greater number of sites for enzymic attack along the main chain, and a higher affinity for the enzyme. Dea and Morrison43reported that, in accordance with the random structure, the action of a-D-galactosidase on the Gleditsia polysaccharide yields a greater amount of insoluble products than its action on carob galactomannan. The action of p-D-mannanase should, therefore, produce the opposite effect. It was shown43that, whatever the initial M/G ratio (1.7-5.3) of the galactomannan, the action of P-D-mannanase always produced final, nondialyzable products having M/G ratios of -1. Thus, lucerne galactomannan (M/G = 1) is resistant to the enzyme. However, the a-D-galactosidase-treated polymer is attacked by the enzyme. It was that the presence of a D-galactosyl group on each of the Dmannosyl residues of the main chain of Dmannan hinders the enzymic cleavage of endo-p-D-mannosidic linkages; for hydrolysis to occur, at least one unsubstituted D-mannosyl residue was required. On the other hand, Reese and Shibata"' found that two such units are necessary for the microbial P-D-mannanase; a similar observation was made by McCleary and coworkers.116It has been found139that, although guar gum (M/G = 2) demonstrates reducing power when it is treated with white-clover @-D-mannanase,oligosaccharides of low molecular weight could not be detected (see

370

PRAKASH M. DEY

Fig. 2,C); this indicates that there are very few points of enzymic attack on the polysaccharide, and, hence, the high K,value. Bourquelot and Hi.rissey266-26s were the first to show the development of p-D-mannanase, then referred to as seminases, during the germination of seeds, notably those of carob, fenugreek, lucerne, and white clover. The activity of this enzyme was also monitored in the germination of the seeds of ivory nut,271barley,272guar,220*236 and konjac.26sIt is now generally established that, in seeds, p-Dmannanase activity increases on germination, with a concomitant depletion of D-mannan and related polysaccharides.'ss~201*204~20s~219~233~ 236.252.254

McCleary and Matheson2s2found that the level of p-D-mannanase reaches a maximum after 2-3 days of germination in lucerne, and after 7-8 days in carob. Similar maxima occurred for guar and honey locust. The authors purified, and resolved, these P-D-mannanaSe activities by ion-exchange chromatography and gel filtration. Honey locust has only one form of the enzyme, namely B; carob and soybean have three forms, and lucerne, four; form B is predominant in all these species. In guar, S . R. Lee23sdetected two forms of the enzyme; these were found only in the endosperm. It was observed252that the enzymes preferentially and rapidly hydrolyze the galactomannans of carob and locust bean, which have a relatively low content of D-gakiCtOSe. However, when the seeds contain polysaccharides having a high content of D-gahCtOSe (lucerne and guar), they also possess a very active a-D-galactosidase. The physiological significance of this enzyme is that an initial, partial removal of D-galaCtOSyl groups could then lead to a rapid depolymerization by p-D-mannanase; this, perhaps, explains why the maximal activity of the latter enzyme is reached at a much later stage in germinating lucerne, compared to carob. The M/G ratio of the polysaccharide also increases in guar and lucerne during germination, but it remains constant in carob and honey locust.2s2 Reid and coworkers204showed that, in fenugreek, P-D-mannanase activity reaches a maximum at 42-48 h of germination, paralleling (266) E. Bourquelot and H. Hhrissey, Compt. Rend., 129,614-616 (1889). (267) E. Bourquelot and H. Hbrissey, Compt. Rend., 130, 42-44, 340-342, 731-733, 1719-1721 (1900). (268) H. HBrissey, Compt. Rend., 133, 49-52 (1901). (269) H. HBrissey, Compt. Rend., 134,721-723 (1902). (270) J. Griiss, Ber. Dtsch. Bot. Ges., 20, 3 6 4 1 (1902). (271) F. J. Paton, D. R. Nanji, and A. R. Ling, Biochem J . , 18, 451-454 (1924). (272) H. Pringsheim and A. Genin, 2.Physiol. Chem., 40,229-234 (1924).

BIOCHEMISTRY OF PLANT GALACTOMANNANS

371

the breakdown of storage galactomannans; this is in accordance with our observations. The enzyme activity increases even when the dryisolated endosperms are incubated under “germination” condit i o n ~The .~~ synthesis ~ of the enzyme takes place in the active, aleurone layer, and can be inhibited by the inhibitors of protein synthesis. 199,201,202,204

The relationship between P-D-mannanase and the germination of lettuce seeds has been examined in detail by Halmer and cow o r k e r ~ . ~ The ” * ~ lettuce-seed ~~ embryo is known to be surrounded by a tough, endospermic wall whose major constituent s ~ g a r s ~ ~ ~ a ~ are D-mannose (58%), D-glucose (lo%), D-galaCtOSe (9.5%), and Larabinose (9.5%).The wall polysaccharide is similar to galactomannar~~~~ (compare, Refs. 80 and 274). The emerging radicle has to penetrate this wall during germination, and an ordered degradation of the wall has, in fact, been demonstrated during this process.273,27s*276 It was suspected that cellulase was p r o d ~ ~ e dwhich ~ ~ might ~ * ~weaken ~ ~ , ~ ~ ~ the endosperm wall, but this would also affect the wall of the emerging radicle. Halmer and coworkers273have, however, shown the production, in lettuce endosperm, of a p-D-mannanase which could degrade this wall; a similar observation was made for ivorynut e n d o ~ p e r m Alternatively, .~~~ it was suggested that the growing radicle accumulates enough mechanical thrust to penetrate the endosperm wa11.280~281 It is possible that the emergence of the radicle is assisted by a combination of the thrust factor and pectinase, cellulase, and P-D-mannanase activities. A low, basal level of the latter enzyme is always present in the endosperm. It is not currently known whether the endosperm wall in the vicinity of the growing radicle-tip is initially degraded because of a local increase in the P-D-mannanase activity. However, the high level of P-D-mannanase produced after the radicle has emerged demonstrates the importance of the enzyme in degrading D-mannose-containing polysaccharides that serve as an energy source in the growing embryo.2s4

(273) P. Halmer, J. D. Bewley, and T. A. Thorpe, Nature, 258,716-718 (1975). (274) G. 0. Aspinall, “Polysaccharides,” Pergamon Press, Oxford, 1970. (275) R. L. Jones, Planta, 121, 133-146 (1974). (276) W. M. Park and S. S. C. Chen, Plant Physiol., 53, 64-66 (1974). (277) H. Ikuma and K. V. Thimann, Plant Cell Physiol., 4, 169-185 (1963). (278) A. D. Pavlista and J. G. Valdovinos, Plant Physiol., 56 (suppl.),83 (1975). (279) H. Meier, Biochim. Biophys. Acta, 28, 229-240 (1958). (280) M. W. Nabors and A. Lang, Planta, 101,l-25 (1971). (281) M. W. Nabors and A. Lang, Planta, 101,26-42 (1971).

372

PRAKASH M. DEY

c. P-D-Mannosidase.-This enzyme (p-D-mannoside mannohydrolase, EC 2.3.1.25) catalyzes the following reaction. CH20H

0

HO

A p-~-mannoside

where

CH20H

H

o

O

O

H

+ ROH

+ H,O D-mannose

R may be an alkyl, aryl or, glycosyl group.

P-D-Mannosidases are exo-hydrolases that have been shown to remove p - ~ 1+4)-linked -( D-mannosyl groups from the nonreducing end of their substrates, for example, D-manno-ohgosaccharides and * * ~presence ~ - ~ ~ ~ of p-DD-mannose-containing g l y ~ o p e p t i d e s . ~ ~The mannosidases has been shown in a range of animal t i s s ~ e s and ~ ~ - ~ ~ ~ Amongst plants, the enzyme has in some micro-organisms.22'*23s~2g8~299 been extracted from fenugreek,2°2,260guar,236*252 pineapple,3"O lucerne,2= honey l o ~ ~ s tPhoenix , ~ ~ canariensis,282 ~ , ~ ~ ~ wheat and malted barley.302On the other hand, extracts of a number of galactomannan-containing leguminous seeds had very little or no p-D-mannosidase There have been only a few kinetic studies on plant p-D-mannosidases, because of the apparently limited distribution of this enzyme in various species, and also because of the paucity of attempts at purification. The enzyme from malted barley has been purified 41-fold by fractionation with ammonium sulfate, and chromatography302on Biogel P-100, DEAE-cellulose, and CM-cellulose. Some properties of p-D-mannosidases from various tissues are summarized in Table 11. (282) J. E. Courtois and P. Le Dizet, Bull. Soc. Chim. Biol., 46,535-542 (1964). (283) K. Sugahara, T. Okumura, and I. Yamashina, Biochim. Biophys. Acta, 268,488-496 (1972). (284) B. A. Bartholemew and A. L. Perry, Biochim.Biophys. Acta, 315,123-127 (1973). (285) S. Toyoshima, M. Fukuda, and T. Osawa, Biochem. Biophys. Res. Commun., 51, 945-950 (1973). (286) K. Sugahara and I. Yamashina, Methods Enzymol., 28, 769 (1972). (287) G . L. E. Koch and C. A. Marsh, Comp. Biochem. Physiol. B , 42,577-590 (1972). (288) T. Nagaoka,]. E x p . Med., 51, 131-138 (1949). (289) T. Muramatsu and F. Egami,]. Biochem. (Tokyo), 62,700-709 (1967). (290) J. C. Steigerwald and B. A. Bartholomew, Biochim.Biophys. Acta, 321,256-261 (1973). (291) T. Sukeno, A. L. Tarentino, T. H. Plummer, Jr., and F. Maley, Biochem. Biophys. Res. Commun., 45,219-225 (1971).

BIOCHEMISTRY OF PLANT GALACTOMANNANS

373

TABLEI1 Kinetic Parameters of Some P-D-Mannosidases

pH Source

Km"

optimum

(mM)

Inhibitor

References

5.0 5.0 4.6

0.29 0.29 4.50

-

252 252 305

Human skin Human synovial fluid Lucerne cotyledon endosperm Malted barley

3.5 3.5-4.0

3.4

p-nitrophenyl P-Dgalactopyranoside

5.0 5.0 5.5

0.29 0.83 0.32

Hg2+,45% inhibition at 1mM Hg2+,42% inhibition at 1mM 2-amino-2-deoxy-Dmannose (competitive, KI = 0.18 mM)

Pineapple Snail

3.5 4.0-5.0

6.5

Carob cotyledon endosperm Hen oviduct

-

-

Ag+,no inhibition at 0.6 mM ~mannono-1,5-lactone (competitive, Ki=17 p M )

-

-

-

290 284 252 252 302 300 286

"The K, values were estimated with respect to p-nitrophenyl P-D-mannopyranoside, except for the snail enzyme, where phenyl P-Dmannopyranoside was used.

(292) T. Sukeno, A. L. Tarentino, T. H. Plummer, Jr., and F. Maley, Biochemistry, 11, 1493-1501 (1972). (293) J. Conchie and T. Mann, Nature, 179, 1190-1191 (1957). (294) T. Muramatsu, Arch. Biochem. Biophys., 115,427-429 (1966). (295) H. B. Bosmann, Biochim. Biophys. Acta, 258,265-273 (1972). (296) A. Ohkawara, K. M. Halprin, J. R. Taylor, and V. Levine, Br.1. Dennatol., 87,450459 (1972). (297) G. A. Levvy, A. J. Hay, and J. Conchie, Biochem. J., 91,378-384 (1964). (298) Y. Hashimoto and J. Fukumoto, Nippon Nogei Kagaku Kaishi, 43,564-569 (1969); Chem. Abstr., 72,51,167d (1970). (299) M. Adams, N. K. Richtmyer, and C. S. Hudson,J. Am. Chem. Soc., 65,1369-1380 (1943). (300) T. T. Li and Y. C. Lee, J. Biol. Chem., 247,3677-3683 (1972). (301) J. W. Lee and J. A. Ronalds, J . Sci. Food Agric., 23, 199-205 (1972). (302) C. W. Houston, S. L. Latimer, and E. D. Mitchell, Biochim. Biophys. Acta, 370, 276-282 (1974). (303) R. Somme, Actu Chem. Scand., 24, 72-76 (1970). (304) R. Somme, Acta Chem, Scand., 25,759-761 (1971). (305) T. Sukeno, A. L. Tarentino, T. H. Plummer, Jr., and F. Maley, Methods Enzymol., 28,777 (1972).

374

PRAKASH M . DEY

There are only limited reports on the substrate specificity of p-Dmannosidases. Reese and Shibata2,1 showed that the fungal enzyme hydrolyzes M3 more rapidly than M2.The reduction of M3 to Dmannotriitol had no effect on the initial rate of hydrolysis, whereas reduced M, was hydrolyzed much more slowly than M, itself. From Rhi-zopus n i v e u was ~ ~ obtained ~~ a purified p-D-mannosidase that was free from p-D-mannanase activity and showed the relative hydrolyses ofthe substrates as: M4 = M3 > M2 > M5 > M6. An enzyme preparation from g ~ a r , described ,~~ as eXO-p-D-( 1 4 ) mannanase, hydrolyzed ivory-nut mannan almost completely to D-mannose. The purified (1,000-fold) enzyme from pineapple300hydrolyzed the substrates in the order: p-nitrophenyl p-D-mannopyranoside >P-D-Man-(GlcNAc),-Asn (a core glycopeptide) > methyl P-D-mannopyranoside > p-D-Man-(1+4)-D-Glc > @-&Man-(1 4 ) - D mannitol. A 10,000-fold purified enzyme from hen oviduct305 hydrolyzed p-D-Man-(GlcNAc),-Asn at one quarter the rate, and P-D-Man(GlcNAc), at one-third the rate, of p-nitrophenyl p-D-mannopyranoside. The snail enzyme286hydrolyzed the core glycopeptide, as well as a tri-D-mannose, a-~-Man-( 1+4)-/3-D-Man-( 1+4)-D-Man, but the latter was treated in the presence of an a-D-mannosidase; here, the action of p-D-mannosidase seems to follow that of a-D-mannosidase. Hylin and S a ~ a i reported ~'~ that the crystalline depolymerase from Leucaena leucocephala converts the galactomannan from the same source into D-galactose and D-mannose. Hence, it is most likely that the enzyme preparation contained p-D-mannosidase activity, in addition to a-D-galactosidase and p-Dmannanase activities. Reid and Meier202showed the presence of p-Dmannosidase in the embryo and the endosperm of fenugreek at all stages of germination. The embryo had a low level of the enzyme, and there was no significant increase in activity on germination. On the other hand, the endosperm possessed low activity up to 30 h, after which it rose steeply and levelled off at 60 h; the rise was approximately 10-fold. This increase coincided with the depletion of the reserve galactomannan. The authors further concluded from in vivo experiments (using inhibitors of protein synthesis) that production of the enzyme was controlled by the aleurone layer. McCleary and Mathe~on,~, showed the presence of two forms of P-Dmannosidase in the seeds of guar, lucerne, carob, and honey locust. Form A (low molecular weight) was located in the cotyledon-embryo part, whereas (the larger) form B was in the endosperm. They suggested25zthat, although the enzyme activity in these

BIOCHEMISTRY OF PLANT GALACTOMANNANS

375

seeds is low, it would be sufficient to degrade the D-manno-oligosaccharides produced by the action of P-D-mannanase on the endogenous galactomannans.

d. Oligo-~-~-mannosyl-(1-,4)-phosphorylase.-In 1970, SOmme3O3 reported that extracts of the germinated, galactomannan-containing seeds of Trifolium repens, T . pratense, Medicago sativa, Anthyllis vulneraria, and Lotus corniculatus contain no free D-mannose. The activity of p-D-mannosidase could not be detected in these extracts, either with M2 or p-nitrophenyl P-D-mannopyranoside as the substrate. The author doubted303that p-D-mannosidase had a role in the mobilization of galactomannan in these seeds. She suggested that phosphorolysis was a possible, alternative pathway. In 1972, Foglietti and Percheron222were successful in preparing a phosphoro1ytic enzyme, namely, oligo-P-D-mannosyl-(1+4)-phosphorylase, from the germinated seeds of fenugreek. The enzyme [(1+4)-P-~mannan:orthophosphate P-D-mannosyl transferase] catalyzes the following reversible reaction, in which the d.p. of the D-mannooligosaccharide must exceed two.

*

(D-Mannose), + orthophosphate (D-mannose),-, + P-D-mannosyl phosphate The D-mannosyl phosphate produced during this reaction can be further transformed into glycosyl esters of nucleoside diphosp h a t e ~ .With ' ~ ~ the participation of a suitable epimerase, this might be a possible pathway for the formation of sucrose and starch in the seed. Courtois and coworkers14shave, in fact, shown a rise in the levels of UDP-D-glucose, UDP-D-galactose, and GDP-D-mannose during the germination of fenugreek seeds. Further work from the same laboratory on fenugreek indicated the presence of a nucleotide pyrophosphorylase capable of converting the glycosyl esters of nucleoside diphosphates into the respective D-hexosyl phosphates, and of an epimerase that can catalyze the conversion of UDP-D-galactose into UDP-D-glucose.14' IV. FUNCTION Seed galactomannans appear to have a double physiological function. Firstly, they retain water by solvation (see Ref. 43 for details on gel formation), and thereby prevent (in regions having high atmospheric temperatures) complete drying of the seeds which

376

PRAKASH M. DEY

would cause protein denaturation, in particular, the denaturation of those enzymes essential for seed germination. In this connection, the D-galactosyl side-branches of the polymer may be regarded as hydrophilic parts of the molecule. Secondly, the galactomannans serve as food reserves for the germinating seeds. Microbial galactomannans whose structures do not conform to those of leguminous seeds have been the subject of a few special ~~~ studies. In Acer plantanoides and A . p s e u d o p l e n t h e n u ~ ,infected with Rhytisma acerinum, a strong inhibitor of tobacco mosaic virus was found. The active compound was water-extractable and was identified as a galactomannan; this is of interest for future studies on the possible inhibition of viruses. Being water-soluble, galactomannans form highly viscous solutions which, on drying, leave a transparent film that can, therefore, adhere to the surface of cells, making them impermeable to viruses. These may also anchor the viruses to the surface of the cell, or immobilize them on its network structure. Also, a galactomannan from Lipomyces starkyi307 was shown to have an interferon-inducing ability in cell cultures. In addition, some microbial galactomannans have been shown to act as serological antigen^.^^^,^^' Some plant galactomannans are also known to interact with milk proteins,310plant lectinsY3" and protein a n t i b o d i e ~ . ~ It' ~is not yet known whether these polysaccharides can exist in the form of complexes with seed proteins. Such complex-formation often results in increased stability of proteins towards heat inactivation, proteolytic degradation, and other unfavorable conditions.

(306) M. Gubanski and M. Saniewski, Actu Microbiol. Pol., 13, 227-232 (1964). (307) L. Borecky, V. Lackovic, D. Blaskovic, L. Masler, and D. Sikl, Acta Virol. Engl. Ed., 11,264-266 (1967). (308) I. Azuma, F. Kanetsuna, Y. Tanaka, Y. Yamamura, and L. M. Carbonell, Mycopathol. Mycol. Appl., 54, 111-125 (1974). (309) W. Lee and K. 0. Lloyd, Arch. Biochem. Biophys., 171, 624-630 (1975). (310) A. S. Ambrose, U.S.Pat. 1,991,189 (1935); Chem. Abstr., 29,2255 (1935). (311) J. P. Van Wauwe, F. G . Loontiens, and C. K. DeBruyne, Biochim. Biophys. Actu, 313,99-105 (1973). (312) M. Heidelberger, J. Am. Chem. SOC., 77,4308-4311 (1958).

BIBLIOGRAPHY OF CRYSTAL STRUCTURES OF POLYSACCHARIDES 1975 BY PUDUPADI R. SUNDARARAJAN AND ROBERT H. MARCHESSAULT Xerox Research Centre of Canada, Mississauga, Ontario L5L 119, Canada; Department of Chemistry, University of Montreal, Montreal, Quebec H3C 3V1, Canada

I. 11. 111. IV.

Introduction .......................................................... Amylose and Other wD-Glycans.. ...................................... Cellulose and Other P-D-Glycans ....................................... Glycosaminoglycans (Amino Polysaccharides) ........................... I.

377 378 379 381

INTRODUCTION

In Volume 33 of this Series, we presented’ a review of the crystalline structures of polysaccharides published during the period 19671974. Detailed accounts of progress in structural studies on specific types of polysaccharides were presented in the Proceedings of the Twenty-sixth Symposium of the Colston Research Society and were subsequently published as a book.2 Precise methods for X-ray diffiaction analysis of biopolymer structures were discussed by H ~ k i n sThe .~ aspects of the structures of cellulose, mannan, and xylan, their organization in the cell wall, and the biosynthesis of cell-wall polysaccharides were described by M a ~ k i e .Work ~ on the structures of the connective-tissue polysaccharides, 0-acetylcellulose, and the various forms of amylose was reviewed by at kin^,^ Chanzy,6 and Sarko,’ (1) R. H. Marchessault and P. R. Sundararajan, Ado. Carbohydr. Chem. Biochem., 33,387-404 (1976). (2) “Structureof Fibrous Biopolymers,” E. D. T. Atkins and A. Keller, eds., Buttenvorth, London, 1975. (3) D. W. L. Hukins, Ref. 2, pp. 293-305. (4) W. Mackie, Ref. 2, pp. 391-416. (5) E. D. T. Atkins, Ref. 2, pp. 35-45. (6) H. D. Chanzy, Ref. 2, pp. 417-434. (7) A. Sarko, Ref. 2, pp. 335-354.

377

378

P. R. SUNDARARAJAN AND R. H. MARCHESSAULT

respectively. An excellent review by Prestons contains an account of X-ray diffraction and conformational studies on agar, alginic acid, carrageenan, cellulose, mannan, pectic acid, and xylan. Two other brief reviews on the structural features of cellulose have also been publi~hed.~*’~ The crystal-structure data reported in 1975 on polysaccharides are given in this article. In addition to the unit-cell dimensions, the significant features of the structures are described. Unless specified otherwise, the chain axis is along the c direction of the unit cell. The helical symmetry is denoted by n(&h),where n specifies the number of repeat units per turn, and h is the projected height, in nanometers, of the repeat unit onto the helix axis. A positive h denotes a righthanded helix, and a negative h, a left-handed helix. As before,’ in the title to each abstract, a common name or descriptive title for the polysaccharide described is given on the left, and the chemical formula on the right. 11. AMYLOSEAND OTHERWD-GLYCANS

1. V-Amylose”

POly[ ( 1-*4)-a-D-Glcp]

The dehydrated form crystallizes in an orthorhombic unit-cell with a = 1.292 nm, b = 2.24 nm, and c = 0.795 nm, with two 6(-0.133)

chains per unit cell. The space group is P2,2,2,. Models constructed with “residue 3” of the cyclohexaamylose structure12were best suited. Two intrachain hydrogen-bonds, Of 2---0i+1-3of length 0.275 nm, and 0f6---0i-6-2 of length 0.284 nm, were proposed. Intermolecular hydrogen-bonds were ruled out. The final error function was 66%. The results were compared with those of Winter and Sarko.I3 2. Mycodextran (nigeran)l 4

Poly[(1+3)-a-~-Glcp-(1+4)-a-~-Glcp]

A method was presented for recording electron diffraction diagrams of hydrated, crystalline biopolymers, and was applied to mycodextran. (8)R. D. Preston, Phys. Lett. C, 21, 185-226 (1975). (9)H. Sihtola and L. Neimo, in “Symposium on the Enzymatic Hydrolysis of Cellulose,” M. Bailey, T. M. Enari, and M. Linko, eds., Biotechnical Laboratory of the Technical Research Centre of Finland, Helsinki, Finland, 1975,pp. 9-21;Chem. Abstr., 83,207,743d(1975). (10)G.La1 and A. Pande, Colourage, 22,21-27 (1975). (11)V. G.Murphy, B. Zaslow, and A. D. French, Biopolymers, 14,1487-1501 (1975). (12)A.Hybl, R. E. Rundle, and D. G. WilliamsJ. Am. Chem. Soc., 87,2779-2788(1965). (13)W. T.Winter and A. Sarko, Biopolymers, 13, 1447-1460 (1974). (14)K.J. Taylor, H. Chanzy, and R. H. Marchessault,j. Mol. Biol., 92,165-167(1975).

BIBLIOGRAPHY OF CRYSTAL STRUCTURES

379

With only the lattice water remaining, and at - lo”,the diffiactogram of the single crystals of mycodextran contained 50 independent reflections, which were indexed with a = 1.76 nm, b = 0.735 nm, and y = 90”. On annealing in the range of 100 to 160”,a dry form was obtained, with a = 1.76 nm, b = 0.51 nm, and y = 90”. It was suggested that the water of hydration is of the sheet type, alternating along the b axis of the crystal. On dehydration, the development in the lamellar crystals of cracks in the direction normal to the b axis was observed. From the difference in the unit-cell volume between the hydrated and dehydrated forms, it was deduced that there are two water molecules per D-glUCOSe residue in the “hydrate.” 111. CELLULOSEAND OTHERP-D-GLYCANS 1. Cellulose1s

Poly[( 1-*4)-p-~-Glcp]

Sodiocellulose I1 crystallizes in a hexagonal unit-cell, with a = 1.0 nm and c = 1.51 nm. The meridional reflection on the third layer-line led to a three-fold helical structure. The unit cell contains two 3(-0.503) chains of sodiocellulose, six sodium ions, six hydroxyl ions, and at least six water molecules. Conformational calculations and packing analysis of the chains led to a structure which is similar in several features to that of poly[(1+4)-p-~-Xylp].’~ 2. Cellulose’7

Poly[(l+ 4)-p-~-Glcp]

X-Ray data on regenerated cellulose (rayon) led to a unit cell with a = 0.801 nm, b = 0.904 nm,c = 1.036 nm, and y = 117.1”.The space group is P21. An antiparallel arrangement of the chains, with a relative stagger of 0.216c, was proposed. The torsion angle x(0-5, (2-5, C-6, 0-6) is different for the “up” and “down” chains. The “down” chain has an 0-2’---0-6 intrachain hydrogen-bond. The intermolecular hydrogen-bonds are: (i) 0-6-03, parallel to the a axis, between adjacent “down” chains; (ii) 0-6-0-2 between adjacent “up” chains along the a axis, and (iii)0-2---0-2’ between the “up” chain at 1,0,0 and the “down” chain at the center.

3. Cellulose’*

Poly[(1+4)-P-~-Glcp]

(15)P. M. Whitaker, I. A. Nieduszynski, and E. D. T. Atkins,PoZymer, 15,125-127 (1974). (16) I. A. Nieduszynski and R. H. Marchessault, Biopolymers, 11, 1335-1344 (1972). (17) F.J. Kolpak and J. Blackwell, Macromolecules, 8,563-564 (1975). (18)A.Koura, B.Philipp, H. Schleicher, and W. Wagenknecht, Fuserforsch. Textiltech., 26, 514-515 (1975);Chem. Abstr., 84, 46,345d (1976).

380

P. R. SUNDARARAJAN A N D R. H. MARCHESSAULT

Aqueous solutions of guanidine cause a structural change in cellulose midway between that produced by aliphatic amines and by alkali hydroxides. The lattice change to a cellulose I1 structure, which occurs with aqueous alkali hydroxide above a certain concentration, does not occur with guanidine. This was attributed to N-H---0 bridges, with the participation of guanidinium ions. 4. C e l l ~ l o s e ' ~

POly[( 1+4)-P-D-Clcp]

The ratio of the intensities of the 020 and 040 reflections for cellulose I11 from various sources leads to two classes. Cellulose I11 from cellulose I has prominent reflections at 0.247 nm on the first layer-line, and 0.28 nm and 0.223 nm on the second. For cellulose I11 from cellulose 11, these are at 0.257 and 0.236 nm on the first layer-line, and at 0.258 and 0.235 nm on the second. It was suggested that cellulose I11 from cellulose I and I1 be termed Cell 1111and Cell IIIn, respectively. The cellulose IV from cell 1111gave reflections at 0.227 nm on the first layer, and at 0.305 and 0.289 nm on the second layer. For cellulose IV from Cell 11111and cellulose 11, the reflections are at 0.251 and 0.298 nm on the first and second layers, respectively. It was suggested that the crystalline modifications of cellulose be classified into two families: Cell I family (I, 1111,and IVJ and Cell I1 (11, IIIn, and IVII).A member of the Cell I1 family cannot be transformed into one of the Cell I family.

X-Ray diagrams of O-p-tolylsulfonylcellulose prepared from cotton slivers showed an amorphous pattern, with a broad maximum at 20 = 21",showing that p-toluenesulfonylation of cotton is an intrafibrillar reaction causing the breakdown of the initial cellulose I lattice. In the initial stages of reaction of O-p-tolylsulfonylcellulose with an anhydrous mixture of potassium fluoride and ethylene glycol at 190", there is a recrystallization into cellulose I1 lattice. The extent of crystallization of cellulose improves with increasing unsaturation up to a certain stage, beyond which the unsaturated cellulose crystallizes in a new, highly crystalline phase. 6. Cellulose2'

Poly[( 1+4)-P-D-Gkp]

(19) J. Hayashi, A. Sufoka, J. Ohkita, and S. Watanabe, J . Polym. Sci., Polym. Lett. Ed., 13,23-27 (1975). (20) H . C. Srivastava, A. K. Kulshreshtha, and V. K. Srivastava,]. Polym. Sci., Polym. Lett. Ed., 13,65-70 (1975). (21) W. Herth, A. Kuppel, and W. W. Franke,]. Ultrastruct. Res., 50,289-292 (1975).

BIBLIOGRAPHY OF CRYSTAL STRUCTURES

38 1

The alkali-resistant material from the cyst walls of the green alga Acetabularia mediterranea was studied by X-ray and other physical methods. The predominant, structural polysaccharide was found to be cellulose I. The stalk and cap walls of the alga contain poly[(l+4)P-D-Manp]. It is possible that the change from mannan to cellulose in Acetabularia is paralleled by a transition from the diploid to the haploid stage. 7. O-AcetylpachymanzZ

POly[(I+ 3)-A~-p-~-Glcp]

The X-ray pattern from O-acetylpachyman derived from the fungus Porio cocos can be indexed either with a hexagonal unit-cell of dimensions a = b = 1.149 nm, c = 1.86 nm, or with an orthorhombic cell having a = 1.149 nm, b = 2.013 nm, c = 1.86 nm. The presence of both 5th- and 6th-order meridional reflections led to the detection of two polymorphs. Films stretched 25 to 50% at 125" led to a unit cell with a = b = 1.099 nm, c = 2.238 nm. Upon further stretching to 300% at 215", the repeat distance was diminished to 1.86 nm. Both polymorphs contain six-fold helices. Conformational and packing analyses with both hexagonal and orthorhombic unit-cells showed that there is parallel packing of the helices, which are right-handed. The mode of transition from one polymorph to the other was discussed. IV. GLYCOSAMINOGLYCANS (AMINO POLYSACCHARIDES) 1. a-Chitinz3

Poly[(1+4-)-p-~-GlcNAcp]

The structures of chitins derived from shrimp and various kinds of crabs are all very similar. For chitin from King crab, the unit cell is orthorhombic, with a = 0.47 nm, b (fiber axis) = 1.05 nm, and c = 1.03 nm. The molecule does not take up water, and the d spacings do not vary on drying or soaking. 2. p-ChitinZ4

PoIY[(1+ ~ ) - ~ - D - G I c N A c ~ ]

The structure of p-chitin from the pogonophore Oligobrachia ivanovi was refined by using X-ray data and a packing of the 2(0.519) chains in a monoclinic unit-cell with a = 0.485 nm, b = 0.926 nm, c = (22) T. L. Bluhm and A. Sarko, Biopolymers, 14, 2639-2643 (1975). (23) B. L. Averbach, Report MITSG 75-17, National Technical Information Service, U. S. Department of Commerce, 1975. (24) K. H. Gardner and J. Blackwell, BiopoZymers, 14, 1581-1595 (1975).

382

P. R. SUNDARARAJAN AND R. H. MARCHESSAULT

1.038 nm, and y = 97.5". The space group is P2,. The analysis indicated "parallel" chain-packing in a sheet arrangement, with an 0-3'---0-5 intramolecular hydrogen-bond (0.275 nm), an N-H---0-7 intrasheet hydrogen-bond (276 pm), and an 0-6'---0-7 intrasheet hydrogen-bond (0.289 nm). The intersheet 0-6-0-7 hydrogen-bond proposed previous1yz5was found to be unacceptable. The R factor is 26.7%. The swelling properties were discussed on the basis of the present structure, and comparison was drawn between the structures of chitin and ~ellulose.'~

3. ChitosanZ3

POly[(~ + ~ ) - P - D - G ~ c N H ~ ~ ]

Crystalline, flake chitosan from King crab exhibited patterns similar to those of chitin, but the 002 peak (b is the fiber axis) shifted from 0.962 nm for chitin to 0.857 nm for flake chitosan. The position of this peak depends on the content of water and is lowered to 0.745 nm on drying at 134". It was postulated that water molecules that enter the lattice are loosely bound between the chains along the 001 direction. 4. Heparinz6

Poly[( 1+4)-a-~-GlcNSO~-p-6SO~--( 1+4)-P-D-GlcpASO3--(1+ ~ ) - C Y - D - G ~ C N S O ~ - ~1+ - ~ S~O )-Q ~-L - (Idop A-2SO3-]

The sodium salt of macromolecular heparin occurs with a periodicity of 1.73 nm if the relative humidity (r.h.) is less than 78%. This

pattern can be indexed with an orthorhombic unit-cell. If the r.h. is increased to 84%, the periodicity decreases, irreversibly, to 1.65 nm. Two geometrically equivalent, triclinic unit-cells were proposed, with a = 1 . 3 7 n m , b = 1 . 1 0 n m , c = 1 . 6 5 n m , a = 116",/3=70",y=123";and w i t h a = 1 . 2 0 n m , b = 1 . 1 0 n m , c = 1 . 6 5 n m , a = 116",/3=9Oo,y=73", with one tetrasaccharide chain segment per unit cell. The dimensions are subject to variations in the r.h. The structure consists of sheets of chains, all at the same relative translation, adjacent sheets being displaced by 0.48 nm along the fiber axis. The merits of the models with "C,(L) and l C 4 ( ~conformations ) for the L-idosyluronic acid residues were discussed. 5. Heparin2'

Poly[( 1~4)-a-D-GlcNS03-p-6S03--( 1-*4)-P-~-GlcpASOa--( ~ + ~ ) - C Y - D - G ~ C N S O ~ - ~1- + ~ 4S )O- a~--~- (Id0p A-2S03-1

(25) N. E. Dweltz, J. R. Colvin, and A. G. McInnes,Can.J. Chem., 46,1513-1521 (1968). (26) I. A. Nieduszynski and E. D. T. Atkins, Ref. 2, pp. 323-334. (27) E. D. T. Atkins and I. A. Nieduszynski, Ado. E x p . Med. Biol., 52, 19-37 (1975).

BIBLIOGRAPHY OF CRYSTAL STRUCTURES

383

At a relative humidity of 80%, the sodium salt of pig-mucosal heparin shows a fiber repeat of 1.65 nm, with a triclinic unit-cell containing one chain. The sodium salt of macromolecular heparin from rat skin, at 78% r-h., showed a repeat of 1.73 nm, and crystallized in an orthorhombic unit-cell. After 24 hours at 84% r.h., the repeat changed to 1.65 nm, the pattern corresponding to that of pigmucosal heparin. The stereochemistry of the chain in terms of the chain repeat and 4, $ maps was discussed. It was suggested that the chemical formula for heparin is poly[( 1+4)-p-D-GlcpA-( 1-4-p-DGlcNAcpl, and a scheme of 5-epimerization of PD-glucuronic acid to a-L-iduronic acid was discussed. 6. Heparan sulfate2*

Poly[(1+4)-a-~-GlcNAcp-GSO,--(1+4)-p-DGlcpA-(~ + ~ ) - ~ - D G ~ c N A c ~ - 1+4)~SO~--( a-L-IdopA]

The sodium salt of heparan sulfate crystallizes in an orthorhombic unit-cell with a = 1.18 nm, b = 1.10 nm, and c = 1.86 nm. The barium and strontium salts exhibit the same value of c. The calcium salt of heparan sulfate (human aorta) crystallizes in a unit cell with a = 1.70 nm, b = 1.27 nm, and c = 1.68 nm. The space group is P2,2,2,. Two parallel-stranded, double helices of opposite polarity pass through the unit cell. Each strand has a pitch of 3.36 nm. The chain conformations were interpreted in terms of a tetrasaccharide having alternating a - ~ (1-4)- and fi-~-(1*4)-linkages, with all the residues in the “C,(D) conformation. 7. HyaluronateZ8

Poly[( 1+4)-P-D-GlcpA-(1*3)-fi-~-GlcNA~p]

Sodium hyaluronate crystallizes with a = b = 1.17 nm, and c = 2.85 nm. The 3(-0.95) helix is favored. The space group is P3,21, with antiparallel chains, and with two hexasaccharide segments per cell. On annealing, the dimensions change to a = b = 1.87nm, and c = 2.85 nm, with six hexasaccharide segments in the unit cell. A new orthorhombic form is found, with a = 3.44 nm, b = 1.17 nm, and c = 2.85 nm. Several two-dimensional packing-arrangements were discussed.

8. HyaluronateZ8

POly[(1+4)-P-~-GlcpA-(1- 3)-p-~-GlcNAcp]

For calcium hyaluronate, at 50 to 60% r.h,, the unit-cell dimensions are a = b = 1.54 nm, and c = 2.85 nm; at 90% r.h., the cell enlarges to (28) J. K. Sheehan, E. D. T. Atkins, and I. A. Nieduszynski,J. Mol. B i d . , 91, 153163 (1975).

384

P. R. SUNDARARAJAN A N D R. H. MARCHESSAULT

b = 1.62 nm, and c = 2.85 nm. After annealing for three weeks under humid conditions, this further changes to a = b = 2.04 nm, and c = 2.85 nm. The 3(-0.95)helix is favored. It was suggested that there is a cooperative interaction between the threefold helices and a network of water molecules that are hydrogen-bonded to one another and to the hyaluronate chains. Various two-dimensional packing-schemes were discussed. a =

9. H yaluronic acidzB

Poly[( 1+4)-P-~-GlcpA-(1-.3)-P-D-GlcNAcp]

Sodium hyaluronate, oriented at 40”and 90% r.h., crystallizes in a trigonal unit-cell with a = b = 1.17 nm, and c = 2.85 nm. Some patterns also showed the presence of a second phase,30having c = 3.39 nm. Two 3(-0.95) helices having an antiparallel arrangement pack in the space group P3z21. In addition, there are about 9 sodium ions, three chloride ions, and 31 to 35 water molecules per unit cell, and one calcium ion for every 9 unit cells. There are two intramolecular hydrogen-bonds, 0-3-0-5’ (0.267 nm), between the residues in (1+3’)-linkage, and 0-4-0-5’ (0.285 nm), between the residues in (1+4’)-linkage. Extensive, interchain hydrogen-bonds through water molecules were found. The acetamido groups are not involved in hydrogen bonding. Each sodium ion is located at the center of a distorted, octahedral, coordination shell involving a carboxylate oxygen atom from one helix, 0-2 (GlcpA) and 0 - 7 (GlcNAcp) from the related helix, and three water molecules. The R factor is 23%. Comparison was made with other polymorphs of h y a l ~ r o n a t e . ~ ~ ~ ~ ~ 10. Hyaluronic acid30

Poly[(1+4)-@~-GlcpA-(1+3)-P-~-GlcNAcp]

Sodium hyaluronate crystallizes in a tetragonal unit-cell, with a = b = 0.989 nm, and c = 3.394 nm. The space group is P432,2. Two 4(-0.85) helices pass through the unit cell, antiparallel to each other. The cell contains 8 sodium ions, and no water molecules. Intrachain hydrogen-bonds of the type NB-H---OA-6(0.278 nm) and OB-4---OA-5 (0.253 nm) were proposed (A = GlcpA, B = GlcNAcp). Adjacent chains along the cell edge are related by hydrogen bonds of the type OB-6---OB-7(0.279 nm) and OB-6---OA-2(0.237 nm). The antiparallel chains are bridged by an OA-3---OA-6(0.259 nm) hydrogen-bond. In addition, octahedrally coordinated sodium ions link the chains through (29) W. T. Winter, P. J. C. Smith, and S. Amott,]. Mol. Biol., 99, 219-235 (1975). (30) J. M. Guss, D. W. L. Hukins, P. J. C. Smith, W. T. Winter, S. Arnott, R. Moorhouse, and D. A. Rees,]. Mol. Biol., 95,359-384 (1975).

BIBLIOGRAPHY OF CRYSTAL STRUCTURES

385

O---Na+---0 bridges. No double-helix model, as originally proposed for this structure, has been found to be in acceptable agreement with the observed data. The R factor is 29%. At higher r.h., for example, 75%, only the a dimension increases, so that the new unit cell has a = 1.153 nm, b = 0.989 nm, and c = 3.386 nm. The space group in this case is P212121.The chain is a 2(1.693) helix, with tetrasaccharide repeat-units. The intrachain hydrogen-bonds are 0$-4---0;4+1-5 (0.269 nm), NE1---Ok1-6(0.257 nm), og14---Of-5(0.292 nm), and Np---0:-6 (0.295 nm). Whereas the OB6---OA-2interchain hydrogen-bond and those between the corner and central chains are the same as in the tetragonal form, the other interchain hydrogen-bonds and the Na---0 bonding are replaced by hydrogen bonds by way of water molecules, which form extensive interchain bridges. The R factor is 29%. A third crystalline form, from rooster-comb sodium hyaluronate, at high r,h., also crystallizes with a = 1.162 nm, b = 0.984 nm, and c = 3.331 nm. Meridional reflections are on every layer line, indicating a perturbation of the 4-fold helix symmetry.

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AUTHOR INDEX FOR VOLUME 35 Numbers in parentheses are reference numbers and indicate that an author’s work is referred to although his name is not cited in the text. A

Abe, Y., 154 Achenbach, H., 89,92,122 Acree, T. E., 33 Adair, W. L., 275,276(666), 322(666), 323, 324, 331(666) Adams, G. A., 349 Adams, M., 372(299),373 Agrawal, B. B. L., 137, 138(134, 135), 140(135),151(134,135), 152(261, 262), 153, 155(262),162, 171(134, 135),179(135),335 Agrawal, K. M. L., 148, 363, 365(233), 370(233),372(235) Ahlgren, E., 366 Ahmad, A., 177, 272, 275(659), 276, 277(669) Ahmed, A. I., 269 Ahmed, 2.F., 344,347 Aida, K., 307,308(778) Ainsworth, C. F., 151, 152(262a), 154(262a),156, 159, 160, 161, 335(262a, 324) Akedo, H., 157, 160,324 Akiya, S., 250,251(594), 253(594) Akiyama, Y.,300,312, 324(740, 741) Alam, M., 345 Albarsheim, P., 147 Alberto, B. P., 172 Allan, D., 294,325 Allen, A. K., 136, 143(128),144(128, 207), 195, 199(213),200(213), 203(213),204(213),205(213),211, 212(207), 213,221, 233,309(213), 336(213),337(128, 207, 213), 338(213),339(128, 207, 213) Allen, H. J., 138, 203, 205, 337(467a) Allen, L. W., 293,294(708, 714), 295(708),335(708, 714) Allen, N. K., 132 Alter, G. M., 156, 158, 161(310) Alving, C. R., 219 Alzer, G., 299,301(735), 302(736)

Amagaeva, A. A., 52, 54(71), 56(71), 57(73), 80(71, 73) Ambrose, A. S., 376 Amen, K.-L., 70 Anagnostopoulos, C., 348, 363(117) Andersen, B. R., 178 Anderson, A. J,, 147 Anderson, B., 253 Anderson, E., 344,348(59) Anderson, L., 102 Anderson, R. L., 232 Andoh, T., 346,367,370(265) Andrews, A. T., 130 Andrews, E. P., 319 Andrews, P., 348(92, 93), 352(92) Ansell, N., 279, 280(673) Anstee, D. J., 131 Aranda, L. H., 132 Archibald, A. R., 176 Amott, S., 384 Aro, H., 294, 295(711), 335(711) Asai, M., 96 Ashwell, G., 131 Aspberg, K., 137, 138(137), 304(137), 306(145) Aspinall, G. O., 343,345,346(85), 347(85), 371 Atkins, E. D. T., 377,379, 382, 383(26), 384(28) Atwood, K. C., 244 Aub, J. C., 130,317(28,29) Auger, J., 325 Aull, F., 139,306(155), 307(155),329, 330(868) Averbach, B. L., 381 Avrameas, S., 139,317 Axelsson, K., 282,291(682) Azavache, V., 166 Azuma, I., 376

B Babczinski, P., 355 Bachhawat, B. K., 141, 168(193), 177,

387

388

AUTHOR INDEX, VOLUME 35

179(193),272,274(193), 275(659), 276,277(669) Bachur, N. R., 138, 296(154),301 Baddiley, J., 98, 99(37), 354 Baenziger, J., 186, 298(432),300(432), 332 Bagchi, P., 343,346(53), 348(53) Bahl, 0. P., 148, 363, 365(233), 370(233), 372(235) Bailey, P. J., 366 Bailey, R. W., 172,343, 345, 356(57), 363(57) Bains, G., 343 Bajpai, K., 108 Baker, B. R., 58 Baker, C. W., 349,350(128) Baker, E. A., 363 Balasubramanian, K. A., 177 Balding, P., 132,250, 252, 253, 254(599), 316, 339(599) Baldo, B. A., 282, 314, 315(800, 807,808) Balint, G. A,, 270 Ballantyne, M. J., 348 Ballas, A., 292 Ballou, C. E., 354 Bamberger, E. S., 363 Banks, J. R., 219 Barber, B. H., 156, 158, 159(311), 160 Barber, G. A., 353 Barker, B. E., 309,311 Barker, R., 136,337(130) Barker, S. A., 49, 63(64),65, 172, 354 Barham, D., 344,345(58) Barkhan, P., 292 Barlow, C. B., 67, 102 Baron, D., 101 Baron, J. M., 294(714),295,335(714) Barondes, S. H., 139, 290(158), 306(159), 307(159),308,309(779,780), 325(159), 336(158),337(158) Barr, R. M., 355 Barrett, J. T., 131 Bartholemew, B. A., 372,373(284,290) Barton, L., 39 Basarab, O., 354 Basham, T. Y., 310 Basu, S., 264 Batrakov, S. G., 68 Bauer, H., 174, 175(388) 178 Bauer, Baues, R. J., l37,262(149b)

s.,

Bausch, J. N., 267, 333 Bazhanova, E. T., 54, 55(77), 80(i'7) Beattie, G., 318 Beaugiraud, S., 367,372(260) Beck, M. L., 206,338(470) Beck, S., 291 Becker, J. W., 137, 138(140), 152, 153(325), 154(268,269, 275, 276), 155(267), 156(268,275), 157(267, 268), 158(267,268, 280), 161(280), 202(140), 203(140), 205(140), 304(276), 335(267),336(140), 337(140), 339(140) Beckman, L., 300 Beevers, H., 149 Behrens, N. H., 354 Beitsch, D. D., 315 Ben-Bassat, H., 150 Benz, G., 342 Beppu, M., 162, 163(332),165(332) Bergel'son, L. D., 68 Bernadac, A., 165 Bernhard, W., 317 Bessler, W., 140, 159(174), 160, 164, 178, 179(174),228(410),246(410), 247(410), 248(342),340(589) BBtail, G., 179, 197, 199(428),210, 339(428) Bettman, B., 36 Beveridge, R. J., 347 Bewley, J. D., 366,370(254), 371(254) Beychok, S., 140, 160, 166(170), 168(319), 176, 179(170), 180(170), 181(170), 184(170),338(170) Bezer, A. E., 176,270,271(644) Bezkorovainy, A., 281,282(679), 335(679) Bhatia, D., 343 Bhatia, H. M., 239,244,245,246 Bhavanandan, V. P., 219 Bielskis, E., 341 Biely, P., 178 Biemann, K., 47,48(56), 55(56), 65(56), 67(56), 68,69(56) Bierry, H., 366 Bird, G. W. G., 129, 132(10), 134, 140(10, 104), 143, 144(206),202(68), 210, 211(68, 206), 212(206), 213(206), 218, 224(502), 226(508d), 227, 257, 258(201), 258(201),267, 268(632), 269(632), 276(104, 119, 206), 297(632), 300(632),307, 311(132,

AUTHOR INDEX, VOLUME 35 510),333(632),338(201,510,511,512, 616, 617), 339(206, 632) Birdsell, D. C., 162, 177(330) Bishayee, S., 177 Bishop, C. T., 353 Bittiger, H., 150 Bjork, I., 240,241(569), 243(569), 335(569) Bjorndal, H., 282, 291(682), 349 Black, M., 360 Black, N. H., 342 Blackwell, J., 379, 381, 382(17) Blake, C. C. F., 221 Blaskovic, D., 376 Blaustein, J., 137, 138(146), 142, 144(146),270(146, 194), 271(146, 194), 272(146),273(146), 274(146), 293(194), 3 17(194), 336(146), 337(146, I%), 339(146, 1%) Bloch, R., 137, 138(133),214(133), 215(133), 224(133), 313 Bluhm, T. L., 381 Blumberg, M., 233,234(541), 283,284, 335(684) Blumson, N. L., 98,99(37) Boeseken, J., 33 Bohlool, B. B., 147 Boldt, D. H., 219 Bomchil, G., 132 Bonner, T. G., 77(117, 118, 119, 120), 78 Borberg, H., 297 Borecky, L., 376 Borjeson, J., 139,292,309,310 Borodulina-Shvetz, V. I., 38, 51(28), 54(28), 80(28) Bosmann, H. B., 372(295), 373 Bottger, M., 239,240(563) Bouchard, P., 214,215(490b), 219(494), 222(494), 223(494) Boundy, J. A., 168, 174(363) Bourne, E. J., 35, 38,42(23), 45, 50(23), 51(23), 53(23,48),54(23), 55(23, 48), 57(23), 58, 60(86), 72(23,48), 77(23, 117, 118, 119, 120, 121), 78(23), 172 Bourquelot, E., 370 Bourrillon, R., 139, 145,227,228(518), 230, 231(523),298, 303, 311(160, 523, 731), 320(731), 322(731), 331(731),335(519), 337(220) Bouveng, R., 292

389

Bowie, R. A., 34,35(13),42(13), 78(13) Bowles, D. J., 139 Bowman, C. M., 32, 35(9), 36(9), 38(9), 41(9), 50(9),52(9), 77(9),78(9) Boyd, W. C., 128, 129, 131, 132(2, 5), 134(62), 140, 147(2,4),202, 224, 226, 239(62), 243, 244(2, 3, 5), 245(575), 246,248(103), 253,258,259(614), 277(13), 289(12, 13), 313, 339(2, 5, 12, 13) Bragg, P. D., 349 Branch, G. E. K., 36 Branstrator, M. L., 231 Braun, C., 250,253(593), 339(593) Bretscher, M. S., 323 Bretthauer, R. K., 354 Bretting, H., 315,316 Brewer, C. F., 155, 156, 157, 158, 161(307,308), 163, 179(308), 184(429),201(308) Brill, W. J., 148 Brilliantine, L., 132 Brillinger, G. U., 83, 84(8) Brimacoinbe, J. S., 48, 52(58), 54(58), 55(60), 57(58), 74(58, 59, 60), 75(58), 85, 89, 95(13) Brodie, G. N., 141 Brown, H. C., 58 Brown, J. C., 329 Brown, J. M., 224 Brown, M. C., 303, 304(762), 330(762), 33l(762) Brown, R., 131, 134(62),239(62) Brown R. D., 111, 156, 157, 179, 184(429) Brownhill, L. E., 309 Browning, W. C., 342 Bruce, G. T., 172 Bruce, R. M., 107 Bruce, W. R., 130, 150(33) Briicher, O., 297 Brunngraber, E. G., 326 Buchala, A. J., 345 Bullis, C. M., 329 Buonassisi, V., 168, 301(366a) BureS, L., 193, 198, 201(446) Burger, M. M., 130, 132, 133, 136, 137, 138(133), 165,212(129), 214(129, 133), 215(129, 133,487), 216,218, 219,224(133), 313,317(30,492), 318(92a), 328, 329(865, 866), 337(129), 339(30), 340(129)

390

AUTHOR INDEX, VOLUME 35

Burke, G. C., 296 Burton, H., 341(9), 342 Bush, D. A., 174, 175(388) Butte, J. C., 122 Bywater, R., 138

C Cabib, E., 354 Cacan, M., 234,235(546), 236, 335(546) Cacan, R., 234,235(546), 236,335(546) Caccam, J. F., 355 Callies, Q. C., 178 Callow, J. A., 132 Caprioli, R. M., 58 Carbonell, L. M., 376 Carchon, H. A., 140, 195(182),197(182), 199(182),200(182) Carlson, W. A,, 341(11),342 Carlsson, H. E., 222 Carter, W. A., 141, 168(192), 178(192), 179(192,418) Carter, W. G., 228,229(520, 521), 234, 235(546),335(520, 521, 546) Carver, J. P., 156, 158, 159(311),160 Catsimpoolas, N., 233 Cawley, L. P., 148,267, 268, 269(635), 339(631,635) Cazal, P., 132,222, 284(81), 289, 339(81) Celano, M. J., 302,339(750) Cerezo, A. S., 344,346(67,68), 348(67, 68), 351(67) Cermakova, M., 199 Chabanier, A. M., 197 Chandra, G. R., 360 Chanzy, H., 377,378 Chapman, H. R., 341(9), 342 Charpentier, M., 367 Chase, P. S., 178 Chassy, B. M., 123 Chattoraj, A,, 244 Chauser, E. G., 38,45(24), 54(24),72(24), 80

Chen, C.-C., 130,254(32), 271(32) Chen, S. S. C., 371 Cherneva, E. P., 55 Chessin, L. N., 139,309(162), 310 Cheung, G., 312 Chien, S. M., 140,251(175),252(184), 253(184),337(184), 339(175, 184, 601)

Child, T. F., 349 Chin, P. S., 48, 54(61),62(61), 79(61) Chittenden, G. J. F., 354 Chiu, T. H., 355 Chopra, A. K., 49 Choudhury, D., 343,346(53), 348(53) Chowdhury, T. K., 132, 139(94) Choy, Y. M., 344, 345(65),346(65), 350(91), 351(91) Chrisp, D. J., 342 Christensen, T. B., 270, 271(651), 272(651),335(651),336(651), 337(651) Christie, D. J., 156 Christner, J. E., 224 Chuba, J. V., 268(637),269, 291, 339(637) Chudzikowski, R. J., 342 Cifonelli, J. A., 134, 140(117),151, 166(117),168(353,354, 355), 169(353, 355), 171(260,355), 173(353),179(117),187(422) Clardy, J., 96 Clark, A. E., 134, 135(116), 139(116),177 Clegg, R. M., 216 Clermont, S . , 360 Clermont-Beaugiraud, S., 366,367(255), 368 Cline, M. J., 317 Closs, O., 276 Coapes, H. E., 176 Coats, J. H., 119 Codington, J. F., 275, 303, 304(762),330, 331 Cohen, D., 360 Cohen, E., 132, 150(132),307 Colburn, P., 168,301(366a) Colvin, J. R., 382 Conard, R. A., 294,335(710), 338(710) Conchi, J., 372(293,297), 373 Condos, R. G., 111 Connett, S. L., 131,308(51) Conrad, H. E., 349 Cooper, A. G., 303,304(762), 330(762), 331(762) Cooper, D. J., 110 Cooper, F. P., 353 Cooper, H. L., 309 Corcoran, J. W., 84, 122 Cote, M.N., 130,317(29) Cottom, G. L., 156

AUTHOR INDEX, VOLUME 35

Coulet, M., 131, 179, 197, 199(428),210, 339(428) Courtois, J. E., 344, 345, 347, 348(72, 107), 350(72, 107), 351(72, 107), 352, 353(145), 356(81), 360(145), 361, 363(81, 108, 117), 364(107, 145), 366(259), 367(110, 112), 369(110, 112), 372,375(145, 146) Cragg, R. H., 70 Creger, W. P., 202,339(462) Cruickshank, C. N. D., 354 Crumpton, M. J., 193, 294, 325(445), 335(709) Cuatrecasas, P., 130, 219, 250(38) Cunningham, B. A., 151, 152, 153(325), 154(263,268,269, 275), 155(263, 267, 270, 271,289), 156(268, 275), 157(267,268), 158(267, 268), 160(289), 161, 162, 165(336), 203(263), 335(271) Cybulska, E. B., 174 Cyr, M. J. S., 168, 175(364), 184(364) Czech, M. P., 130, 150(39) Czonka, F. A., 155

D Dahlgren, K., 297 Dahlhoff, W. V., 36,39,40(17), 41(33a, 41,42), 43(39), 46(33a), 47(33a), 48(33a), 52, 53(41), 58(17), 66(40), 70(17,33a, 40,41,42), 71(17, 33a, 40,41,42), 74(33a), 75(33a), 76(33a), 77(42), 78(40,41) Dahr, W., 259,303,304(761), 305(761), 306(761), 338(617, 618) Dale, J., 33,35 Daniel, T. M., 179, 184(424) Daniels, P. J. L., 110, 111 Dankert, M., 355 Danon, D., 143, 258(202), 259(202), 261(202), 335(202), 338(202) Daoud, K. M., 348 Davey, M. W., 141, 168(192), 178(192), 179(192,418) Davidson, E. A., 219 Davies, C., 356(204),357, 366(204), 370(204), 371(204) Davies, D. R., 358 Dazzo, F., 147, 148 Dea, I. C. M., 343,345, 347(43), 349,

391

350(43), 356(43), 363(43), 364(43), 369, 375(43) Dean, B. R., 162 Debray, H., 234, 235(546), 236, 335(546) De Bruyne, C. K., 134(124), 135, 140(124), 145, 160, 188(181, 323), 189(181,323), 195(182), 197(182), 199(182,211),200(182, 211), 216, 273(124), 274(124),339(124,211), 340(124), 376 Dechary, J. M., 132 De Fekete, M. A. R., 358 Degand, P., 270, 271(650), 272(650), 337(650) De Gussem, R., 140, 188(181), 189(181) Dekker, R. F. H., 366, 367(243), 368(243), 369(243) de la Chapelle, A., 292, 293(703) Delmotte, F., 134, 135(115),211, 212(483), 213(483),215, 216(115), 217(115), 219(115,494,497), 222(115,494), 223(494), 339(483), 340(115) de Maelenaere, H . J. H., 130, 150(41), 232 Demain, A. L., 104, 106, 108(50) Den, H., 131 Denborough, M. A., 177 De Neve, R., 191, 198(442b) Desai, N. N., 144, 195(213), 199(213), 200(213), 203(213), 204(213), 205(213), 309(213),336(213), 337(213), 338(213),339(213) Desai, P. R., 131,227,278(674, 676), 279, 280(66,671, 674, 676), 281(66, 671, 677), 282(679, 680), 284(674), 303(678), 304(678),318(678), 338(674,676) Deuel, H., 64,348 de Waard, A., 131 Dey, P. M., 344, 345(58), 350,351(139), 362, 363(233),365(139, 223), 366(223,224), 367(139), 368(139, 140), 369(139, 140) Diamond, M. A., 246 Di Ferrante, N., 168 Di Girolamo, A., 98 Di Girolamo, M., 98 Dillner, M.-L., 243 Dixon, M., 148 Dixon, T., 270

392

AUTHOR INDEX, VOLUME 35

Dodd, R. Y.,315 Dolhun, J. J., 38,68(29) Dong, T., 366(259),367 Donnelly, A. J., 291 Donnelly, E. H., 173 Dorset, M., 129,291 Doty, D. M., 362,370(220) Douglas, S. D., 139, 309(162) Doyle, R. J., 156, 159, 160(297),161, 162, 165, 168, 177(330),179(366) Drabik, J. S., 258, 259(614) Drewes, L. R., 354 Duke, J., 173, 179, 182(381),187(422) Durand, R., l49,297(243e) Durso, D. F., 347 Dutcher, J. D., 81 Dweltz, N. E., 382 Dyckes, D. F., 190, 191(441),192(441), 193,335(441) Dymova, S. F., 38,42(26),44(26), 70(26), 72(26), 73(26),78(26)

E Eagles, J., 48 Ehisu, S., 206, 208, 224 Ebisu, T., 262, 266(621) Eckhardt, A. E., 266,295(629) Edelev, M. G., 44,45(45), 70(45) Edelman, G. M., 137, 138(140),139, 151, 152, 153(325),154(263,268,269, 275, 276), 155(263,267, 270,271, 280,289), 158(267,),160(283,289), 161(280),162, 163(333),165(333, 336), 202(140), 203(140,263), 205(140),224(156),296(156), 304(276),306, 307(769),335(267, 271, 769), 336(140), 337(140), 339(140) Edmundson, A. B., 151, 152(264) Edwards, J. O., 32,42(6), 49(6) Egami, F., 372 Eginitis-Rigas, C., 293, 335(706) Egorin, M. J., 296 Eguchi, Y., 86, 87(16),88(16) Ehrlich, P., 128, 129 Eichmann, K., 314, 315(808) Einstein, J. R., 255,256, 257(610), 334(610),335(610) Eisenherg, F., 65

Ekstedt, R. D., 176 Elbein, A. D., 353 El Khadem, H. S., 343,346(54) Ellestad, G. A., 89,91(21) Elting, K. A., 342 Ely, K. R., 151, 152(264) Emi, S., 366,367(251) Emoto, S., 74(111),77 Engvall, E., 178, 301(413),330(413) Ensgraher, A., 147, 148(228) Entlicher, G., 134(122),135, 137, 138(141, 142), 141, 155, 190(142), 191(142,442), 192(442),193(142), 194(440,442), 196(141),197(141, 440), 198(454,455), 199(122,440), 201(122,446), 321(185),322(185), 325(844),326, 335(141, 142,442), 337(141,454,455), 388(142), 339(122, 141,440) Eoff, W. E., 363,370(220) Eriksson, K. E., 366 Eriksson-Quensel, I.-B., 151, 152(257, 258), 335(258) Ermishkina, S. A., 38, 53(30), 54(30), 80(30) Erskine, A. J., 346 Ersson, B., 137, 306 Eshimoto, N., 108 Estola, E., 131 Eto, E., 108 Etzler, M. E., 134, 137, 138(108, 146), 140(108),142(108), 144(108, 146), 149, 162,215, 223(335),227(108), 228,229(520, 521), 231, 249, 270(146),271(146),272(146), 273(146), 274(146), 335(108, 520, 521), 336(146), 337(146, 335,495), 338(108), 339(146) Evans, P. J., 355 Evans, P. M., 165 Evans, V. J., 317 Evdokimova, G. S., 54, 55(77),80(77) Everhart, D. L., 147,288 Eylar, E. H., 355

F Fahn,A., 356 Fairbanks, G., 323 Fanger, H., 309

AUTHOR INDEX, VOLUME 35

Farkas, E. H., 341 FarkaS, J., 75(112, 113),76(114), 77 Farmer, P. B., 123, 124(86) Farnes, P., 309, 311 Fath, J., 342 Feeney, R. F., 268(637),269, 339(637) Feizi, T., 253, 285, 288(686) Feldman, J. D., 267,268(633) Felsted, R. L., 138, 296(154), 301 Fennessey, P., 355 Feniindez-Moran, H., 306 Ferrier, R. J., 32, 37,38(3), 46(22), 47(3), 48(3), 50(55), 51, 52(3), 53(3, 22, 51, 55, 69), 54(22, 51, 69), 55(22, 52), 57(3,22, 55), 58(69), 62, 73(69, 70), 74(3, 51,55), 75(3, 51, 55), 76(22, 52, 69, 115, 116), 77, 79(3, 55) Ferris, B., 325 Ferris, C., 178, 179(416), 186(416), 187, 312(416) FialovB, D., 193, 194(447) Filippova, T. M., 44,45(45), 70(45) Finch, A., 34, 35(11) Findlay, J. B. C., 323,324 Finstad, C. L., 306,307(771), 309(771), 335(771) Fishel, C. W., 168, 179(366) Fliegerovi, O., 192,335(444) Foglietti, M. J., 360, 362, 375 Folling, I., 276 Font, J., 139, 227, 228(518), 311(160), 335(519) Fontand, J., 227 Ford, W. W., 131 Fordom, M. T., 366 Foriers, A., 191, 198(442a,442b, 442c), 235(442c) Fornstedt, N., 311 Foster, A. B., 33,45, 50, 51(67), 53(67), 54(67), 56(67), 70, 72(49), 77(67), 78(67) Foster, D. M., 132 Fountain, D. W., 239 Franke, W. W., 380 Franks, D., 218, 339(501) Fraser, A. R., 162, 163(333), 165(333) Fraser-Reid, B., 96 Frkchet, J. M. J., 53 Freedman, S. O., 330 French, A. D., 378 French, D., 363

393

Frerman, F. E., 354,355 Fridman, C., 232, 235(539) Fried, W., 104 Friedman, B. A., 263 Frost, R. G., 290, 336(691), 337(691), 339(691) Fmmin, A. M., 202, 203, 205(468,469), 339(468,469) Fuhr, B. J., 156, 158, 159(311) Fujinaga, D. M., 258,259(614) Fujita, T., 189 Fujita, Y.,131,307(52),308(52, 778) Fukuda, M., 178, 187,297(417), 300(417), 301(417),304(417), 319, 323,324,374 Fukumoto, J., 366,367(251), 372(298), 373,374(298) Funabashi, M., 85, 95(14) Funatsu, G., 270, 271(642), 272(642), 339(641, 642) Funatsu, M., 270,271(642), 272(642), 339(641, 642) Furihata, K., 124 Furthmayr, H., 323,324(845)

G Gabriel, O., 89, 99(17), lOO(17) Gachelin, G., 156 Gaillard, B. D. E., 345 Galbraith, W., 138, 142, 144(199), 164(199), 235(151),246(151, 199), 247(151, 199), 248(151, 199), 249(199), 295(151),335(151, 199), 339(151, 199) Galun, E., 147 Gander, J. E., 354 Gantt, R. R., 317 Gardell, S., 292 Gardner, D. A., 264 Gardner, K. H., 381 Gardner, P. J., 34,35(11) Garegg, P. J., 62, 71 Garrido, J., 317 Gatt, S., 363 Gaugler, R. W., 89, 99(17), 100(17) Gebb, C., 101 Gellhorn, E., 139 Genaud, L., 179, 199(428),339(428) Genin, A., 370

AUTHOR INDEX, VOLUME 35

394

Gepner, I. A., 165 Gerrard, W., 32 Geserick, G., 131 Gesner, B., 297 Giaja, J., 366 Gibson, W. A., 317 Gielen, W., 218,239 Gifford, H., 202,339(462) Gilboa-Garber, N., 131 Gilham, P. T., 63 Gilliam, E. B., 327,328(862, 863) Gillies, D. G., 78 Ginsburg, V., 100 Glaser, C., 139 Glaser, L., 89,99(18),100(18) Glaudemans, C. P. J., 317 Glew, R. H., 156,160(297),163 Glicksman, M., 341(10),342 Goddard, V. R., 292 Goffart, P. R., 342 Gold, E. R., 132,250,252, 253,254(599),

315,316,339(599) Gold, P., 330 Goldberg, A. R., 130,214,218,219,

317(30),339(30) Goldberg, M. L., 296 Goldman, D. S.,355 Goldstein, A. W., 118,119(77) Goldstein, I. J., 129,134(121),135(112,

113,120),136,137(128),138(125, 134,155),140(113,120,121,135), 141,142(125,131,168,197), 143(131,168,169),144(131,168, 169,199),146(131),150(121,168), 151(120,121,134,135,168), 152(261,262),153(172),154, 155(173,262), 156, 157(180), 159(173,174),160(173,174),161, 162(326),163(329),164(173,199, 339),165(173,204,290),166(121, 320),167(120,121,168,197,204, 359),168(120,204,209, 215,240, 320,359),169(320),170(359), 171(120,121,134,135,359,369), 172(320,362),173(121,320,362, 369),174(120,204,365), 175(204, 215,320),178(240),179(112,113, 120,121,126,135,168,169,173, 179,197,204,209, 215,240), 180(168,169,197,204,215,365), 181(118,169,197,204,215,240,

365),182(169,209, 240, 365,381), 183(197,240,365), 184(120,168, 169,197,209,215,240,423,430), 185(168,197,204,240,365,430), 186(209,240,431), 187(197,209, 422,430),188(180,365),189(113, 180,436),190(204),200(169,197, 240, 365),206(125),207(125),208, 212(204),215(131),216,217(498), 218(498),219(498),222(498),224, 228(410),235(151),246(151,199, 410),247(151,199,240,410), 248(151,199,342),249(199,587, 591),262(125),263(132,622), 264(126,131,625), 265(125), 266(191,621),295(157,628,629), 30l(413),322(209),330(413), 335(125,131,151,199,261,262), 338(125,131,168,169,204,215, 365,470,622,625,626), 339(151, 199,498), 340(589,626) Goldstein, L., 156 Goldwasser, S.M., 132,313(85) Gombos, G., 326,327(857) Gonatas, N. K., 317 Gonzales, J. I., 342 GonzPlez, D . I., 178 Gonzalez-Porque, P., 95 Good, R. A., 306,307(771),309(771), 335(771) Gordon, J. A., 133,140,233,234,238(99) Gorin, P. A. J., 71,143,168(209), 179(209),182(209),184(209), 186(209),187(209),322(209),343, 354(41) Goring, H., 360 Gottlieb, D., 104,107(54),108(54) Gould, N.R., 246,247(586),335(586, 588) Goussault, Y., 311 Graham, V. A., 151 GralBn, N., 151,152(257,258),335(258) Grasbeck, R., 292,293(703,704), 294(716),310(703),335(716,718) Gray, D., 163 Gray, G. R., 137,262(149b) Gray, R. D., 156,160(297) Green, D. M., 258,259(614) Green, J. W., 33 Greenaway, P. J., 219 Greer, J., 152,154(274a)

AUTHOR INDEX, VOLUME 35 Gregory, W. T., 187, 298, 301(733), 320(434),331(434) Griffiths, D., 344, 345(58) Grimaldi, J. J., 163, 164 Grimmett, M. R., 347 Grisebach, H., 82,83(4, 5, 6), 84,89, 90(24), 91(24), 92, 95(28), 98(4, 6), 99, 100(5), 101, 102, 109(40), 122 Grollman, A. P., 155, 158, 161(307,308), 163, 179(308),201(308) Gross, R., 299,307(735) Grubb, R., 131,277(65), 338(65) Grundbacher, F. J., 208,288 Griiss, J., 370 Gubanski, M., 376 Giirtler, L. G., 270,271(648), 272, 336(648), 337(648) Guilbert, B., 139 Guillot, J,, 131, 197 Gul, B., 254 Gumpf, D., 314 Gunja Smith, Z. H., 166, 167(359), 168(359), 170(359), 171(359) Gunther, G. R., 152, 154(268), 156(268), 157(268), 158(268),162, 165(336) Gupta, D. S., 347 Gupta, P. C., 343, 344,345(45) Curd, J. W., 326 Guseva, A. S., 38, 39,42(26), 44(26), 45(45), 51(36), 53(36), 54(46), 56(46), 70(26,45), 72(26,46), 73(26), 80(36) Guss, J. M., 384 Gussin, A. E. S., 214, 215(491) Guthrie, R. D., 67, 110 Gutz, C. G., 156 Gyaw, M. O., 353

H Haak, W. J., 104, 107(53), 115(53), 116(53), 117(53), 119 Haddock, J. W., 354 Haglid, K. G., 326 Hague, D. R., 157 Haines, A. H., 50, 51(67). 53(67), 54(67), 56(67), 77(50), 78(50) Hakomori, S., 216, 217(499), 315 Hall, J. L., 313 Hall, L. D., 70 Hall, S . , 347

395

Halmer, P., 366, 370(254), 371(254) Halpern, B., 307 Halprin, K. M., 372(296), 373 Hamblin, J., 147 Hammarstrom, S., 131, 134(63), 138(63), 140, 141, 144(63),154, 165(290), 178, 216, 217(498),218(498), 219(498), 222(498), 239(63), 240(63, 561,562), 241(63, 561, 562, 564), 242(63, 178, 562, 563), 243(63, 189, 569), 249, 282, 291(682),301(413), 330(1), 335(562, 569, 570), 338(63, 189, 562, 563), 339(498),340(570) Hannaford, A. J., 47, 50, 53(55, 68), 57(53), 74(53), 75(53), 79(53) Hannig, K., 297 Hansch, C., 189 Hara, K., 271 Hara, M., 366 Haratz, A., 312 Harboe, M., 276 Hardman, K. D., 151, 152(262a), 154(262a, 274), 156, 159, 160, 161(274),335(262a, 324) Harper, A. A., 271,272(658) Harris, H., 134, 177(106) Hartigay, B., 35 Hartman, F. C., 255, 334(607), 335(607) Hanvood, S. E., 77(119, 120), 78 Hashem, N., 202 Hashimoto, Y.,366, 367,372(298), 373, 374(298) Haikovec, C., 325,326(851) Hassid, W. Z., 353,355 Hassing, G. S., 140, 155(173), 159(173), 160(173), 161, 162(326), 163(329), 164(173), 165(173),179(173) Hatano, M., 366 Hatt, B. W., 49,63(64), 65(64) Hatton, L. R., 51, 73(70), 76(115, 116), 77 Hawkins, D. C., 315 Hay, A. J., 372(297),373 Hayashi, J., 380 Hayashi, K., 270, 271(642), 272(642), 339(642) Hayes, C. E., 136, 141, 142(131), 143(131), 144(131), 146(131), 148(131),215(131), 263(131), 264(131,625), 266(191), 295(629), 335(131), 338(131, 625, 626), 340(626)

396

AUTHOR INDEX, VOLUME 35

Hayman, M. J., 193, 325(445) Head, C., 293,335(705, 706) Healey, P. L., 356 Heath, E. C., 354,355 Heath, M. F., 211 Heding, H., 108 Hedrick, J. L., 97 Heggen, M., 267,268(632), 269(632), 297(632), 300(632),333(632), 339(632) Hehre, E. J., 150, 151(253), 168(253), 171(253),173(371), 186(253,371, 372) Heidelberger, M., 270, 271(644),376 Heinrikson, R., 267,294(630), 295(630, 712), 335(630,712) Hellin, H., 254, 259(604) Hellstrom, U., 243 Helman, J. R., 177 Hemperly, J. J., 162, 163(333), 165(333) Hems, R., 45, 70, 72(49) Henderson, M. E., 352 Henley, R. R., 129,291 HBrissey, H., 370 Hermetet, J. C., 326 Herth, W., 380 Heyne, E., 348 Hicklin, B. L., 206, 338(470) Hickman, S., 131 Higashi, Y.,355 Higuchi, T., 355 Hildesheim, J., 283,284(684), 335(684) Hill, R. L., 136, 337(130) Hirschhom, R., 297 Hirschmann, W. D., 299,301(735), 302(736) Hirst, E. L., 343, 347, 348(100), 351(100), 352(100) Hitchcock, C., 347 Hitz, W. D., 58 Hiyama, K., 367 Hoglund, S., 297 Hoeksema, H., 119(80), 120 Hof, H. I., 326 Hoffman, J., 349 Hoffman, M. K., 58 Hoffmann-Ostenhof, O., 104 Hofheinz, W., 84, 122 Hofnung, D., 156 Hogenkamp, H. P. C., 123, 124(86) Holland, N. H., 300

Holland, P., 300 Hollerman, C. E., 134(121),135, 140(121), 142(168),143(168), 150(121, 168),151(121, 168), 166(121), 167(121, 168), 168(121), 169(121), 171(121),173(121), 179(121, 168), 180(168),181(168), 184(168), 185(168),338(168) Holmen, H., 137, 138(137), 304(137) Holt, P. D., 131 Homer, B. R., 162 Horecker, B. L., 355 HoiejHi, V., 135, 136, 137, 289, 290, 291(690), 333, 336(690), 337(690), 339(690) Horii, S., 120, 122(81) Horisberger, M., 138, 174, 175(388), 262(150c), 264 Homer, V. V., 342 Hornick, C. L., 141 Horowitz, P., 163 Horstmann, H. J.,270,271(648), 272, 336(648), 337(648) Horton, C. B., 148, 149(243) Horvei, K. F., 345,348(86) Hossaini, A. A., 132 Hotta, K., 302,339(757) Hough, L., 346,348(92, 93), 349, 352(92) Houston, C. W., 372(302),373 Howard, I. K., 134(123),135, 137, 138(138), 141, 142(123), 144(123), 148, 149(243),190(138), 191(441), 192(441), 193(138,441), 194(123, 138, 186), 195(125),202(138), 335(138,441),338(125, 138) Howe, M. L., 131 Howell, S. F., 134, 136(102), 140(102), 150, 151, 155, 166(102), 168(102), 169(102), 173(102), 177(102), 179(102), 338(102,246, 247) Howes, F. N., 356 Hiebabeckf, H., 75(112),77 Hrgovcic, R., 168 Hsu, R., 267, 294(630),295(630), 335(630) Huang, A. H. C., 149 Huang, J. W., 141, 168(192), 178(192), 179(192) Hubbard, A., 325 Hubbell, D., 147, 148 Hubbell, W., 318

AUTHOR INDEX, VOLUME 35 Huber, G., 348 Hubert, A. J., 35 Hudgin, R. L., 131 Hudson, C. S., 372(299),373 Huet, C., 165,317 Huet, M.,152,165 Hui, P. A., 347,348,350(109,120),363 Hukins, D. W. L., 377,384 Hunedy, F., 48,52(58),54(58), 57(58),

74(58,59),75(58) Hungerford, D.A., 291 Hunt, R. C., 329 Huprikar, S. V., 145,196,197(448),

303(219),339(448) Husain, A., 48,52(58),54(58),57(58),

74(58,59),75(58) Hutson, D. H., 172 Hybl, A., 378 Hylin, J. W., 361,370(219), 374 I Ichihara, N., 108 Ichiki, N.,137,138(144),254(144),

270(144),271(144) Ikuma, H., 371 Il’ina, E. F., 68 Imahori, K., 131,307(52),308(52) Inamine, E., 104, lOS(50) Inbar, M.,130,150(31),179(250),288,

397

Iwasa, J., 189 Iwen, M. H., 347 Iyer, R. N., 134, 138(112,113),140(113),

179(112,113),181,184(430), 185(430),189(113)

1 Jack, A., 156,161(296) Jackson, J. J., 355 Jackson, R. L., 319,320,321, 324(843) Jacoby, M., 272 Jaffe, M., 166 Jaff6, W. G., 130,132(44),150,159,166, 178,297 Jamieson, G. A., 300 Janata, J., 150 Jansons, V. K., 328,329(865,866) Janzen, D. H., 149 Jaumatte, J., 226 Javaid, J. I., 326 Jayne-Williams, D.J., 130,341(9),342 Jeanes, A., 354 Jeanloz, R. W., 143,186(210),187(210),

195(210),196(210),275,303, 304(762),322(210),330(762), 331(762) Jenkins, J., 313 Jentoft, N. H., 354 Jermyn, M. A., 134,135(116),139(116) 317(31) Inch, T. D., 50,51(67),53(67),54(67), Jindal, V. K., 343 Jirgensons, B., 215,223(495a),230, 77(67),78(67) Innawi, S., 366 231(523),272,281,282(680), Inuzuka, T., 366 31l(523) Irimura, T., 140,238,251,252(183),254, Johansson, B. G., 178,301(413),331(413) Johnson, E. A., 292,293(697, 698), 258,259(616),261(616),273(183), 294(697,698),335(697,698,707) 275,297,300(183),304(183),305, Johnson, E. A. Z., 138,203,205, 318(183),331(183),335(616), 337(467a) 339(183) Johnson, L.N., 221 Isaacs, R., 330 Jones, B . M.,165 Ischiguro, S., 271 Jones, D. A., 147 Ishiguro, M., 270,271(642),272(642), Jones, D. B., 155 339(642) Ishii, S.-I., 154 Jones, J. K. N., 179,346,347,348(92,93, loo),351(100),352(92) Ishiyama, I.,239,240(559, 560),241(560, Jones, J. M., 148,267,268(633), 567),242(567),243(559,567) Isono, K., 126 269(635),339(631,635) Ivanova, E. A., 39,49,51(36),53(36),63, Jones, Q., 344,348(61) 64,80(36) Jones, R. D., 348,350(121) Iwabuchi, M., 154 Jones, R. L., 371

398

AUTHOR INDEX, VOLUME 35

Jones, R. T., 292, 293(698),294(698), 335(698) Jonsson, B., 131,277(64),338(64) Joseph, J. P., 58 Jourdain, G. W., 224 Judd, W. J., 206,263,338(470) Juergens, W. G., 176 Jung, P., 355 Jurgelsky, W., 244 Juster, H. B., 149

K Kabarity, A,, 202 Kabat, E. A,, 131, 133, 134(63), 138(63, 108), 140(108),142(108), 144(63, 108, 198, 200), 154, 156(170), 176, 178, 179(170),180(170), 181(170), 184(170),208,227( 108),228,229,231, 236(552), 237, 238(552),239(63), 240(63), 241(63), 242(63), 243(63), 253,259(203), 260(198), 261(203), 270, 271(644),284, 285, 286, 287, 288(200, 526), 313, 314(803),316, 335(108,200,570), 338(63, 108, 180, 552), 340(570) Kafka, J. A., 308,309(779) Kahane, I., 319,320(843), 321(843), 323, 324(843) Kahlem, G., l49,297(243e) Kahn, A. H., 254 Kaifu, R., 143, 186(210),187(210), 195(210),196(210),302,322(210) Kaiser, R., 361 Kalb, A. J., 138, 152, 153, 154(274a), 155(278),156, 161(296),179(278), 283(153),285(683), 335(265, 683), 340(683) Kalvoda, L., 75(114), 77 Kamata, T., 346 Kameda, Y., 120, 122(81) Kan, T.-J., 168, 177 Kanarek, L., 191, 198(442a,442b) Kandler, O., 352 Kanellakes, T. M., 264 Kanetsuna, F., 376 Kaplan, M. J., 214, 215(488),216(488) Kaplan, N. O., 294(717),295 Kaplan, R., 306 Kapoor, V. P., 344, 346(66), 347,348, 351(124)

Kargin, V. A., 55 Karl, W., 89, 90(24), 91(24), 92 Karlsson, B., 326 Karlstrom, B., 156, 193(301) Karush, F., 141 Katar, M., 312 Katchalski, E., 138,232,233(538), 234(538), 235(537, 538, 539), 238, 335(538) Kates, M., 363 Katsuno, A., 177 Katzen, H. M., 130, 150(40) Kaufman, H. W., l52,154(274a) Kaufman, S. J., 257, 335(612) Kaul, R. K., 344 Kauss, H., 139,355 Kawaguchi, H., 102 Kawaguchi, T., 140, 238(183), 251(183), 252(183), 254(183), 261(183), 273(183),275(183),297(183), 300(183), 304(183), 313,331(183), 339(183) Kawamura, T., 108 Kawasaki, T., 131 Kawauchi, H., 315 Kay, C. M., 153, 154, 160(285), 163(285a) Keen, J. L., 342 Kehoe, J. M., 306 Keilich, G., 366 Keith, C., 215 Kelkar, P. S., 347 Keller, J., 186,298(432),300(432) Keller, L., 270,271(646, 647) Keller-Schierlein, W., 81, 122(2) Kent, S. P., 147 Keough, A. H., 32, 38(8), 50(8), 51(8), 77(8), 78(8) Khalap, S., 315, 316 Khanna, S. N., 343,345(45) Kieda, C., 211, 212(483),213(483), 339(483) Kiehs, K., 189 Kiernan, J. A., 317 Kikuchi, T., 173 Kim, Y. C., 244,245(575) Kim, Y. S., 330 Kim, Z., 239,242(556) Kimata, K., 86,87(16), 88(16) Kingdon, H . S., 294, 295(712), 335(712) Kiyohara, H., 109 Klages, F., 343

AUTHOR INDEX, VOLUME 35

Kniep, B., 109 Knight, J. C., 119(80), 120 Knoboch, I., 239, 240(563) Knoboch, W., 239,240(563) Knop, F. B., 342 Knox, R. B., 134, 135(116), 139(116) Kobayashi, M., 160, 173 Koch, G. L. E., 372 Kocourek, J., 134(122), 135, 136, 137, 138(141), 141, 145, 155, 190, 191(442), 192(442), 193, 194(440, 442,447), 196(141),197(141,440), 198(454,455), 199(122), 201(122), 289, 290, 291(690),313, 314, 321(185), 322(185), 325(844), 326(851),333,335(141,142,442,444), 336(690),337(142,454,455, 690), 338(142), 339(122, 141,440, 690) Kohler, W., 133, 145, 227, 239(loo), 240(558), 241(100), 242(100), 243(100, 558), 338(100) Koenig, S. H., 156, 157, 179, 184(429) Kossel, H., 65 Koster, R., 36, 39,40,40(17), 41(33a, 41, 42), 43(39), 46(33a), 47(33a), 48(33a), 52, 53(41), 58(17), 66(40), 70(17,33a, 39, 40,41,42), 71(17, 33a, 40,41,42) 74(33a), 75(33a), 76(33a), 78(40,41, 42) Kottgen, E., 132 Koh, C., 256, 257(610), 334(610), 335(610) Kolecki, B. J., 278(674, 676), 279, 280(674,676), 338(674, 676) Kolodkina, I. I., 37, 38, 39,49, 51(27,28, 36), 53(27,36),54(27, 28, 35), 55(77), 63, 64,80(27, 28,35, 36, 75, 76, 77) Kolpak, F. J.,379,382(17) Kooiman, P. J., 343, 347(46), 366(257), 367 Kornfeld, R., 145, 178, 179(416), 186(416), 197, 297(216), 298(216, 432), 299(216, 730, 732), 300(432, 732), 301(216, 733), 304(216), 311(416),318(216, 730, 747), 319(216,730, 747), 320(216), 321(216, 730, 747), 322(730, 747), 324(216, 730, 747), 331(216, 730, 747) Kornfeld, S., 131, 145, 186, 187, 275,

399

276(666), 297(216),298(216, 432), 299(216, 730, 732), 300(432, 732), 301(216,733),304(216),308,311(731), 318(216,435, 747), 319(216,435, 730, 747), 320(216,434, 731), 321(216,435, 730, 747), 322(435, 666, 730, 731, 747), 323, 324(216, 730, 747), 331(216, 434, 435, 666, 730, 731, 747), 332 Koshiyama, H., 102 KoStif, J. V., 134(122), 135, 137, 138(141, 142), 155, 190(142), 191(142), 193(142), 196(141), 197(141), 199(122),201(122), 335(141, 142), 337(141), 338(142), 339(122, 141) Koulumies, R., 208,338(472,473) Koura, A., 379 Kovacs, P., 341 Kozak, L. P., 354 KritkL, Z., 178 Kreger, D. R., 343, 347(46) Krishnaswami, N. R., 345 Kristenko, L. V., 52, 54(71), 56(71), 80(71) Kristiansen, T., 129,304 Kriipe, M., 132, 133(77), 139(77), 140(77), 141, 142, 146, 147,208,210,212(77, 480), 224,226,244, 250, 252(195), 253(593), 263(195), 264(195), 265(195), 282(77), 284(77), 289(77), 290, 302, 303(754),304(77, 755), 305(77),313(77),338(77,195), 339(77, 480, 593,754,755, 756) Kubknek, J., 321,325 Kiihnemund, O., 227, 239, 240(588), 243(558) Kuhn, R., 277, 283, 284(672), 338(672) Kuhns, W. J., 268(637), 269, 291, 339(637) Kuivila, H. G., 32,38(8), 50(8), 51(8), 7 7 W 78(8) Kulshreshtha, A. K.,380 Kunitz, M., 270,271(645) Kunstmann, M. P., 89, 91(21) Kuppel, A., 380 Kurokawa, T., 137, 138(144),202, 254(144), 270(144), 271(144), 312, 340(796) Kustanovich, I. M., 52, 54(71), 56(71), 80(71) Kuzuhara, H., 74(111), 77)

AUTHOR INDEX, VOLUME 35

400

L Lackovic, V., 376 Lal, B. M., 356, 361(200) Lal, G., 378 Lalaurie, M., 132,224,284(81),289, 339(81) Lamblin, G., 270,271(650), 272(650), 337(650) Lampen, J. O., 174 Lamport, D. T. A,, 211 Lancaster, J. E., 89, 91(21) Landsteiner, K., 129, 139, 190, 291 Lang, A,, 371 Langridge, R., 215 Lankester, A., 130 Lantz, R. S., 327 Lappert, M. F., 32 L a m , O., 349 Larson, E. B., 345, 346(84) Latimer, S. L., 372(302),373 Lau, P. Y., 96 Leavitt, R. D., 138,296(154),301 Lechevallier, D., 365 Le Dizet, P., 344, 347,348(72, 107), 350(72, 107), 351(72, 107), 354, 361(112),363,364(107), 367(110, 112), 369(110, 112), 372 Lee, B. K., 110, 111 Lee, J. K. N., 203, 205(469),339(469) Lee, J. W., 372(301),373 Lee, S. R., 364, 367,370(236), 372(236), 374(236) Lee, W., 376 Lee, Y. C., 138,258(150d), 333, 372(300), 373, 374(300) Lees, E. M., 38,42(23), 50(23), 51(23), 53(23), 54(23), 55(23), 57(23), 58, 60(86), 72(23), 77(23), 78(23) Lemanski, T., 253,339(601) Lemonnier, M., 311 Lennarz, W. J., 355 Leo, A. J., 341 Leon, M. A., 134(123),135, 138, 141, 142(123), 144(123), 150, 178(109), 179(249),190, l91(441), 192(441), 193(441),194(109, 123), 195(123), 224,338(109, 123) Leschziner, C., 344, 346(68), 348(68) Leseney, A. M., 227, 228(518), 298, 311(73l),320(731),322(73l), 33l(731)

Letsinger, R. L., 35 Leunberger, R., 348 Le Vine, D., 214,215(488), 216,219 Levine, P., 302,339(750) Levine, V., 372(296),373 Levitzki, A., 153, 155(278), 179(278) Levvy, G. A., 372(297),373 Levy, A., 150, 166, 178 Lewis, B. A., 151, 168, 171(260), 175(364), 184(364) Lewis, D., 77(117, 118, 119, 120), 78 Lewis, S. D., 163, 164(339) Lhermitte, M., 270,271(650), 272(650), 337(650) Li, J. G., 129, 292 Li, S. C., 363 Li, S. S.-L., 271, 306 Li, Y.-T., 363,372(300), 373,374(300) Liao, J., 316 Liener, I. E., 130, 132(45),134, 137, 138(136), 149, 151, 152(136, 264), 154, 171(136),231,232(529), 233, 234(534), 235(532),296, 301, 335(136) Liese, W., 366 Lightbody, J. J., 224 Lin, C. M., 110 Lin, H., 130, 150(33) Lin, J.-Y., 130, 254(32),255, 271(32) Lin, K., 271 Lin, L.-T., 130,254(32), 271(32) Lindahl-Kiessling, K., 297 Lindberg, A. A., 140, 241(178), 242(178) Lindberg, B., 174,349 Lindberg, Bengt, 46, 53(53), 57(53), 62 Lindberg, Boje, 62 Linden, J. C., 355 Lindstrom, K., 71 Ling, A. R., 370 Ling, N. R., 130,291(36) Linsley, K. B., 275 Lipscomb, W. N., 152 Lis, H., 129, 130(18,26), 132(18, 26, 37b), 133, 134(97),136(18), 137(18, 132), 144, 150(72),164, 208(18), 209(18), 214,215(491), 232(97), 233(538), 234(538,541), 235(537, 538, 539), 236(212),238(99,212), 239(343,343a), 296, 317(354), 318(18), 335(538),338(212) Liske, R., 218,339(501) Lisowska, E.,302,303(759), 339(753)

AUTHOR INDEX, VOLUME 35 Litman, G. W., 306,307(771), 309(771), 335(771) Little, L. L., 341(8), 342 Livingston, D. C., 317 Lloyd, K. O., 140, 166(170), 172, 175, 176, 179(170), 180(170), 181(170), 184(170),338(170),354,376 Lockhart, J. C., 34,70 Lohmar, R. L., 344, 348(61) Lonchampt, M., 165 Longcor, F., 39 Lonngren, J., 135, 138, 179(126), 224, 262, 263(622), 264(126, 622), 338(622) Loontiens, F. G., 134(124), 135, 140(124), 144, 160, 188(181,323), 189(181, 323), 195(182),197(182), 199(182, 211), 200(182,211), 216, 273(124), 274(124),339(124,211),340(124),376 Lorand, J. P., 32,42(6), 49(6) Lotan, R., 136, 137(132), 143, 147, 164, 214, 215(491),218, 219(504), 223, 234, 235(544),236,239(343, 343a), 258, 259(202,203), 261(202,203), . 262(619), 318,335(202, 544, 546), 338(202, 619), 340(544) Lucas, J. J., 355 Luecker, P., 341(17), 342 Luisada, A., 202 Lundeen, D. E., 164,248(342) Lundstrplm, H., 349 Lustig, A., 152,335(265) Lynn, W. S., 130, 150(39) Lyr, H., 366

M McAlpine, T. S., 84 McCleary, B. V., 347,356,(205), 357, 364(205), 365,366, 369,370(205), 372(252), 373(252), 374 McClendon, J. H., 32,49(5) McCormick, J. E., 58 McCredie, R. J., 348 McCubbin, W. D., 153, 154, 160(285), 163(285a) McDaniel, L., 110 McDannel, M. L., 177 McDermed, J. D., 292, 293(698), 294(698),335(698) McDonald, M. R., 270, 271(645)

401

McElhinney, R. S., 58 McGinnis, G. D., 48, 54(61), 62(61), 79(61) McInnes, A. G., 382 McKenzie, G. H., 152, 153, 154(279), 160(266), 164(279),165 Mackie, W., 377 McKinley, I. R., 35,42,43, 45, 53(43, 48), 55(43,48),66, 67(lo), 72(48), 77(43),78(43) MacLennan, A. P., 315 McMaster, M., 134, 147, 244(103), 245(103), 248(103) McNamara, P. M., 34, 35(11), McPherson, A., 257, 335(612) Makela, O., 129, 132, 133(78, 79), 134(78), 140(78),141, 142(20), 144(20), 146, 147, 148(20), 199(20), 202, 208, 209,(20, 78), 210, 226, 244, 250(78), 252(195), 253(78), 262, 263(195, 620), 264(195), 265(195), 282(78), 284(78), 290, 304(78), 305(78), 306(78), 313(78),338(20,78, 95), 339(78) Makela, P., 142,252(195),262, 263(195, 620), 264(195),265(195), 338(195) Magnuson, J. A., 156, 158, 161(310) Mahler, H. R., 326 Mahmood, S., 85(13), 95(13) Maier, S., 102, 109 Mair, G. A., 221 Maisonrouge-McAuliffe,F., 178 Maiti, B. C., 56 Majems, P. W., 141 Majumdar, M. K., 117, 118 Majumdar, S. K., 117, 118 Malcolm, E. W., 33 Maley, F., 372(292),373 Malik, J. M., 104, 107(53), 115(53), 116(53), 117(53) Malinzak, D. A,, 131 Mann, T., 372(293),373 Manners, D. J., 166, 168(357, 358), 169 Mannschreck, A,, 96 Marchalonis, J. J.,306, 307(769), 335(769) Marchesi, V. T., 214, 317(493), 319, 320(843), 321(843), 323, 324(843, 845) Marchessault, R. H., 377, 378(l),379 Marcus, D. M., 155, 158, 161(307,308), 163, 179(308),201(308)

402

AUTHOR INDEX, VOLUME 35

Marcusson-Bequn, H., 210 Maiik, T., 197, 198(454),337(454) Marini, M., 161, 162(326) Marinkovich, V. A., 210,211(482), 339(482) Markowitz, H., 140, 177(177) Marquardt, M. D., 140 Marsh, C. A., 372 Marsh, W. L., 253 Marshall, R. D., 211 Marshall, W. H., 300 Martin, C. R., 344, 348(62) Martin, J. R., 118, 119(77),317 Martin, L. L., 118, 119(77) Martin, T., 132 Masler, L., 376 Massaro, E. J., 307 Matern, H., 83, 84(8),92, 93, 95(28) Matern, U., 99, 101, 102, 109(40) Matheson, N. K., 347,356(205), 357, 364(205),365,366, 369(116), 370(205), 372(252), 373(252),374 Mathews, K. P., 264 Matsubara, M., 366 Matsubara, S., 245, 246 Matsuda, K., 173 Matsumoto, I., 137, 138(149), 142, 143, 144(208),145(208),208(196,208), 209(149, 196), 210(196), 212, 225(149, 196,208, 226), 226, 262(149),282(476), 289(149, 196, 226), 290(196), 291(196), 296(149), 305(196),313,335( 149,476), 336(226),337(196,208,226, 509), 338(476),339(196, 509, 692) Matsumoto, T., 156, 193(300) Matsuzaki, M., 102 Mayer, M. M., 133 Meier, H., 345,346,352, 353(143),355, 356(204),357, 360,361(201), 364(202), 365, 366(204),370(201), 371(201, 202,204), 372(202),374 Meir, H., 344, 345(70),348(70) Mekinnon, A. A., 349 Melo, A., 89,99(18), lOO(18) Meloche, H. P., 123 Melton, L. L., 342 Merdel, L. B., 231,292 Merlis, N. M., 55 Merrick, J. M.,134(121), 135, 140(121), 150(121), 151(121),166(121),

167(121), 168(121), 169(121), 171(121),173(121),179(121) Meyer, E. W., 233 Meyer, H . W., 317 Meyers, F. L., 366(258),367 Mialonier, G., 149, 215, 219(494), 222(494),223(494),297(243e) Michelson, A. M., 94 Miescher, P., 297 Miki, T., 248 Miller, A. L., 290, 336(691),337(691), 339(691) Miller, F., 178, 186, 187(433) Miller, J. B., 267,294(630),295(630,712), 335(630, 712) Miller, J. T., 246, 253 Miller, S. E., 347 Minshall, J., 89 Mirelman, D., 147,218 Misaki, A., 143, 148, 168(209,240), 172, 173, 174(240),178(240),179(209, 240), 181(240),182(209,240, 381), 183(240),184(209,240,423), 185(240),186(209,240), 187(209), 200(240),247(240),322(209) Misawa, Y.,366 Mitchell, E. D., 372(302),373 Mitscher, L. A., 89, 91(21), 118, 119(77) Mizoguchi, T., 109 Moe, 0. E., 347 Moller, G., 130, 154, 165(290),291(37) Mogel, L. G., 45, 52, 54(47),56(72), 72(47) Moldow, C. F., 300 Monsigny, M., 134, 135(115),149, 211, 212(483),213(483),214,215(490b), 216(115),217(115),219(115,494, 497), 222(115,494), 223(494), 297(243e),306, 307(770),335(770), 339(483),340(115) Montgomery, R., 134, 140(117),151, 166(117),168(354,356), 169(117, 356), 171(260),179(117),343 Moore, L., 189 Moorhouse, R., 384 Mordman, C. T., 292,293(703), 310(703) Morel], A. G., 131 Moreno, C., 178 Morgan, I. G., 326, 327(857) Morgan, W. T. J., 129, 131(21, 22), 140(21,22), 141, 142(22),144(22),

AUTHOR INDEX, VOLUME 35

207,208(22,471),209(471,475), 210(471), 226(476), 244, 248(22), 250, 253(22), 277(21), 282(22), 283, 284(22), 305(471), 338(21, 22,471, 475,477), 339(22) Morgenstem, M., 177 Morgenthaler, W. W., 342 Mori, Y., 157, l60,324(306b) Morinioto, J. Y., 344 Morita, K., l57,324(306b) Morley, R. G., 346 Moroux, Y., 214,215(490b) Morrison, A,, 343,345,347(43), 349, 350(43), 356(43), 363(43), 364(43), 369, 375(43) Morrison, J. D., 35 Morse, J. H., 134, 177(105), 178(105), 300(105), 301 Morse, S. I., 176 Morton, G., 89, 91(21) Moscona, A. A., 317 Moskal, J. R., 264 Mountfield, B. A., 32 Mueller, G. W., 292 Mueller, H., 341(16, 17), 342 Mukaida, M., 293, 240(559, 560), 241(561), 243(560) Mukherjee, A. K., 343,346(52), 348(52) Mukhejee, S., 343,347, 348, 351(124) Munro, M. H. G., 104, 107(54), lOB(54) Murachi, T., 333 Murakami, M., 54, 79(78) Murakawa, S., 134, 177(107), 300(107)’ Muramatsu, T., 372(294), 373 Murase, K., 54,79(78) Murawski, A., 111 Murphy, J. C., 155 Murphy, L. A., 248,249(591), 262, 263, 264(625), 266(621), 295(628),338(625) Murphy, V. G., 378 Musgrave, 0. C., 34 35(13), 42(13), 56, 78(13) Mustier, J., 131 Mustier, M., 197 Muzurek, M., 71 N

Nabors, M. W., 371 Nachbar, M. S., 139,291, 306(155), 307(155), 329,330(868)

403

Nachman, R. L., 325 Nadelmann, H., 358 Nagai, Y., 302, 303(758),304(758), 339(752, 758) Nagaoka, K., 106 Nagaoka, T., 372 Nagata, Y.,136,212(129), 214(129), 215(129,487), 216,337(129), 340(129) Nainawatee, H. S., 356, 361(200) Nakamura, S., 134, 157, 177(107), 300(107) Nakano, K., 312 Nanji, D. R., 370 Nanno, S., 271 Napier, P. W., 288 Napoli, C., 147, 148 Naspitz, C. K., 130, 291(35) Natarajan, C., 343 Nathenson, S. G., 176 Neely, W. B., 172 Neimo, L., 378 Neri, G., 272,327, 328(861, 862, 863) Neter, E., 131 Neuberger, A., 136, 143(128), 144(128), 195(213), 199(213),200(213), 203(213), 204(213),205(213), 211, 212(207), 213,221, 233,309(213), 336(213), 337(128,207, 213), 338(213), 339(128,207, 213) Neukom, H., 348,350(120) Neupert, G., 317 Newman, A. D., 156 Nichol, L. W., 152, 160(266), 165 Nicholson, S. K., 160 Nicolson, G. L., 129, 130(25), 132(25), 133(25), 137, 138(146), 144(146), 142, 150(25),270(146, 194), 271(146, 194), 272(146), 273( 146), 274(146), 293(194), 317(194),318(25), 336(146), 337(146, 194),339(146, 194), 340(146) Niederhuber, J. E., 135, 179(126), 264(126) Nieduszynski, I. A., 379,382,383(26), 384(28) Nigrelli, R. F., 268(637),269, 291, 339(637) Nijenhuis, L. E., 302 Nikaido, H., 355 Nikaido, K., 355

AUTHOR INDEX, VOLUME 35

404

Nimi, O., 109 Nisizawa, K., 367 Noguchi, H., 307 Nomi, R., 109 Noonan, K. D., 132, 165,318 Nordbn, A., 292 Nordgren, R.,342 Nordman, C. T., 292,293(704), 294, 295(71l), 335(711) Norins, L. C., 300 North, A. C. T., 221 Northcote, D. H., 211, 366(258), 367 Northrup, R. L., 301 Novak, E., 366 Novogrodsky, A., 164, 238, 239(343, 343a), 261, 262(619),338(619) Nowak, H., 341(17),342 Nowak, T. P., 139,306(159), 307(159), 325(159) Nowakova, N., 145 Nowell, P. C., 130, 291, 291(34) Noyes, C., 294,295(712), 335(712) Nuernberg, E., 341(12, 13, 14, 16, 17), 342 Nystrom, R. F., 104, 107(53), 115(53), 116(53), 117(53)

0

Oblin, A,, 214 Obrenovitch, A., 214 O’Brien, J. S., 290, 336(691),337(691), 339(691) Oerkermann, H., 299, 301(735), 302(736) Oh, Y. H., 294,335(710), 338(710) Ohashi, S., 102 Ohata, Y., 109 Ohkawara, A., 372(296),373 Ohkita, J., 380 Ohmari, T., 102 Oikawa, K., 153, 154, 160(285),163(285a) Oishi, K., 131,307(52),308(52, 778) Okada, M., 160 O’Kane, D. J., 166, 168(352) Okanishi, M., 102 Okazaki, R., 94 Okazaki, T., 94 Okuda, S., 85,86,87(16), 88(16) Okumura, T., 372 Olsnes, S., 137, 138, 254(147, 150), 255, 256, 257,270(147, 150), 271(147,

150, 639, 649, 651), 272(639, 651, 658), 273,274( 147), 276,334(147, 150), 335(147, 150, 651), 336(147, 150, 649, 651), 337(147, 150, 651), 338(147, 649), 339(147), 340(147) Olson, M. 0. J., 137, 138(136), 151, 152(136), 154, 171(136), 335(136) Onodera, K., 197, 199(452),200(452), 339(452) Onozaki, K., 137, 138(144),202,254(144), 270(144), 271(144) Opie, J. W., 342 Oppenheim, J. D., 139, 291, 306(155), 307(155), 329, 330(868) Orenstein, N. S., 100 Ortmann, R.,99 Osawa, T., 129, 132(19), 137, 138(139, 143, 144, 149), 140(19), 142(143), 143, 144(143,208), 162, 163(332), 165(332), 178, 186(210), 187(210), 190(143), 191(143),192(143), 194(143), 195(143,210), 196(210), 202(139), 205(139),207(19), 208(195, 208), 209(149, 195),210(195), 212, 225(149, 195, 208,226), 226, 238(183), 250,251( 183, 594), 252(183), 253(594),254(144, 183), 258,259(616), 261(183, 616), 262(149), 270(144), 271(144), 275(183,289), 282(476),289(149, 226, 290,291), 290(196), 291(196), 296(149), 297(183,417), 300(183, 417), 301(417), 302,304(183,417), 305(19, 96),310,312, 313, 318(183), 319, 321(210), 323,324(740, 741), 331(183), 335(143, 149,476, 616), 336(226), 337(196,208, 226, 509), 338(19, 143,476), 339(183, 196, 509, 692), 372 Osborn, M. J., 355 Osborne, T. B., 231 Osgood, E. E., 129,292, 339(696) Osman, H. G., 277,283,284(672), 338(672) Osuga, D. T., 268(637),269, 339(637) Ottensooser, F., 148,302,339(750) Overend, W. G., 47, SO(SS), 53(55), 57(55),58,62,74(55), 75(55), 76(115), 77, 79(55) Overton, J. D., 341(11), 342 Oyen, R.,297 Ozanne, B., 214,318(489)

AUTHOR INDEX, VOLUME 35

P Pacak, F., 145, 313, 314 Pachtman, E. A., 202,205(469), 339(469) Painter, T. J., 208, 209(475), 226(475), 338(475, 476), 349, 352 Paley, L. G., 360 Pallansch, M. J., 231, 232(529), 233, 234(534) Palmer, K. J., 348 Palozzo, A., 159, 166, 297 Panchenko, S. I., 64 Pande, A., 378 Pandolfino, E. R., 156 Pape, H., 83,84(8) Pappenheimer, A. M., 271, 272(658) Pardoe, G . I., 131, 133, 143, 144(206), 211,212(206), 213(206), 218, 224(502), 258(201), 259(201), 267, 268(632, 636), 269, 276(206), 299, 300(632), 301(735), 307, 333(632), 338(201), 339(206, 632, 636) Park, R. B., 363 Park, W. M., 371 Partridge, J., 314 Paton, F. J., 370 Paulovi, M., 134(122), 135, 190, 191(442),192(442), 194(442), 197, 199(122),201(122), 335(442), 339(122) Paulsen, H., 89,99 Pavlista, A. D., 371 Pecht, I., 155 Pelly, R., 163 Pemberton, R. T., 239(566), 240 Percheron, F., 352,353(145), 360(145), 362,363, 364(145), 366, 367(255), 368(255), 372(260), 375(145) Percival, E. G. V., 343 Perdomo, J. M., 330 Pere, M., 228,230, 231(523), 311(523), 335(519) Pereira, M. E. A., 142, 144(198, 200), 231, 236(552), 237, 238, 259, 260(198), 261(203), 284, 285, 286, 287, 288(200, 526, 686), 313, 314(803), 316,335(200), 338(552) Perera, C. B., 203, 205(468), 339(468) Perila, O., 353 Perkins, H. R., 353 Perlmann, H., 243 Perlmann, P., 243

405

Perrodon, Y., 307 Perricone, A. C., 342 Perry, A. L., 372, 373(284) Perry, M. B., 179 Petek, F., 348,350, 363(117), 366(259), 367, 368(140, 253), 369(140, 253) Petek, P., 347, 363(108) Petryniak, J., 313, 314 Pettitt, D. J., 342 Pezzanite, J. O., 96 Pfeiffer, V. F., 344, 348(62) Pflumm, M. N., 153,154, 155(289), 160(283,289), 168(319) Pfuderer, P., 255, 256, 257(610), 334(607, 610), 335(607, 610) Phelps, C. F., 316 Philipp, B., 379 Phillips, D. C., 221 Pichuzhkina, E. I., 64 Pihl, A., 137, 138, 254(137, 150), 255(137), 256(137), 257(137), 270(137, 155), 271(137, 155, 639, 649, 651), 272(137, 639, 651), 273(137), 274(137), 334(137, 150), 335(137, 150, 651), 336(137, 150, 649, 651), 337(137, 150, 651), 338(137, 649), 339(137), 340(137) Pittner, F., 104 Pittz, E. P., 159 Plow, E. F., 178, 182(419) Plummer, T. H., Jr., 372(292), 373, 374(305) Podder, S. K., 141, 168(193), 177, 179(193), 274(193) Poleflca, T. G., 219 Pollitzer, W., 302, 339(750) Porath, J., 137, 138(137),297, 304(137), 306(145), 311 Poretz, R. D., 140, 151, 157(180), 166, 168, 172(362),173(362), 174(365), 180(365), 181(365),182(365), 183(365), 185(365), 188(180, 365), 189(180,436), 190(365),200(365), 250, 251(175, 184, 598), 252(184), 253(184), 267, 312, 333, 337(184, 598), 338(365), 339(175, 184, 601) Portsmouth, D., 48, 55(60), 74(60) Pospelova, T. A., 54, 56(74), 80(74) PospiSilovii, J., 141, 321, 322(185),325, 326(851) Powell, A. E., 150,179(249) Powers, D. A., 151, 152(264)

406

AUTHOR INDEX, VOLUME 35

Prasad, D., 38,46(22),51, 53(22, 51, 69), 54(22, 51,69), 55(22, 52), 57(22), 58(50, 69), 73(69), 74(51), 75(51), 76(22, 50, 52, 69) Pratt, R. M., Jr., 317 Preobrazhenskaya, M. E., 168, 171 Preobrazhenskii, N. A., 38,45(24) 51(27, 28), 52, 53(27), 54(24, 25, 27,28,71), 55(77), 56(71), 57(73), 72(24, 25), 80(27, 28, 71, 73, 75, 76, 77) Presant, C. A., 187,308, 318(435), 319(435),320, 321(435), 322(435), 331(435) Preston, R. D., 378 Pricer, W. E., Jr., 131 Pridham, J. B., 344,345(58), 358,362, 363(223),365(223),366(223, 224) Prigent, M. J., 145,303,337(220) Pringsheim, H., 370 Prior, A. M., 110 Privat, J.-P., 134, 135(115), 149,214, 215(490b),216,217,219(115,494, 497), 222(494),223(494), 297(243e), 340(115) Prokop, O., 129, 131, 133, 145(14a),227, 239(58, 60, 61, loo), 241(60, loo), 242(61, 100, 556), 243(100), 338(60, 61, 100) Pryce, N. G., 349 Punin, W., 147 Punnett, H. H., 292 Punnett, T., 292 Pusztai, A., 297,301

Q Quicke, G. V., 232 Quiocho, F. A., 152 Quirt, A., 158, 159(311)

R Race, R. R., 129, 133(14),226(14), 289(14), 339(14) Rackis, J. J., 232 Rackwitz, A., 131, 239(58, 60), 241(60), 338(60) Rashen, V., 295, 335(718) Rafestin, M. E., 214 Rdferty, G. A., 58,62 Rafique, C. M., 346,347(90)

Ragheb, H . S., 107 Rahman, M. A., 254 Ramachandra, G., 365 Ramachandramurthy, P., 296 Ramirez, G., 326 Randall, M. H., 50,51(67), 53(67), 54(67), 56(67), 77(67), 78(67) Rao, C. V. N., 343,346(53), 348(53) Rao, M. V. L., 345 Rapin, A. M. C., l32,318(92a) Raubitshek, H., 190,291 Ravid, A., 261, 262(619),338(619) Rebers, P., 179 Reckin, E., 360 Redlich, H., 89 Redwood, W. R., 219 Reeber, A., 326 Reeder, W. J., 176 Reeke, G. N. Jr., 137, 138(140), 152, 153(325), 154(268,269, 275,276), 155(267), 156(268,275), 157(267, 268,280), 158(267,268,280), 161(280),202(140), 203(140), 205(140), 304(276),335(267), 336(140), 337(140),339(140) Rees, D. A., 341,349,384 Reese, E. T., 362, 369,372(221), 374 Refsnes, K., 255, 270,271(651), 272(651), 335(651), 336(651),337(651) Rege, V. P., 208,209(475), 226(475), 338(475,477) Reguera, R. M., 128, 147(4),243(4) Reichert, C. M., 143, 148, 168(209,240), 174(240), 178(240),179(209, 240), 181(240), 182(209),183(240), 184(209,240), 185(240), 186(209, 240), 187(209),200(240), 247(240), 322(209) Reid, J. S. G., 344,345(70), 346(199), 348(70), 352, 353(143),355, 356(204), 357, 359(199),360,361(201), 364(202), 365, 366(204),370(199, 201), 371(199, 201,202, 204), 372(202), 374 Reifenberg, U., 299 Reiner, R., 102 Reinhold, V. N., 47,48(56), 55(56), 65(56), 67(56),68(56), 69(56) Reisfeld, R. A., 139, 309(162), 310 Reitherman, R. W., 139, 290(158),

AUTHOR INDEX, VOLUME 35 336(158, 691), 337(158, 691), 339(691) Rekunova, V. N., 39, 54(37), 80(37) Renkonen, K. O., 128, 132(6),208(6), 282, 283,304(6), 338(6) Renwrantz, L., 316 Resheff, G., 315 Resnick, H., 178, 182(419) Rettig, E., 341(12, 13, 16), 342 Revel, J.-P., 317 Rice, R. H., 162, 215, 223(335), 337(335, 495) Richards, E. L., 347 Richards, G. N., 345,366, 367(243), 368(243), 369(243) Richards, J. B., 355 Richards, R. L., 219 Richter, M., 130, 291(35) Richtmyer, N. K., 372(299), 373 Rick, P. D., 354 Rigas, D. A., 292, 293(697,698), 294(697, 698), 335(697,698, 705, 706, 707), 339(696) Rinehart, K. L., Jr., 102, 104, 107(53,54), 108(54), 115(53), 116(53), 117(53), 119 Riss, H., 140, 251(184),252(184), 253(184), 337(184),339(184) Rist, C. E., 172 Rivera, A., 292 Rizk, A. M., 344 Rizvi, S. A. I., 344 Robbins, P., 355 Robertson, E. S., 140, 241(178), 248(178) Robinson, D. S., 48 Robinson, R., 174 Robos, V. N., 44,45(45), 70(45) Robson, E. B., 134, 177(106) Roche, A.-C., 306,307(770), 335(770) Rochmilevitz, T., 356 Ronnback, L., 326 Rogers, H. J., 353 Rogers, J., 187,320(434),331(434) Rogers, T. O., 104, 107(54), 108(54) Roguet, R., 231 Roholt, 0. A., 161, 162 Roland, J.-C., 356 Rollins, A. J., 85(13), 95 Rolls, J. P., 104, 107(53), 115(53), 116(53), 117(53), 119

407

Romanowska, E., 303 Ronalds, J. A,, 372(301),373 Rose, A. W., 307 Rose, J. E., 709 Rosen, M., 342 Rosen, S. D., 139,290(158), 308, 309(779,780), 336(158), 337(158) Rosenau, W., 296 Rosenfel’d, E. L., 168, 171 Rosenfeld, L., l38,258(150d) Rosenthal, A. S., 317 Rosenwasser, A., 164,239(343) Ross, T. T., 266 Rosset, J., 264 Roth, J., 313,317 Roth, K. L., 202 Rothfield, L., 355 Rougier, M., 356 Roussel, P., 270,271(650), 272(650), 337(650) Rovis, L., 285, 288(686) Rowlands, D. T., Jr., 313 Roy, J., 256,257(611), 334(611), 335(611) Roychoudhury, R., 65 Rozenberg, M., 307 Rozynov, B. V., 68 Ruckel, E. R., 172 Rudakova, I. P., 38, 39,44, 51(28), 52, 54(28, 37,46, 71), 56(46, 71, 74), 57(73), 72(46),80(28,37, 71, 73, 74) Ruddon, R. W., 164,248(342) Rudloff, E., 65 Rudowski, A., 38,46(22), 51, 53(22, 69), 54(22, 69), 55(22), 57(22), 58(50, 69), 76(22, 50, 69) Riidiger, H., 305 Ruelius, H. W., 279, 280(673) Ruff, B. A., 104, 107(53), 115(53), 116(53), 117(53), 119 Rule, A. H., 140 Rundle, R. E., 378 Rydlund, P. H., 342 Rzedowski, W., 366

S Saarnio, J., 344 Sachs, L., 130, 144, 150(31),236(212), 238(212), 288, 296, 317(31,250, 554), 338(212),339 Sadaksharaswami, M., 365

408

AUTHOR INDEX, VOLUME 35

Sadovskaya, V. L., 68 Sage, H. J., 131, 134(123), 135, 137, 138(138), 141, 142(123),144(123), 145, 148, 149(243), 190(138), 191(441),192(441), 193(138,441), 194(123, 138, 186), 195(123), 202(138),308(51, 221), 335(138,441), 338(123, 138),340(186) Saint-Paul, M., 147 Saito, N., 46, 76(54) Sakakibara, F., 315 Sakakibara, K., 172 Sakamoto, C. K., 328, 329(866) Sakurai, Y., 137, 138(139),202(139), 205(139) Salfner, B., 299, 302(736) Sallach, H. J., 97 Sallam, M. A. E., 343, 346(54) Saltmarsh-Andrew, M., 355 Salton, M. J. R., 139,306(155),307(155) Saltvedt, E., 137,254(147),255(147), 256(147), 257(147), 270(147), 271(147), 272,273( 147), 274(147), 276,334( 147),335(147), 336(147), 337(147),338(147), 339(147), 340(147) SalvetovL, A., 192,335(444) Sambrook, J., 214,318(489) Sandermann, H., 355 Sanderson, A. R., 176 Sandoz, D., 356 Sanford, B. H., 130,317(29) Sanger, R., 129, 133(14), 226(14), 289(14), 339(14) Sangster, I., 38,46(22), 53(22), 54(22), 55(22),57(22), 76(22) Saniewski, M., 376 Sarko, A., 377, 378, 381 Sarma, V. R., 221 Sasada, Y., 46, 76(54) Sasame, H. A., 232 Sastry, P. S., 363 Sawai, K., 361, 370(219),374(219) Sawyer, W. H., 152, 153, 154(279), 160(266), 164(279),165(290) Schaffer, J. W., 172 Schaffner, C., 110 Schaub, R. E., 58 Schechter, B., l64,239(343a) Scheinberg, I. H., 131 Scheinberg, S. L., 244,246, 247(586), 335(586, 588)

Scher, M., 355 Schertz, K. F., 244 Schiffer, M., 152 Schilling, E. D., 341,342 Schleicher, H., 379 Schlesinger, D., 131, 239(58, 60), 241(60), 242(556),338(60) Schmid, R., 82,83(5), 89, 90(24), 91(24), lOO(5) Schmidt, E. L., 147 Schmidt, P., 65 Schnebli, H. P., 150 Schnitzler, S., 239, 240(563) Schott, H., 64,65 Schuerch, C., 172 Schulze, R. E., 342 Schumacher, K., 299,301(735), 302(736) Schuppner, H. R., 341 Schussler, W., 40, 41(41), 52(41), 53(41), 70(41), 71(41), 78(41) Scocca, J. R., 333 Segrest, J. P., 319, 320(843), 321(843), 824(843) Sehgal, K., 356,361(200) Seib, P. A., 58 Seidl, D. S., 166 Seiler, A., 356(203), 357, 365, 372(203) Sela, B.-A., 139, 144,224(156),236(212), 238(212), 239, 296(156), 317(554), 338(212) Self, R., 48 Sen, A., 256,257(611), 334(611), 335(611) Seshadri, T. R., 345 Seto, H., I24 Seymour, E., 53 Shafer, J. A., 140, 159(174), 160, 163, 164(339), 179(174), 185 Shafizadeh, F., 48, 54(61), 62(61), 79(61) Shankar Iyer, P. N., 135, 137(125), 138(125), 142(125), 146(125), 206(125), 207(125), 262(125), 265(125), 335(125), 338(125, 470) Shannon, L., 314 Shaper, J. H., 136, 337(130) Shapleigh, E., 128, 129, 131, 132(2), 134, 147(2),224,243(2), 244(2, 3, 103), 245(103), 248(103), 289(12, 13), 339(2, 12, 13) Sharma, B. R., 345 Sharon, N., 129, 130(18,26,27), 132(18, 26, 37b), 133, 134(97), 136(18), 137(18, 132), 142, 143(128), 144(128),

AUTHOR INDEX, VOLUME 35 147, 150(72), 164, 191, 198(442c), 208(18), 209(18),214, 215(491), 216, 218, 219(504),222(504), 223, 232(97), 233(538), 234(538, 541), 235(442c, 537, 538, 539, 544, 546), 236(212, 552), 237, 238(99, 212, 552), 239(343, 343a),258(202), 259(202, 203), 260(198), 261(202, 203). 262(619), 291(37a), 296, 317(554), 318(18), 335(202, 538, 544, 546), 337(128), 338(202, 212, 552, 619), 339(128), 340(544) Shaw, Y.-S., 255,271 Sheehan, J. K., 383, 384(28) Sheichenko, V. I., 56 Sherman, W. R., 69,70 Sherry, A. D., 156 Shibata, Y.,362, 369, 372(221), 374 Shier, W. T., 134,206(114), 216,222 Shimahara, H., 346,367, 370(265) Shimanouchi, H., 46, 76(54) Shimazaki. K., 272 Shinkai, K., 157, 324(306b) Shinohara, T., 197, 199(449,452), 200(452), 339(449,452) Shiroya, T., 360,365(210) Shishido, K., 173 Shmyrev, I. K., 44,45(45), 70(45) Shoham, J., 150 Shoham, M., 155 Siddiqui, I. R., 38,43,45(32), 53(32), 65, 67(32, 44, 99), 70(32, 44), 72(32, 44), 73(32,44) Sidebotham, R. L., 171 Siegelman, H. W., 234,235(544), 335(544),340(544) Sigarlakie, E., 174, 175(388) Sihtola, H., 378 Sikl, D., 376 Silber, R., 297,300 Silberschmidt, K., 302 Silman, I., 315 Simpson, D. L., 308, 309(779,780) Singer, S. J., 317 Singh, G., 210 Singla, S., 140, 251(175), 339(175) Sinha, M. P., 344 Sinnwell, V., 99 Sioufi, A., 352, 353(145), 360(145), 364(145),375(145), Skorvaga, M., 109 Skoyles, D., 56

409

Skutelsky, E., 143, 258(202), 259(202), 261(202), 335(202), 338(202) Slechta, L., 119 Slessor, K. N., 46, 53(53),57(53) Slodki, M. E., 168, 174, 354 Sly, D. A., 151, 152(264) Small, D. M., 347,369(116) Small, P. A., 310 Smith, A. K., 232 Smith, B. C., 47, 50(55), 53(55), 57(55), 74(55), 75(55), 79(55) Smith, C. W., 89 Smith, D. F., 327, 328(861, 862, 863) Smith, E. E., 140, 142(168), 143(168, 197), 144(168),150(168), 151(168), 166, 167(168, 197, 359), 168(359), 170(359),171(359), 173, 179(168, 197), 180(168, 197), 181(168, 197), 183(197), 184(168, 197,430), 185(168, 197,430), 187(197,430), 200(197), 338(168) Smith, F., 134, 140(117), 151, 166(117), 168(353,354,355),169(353), 171(260), 173(353), 175(364), 179(117), 184(364),343, 345, 346(84), 347(90), 348(99),350(99) Smith, P. J. C., 384 Smith, S. B., 317 So, L. L., 134, 135(120), 140(120), 143(169), 144(169), 151(120), 153(172), 160, 162, 165(204), 166(320), 167(120,204), 168(120, 169, 204, 215, 320), 169(320), 170, 171(120,369), 172(320,362), 173(320,362,369), 174(120, 204), 175(204,215,320), 178, 179(120, 169, 172,215), 180(169,204,215), 181(169,204,215), 182(169), 184(120, 169, 215, 430), 185(204, 430), 187(430),196(204),200(169), 212(204), 338(169, 204, 215), 340(172) Soboczenski, E. J., 32, 38(8), 50(8), 51(8), 77(8), 7863) Soderman, D. D., 130, 150(40) Somme, R., 344, 345(76), 372(303, 304), 373,375 Sohonie, K., 196, 197(448),339(448) Solms, J., 32, 38(7), 43(7), 44(7), 50(7), 52(7), 64,72(7), 73(7) Som, S., 256, 257(611),334(611), 335(611)

410

AUTHOR INDEX, VOLUME 35

Somers, G. F., 32,49(5) Somers, P. J., 49,63(64),65(64) $om, F., 75(114),77 Southworth, D., l49,229(243a) Speckart, S. F., 219 Spencer, J. F. T., 343,354(41) Spiro, R. G., 300 Sprenger, I.,227,267, 268(632,636),

269(632),297(632),300(632), 333(632),339(632,636) Springer, G . F., 131,134,140,144(167), 145,277,278(167,676), 279(118, 167),280(66,167,671,673,676), 281(66,671,677),282(679),284,285, 302,303(219,678,758),304(678, 758),318(678),335(679),338(167, 674,676),339(752,757,758) Srivastava, H.C., 380 Srivastava, V. K., 380 Stacey, B. E., 39,45(33), 70(33),72(33), 73(33) Stacey, M., 48,74(59),172 Stadler, P., 99 Staub, A. M., 179 Stead, R. H., 232 Steck, T. L., 323 Steigerwald, J. C., 372,373(290) Stein, J. Z., 347 Stein, M. D., 141,190,191(441), 192(441),193(441),194(186), 335(441),340(186) Steinberg, M. S., 165 Steiner, E. A., 263 Steinhausen, G., 282,314, 315(806) Stepanenko, B. N., 343 Sternlicht, H., 155,158,161(307,308), 163,179(308),201(308) Stillmark, H., 128,254,258(603), 270, 338(603),339(603) Stobo, J. D., 317 Stocked, R. J., 131 Stoddart, R. W., 317 Stone, K.J., 355 Stoudt, T. H., 104, 107(54),108(54) Strauchen, J. A., 300 Streips, U. N.,177 Strominger, J. L., 94,176,355 Strosberg, A. D., 191,198(442a,44213, 442c),235(442c) Stroshane, R. M., 102,104,107(53), 115(53),116(53), 117,119 Subbarao, P. V., 345

Subrahamnyan, V., 343 Suescun, E., 202 Sufoka, A., 380 Sugahara, K., 372,373(286), 374(286) Sugihara, J. M.,32,35(9), 36(9),38(9),

41(9),50(9),52(9),77(9),78(9) Sugimori, T., 123 Sugino, Y., 312,340(796) Sugishita, S., 277,338(670) Sugiyama, N., 346,367,370(265) Suhadolnik, R. J., 122,123,124(86),126 Sukeno, T., 372(292),373,374(305) Sulkowski, E., 141,168(192),178(192),

179(192,418) Sumner, J. B., 134,136(102),140(102),

151,152(257,258),155,166(102), 168(102,352),169(102),173(102), 177(102),179(102),335(258), 338(102,246,247) Sundberg, L., 304 Sundblad, G., 178,216, 217(498),218(498), 219(498),222(498),301(413), 33l(413),339(498) Surolia, A., 141,168(193),177,179(193), 272,274(193),275(659),276, 277(669) Susz, J. P., 326 Sutoh, K., I38,258(150d) Suzuki, H., 171,186(372),363,367 Suzuki, K.,131,307(52),308(52) Suzuki, N., 85,86,87(16), 88(16),95 Suzuki, S., 85,86,87(16), 88(16) Suzuno, R., 157 Svenson, R. H., 293,294(708, 714), 295(708),335(708,713,714) Svensson, S., 62,174,178,282,291(682), 301(413),331(413),349 Sweeley, C. C., 355 Swenson, H. A., 33 Sykes, B. D., 163,164

T Takagi, M., 86, 87(16),88(16) Takahashi, N.,333 Takahashi, T., 134(123),135,142(123),

144(123),194(123),195(123),270, 271(642),272(642),278(676),279, 280(676),296,338(123, 676), 339(641,642) Takatsu, A., 239,240(560),241(560)

AUTHOR INDEX, VOLUME 35 Takayama, K., 355 Takayanagi, G., 315 Takemoto, M., 346,367, 370(265) Takenishi, S., 367 Talbot, C. F., 149,229 Tanaka, K., 134, 177(107),300(107) Tanaka, Y., 376 Tanigaki, Y., l57,324(306b) Taniguchi, M., 104, 107(53,54), 108(54), 115(53),116(53), 117(53),119 Tanner, W., 352,355 Tarentino, A. L., 372(292),373, 374(305) Taylor, J. R., 372(296),373 Taylor, K. J., 378 Tegtmeyer, H., 145,302, 303(219, 758), 304(758),339(758) Teichberg, V. I., 315 Tell, G. P. E., 130, 150(38) Terao, T., 140, 162, 163(332), 165(332), 238(183), 250,251(183), 252(183), 254(183),258, 259(616),261(183, 616), 273(183),275(183), 297(183), 300(183),304(183),310, 318(183), 33 1(183), 335(616), 339(183) Teresa, G. W., 148,267, 268, 269(635), 339(631, 635) Testa, R. T., 110, 112, 113(71) Thambi-Dorai, D., 177 Thierne, T. R., 354 Thimann, K. V., 371 Thomas, D. B., 319,321 Thomas, M. W., 215,223(495a) Thomasson, D. L., 156, 160(297),165 Thompson, T. E., 315 Thorpe, T. A., 366,370(254), 371(254) Thoss, K., 317 Thunell, S., 292 Tichi, M., 134(122),135, 136, 137, 138(142),190(142),191(142,442), 192(442),193(142),194(440,442, 447), 196, 197(440),199(121,440), 201(121,446), 335(142,442,444), 338(142), 339(122,440) Tichf, M., 192 Tierney, B., 39,45(33), 70(33), 72(33), 73(33) Tieslau, C., 130,317(28) Tilley, B. C., 112, 113(71) Timberlake, J. W., 140, 251(184), 252( 184),253(184), 337(184), 339(184) Timell, T. E., ,345,353,367(150)

411

Tisdale, V. V., 292,293(698), 294(698), 335(698) Tiwari, R. D., 344 Tixier, R., 214,215(490b) Tkacz, J. S., 174 Tobiska, J., 132,313(80) Todd, L. S., 179 Tokuyama, H., 310,311(788) Tominaga, S., 177 Tomita, M., 137, 138(139, 144), 202(139), 205( 139), 254(144), 270(144), 271( 144) Toms, G. C., 132 Tonornura, A., 137, 138(143),142(143), 144(143),190(143),191(143), 192(143), 194(143), 195(143),312, 335(143), 338(143) Tookey, H. L., 344,348(61, 62) Torii, M., 172, 176 Torssell, K., 32,48, 49(5) Toyoshima, S., 137, 138(143),142(143), 144(143), 178, 190(143),191(143), 192(143), 194(143), 195(143), 2 97(417), 300(417), 301(417), 304(417),312,335(143), 338(143), 372 Trejo, G., 354 Treska-Ciesielski, J., 326 Trowbridge, I. S., 197, 198(453), 199(453),337(453),340(453) Troy, F. A., 354,355 Tmitt, S. G., 104, 107(53), 115(53), 116(53), 117(53) Tschirch, A., 345, 356(82) Tserng, K.-Y., 130,254(32),271(32) Tsuda, M., 312, 340(796) Tsujisaka, Y., 367 Tully, R. E., 149 Tung, T.-C., 130, 254(32), 255,271(32) Tunis, M., 245, 247(579) Turner, R. H., 130 Tyminski, A., 353

U Uchida, T., 156, 193(300) Uda, F., 86,87(16), 88(16) Uernatsu, T., 123, 124(86) Uhlenbruck, G., 129, 131, 133, 143, 144(206),145(14a),211(206), 212(206),213(206),218,224(502),

412

AUTHOR INDEX, VOLUME 35

227, 239(61, loo), 240, 241(100, 567), 242(61, 100, 556, 567), 243(100, 567), 258(201),259(201), 267, 268(632, 636), 269(632),276(206), 282, 299, 300(132),301(735), 302(736), 303(754),304(755, 761), 305(761), 306(761),307, 314,315(806, 807, 808), 333(632), 338(61, 100, 201, 617, 618), 339(206, 632, 636, 754, 755, 756) Ukita, T., 137, 138(139, 144), 202(139), 205(139), 254(144), 270(144), 271(144) Ulevitch, R. J., 267, 268(633) Ullrich, J., 96 Umemoto, J., 219 Umezawa, S., 81, 102(1) Unrau, A. M., 344,345(65), 346(65), 347(90), 361(69) Unrau, I. C. J., 344 Unzelman, J. M., 356 Uy, R., 138

Vesterberg, O., 366 Vicari, G., 253 Vieweg, H. G., 358 Villafranca, J. J., 157, 158, 161(309), 165(309), 167(309),179(309) Villarroya, E., 363 Villarroya, H., 350, 366, 367, 368(140, 253), 369(140, 253) Villiers, T. A., 358 Viola, R. E., 157, 158, 161(309), 165(309), 167(309), 179(309) Vlodavska, I., 288 VO@, W.-E., 239, 240(563) Vretblad, P., 137 W

Wada, S., 235 Waechter, C. J., 355 Wagenknecht, W., 379 Wagman, G. H., 110, 111 Wagner, M., 317 Wahl, H. P., 99, 101, 109(40) Walborg, E. F., Jr., 215, 223(495a), 272, V 327,328(861, 862,863) Valdovinos, J. G., 371 Waleroft, M. J., 130, 150(33) Valueva, S. P., 55 Waldschmidt-Leitz, E., 270 271(646,647) Van Cleve, J. W., 172 Walker, D. L., 96 Walker, J. B., 98, 104(36), 106, 108(36), Vani.urovi, D., 196 Vandenheede, J. R., 268(637),269, 109(36) 339(637) Walker, M. S., 109 Van Driessche, E., 191, 198(442a,442b) Walker, R. E., 342 Van Landschoot, A,, 216 Wall, H. M., 58,59(84), 62 Van Wageningen, T., 302 Wallach, D. F. H., 323 Van Wauwe, J. P., 134(124),135, Wallenfels, K., 363 140(124),144, 160, 188(181, 323), Walter, M. W., 360 189(181,323), 195, 197(182), Wang, J. L., 137, 138(140), 139, 151, 152, 199(182,211),200(211),273,274(124), 153, 154(263,268,269, 275), 339(124, 211), 340(124),376 155(263, 267,270, 271, 289), 156(268,275), 157(267,268), Vargha, L., 31 Varner, J. E., 360 158(267,268), 160(283,289), 162, Varsharskaya, L. S., 38, 39, 51(27,28, 163(333), 165(333,336), 202(140), 36), 53(27,36), 54(27, 28, 35), 80(27, 203(140,263), 205(140), 224(156), 28, 35, 36, 76) 296(156), 335(267, 271), 336(140), Vartia, K. O., 131 337(140), 339(140) Vasquez, J. J., 145,308(221) Wgngstrbm, B., 349 Verenikina, S. G., 37,38,42(26), 44(26), Ward, R. M., 168, 174(363) 45(24), 54(28,25,46), 56(46), 70(26), Waszczenko-Zacharczenko, E., 132,258, 72(28,25,26,46), 73(26), 78(26), 80 259(614), 313(85) Verma, S. D., 210 Watanabe, K., 216,217(499), 315 Watanabe, S., 380 Vesel?, P., 201

AUTHOR INDEX, VOLUME 35 Watkins, W. M., 129, 131(21, 22), 140(21, 22), 141, 142(22),207, 208(22, 24, 471), 209(471,475), 210(471), 225(24), 226(475), 244, 248(22), 250, 253(22), 277(21), 282(22), 283, 284(22),305(471), 338(21, 471, 475), 339(22) Watson, P. R., 354 Watson, R. R., 100 Watt, W. B., 297 Waxdal, M. J., 152, 155(267, 270, 271), 157(267), 158(267), 310, 335(267, 271) Webb, E. C., 148 Webber, J. M., 45, 50, 51(67), 53(67), 54(67), 56(67), 72(49), 77(67), 78(67) Weber, T. H., 292, 293, 294(715, 716), 295, 335(715, 716) Wecksler, M., 150 Wei, C. H., 255, 256, 257, 271, 334(160, 607), 335(607, 610) Weigel, H., 35, 38,42(23), 43, 45, 50(23), 51(23), 53(23,43,48), 54(23), 55(23, 43, 48), 57(23), 58,60(86), 66, 67(10), 72(23, 48), 77(23,43), 78(23,43), 172 Weinbaum, G., 123 Weiner, H., 107 Weiner, I. M., 355 Weinstein, B., 341(7), 342 Weinzierl, J., 156, 161(296) Weisenthal, L. M., 164, 248(342) Weiss, A. K., 132, 139(94) Weisz, J., 65 Weith, H. L., 63 Wellum, G. R., 34,35(11) Welsch, P. D., 139,309(162) Wester, D. W., 39 Western, A., 132 Westholm, F. A., 151, 152(264) Westoo, A., 240, 241(569), 243(569), 335(569) Whistler, R. L., 343,344,347, 348, 349, 350( 128), 362, 370(220) Whitaker, P. M., 379 Whyte, J. N. C., 345,346(85), 347(85) Wickstrbm, A., 345, 348(86) Wiebers, J. L., 38, 63, 68(29) Wiecko, J., 69, 70 Wiener, A. S., 145 Wilkinson, K. D., 135, 137(125), 138(125), 142(125), 146(125),

413

206(125), 207(125), 262( 125), 265( 125),335(125), 338(125) Williams, D. G., 378 Williams, J., 350, 368(140), 369(140) Williams, J. H., 58 Williams, N. R., 58, 62 Williams, P., 140, 144(167), 278(167), 279( 167), 280(167), 284( 167), 285( 167), 338(167) Williams, T. J., 185 Williamson, J. R., 343 Williamson, P., 144 Willoughby, E., 355 Wilson, K., 283, 284(684), 335(684) Wingham, J., l32,224,226(508d),257 Winter, W. T., 378,384 Wintzer, G., 299 301(735), 302(736) Winzler, R. J., 319,321 Wirtz-Peitz, F., 47,48(56), 55(56), 65(56), 67(56), 68(56), 69(56) Wisnieski, J. J., 179, 184(424) Wissler, F. C., 307 Wold, F., 138 Wolfrom, M. L., 32, 38(7), 43(7), 44(7), 50(7), 52(7), 72(7), 73(7) Wolpert, J. S., 147 Wood, C., 208 Wood, G., 96 Wood, M. K., 152 Wood, P. J., 38,43,45(32), 53(32), 65, 67(32,44, 99),70(32, 441, 72(32,44), 73(32,44) Woodbury, R. R., 49,63(64), 65(64) Woodruff, J., 297 Woodside, E. E., 159, 168, 179(366) Worth, H. G. J., 360 Wray, V. P., 327 Wright, A., 166, 168(357, 358), 169,355 Wright, C. S., 215, 216(496a) Wright, D. C., 58 Wu, T. T., 154 Wuilmart, C., 191, 198(442c), 235(442c)

Y Yabroff, D. L., 36 Yachnin, S., 134, 267,294(630, 714), 295(630, 712), 301(111), 335(630, 712, 713,714) Yahara, I., 152, 154(269), 162, 165(336) Yamamoto, T., 366,367(251)

4 14

AUTHOR INDEX, VOLUME 35

Yamamura, Y., 376 Yamano, T., 120, 122(81) Yamashina, I., 372, 373(286),374(286) Yamazaki, S., 85, 95(14) Yancik, J. J., 342 Yang, C. K., 246 Yang, W.-K., 239, 255,334(607), 335(607) Yang, Y., 168, 172(362),173(362),178 Yano, O., 310 Yariv, J., 138, 153, 155(278), 179(278), 283, 284(684),335(684) Yasuda, Y., 333 Yesner, I., 297 Yokoyama, K., 310 Yomaguichi, T., 239 Yomo, H., 360 Yonehara, H., 124 Yoshimura, J., 85, 95(14) Yosizawa, Z., 248,250,253(596) Youle, R. J., 149 Young, F. E., 177 Young, N. M., 134(123),135, 142(123), 144(123), 162, 178(log), 190, 191(441), 192(441),193(441),

194(109, 123), 195(123),335(441), 338(109, 123) Yudis, M. D., 110 Yueh, M. H., 341,342 Yurkevich, A. M., 37, 38,39, 42(26), 44(26), 45(24), 46,49, 51(27,28, 36), 52, 53(27,30, 36), 54(24,25, 27, 28, 30, 35,37,46,47, 71), 55(77), 56(46, 71, 72, 74), 57(73), 63, 64,70(26), 72(24, 25,26,46,47), 73(26), 78(26), 80(27, 28,30, 35, 36, 37, 71, 73, 74, 75, 76, 77)

2

Zand, R., 153,263 Zanetta, J.-P., 326, 327(857) Zaslow, B., 378 Zeissig, A., 150, 338(246) Ziaya, P. R., 229 Ziegenfuss, E. M., 341(11), 342 Ziska, P., 205 Zweifel, G., 58

SUBJECT INDEX FOR VOLUME 35

A

Aaptos papillata lectins, isolation and properties, 316 Abrin carbohydrate-binding specificity, 255 immunization to toxic, 129 isolation and purification, affinity chromatography, 138 purification and properties, 254-257 Abrus precatorius, seed-extracts, hemagglutinating and toxic activities, 254-257 Abrus precatorius lectin, see Abrin Acetylation, of carbohydrate boronates,

53 Actinamine, biosynthesis, 119 Actinospectinoic acid, structure, 119 Adenine 9-P-D-arabinofuranosyl-,biosynthesis, 123, 124 arabinosyl-, biosynthesis, 124 Adenosine complex formation with phenylboronic acids, 49 5'-phosphate, preparation, 54 -, 3'-amino-3'-deoxy-, biosynthesis, 123 -, 5'-0-trityl-, preparation, 55 Agaricus bisporus lectin, see Mushroom lectin Agglutinins, see also Lectins plant, history, 128 Aldgamycin C , periodate oxidation, 90 Aldgamycin E, components, and degradation, 89-91 Aldgarose, D, biosynthesis, 89-91 Alditols borinate-boronates, preparation, 40 boronates, acetates and benzoates, 53 preparation and structure of, 42,43 properties, of, 77,78 column chromatography, boronic acids in, 63 electrophoresis, sulfonylated phenylboronic acids in, 62 gas-liquid chromatography, boronic acids in, 65

interaction with lectins, 180 paper chromatography, phenylboronic acid in, 60 Aldohexose 2,3,6-trideoxy-46-(2-hydroxyacetyl)L-threo-, 96 Algae, polysaccharides of' marine, 8, 9 Alginic acid, structure, 7, 8, 10 Allopyranoside methyl 6-deoxy-p-~-,2,4-phenylboronate, oxidation, 57 preparation, 48 Amino acids of asparagus-pea lectin, 284 of Bauhinia purpurea alba lectin, 305 of castor-bean lectin, 272 of concanavalin A, 162 of discoidin, 309 of eel-serum lectin, 281 of horse-shoe-crab lectin, 306 of lectins, 335,337 of lentil lectin, 191-193 of lima-bean lectin, 247 of Osage-orange lectin, 267 of pea lectins, 197 of peanut lectin, 258 of potato lectin, 211 of red kidney-bean lectin, 292,293 of ricin, 271 of snail lectin, 240 of soybean lectin, 234 of sun-hemp lectin, 306 of Ulex europeus lectin, 289 of wheat-germ lectin, 215 Amino acid sequence, of concanavalin A, 152 Amylopectin protozoal, 11 reaction with concanavalin A, 169-171 Amylose, V-, crystal structure bibliography, 378 Anguilla anguilla serum lectin, see Eelserum lectin Anhydro sugars boronates, properties, 79 paper chromatography, phenylboronic acid in, 60

415

SUBJECT INDEX, VOLUME 35

416

Antibiotics aminocyclitol, biosynthesis, 81, 102-122 anthracycline, 91-96 biosynthesis of sugar components, 81-126 macrolide, glycosidic, 81 nucleoside, biosynthesis, 122-126 Antibodies, determination, 129 Antibody activity, of lectins, 147 Antigens, lectins and blood-group, 129 Apiose, D-, biosynthesis, 100, 101, 125 Arabinitol, 1,5-dideoxy-~-, phenylboronate, structure, 43 Arabinogalactans, interaction with concanavalin A, 179 Arabinomannans, interaction with concanavalin A, 179 Arabinopyranose, 1,2-O-isopropylidenep-L-,3,4-phenylboronate, preparation, 39 Arabinose, L-, butylboronates, hydrolysis, 51 preparation, 43 phenylboronates, hydrolysis, 50, 51 preparation, 43 Arachis hypogaea lectin, see Peanut lectin Ascorbic acid, history, 5, 6 Asparagus-pea lectin carbohydrate-binding specificity, 285-288 composition, 284 isolation, 138 purification, 283 Axinella polypoides lectins, isolation and properties, 316

B Bandeiraea simplicifolia lectin, isolation of, 138 Bandeiraea simplicifolia I lectin carbohydrate-binding specificity, 264-266 purification and properties, 262 Bandeiraea simplicifolia I1 lectin carbohydrate-binding specificity, 206 isolation, properties, and structure, 206-208 Bandeiraea simplicifolia seeds, lectin isolation, 137

Barley lectin, isolation and properties, 314 Barry degradation, of carbohydrates, 9 Bauhinia purpurea alba lectin carbohydrate-binding specificity, 305 composition and purification, 305 isolation, 138 Beans, lectins and toxic properties, 130 Benzoylation, of carbohydrate boronates,

53 Bibliography of crystal structures of polysaccharides, 377-385 of Edmund L. Hirst and colleagues publications, 17-29 Biochemistry, of plant galactomannans, 34 1-376 Biosynthesis of antibiotic sugar components, 81-126 of plant galactomannans, 352-356 Black-locust lectin, see Robinia pseudoaccacia lectin Blasticidin S, biosynthesis, 124, 125 Bluensidine, biosynthesis, 105, 107 Bluensomycin, structure, 102, 103 Boranediyl, nomenclature, 36 Boric acid, reaction with D-glucose, 31 Borinates boronates from, 39-41 carbohydrate, preparation, 70 Boronates in aqueous solutions, 48-52 carbohydrate, 5, 31-80 esterification, 53 etherification, 55 hydrolysis, 50-52 mass spectrometry, 65-70 nomenclature, 36 nuclear magnetic resonance spectroscopy, 70 nucleophilic displacement reactions,

55-57 oxidation, 57 preparation and structure, 43-45 properties, 72-80 removal of boronate group, 52 in separation of carbohydrates, 57,

58 stability, 35, 53-55 structure, 33,41-48 synthesis, 37-41

SUBJECT INDEX, VOLUME 35 Boronic acids in biochemistry, 32 in column chromatography, 63-65 in gas-liquid chromatography, 65 interactions with carbohydrates in aqueous solutions, 48-52 Borylene, nomenclature, 36

C Calcium hyaluronate, crystal structure bibliography, 383 Canavalia ensifomis, see Jack bean Canavalia ensifomis lectin, see Concanavalin A Cancer therapy, lectins in, 130 Carugana uborescens lectins, purification, composition, and properties of,

3 13 Carbamic acid, N-phenyl-, esterification of carbohydrate boronates by, 54 Carbohydrate-binding specificity, of lectins, 139-145,331-333, see also specific lectins Carbohydrates boronates, see Boronates interaction with boronic acids in aqueous solutions, 48-52 phosphates, mass spectrometry of boronates, 69 separation, by use of boronates, 57, 58 Carob galactomannan, structure, 349 Carob gum, structure, 349 Castor-bean extracts, agglutinating action, 128 Castor-bean lectin, see also Ricin carbohydrate-binding specificity,

273-276 interaction with cellular structures,

317 isolation, purification, and properties of, 137,270,272 Cellobiose, and cellulose structure, 5 Cells, see also Neuronal cells; Tumor cells interaction with lectins, 317-333 Cellulose regenerated, crystal structure bibliography, 379 structure, 5 -, sodio-, crystal structure bibliography,

379

417

-, Op-tolylsulfonyl-, crystal structure bibliography, 380 Cellulose I, green algae, crystal structure bibliography, 381 Cellulose 11, crystal structure bibliography, 379,380 Cellulose 111, crystal structure bibliography, 380 a-Chitin, crystal structure bibliography,

38 1 P-Chitin, crystal structure bibliography,

38 1 Chitosan, crystal structure bibliography,

382 Chloroacetylation, of carbohydrate boronates, 54 Chromatography affinity, of concanavalin A, 157 in lectin isolation and purification,

137 column, boronic acids in, 63-65 gas-liquid, boronic acids in, 65 history, 6 paper, phenylboronic acid in, 58-62 Cladinose, L-, biosynthesis of, 82 Cobalt compounds, carbohydrate, preparation, 56 Coffee beans, a-D-galactosidase from, 363 Concanavalin A acetylation and succinylation, 162 amino acids, 161, 162 amino acid sequence, 152 carboh ydrate-binding specificity, 142,

143,157-164,179-190,204 complex formation, 163, 168, 178 crystal structure, 152 hemagglutinating activity, 201 interaction with amylopectin, 169-171 with cellular structures, 317 with dextrans, 166, 171-173 with D-fnictans, 175 with glycogen, 169-171 with glycoproteins, 177-179 with mannans, 173-175 with polysaccharides, 166-169, 179 with teichoic acids, 175-177 isolation, 136, 138 of jack bean, physical and chemical characterization, 150-157 mono-, di-, and tetra-valent, preparation and biological activities, 164,

165

418

SUBJECT INDEX, VOLUME 35

preparation and properties, 150, 151 structure, 153-155 Cordycepin, biosynthesis, 123 Cosmetics, plant galactomannans, 342 Cotyledon, galactomannans, 346, 356-361 Crotalaria juncea lectin, isolation, purification, and properties of, 306 Crystal structure of concanavalin A, 152 of polysaccharides, bibliography, 377-385 Cucumber, wild, lectin, isolation, 138 Cyclitol, amino-, antibiotics, biosynthesis, 81, 102-122 1,3-cis-Cyclohexanediol,cyclic boronates, preparation, 36 1,4-Cyclohexanediol, cyclic boronates, preparation, 36 Cycloheximide, inhibitor of galactomannan degradation, 360 Cytidine, 5'-phosphate, preparation, 54 Cytisus sessilifolius lectin carbohydrate-binding specificity of, 209 hemagglutinating activity, 208 Cytosine, 1-(P-D-glUCOpyranOSylUrOniC acid)-, formation, 124 isolation, 124

D Deboronation, of boronates, 52 Decoyinine, biosynthesis, 123 Degradation, see also Barry degradation; Smith degradation biochemical, of galactomannans, 356-375 Desosamine, D-,biosynthesis, 84, 122 Dextrans interaction with concanavalin A, 166, 171-173 with lentil lectins, 194 Dictyostelium discoideum lectin, isolation and properties, 308 Dinucleoside phosphates, synthesis, 54 1,3,2-Dioxaborinane, nomenclature, 36 -, 5-hydroxy-2-phenyl-, nomenclature, 36 1,3,2-Dioxaborolane, nomenclature, 36 -, 2-methyl-, preparation, 39 1,3,5,2,4-Dioxazadiborepane,2,4-

diphenyl-, formation, 67 Disaccharides interaction with concanavalin A, 184 with lectins, 142 structure, 5 Discoidin, preparation, composition, and properties of, 309 Dolichos bijlorus lectin carbohydrate-binding specificity, 229 circular dichroic spectrum, 230 hemagglutinating activity, 226 isolation, 138 purification and properties of, 227-229 uses, 231 Drilling, well, plant galactomannans in, 342

E Echinocystis lobata lectin, isolation, 138 Eel, electric, electrolectin from, 315 Eel-serum lectin agglutinating activity, 277 carbohydrate-binding specificity, 277-279 purification, 281,282 Ehrlich cells, lectin-reactive, 329,330 Electrolectin, isolation and properties, 315 Electrophoresis affinity, for lectin detection, 135 sulfonylated phenylboronic acid in, 62 Electrophorus electricus, electrolectin, properties, 315 Endo-p-D-mannanase, in galactomannan degradation during germination of seeds, 361 Endosperm, galactomannans in, 345, 355,356-361 Enzymes in biosynthesis of tylosin, 84 for galactomannan degradation, 361-375 Enzymic analysis, of carbohydrates, 11 Erythritol, 4-deoxy-~-,phenylboronate, structure, 43 Erythrocytes lectin-reactive glycoproteins, 318-325 lentil lectin in hemagglutination, 193 Erythromycin A, formation, 85 Erythromycin C, biosynthesis, 84

SUBJECT INDEX, VOLUME 35 Esterification of carbohydrate boronates, 53 l,e-Ethanediol, reaction with trimethylborane, 39 Etherification, of carbohydrate boronates, 55 Euonymus europeus lectin carbohydrate-binding specificity, 145 purification and properties, 313 Explosives, plant galactomannans in, 342

F Fava-bean lectin agglutinating activity, 201,202 carbohydrate-binding specificity, 203-205 isolation, 138 purification and properties, 202 Favism, fava-bean, 202 Fenugreek seeds components of immature, 352,355 galactomannan biosynthesis in, 352,

355 galactomannan location in, 345 a-D-galactosidase in, 365 germination, galactomannan degradation during, 356-361 P-”mannanase from, 367 P-D-mannosidase in, 374 oligo-P-Dmannos yl-( 1+4)phosphorylase from, 375 Fetuin, separation, 277 Fire-fighting, plant galactomannans in, 342 Food, plant galactomannans in, 341 Frog-egg lectin, isolation and properties, 315 Fructans interaction with concanavalin A, 175 structure, 7 Fructopyranose, P-D-, 2,3:4,5-bis(phenylboronate), preparation, 45 Fructosans, review, 8 Fructose, D-, complex formation with phenylboronic acid, 48 polymers, 7 -, 3,4- and 4,5-dia-methyl-~-,synthesis, 11 -, 4-0-methyl-D-, synthesis, 11

419

-, tri-0-methyl-D-, synthesis, 11 L-Fucose-binding lectins, 277-291 Fucoxylomannan, isolation, 282 Furze seed, see Uler europeus I1

G Galactan, e-, from larch, 10 Galactitol 1,6-bis(diethylborinate) 2,3:4,5-bis(ethylboronate), preparation, 40 2,3:4,5-his(ethylboronate),preparation, 41 1,6:2,3:4,5-tris(ethylboronate),preparation, 40, 43 tris(phenylboronate), preparation, 4 1 Galactomannan, peptido-0-phosphono-, biosynthesis, 354 Galactomannan depolymerase, in seed germination, 361 Galactomannans biochemistry of plant, 341-376 hiosynthesis, 352-356 function, 375,376 isolation, 345 location in oiuo, 345 metabolism during seed germination, 356-361 in micro-organisms, 354 occurrence, 343-345 structure, 347-351 uses, 341,342 Galactopyranoside, methyl 6-deoxy-a-~-, 3,4-phenylboronate, preparation, 48 -, methyl 2,3-di-O-benzoyl-a-~-,preparation, 53 Galactose, D-,

in endosperm during germination, 357 lectins, 254-277 -, 2-acetamido-2-deoxy-D-, lectins, 226-254 -, 6-deoxy-a-~-,1,2:3,4-bis(phenylboronate), preparation of, 45 -, 6-0-methyl-D-, isolation of, 7 a-DGalactosidase in galactomannan degradation during germination of seeds, 362-366 multimolecular forms, 365 specificity, 364

SUBJECT INDEX, VOLUME 35

420

a-D-Galactoside galactohydrolase, in galactomannan degradation, 362 Carosamine, L-, biosynthesis, 112 Centarnicins, structure, 110-115 Germination galactomannan degradation during, of seeds, 356-361 lectins during, 149 Glebomycin, biosynthesis, 102 p-D-Glucan, structure, 7, 8, 10 Glucofuranose, 3-deoxy-3-fluoro-1,2-0isopropylidene-a-D-, 5,6-phenylboronate, preparation, 4 5 -, 1,2-0-isopropylidene-a-~3,5-borate, preparation, 31 3- and 6-chloroacetates, preparation,

54 3,5-phenylboronate, preparation and use, 32 Glucomannan, biosynthesis, 353 Glucono-l,4-lactone, 6-O-(N,N-dimethylglycyl)-D-, synthesis, 54 Glucopyranose, 1,6-anhydro-P-~-,2,4phenylboronate, preparation, 48 Glucopyranoside, methyl a - ~ 4,6-phenylboronate 2,3-(diphenylcyclodiboronate), hydrolysis, 52 preparation, 47 phenylboronate, preparation, 38,46, 52 -, methyl 6-deoxy-a-, 2,4-phenylboronate, preparation, 48 -, methyl 2,3-di-O-benzoyl-a-~-,preparation, 53 Glucopyranosides, interaction with concanavalin A, 188-190 Glucose D-,

-, -, -, -,

1,2:3,5-bis(phenylboronate),preparation, 45 complex formation with phenylboronic acid, 49 reaction with boric acid, 31 2-acetarnido-2-deoxy-~-, lectins, 206-226 1,8anhydro-n-, 2,4-phenylboronate, reaction with rnethacrylic anhydride, 55 6-bromo-6-deoxy-D-, preparation from boronate, 56 6-chloro-6-deoxy-D-,preparation from boronate, 56

-, 2-deoxy-2-(rnethylamino)-~-, biosynthesis, 107-109 -, 6-O-(N,”-dimethylglycyl)-~-, synthesis, 54

-, 1,2-O-isopropylidene-a-D5,6-diphenylcyclodiboronate, preparation, 45 3,5-phenylboronate, preparation, 45 -, 6-O-methyl-D-, preparation, 55 Glucuronic acid, D-, methyl ethers, synthesis, 11 Glutarimide, 3-[2-(3,5-dimethyl-2oxocyclohexyl)-2-hydroxyethyl]-, inhibitor of galactomannan degradation, 361 a-D-Glycans, crystal structure bibliography, 378 P-D-Glycans, crystal structure bibliography, 379-381 Glycerol ethylboronate, structure, 4 3 phenylboronate, nomenclature, 36 properties and structure, 42 -, 3-O-glycopyranosyl-, mass spectrometry of phenylboronates, 68 -, 1,3-O-(phenylboranediyl)-, nomenclature, 36 -, 1,3-O-(phenylborylene)-, nomenclature, 36 Glycine mox lectin, see Soybean lectin Glycogen end-group assays, 8 reaction with condanavalin A, 169-171 separation from guaran, 277 structure, 5 Glycolipids, Dolichos biflorus lectin in study of, 231 Glycopeptides concanavalin A-reactive, from calf thymocytes, 325 interaction with concanavalin A, 187 with lectins, 140 lectin-reactive, from human erythrocytes, 319 from tumor cells, 327-333 from neuronal cells, lectins in isolation of, 326 pea lectin-reactive, structure, 321 Glycoproteins, see also Lectins cell-surface lectin-reactive, 205, 317-333 distribution and mobility, 129

SUBJECT INDEX, VOLUME 35

Dolichos biflorus lectin in study of, 23 1 interaction with concanavalin A, 177-179 with lectins, 140, 141 with lentil lectins, 184 lectin-reactive, of erythrocyte membrane, 318-325 from tumor cells, 328-333 lentil lectin-reactive, from pig lymphocyte, 325 from neuronal cells, lectins in isolation, 326 from platelet membrane, 325 separation of partially sialated, 277 Glycopyranosides, ethylhoronates, preparation, 41 Glycosaminoglycans, crystal structure bibliography, 38 1-385 Gl ycosides horonates, acetates and henzoates, 53 preparation, 45-48 properties, 72-76 reaction with lectins, 140 with phenylboronic acid, 32 Glycosid-3-uloses, preparation, 46 Glycosylation, of phenylboronates, 55 Gorse-seed extract, see Ulex europeus I1 Guanosine, 5’-phosphate, preparation, 54 Guaran separation from glycogen, 276 structure, 349 Guar gdactoniannan, structure, 349 Guar seeds enzymes in galactomannan degradation during germination, 361 a-D-galactosidase from, 364 P-D-mananase from, 367 Guluronic acid, L-, of alginic acid, 8, 10 Gums plant, constitution, 6 review, 8 sbucture, 9 H Helix pomatiu lectin, see Snail lectin Hemagglutination tests, for lectins, 133, 140 Hemagglutinins, see Lectins Hemicelluloses, problems, 8, 10

42 I

Heparan sulfate, crystal structure hibliography, 383 Heparin, crystal structure bibliography,

382 1,6-Hexanediol, cyclic phenylhoronate, preparation, 35 Hexopyranose, 2,6-dideoxy4C-acetyI-~xylo-, component of quinocycline B and isoquinocycline B, 92 -, 2,6-dideoxy4C-(l-hydroxyethyl)-~xylo-, component of quinocycline A and isoquinocycline A, 91 Hexose, 2,3,6-trideoxy-46-(2-hydroxyacety1)-L-threo-, see Pillarose Hirst, Edmund Langley, obituary, 1-29 Hoinarus americanus lectins, isolation,

3 13 Horse gram, see Dolichos biflorus Horseshoe-crab lectin, isolation, purification, and composition, 306 Hyaluronates, crystal structure hihliography, 383-385 Hyaluronic acid, and salts, crytal structures of, 384,385 Hydrolysis, of carbohydrate horonates, 50-52

I Immunoglobulins, myeloma, interactions with polysaccharides, 317 Immunology, lectins role in, 128 Infrared spectroscopy, and carbohydrate boronate structure, 42 Inositols, paper chromatography, phenylhoronic acid in, 60 Inulin, structure, 7 Isolectins of Bandeiraea simplicifolia I, 266 from haricot kidney-bean, 297 lentil, properties, 190,192 pea, 197 ofPhaseolus vulgaris, 294 Isoquinocyclines A and B, components of, 91

J Jack-bean lectin, see Concanavalin A Jack beans, lectin isolation from, 137 Japanese pagoda tree, see Sophora japonicu Jequirity bean, see Abrus precatoriiis

SUBJECT INDEX, VOLUME 35

422

K

lectin isolation from, 137, 138 Lentil lectin carbohydrate-binding specificity, 194, 204,205 hemagglutinating activity, 190,201 L interaction with cellular structures, Laburnum alpinum lectin, 208 317 carbohydrate-binding specificity of, 305 with erythrocyte glycopeptide, 320 Landsteiner hapten-inhibition techisolation and properties, 138, 190-196 nique, for lectins, 139 structure, 190-192 Lectins, 127-340, see also Isolectins and Lettuce seeds, p-D-mannanase and gerspecific lectins mination, 371 2-acetamido-2-deoxy-~-galactoseLeucoagglutinin, purification and binding, 226-254 composition of, from Phaseolus 2-acetamido-2-deoxy-~-glucose-binding, vulgaris, 295 206-226 Levans antibody activity, 147 interaction with concanavalin A, 175 applications in serological laboratories, plant, structure, 7 129 Lima-bean extracts, agglutinating action, blood-group and carbohydrate-binding 128 specificities of purified, 338,339 Lima-bean lectin carbohydrate association, 340 agglutinating activity, 243,244,247 carbohydrate-binding specificity, carbohydrate-binding specificity, 249 139-145,331-333 composition, 247 classification, 133, 141, 146 isolation, 138 definition, 128, 131 purification, 245,246 detection, 133-136 Limulus polyphemus lectin, see L-fucose-binding, 277-291 Horseshoe-crab lectin functions, 146-149 Lipopolysaccharides, interaction with D-galactose-binding, 254-277 concanavalin A, 179 interaction with cellular structure, Lotus tetragonolobus lectin, see 317-333 Asparagus-pea lectin with glycosides, 140 Lymphocytes, lectin-reactive with polysaccharides, glycoproteins, glycoproteins from, 325 and glycolipids, 140 isolation and purification, 136-139 in life cycle of plant, 148 Kidney-bean extracts, effect on lymphocyte division, 130

D-mannose(D-glucose)-binding,

150-205 nomenclature, 145, 146 physical and chemical properties, 133, 334-337 reviews, 132 sources, 139 toxicity, 149 uses, 129 Lens culinaris, see Lentil Lens esculenta, lentil lectin from, 193 Lentil large-seed and small-seed, lectins from, 193

M

Maackia amurensis lectins, isolation, purification, and properties, 313 Maclura pomifera lectin, see Osageorange lectin Macrolide antibiotics, glycosidic, 81 Maleylation, of lentil lectin, 196 P-D-Mannanase action on galactomannans, 350 activity, occurrence, and properties, 366-371 Mannans interaction with concanavalin A, 173-175

SUBJECT INDEX, VOLUME 35 structure, from ivory nuts, 7 Mannitol

423

Mining, plant galactomannans in, 342 Mitogenesis, lectin-induced, 291 Mitogenic activity D-, 3,4-ethylboronate, preparation, 41 of Phytolacca lectins, 309,311 1,2,5,6-tetrakis(diethylborinate)3,4of Wistaria floribunda lectins, 311, ethylboronate, preparation, 40 312 1,2:3,4:5,6-triboronate,preparation, Molecular weight 40 of asparagus-pea lectin, 284 tris(phenylboronate), hydrolysis, 50 of castor-bean lectin, 272 Mannofuranose, 2,3-O-isopropylidene-D-, of concanavalin A, 152 5,6-phenylboronate, preparation, 39 of fava-bean lectin, 203 Mannopyranoside, methyl a-D-, 2,3- and of lectins, 334, 336 4,6-phenylboronates and 2,3:4,6-bisof Osage-orange lectin, 268 (phenylboronate), preparation, 48 of ricin, 271 methyl 6-deoxy-a-~-,2,3-phenylof Ulex europeus lectin, 289 boronate, preparation, 48 Monosaccharides Mannopyranosides, alkyl and aryl, interaction with concanavalin A, interaction with concanavalin A, 188 181-184 Mannose, lectin-reactive, classification, 141 D-, complex formation with phenylstructure, 5,7 boronic acid, 49 Mucilages structure, history, 5 constitution, 6 P-D-Mannosidase structure of plant, 9 in galactomannan degradation during Mucoprotein, from Phaseolus vulgaris, germination of seeds, 362, 292 366-371 Mung-bean seedlings, enzyme from, 353 occurrence, purification, and properMushroom lectin ties, 372-375 carbohydrate-binding specificity, 145 Mannoside, methyl a-D-,phenylboronate, interaction with cellular glycopeptides, preparation, 38 318 Mannuronic acid, with erythrocyte glycopeptide, 320 D-, of alginic acid, 10 isolation, purification, and structure, methyl ethers, synthesis, 11 308 Mass spectrometry, of carbohydrate Mycaminose, D-, biosynthesis, 122 boronates, 41,65-70 Mycarose, L-, biosynthesis, 82,83, 88 Meadow-mushroom lectin, see Mycodextran, crystal structure bibliograMushroom lectin phy, 378 Melibiase, in galactomannan degradation during germination of seeds, N 362-366 Neomycins, biosynthesis, 115-118 Methacrylic anhydride, reaction with Neuronal cells, glycoproteins and 1,6-anhydro-D-glucose 2,4-phenylglycopeptides from, isolation and boronate, 55 Methylation properties of, 326 Nigeran, crystal structure bibliography, of carbohydrate boronates, 42, 55 378 carbon, of sugars, 83 Nomenclature in structural analysis, 5 of carbohydrate boronates, 36 Microanalysis, of sugars, 5 furanose and pyranose, 5 Micro-organisms of lectins, 145, 146 galactomannans in, 354, 376 Noviose, L-, biosynthesis, 82 p-D-mannanase in, 366

SUBJECT INDEX, VOLUME 35

424

Nuclear magnetic resonance spectroscopy, of carbohydrate boronates, 41, 70 Nucleoside antibiotics, see Antibiotics Nucleosides arabinosyl, biosynthesis, 123 boronates, acetates and benzoates of,

53 preparation, 38,45-48 properties, 80 cobalt-containing, preparation, 56 column chromatography, boronic acids in, 63,64 complex formation with boronic acids,

49 cytosine, isolation, 124 phenylboronates, hydrolysis, 51 mass spectrometry, 68 Nucleotides column chromatography, boronic acids in, 64 complex formation with boronic acids,

49 phenylboronates, mass spectrometry,

68 0 Obituary, Edmund Langley Hirst, 1-29 Oligo-P-D-mannosyl-(1+4)-phosphorylase, in galactomannan degradation during germination of seeds, 362,375 Oligosaccharides interaction with asparagus-pea lectin, 286-288 with concanavalin A, 181-186 with lectins, 142 with wheat-germ lectin, 219 structure, 5 Optical rotatory dispersion, of sugars, 5 Osage-orange lectin carbohydrate-binding specificity, 268-270 isolation, purification, and properties, 267 1,3,2-Oxazaborolane, 2-phenyl-, formation, 67 Oxidation, of carbohydrate boronates, 57

P Pachyman, 0-acetyl-, crystal structure bibliography, 381

Paints, plant galactomannans in, 342 Pangamic acid, synthesis, 54 Pangamolactone, synthesis, 54 Paper products, plant galactomannans in,

342 Paromamine, formation, 113, 114 Pea lectin, carbohydrate-binding specificity, 199, 204 hemagglutinating activity, 197, 201 interaction with erythrocyte glycopeptides, 321 with glycopeptides, 141 isolation, 138, 196 purification, 196 Peanut lectin, 257-262 biological activity, 261 carbohydrate-binding specificity,

259-261 isolation, 138 purification and properties, 258 Pea-tree lectin, composition, purification, and properties, 313 Pectic substances, constitution, 6 1,5-Pentanediol, cyclic phenylboronate, preparation, 35 Pentopyranosid3-ulose, methyl (Y-Dand P-D-erythro-, preparation by oxidation of boronate, 57 P e p t i d o e -phosphonogalactomannan, biosynthesis, 354 Periodate oxidation of carbohydrate phenylboronates,

57 in structural analysis of polysaccharides, 8 Pharmaceuticals, plant galactomannans in, 341 Phaseolus coccineus lectin, carbohydrate-binding specificity, 145 Phaseolus lunatus lectin, see Lima-bean lectin Phaseolus uulgaris lectin, see Red kidney-bean lectin Phenol, ep-dinitro-, inhibitor of galactomannan degradation, 361 Phenylboronates, stability to hydrolysis,

50 Phenylboronic acid complex formation with carbohydrates,

48,49 effect in paper chromatography, 59-62 reaction with glycosides, 32

SUBJECT IND,EX, VOLUME 35 sulfonylated, in electrophoresis, 62 Phosphates, carbohydrate, mass spectrometry of boronates of, 69 Phosphorylation, of nucleoside boronates, 54 Phytohemagglutinin, *seealso Lectins from red kidney-beans, 291, 293 Phytolacca americanum, see Poke-weed Phytolacca esculenta lectin, isolation and properties, 310 Pillaromycin A, structure, 96 Pillarose, structure, 96 Pisum satious lectin, see Pea lectin Plant life-cycle, lectins in, 148 Plasters, plant galactomannans in, 342 Platelet membrane, glycoproteins from, 325 Poke-weed extract, mitogenic activity, 309,310 Poke-weed lectin, isolation and properties, 309 Polyagglutinability, in serology, 257 Polyoxins, biosynthesis, 125, 126 Pol ysaccharides amino, crystal structure bibliography,

381-385 conformational analysis, 8 crystal structure bibliography, 377-385 interaction with castor-bean lectin, 274 with concanavalin A, 166-169, 179 with lectins, 140 with lentil lectins, 1% with myeloma immunoglobulins, 317 plant, structure, 10 structure, 5, 7 Polysphondylium pallidum lectin, isolation and properties, 309 Potato lectin amino acids, 211 carbohydrate-binding specificity, 212 hemagglutinating activity, 210 isolation, 138 purification, 210 Prolectin, isolation, 149, 229 Proteins, carbohydrate-binding, of plants and animals, see Lectins Psicofuranine, biosynthesis, 123

Q Quinocyclines A and B, biosynthesis, 91-96

425

R Raffinose, structure, 5 Rana catesbiana lectin, isolation and properties, 315 Red kidney -)bean, phytohemagglutinin, 291,293 Red kidney-bean lectin carbohydrate-binding specificity, 145, 297-302 composition, 292 hemagglutinating and mitogenic activity, 291, 292 interaction with cellular glycopeptides, 318-320 isolation, 138,296,297 purification, 292 structure, 267 Ribitol, 1,5-dideoxy-, phenylboronate, structure, 43 Ribofuranose, p-D-, 1,5:2,3bis(phenylboronate), preparation and structure, 44 Ribonucleosides, reaction with isobutyl diphenylborinate, 39 Ribopyrdnosylamine, N-( p-bromopheny1)-a-D-, 2,4-phenylboronate, preparation and structure, 46 Ribose, CY-D-,2,4-phenylboronate, preparation and structure, 44 Ricin carbohydrate-binding specificity, 274 immunization, 129 isolation, 270 purification, 138, 271 toxicity, 271 Ricinus communis lectin, see Castor-bean lectin; Ricin Robinia pseudoaccacia lectin robin, interaction with erythrocyte glycopeptide, 320 isolation and properties, 311

S Scarlet runner-bean lectin, carbohydratebinding specificity, 145 Serology Dolichos bijlorus lectin in, 226 Helix pomatia lectin in, 226, 239, 241 lectins in, 129 polyagglutinability in, 257 Ulex europeus seed extract in, 224,289

SUBJECT INDEX, VOLUME 35

426

Sheep’s rumen, carbohydrates, 11 Sialoglycopeptide, from tumor cells, 327 Sialoglycoprotein, properties of, of erythrocyte membrane, 318, 319 Smith degradation, of carbohydrates, 9 Snail lectin carbohydrate-binding specificity, 242 composition, 240 hemagglutinating activity, 239 isolation, 138 purification and properties, 240 Sodiocellulose 11, crystal structure bibliography, 379 Sodium hyaluronate, crystal structure bibliography, 383-385 Solanum tuberosum lectin, see Potato lectin Sophora japonica lectin agglutinating activity, 250-254 carbohydrate-binding specificity, 251 isolation, 138 purification, 250 Sophorose interaction with concanavalin A,

184- 186 structure, 186 Soybean lectin amino acids, 234 biological activity, 238 biophysical characteristics, 233,234 carbohydrate-binding specificity,

236-238 hemagglutinating activity, 23 1 interaction with cellular structures, 317 isolation, 138 purification, 232,233 toxicity, 232 Spectinomycin, biosynthesis and structure, 118-121 -, dihydro-, formation, 119, 121 Spectrophotometric assay, of lectins, 134 Spindle-tree lectin, carbohydratebinding specificity, 145 Sponge Iectins, isolation and properties,

315,316 Stachyose biosynthesis in fenugreek seeds, 352 in endosperm during germination, 357 Starch in endosperm and cotyledon during germination, 356-361

end-group assays, 8 structure, 10 Stereochemistry, of sugars, 5 Streptarnine, biosynthesis, 117 -, deoxy-, biosynthesis, 104, 117 Streptidine biosynthesis, 102-107, 109 6-phosphate, biosynthesis, 103-106,

109 Streptomutin A, production and structure, 106 Streptomyces 2755 lectin, isolation, purification, and composition, 307 Streptomycin biosynthesis and structure, 102-110 6-phosphate, biosynthesis, 109 -, dihydro-, biosynthesis, 102 Streptose, L-, biosynthesis, 98-102 -, dihydro-L-, biosynthesis, 98-102 Sucrose in endosperm and cotyledon during germination, 357 structure, 5 Sugars C-acetyl-branched, 92,94, 95 anhydro, boronates, properties, 79 paper chromatography of, phenylboronic acid in, 60 biosynthesis of, of antibiotic substances, 81-126 boronates, see Boronates branched-chain, antibiotic components, 82-102 column chromatography, boronic acids in, 64 electrophoresis of, sulfonylated phenylboronic acids in, 62 formyl- or hydroxymethyl-branched, 98 gas-liquid chromatography of, boronic acids in, 65 C-methyl-branched, biosynthesis, 97 paper chromatography of, phenylboronic acid in, 59, 61 structure, 5 Sunn-hemp lectin, isolation, purification, and properties, 306

T Teichoic acids, interaction with concanavalin A, 175-177

SUBJECT INDEX, VOLUME 35 Tetritol, 3-deoxy-D~-glyceroethylboronate, structure, 43 phenylboronate, struchire, 43 cis-3,4-Thiolanediol 1-oxide, phenylboronates, separation of stereoisomers, 58 p-Toluenesulfonylation, of carbohydrate boronates, 53 p-Tolylboronates, stability to hydrolysis, 50 Tridncna marima lectin, purification and properties, 314 Tridacnin, purification and properties, 314 1,3,5,2,4-Trioxadiborepane, nomenclature, 36 Trisaccharides interaction with lectins, 142 structure, 5 Triticum vulgaris lectin, see Wheat-germ lectin Tritylation, of nucleoside boronates, 55 Tumor cells lectin-binding, 205 lectin-reactive, 327-333 Tylosin, biosynthesis, 83

U Ulex europeus I lectin carbohydrate-binding specificity, 290 hemagglutinating activity, 289 isolation and purification, 289, 290 Ulex europeua 11, extract, carbohydratebinding specificity, 224-226 Ultraviolet absorption spectra, of sugars, 5 Uridine, 5’-phosphate, preparation, 54

V Validamycins, biosynthesis and structure, 120-122 Validoxylamine, structure, 120-122 Verbascose, in endospem during gerniination, 357 Vicia crucca lectin, isolation and properties, 138,304 Viciu eruilia lectin, isolation, composition, and properties, 311 Vicia faho lectin, see Faba-bean lectin Vicia graminen lectin

427

carbohydrate-binding specificity, 145, 303 isolation, 302 purification and composition of, 303 Vinelose, L-, biosynthesis, 85-89

W Wax-bean lectin, purification and composition, 296 Well-drilling, plant galactomannans in, 342 Wheat-germ lectin agglutinating activity, 214 carbohydrate-binding sites, 216, 222 carbohydrate-binding specificity, 220 interaction with cellular structures, 317 isolation, 137, 214 mitogenic activity, 224 precipitation, 216, 224 purification, and properties, 214 structure, 215 Wistaria Joribunda lectin isolation, 138,312 purification and properties, 312

X X-ray crystallography, of sugars, 5 X-ray diffraction, and carbohydrate boronate structure, 41 Xylan, structure, 7 Xylitol 2-diethylborinate 1,3:4,5-bis(ethylboronate), hydrolysis, 52 -, 1,5-dideoxy-, phenylboronate, structure, 43 Xylofuranose, l,Z-O-isopropylidene-a-D-, 3,5-phenylboronate, preparation, 38 Xylofuranoside methyl, isolation of anomeric phenylboronates, 58 -, methyl p-D, 3,5-phenylboronate, hydrolysis, 51, 52 Xylopyranoside, methyl CY-D2,4-phenylboronate, oxidation, 57 preparation, 46 separation from isomers, 58 -, methyl p-D2,4-phenylboronate, oxidation, 57 preparation, 37

SUBJECT INDEX, VOLUME 35

428

Xylopyranoside-5-W, methyl (Y-D-and p-D-, preparation, 58 Xylose, D-

boronates, hydrolysis, 51 structure, 4 sulfur-containing,8

(Y-D-, 1,2:3,5bis(phenylboronate)and

1,2:3,5-bis(butylboronate),preparation, 43 -, 3-0-~-D-glucopyranosy~-D-, synthesis, 55 -, 3-0-a- and -P-D-xylopyranosyl-D-, synthesis, 55

CUMULATIVE AUTHOR INDEX FOR VOLS. 31-35* B

G

BALLOU,CLINTONE., and BARKER, HORACEA., [Obituary ofl WilliamZev Hassid, 32, 1-14 BARKER, HORACEA. See Ballou, Clinton E. BARNETT,JAMESA., The Utilization of Sugars by Yeasts, 32, 125-234 BUSHWAY, ALFREDA. See Whistler, Roy L.

GELPI,MARIAE., and CADENAS, RAUL A., The Reaction of Ammonia with Acyl Esters of Carbohydrates, 31,81-134 GLAUDEMANS, CORNELISP. J., [Obituary ofl Hewitt Grenville Fletcher, Jr., 31, 1-7 GLAUDEMANS, CORNELISP. J., The Interaction of Homogeneous, Murine Myeloma Immunoglobulins with Polysaccharide Antigens, 31,313346 GOLDSTEIN,IRWINJ., and HAYES, COLLEENE., The Lectins: Carbohydrate-binding Proteins of Plants and Animals, 35, 127-340 GRISEBACH, HANS,Biosynthesis of Sugar Components of Antibiotic Substances, 35,81-126

C CADENAS, RAUL A. See Gelpi, Maria E. CERNY,MILOSLAV, and S T A N ~ K JAN, , JR., 1,6-Anhydro Derivatives of Aldohexoses, 34,23-177 CHEN, MINSHEN,and WHISTLER,ROY L., Metabolism of D-Fructose, 34, 285-343

H D DAX,KARL, and WEIDMANN, HANS,Reactions of ~-Glucofuranurono-6,3Iactone, 33,189-234 DEA, IAINC. M., and MORRISON, ANTHONY,Chemistry and Interactions of Seed Galactomannans, 31, 241312 DE BELDER,ANTHONY N., Cyclic Acetals of the Aldoses and Aldosides: Highlights of the Literature Since 1964, and a Supplement to the Tables, 34, 179-241 DEKKER,ROBERTF. H., and RICHARDS, GEOFFREYN., Hemicellulases: Their Occurrence, Purification, Properties, and Mode of Action, 32,277452 DEY, P%UCASHM., Biochemistry of Plant Galactomannans, 35, 3 4 1 3 7 6 F FERRIER, ROBERTJ., Carbohydrate Boronates, 3 5 , 3 1 4 0

HAINES,ALANH., Relative Reactivities of Hydroxyl Groups in Carbohydrates, 33,ll-109 HANESSIAN,STEPHEN,and PERNET, ANDRE G., Synthesis of Naturally Occurring C-Nucleosides, Their Analogs, and Functionalized C-GIycosyl Precursors, 33, 111-188 HAYES,COLLEENE. See Goldstein, Irwin J. HORTON,DEREK.See Wander, Joseph D. I IGARASHIY KrKvO, The Koenigs-Knorr Reaction, 34,243-283

J JEFFREY,GEORGEA., and SUNDARALINGAM, MUTTAIYA,Bibliography of Crystal Structures of' Carbohydrates, Nucleosides, and Nucleotides (1973), 31,347371; (1974), 32,353384; (1975),3 4 , 3 4 5 3 7 8

* Starting with Volume 30, a Cumulative Author Index covering the previous 5 volumes will be published in every 5th volume. That listing the authors of chapters in Volumes 1-29 may b e found in Volume 29. 429

430

CUMULATIVE AUTHOR INDEX FOR VOLS. 31-35 K

KHAN,RIAZ,The Chemistry of Sucrose, 33,235-294 L LAM, OLLE, and LINDBERG, BENGT,The Pneumococcal Polysaccharides: A Re-examination, 33,295-322 LINDBERG,BENGT,LONNGREN, JORGEN, and SVENSSON, SIGFFUD,Specific Degradation of Polysaccharides, 31, 185-240 LINDBERG,BENGT.See also, Larm, Olle. LONNGREN, JORGEN.See Lindberg, Bengt. M MANNERS,DAVIDJOHN.See Stacey, Maurice. MARCHESSAULT, ROBERTH., and SUND A R A R A JPUDUPADI AN, R., Bibliography of Crystal Structures of Polysaccharides (1967-1974), 33,387404 MARCHESSAULT, ROBERTH. See also, Sundararajan, Pudupadi R. MARKOVIC, OSKAR.See Rexovi-Benkova, iubomira. MOHNSON,ANTHONY. See Dea, Iain C. M. N

NAKAHARA, WARO.See Whistler, Roy L. NEUBERGER, ALBERT,[Obituary ofl Alfred Gottschalk, 33, 1-9 0

ORENSTEIN,NEIL S. See Watson, Ronald R.

P PERNET,AND& G. See Hanessian, Stephen.

R REXOVA-BENKOVA, ~ U B O M ~and R A MARK, V I ~ OSKAR, , Pectic Enzymes, 33, 323-385 RICHARDS, GEOFFREYN. See Dekker, Robert F. H. S SINGH,PREMP. See Whistler, Roy L. STACEY,MAURICE,and MANNERS, DAVIDJ., [Obituary ofl Edmund Langley Hirst, 35,"1-29 STANEK,JAN,JR. See Cernf, Miloslav. See Jeffrey, SUNDARALINGAM, MUTTAIYA. George A. SUNDARARAJAN, PUDUPADIR., and MARCHESSAULT, ROBERTH., Bibliography of Crystal Structures of Polysaccharides (1975), 35,377-385 SUNDARARAJAN, PUDUPADIR. See also, Marchessault, Robert H. SVENSSON, SIGFRID. See Lindberg, Bengt. T

TOKUZEN,REIKO. See Whistler, Roy L. W

WANDER,JOSEPHD., and HORTON, DEREK,Dithioacetals of Sugars, 32, 15-123 WATSON,RONALDR., and ORENSTEIN, NEIL S., Chemistry and Biochemistry of Apiose, 31, 135-184 WEIDMA", HANS.See Dax, Karl. WEIGEL,HELMUT,[Obituary ofl Edward John Bourne, 34,l-22 WHISTLER,ROYL., BUSHWAY, ALFRED A., SINGH,PREM P., NAKAHARA, WARO,and TOKUZEN,REIKO,Noncytotoxic, Antitumor Polysaccharides, 32,235-275 WHISTLER,ROYL. See also, Chen, Minshen. WILLIAMS,J. MICHAEL,Deamination of Carbohydrate Amines and Related Compounds, 31,9-79

CUMULATIVE SUBJECT INDEX FOR VOLS. 31-35* A Acyl esters, of carbohydrates, reaction of, with ammonia, 31,81-134 Aldohexoses, 1,6-anhydro derivatives of, 34,23-177 Aldoses and aldosides, cyclic acetals of, 34, 179-241 Ammonia, the reaction of, with acyl esters of carbohydrates, 31, 81-134 1,6-Anhydro derivatives, of aldohexoses, 34,23-177 Animals, carbohydrate-binding proteins of, 35,

127-340

Biosynthesis, of sugar components of antibiotic substances, 35,81-126 Boronates, of carbohydrates, 35,3140 Bourne, Edward John, obituary of, 34,l-22 C Carbohydrate-binding proteins (the lectins), of plants and animals, 35, 127-340 Carbohydrates. See also, Polysaccharides, Sugars. acyl esters of, reaction with ammonia,

31,81-134

Antibiotic substances, biosynthesis of sugar components of,

amines of, and related compounds, deamination of, 31,9-79 bibliography of crystal structures of,

35,81-126 Antigens, polysaccharide, interaction of, with homogeneous, murine myeloma immunoglobulins, 31,313-346 Antitumor polysaccharides, noncytotoxic, 32,235-275 Apiose, chemistry and biochemistry of, 31,

135-184

B Bibliography, of crystal structures of carbohydrates, nucleosides, and nucleotides,

(1973),31,347471 (1974),32,353484 (1975),34,345378 boronates of, 35,3140 relative reactivities of hydroxyl groups in, 33, 11-109 Chemistry, of apiose, 31, 135-184 of seed galactomannans, 31,241-312 of sucrose, 33,235-294 Crystal structures, bibliography of, of carbohydrates, nucleosides, and nucleotides,

(1973), 31,347471 (1974),32,353-384 (1975).34,345478

(1973),31,347-371 (1974),32,353-384 (1975),34,345-378 of crystal structures of polysaccharides, (1967-1974), 33,387404 (1975), 35,377485 Biochemistry, of apiose, 31, 135-184 of plant galactomannans, 35,341476

e

of polysaccharides,

(1967-1974), 33,387404 (1975),35,377-385 Cyclic acetals, of the aldoses and aldosides, 34,

179-241

* Starting with Volume 30, a Cumulative Subject Index covering the previous 5 volumes will be published in every 5th volume. That listing the chapters in Volumes 1-29 may be found in Volume 29. 431

432

CUMULATIVE SUBJECT INDEX FOR VOLS. 31-35 D

Deamination of carbohydrate amines and related compounds, 31,9-79 Degradation, specific, of polysaccharides, 31, 185-240 Dithioacetals, of sugars, 32, 15-123 E Enzymes. See also, Hemicellulases. pectic, 33,323385

F Fletcher, Hewitt Grenville, Jr., obituary of, 31,l-7 D-Fructose, metabolism of, 34,285443 G Galactomannans, of plants, biochemistry of, 35,341-376 of seeds, chemistry and interactions of, 31,241-312 D-Glucofuranurono-6,3-lactone, reactions of, 33, 189-234 Gottschalk, Alfred, obituary of, 33, 1-9

H Hassid, William Zev, obituary of, 32, 1-14 Hdnicellulases, mode of action, occurrence, properties, and purification, 32,277-352 Hirst, Edmund Langley, obituary of, 35, 1-29 Hydroxyl groups, relative reactivities of, in carbohydrates, 33,ll-109 I Immunoglobulins, homogeneous, murine myeloma, the interaction of, with polysaccharide antigens, 31,313346

Interactions, of homogeneous, murine myeloma immunoglobulins, with polysaccharide antigens, 31,313346 of seed galactomannans, 31,241-312 K Koenigs-Knorr reaction, the, 34,243-283 1

Lectins, 35,127-340 M Metabolism, Of D-fructose, 34,285343 Mode of action, of hemicellulases, 32,277-352

N C-Nucleosides, naturally occurring, and their analogs, and functionalized C-glycosyl precursors, synthesis of, 33, 111-188 Nucleosides and nucleotides, bibliography of crystal structures of, (1973),31,347371 (1974),32,353384 (1975),34,345-378 0 Obituary, of Edward John Bourne, 34, 1-22 of Hewitt Grenville Fletcher, Jr., 31, 1-7 of Alfred Gottschalk, 33, 1-9 of William Zev Hassid, 32, 1-14 of Edmund Langley Hirst, 35, 1-29 Occurrence, of hemicellulases, 32,277352 P Pectic enzymes, 33,323485 Plants, carbohydrate-binding proteins of, 35, 127340 galactomannans of, biochemistry of, 35, 341-376

CUMULATIVE SUBJECT INDEX FOR VOLS. 31-35 Pneumococcal polysaccharides, a re-examination, 33,295-322 Polysaccharides. See also, Carbohydrates, Galactomannans. antigens, interaction of, with hornogeneous, murine myeloma immunoglobulins, 31,313-346 bibliography of crystal structures of,

(1967-1974), 3 3 , 3 8 7 4 0 4 (1975), 35,377-385 noncytotoxic, antitumor, 32,235-275 the pneumococcal, a re-examination of,

33,295-322 specific degradation of, 31, 185-240 Properties of hemicellulases, 3 2 , 2 7 7 4 5 2 Proteins, carbohydrate-binding, of plants and animals, 35, 127-340 Purification, of hemicellulases, 32, 277-352

R Reaction, of ammonia with acyl esters of carbohydrates, 31,81-134 the Koenigs-Knorr, 34, 243-283 Reactions, of D-g~ucofuranurono-6,3-~actone, 33,

189-234 Reactivities, relative, of hydroxyl groups in carbohydrates, 33, 11-109

433

S Striictures, crystal, of carbohydrates, nucleosides, and nucleotides,

(1973), 3 1 , 3 4 7 4 7 1 (1974), 32,353484 (1975), 34,345-378 of pol ysaccharides, (1967-1974), 3 3 , 3 8 7 4 0 4 (1975), 35,377485 Sucrose, the chemistry of, 33,235-294 Sugar components, of antibiotic substances, biosynthesis of,

35,81-126 Sugars, dithioacetals of, 32, 15-123 the utilization of, by yeasts, 32,

125-234 Synthesis, of naturally occurring C-nucleosides, their analogs, and functionalized C-glycosyl precursors, 33, 111-188

U Utilization, of sugars by yeasts, 32, 125-234

Y Yeasts, utilization of sugars by, 32, 125-234

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