Lectins: Biomedical Perspectives

Lectins Biomedical Perspectives

Lectins Biomedical Perspectives Edited by

Arpad Pusztai and Susan Bardocz The Rowett Research Institute Greenburn Road Bucksburn Aberdeen AB2 9SB

UK Taylor & Francis Ltd, 4 John St, London WC1N 2ET This edition published in the Taylor & Francis e-Library, 2005. “To purchase your own copy of this or any of Taylor & Francis or Routledge’s collection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.” USA Taylor & Francis Inc., 1900 Frost Road, Suite 101, Bristol PA 19007 Copyright © Taylor & Francis Ltd 1995 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, electrostatic, magnetic tape, mechanical, photocopying, recording or otherwise, without the prior permission of the copyright owner. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library. ISBN 0-203-98375-0 Master e-book ISBN

ISBN 0 7484-0177 6 (Print Edition) Library of Congress Cataloging in Publication Data are available Cover design by Hybert Design and Type.

Contents

Preface

vi

Acknowledgements

ix

List of Contributors

x

Chapter 1.

Lectins as Plant Defence Proteins W.J.Peumans and E.J.M.Van Damme

1

Chapter 2.

Possible Mechanism of Action for the Bean (Phaseolus vulgaris) -amylase Inhibitor: A Molecular Modelling Approach P.Rousseau, A.Barre, H.Causse, C.Chatelain, G.Porthé and P.Rougé

19

Chapter 3.

Insecticidal Properties of Plant Lectins: Their Potential in Plant Protection A.M.R.Gatehouse, K.S.Powell, W.J.Peumans, E.J.M.Van Damme and J.A.Gatehouse

29

Chapter 4.

The Mannose-binding Monocot Lectins and Their genes E.J.M.Van Damme, K.Smeets and W.J.Peumans

49

Chapter 5.

Enterocyte-like CACO-2 Cells as a Tool to Study Lectin Interaction J.F.J.G.Koninkx

67

Chapter 6.

Lectins as Growth Factors for the Small Intestine and the Gut S.Bardocz, S.W.B.Ewen, G.Grant and A.Pusztai

86

Chapter 7.

The Potential of Bioadhesive Lectins for the Delivery of Peptide and Protein Drugs to the Gastrointestinal Tract C-M.Lehr and A.Pusztai

98

Chapter 8.

Lectins Binding to the Gut Wall are Growth Factors for the Pancreas: Nutritional Implications for Transgenic Plants A.Pusztai, G.Grant, D.S.Brown, S.Bardocz, S.W.B.Ewen, K. Baintner, W.J.Peumans and E.J.M.Van Damme

119

Chapter 9.

Lectins in Immunology D.C.Kilpatrick

131

Chapter 10.

Lectin Cytochemistry and Intestinal Epithelial Cell Biology T.P.King

155

Chapter 11.

Lectins and Cancer-An Old Field Revisited U.Schumacher

178

v

Chapter 12.

Dietary Galactose-Binding Lectins and Their Effects On Human Colonic Epithelial Cells J.D.Milton and J.M.Rhodes

190

Chapter 13.

Enterotoxigenic Fimbrial E. coli Lectins and Their Receptors: Targets for Probiotic Treatment of Diarrhoea E.Van Driessche, R.Sanchez, I.Dieussaert, L.Kanarek, P.Lintermans and S.Beeckmans

197

Chapter 14.

Identification of the F17 Gene Cluster and Development of Adhesion Blockers and Vaccine Components P.Lintermans, A.Bertels, E.Van Driessche and H.De Greve

246

Chapter 15.

Infection of the Gut by Pathogenic Bacteria is Inhibited by Dietary Lectins. Chemical Probiosis A.Pusztai, G.Grant, S.W.B.Ewen, W.J.Peumans, E.J.M.Van Damme and S.Bardocz

262

Index

270

Preface

It has become clear from fundamental studies, carried out since the discovery of lectins by Stillmark in 1888, that lectins form a ubiquitous and important class of natural carbohydrate-binding information proteins. Although the main scientific interest was originally focused on toxic lectins such as ricin, recent emphasis has shifted to exploring their involvement in cell-to-cell communication and recognition in microorganisms, plants and animals. As knowledge of the basic properties and biological activities of lectins has increased, exciting opportunities have arisen for the exploitation of some of the advantageous aspects of fundamental lectinology. Lectins are now recognized as natural components of our diet. Our digestive system has been exposed to lectins throughout our evolution and we have learnt by experience to select foods that are not toxic, and may indeed be beneficial to the development of an efficient digestive system and the maintenance of a high standard of health. Accordingly, it is quite natural that some of the biological knowledge gathered by basic and fundamental science may have nutritional and medical applications. This was recognized at an early stage by authorities within the Commission of European Communities who were concerned with commissioning and supporting emerging areas of research aimed at promoting new ways of scientific thinking, and consequently to increase the competitiveness of European industry. A new and important scientific challenge in Europe is to increase the nutritional value and safety of both food and foodstuff to obtain greater efficiency of utilization within the Community; this should be achieved without resorting to the use of antibiotics and other undesirable antibacterial agents and without harming the environment. Because of the emergence of new technologies, such as the development of transgenic plants, the use of ‘beneficial’ probiotic bacterial supplements in the diet to prevent pathogenic infections, and the formulation of new diet-based cancer therapies, the need for new scientific approaches to safety has become urgent. The realization that many European laboratories engaged in lectin research could make major contributions to these central aims of the Community led the EC to award a generous grant of 200k ECUs to support the setting up of a FLAIR (food-linked agro-industrial research) Concerted Action Programme (Group No. 9) on ‘Improvement of Food Safety and Quality through the Use of Interactive and Competitive Bindings of Food Lectins and Bacterial Adhesins in the Gut’. The grant was for the co-ordination of lectin research in European laboratories and to facilitate regular contacts, workshops and training visits between scientists participating in the Action for up to 4 years. The participants shared a common objective of improving food safety and had already received core funds for their research from the appropriate national agency. The whole Action was co-ordinated from the Rowett Research Institute in Aberdeen, the Lead Centre of the project, in such a way that the complementary skills and expertise available in the respective laboratories were used to the maximum benefit of the Action. By the end of the Programme, over 25 laboratories had participated and had made valuable contributions to its successful outcome.

vii

This book, in the view of its editors, records most of the major achievements of this European Action but, in addition, sets out at an advanced level new biomedical and biotechnological perspectives which are at the cutting edge of lectin research and could benefit the wider scientific community. Accordingly, where necessary, the editors have included contributions from experts who were not part of the Concerted Action but whose knowledge has complemented that of the group and increased the scientific value of the book. In keeping with the emerging importance of transgenic plants with increased insect resistance, the rationale of using lectin genes in transgenic research is discussed in detail in the first four chapters. Evidence is also presented (Chapter 2) that transfection with the gene of kidney bean -amylase inhibitor, which may have been derived by mutation from the lectin gene of the same seed during evolution, could be used to increase insect resistance in sensitive plants. Indeed, protease inhibitor genes can also be used for the same purpose. Although this book is not intended to concentrate on the technical aspects of transgenic research, several examples of successful transfections of plants with lectin genes are described in Chapter 3. Aspects of the molecular biology of genes coding for mannose-specific lectins in plants, which have potential for use in future gene transfers, are discussed in Chapter 4. The gap from plants to animals is bridged by a detailed discussion of the in vitro interactions between lectins of different specificities and gut cells in culture (Chapter 5). Next, it is shown that dietary lectins, which bind avidly and are endocytosed by cells of the brush border epithelium, are powerful growth factors for the gut, induce changes in its digestive/absorptive functions, modify the state of glycosylation of luminal receptors, alter the expression of genes coding for digestive enzymes, transport and structural proteins and interfere with both the bacterial ecology and the immune response of the gut to food antigens. Of the several potential applications of gut-food-lectin interactions particular attention has been given to lectintargeted oral drug delivery systems (Chapter 7). Furthermore, as stimulation of pancreatic growth by lectins has hitherto been largely ignored as a possible harmful consequence of transfecting plants with genes of insecticidal lectins, the reaction mechanisms of the growth stimulation by lectins and its inhibition by CCKA receptor antagonists are also described. It is apparent from the detailed description of the role of lectins in immunology (Chapter 9) that lectins have profound effects on the immune system and that the plasma cells involved in a multiplicity of immune functions express high and variable levels of endogenous membrane lectins, most of which are used in cellto-cell communication. Next, one of the best known applications of lectins, their exten sive and important use for the histological detection of glycosyl residues of receptors and other structural or functional glycans of cells and tissues is described. This is then followed by a description of the use of some of these techniques in cancer research, including the use of the binding of peanut lectin to human colonic mucosa in vivo as a possible marker of neoplastic transformations in this tissue (Chapter 12). Indeed, there are indications that the consumption of large amounts of peanut lectin may itself promote such a transformation, whereas the common mushroom lectin and others may have an inhibitory effect. As it is now recognized that the adherence of micro-organisms to tissues and cells of micro-organisms is mediated mainly by their fimbrial and/or surface adhesin-lectins, the final three chapters deal with the microbiological aspects of our Programme. First, there is a major review on the fimbrial adhesins of various species of Escherichia coli and their involvement in adherence and pathogenicity. This includes discussion of the use of lectins to inhibit bacterial binding to the gut surface and their applications in medical and veterinary practice to prevent or treat disease. Next, because fimbrial (or other) adhesins are usually expressed at very low levels by bacteria, it is very difficult to establish their binding specificity and other properties by conventional protein chemistry methods. As this is vital for the development of strategies to prevent bacterial binding, the application of molecular biology techniques to resolve the numerous components of the F17 fimbriae of E. coli and to clone its adhesin is described. In the final chapter, it is

viii

shown that knowledge of the binding specificity of the bacterial adhesin can lead to the use of dietary supplements of specific non-toxic lectins to compete with the bacteria and prevent their binding to the gut wall (chemical probiosis). An example is the use of mannose-specific lectins to prevent the binding of mannosesensitive Type 1 fimbriated E. coli. The description of this simple but powerful dietary method for controlling pathogenic infections vividly demonstrates the successful achievement of the main objective of Concerted Action Group No. 9 and rather appropriately ends this book on Lectins: Biomedical Perspectives.

Acknowledgements

We are grateful to the Commission of European Communities for their support of the European FLAIR Concerted Action Programme (No. 19) and Concerted Action on Polyamines (No AIRIl-CT92-0569), and also to the Scottish Office Agriculture and Fisheries Department. We are indebted to the Rowett Research Institute, the Lead Centre for these EC Concerted Actions, its Director, Professor W.P.T.James, and staff for the scientific and administrative facilities put at our disposal and without which the coordination of the Programmes would have been impossible. Finally, we wish to express our sincerest thanks to Mrs Ann White for her meticulous care, attention and enthusiasm in helping us with the editing of this book.

List of Contributors

Dr. W.J.Peumans and E.J.M.Van Damme Katholieke Universiteit Leuven Laboratorium voor Fytopathologie en Plantenbescherming Willem De Croylaan 42 B-3001 Leuven, Belgium P.Rousseau, A.Barre, H.Causse, C.Chatelain, G.Porthé and Dr. P.Rougé Département de Biologie Structurale et Ingénierie des Protéines Laboratoire de Pharmacologie et Toxicologie Fondamentales UPR CNRS no. 8221 Faculté des Sciences Pharmaceutiques, 35 chemin des Maraîchers 31062 Toulouse, France Dr. A.M.R.Gatehouse, K.S.Powell, W.J.Peumans, E.J.M.Van Damme and J.A.Gatehouse University of Durham Department of Biology Science Laboratories South Road Durham DH1 3LE, UK Dr. E.J.M.Van Damme, K.Smets, W.J.Peumans Katholieke Universiteit Leuven Laboratorium voor Fytopathologie en Plantenbescherming Willem De Croylaan 42 B-3001 Leuven, Belgium Dr. J.F.J.G.Koninkx

xi

Department of Veterinary Pathology Faculty of Veterinary Medicine University of Utrecht Yalelaan 1, P.O. Box 80.158 3508 TD Utrecht The Netherlands Dr. S.Bardocz,1 S.W.B.Ewen,2 G.Grant1 and A.Pusztai1 1 The Rowett Research Institute Bucksburn, Aberdeen AB2 9SB Scotland UK 2 Department of Pathology University of Aberdeen Medical School Foresterhill, Aberdeen AB9 2ZD Scotland UK Dr. C.-M.Lehr1 and A.Pusztai2 1 Department of Pharmaceutical Technology and Biopharmaceutics Phillipps-University Marburg Ketzerbach 63, 35037 Marburg Germany 2 The Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB Scotland UK Dr. A.Pusztai,1 G.Grant,1 D.S.Brown,1 S.Bardocz,1 S.W.B.Ewen,2 K.Baintner,3 W.J.Peumans4 and E.J.M.Van Damme4 1 The Rowett Research Institute Bucksburn, Aberdeen AB2 9SB Scotland UK 2 Deparment of Pathology University of Aberdeen Aberdeen AB1 2ZX Scotland UK 3 Department of Physiology Pannon University of Agriculture 7400 Kaposvár, Hungary 4 Catholic University of Leuven, Laboratory of Fytopathology and Plant Protection 3001-Heverlee, Belgium Dr. D.C.Kilpatrick Edinburgh and SE Scotland Blood Transfusion Service Cellular Immunology Laboratory 2 Forrest Road Edinburgh EH1 2QN Dr. T.P.King The Rowett Research Institute Bucksburn, Aberdeen AB2 9SB Scotland UK Dr. U.Schumacher Human Morphology University of Southampton Bassett Crescent East Southampton SO9 3TU England UK

xii

J.D.Milton and Dr. J.M.Rhodes Department of Medicine Liverpool University PO Box 147, L69 3BX England UK Dr. E.Van Driessche, R.Sanchez, I.Dieussaert, L.Kanarek, P.Lintermans and S.Beeckmans Laboratorium voor Chemie der Proteïnen Vrije Universiteit Brussel Paardenstraat 65 B-1640 St-Genesius-Rode Belgium Dr. P.Lintermans,1 A.Bertels,1 E.Van Driessche2 and H.De Greve2 1 SmithKline Beecham Animal Health Place de L’Université 16 Louvain-La-Neuve, Belgium 2 The Vrije Universiteit van Brussel Paardenstraat 65 B-1640 St-Genesius-Rode Belgium Dr. A.Pusztai, G.Grant, S.W.B.Ewen W.J.Peumans, E.J.M.Van Damme and S.Bardocz The Rowett Research Institute Bucksburn, Aberbeen AB2 9SB Scotland UK

Chapter 1 Lectins as Plant Defence Proteins Willy J.Peumans and Els J.M.Van Damme

Introduction More than a century after the initial detection of plant lectins by Stillmark, the physiological role of this particular group of plant proteins is still poorly understood. Obviously, the present lack of insight into their possible function(s) cannot be ascribed to disinterest in these carbohydrate-binding proteins because many of them have been studied in detail at the biochemical, physiological and molecular levels (Goldstein and Poretz, 1986). Similarly, as the search for the role of plant lectins has been one of the most important aspects of lectin research during the last decade, the present lack of understanding of their natural function is certainly not due to an insufficient interest in this matter. On the contrary, based on the results of intensive studies, a number of possible physiological roles have been proposed for plant lectins. Clearly, the carbohydrate-binding properties and specificities of the lectins can be regarded as determining factors in the different proposed functions since they enable the phytohaemagglutinins to serve as recognition molecules. Recognition between lectins and receptor molecules can occur at three distinct levels, namely, within the cell, between different cells of the same organism, or between different organisms. As a result, lectins can play a role in the plant itself, in interactions between plants and micro-organisms, or outside the plant. In the plant itself, lectins are supposed either to be involved in processes such as transport of carbohydrates, cell wall elongation, cell-cell interactions, growth regulation, recognition of receptors in membranes, self incompatibility, or to function as enzymes or storage proteins. A possible involvement of lectins in interactions between plant and micro-organisms has been proposed for the recognition and/or binding of symbiotic bacteria to the roots of legumes and for the defence of plants against bacteria and fungi. Finally, outside the plant, lectins are supposed to play a role in protecting the plants against either micro-organisms or predators. A variety of hypotheses have been put forward by lectinologists to explain the occurrence of lectins in plants. Unfortunately, no conclusive evidence has been obtained to support these proposed functions, so the exact role of plant lectins is still controversial. Nevertheless, most lectinologists now agree that most, if not all, plant lectins play important biological roles both within and outside the plant and that different types of lectins may fulfil different physiological functions. Another important development in the study of the role of plant lectins is due to the changing attitude of the lectinologists towards this problem. In the past, most of the effort was concentrated on proving or disproving one or another of the classical hypotheses which were put forward in the early days of the research. As a result, little attention was given to the development of alternative hypotheses, so much so that it became difficult to introduce new ideas because most lectinologists were reluctant to accept novel concepts. Fortunately, progress made in the biochemistry, physiology and molecular biology of plant lectins during the last few years and especially in unravelling their

2

LECTINS: BIOMEDICAL PERSPECTIVES

biological activities in cell-free systems and against foreign organisms, has led to new insights into the possible functions of these proteins. One such novel concept, the idea that at least some plant lectins are genuine storage proteins which, if needed, can be used by the plant as protective molecules against their attack by micro-organisms or herbivores, is critically assessed in this contribution. Before discussing the arguments in favour of this double role, a brief summary is given of the heterogeneity of plant lectins and its further implications for possible or presumed physiological functions. Plant lectins are a heterogeneous group of proteins It is obvious that the group of plant proteins that fall within the limits set by the definition of carbohydratebinding proteins, comprises many types of proteins with different properties. Indeed, even when we take into consideration only the biochemical and carbohydrate-binding properties of the plant lectins which have been characterized in detail, it becomes evident that they represent a heterogeneous group of proteins with only one property in common, namely, their ability to recognize and bind sugars (Goldstein and Poretz, 1986). A comparison of the most important biochemical and physico-chemical properties of plant lectins which have been isolated and characterized to date suggests that they may be divided into different classes of proteins with distinct properties. In general, related plant species contain more or less closely similar lectins, which clearly belong to the same natural family of proteins. Well-known examples of lectin families are, for instance, the N-acetylglucosamine-binding Gramineae lectins (Stinissen and Peumans, 1985), the chitin-binding Solanaceae lectins (Allen, 1983), and a wider group of legume lectins with different specificities (Van Driessche, 1988). In some instances, closely related lectins appear to occur in species belonging to different families such as the mannose-binding lectins from Amaryllidaceae, Alliaceae and Orchidaceae species, which undoubtedly represent a single superfamily of evolutionary well-conserved proteins (Van Damme et al., Chapter 4). As well as these, dozens of other types of agglutinins are known. In fact, there are a number of lectins which are unique in a sense that no related proteins are known. Some examples of these lectins are, for instance, the agglutinins from Aegopodium podagraria (Peumans et al., 1985), Eranthis hyemalis (Cammue et al., 1985) and Urtica dioica (Peumans et al., 1984a). Plant lectins not only differ in their molecular structure and biochemical properties but also exhibit a wide variety of sugar-binding specificities. Usually, phytohaemagglutinins are classified according to their specificity towards monosaccharides and are divided into a number of artificial groups such as mannose/ glucose-binding-, N-acetylglucosamine-binding-, N-acetylgalactosaminebinding-, galactose-binding-, fucose-binding- and sialic-acid-binding lectins. All other lectins, which do not bind to any simple sugar, are regarded as having a ‘complex’ specificity. In some cases, this so-called complex specificity has been elucidated (e.g. the Phaseolus vulgaris agglutinins), but for most the exact structure of the complementary oligosaccharide is still unknown. Irrespective of differences in their molecular structure and specificity, plant lectins are also heterogeneous from the point of view of their occurrence within the plant kingdom and in their distribution in different tissues of plants. In the early days of lectinology, almost all the research in this particular area was concentrated on the seed lectins of legumes, and phytohaemagglutinins were considered as typical seed proteins. It was evident from the beginning, however, that seeds from non-legume species, such as castor bean (Ricinus communis) and wheat (Triticum aestivum), were also rich sources of lectins. Moreover, as more and more plants were tested for the presence of lectins in their seeds, it became apparent that many species belonging to different families possess agglutinins in them. In addition, lectins were also detected in vegetative tissues. Although, until quite recently, only a few lectins have been isolated from non-seed

LECTINS AS PLANT DEFENCE PROTEINS

3

materials, such as those from potato tubers or tomato fruits, further exploration of the plant kingdom has revealed that lectins are found in all types of vegetative tissues, leaves, stems, bark, bulbs, tubers, corms, root stocks, rhizomes, phloem, fruits and flower tissues. Therefore, plant lectins cannot be regarded any longer as typical seed proteins. On the contrary, it appears that with the exception of the legume family, plant lectins are more widespread in vegetative tissues than in seeds. Moreover, as will be discussed in more detail below, some vegetative (storage) tissues contain at least as high a concentration of lectins as the most lectin-rich legume seeds. This observation is important because the concentration of lectins in different plants may vary greatly. Thus, whereas some lectins occur in very large quantities (up to 50 per cent of the total protein content) both in seeds (e.g. of legumes such as Phaseolus vulgaris and Canavalia ensiformis; Van Driessche, 1988) and in vegetative storage tissues (e.g. in bark tissue of Robinia pseudoacacia and in bulbs of Allium sativum; Peumans et al., 1986b; Van Damme et al., 1991) lectins can be barely detectable in other plants. The major conclusion to be drawn from the data discussed in this section is that plant lectins are indeed a heterogeneous group of proteins. Moreover, as the individual members of this large group of carbohydratebinding proteins differ not only in their biochemical and physico-chemical properties, but also in their sugar-binding specificity, tissue distribution and abundance, it is evident that the physiological role which can be attributed to plant lectins may also be different with individual lectins and that various types of lectins may fulfil different functions. Nevertheless, lectins which at first glance appear to be totally different, can also show similarities. Indeed, as well as their carbohydrate-binding activity, a surprisingly high number of lectins behave as typical plant storage proteins. Thus, in the next section, the possible storage role of lectins is discussed in detail. Plant lectins as storage proteins A general survey of the occurrence of lectins in different plant tissues indicates that the majority of these proteins are found in typical storage tissues. It is striking that most seed lectins are principally located in the parenchyma of the seed storage organs, such as the cotyledons (e.g. all legume lectins) and the endosperm (e.g. the lectins from Ricinus communis and Datura stramonium). Similarly, most of the non-seed lectins occur, in what can be called, ‘vegetative storage’ tissues, such as tubers (e.g. potato), bulbs (e.g. tulip, Amaryllidaceae and Alliaceae species), corms (e.g. Araceae species), rhizomes (e.g. Aegopodium podagraria, Urtica dioica), root stocks (e.g. Phytolacca) and bark (e.g. elderberry, black locust). Moreover, since the leaves and stems of some plant species are also capable of accumulating storage proteins, they have to be considered as potential storage tissues. This is clearly the case for a plant like mistletoe (Viscum album), which accumulates large quantities of lectins in its leaves and (green) stems (Franz, 1989). The fact that plant lectins are usually associated with storage tissues does not necessarily imply that they are storage proteins although quite a few of them in seeds or vegetative tissues behave exactly as can be expected of genuine plant storage proteins. To demonstrate the similarities between plant lectins and storage proteins, a comparison will be made of the most important biochemical, physiological and molecular biological aspects of both groups of proteins. Since most lectinologists are not familiar with plant storage proteins, a brief overview of this important class of proteins is given here. Plant storage proteins Usually, a plant storage protein is defined as a protein that is found in large amounts in cells of a reserve tissue and to which no other function can be attributed other than to serve as a source of nitrogen. Most

4

LECTINS: BIOMEDICAL PERSPECTIVES

research on these proteins has been concentrated on the different types of globulins and albumins found in dry seeds of legume and cereal species (although virtually all plant species contain these types of proteins in their seeds). However, seeds are not the only source of storage proteins. On the contrary, similar or at least functionally similar proteins have also been found in vegetative storage organs such as tubers, root stocks, rhizomes, bark, bulbs, leaves and stems. Plant storage proteins are a large and heterogeneous group comprising different subgroups of more or less closely related proteins. In spite of their apparent heterogeneity, however, storage proteins share several important properties. From a biochemical point of view, they all have an amino acid composition characterized by high contents of glutamine/glutamic acid, asparagine/aspartic acid, serine and glycine and low contents of lysine and sulphur-containing amino acids, methionine and cysteine. Furthermore, at the cellular level, storage proteins resemble each other because they are all synthesized, processed, and transported as secretory proteins. They are synthesized on the rough endoplasmic reticulum and subsequently accumulate in vacuoles or vacuole-like organelles called ‘protein storage vacuoles’ (formerly referred to as ‘protein bodies’). Plant storage proteins are also quite similar with respect to the developmental regulation of the expression of their genes. Their genes become activated in the storage parenchyma cells of the relevant tissue at a time when the environmental conditions or the development of the plant, or a combination of both, triggers the induction of a massive supply of amino acids (or possibly another form of readily convertible nitrogen) in these cells. In order to store this supply of nitrogen in a harmless and non-toxic form, the cells synthesize large amounts of storage proteins and sequester them in specialized organelles. Generally speaking, the accumulation of storage proteins continues until the storage tissue begins to desiccate (as in the case of ripening seeds) or enters a resting phase (e.g. in bulbs, tubers or bark). After the completion of the accumulation, the storage proteins remain in an unchanged form in the protein storage vacuoles until the parenchyma cells are triggered by external or internal factors (e.g. seed imbibition, shoot growth, increasing daylight or temperature, phytohormones) to mobilize their nitrogen reserve. When this happens the storage protein vacuoles turn into autophagic organelles and progressively hydrolyse their proteins by specific proteases, a process which continues until the storage proteins are completely degraded. Studies of cell biological, physiological, developmental and molecular biological aspects of the properties of phytohaemagglutinins have supplied a wealth of information about the spatial and temporal developmental control of their biosynthesis and cellular and subcellular distribution in different species. Although there is no doubt that there are exceptions, most plant lectins which have been studied in detail, behave exactly as can be expected for classical storage proteins. Lectins, which are present in large quantities within seeds or within vegetative storage tissues, exhibit particularly distinct storage protein-like behaviour. Thus, it is clear that most plant lectins are also storage proteins.· Lectins Seed lectins Virtually all seeds lectins that have been studied at the physiological and cellular level are secretory proteins. This means that they are synthesized on the endo-plasmic reticulum and eventually accumulate within storage protein vacuoles. Usually, lectins are predominantly located in the storage parenchyma cells of the seed’s reserve tissue, which is mostly the cotyledon (e.g. all legume seeds) and in some rare cases the endosperm (e.g. in castor bean and thorn apple). Irrespective of their exact location within the seed, most of the well-known seed lectins can be considered as abundant proteins (as they usually represent between 1

LECTINS AS PLANT DEFENCE PROTEINS

5

Figure 1.1. Schematic representation of the developmental regulation of lectin and storage proteins in seeds.

and 10 per cent of the total protein content) which accumulate in the storage protein vacuoles together with other genuine storage proteins. Moreover, seed lectins and storage proteins are not only found in close association with each other but are also developmentally regulated in a markedly similar way (Figure 1.1). For instance, legume seed lectins are synthesized during seed development and degraded during germination and seedling growth, together with the major storage proteins. In conclusion, there is little doubt that particularly legume seed lectins behave very much like storage proteins. Moreover, since they also meet all the criteria of plant storage proteins, most seed lectins have to be regarded as genuine storage proteins which differ from classical storage proteins only by their carbohydrate-binding activity. Lectins in vegetative plant storage organs Plant lectins have for a long time been considered as typical seed proteins. Obviously, this misconception cannot be sustained any longer for the majority of the currently known phytohaemagglutinins has been isolated from non-seed tissues and different types of vegetative storage organs are rich, and in some instances extremely rich, sources of lectins. To illustrate this point, some representative examples of lectins from vegetative reserve tissues are listed in Table 1.1. The data clearly demonstrate that different types of vegetative storage organs of species belonging to different taxonomic groupings contain high concentrations of lectins (which in some instances surpass the highest lectin levels found in seeds). In contrast to legume seed lectins, whose biosynthesis, subcellular localization and developmental regulation have been studied in detail, present knowledge of lectins from vegetative storage tissues is rather

6

LECTINS: BIOMEDICAL PERSPECTIVES

scarce and incomplete. Nevertheless, the information that is available indicates that they behave very much like storage proteins, as, unlike seeds which are structurally and functionally similar, the different vegetative storage tissues can be dissimilar in their (microscopic and macroscopic) structure, development as a function of the life cycle of the plant, composition of the reserve materials, exposure to the environment and longevity. Thus, a discussion on the storage role of the lectins from different tissues requires a description of some of the major types of vegetative storage organs rich in lectins such as bulbs, rhizomes and bark. Table 1.1 Some examples of abundant lectins in vegetative storage tissues Lectina

Molecular structure (kDa)

Specificity

Relative concentration (% of total protein)

Bark SNA I 2×32+2×37 Neu5Ac 2,6Gal 2–3 SNA II 2×30 GalNAc 1,3Gal 2–3 RPA 2×29+2×31 Complex 20–40 Bulb TL 4×28 Complex 5 GNA 4×12.5 Mannose 10 NPA 212.5 Mannose 10 ASA 2×12.5 Mannose 30 Rhizome APA 8×60 GalNAc 50 Tuber CAA 4×10+4×15 Complex 1 EHL 32+30 GalNAc 0.5 Root stock BDA 32+30 GalNAc 0.5 a SNA Sambucus nigra agglutinin (Broekaert et al., 1984; Kaku et al., 1990) RPA Robinia pseudoacacia agglutinin (Peumans et al., 1986b) TL tulip lectin (Cammue et al., 1986) NPA Narcissus pseudonarcissus agglutinin (Van Damme et al., 1991) ASA Allium sativum agglutinin (Van Damme et al., 1991) APA Aegopodium podagraria agglutinin (Peumans et al., 1985) CAA Colchicum autumnale agglutinin (Peumans et al., 1986a) EHL Eranthis hyemalis lectin (Cammue et al., 1985) BDA Bryonia dioica agglutinin (Peumans et al., 1984b) Table 1.2 Amino acid composition of lectins and storage proteins Amino acid

PHA

SNA I

RPA

TL

NPA

ASA

APA

CAA

IIS

Asx Thr Ser Glx Pro Gly

13.4 7.6 7.5 7.0 3.1 4.0

10.9 6.6 10.5 9.5 5.2 7.3

14.2 8.4 13.5 7.3 2.9 11.8

16.0 5.8 7.5 6.9 5.5 9.7

16.8 7.6 7.1 8.0 3.7 12.9

14.0 4.2 12.0 11.7 2.7 11.6

13.7 7.5 9.4 8.6 4.9 5.6

12.9 7.0 10.6 8.4 2.2 11.9

12.1 5.0 8.5 19.2 5.7 7.2

LECTINS AS PLANT DEFENCE PROTEINS

Amino acid

PHA

SNA I

RPA

TL

NPA

Ala 4.6 6.3 7.3 5.6 5.0 Cys 0.4 1.7 ND 1.5 3.0 Val 6.8 10.0 9.2 6.9 3.9 Met 0.3 0.6 0.0 1.1 1.8 Ile 4.7 5.0 3.7 3.7 5.9 Leu 9.4 10.0 5.5 11.0 8.0 Tyr 3.2 3.0 1.6 4.3 5.1 Phe 7.9 3.6 7.1 2.4 1.7 Lys 4.1 2.1 4.9 3.4 2.9 His 1.0 1.2 0.3 2.0 0.9 Arg 4.0 6.4 2.5 4.5 3.8 Trp 2.7 ND ND 2.2 2.0 % sugar 7.6 16.0 2.7 0.0 0.0 PHA Phaseolus vulgaris agglutinin (Van Driessche, 1988) SNA I Sambucus nigra I agglutinin (Broekaert et al., 1984) RPA Robinia pseudoacacia agglutinin (Peumans et al., 1986b) TL Tulipa sp lectin (Cammue et al., 1986) NPA Narcissus cv Carlton agglutinin (Van Damme et al., 1991) ASA Allium sativum agglutinin (Van Damme et al., 1991) APA Aegopodium podagraria agglutinin (Peumans et al., 1985) CAA Colchicum autumnale agglutinin (Peumans et al., 1986a) 11S 11 S seed globulin from Glycine max (Derbyshire et al., 1976)

ASA

APA

CAA

IIS

6.2 1.8 6.4 0.9 3.3 7.0 4.7 1.9 3.6 2.9 3.8 1.5 0.0

5.4 2.7 7.5 2.1 6.0 7.7 4.2 2.6 5.4 1.0 3.9 1.7 11.0

8.6 1.7 8.3 0.6 3.4 8.5 2.3 2.6 1.4 1.4 5.6 2.6 4.4

5.4 1.5 4.9 1.1 5.1 6.3 2.7 3.8 3.8 1.9 5.1 0.8 <1.0

7

BULBS Bulbs can be defined as underground storage organs composed of fleshy leaves. They are typical for perennial plants and are most widespread within the order of the Liliales, which comprises important plant families such as the Liliaceae, Amaryllidaceae and Alliaceae. Bulbs accumulate large amounts of storage carbohydrates and proteins at the end of the growing period when the upper parts of the plants die off and their nitrogen content has to be salvaged and stored in underground tissues. When the new plant emerges after the resting (dry or cold) season, the old bulb converts its storage macromolecules into simple carbohydrates and free amino acids and makes them available to the rapidly growing and developing shoot (s). Based on their structure and mode of renewal, several types of bulbs can be distinguished. Some species form bulbs that last for only one resting period and hence have to be renewed yearly. Well-known examples of this type of bulb are tulip, lily and garlic. Other plants, such as snowdrop, daffodil and amaryllis, have perennial bulbs. In these plants, a number of new scales are formed in the innermost part every year at the end of the growing season, whereas the outer scales become progressively depleted and eventually die, giving rise to the brown, papery scales surrounding the living parts. Several bulb lectins (especially from Liliaceae, Amaryllidaceae and Alliaceae species) have been isolated and characterized (Van Damme et al., Chapter 4). Based on their abundance and biochemical properties, such as their amino acid composition (Table 1.2), and the fact that they follow the secretory pathway in their biosynthesis, processing and transport, they resemble storage proteins. In addition, detailed studies of changes in the lectin content during the life cycle of tulip (Van Damme and Peumans, 1989), snowdrop and

8

LECTINS: BIOMEDICAL PERSPECTIVES

daffodil (Van Damme and Peumans, 1990) have shown that these lectins accumulate and disappear during bulb formation and depletion respectively, which demonstrates that these bulb lectins are also developmentally regulated like storage proteins (Figure 1.2). In summary, bulb lectins meet the definition of plant storage proteins and hence have to be considered as such. Importantly, however, they also have a welldefined carbohydrate-binding specificity, which distinguishes them from other types of storage proteins such as globulins or albumins. RHIZOMES According to their definition, rhizomes are underground horizontal stems. They represent another common type of vegetative storage organ of perennials that accumulates substantial amounts of both carbohydrate and proteins when the upper parts of the plants undergo senescence. Like bulbs, the rhizomes mobilize their reserves at the beginning of the next growing season to provide the rapidly growing shoots with free amino acids and carbohydrates. Only a few rhizome-specific lectins have been isolated to date, such as those from stinging nettle (Urtica dioica) and ground elder (Aegopodium podagraria). Rhizomes of the ornamental plant Clivia miniata also contain a lectin but the lectin in this species is not confined to the rhizomes, rather, it occurs throughout the plant (Van Damme et al., Chapter 4). It is interesting to note, however, that the Clivia lectin is almost identical to the lectins from the bulbs of other Amaryllidaceae species like snowdrop, daffodil and amaryllis. Of the two rhizome lectins, that of the stinging nettle is probably one of the most unusual plant lectins (Peumans et al., 1984a). It is a very small protein composed of a single polypeptide chain of 89 amino acids. Its amino acid composition is unusual due to high contents of glycine and cysteine (Beintema and Peumans, 1992). In addition, its biosynthesis is unique because the mature lectin is only a small remnant of the primary translation product, which as well as a lectin domain contains a much larger domain that exhibits a marked sequence homology to the potato WIN proteins (wound inducible proteins; Lerner and Raikhel, 1992). Accordingly, as nettle lectin has no similarity to other plant storage proteins, it cannot be regarded as such. However, the other rhizome-specific lectin, that from ground elder, is more in keeping with what is expected of a storage protein. Its distribution in the plant and seasonal changes of its concentration in the rhizomes have been studied in detail (Peumans et al., 1985). Thus, it has been shown that the ground elder lectin is exclusively expressed in the rhizomes, where it is the major protein representing over 50 per cent of the total protein content of this tissue, at least in the resting plant. Although the rhizomes contain some lectin throughout the year, there is a spectacular increase in lectin content during early autumn when the storage tissue accumulates starch and proteins at the expense of the aging above-ground parts of the plants. As soon as the plants resume their growth during early spring, virtually all the lectin disappears from the rhizomes within about 6 weeks (Figure 1.2). Evidently, the ground elder rhizome lectin behaves like a typical storage protein in its developmental regulation. This observation, taken together with the abundance of the protein and its storage protein-like amino acid composition, suggests that the ground elder rhizome lectin is truly a storage protein with carbohydrate-binding activity. BARK The living bark tissue of deciduous trees plays an important role in the nitrogen metabolism of these perennial plants. Although it sounds somewhat artificial to compare this particular storage tissue with seeds, there is certainly an analogy between the storage role of the bark of trees and that of seed cotyledons. As a matter of fact, plants use both types of storage organs to accumulate carbohydrates and proteins for their own

LECTINS AS PLANT DEFENCE PROTEINS

9

Figure 1.2. Schematic representation of the developmental regulation of lectins and storage proteins in bark (a), rhizomes (b) and bulbs (c, d).

10

LECTINS: BIOMEDICAL PERSPECTIVES

survival or that of their progeny, and there is little difference between the accumulation of storage compounds in the parenchyma cells of the bark or in the cotyledons. Although depletion of the storage compounds in these organs is similar, the cotyledons are almost completely used up and eventually die whereas the storage parenchyma of bark degrades and exports its content but remains functional. Several bark lectins have been isolated and characterized. Well-known examples of typical bark lectins are those from the elderberry (Sambucus nigra) and black locust (Robinia pseudoacacia), which have been intensively studied at the biochemical and plant physiological level (Broekaert et al., 1984; Nsimba-Lubaki and Peumans, 1986). Several other tree species have been found to contain lectins in their bark tissue, such as the dwarf elder (Sambucus ebulus), the red berried elder (Sambucus racemosa; Nsimba-Lubaki et al., 1986), a japanese elder species (Sambucus sieboldiana; Tazaki and Shibuya, 1989), and the legume tree Sophora japonica (Hankins et al., 1988; Baba et al., 1991). Biochemical studies of bark lectins have shown that they resemble the classic plant storage proteins in their amino acid composition (Table 1.2). Moreover, because they all occur in large quantities, and some of them in very large quantities (e.g. in black locust) in the resting bark tissue, they certainly meet the criterion of abundancy set by the definition of a plant storage protein. In addition, studies of seasonal changes of the lectin content in the bark of elderberry, black locust and Sophora japonica have shown that their developmental control is similar to that of a classic storage protein (Nsimba-Lubaki and Peumans, 1986; Baba et al., 1991). It is striking indeed, that the lectin levels in the bark increase dramatically (about 100fold) during autumn within a period of 6 weeks, which coincides with a rapid accumulation of storage compounds in the bark parenchyma. During this period, amino acids generated by the endogenous degradation of the proteins from the withering leaves are transported to the storage tissues of the bark and subsequently incorporated into storage proteins. During the resting season, the lectin levels remain invariably high. As soon as the trees resume their growth at the onset of the spring, however, the lectin content of the bark rapidly decreases as the storage macromolecules of the bark are hydrolysed in order to satisfy the metabolic demands of the rapidly growing young shoots (Figure 1.2). There is little doubt, therefore, that the bark lectins are developmentally regulated in the same way as storage proteins. Moreover, since at least in elderberry and Sophora japonica the lectin is located in the protein bodies of the parenchyma cells of the bark (Greenwood et al., 1986; Baba et al., 1991), it is evident that they reside in the same intracellular compartment as storage proteins. Therefore, it can be concluded that bark lectins are a special class of storage proteins, which in comparison with classical types of storage proteins have an important additional property, namely, their ability to recognize and bind carbohydrates. OTHER VEGETATIVE STORAGE ORGANS As well as in bulbs, rhizomes and bark, lectins have also been isolated from root stocks (Bryonia dioica; Peumans et al., 1984b), corms (e.g. Araceae species; Sandhu et al., 1990) and tubers (e.g. potato, Colchicum autumnale; Desai and Allen, 1979; Peumans et al., 1986a). Some of these lectins have been characterized in detail at the biochemical level but virtually no information is available about their physiology and developmental regulation. Nevertheless, on the basis of their abundance and amino acid composition (Table 1.2) it can be expected that, like their counter-parts in other vegetative storage tissues, most of these lectins are storage proteins with the extra property of sugar-binding activity. A general conclusion to be drawn here is that plant lectins, irrespective of whether they occur in seeds or in vegetative tissues behave as can be expected for storage proteins. A major question, then, is why do these putative storage proteins exhibit such a highly specific carbohydrate-binding activity? Although, as outlined above, many different roles have been proposed for plant lectins, we believe that apart from a storage role,

LECTINS AS PLANT DEFENCE PROTEINS

11

the most likely function of the majority of plant lectins is related to the defence of plants. This, however, does not imply that all plant lectins must play a role in the plant’s defence. On the contrary, it can be expected that some of them, particularly those occurring in low concentrations, may be involved in other recognition processes. The role of lectins in plant defence Before discussing the possibility of plant lectins taking part in the defence of plants, it is useful to reflect upon the major problems that plants are confronted with when they are attacked by micro-organisms or predators and the solutions that they have developed during evolution. Problems and solutions in plant defence Unlike the higher animals, plants do not have an immune system to defend themselves against viruses or micro-organisms and, because of their immobility, plants cannot escape from attacking predators. As a result, they must rely on other defen sive systems, which are either constitutive or inducible. It is not surprising, therefore, that during their evolution plants developed a variety of more or less specific protection mechanisms that enable them to survive in a hostile environment. Their simplest form of defence is based on non-specific physical barriers such as thorns, hairs, seed coats, thick cell walls, etc. Obviously, these cannot offer a complete protection, which implies that plants must have other and more specific strategies at their disposal. One of the most important protective mechanisms is based on the constitutive or inducible accumulation of toxic low molecular weight compounds of secondary metabolites such as antibiotics (e.g. phytoalexins), saponins, alkaloids, glycosides, phenolics, etc. In addition, plants have also developed resistance mechanisms that rely on a constitutive or an inducible accumulation of proteins with toxic properties. Some of these are potent toxins with a very broad spectrum of action, such as the ribosome-inhibiting proteins and lectin-like toxins (e.g. ricin, abrin and modeccin), whereas others exhibit a more selective toxicity against a particular group of organisms. Wellknown examples of the latter group are the fungitoxic proteins (e.g. chitinases, glucanases, pathogenesisrelated proteins and antifungal proteins), insecticidal proteins and antinutrients (e.g. protease inhibitors, amylase inhibitors and lectins). Some of these proteins are constitutively accumulated by the plant (e.g. lytic enzymes such as some types of chitinases, protease inhibitors, -amylase inhibitors), whereas others are synthesized in response to an attack by foreign organisms or to wounding (e.g. pathogenesisrelated proteins, protease inhibitors). Finally, some of the defensive proteins can be induced in a plant by volatile substances (e.g. ethylene, jasmonic acid) produced by neighbouring individuals of the same or different species. A classical example of such an inter-individual or inter-species warning and induction system is the enhanced production of fungitoxic enzymes, e.g. chitinase and glucanase, in ethylene-treated bean and tobacco plants (Boller et al., 1983). However, the question still has to be answered as to whether or not lectins are a part of the general defensive strategy of the plants, and if so, how they act and what is their evolutionary advantage for the plant. Plant lectins are unique proteins because of their carbohydrate-binding activity When discussing the possible physiological role of plant lectins it is important to realize that, with the exception of some enzymes such as chitinases, glucanases and glycosidases, phytohaemagglutinins are one of those few classes of plant proteins which are capable of recognizing and binding glycoconjugates present

12

LECTINS: BIOMEDICAL PERSPECTIVES

on the external surface of micro-organisms or on the luminal surface of the gastrointestinal tract of plant predators. In fact, predators are a heterogeneous group of all plant-eating organisms such as nematodes, insects, other invertebrates and higher animals. It is also important to realize that the structures of surface glycoconjugates, with which lectins can interact, are manyfold and vary from homopolymers of simple sugars (e.g. chitin in the cell wall of fungi) to complex oligosaccharide side chains of, for example, stomach or intestinal mucins. Considering the wide range of sugar-binding specificities of the known different lectins, however, there is no doubt that virtually all surface carbohydrates can be recognized by one or more phytohaemagglutinins. It is therefore possible that most lectins can play a protective role outside the plant. It is remarkable that although in the past a great deal of work has been carried out to find evidence for a specific endogenous role of plant lectins but without success, this seemingly obvious function has only recently emerged. Indeed, on close examination the abundance of most plant lectins, especially in seeds and vegetative storage tissues, argues against an endogenous lectin-functional role in the plant. It is hard to imagine that, apart from a storage role, a lectin which may represent a considerable fraction of the total tissue protein, can play a fundamental role in cell metabolism or recognition of and interaction with specific receptor molecules. Moreover, as most lectins are vacuolar proteins, it is highly questionable as to whether or not they can exhibit any carbohydrate-binding activity at all in situ. A second, and maybe even a more important argument against an endogenous role of most plant lectins is that their specificity is not directed towards carbohydrate molecules present in the plant cells or, more precisely, in their vacuolar compartment. Although there is no doubt that several groups of plant lectins bind to simple sugars such as glucose, mannose or galactose, they usually have much higher affinities for oligosaccharides which are not common in plants. Indeed, a number of lectins exhibit specificity towards carbohydrates that have not been found in plants. For example, all lectins that bind exclusively to chitin recognize a carbohydrate that does not occur in plants but is only found in the cell wall of fungi and in some structures of invertebrates. Similarly, the bark lectins from elderberry (Sambucus sp.; Shibuya et al., 1987) and Maackia amurensis (Knibbs et al., 1991), which exhibit an exclusive specificity towards Neu5Ac -2,6Gal/GalNAc and Neu5Ac -2,3-Gal/GalNAc, respectively, cannot bind to any plant glycoconjugates simply because sialic acid does not occur in plants. The same reasoning holds true for all other plant lectins with ‘complex’ specificity. Most of these do not bind to simple sugars but have high affinities for oligosaccharides that are not found in plants but are common in animal glycoproteins. Furthermore, although some lectins (e.g. those from Aegopodium podagraria rhizomes, tulip bulbs, Bryonia dioica root stocks) are inhibited by simple sugars such as galactose or N-acetylgalactosamine, they cannot be retained and purified by affinity chromatography using these sugars immobilized on an inert matrix but require more complex glycans present on animal glycoproteins (mucins, fetuin, etc.; Peumans et al., 1984b, Peumans et al., 1985, Cammue et al., 1985). Thus, if the biological function of plant lectins is determined by their carbohydrate-binding activity, and if this is not used for endogenous roles, it is possible that these proteins are truly destined to fulfil a role outside the plant. Plant lectins must come in contact with potential receptor molecules on the cell surface of microorganisms, such as chitin in the fungal cell wall or glycoconjugates of surface membranes in the intestinal tract of predators. As such contacts must occur when seeds or other plant organs are attacked by microorganisms invading the cells of the plant and liberating their contents, including the vacuolar lectins, or are eaten by predators whereby the lectins are released from the disrupted cells, there is at least an opportunity for plant lectins to fulfil this putative protective role. However, one of the essential points of this reasoning is that plant lectins can fulfil this role only after the plant has been attacked. Consequently, they have to be considered as non-specific defensive agents which are accumulated by the plants to anticipate a possible

LECTINS AS PLANT DEFENCE PROTEINS

13

attack by predators or microorganisms. As long as the plant or the seed is not challenged (which is rather unlikely under natural conditions), the lectin does not fulfil any defensive role but is simply used as a storage protein. Therefore, most plant lectins can be regarded as storage proteins which, in addition, have a potential defensive role when the plant or the seed is attacked. Such a double role is certainly advantageous for the plant from an evolutionary point of view. It makes sense, indeed, to accumulate storage proteins that are not only a source of readily available nitrogen but, in addition, can also be used as defence compounds when necessary. The assumption that lectins play a role in plant defence is no longer a matter of speculation. Indeed, there is sufficient experimental evidence to support the idea that they are truly involved in the plant’s defensive strategy. Experimental evidence for a defensive role of plant lectins The steadily increasing interest in the possible defensive role of plant lectins during the last two decades has led to some important conclusions. To give an overall picture of the most relevant data, plant lectins with toxic properties are divided into four groups according to their target organisms. The first group comprises all lectins with powerful but non-selective toxicity. Groups two, three and four include lectins with a selective toxicity against fungi, insects and higher animals, respectively. Plant lectins with high, non-selective cytotoxic properties A first group of lectins that undoubtedly play a role in the defence of plants are the extremely cytotoxic proteins such as ricin, abrin, mistletoe lectin I and modeccin. The B-chains of these lectins bind to receptors on the surface of cells and promote the uptake of the A-chain into the cell. After the A-chain enters the cell, it inactivates catalytically all eukaryotic ribosomes by its highly specific N-glycosidase activity which cleaves the N-glycosidic bond of the adenosine residue at position 4324 of the 28S rRNA (Endo, 1989). Plants that accumulate proteins of this type are extremely toxic for all eukaryotes and, in principle, are completely protected against attack by any eukaryotic organism. There is a possibility, however, that some fungi or insects are able to inactivate these toxic lectins by proteolysis well before they can do any significant harm. Lectins with fungitoxic properties Because of their specificity, chitin-binding lectins seem to be predestined to play a protective role against fungal attack. As early as 1975, it was observed that wheat germ agglutinin (WGA) inhibits spore germination and hyphal growth of Trichoderma viride, a finding that led to the hypothesis that WGA may protect the plant against fungi (Mirelman et al., 1975). Although this hypothesis was later supported by the results of localization studies, which demonstrated that WGA accumulates in tissues in direct contact with the environment during germination and seedling growth (Mishkind et al., 1982), its role in the defence of the wheat plant could not be sustained because chitinase-free WGA preparations were shown to have only slight effects on the fungi. It was suggested, therefore, that the earlier positive results were most probably due to a chitinase contamination in the lectin preparations used in these studies (Schlumbaum et al., 1986). Since then, not only WGA but also most other chitin-binding lectins were shown to be essentially devoid of antifungal activity. More recently, however, conclusive evidence has been obtained that a chitinase-free preparation of nettle (Urtica dioica) lectin had potent antifungal properties (Broekaert et al., 1989). In fact,

14

LECTINS: BIOMEDICAL PERSPECTIVES

with some fungi (e.g. Botrytis cinerea) this lectin was more effective than chitinase, but with other fungi (e.g. Trichoderma bamatum) the reverse was true. Although at present the exact mechanism action of the nettle lectin is not fully understood, the lectin appears to affect the synthesis of the fungal cell wall (Van Parijs et al., 1992). Apart from the nettle lectin, hevein, a small (43 amino acids) chitin-binding protein of the latex of the rubber tree (Hevea brasiliensis) was shown to inhibit fungal growth (Van Parijs et al., 1991). Its effect on the morphology of the fungi was similar to that of nettle lectin although it was about five times less active. The two small chitin-binding lectins, UDA and hevein, have thus been shown to possess, at least in vitro, antifungal properties and therefore can be considered as plant protective agents. It is also worth noting that a small (30 amino acid residues) chitin-binding protein from Amaranthus caudatus seeds, with a striking sequence homology to both hevein and the nettle lectin, is also a potent antifungal protein (Broekaert et al., 1992). This small antifungal protein has strong affinity for chitin but, because it has only one binding site per molecule (like hevein), is not considered as a lectin. Lectins with anti-insect properties A potential role for lectins in the defence of plants against insect attack has been recognized since 1976 (Janzen et al., 1976). However, intensive screening of lectins from a wide variety of sources for their antiinsect properties has only started in the last few years. Feeding trials with both artificial seeds and diets containing varying levels of different lectins have demonstrated that many lectins are toxic for one or more insect species and that the spectrum of insects that are sensitive to a particular lectin is highly variable. Some representatives of chitin-binding lectins (e.g. WGA) are particularly active against cowpea weevil (Callosobruchus maculatus) and against two important maize pests, the European corn borer (Ostrinia nubilalis) and Southern corn rootworm (Diabrotica undecimpunctata); (Murdock et al., 1990; Czapla and Lang, 1991). A very interesting plant lectin, from the point of view of its anti-insect properties, is the mannose-binding snowdrop (Galanthus nivalis) lectin (Hilder et al., 1991). This lectin is not only toxic for some chewing insects but also for sucking insects like aphids and leaf hoppers. Interestingly, the snowdrop lectin is active not only when included in an artificial diet but also when the insects are feeding on transgenic tobacco plants expressing the lectin gene. Plant lectins toxic for higher animals It has been known for a long time that seeds of the common bean (Phaseolus vulgaris) are toxic to a variety of animals when eaten raw. Feeding trials with purified PHA, the lectin from Phaseolus vulgaris, have demonstrated that the toxicity can be ascribed to this lectin, which is abundantly present in the seeds (Pusztai et al., 1979). The striking biological effects of PHA are due to its resistance to breakdown by the digestive enzymes in the gut and its specific recognition of and binding to the brush-border cells of the intestine (Pusztai et al., 1990). PHA is a strongly mitogenic lectin and, therefore, a powerful growth factor for the small intestine. Attachment to epithelial cell membranes is immediately followed by endocytosis. This intracellular delivery of the lectin results in an enhanced metabolic activity and eventually leads to both hyperplasia and hypertrophy of the small intestine. It is not difficult to imagine that the accumulation of large quantities of a lectin that causes severe effects in the intestine (thus bringing discomfort for the animal) makes the common bean much less palatable to possible predators. Thus, instead of eating these beans, they will look (in a reaction of avoidance) for another source of food.

LECTINS AS PLANT DEFENCE PROTEINS

15

Recent work by Pusztai and co-workers has demonstrated that not only PHA but most plant lectins survive well in the gut and are able to bind to the brush-border membranes (Pusztai et al., 1990). Although not all these lectins are as potent growth factors as PHA, many do seriously disturb the proper functioning and the growth of the intestine. Consequently, seeds or vegetative tissues that contain such antinutritive lectins are virtually unpalatable and, hence, will be avoided. Thus, the expression of these moderately toxic proteins, which nevertheless cause severe discomfort for animals eating them as a part of their diet, has to be considered as an evolutionary adaptational strategy by plants to ensure the survival of the species. Indeed, because of their moderate toxicity and delayed discomforting effects, the presence of lectins cannot prevent the seed or the plant (or part of a plant) being eaten. However, the reaction of avoidance will be beneficial for the plant species. At present, several lectins isolated from vegetative storage tissues are being tested for their possible toxicity. The rationale behind these experiments is that particularly those lectins which exhibit a high affinity for complex oligosaccharides such as carbohydrates on intestinal mucins and epithelial cell membranes, are potential defence molecules against predators. Recent developments and future prospects The discovery that lectins fulfil an important role in the defence of plants has stimulated research on possible applications of these proteins in the protection of crop plants. Research has intensified in many laboratories to test as many lectins as possible on all major pest insects to find effective and selective antiinsect proteins. As toxicity tests on animals are complex and time-consuming, studies with rats (or other laboratory animals) cannot keep pace with insect-testing. This is rather unfortunate because it is imperative to know whether (and to what extent) lectins which exhibit promising anti-insect properties, are toxic for higher animals. It is important to note here that recent feeding experiments have indicated that most plant lectins are toxic for rats to a certain degree. Lectins, particularly from vegetative storage tissues (bark, tubers, root stocks) appear to be toxic possibly because of their high affinity for glycoconjugates present on brush-border membranes of the intestine (Pusztai et al., 1990). In addition, even WGA, which was considered safe because it is present in a health food (wheat germs; Pusztai et al., 1993) appeared to be fairly toxic for rats. This finding is very important in itself, but becomes even more important in view of the proposed use of the WGA gene in crop protection against insects. Because of its strong insecticidal activity, WGA is considered to be a good candidate for the control of insects in crop plants by the transfer and expression of its gene into sensitive plants. As the presence of WGA in the diet may harm higher animals at the concentration required to be effective against the target pests, its use as a natural insecticide is not without health risk for man. Fortunately, there are also exceptions such as the mannose-binding Amaryllidaceae and Alliaceae lectins, which apparently have no toxic effects in higher animals (Pusztai et al., 1990) but nevertheless exhibit interesting anti-insect properties. In spite of the apparent toxicity of most plant lectins, the search for possible applications of these proteins in plant protection will obviously go on. The basic idea is that once lectins with interesting protective properties (e.g. antifungal or anti-insect activity) are found, their genes can be cloned and transferred into crop plants in order to make them resistant against a particular pest. At present, the technology of isolation and transfer of genes is readily available, and so it is the detection of ‘useful’ lectins that is really the limiting factor. In order to be ‘useful’ for plant protection purposes, a lectin should meet several criteria. First, it must have anti-insect or antifungal activity. Second, it must be sufficiently active at concentrations

16

LECTINS: BIOMEDICAL PERSPECTIVES

below the maximum levels that can be reached in transgenic plants without altering too much the vigour of the plant. Third, it should not be toxic for the consumer (humans or life stock). It is expected, with some confidence, that some plant lectins will meet all three criteria. Therefore, it is certainly worthwhile to extend the present toxicity studies to as many pests as possible, although to isolate sufficient amounts of lectins for these studies will take a considerable effort. In addition, new lectin sources should be explored and new lectins isolated and tested for their possible selective toxicity against a particular group of organisms. Although it is true that many lectins have been described, this does not imply that all useful plant lectins are already known. On the contrary, it is our belief that many interesting plant lectins await to be discovered and characterized, and, maybe at a later stage, used for the protection of our crops against their most important pests and predators. Acknowledgments This work was supported in part by grants from the KULeuven (OT/90/19) and the National Fund for Scientific Research (Belgium, FGWO grant 2005989). The collaborative work is part of a European FLAIR concerted Action Programme (No. 9) co-ordinated by A.Pusztai with financial support from the Commission of European Communities. E.V.D is a Senior Research Assistant and W.P. a Research Director of the National Fund for Scientific Research (Belgium). References Allen, A.D., 1983, Potato lectin A glycoprotein with two domains, in Goldstein, I.J. and Etzler, M.E. (Eds) Chemical Taxonomy, Molecular Biology, and Function of Plant Lectins, pp. 71–85, New York: Alan R Liss. Baba, K., Ogawa, M., Nagano, A., Kuroda, H. and Sumiya, K., 1991, Developmental changes in the bark lectin of Sophora Japonica L., Planta, 183, 462–70. Beintema, J.J. and Peumans, W.J., 1992, The primary structure of stinging nettle (Urtica dioica) agglutinin: a twodomain member of the hevein family, FEBS Letters, 299, 131–34. Boller, T., Gehri, A., Mauch, F. and Vögeli, U., 1983, Chitinase in bean leaves: induction by ethylene, purification, properties and possible function, Planta, 157, 22–31. Broekaert, W.F., Nsimba-Lubaki, M., Peeters, B. and Peumans, W.J., 1984, A lectin from elder (Sambucus nigra) bark, Biochemical Journal, 221, 163–69. Broekaert, W.F., Van Parijs, J., Leyns, F., Joos, H. and Peumans, W.J., 1989, A chitin-binding lectin from stinging nettle rhizomes with antifungal properties, Science, 245, 1100–2. Broekaert, W.F., Mariën, W., Terras, F.R.G., De Bolle, M.F.C., Proost, P., Van Damme, J., Dillen, L., Claeys, M.Rees, S.B., Vanderleyden, J. and Cammue, B.P.A., 1992, Antimicrobial peptides from Amaranthus caudatus seeds with sequence homology to the cysteine/glycine-rich domain of chitin-binding proteins, Biochemistry, 31, 4308–14. Cammue, B.P., Peeters, B. and Peumans, W.J., 1985, Isolation and partial characterization of an N-acetylgalactosaminespecific lectin from winter aconite (Eranthis hyemalis) root tubers, Biochemical Journal, 227, 949–55. Cammue, B.P., Peeters, B. and Peumans, W.J., 1986, A new lectin from tulip bulbs, Planta, 169, 583–88. Czapla, T.H. and Lang, B.A., 1991, Effect of plant lectins on the larval development of European corn borer (Lepidoptera: Pyralidae) and southern corn rootworm (Coleoptera: Chrysomelidae), Journal of Economic Entomology, 83, 2480–85. Derbyshire, E., Wright, D.J. and Boulter, D.J., 1976, Legumin and vicilin storage proteins of legume seeds, Phytochemistry, 15, 3–24. Desai, N.N. and Allen, A.K., 1979, The purification of potato lectin by affinity chromatography on an N-,N -,N triacetylchitotriose-Sepharose matrix, Analytical Biochemistry, 93, 88–90.

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Endo, Y., 1989, Mechanism of action of ricin and related toxic lectins on the inactivation of eukaryotic ribosomes, in Franz, H. (Ed.) Advances in Lectin Research, vol. 2, pp. 60–73, Berlin: VEB Verlag. Franz, H., 1989, Viscaceae lectins, in Franz, H. (Ed.) Advances in Lectin Research, vol. 2, pp. 28–59, Berlin: VEB Verlag. Goldstein, I.J. and Poretz, R.D., 1986, Isolation, physicochemical characterization and carbohydrate-binding specificity of lectins, in Liener, I.E., Sharon, N. and Goldstein, I.J. (Eds) The Lectins: Properties, Functions, and Applications in Biology and Medicine, pp. 33–248, New York: Academic Press. Greenwood, J.S., Stinissen, H.M., Peumans, W.J. and Chrispeels, M.J., 1986, Sambucus nigra agglutinin is located in protein bodies in the phloem parenchyma of bark, Planta, 167, 275–78. Hankins, C.N, Kindinger, J.I. and Shannon, L.M., 1988, The lectins of Sophora japonica. II Purification, properties and N-terminal amino acid sequences of five lectins from bark, Plant Physiology, 86, 67–70. Hilder, V.A., Gatehouse, A.M.R., Gatehouse, J.A. and Boulter, D., 1991, Beyond Bt toxin; Higher plant genes which enhance insect resistance in transgenic plants, Third International Congress for Plant Molecular Biology, abstract no. 734, Tuczon, AZ, USA. Janzen, D.H., Juster, H.B. and Liener, I.E., 1976, Insecticidal action of the phytoheamagglutinin in black beans on a Bruchid beetle, Science, 192, 795–96. Kaku, H., Peumans, W.J. and Goldstein, I.J., 1990, Isolation and characterization of a second lectin (SNA-II) present in elderberry (Sambucus nigra L.) bark, Archives of Biochemistry and Biophysics, 277, 255–62. Knibbs, R., Goldstein, I.J., Ratcliff, R.M. and Shibuya, N., 1991, Characterization of the carbohydrate binding specificity of the leukoagglutinating lectin from Maackia amurensis, Journal of Biological Chemistry, 266, 83–88. Lerner, D.R. and Raikhel, N.V., 1992, The gene for stinging nettle lectin (Urtica dioica agglutinin) encodes both a lectin and a chitinase, Journal of Biological Chemistry, 267, 11085–91. Mirelman, D., Galun, E., Sharon, N. and Lotan, R., 1975, Inhibition of fungal growth by wheat germ agglutinin, Nature, 256, 414–16. Mishkind, M., Raikhel, N.V., Palevitz, B.A. and Keegstra, K., 1982, Immunocytochemical localization of wheat germ agglutinin in wheat, Journal of Cell Biology, 92, 753–64. Murdock, L.L., Huesing, J.E., Nielsen, S.S., Pratt, R.C. and Shade, R.E., 1990, Biological effects of plant lectins on the cowpea weevil, Phytochemistry, 29, 85–89. Nsimba-Lubaki, M. and Peumans, W.J., 1986, Seasonal fluctuations of lectin in bark of elderberry (Sambucus nigra) and black locust (Robinia pseudoacacia), Plant Physiology, 80, 747–51. Nsimba-Lubaki, M., Allen, A.K. and Peumans, W.J., 1986, Isolation and characterization of glycoprotein lectins from the bark of three species of elder, Sambucus ebulus, Sambucus nigra and Sambucus racemosa, Planta, 168, 113–18. Peumans, W.J., De Ley, M. and Broekaert, W.F., 1984a, An unusual lectin from stinging nettle (Urtica dioica) rhizomes, FEBS Letters, 177, 99–103. Peumans, W.J., Nsimba-Lubaki, M., Carlier, A.R. and Van Driessche, E., 1984b, A lectin from Bryonia dioica root stocks, Planta, 160, 222–28. Peumans, W.J., Nsimba-Lubaki, M., Peeters, B. and Broekaert, W.F., 1985, Isolation and partial characterization of a lectin from Aegopodium podagraria rhizomes, Planta, 164, 75–82. Peumans, W.J., Allen, A.K. and Cammue, B.P., 1986a, A new lectin from meadow saffron (Colchicum autumnale), Plant Physiology, 82, 1036–39. Peumans, W.J., Nsimba-Lubaki, M., Broekaert, W.F. and Van Damme, E.J.M., 1986b, Are bark lectins of elderberry (Sambucus nigra) and black locust (Robinia pseudoacacia) storage proteins?, in Shannon, L.M. and Chrispeels, M.J. (Eds) Molecular Biology of Seed Storage Proteins and Lectins, pp. 53–63, Proceedings of the 9th Annual Symposium in Plant Physiology, UCR Riverside. Pusztai, A., Clarke, E.M.W. and King, T.P., 1979, The nutritional toxicity of Phaseolus vulgaris lectins, Proceedings of the Nutrition Society, 38, 115–20.

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Pusztai, A., Ewen, S.W.B., Grant, G., Peumans, W.J., Van Damme, E.J.M., Rubio, L. and Bardocz, S., 1990, The relationship between survival and binding of plant lectins during small intestine passage and their effectiveness as growth factors, Digestion, 46, 308–16. Pusztai, A., Ewen, S.W.B., Grant, G., Brown, D.S., Stewart, J.C., Peumans, W.J., Van Damme, E.J.M. and Bardocz, S., 1993, Antinutritive effects of wheat germ agglutinin and other N-acetylglucosamine specific lectins, British Journal of Nutrition, 70, 313–21. Sandhu, R.S., Arora, J.S., Chopra, S.K., Pelia, S.S., Kamboj, S.S., Naidu, Y.C. and Nath, I., 1990, New sources of lectins from Araceous Indian plants, in Kocourek, J. and Freed, D.L.J. (Eds) Lectins, Biology, Biochemistry, Clinical Biochemistry, vol. 7, pp. 19–26, St. Louis, USA: Sigma Chemical Company. Schlumbaum, A., Mauch, F., Vögeli, U. and Boller, T., 1986, Plant chitinases are potent inhibitors of fungal growth, Nature, 324, 365–67. Shibuya, N., Goldstein, I.J., Broekaert, W.F., Nsimba-Lubaki, M., Peeters, B. and Peumans, W.J., 1987, The elderberry (Sambucus nigra) bark lectin recognizes the Neu5Ac ( 2 6)Gal/GalNac sequence, Journal of Biological Chemistry, 262, 1596–601. Stinissen, H. and Peumans W.J., 1985, Recent advances in the biochemistry, cell biology, physiology, biosynthesis and genetics of Gramineae lectins, Biochemie und Physiologie der Pflanzen, 180, 85–106. Tazaki, K. and Shibuya, N., 1989, Purification and partial characterization of a lectin from the bark of Japanese elderberry (Sambucus sieboldiana), Plant Cell Physiology, 30, 899 903. Van Damme, E.J.M. and Peumans, W.J., 1989, Developmental changes and tissue distribution of lectin in Tulipa, Planta, 178, 10–18. Van Damme, E.J.M. and Peumans, W.J., 1990, Developmental changes and tissue distribution of lectin in Galanthus nivalis L. and Narcissus cv. Carlton, Planta, 182, 605–9. Van Damme, E.J.M., Goldstein, I.J. and Peumans, W.J., 1991, Comparative study of related mannose-binding lectins from Amaryllidaceae and Alliaceae species, Phytochemistry, 30, 509–14. Van Driessche, E., 1988, Structure and function of leguminosae lectins, in Franz, H. (Ed.) Advances in Lectin Research, vol. 1, pp. 73–134, Berlin: VEB Verlag. Van Parijs, J., Broekaert, W.F., Goldstein, I.J. and Peumans, W.J., 1991, Hevein: an antifungal protein from rubber-tree (Hevea brasiliensis) latex, Planta, 183, 258–62. Van Parijs, J., Joosen, H.M., Peumans, W.J., Geuns, J.M. and Van Laere, A.J., 1992, Effect of the lectin UDA (Urtica dioica agglutinin) on germination and cell wall formation of Phycomyces blakesleeanus Burgeff, Archives of Microbiology, 158, 9–25.

Chapter 2 Possible Mechanism of Action for the Bean (Phaseolus vulgaris) -amylase Inhibitor: A Molecular Modelling Approach Patrice Rousseau, Annick Barre, Henri Causse, Christian Chatelain, Gilberte Porthé and Pierre Rougé

Introduction Predatory insects are responsible for severe losses in agricultural production of crop plants destined for consumption by both humans and animals, whether they attack the plants themselves or their harvested products, e.g. seeds. For many years, the indiscriminate use of chemical pesticides on cultivated lands was the only way to reduce the destruction caused by these insects. As a result, many insect species have developed resistance to various pesticides and, because these chemicals are also harmful for humans, their extensive use is now considered a serious health hazard. In addition, the protective effect of pesticides on some plants cultivated on large-scale (e.g. rice) has now become questionable (Hadfield, 1993). More recently, as an alternative to the chemical control of insects, the attention has turned to the exploration of the use of more consumer- and environmentally friendly biological methods for plant protection. Thus, the introduction and subsequent expression of genes in sensitive plants, coding for molecules that are toxic for insects, is now considered to be one of the best methods to protect cultivated plants against these predators. There are several reasons for this: the introduction of a single foreign gene in a transgenic plant may not require considerable genetic changes in comparison with other genetic manipulations such as crossbreeding; the introduced gene can provide continuous protection for the plants; and this protection is independent of the weather; plants which would be difficult to spray with insecticides can be readily protected by this method. Conversely, the protective molecule must fulfil certain conditions, first of all that it must be totally innocuous for higher animals and man. Moreover, the protective molecule must be active at a concentration far below that tolerated by the transgenic plant, in order to be expressed without any deleterious effect on the host plant. Among the protective molecules that can be used for such a purpose, some of the early candidates selected were the plant lectins (Janzen et al., 1976) and lectin-like proteins (arcelins, -amylase inhibitors) because their biological properties were well-known (Rougé et al., 1991). Although some of these proteins are toxic for insects (Gatehouse et al., 1984), it is not always so for higher animals and man (Pusztai, 1991). Furthermore, genes of some of these potentially protective proteins have already been isolated and characterized (Chrispeels and Raikhel, 1991). Seeds of the common bean (Phaseolus vulgaris L.) are known to contain an -amylase inhibitor ( -AI) protein with powerful insecticidal properties against the larvae of bruchid pests. This -AI has been isolated and partially characterized from different varieties and cultivars of Phaseolus vulgaris (Marshall and Lauda, 1975; Powers and Whitaker, 1977; Pick and Wöber, 1978; Lajolo and Finardi-Filho, 1985; Moreno and Chrispeels, 1989). It inhibits the activity of both insect and mammalian -amylases (Ishimoto and Kitamura, 1989), and therefore its presence may explain the resistance of the common bean against a

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bruchid pest, the cowpea weevil (Moreno et al., 1990). The -AI was shown to be identical to LLP (Moreno and Chrispeels, 1989), a lectin-like protein encoded by a gene of Phaseolus vulgaris cv tendergreen, the cDNA of which had been previously isolated by Hoffman et al. (1982). Indeed, it has been suggested that the -AI, which is a contaminant in PHA preparations, is the factor responsible for the resistance of the common bean seeds to the cowpea weevil (Huesing et al., 1991) and not the seed lectin as originally proposed by Gatehouse et al. (1984). The optimal inhibitory activity of -AI occurred at acidic pH (5.0–5.5) and measurement of the stoichiometry of inhibition showed the formation of a 1:1 complex of -amylase and inhibitor (Marshall and Lauda, 1975). The dissociation constant of the enzyme-inhibitor complex was 3. 5×10−11 M at a neutral pH (pH 6.9; Powers and Whitaker, 1977). However, the Lineweaver-Burk plot of its inhibitory effect on hog pancreatic -amylase clearly showed that -AI behaved as a non-competitive inhibitor (Marshall and Lauda, 1975). In view of the properties of -AI, it has been considered by many that this protein may be a suitable and effective insecticidal molecule and that once its gene has been introduced by DNA recombinant techniques in a previously non-resistant plant, the -AI product could contribute to the protection of the transgenic plant against some insects. Indeed, the gene encoding -AI has now been successfully expressed in tobacco seeds allowing this plant to produce -AI protein in its tissues (Altabella and Chrispeels, 1990; Pueyo et al., 1993). However, as a prerequisite to these genetic manipulations to provide protection for the cultivated plants, the mechanism of the action of this inhibitor must be clarified. According to Pueyo et al. (1993), -AI is synthesized as a pre-proprotein, which is subsequently processed to give two polypeptides of smaller sizes. The processing was previously shown to occur at residue Asn77 producing two polypeptides of 10 and 14.6 kDa, respectively (Santino et al., 1992). This proteolytic cleavage is probably responsible for the removal of conformational constraints on the precursor that is necessary to produce an active inhibitor. From a comparison with the -AI called tendamistat (Hoe-467A) from Streptomyces tendae (Pflugrath et al., 1986; Kline et al., 1986, 1988), whose active site consists of the triad Trp18– Arg19–Tyr20, it was suggested that because the folding of the polypeptide chain of -AI was similar to that of legume lectins (Pueyo et al., 1993), its active site could be created by the confluence of three distant but similar residues (Trp189, Arg75 and Tyr191). As the three-dimensional structure of -AI and arcelin (another insecticidal protein from Phaseolus vulgaris) has been shown recently by molecular modelling computations to be similar to truncated lectins (Rougé et al., 1994), a model of AI could be compared with that of tendamistat and checked whether these two different inhibitors react with amylase by a common mechanism of action. Materials and methods The co-ordinates of the -AI Hoe-467A (tendamistat) from Streptomyces tendae (Pflugrath et al., 1986), were taken from the Protein Data Bank (code 1HOE, PDB, Brookhaven National Laboratory, New York, USA). The three-dimensional model of the bean -AI was previously modelled (Rougé et al., 1994) from the coordinates of both the Lathyrus ochrus isolectin I (LoLI; Bourne et al., 1990) and the pea lectin (PsA; Einspahr et al., 1986). The amino acid sequences of PHA-L and PHA-E, the leuco- and erythroagglutinating subunits of the common bean lectin (Hoffman and Donaldson, 1985), arcelin-1 (Osborn et al., 1988), the bean -AI (Hoffman et al., 1982) and that of tendamistat (Pflugrath et al., 1986) were compared with the ialign program (PIR-NBRF, Washington DC, USA), using the evolutionary matrix of Dayhoff et al. (1972), on a Micro Vax 3100 station (Digital). The Hydrophobic Cluster Analysis (HCA) method (Gaboriaud et al., 1987) was run (HCA-plot-V2 program, Doriane, Paris, France) using a Macintosh LC computer. The computer program Turbo Frodo (Roussel and Cambillau, Marseille, France) run on a Silicon

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Graphics Iris 4D25G station was used to generate and compare the three-dimensional models of the amylase inhibitors of plant and microbial (Hoe) origins.

Figure 2.1. Comparison of the amino acid sequences of the leuko-agglutinating subunit (PHA-L) of the common bean lectin and the bean -amylase inhibitor ( -AI). Dashes (—) indicate identical residues, and gaps (*) were inserted to maximize the homology.

Results and discussion Due to the high homology of the amino acid sequence of the bean -AI with those in PHA-L or PHA-E, the two subunits of bean lectins (Moreno and Chrispeels, 1989), or with other legume lectins (Sharon and Lis, 1990), -AI could be easily modelled from the co-ordinates of both LoLI and PsA (Rougé et al., 1994). All

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of these proteins share a similar folding but, by comparison with lectins, the bean -AI lacks two loops of 8 and 15 residues, respectively (Figure 2.1). In order to explain the mechanism by which the bean -AI could exert its insecticidal activity against the bruchid pest, Pueyo et al. (1993) have recently proposed a mechanism for its action by analogy to that proposed for tendamistat, Hoe-467A, the bacterial -AI from Streptomyces tendae (Pflugrath et al., 1986). Accordingly, if the active site of the bean -AI is similar to that of Hoe, which consists of three adjacent residues forming the triad Trp18–Arg19–Tyr20, it is possible that the three residues in -AI are the residues of Trp189, Arg75 and Tyr191. Although these are located in two different regions of the polypeptide chain of the plant inhibitor, because of its folding, they could be closely associated and form the active triad. In addition, the processing occurring at residue Asn77 of the bean -AI precursor, could bring these three residues closer to create the active site. According to spectroscopic studies performed with a complex of Haim II, an inhibitor similar to Hoe-467A, with pig pancreatic -amylase (Goto et al., 1985), the two aromatic residues of the triad in the putative active site are thought to play a major role. When superimposed on the triad Trp18–Arg19–Tyr20 of tendamistat, residues forming the cluster Trp189–Arg75–Tyr191 of -AI (Trp184, Arg74 and Tyr182 in our -AI model) were shown to be only distantly related and exhibited side-chains that were oriented differently (Figure 2.2). Although some torsion of their C -carbons could occur, this is thought to be insufficient to bring together the aromatic rings of the Trp184 and Tyr182 residues and the side chain of Arg74. However other triads, very similar to that of tendamistat, also exist in the bean -AI. They are the clusters of Tyr173−Asp174−Trp175, Tyr187−Gln188−Trp189, Trp189− Ser190−Tyr191 or Trp199−Ser200−Phe201 in its polypeptide chain (Figure 2.3). By a different numbering of these residues, they correspond to the clusters of Tyr168− Asp169−Trp170, Tyr182−Gln183−Trp184, Trp184−Ser185−Tyr186, and Trp194−Ser195− Phe196 in the three-dimensional model of -AI presented by us. Three of them, Tyr168−Asp169−Trp170, Tyr182−Gln183 −Trp184 and Trp184−Ser185−Tyr186, are sufficiently exposed on the surface of the protein to behave similarly to the tendamistat triad (Figure 2.4). However, superimposition of these three clusters with that of tendamistat, allowing the carbons of the central residue in the triads (Asp169, Gln183, Ser185 or Ser195) to overlap that of tendamistat (Arg19), resulted in a somewhat different orientation of the two aromatic residues Trp170/Trp184 and Tyr168/182/186 (Figure 2.5). Nevertheless, according to the possible torsion of the C -carbons of these residues, their aromatic rings could become similarly oriented. This is particularly true for the cluster Tyr182−Gln183−Trp184 and, to a lesser extent, for the cluster Tyr168−Asp169−Trp170 (Figure 2.6). Accordingly, these clusters could mimic the active site of tendamistat and, therefore, act as a main structural feature responsible for the inhibitory potency of the bean -AI. Both the lectin (PHA-L and PHA-E) and arcelins lack these clusters (Figure 2.1). These findings suggest that a common mechanism could explain the inhibitory character of both the bean -AI and tendamistat towards -amylase. Although the -amylase inhibitors from the bean and tendamistat have originated from very distantly related groups, both proteins consist of strands of nonparallel sheets interconnected by turns and loops. As well as these structural similarities, these proteins exhibit a rather high degree of both identity (up to 23 per cent) and similarity (up to 45 per cent), when their amino acid sequences are compared. However, these percentages vary according to which section of the amino acid sequence of -AI is compared to the whole amino acid sequence of tendamistat. Moreover, the HCA plots of both proteins share a degree of similarity, especially when residues of 75–140 in the polypeptide chain of -AI are compared to the whole sequence of tendamistat. These similarities cannot be explained unequivocally. They could reflect a distant but common origin for both proteins but, conversely, they could result from convergent evolution of distantly related proteins with a similar function.

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Acknowledgements This work was supported in part by grants from the Conseil Régional de Midi-Pyrénées. P Rousseau has a fellowship from AGEFIPH

Figure 2.2. Superimposition of the triad Trp18−Arg19−Tyr20 of tendamistat (thin) with residues Trp184, Arg74 and Tyr182 of the bean -AI (thick). Residues Arg19 (tendamistat) and Arg74 ( -AI) were used for the superimposition of the two triads.

Figure 2.3. Localization of the four triads Tyr168−Asp169−Trp170, Tyr182−Gln183−Trp184, Trp184−Ser185− Tyr186 and Trp194−Ser195−Phe196 (bold, underlined) along the amino acid chain of the bean -AI, which could mimic the triad Trp18 −Arg19−Tyr20 of tendamistat.

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Figure 2.4. -carbon tracings of the bean -AI (thin), showing the localisation (bold) of the four triads Tyr168−Asp169 −Trp170 (A), Tyr182−Gln183−Trp184, Trp184−Ser185−Tyr186 (B) and Trp194−Ser195−Phe196 (C) susceptible to act as the triad Trp18−Arg19−Tyr20 of tendamistat.

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Figure 2.5. Superimposition of the triad Trp18−Arg19−Tyr20 of tendamistat (thick) with the triads (thin) Tyr168−Asp169 −Trp170 (A), Tyr182−Gln183−Trp184 (B) and Trp194−Ser195−Phe196 (C) of the bean -AI. In all cases, the central residue Asp169, Gln183 or Ser195, was superimposed to Arg19 of tendamistat.

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Figure 2.6. Stereoscopic view of the superimposition of the triad Trp18−Arg19−Tyr20 of tendamistat (thick) with the triads (thin) Tyr168−Asp169−Trp170 (A) and Tyr182−Gln183−Trp184 (B) of the bean -AI, after proper torsion of the Tyr168/ 182 and Trp170/184 side chain residues in order to improve the superimposition with the Tyr20 and Trp18 residues of tendamistat.

References Altabella, T. and Chrispeels, M.J., 1990, Tobacco plants transformed with the bean AI gene express an inhibitor of insect -amylase in their seeds, Plant Physiology, 93, 805–10. Bourne, Y., Abergel, C., Cambillau, C., Frey, M., Rougé, P. and Fontecilla-Camps, J.C., 1990, X-ray crystal structure determination and refinement at 1.9 resolution of isolectin I from the seeds of Lathyrus ochrus, Journal of Molecular Biology, 214, 571–84.

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Chrispeels, M.J. and Raikhel, N.V., 1991, Lectins, lectin genes, and their role in plant defense, Plant Cell, 3, 1–9. Dayhoff, M.O., Eck, R.V. and Park, C.M., 1972, Atlas of Protein Sequence and Structure, vol. 5, Washington DC, USA: National Biomedical Research Foundation. Einspahr, H., Parks, E.H., Suguna, K., Subramanian, E. and Suddath, F.L., 1986, The crystal structure of pea lectin at 3. 0-resolution, Journal of Biological Chemistry, 261, 16518–27. Gaboriaud, C., Bissery, V., Benchetrit, T. and Mornon, J.P., 1987, Hydrophobic cluster analysis: an efficient new way to compare and analyse amino acid sequences, FEBS Letters, 224, 149–55. Gatehouse, A.M.R., Dewey, F.M., Dove, J., Fenton, K.A. and Pusztai, A., 1984, Effect of seed lectins from Phaseolus vulgaris on the development of Callosobruchus maculatus, mechanism of toxicity, Journal of the Science of Food and Agriculture, 35, 373–80. Goto, A., Matsui, Y., Ohyama, K., Arai, M. and Murao, S., 1985, Inhibitory effects of the proteinaceous -amylase inhibitor haim on animal -amylase, Agricultural and Biological Chemistry, 49, 435–39. Hadfield, P., 1993, Japanese aid may upset Cambodia’s harvests, New Scientist 137(1864), 5. Hoffman, L.M. and Donaldson, D.D., 1985, Characterization of two Phaseolus vulgaris phytohaemagglutinin genes closely linked on the chromosome, EMBO Journal, 4, 883–89. Hoffman, L.M., Ma, Y. and Barker, R.F., 1982, Molecular cloning of Phaseolus vulgaris lectin mRNA and use of cDNA as a probe to estimate lectin transcript levels in various tissues, Nucleic Acids Research, 10, 7819–28. Huesing, J.E., Shade, R.E., Chrispeels, M.J. and Murdock, L.L., 1991, -Amylase inhibitor, not phytohaemagglutinin explains the resistance of common bean seeds to cowpea weevil, Plant Physiology, 96, 993–96. Ishimoto, M. and Kitamura, K., 1989, Growth inhibitory effects of an -amylase inhibitor from the kidney bean (Phaseolus vulgaris L.) on three species of bruchids (Coleoptera: Bruchidae), Applied Entomology and Zoology, 24, 281–86. Janzen, D.H., Juster, H.B. and Liener, I.E., 1976, Insecticidal action of phytohaemagglutinin in black beans on a bruchid beetle, Science, 192, 795–96. Kline, A.D., Braun, W. and Wüthrich, K., 1986, Studies of 1H nuclear magnetic resonance and distance geometry of the solution conformation of the -amylase inhibitor tendamistat, Journal of Molecular Biology, 189, 377–82. Kline, A.D., Braun, W. and Wüthrich, K., 1988, Determination of the complete three-dimensional structure of the amylase inhibitor tendamistat in aqueous solution by nuclear magnetic resonance and distance geometry, Journal of Molecular Biology, 204, 675–724. Lajolo, F.M. and Finardi-Filho, F., 1985, Partial characterization of the -amylase inhibitor of black beans (Phaseolus vulgaris) variety Rico23, Journal of Agricultural and Food Chemistry, 33, 132–38. Marshall, J.J. and Lauda, C.M., 1975, Purification and properties of phaseolamin, an inhibitor of -amylase, from kidney bean, Phaseolus vulgaris, Journal of Biological Chemistry, 250, 8030–37. Moreno, J. and Chrispeels, M.J., 1989, A lectin gene encodes the -amylase inhibitor of the common bean, Proceedings of the National Academy of Sciences of the United States of America, 86, 7885–89. Moreno, J., Altabella, T. and Chrispeels, M.J., 1990, Characterization of -amylase-inhibitor, a lectin-like protein in the seeds of Phaseolus vulgaris, Plant Physiology, 92, 703–9. Osborn, T.C., Alexander, D.C., Sun, S.S.M., Cardona, C. and Bliss, F.A., 1988, Insecticidal activity and lectin homology of arcelin seed protein, Science, 240, 207–10. Pflugrath, J.W., Wiegand, G., Huber, R. and Vertesy, L., 1986, Crystal structure determination, refinement and the molecular model of the -amylase inhibitor Hoe-467a, Journal of Molecular Biology, 189, 383–86. Pick, K.H. and Wöber, G., 1978, Proteinaceous -amylase inhibitor from beans (Phaseolus vulgaris). Purification and characterization, Hoppe-Seyler’s Zeitschrift für Physiologische Chemie, 359, 1371–77. Powers, J.R. and Whitaker, J.R., 1977, Purification and some physical and chemical properties of red kidney bean (Phaseolus vulgaris) -amylase inhibitor, Journal of Food Biochemistry, 1, 217–38. Pueyo, J.J., Hunt, D.C. and Chrispeels, M.J., 1993, Activation of bean (Phaseolus vulgaris) -amylase inhibitor requires proteolytic processing of the proprotein, Plant Physiology, 101, 1341–48. Pusztai, A., 1991, Plant Lectins, Cambridge, UK: Cambridge University Press.

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Rougé, P., Cambillau, C. and Bourne, Y., 1991, The three- dimensional structure of legume lectins, in Kilpatrick, D.C., Van Driessche, E. and Bog-Hansen, T.C. (Eds) Lectin Reviews, vol. 1, pp. 143–59, St-Louis, Missouri, USA: Sigma Chemical Co. Rougé, P., Barre, A., Causse, H., Chatelain, C. and Porthé, G., 1994, Arcelin and -amylase inhibitor from the seeds of common bean (Phaseolus vulgaris L.) are truncated lectins, Biochemical Systematics and Ecology, in press. Santino, A., Daminati, M.G., Vitale, A. and Bollini, R., 1992, The -amylase inhibitor of bean seed: two step proteolytic maturation in the protein storage vacuoles of the developing cotyledon, Physiologia Plantarum, 85, 425–32. Sharon, N. and Lis, H., 1990, Legume lectins, a large family of homologous proteins, FASEB Journal, 4, 3198–208.

Chapter 3 Insecticidal Properties of Plant Lectins: Their Potential in Plant Protection Angharad M.R.Gatehouse, Kevin S.Powell, Willy J.Peumans, Els J.M.Van Damme and John A.Gatehouse

Role of lectins in plant defence The plant kingdom provides a rich and diverse source of secondary compounds. A protective role against various pests, pathogens and competitors has been established for many of these and in recent years the utilization of such compounds in crop protection, either by conventional plant breeding or by genetic engineering has been, and is being, investigated (Gatehouse et al., 1990). Amongst these compounds are the lectins. There are now several clear examples of lectins providing a protective role. The first report of plant lectins being toxic to insects was in 1976 when Janzen and co-workers attributed the inability of the cowpea bruchid beetle, Callosobruchus maculatus, to attack the seeds of Phaseolus vulgaris to the presence of the lectin (PHA). In these studies the purified lectin was added at a range of concentrations (0.1–5 per cent) to artificial beans. At the highest level there was no insect survival, and even at 0.1 per cent the lectin was found to cause a significant reduction in the number of larvae developing to adulthood. Gatehouse et al., (1984) subsequently confirmed the toxicity of these lectins towards developing larvae and further suggested the possible mechanism through which these molecules were able to exert their toxic effects. PHA lectin is composed of a mixture of E type and L type subunits and although the ratio of these subunits does vary, generally the E2-L2 form predominates. Interestingly, when either the purified E type or L type subunits on their own were tested in artificial diets, neither had any significant detrimental effects upon development of C. maculatus (Boulter and Gatehouse, 1986). More recent results have suggested that the toxicity of PHA preparations towards bruchids is due to a contaminating -amylase inhibitor which, although it is without lectin functionality, has sequence homology to the lectins (Altabella and Chrispeels, 1990). However, this observation appears to be specific to P. vulgaris and is not the case with lectin preparations from other species. Lectins present in the mature seeds of the winged bean Psophocarpus tetragonolobus D C have also been shown to be involved in seed resistance to non-pest species (Gatehouse et al., 1991), again indicating a protective role (Figure 3.1). The LC50 value for the basic form of the lectin was found to be 0. 35 per cent, which approximates to the physiological levels present in the mature seeds. Another such example is provided by the seed protein arcelin, (Mr 35000– 38000) present in several resistant wild lines of P. vulgaris (Romero Andreas et al., 1986), that constitutes the major seed storage protein at the expense of phaseolin (Gatehouse et al., 1990). Backcrossing experiments showed a strong correlation between the presence of this protein and resistance to a major storage pest of beans, Zabrotes subfasciatus (Osborn et al., 1989) and in subsequent feeding trials when the purified protein was added to artificial seeds at levels significantly lower than the physiological concentration present in the wild resistant seed, adult survival was reduced by 52 per cent (Minney et al., 1990).

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Figure 3.1. Effect of different concentrations of purified basic lectin from winged bean seeds on adult emergence of Callosobruchus maculatus.

Despite the reservations by some workers about the involvement of PHA in plant protection, these early studies paved the way for exploiting lectins in crop protection, particularly by means of plant genetic engineering (reviewed by Gatehouse et al., 1992a). Crop protection Crop protection plays a vital and integral role in modern-day agricultural production. The ever-increasing demands on yield and the projected shortfall in pro duction relative to demand has led to an intensification of farming practice worldwide. This in itself has increased the potential for pest damage, and hence the requirements for control. Problems with pest damage caused by intensive farming regimes can be illustrated by some of the highyielding dwarf rice varieties to come out of the ‘Green Revolution’, which require high fertiliser levels to fulfil their yield potential. This not only results in increased vegetative tissue, but also alters the amino acid composition of phloem sap, thus making the plants more attractive to sap-sucking pests such as the rice brown planthopper (Nilaparvata lugens). Other agricultural practices, such as monoculture and continuous cropping (Strong, 1979) can also cause the build-up of a pest population, thereby exacerbating the problem of control. Even multiple cropping can be detrimental if the crops act as hosts for a single pest, such as cotton and maize, both of which are attacked by the corn earworm (Heliothis zea). At present, crop protection in intensive agricultural systems relies almost exclusively on the use of agrochemicals, although a few specific cases do exist where inherent varietal resistance and biological

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Figure 3.2. Total world insecticide usage, divided according to crops. Total: US $6.075 billion. (County NatWest Woodmac, 1988 figures).

control have been successfully employed. A breakdown of the use of insecticides per pest order is illustrated in Figure 3.2. Exclusive use of chemical pesticides has resulted in the rapid build-up of resistance to such compounds. For example, the cotton bollworm (Heliothis virescens) has rapidly developed resistance to organochlorine insecticides, and the Colorado beetle to synthetic pyrethroids. Examples have been observed of pest species developing resistance to new insecticides within their first year of field use (Metcalf, 1986). The non-selective toxicity of pesticides also affects the balance between pests and their natural predators, in favour of the pest (which develops resistance quicker). For example, two outbreaks of rice brown planthopper infestations in Thailand, in the late 1970s and again in the late 1980s, each were preceded by an increase in the use of pesticides (Hadfield, 1993). A decrease in pesticide usage has been shown in certain cases to have beneficial results. Following the introduction of an integrated pest management (IPM) programme in Indonesia in the 1980s, pesticide application fell by more than 50 per cent, and this was accompanied by a decrease in planthopper infestations and a 12 per cent increase in rice yields (Hadfield, 1993; Figure 3.3). The ‘blanket’ approach to pesticide application employed until now has not only reduced the effective life-span of a given compound, but has also had serious environmental consequences. It is enormously wasteful, estimates have suggested that only about 1 per cent of the applied chemical actually comes into direct contact with the plant itself, let alone the pest that it is meant to control. Human health concerns, either as a result of direct or indirect contact with pesticides also cannot be ignored. World Health Organisation estimates put the number of accidental deaths attributable to pesticide poisoning at about 20000 per year, mostly in developing countries (Dirham, 1993). Despite an annual insecticide expenditure of approximately US $ 7.5 billion, it is estimated that 37 per cent of all crop production is lost worldwide to pests and diseases, with at least 13 per cent lost directly to insects (Figure 3.4). There is, therefore, a great need not only to provide a greater level of protection to our crops, but also for a more efficient and safer method of pest control. Although complete elimination of chemical control agents is not realistic in the foreseeable future, a significant reduction in their use is both necessary and desirable. An integrated pest control programme, comprising a combination of practices including the judicious use of pesticides, crop rotation, field sanitation, but above all exploiting inherently resistant plant varieties, would appear to provide the best option (Meiners and Elden, 1978). Within this last category the use of genetically engineered crops expressing selected insecticidal lectins may be included.

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Figure 3.3. The effects of pesticide application on rice production in Indonesia (Hadfield, 1993; courtesy of New Scientist).

Effect of lectins on phytophagous insects Not only have certain lectins been identified as insecticidal by demonstrating a protective role within the seed, as typified by the bean lectins and winged bean lectins (Janzen et al., 1976; Gatehouse et al., 1984, 1991), but also several studies have been carried out involving the screening of purified lectins against insect pests of economically important crop plants in an attempt to identify insecticidal proteins (Murdoch et al., 1990), and hence isolate the genes encoding them for subsequent plant transformation. Work carried out to date in an attempt to identify suitable insecticidal lectins will be discussed by insect order. Coleoptera This is the largest order in the animal kingdom, numbering approximately 220000 species, all of which are beetles. Many of this order of species represent serious pests of agricultural crops, both in the field, and of stored products. Although in some cases it is only the larval form which causes severe damage, with others it can be both the larval and adult stages. Members of this order account for approximately 11.0 per cent of the insecticide market (Figure 3.2). The first lectin with reported insecticidal activity was that from P. vulgaris (PHA) although its effectiveness against the cowpea bruchid, C. maculatus is now seriously questioned (Janzen et al., 1976; Gatehouse et al., 1984). Apart from this, further studies have been carried out to identify lectins which may be used in plant protection. Although both PHA and winged bean lectin are insecticidal towards certain

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Figure 3.4. The World Agricultural Cake.

insect pests, their potential is limited as they are also highly toxic to mammals (Pusztai et al., 1979; Higuchi et al., 1984). The potential of arcelin may also be of little value, but for different reasons. This protein has been shown to be resistant to digestion by insects in vitro (Figure 3.5; Minney et al., 1990) and, because it replaces the major storage protein when present, if it is expressed in a background of readily digestible proteins, as is required in food crops, it may not prove to confer resistance. Although arcelin shows a high level of sequence homology with PHA, unlike PHA it does not possess agglutination activity, neither does it appear to be toxic to mammals (Pusztai et al., 1993a). In an attempt to identify further lectins toxic to C. maculatus, a major storage pest of cowpeas in many parts of the developing world, and responsible for an estimated annual loss in excess US$50 million (Shade et al., 1986), Murdoch and co-workers screened 17 commercially available plant lectins for insecticidal activity against this pest (Murdoch et al., 1990). Five lectins were found to cause a significant delay in larval development at dietary levels of 0.2 per cent and 1.0 per cent (w/w) and all fell within one of two groups, those with specificity for N-acetylgalactosamine/galactose (GalNAc) and those for N-acetylglucosamine (GlcNAc). The rationale for using lectins with specificity for GlcNAc is based on the fact that the insect mid-gut contains chitin, a polymer of N-acetylglucosamine, in the peritrophic membrane (Richards and Richards, 1977). Of all those tested against this particular insect in the present study, wheat germ agglutinin (WGA) was found to be the most potent. Not only did each successive 0.1 per cent increase in dose delay development by 1.47 days, but it also gave a corresponding increase in mortality of 2.79 per cent. These same workers later identified rice and stinging nettle lectins (UDA) as being toxic to C. maculatus, exhibiting similar levels of toxicity to WGA; like WGA they are specific for GlcNAc (Huesing et al.,

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Figure 3.5. In vitro digestion of purified proteins from Phaseolus vulgaris by Zabrotes subfasciatus larval gut proteases. Phaseolin, major storage protein present in P. vulgaris seeds, was moderately digestible, whereas arcelin was resistant to proteolysis.

1991). However, as with PHA and winged bean lectin, these three lectins may also be of limited value with respect to their use in crop protection as WGA and UDA are nutritionally toxic for mammals at the levels required in crop protection (Pusztai et al., 1993b). Moreover, as these lectins are resistant to heat treatment they retain their deleterious effects after normal cooking. Czapla and Lang (1990) took a similar approach when they screened a range of lectins for activity against the Southern corn rootworm (Diabrotica undecimpunctata), a major economic pest of corn. The lectins divided into three groups based on their specificity namely GalNAc, GlcNAc and mannose/glucose. Of the lectins tested, three, from caster bean, pokeweed and green marine algae, were found to be toxic to the neonate larvae when applied topically (2 per cent) to the artificial diet. Several others, including WGA, were found to inhibit larval growth by at least 40 per cent when compared with larvae fed on control diet. Similar to findings by Murdoch et al. (1990) with C. maculatus, all those lectins with insecticidal activity against corn rootworm were either specific for GalNAc or GlcNAc. In contrast to the insecticidal lectins described above, the lectin isolated from the pea, Pisum sativum L, has been shown to be innocuous to mammals as it is readily broken down in the gut (Begbie and King, 1985). This lectin (specific for mannose/glucose) whilst ineffective against Southern corn rootworm, was toxic to C. maculatus with an LC50 value of 1.5, as was the mannose-specific lectin from snowdrop (Galanthus nivalis; GNA) with an LC50 value of 0.9. As with the pea lectin, GNA does not appear to exhibit mammalian toxicity (Pusztai et al., 1990) and in this respect the encoding genes are ideal candidates to transfer for crop protection. Two lectins have been isolated from elderberry (Sambucus nigra; SNA) SNA-I,

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specific for 2,6-neuraminyl-gal/GalNAc and SNA-II which is specific for GalNAc, and whilst the former was found to be extremely insecticidal (Gatehouse et al., in preparation) the latter was ineffective; this is contrary to their mammalian toxicity: SNA-I was found not be to toxic to rats whereas SNA-II did exhibit some toxicity. This information would suggest that genes encoding SNA-I may also be of value in order to confer insect resistance in crops. Although the specificity of a given lectin could be a good indicator of its potential insecticidal properties, in practice this does not always appear to be the case and it is still necessary to test each lectin against the target pest on a case by case basis. For example, although SNA-I was found to be extremely potent to C. maculatus, the seed lectin from Maackia, which exhibits similar specificity, was relatively innocuous to this same insect. Again, whilst the lectins from the seeds of the Brazilian plant species Canavalia brasiliensis, Dioclea paraguariensis, D. rostrata and Cratylia floribunda have identical specificities (Man/Glc) their insecticidal properties differ from one another (Gatehouse et al., in preparation) with the two former being totally ineffective and the two latter being toxic. The prediction of toxicity being based on specificity is further complicated by the recent finding that the insecticidal properties of a range of mannose-specific lectins is influenced by the subunit number (Powell et al., in press). For example, the lectins from snowdrop, daffodil and garlic are all toxic to rice brown planthopper (see section on Homoptera), but snowdrop (tetramer) was the most effective, garlic (dimer) the least effective, and daffodil (trimer) exhibited intermediate levels of toxicity. Lepidoptera Lepidoptera are moths and butterflies, and it is the larval stage, the caterpillars, which cause crop damage. This order includes both field and storage pests and accounts for approximately 37.0 per cent of the insecticide market. Unlike for the Coleoptera, comparatively few lectins have been tested in artificial diets and found to be toxic to members of this economically important insect order. Czapla and Lang (1990) tested a range of lectins for insecticidal activity against the European cornborer, Ostrinia nubilalis. Of those tested, the lectins from caster bean, Ricinus communis, RCA, Camel’s foot tree, Bauhinia purpurea, BPA, both specific for GalNAc, and wheat germ, Triticum vulgare, WGA, specific for GlcNAc, were found to give 100 per cent mortality after 7 days when administered to neonate larvae as a 2 per cent topical application. WGA and RCA were also found to inhibit larval weight gain by >50 per cent at 0.1 per cent topical applications. The LC50 values for RCA, WGA and BPA against this particular insect pest were 0.29, 0.59 and 0.73 mg g–1 of diet respectively. Based upon these results it was suggested that transformation with the genes encoding these particular lectins could be beneficial in the development of insect resistance in important agronomical crops. In contrast, the soya bean lectin, SBA, actually increased larval weights of O. nubilalis by >25 per cent compared with control larvae. This is in contrast to earlier reports where addition of this particular lectin at the 1 per cent level was found to be detrimental to the larval growth of Manduca sexta, the tomato hornworm. Weight differences between treated and control larvae were seen at 4 days and became significant by 8 days (Shukle and Murdock, 1983). Homoptera Sucking insect pests cause serious crop damage, both directly and by acting as vectors for plant pathogens. Furthermore, they are difficult to control using con ventional pesticide regimes due to their rapid adaptation, resulting in insecticide resistant phenotypes. At present, an estimated 26 per cent of the

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Figure 3.6. Effects of a range of plant lectins on mortality of third instar nymphs of brown planthopper (Nilaparvata lugens). All lectins were tested at a single concentration of 0.1 per cent (w/v). (Lectins tested Galanthus nivalis agglutinin (GNA), wheat germ agglutinin (WGA), Phaseolus vulgaris agglutinin (PHA), Pisum sativum agglutinin (PSA), Lens culinaris agglutinin (LCA), horse gram agglutinin (HGA), jacalin agglutinin (JCA), Concanavalin (Con A) and potato lectin (PPL).)

insecticide market is spent on the control of these pests. Whereas considerable success in combating these insects has been achieved in glasshouse crop production using biological control, such measures are not in general as effective in the field, and there is a need for improving the endogenous resistance of host plants. Most work on improving the resistance of plants to sucking pests has concentrated on the role of semiochemicals, and plant secondary metabolites as feeding deterrents. Due to the complexity and speciesspecificity of the biochemical pathways involved, the feasibility of engineering transgenic plants to confer

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Figure 3.7. Effects of wheat germ agglutinin (WGA) and Galanthus nivalis agglutinin (GNA) on survival of third instar nymphs of brown planthopper (Nilaparvata lugens) with time. Each lectin was incorporated into the artificial diet (MMD-1) at a concentration of 0.1 per cent (w/v).

the ability to produce secondary metabolites has yet to be demonstrated, and the ability to do this on a routine basis for given secondary compounds is some way in the future, although this approach is now being addressed (Hallahan et al., 1992). In order to attempt to tackle the problem of producing transgenic plants with inherent resistance to sucking pests, suitable insecticidal proteins had first to be identified, but this did not happen until 1991 when preliminary results from this laboratory were reported (Shi et al., 1991). Powell et al. (1993), tested a series of lectins against the rice brown planthopper (Nilaparvata lugens) an important pest of rice in SE Asia, and although some (for example pea lectin and potato lectin) had no significant effect on insect survival, others did (Figure 3.6). The two most effective proteins tested were GNA and WGA (Figure 3.7), which gave approximately 80 per cent corrected mortality at a concentration of 0.1 per cent w/v in the diet against both first and third instar nymphs. The LC50 value of GNA against this particular pest was found to be 0.02 per cent. GNA was also found to be toxic to another sucking pest of rice, the rice green leafhopper, Nephotettix cinciteps.

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The possibility of controlling homopteran pests by using suitable insecticidal lectins in transgenic crops is now receiving much attention. Habibi et al. (1992; 1993) have carried out similar bio-assays in order to identify lectins which may be suitable in the control of the potato leafhopper (Empoasca fabae). Of the lectins tested (either specific for GlcNAc or GalNAc), six were found to cause a significant reduction in insect survival at dietary levels of 0.2 per cent to 1.5 per cent (w/w). Those found to be effective were from Jackfruit, pea, lentil and horse gram and also PHA and WGA. Rahbé and Febvay (1993) demonstrated that the lectin from Canavalia ensiformis (Con A) was a potent toxin of the pea aphid Acyrthosiphon pisum, having a significant effect upon both survival and growth. In comparison, WGA was relatively ineffective. As Con A does not appear to be toxic to mammals, at least at low levels of dietary inclusion, the genes encoding this protein should be suitable for transfer. In collaboration with Rahbé and co-workers we have extended these studies to examine the effects of a range of different plant lectins, including GNA, on the pea aphid (Rahbé et al., in press) and on the peach/potato aphid Myzus persicae (Sauvion et al., in preparation). Not only did GNA cause a significant reduction in growth of M. persicae, but it also significantly reduced female fecundity, a very important parameter when trying to prevent the build-up of an insect population. Diptera With one exception, all reported studies to date on the effects of plant lectins on insects have been on phytophagous insects. Very recently a study has been carried out to examine the effects of plant lectins on larvae of the blowfly, Lucilia cuprina, in an attempt to identify possible control strategies for this pest (Eisemann et al., 1994). The larvae of this pest feed on tissue and tissue fluids of susceptible sheep ultimately leading to conditions which can cause the death of the animal and hence severe economic losses to the sheep and wool industries. In this study, Eisemann and co-workers demonstrated that both WGA and Con A caused strong concentration-dependent inhibition of larval growth and substantial mortality. Of these two, WGA was the most potent resulting in 50 per cent inhibition of larval growth at a concentration of 2 µ M and 100 per cent mortality at 25 µ M. The fact that these deleterious effects could be prevented by the presence of the appropriate sugars, suggests a highly specific interaction. Mechanism of lectin toxicity Of fundamental importance is the means through which insecticidal lectins exert their toxic effects. For a given lectin to be toxic it must survive passage through the gut for a sufficiently long period in order to exert its effect. In an attempt to address the problem as to why relatively few lectins tested so far are toxic to Lepidoptera, the survival of several different lectins in the larval gut of Heliothis virescens was investigated and compared with their survival in Diabrotica (Coleopteran). Of those tested, apart from the lectin from Pisum sativum which was readily hydrolysed by H. virescens, the others were stable although not necessarily toxic (Table 3.1). These results suggest that stability of the protein alone is not sufficient and that some positive mechanism(s) must be operational. It is only relatively recently that detailed investigations as to the possible mechanisms of lectin toxicity in insects have begun. The first such study was in 1984, when Gatehouse and co-workers were able to demonstrate by immunofluorescence microscopy, PHA-binding to the mid-gut epithelial cells of C. maculatus (Figure 3.8). In contrast, there was no binding of the lectin molecules to mid-gut epithelial cells of Acanthoscelides obtectus (Figure 3.8). This insect is a storage pest of P. vulgaris seeds and is therefore

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able to tolerate moderately high levels of PHA (Gatehouse et al., 1989). It would appear that this lack of lectin-binding to the mid-gut cells of A. obtectus enables the insect to feed on P. vulgaris seeds with no detrimental effects. However, the effects of this binding on the mid-gut epithelial cells of C. maculatus are not known. The bound lectin may inhibit the absorption of nutrients or may disrupt the mid-gut cells by stimulating endocytosis of not only the lectin, but possibly other toxic metabolites and bacterial toxins present in the mid-gut. Another possibility is that lectins may bind to the peritrophic membrane, as opposed to the epithelium, in the mid-gut region. This membrane exists in most phytophagous insects protecting the mid-gut epithelial cells from abrasive food particles. Since the membrane is composed primarily of chitin, which in turn is composed of GlcNAc residues (Richards and Richards 1977), it is not unreasonable to postulate that certain lectins exert their toxic effects through binding to this membrane, particularly as many of the insecticidal lectins are specific for GlcNAc (Czapla and Lang, 1990; Huesing et al., 1991). Apart from the work of Eisemann and co-workers, however, there has been little other direct evidence reported to support this hypothesis. Perhaps one of the most comprehensive studies on the possible mechanisms of lectin toxicity to be reported to date involves the effects of WGA, Con A and Table 3.1. Stability of plant lectins in in vitro proteolysis Lectin

Diabrotica undecimpunctata (Coleoptera)

Heliothis virescens (Lepidoptera)

PHA (Phaseolus vulgaris) ++++ n.d. GNA (Galanthus nivalis) ++++ ++++ WGA (Wheat germ) ++ ++++ SBL (soya bean) ++ ++++ PSL (Pisum sativum) ++++ + Lectins resistant to proteolysis in vitro by an insect gut enzyme preparation are denoted + + + +, those partially hydrolyzed are denoted + + and those readily hydrolyzed are denoted +. n.d.=not determined.

lentil lectin on larvae of the blowfly, Lucilia cuprina (Eisemann et al., 1994). These authors propose that the insecticidal effects on blowfly larvae is caused by at least three different mechanisms of action, namely a reduced intake of diet, a partial blockage of the pores of the peritrophic membrane and the direct binding of specific lectins to the mid-gut epithelial cells. The first two mechanisms of action could cause a restriction in the nutrients available to the digestive cells and a subsequent general starvation effect in the larva. This conclusion is consistent with the observation that ingested WGA, Con A or lentil lectin cause no obvious damage to the larvae and that larvae can have their weight reduced by up to 80–90 per cent by these lectins before there is substantial mortality. The feeding deterrence caused by the ingested lectins, if mediated by gustation, may be due to the binding of lectins to glycoproteins situated on dendrites of chemoreceptor neurones near the mouth parts of the larvae. A consequent disruption of the normal functioning of these neurones may give rise to abnormal sensory input to the central nervous system, resulting in a partial inhibition of feeding. The apparent blockage of the pores of the peritrophic membrane by ingested lectins may not be a simple event. The large amount of undefined material localized on the gut lumen side of this membrane after a larva has fed on growth medium containing any of the lectins Con A, lentil lectin or WGA suggests that the binding of these lectins to the peritrophic membrane induces the aggregation of ingested material (probably protein). This feeding deterrency of lectins has also been observed in other studies. Blowfly

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Figure 3.8. Immunofluorescence micrographs of part of a transverse section through the mid-gut of Callosobruchus maculatus larva (Ai) and Acanthoscelides obtectus larva (Bi) fed on a diet containing Phaseolus vulgaris lectin (PHA) for 48 h. Incubation with rabbit anti-lectin immunoglobulin and fluorescein isothiocyanate-conjugated IgG showing immunofluorescence in the cell surfaces adjacent to the gut lumen in Ai and in parts of the ingested food in Ai and Bi; Aii and Bii are the corresponding light micrographs (Gatehouse et al., 1989).

larvae, when offered a free choice between pads containing either bovine serum albumin (5 mg ml−1) in the presence of 50 M WGA, or bovine serum albumin alone, were nine times more likely to choose the latter

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Figure 3.9. Effects of Galanthus nivalis agglutinin (GNA) on the rate of honeydew production on adult brown planthoppers (Nilaparvata lugens) over a period of 48 h. GNA was administered at a single concentration of 0.1 per cent (w/v).

(Eisemann, personal communication). These preliminary observations suggest that some lectins can directly stimulate avoidance responses. Reduced food intake may also be a mechanism by which certain lectins exert their deleterious effects on the brown planthopper (Powell et al., in press). When adults of this insect were fed a diet containing either GNA (0.1 per cent w/v) or WGA (0.1 per cent w/v) for 24 h, the volume of honeydew produced was significantly reduced compared to that collected from control diet fed adults (Figure 3.9). Since the volume of honeydew excreted is roughly proportional to the volume of fluid ingested it would appear that both lectins, which are known to be toxic to brown planthopper (Powell et al., 1993) have a marked feeding deterrent effect. Since GNA had a greater effect than WGA in terms of reduced feeding, experiments were carried out to see whether this deterrent effect was overcome with time. Although there was some recovery in the amount of honeydew produced over a time period of 48 h, this never reached the levels produced by control insects (Figure 3.9). Powell also investigated possible GNA-binding to the mid-gut epithelial cells of rice brown planthopper, using the Avidin/biotin technique. As with other workers (Gatehouse et al., 1984, 1989; Eisemann et al., 1994) he found that there was a positive binding of the lectin. Very recently Rahbé and co-workers have clearly demonstrated binding of Con A to the mid-gut epithelium of another sap sucking insect, the pea aphid. From studies reported so far, there is little doubt that there are several mechanisms involved in the toxicity of certain lectins towards insects. Whether one is a consequence of another, or whether they operate independently of each other, and indeed which, if any, is the most important, has yet to be clarified.

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Transgenic plants expressing lectin encoding genes Expression of Pisum sativum lectin Genes encoding the pea lectin (P-Lec) have been expressed at high levels in transgenic tobacco plants from the CaMV 35S promoter by Agrobacterium transformation (Edwards, 1988). P-Lec expressing plants were then tested in bioassay for enhanced levels of resistance/tolerance to Heliothis virescens (tobacco budworm). The results showed that not only was larval biomass significantly reduced on the transgenic plants compared with that from control plants, but leaf damage, as determined by computer-aided image analysis, was also reduced (Boulter et al., 1990). Transgenic tobacco plants containing both the cowpea trypsin inhibitor gene (CpTI) and the P-Lec gene were obtained by cross-breeding plants derived from the two primary transformed lines. These plants expressing the two insecticidal genes, each at approximately 1 per cent of the total soluble protein, were also screened for enhanced resistance to H. virescens. Although the insecticidal effects of the two genes were not synergistic, they were additive with insect biomass on the double expressers being only 11 per cent compared with those from control plants and 50 per cent of those from plants expressing either CpTI or P-Lec alone (Figure 3.10). Leaf damage was also the least on the doubleexpressing plants (Figure 3.11). Not only is this the first example of a lectin gene being successfully transferred to another plant species resulting in enhanced insect resistance, but it is also the first demonstration of additive protective effects of different plant-derived insect-resistance genes. Expression of a lectin-like protein from Phaseolus vulgaris Moreno and Chrispeels (1989) presented strong circumstantial evidence that an -amylase inhibitor present in the seeds of P. vulgaris and active against mammalian and insect, but not plant, -amylases was encoded by an already identified lectin gene, whose product is referred to as lectin-like protein (LLP). A chimeric gene, consisting of the coding sequence of the lectin gene that encodes LLP and the 5 and 3 flanking sequences of the lectin gene that encode phytohaemagglutinin-2, has been made and expressed in transgenic tobacco (Altabella and Chrispeels, 1990). Subsequent analysis of the seeds obtained from these transgenic plants demonstrated the presence of a series of polypeptides (Mr=10000−18000) which cross-reacted with antibodies to the bean -amylase inhibitor. As seed extracts from these plants inhibited not only pig pancreatic -amylase activity but also the -amylase activity present in the mid-gut of Tenebrio molitor (mealworm), this led the authors to suggest that introduction of this lectin gene ( ai) into other leguminous plants may be a strategy to protect the seeds from the seed-eating larvae of Coleoptera. Although transgenic tobacco plants expressing this gene are available, no insect bioassays on these plants appear to have been reported. The authors do, however, express reservations as to the usefulness of this particular inhibitor to protect plants against attack by lepidopteran insects, as the pH optimum for the formation of the protein complex between -amylase and the inhibitor is pH 5–6 (Powers and Whitaker, 1977). It is known that insects in this order have a basic pH, whilst Coleoptera have an acid pH in their mid-gut (Dow, 1986). Expression of Galanthus nivalis lectin (GNA) On the basis of bioassays with both brown planthoppers and several different species of aphid, a gene encoding GNA could be identified as having potential in engineering plants for resistance to sucking insects. GNA is a tetrameric protein with a subunit molecular weight of approximately 13000 and has a binding specificity to D-mannosyl residues (Van Damme et al., 1987). The cDNA clone (obtained from the laboratory

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Figure 3.10. Insect survival and biomass and leaf damage of CpTI/P-Lec expressing tobacco plants against Heliothis virescens. C−L+, plants express pea lectin but not CpTI; C+L−, plants express CpTI but not pea lectin; C+L+, plants express both CpTI and pea lectin.

of W.Peumanns) contained the complete coding sequence of the mature protein, plus an N-terminal leader sequence, and a C-terminal extension. GNA is a heterogeneous protein, isolated from snowdrop bulbs and is apparently encoded by a large gene family. The use of a cDNA clone, however, ensured that the sequence used was expressed in the source plant. Initial experiments placed the GNA coding sequence under control of the CaMV 35S promoter and examined the expression in transgenic tobacco plants. As was the case for other plant proteins, achieving reasonable levels of expression of GNA proved to be straightforward. The complete coding sequence gave rise to levels of GNA protein up to 1 per cent of total protein in leaf tissue of primary transformants, as determined by quantitative dot-blot immunoassay using anti-GNA primary antibodies. Higher levels of GNA (up to 1.5 per cent) were observed in progeny plants produced by selfing the primary transformants, although the segregation pattern was not consistent with a single gene locus for GNA. The functional integrity of GNA expressed in the transgenic tobacco was

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Figure 3.11. Bioassay of control and CpTI/P-Lec expressing transgenic tobacco plants against larvae of Heliothis virescens (tobacco/cotton budworm). (A) control plants showing high levels of leaf damage; (B) transgenic plants showing minimal damage.

demonstrated by haemagglutination assay. In this assay the highest dilution to agglutinate erythrocytes was consistent with the level of GNA expression deter mined for the tissue, and with the known

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haemagglutination activity of pure GNA. Although immunolocalization of GNA in transgenic tobacco plants expressing the CaMV-GNA construct showed that GNA was present in phloem cells, as well as in all other tissues, a phloem-specific promoter would possibly lead to higher levels of the protein in phloem tissue, and might be advantageous under some circumstances in confining expression to the site of insect attack. As rice is one of our target crops, particularly in relation to control of sap sucking pests, and because the maize sucrose synthase gene (Sh-l) is specifically expressed in phloem (and in developing grains) the promoter from the corresponding rice gene was chosen as optimal for the ultimate aim of engineering resistance to sucking pests into rice. The ricesucrose synthase gene RSSI has been isolated, characterized and fully sequenced (Wang et al., 1992), and its promoter (1.9 kb of 5 flanking sequence, plus the transcription start, first exon and first intron of the gene) has been tested in transgenic tobacco by means of a suitable reporter gene (gus) fusion. The RSSI-gus chimaeric gene directs expression of GUS protein in the phloem tissue of leaves, stems, petioles and roots of transgenic plants with no detectable expression in other tissues, as required (Shi et al., 1994). A similar RSSI-promoter fusion to the GNA coding sequence was used to direct the phloem specific expression of GNA. The fusion contained 13 extra N-terminal amino acids, 6 from the N-terminus of ricesucrose synthase and 7 from a multipurpose cloning site, fused to the complete GNA preprotein coding sequence. Transgenic tobacco plants expressing this construct were shown to be expressing GNA in phloem cells by immunolocalization, and a novel method was used to demonstrate that the foreign protein was present in the phloem sap (Shi et al., 1994). Peach-potato aphids (Myzus persicae) were allowed to feed on the phloemspecific GNA-expressing transgenic tobacco plants, and the excreted honeydew from the insects (in amounts of <1 µ l) was collected on nitrocellulose membranes. The drops of honeydew were then tested by blot-immunoassay, using anti-GNA antibodies, for the presence of GNA. Honeydew from aphids fed on transgenic plants gave a positive reaction, whereas that from control plants was negative. This experiment proved that GNA must have been present in the phloem sap ingested by the aphids, and proves the viability of the promoter-coding sequence fusion used for the transformation. Besides promoter-determined tissue specific expression, it shows that the GNA protein is transported to the phloem sap from its site(s) of synthesis in the companion cells and immature sieve elements, and that the protein is not subject to rapid proteolytic degradation, or immobilization on phloem structures, once there. Furthermore, these experiments also demonstrated that, as with the brown planthopper in artificial diet studies, there was a significant reduction in honeydew production compared with insects on control plants. Thus, a possible mechanism for lectin toxicity proposed on the basis of artificial diet studies appears to hold for studies in planta. Not only have tobacco plants expressing GNA shown a significant degree of resistance to aphids (Hilder et al., 1994) but very recently we have also demonstrated a similar effect in potato plants expressing GNA. In these studies GNA had a marked deleterious effect upon female fecundity, as was also the case in artificial diet studies. Fecundity is a very important parameter in the field in that it determines the rate of population build-up. In addition to these transgenic potato plants being effective against the peachpotato aphid, they were also shown to be significantly resistant to attack by larvae of the lepidopteran Lacanobia oleraceae in terms of survival, larval biomass and area of leaf damage. This particular example is the first, as far as we are aware, of lectins being expressed in an agronomically important crop with significantly increased levels of insect resistance.

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Conclusion There have been several clear examples that certain lectins do have a protective role in plants, particularly within storage tissues such as seeds. In addition to these, a range of commercially available lectins have also been shown to be insecticidal to economically important insect pests both in artificial diet and, in a few limited examples, in transgenic plants. Thus the use of genetically engineered crops expressing lectin genes appears to be a viable means of producing crops with significantly enhanced levels of resistance which can then be incorporated into an IPM programme. The strategy employed by the authors is not only to use single genes, but also to use gene combinations whose products are targetted at different biochemical and physiological processes within the insect. These packages will not only contain lectin genes but also genes encoding other demonstrated insecticidal proteins such as enzyme inhibitors (Gatehouse et al., 1992a, b). In this way, it is hoped to provide a multimechanistic form of resistance that can be tailored to the different crops and prevailing insect pests at a given time. Acknowledgements The authors would like to acknowledge financial support of some of the work presented here from the Agricultural Genetics Company Ltd, the Rockefeller Foundation and the British/French Alliance Scheme. Work on licensed insect pests was carried out on a MAFF Licence (No. PHF 346A/410/23). Our grateful thanks to Ms Elaine Williams for the typing of this chapter. References Altabella, T. and Chrispeels, M.J., 1990, Tobacco plants transformed with the bean ai gene express an inhibitor of insect -amylase in their seeds, Plant Physiology, 93, 805–10. Begbie, R. and King, T.P., 1985, The interaction of dietary lectin with porcine small intestine and the production of lectin-specific antibodies, in Bog-Hansen T.C. and Breborowicz J. (Eds) Lectins, vol. IV, pp. 15–17, Berlin: Walter de Gruyter and Co. Boulter, D. and Gatehouse, A.M.R., 1986, Isolation of genes involved in pest and disease resistance, in Magnien E. (Ed.) Biomolecular Engineering in the European Community, pp. 715–25, Dordrecht: Martinus Nijhoff. Boulter, D., Edwards, G.A., Gatehouse, A.M.R., Gatehouse, J.A. and Hilder, V.A., 1990, Additive protective effects of incorporating two different higher plant derived insect resistance genes in transgenic tobacco plants, Crop Protection, 9, 351–54. Czapla, T.H. and Lang, B.A., 1990, Effect of plant lectins on the larval development of European corn borer (Lepidoptera: Pyralidae) and Southern corn rootworm (Coleoptera: Chrysomelidae), Journal Economic Entomology, 83, 2480–85. Dirham, B., 1993, The Pesticides Hazard, London: The Pesticides Trust. Dow, J.A.T., 1986, Insect midgut function, Advances in Insect Physiology, 19, 187–328. Edwards, G.A., 1988, ‘Plant transformation using an Agrobacterium tumefaciens’, PhD thesis, University of Durham, UK. Eisemann, C.H., R.A.Donaldson, R.D.Pearson, L.C.Cadagon, T.Vuocolo & R.L. Tellam, 1994. Larvicidal activity of lectins on Lucilia cuprina: Mechanism of action. Entomologia experimentalis et Applicata 72:1–11. Gatehouse, A.M.R., Dewey, F.M., Dove, J., Fenton, K.A. and Pusztai, A., 1984, Effect of seed lectin from Phaseolus vulgaris on the development of larvae of Callosobruchus maculatus; mechanism of toxicity, Journal of the Science of Food and Agriculture, 35, 373–80.

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Gatehouse, A.M.R., Shackley, S.J., Fenton, K.A., Bryden, J. and Pusztai, A., 1989, Mechanism of seed lectin tolerance by a major insect storage pest of Phaseolus vulgaris, Acanthoscelides obtectus, Journal of the Science of Food and Agriculture, 47, 269–80. Gatehouse, A.M.R., Minney, B.H., Dobie, P. and Hilder, V.A., 1990, Biochemical resistance to bruchid attack in legume seeds: Investigation and exploitation, in Fujii, K., Gatehouse, A.M.R., Johnson, C.D., Mitchel, R. and Yoshida, T. (Eds) Bruchids and Legumes: Economics, Ecology and Coevolution, pp. 241–56, Amsterdam: Kluwer Academic. Gatehouse, A.M.R., Howe, D.S., Flemming, J.E., Hilder, V.A. and Gatehouse, J.A., 1991, Biochemical basis of insect resistance in winged bean seeds (Psophocarpus tetragonolobus) seeds, Journal of the Science of Food and Agriculture, 55, 63–74. Gatehouse, A.M.R., Boulter, D. and Hilder, V.A., 1992a, Potential of plant-derived genes in the genetic manipulation of crops for insect resistance, in Gatehouse, A.M.R., Hilder, V.A. and Boulter, D. (Eds) Plant Genetic Manipulation for Crop Protection Biotechnology in Agriculture, No. 7, pp. 155 81, Wallingford, UK: CAB International. Gatehouse, A.M.R., Hilder, V.A., Powell, K.S., Boulter, D. and Gatehouse, J.A., 1992b, Potential of plant derived genes in the genetic manipulation of crops for insect resistance, in Menken, S.B.J., Visser, J.H. and Harrewijn, P. (Eds) Proceedings of the 8th International Symposium on Insect-Plant Relationships, Dordrecht, pp. 221–33, Amsterdam: Kluwer Academic. Habibi, J., Backus, E.A. and Czapla, T.M., 1992, ‘Effect of plant lectins on survival of potato leafhopper’, Proceedings of the XIX International Congress Entomology, p. 373, Beijing, China. Habibi, J., Backus, E.A. and Czapla, T.M., 1993, Plant lectins affect survival of the potato leaf-hopper (Homoptera Cicadellidae), Journal of Economic Entomology, 86, 945–51. Hadfield, P., 1993, Japanese aid may upset Cambodia’s harvest, New Scientist, 137(1864), 5. Hallahan, D.L., Pickett, J.A., Wadhams, L.J., Wallsgrove, R.M. and Woodcock, C.M., 1992, Potential of secondary metabolites in genetic engineering of crops for resistance, in Gatehouse, A.M.R., Hilder, V.A. and Boulter, D. (Eds) Plant Genetic Manipulation for Crop Protection—Biotechnology in Agriculture, No. 7, pp. 215–48, Wallingford, UK: CAB International. Higuchi, M.I., Tsuchiga, I. and Iwai, K., 1984, Growth inhibition and small intestinal lesions on rats after feeding with isolated winged bean lectin, Agricultural and Biological Chemistry, 48, 695–701. Hilder, V.A, Powell K.S, Gatehouse, A.M.R., Gatehouse, J.A., Gatehouse, L.N, Shi, Y., Hamilton, W.D.O., Merryweather, A., Newell, C.A., Timans, J.C., Peumans, W.J., Van Damme, E. & Boulter, D. 1994. Expression of snowdrop lectin in transgenic tobacco plants results in added protection against aphids. Transgenic Research 3 (in press). Huesing, J.E., Murdock, L.L. and Shade, R.E., 1991, Rice and stinging nettle lectins: Insecticidal activity similar to wheat germ agglutinin, Phytochemistry, 30, 3565–68. Janzen, D.H., Juster, H.B. and Liener, I.E., 1976, Insecticidal action of the phytohaemagglutinin in black bean on a bruchid beetle, Science, 192, 795–96. Meiners, J.P. and Elden, T.C., 1978, Resistance to insects and diseases in Phaseolus, in Summerfield, R.S. and Bunting, A.H. (Eds) Advances in Legume Science, pp. 359–64, Kew: International Legume Conference. Metcalf, R.L., 1986, The ecology of insecticides and the chemical control of insects, in Kogan, M. (Ed) Ecological Theory and Integrated Pest Management, pp. 251–97, New York: John Wiley & Sons. Minney, B.H.P., Gatehouse, A.M.R., Dobie, P., Dendy, J., Cardona, C. and Gatehouse, J.A., 1990, Biochemical bases of seed resistance to Zabrotes subfasciatus (bean weevil) in Phaseolus vulgaris (common bean); A mechanism for arcelin toxicity, Journal of Insect Physiology, 36, 757–67. Moreno, J. and Chrispeels, M.J., 1989, A lectin gene encodes the -amylase inhibitor of the common bean, Proceedings of the National Academy of Sciences of the United States of America, 86, 7885–89. Murdoch, L.L., Huesing, J.E., Nielsen, S.S., Pratt, R.C. and Shade, R.E., 1990, Biological effects of plant lectins on the cowpea weevil, Phytochemistry, 29, 85–89. Osborn, T.C., Alexander, D.C., Sun, S.S.M., Cardona, C. and Bliss, F.A., 1989, Insecticidal activity and lectin homology of arcelin seed protein, Science, 240, 207–10.

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Powell, K.S., Gatehouse, A.M.R., Hilder, V.A. and Gatehouse, J.A., 1993, Antimetabolic effects of plant lectins and fungal enzymes on the nymphal stages of two important rice pests, Nilaparvata lugens and Nephotettix cinciteps, Entomologia Experimentalis et Applicata, 66, 119–26. Powell, K.S., Gatehouse, A.M.R., Hilder, V.A., Van Damme, E.J.M., Peumans, W.J., Boonjawat, J., Horsham K. & Gatehouse, J.A. 1995. Different antimetabolic effects of related lectins towards nymphal stages of Nilaparvata lugens. Entomologia Experimentalis et Applicata, (in press). Powell, K.S., Gatehouse, A.M.R., Hilder, V.A., & Gatehouse, J.A. 1995. Antifeedant effects of plant lectins and an enzyme on the adult stage of the rice brown planthopper, Nilaparvata lugens. Entomologia Ecperimentalis et Applicata, (In press). Powers, J.R. and Whitaker, J.R., 1977, Effect of several experimental parameters on combination of red kidney bean (Phaseolus vulgaris) -amylase inhibitor with porcine pancreatic -amylase, Journal of Food Biochemistry, 1, 239–60. Pusztai, A., Clarke, E.M.W. and King, T.P., 1979, The nutritional toxicity of Phaseolus vulgaris lectins, Proceedings of the Nutrition Society, 38, 115 20. Pusztai, A., Ewen, S.W.B., Grant, G., Peumans, W.J., Van Damme, E.J.M., Rubio, L. and Bardocz, S., 1990, Relationship between survival and binding of plant lectins during small intestinal passage and their effectiveness as growth factors, Digestion, 46 Supplement 2, 308–16. Pusztai, A., Grant, G., Stewart, J.C., Bardocz, S., Ewen, S.W.B. and Gatehouse, A.M.R., 1993a, Nutritional evaluation of RAZ-2, a new Phaseolus vulgaris bean cultivar containing high levels of the natural insecticidal protein arcelin, Journal of Agricultural and Food Chemistry, 41, 436–40. Pusztai, A., Ewen, S.W.B., Grant, G., Brown, D.S., Stewart, J.C., Peumans, W.J., Van Damme, E.J.M. and Bardocz, S., 1993b, Antinutritive effect of wheat germ agglutinin and other N-acetylglucosamine-specific lectins, British Journal of Nutrition, 70, 313–21. Rahbé, Y. and Febvay, G., 1993, Protein toxicity to aphids: an in vitro test on Acyrthosiphon pisum, Entomologia Expermentalis et Applicata, 67, 149–60. Richards, A.G. and Richards, P.A., 1977, The peritrophic membranes of insects, Annual Review of Entomology, 22, 787–91. Romero Andreas, J., Yandell, B.S. and Bliss, F.A., 1986, Bean arcelin 1. Inheritance of novel seed protein of Phaseolus vulgaris L., and its effects on seed composition, Theoretical and Applied Genetics, 72, 123–28. Shade, R.E., Murdock, L.L., Foard, D.E. and Pomeroy, M.A., 1986, Artificial diet seed system for bioassay of cowpea weevil (Coleoptera: Bruchidae), Environmental Entomology, 15, 1286–91. Shi, Y., Powell, K.S., Wang, M.B., Hilder, V.A., Gatehouse, A.M.R., Gatehouse, J.A. and Boulter, D., 1991, ‘Genetically engineered rice resistance to the brown planthopper’, Abstracts of the 5th Annual Meeting of the International Programme of Rice Biotechnology, USA, 2–5 October. Shi, Y., Wang, M.B., Powell, K.S., Van Damme, E.J.M., Hilder, V.A., Gatehouse, A.M.R., Boulter, D. and Gatehouse, J.A., 1994, Phloem-specific expression of GUS and GNA directed by RSSI promoter in transgenic tobacco plants, Journal of Experimental Botany, in press. Shi, Y., M.B.Wang, K.S.Powell, E.J.M.Van Damme, V.A.Hilder, A.M.R.Gatehouse, D.Boulter, & J.A.Gatehouse, 1994. Use of the rice sucrose synthase-1 promoter to direct phloem-specific expression of -glucuronidase and snowdrop lectin genes in transgenic tobacco plants. Journal of Experimental Botany 45, 623–631. Shukle, R.H. and Murdock, L.L., 1983, Lipoxygenase, trypsin inhibitor and lectin from soybeans: effects on larval growth of Manduca sexta (Lepidoptera: Sphingidae), Environmental Entomology, 12, 787–91. Strong, D.R., 1979, Biogeographic dynamics of insect-host plant communities, Annual Review of Entomology, 24, 89. Van Damme, E.J.M., Allen, A.K. and Peumans, W.J., 1987, Isolation and characterization of a lectin with exclusive specificity towards mannose from snowdrop (Galanthus nivalis) bulbs, FEBS Letters, 215, 140–44. Wang, M.B., Boulter, D. and Gatehouse, J.A., 1992, A complete sequence of the rice sucrose synthase-1 (RSsI) gene, Plant Molecular Biology, 19, 881–85.

Chapter 4 The Mannose-Binding Monocot Lectins and Their Genes Els J.M.Van Damme, Koen Smeets, Willy J.Peumans

Introduction At present, over two hundred plant lectins have been isolated and characterized in some detail with respect to their molecular structure, biochemical properties and carbohydrate-binding specificity. Although in the past most interest in plant lectins has been focused on lectins in dry seeds, especially from leguminous species, evidence has been accumulating that lectins also occur in vegetative tissues and are widespread in a large number of plant families belonging to all major taxonomical groupings (Etzler, 1986). Research on lectins in monocotyledonous species has for a long time concentrated on Gramineae (Van Damme and Peumans, 1991). However, during the last 5 years it has been established clearly that other monocot species are also a rich source of plant lectins with interesting properties. At present plant lectins have been reported in the families Gramineae (for review see Stinissen and Peumans, 1985), Liliaceae (Cammue et al., 1986; Oda and Minami, 1986; Peumans et al., 1986), Orchidaceae (Van Damme et al., 1987b), Amaryllidaceae (Van Damme et al., 1987a; 1988), Alliaceae (Van Damme et al., 1991a) and Araceae (Sandhu et al., 1990) of the class Liliatae. The observation that agglutinins occur in numerous plant families of the Monocotyledonae acknowledges once more that lectins are indeed ubiquitous within the plant kingdom. The isolation and characterization of new monocot lectins has revealed several distinct classes of agglutinins within this taxonomic group which not only differ in their molecular structure, biochemical properties and carbohydrate-binding specificity, but also regarding their abundance, distribution throughout the plant, and control of expression (Van Damme and Peumans, 1991). Characterization of some of these lectins has demonstrated that they differ from dicotyledonous lectins and exhibit several unique properties particularly in carbohydrate-binding specificity. For instance, the Amaryllidaceae and Alliaceae species contain appreciable amounts of lectins with exclusive specificity towards mannose, which makes them different from the mannose/glucose-binding lectins isolated from leguminous species (Goldstein and Poretz, 1986). Indeed, in contrast to concanavalin A, pea or lentil lectins, the Amaryllidaceae and Alliaceae lectins do not recognize glucose or N-acetylglucosamine. Moreover, since Amaryllidaceae lectins can easily be purified in large amounts they are promising tools for glycoconjugate research. The work presented here gives an overview of different aspects of the mannose-binding lectins from Amaryllidaceae, Alliaceae and Orchidaceae species. It is shown that despite their similar carbohydrate binding specificity the lectins from these three plant families clearly differ from each other in molecular structure and biological activity. Furthermore, when compared at the molecular level, these differences have been clearly shown to be reflected in the amino acid sequences of these lectins.

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Biochemical and biophysical properties, and biological activities of Amaryllidaceae, Alliaceae and Orchidaceae lectins A few years ago, the isolation and characterization of an apparently new type of plant lectin, GNA, from the bulbs of Galanthus nivalis (snowdrop), a representative of the plant family Amaryllidaceae was reported (Van Damme et al., 1987a). Because of its absolute specificity towards mannose GNA soon became a powerful tool for glycoconjugate research (Shibuya et al., 1988b). To find out whether it was a unique lectin or was a typical representative of a family of agglutinins, we searched for the occurrence of similar lectins in related species. It soon became evident that within Amaryllidaceae, lectins occur in the bulbs of species that belong to three genera indigenous to Mediterranean Europe namely Galanthus, Narcissus and Leucojum and to two genera Hippeastrum and Clivia common in America and Southern Africa, respectively, but are also often cultivated in Western Europe (Van Damme et al., 1988). As mannose-binding lectins occurred generally within the family Amaryllidaceae, we looked for the presence of similar lectins in species of the family Alliaceae because these two plant families are closely related taxonomically (Dahlgren et al., 1985) and also because a non-specific agglutinating activity in bulbs and leaves of several Allium species has already been reported by Sun and Yu (1986). Very soon it became evident that mannose-binding lectins similar to Amaryllidaceae lectins also occur within the tribe Allieae of the family Alliaceae, all of which are perennial herbs with bulbs or, more rarely, rhizomes. As the genus Allium is widely distributed and contains both wild species and a large number of economically important crops, it was interesting to see whether the lectins of Alliaceae and Amaryllidaceae species were related. Apart from species within Amaryllidaceae and Alliaceae, representatives of the Orchidaceae family were also found to contain mannose-binding lectins. In 1987, the isolation and characterization of a lectin with specificity for mannose from the leaves of the orchid species Listera ovata (twayblade), LOA was reported (Van Damme et al., 1987b). However, LOA also strongly reacted with glycoproteins, such as fetuin and thyroglobulin, suggesting that the fine carbohydrate binding specificity of the orchid lectin was different from that of the Amaryllidaceae and Alliaceae lectins. A study of the occurrence of orchid lectins in different species of Orchidaceae has shown that lectins occur widely in this plant family (Van Damme, unpublished results). Purification and molecular structure of Amaryllidaceae, Alliaceae and Orchidaceae lectins Preliminary experiments with crude extracts from different species of these families indicated that they contained an agglutinating factor that could only be inhibited by mannose. Hence, a purification scheme based on affinity chromatography on immobilized mannose was developed. Although the affinity purified lectins were virtually pure (as could be judged from SDS-PAGE), additional steps of hydrophobicinteraction chromatography and ion-exchange chromatography were included to ensure the complete purity of the lectins. The molecular structure of the mannose-specific lectins was determined using SDS-PAGE, gel filtration and ultracentrifugation. On SDS-PAGE (Table 4.1), the lectins isolated from Galanthus nivalis (snowdrop), GNA; Narcissus cv Carlton (daffodil), NPA; Listera ovata (twayblade), LOA; Epipactis helleborine (broad leaved helleborine), EHA, and Cymbidium hybrid, CHA, yielded a single polypeptide band of 12.5 kDa, while lectin polypeptides of Hippeastrum (amaryllis), HHA, and Clivia miniata, CMA, were of a slightly higher molecular mass (c. 14 kDa). Furthermore, the Allium porrum (leek) lectin, APA, migrated as a polypeptide of 13 kDa, while lectins from A. ascalonicum (shallot), AAA, and A. cepa (onion), ACA, yielded polypeptides of 12.5 kDa. A detailed study of A. sativum (garlic) lectin, ASA, and

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Table 4.1. Molecular structure of Amaryllidaceae, Alliaceae and Orchidaceae lectins Species

Molecular mass (kDa) determined by

SDS-PAGE

gel filtration

ultracentrifugation

12.5 12.5 14 14

50 25 50 25

49 36 51 ND*

11.5 and 12.5 12 11.5 and 12.5 12 12.5 12.5 13

25 25 25 25 25 50 50

25 29 25 25 24 ND* 35

12.5 12.5 12.5

25 50 25

27 52 28

AMARYLLIDACEAE Galanthus nivalis Narcissus cvs. Hippeastrum hybrid Clivia miniata ALLIACEAE Allium sativum lectin I lectin II Allium ursinum lectin I lectin II Allium ascalonicum Allium cepa Allium porrum ORCHIDACEAE Listera ovata Epipactis helleborine Cymbidium hybrid * Not determined

A. ursinum (ramsons) lectin, AUA, preparations revealed that these species contained two types of lectins with different molecular structures. Whereas the first lectin was composed of 11.5 and 12.5 kDa subunits, the second lectin yielded subunits of 12 kDa. These results were not altered by the presence or absence of mercaptoethanol indicating that subunits are not held together by disulphide bonds. Gel filtration experiments on a Superose 12 column in non-dissociating media demonstrated that lectins, including NPA (Narcissus), LOA (Listera ovata), CHA (Cymbidium), ASA (garlic), AUA (ramsons), AAA (A. ascalonicum) and CMA (Clivia miniata) all had an apparent molecular mass of 25 kDa indicating that the native lectin molecules were dimers. In contrast, GNA, HHA (amaryllis), EHA (Epipactis helleborine), ACA (onions) and APA (leek) were tetramers with an apparent molecular mass of 50 kDa (Table 4.1). As non-specific interactions of the lectins with the gel filtration matrix often occur and cannot be abolished completely by the addition of the specific sugar to the running buffer, the molecular mass of some lectins was also determined by ultracentrifugation, which showed (Table 4.1) that, apart from dimeric and tetrameric forms, some of the lectins were trimeric. Purified lectins from Amaryllidaceae and Alliaceae species were shown by high resolution ion-exchange chromatography to give complex elution patterns indicating that the lectin preparations contained mixtures of isoforms (Van Damme et al., 1991a). The occurrence of multiple isolectins was confirmed by isoelectric focusing which also indicated that the concentration of the different isolectins was highly variable in different pure lectin preparations. Furthermore, in different species and cultivars of Narcissus there were pronounced inter- and intraspecies differences in isolectin patterns. Lectin preparations isolated from various tissues at different developmental stages were quite different indicating that isolectin composition was both tissue-specific and developmentally regulated (Van Damme and Peumans, 1990a).

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Amino acid and carbohydrate analyses Lectins of Amaryllidaceae, Alliaceae and Orchidaceae had a similar but not identical amino acid composition with characteristically high contents of asparagine/ aspartic acid, threonine, glycine, serine, glutamine/glutamic acid and leucine. None of the lectins contained amino sugars. Their neutral sugar content was also low, most of it was probably contaminants, indicating that these lectins are not glycosylated (Van Damme et al., 1987b, 1991a). Carbohydrate-binding specificity The carbohydrate-binding specificity of these lectins was assessed by hapten inhibition assays using a whole series of simple sugars. Of all the monosaccharides tested, only D-mannose was inhibitory. A more detailed study of the carbohydrate binding specificity of GNA, NPA and HHA revealed that GNA was highly spe cific for terminal -1,3-linked mannose oligomers whereas NPA showed the highest affinity for both terminal and internal -1,6-linked mannosyl residues. Oligosaccharides containing either -1,3- or -1, 6-linked mannosyl units were bound by HAA (Shibuya et al., 1988a; Kaku et al., 1990). Although Alliaceae lectins were more difficult to inhibit, the carbohydrate-binding specificity of ASA was similar to GNA interacting strongly with -1,3-linked mannosyl units whereas AUA was similar to the amaryllis lectin (Kaku et al., 1992). Detailed carbohydrate binding studies with LOA showed that it was highly specific for -1,3 mannose oligomers, similar to GNA. However, LOA was also somewhat different from GNA because it did bind to glycoproteins such as fetuin and thyroglobulin (Saito et al., 1993). Biological activities and applications of mannose-specific lectins Purified preparations of GNA, NPA, HHA, LOA and ASA were assayed in a biological test system to study their effect on the induction of interleukin-6, IL-6, in human leucocytes, the infection of target cells by retroviruses and the induction of mitogenicity of T-lymphocytes. Although the Amaryllidaceae lectins were able to induce the production of IL-6 by human leucocytes, these lectins were virtually non-mitogenic to lymphocytes whereas LOA exhibited a modest mitogenic activity (Kilpatrick, 1990). Furthermore, these lectins were shown to inhibit markedly the infection of target cells by some retroviruses (e.g. HIV) and cytomegalovirus in vitro (Weiler et al., 1990; Balzarini et al., 1991). Surprisingly, ASA was inactive in all three bioassays. However, lectins from other Allium species, such as ACA, AAA and APA, strongly inhibited the infection of target cells by retroviruses in vitro although AUA was inactive (Balzarini et al., 1992). It appears, therefore, that the Alliaceae lectins can be divided into two groups with respect to their antiviral activity. One group, comprising the garlic species (A. sativum and A. ursinum), is completely inactive whereas the second group, comprising species related to onions and leeks (A. cepa, A. ascalonicum and A. porrum), exhibits a pronounced protective effect against animal and human viruses. Incorporation of GNA in the diet of rats has shown that although the lectin does not bind to the brush border on a first exposure, it does bind after a chronic (10 d) exposure, indicating that GNA may have induced the synthesis of new receptors containing terminal mannose residues in the gut. In addition to binding, it was also shown that the lectin was endocytosed by the epithelial cells (Pusztai et al., 1990). Interestingly over 90 per cent of GNA remained intact after passage through the intestinal tract, thereby confirming the stability of the lectin. Whereas GNA appears to be relatively harmless to mammals, at least in feeding experiments lasting for ten days (Pusztai et al., 1990), it has recently been shown to be very toxic to insects (Hilder et al., 1991).

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Incorporation of GNA in the diet of both chewing and sucking insects led to almost 100 per cent mortality of the insect population. Due to their unique and absolute specificity towards mannose, the Amaryllidaceae lectins are useful tools in glycoconjugate research. This has already been exploited successfully in the one-step purification of IgMmonoclonal antibodies from murine serum and 2-macroglobulin from human serum by affinity chromatography on a column of immobilized GNA (Shibuya et al., 1988b) and the isolation of haptoglobin and -lipoprotein on immobilized NPA and HHA. Furthermore, these lectins were also useful tools in the structural characterization of glycoprotein carbohydrate chains (Haselbeck et al., 1990; Mahmood and Hay, 1992). Biosynthesis and molecular cloning of Amaryllidaceae lectins Since the first report on the isolation and characterization of GNA from snowdrop bulbs with specificity for mannose (Van Damme et al., 1987a), there has been an increasing interest in this lectin. However, GNA is not unique in this respect; it is a member of a family of proteins that all recognize mannose and occur widely in the family Amaryllidaceae, at least during certain developmental stages (Van Damme and Peumans, 1990b). Despite their exclusive specificity towards mannose, the lectins, GNA, NPA and HHA also showed several marked differences in molecular structure, biological activity and fine carbohydratebinding specificity. First, there were obvious differences in molecular size as GNA and HHA were tetramers whereas NPA was a trimer (Table 4.1). Second, the fine specificity of the three Amaryllidaceae lectins was also different (Shibuya et al., 1988a; Kaku et al., 1990). Third, the anti-retroviral activity of these lectins were definitely different and the relative inhibitory potency of the three agglutinins varied as a function of the virus and the assay system (Balzarini et al., 1991). It is evident, therefore, that there are differences in the structure of the carbohydrate-binding site of the three lectins, probably as a result of the differences in the amino acid sequences of the respective lectin polypeptides. Although individual members of the Amaryllidaceae lectins are somewhat different, for a number of reasons there is an increasing interest in the exploitation of this group of lectins. First, because of their unique carbohydrate-binding specificity Amaryllidaceae lectins can be used for the purification and characterization of glycoconjugates (Shibuya et al., 1988b, Haselbeck et al., 1990). Second, since they are potent inhibitors of human and animal retroviruses including HIV, the Amaryllidaceae lectins have become important tools in AIDS research (Balzarini et al., 1991). Third, because of the recent discovery that GNA is a potent toxin for several species of both chewing and sucking insects (Hilder et al., 1991), this has raised the hope of their possible use for plant protection. Unlike most plant agglutinins, incorporation of the Amaryllidaceae lectins in the diet has no apparent toxic effects for rats, and this makes their use in food/ feed a definite practical possibility (Pusztai et al., 1990). Fourth, as inclusion of GNA in the rat diet significantly reduces the extent of overgrowth of the gut by mannose-sensitive, Type-1 fimbriated Escherichia coli, GNA and perhaps other mannose-specific lectins may possibly be used to specifically block the proliferation of E. coli in the small intestine (Pusztaie et al., 1993). It is because of the potential use of the Amaryllidaceae lectins in practice, that their molecular biology and possible role in cell biology have been studied extensively. Indeed, the results of these studies in combination with those of plant physiology (e.g. the occurrence and abundance of the lectins in different tissues throughout the life cycle of the plant) have been expected to yield important information about the normal biological function(s) of these lectins. Accordingly, in this section the synthesis and processing of GNA are described. In addition, the complete amino acid sequence of the mature protein by structural analysis and the sequence of the lectin precursor as deduced from the nucleotide sequence of its cloned

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cDNA, are also given. Finally, by molecular cloning of the Amaryllidaceae lectins, the origin of multiple isoforms of these lectins at the molecular level has been unravelled. Biosynthesis and cloning of GNA A detailed study of the in vivo synthesis and processing of GNA in young developing ovaries revealed that the lectin is synthesized on the endoplasmic reticulum where it occurs as a 15 kDa precursor, which is then post-translationally converted into the 12.5 kDa mature lectin polypeptide (Van Damme and Peumans, 1988). As the in vitro translation of poly(A)-rich RNA isolated from ripening ovaries of the snowdrop in a wheat germ cell-free system yielded a single 17 kDa lectin polypeptide, whereas translation of the same mRNA in Xenopus leavis oocytes resulted in a lectin polypeptide, which was about 2 kDa smaller than the in vitro synthesized precursor, it is clear that the primary translation product contains a 2 kDa signal peptide. However, as both the in vivo synthesized precursor and the Xenopus translation products were about 2 kDa larger than the mature lectin polypeptide, a second post-translational processing step is likely to be involved. The lack of evidence for the glycosylation of the precursor forms and the identity of the N-terminal amino acid sequence of the in-vivo-produced lectin precursor with that of the mature lectin suggests that this had an additional sequence at the C-terminus, which accounted for the 2 kDa difference in molecular mass between the mature lectin polypeptide and both the in vivo and in vitro synthesized translation products. Structural analysis of the amino acid sequence of the mature protein and the cloned cDNA have confirmed the postulated existence of a pre-prolectin (Figure 4.1). Edman degradation and carboxypeptidase Y digestion of the mature protein, and structural analysis of the peptides obtained after chemical cleavage and modification allowed us to determine the complete amino acid sequence (105 residues) of GNA. The lectin is rich in asparagine, glycine and leucine, and contains three cysteine residues, two of which are involved in an internal disulphide bond. However, this bond does not appear to be necessary for biological activity because GNA did not lose its activity to precipitate yeast mannans after treatment with dithiothreitol (Van Damme et al., 1991b). Amino acid sequencing of GNA revealed several instances of microheterogeneity in the lectin sequence. However, this is not surprising since the lectin is a complex mixture of isolectins as shown by ion-exchange chromatography or isoelectric focusing of purified preparations of GNA (Van Damme et al., 1988; 1991a). A cDNA library, constructed using poly(A)-rich RNA isolated from developing snowdrop ovaries, was first screened for lectin cDNA clones by a degenerate oligonucleotide probe derived from the N-terminal amino acid sequence of the mature lectin. The resulting lectin cDNA clone contained an open reading frame of 488 base pairs encoding a polypeptide of 157 amino acids with a calculated molecular mass of 16917 Da (Figure 4.2). This corresponded in size to the in vitro translation product of the GNA mRNA. A comparison of the amino terminal sequence with the deduced amino acid sequence of the lectin cDNA clone showed that GNA is synthesized with a signal sequence of 23 amino acids. This accounts for a decrease of 2315 in molecular mass upon transport of the polypeptide across the endoplasmic reticulum. As the carboxyl terminus of the lectin clone extends 29 amino acids (2944 Da) beyond the C-terminal amino acid of the mature lectin as determined by carboxypeptidase Y treatment of the protein (Thr-Gly), a second processing step is probably involved resulting in the removal of a C-terminal extension of 2944 Da during this posttranslational processing of the protein. Fur thermore, the hydrophobic character of this carboxyl terminal peptide is consistent with the possibility that it is removed post-translationally. A similar loss of C-terminal extensions has been reported for several plant lectins (wheat germ agglutinin B [Raikhel and Wilkins, 1987]; rice lectin [Wilkins and Raikhel, 1989]; barley lectin [Lerner and Raikhel, 1989]; concanavalin A [Carrington et al., 1985]; pea lectin [Higgins et al., 1983]) as well as plant storage proteins such as

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Figure 4.1. (A) Schematic representation of the coding region of the cDNA clones encoding the Amaryllidaceae and Orchidaceae lectins as well as the Alliaceae lectins from A. ursinum, A. cepa, A. ascalonicum, A. porrum and the A. sativum lectin ASAII. S refers to the signal peptide and C to the C-terminal peptide. Depending on the species possible glycosylation sites occur in the C-terminal peptide. The bar represents the mature lectin polypeptide. (B) Schematic representation of the coding region of the cDNA clone encoding the A. sativum lectin ASAI. S refers to the signal peptide. Domain 1 and domain 2 represent the 12.5 and 11.5 kDa mature lectin polypeptides, respectively. The glycosylation site is indicated by an asterisk.

thaumatin (Edens et al., 1982), napin (Ericson et al., 1986) and 2S albumin (Krebbers et al., 1988), and -1, 3-glucanase (Shinshi et al., 1988). Moreover, Bednarek et al. (1990) have shown that the C-terminal peptide of barley lectin is necessary for the efficient sorting of this protein to the plant cell vacuoles. However, despite all these results the precise function of these C-terminal peptides remains unclear and no consensus sequence has been identified. Multiple screenings by other oligonucleotide probes or cDNA inserts from previously isolated lectin clones resulted in the isolation of multiple GNA cDNA clones which clearly differed from each other in both their nucleotide and amino acid sequences (Van Damme et al., 1991c). An alignment of the different GNA sequences revealed that certain regions within the mature lectin sequence are more conserved than others. Furthermore, differences in charge distribution due to amino acid replacements within the peptide chain in the poorly conserved regions gave a ready explanation for the existence of multiple isoforms of GNA. Computer searches of the EMBL DNA sequence databases have not revealed any significant homology of GNA to other proteins. Thus, a comparison of the sequence of GNA with several other mannose-binding lectins isolated from plants (e.g. Canavalia [Wang et al., 1975], pea [Higgins et al., 1983], lentil [Foriers et al., 1981]) or rat serum and liver (Drickamer et al., 1986, Ikeda et al., 1987) or E. coli (Klemm, 1984) showed only about 20 per cent identity.

56

LECTINS: BIOMEDICAL PERSPECTIVES

Figure 4.2. Deduced amino acid sequences of lectin cDNA clones from different Amaryllidaceae, Alliaceae and Orchidaceae species: Narcissus cv. Fortune (LECNPA), Hippeastrum hybr. (LECHHA), Galanthus nivalis (LECGNA), Clivia miniata (LECCMA), Listera ovata 1 (LECLOA1), L. ovata lectin 2 (LECLOA2), Epipactis helleborine (LECEPA), Cymbidium hybrid (LECCHA), A. sativum lectin I (LECASAI), A. sativum lectin II (LECASAII), A. ursinum lectin I and lectin II (LECAUA GO, LECAUA G1 and LECAUA G2), A. cepa (LECACA), A. ascalonicum (LECAAA) and A. porrum (LECAPA). In the case of LECASAI only the sequence of the two domains within the sequence is shown. The arrow head indicates the processing site for the cleavage of the signal peptide. (−) denotes sequence identity with LECNPA. Dots represent gaps introduced for maximal alignment. Putative glycosylation sites are underlined.

Molecular cloning of other Amaryllidaceae lectins Using a cDNA clone of GNA as a probe, multiple homologous lectin cDNA clones were obtained from cDNA libraries constructed from poly(A)-rich RNA isolated from young developing ovaries of Narcissus cv Fortune, Hippeastrum hybr. and Clivia (Figure 4.2; Van Damme et al., 1992a). Although these lectin clones showed a high degree of homology in their coding region, they clearly differed from each other at some positions in their nucleotide sequences and the corresponding deduced amino acid sequences. Moreover, these differences resulted in different charge distribution within the polypeptide chains. Accordingly the lectins encoded by the different cDNA clones had different isoelectric points, explaining the occurrence of multiple lectin isoforms in Amaryllidaceae. Furthermore, Southern blot analysis of genomic DNA isolated from the different Amaryllidaceae species yielded numerous restriction fragments hybridizing with lectin cDNA probes, leaving no doubt that the Amaryllidaceae lectins are encoded by families of closely related lectin genes (Van Damme et al., 1991c, 1992a). As shown by northern blot hybridization all these lectins are translated from mRNAs of approximately 800 nucleotides (Van Damme et al., 1992a). The cDNA clones of Amaryllidaceae lectins contained an open reading frame encoding a pre-prolectin which, apart from the coding sequence of the mature lectin also comprised a signal peptide and a C-terminal peptide. These were removed co- and post-translationally, respectively (Figure 4.1). Because of the high sequence homology between the different isolectin clones of all Amaryllidaceae species, the C-terminal peptide of the daffodil, amaryllis and Clivia clones might also be cleaved at the same position (after the

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amino acid sequence Thr-Gly of the pre-proprotein) as in GNA. A sequence comparison of the different Amaryllidaceae lectin clones revealed that there was a 60–80 per cent sequence similarity within the coding region of their cDNA clones depending on the isolectin under study. Biosynthesis and molecular cloning of Alliaceae lectin genes Lectins with exclusive specificity towards mannose have also been isolated from species belonging to the tribe Allieae of the family Alliaceae. Despite the strong resemblance between the Amaryllidaceae and Alliaceae lectins in carbohydrate-binding specificity and a close serological relationship, they were also different in some properties. The different Alliaceae lectins were shown by biochemical and physicochemical methods to be a heterogeneous group of agglutinins. Thus, both homomeric (dimers and trimers), and heterodimeric lectins were isolated from different species within the same plant family. Furthermore, some species e.g. A. sativum and A. ursinum contained two different lectins within the same tissue. In addition, on the basis of their antiviral activity the Alliaceae lectins could be divided into two groups. Thus, onion, leek and shallot contained mannose-binding lectins that, similar to the Amaryllidaceae lectins, were potent inhibitors of the infection of target cells with human and animal retroviruses. In contrast, the two different lectins from garlic and ramsons were both inactive. As differences in molecular structure and biological activities within the Alliaceae lectins and also the similarities or differences between Amaryllidaceae and Alliaceae lectins were expected to be reflected in amino acid sequences, cDNA clones encoding the different Alliaceae lectins were isolated and their sequences analysed. As expected, molecular cloning of these lectin genes showed that their organization at the molecular level was complex. Indeed, the structure, the biosynthesis and processing of precursors of the homomeric garlic, ramsons and onion lectins were closely similar to that of Amaryllidaceae lectins, while the heterodimeric lectins from garlic and ramsons were synthesized and processed differently. Biosynthesis and cloning of ASA Molecular cloning of ASA genes has revealed that this species contains two closely related lectins, a homodimer and a heterodimer. Of these the homodimer is encoded by a one-domain gene while the heterodimer by a two-domain lectin gene (Van Damme et al., 1992b). Thus, ASAI is a heterodimer composed of two different subunits of 11.5 and 12.5 kDa. It is apparently translated from an mRNA of 1400 nucleotides encoding a polypeptide of 306 amino acids (33020 Da). A more detailed study of the cDNA sequence of this lectin revealed that the lectin clone consisted of two similar domains which shared 84 per cent homology at the amino acid level. Both domains are separated by a 30 amino acid sequence containing a putative glycosylation site (Figure 4.1). N-terminal sequencing of the two mature lectin polypeptides confirmed that both subunits were derived from the same precursor and corresponded to one of the two domains in the sequence. The existence of a large precursor was confirmed by studies on the in vivo biosynthesis of ASAI, which demonstrated the occurrence of a glycosylated 38 kDa primary translation product in the endoplasmic reticulum. After this glycosylated precursor leaves the endoplasmic reticulum, it undergoes a complex processing resulting in the formation of 11.5 and 12.5 kDa mature lectin polypeptides. Therefore the signal peptide and a C-terminal peptide are removed cotranslationally and post-translationally, respectively. In addition, the intervening sequence of 30 amino acids between the two domains is also removed. In contrast to ASAI, the second garlic lectin, ASAII, is composed of two identical 12 kDa subunits. It is translated from an mRNA of 800 nucleotides encoding a polypeptide of 154 amino acids (16743 Da).

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Compared to the cDNA clones of ASAI, the cDNA encoding ASAII contains a signal sequence flanked by a sequence almost identical to that of the second domain, including the C-terminal extension of ASAI lectin clones. Studies on in vivo biosynthesis and in vitro translation indicated that the post-translational modification of the primary ASAII translation products was similar to those of GNA. Cotranslational removal of the signal peptide and post-translational cleavage of a C-terminal peptide from the preproprotein of 154 amino acids yields the mature 12 kDa ASAII polypeptide, which is almost identical to the 11.5 kDa subunit of ASAI. Digestion of genomic DNA isolated from A. sativum bulbs using various restriction enzymes revealed a complex pattern of bands after hybridization with a lectin cDNA insert, indicating that the garlic lectins are encoded by multiple genes. Furthermore, starting from the genomic DNA, PCR amplification of the coding region of the two garlic lectins demonstrated that there were no introns present in the coding region of these proteins. This suggested that the coding region of the lectin was entirely encoded by one single exon. Hence the two mannose-binding lectins from garlic are the result of the expression of two different genes. Similar to the Amaryllidaceae lectins, several lectin cDNA clones with slightly different nucleotide and amino acid sequences have been isolated from the cDNA library constructed from poly(A)-rich RNA isolated from young and old garlic bulbs. However, in contrast to the Amaryllidaceae lectin clones, the garlic lectin clones had a high degree of homology both in their coding region and 3 untranslated region. These taken together with the results from Southern blot analyses indicated that the garlic lectins were also encoded by a family of closely related genes. Compared to the previously cloned mannose-specific lectins from Amaryllidaceae, both ASAI and ASAII showed only about 40 per cent homology in their amino acid sequences. Computer searches in the EMBL DNA sequence databases revealed no significant homology in the cDNA sequences of the garlic lectins to any other known sequences (except of course to the Amaryllidaceae lectins). Molecular cloning of AUA Similar to the garlic cloves, bulbs of ramsons (A. ursinum) contained two mannose-binding lectins with different molecular structures. The first lectin, AUAI, was a heterodimer composed of 11.5 and 12.5 kDa subunits, and the second, AUAII, was a homodimer containing 12 kDa lectin polypeptides (Van Damme et al., 1993a). Despite these similarities with the garlic lectins, molecular cloning of the two ramsons lectins showed that the lectin genes in ramsons were organized differently. Whereas the ASAI lectin polypeptides were encoded by one large precursor, the AUAI lectin polypeptides were derived from two different precursors. These results were confirmed by northern blot hybridization of A. ursinum RNA which, after hybridization with a labelled lectin cDNA, revealed only one band of 800 nucleotides in contrast to garlic RNA which yielded two bands of 1400 and 800 nucleotides, respectively. Screening of cDNA libraries constructed from poly(A)-rich RNA isolated from young ramsons bulbs using the garlic cDNA-insert as a probe resulted in the isolation of three types of lectin cDNA clones which were not only different in their sequences but also in the number of potential glycosylation sites (Van Damme et al., 1993a). Type LECAUA G2 contained two possible glycosylation sites within its sequence encoding a pre-proprotein of 174 amino acids with a calculated molecular mass of 18179 Da (Figure. 4.2). Upon transportation of the precursor polypeptide across the endoplasmic reticulum a 2481 Da signal peptide is cleaved off, resulting in a 15698 Da propeptide. The second type of lectin cDNA clone, LECAUA G1, contained only one putative glycosylation site the position of which coincided with the first glycosylation site in LECAUA G2. LECAUA G1 encodes a 17227 Da precursor polypeptide of 165 amino acids containing a 1497 Da signal peptide. Cotranslational cleavage of this signal sequence results in a 15730 Da

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propeptide. Studies of the in vivo biosynthesis of the ramsons lectins showed the presence of different glycosylated lectin precursors in the organelle fraction of radioactively labelled ramsons bulbs indicating that the glycosylation sites in LECAUA G1 and LECAUA G2 are actually used. In contrast to LECAUA G2 and LECAUA G1, a third type of cDNA clone encoding the ramsons lectin, LECAUA GO, contained no glycosylation sites within its sequence. This encodes a 15082 Da precursor, which is converted into a 13613 Da propeptide after the removal of the signal peptide. In contrast to garlic lectins, the ramsons lectins contained within their C-terminal part a sequence of Thr-Gly, which was also found to be the processing site for the cleavage of the C-terminal extension in the Amaryllidaceae lectins (Van Damme et al., 1991b). Experiments to determine the C-terminal sequence of the mature lectin polypeptides of the ramsons lectins were unsuccessful. It appears, however, that a second post-translational modification (removal of a Cterminal peptide) may be involved in the processing of the lectin precursor as (1) it was shown in preliminary experiments by SDS-PAGE that the polypeptides produced in both in vivo synthesis and in vitro translation were larger than the mature lectin polypeptides and (2) the precursor polypeptides encoded by the lectin cDNA clones were about 2 kDa larger than the mature lectin polypeptides after the removal of the signal peptide. Assuming that the sequence Thr-Gly was also the processing site for the cleavage of a Cterminal peptide in AUA precursors, the putative glycosylation sites in the lectin clones LECAUA G2 and LECAUA G1 could be located in the C-terminal peptide. This is consistent with the observation that the mature ramsons lectins are not glycosylated. An alignment of sequences of the different AUA cDNA clones revealed that LECAUA G2 and LECAUA G1 had about 90 per cent homology in the deduced amino acid sequence of the total coding region, whereas the sequence similarity between lectin clones LECAUA G2 and LECAUA G0, and LECAUA G1 and LECAUA G0 was only 76 per cent. Taking into consideration that ASAI is composed of two different though homologous lectin polypeptides it may be assumed that the clones LECAUA G2 and LECAUA G1 encode the lectin polypeptides of AUAI whereas cDNA clone LECAUA G0 encodes that of AUAII. The overall homology of the coding sequence of the ramsons lectin clones with previously isolated lectin cDNA clones from Amaryllidaceae species and A. sativum is low being only 42–62 per cent. Molecular cloning of Alliaceae lectins from A. cepa, A. ascalonicum and A. porrum Screening of cDNA libraries constructed from poly(A)-rich RNA isolated from young shoots of these Alliaceae species resulted in the isolation of their lectin cDNA clones (Van Damme et al., 1993b). In contrast to the cDNA libraries constructed for ramsons and garlic only a small number of positive colonies were detected in the libraries for onion, shallot and leek. However, this was not surprising because the lectin concentration in these species is at least 100-fold lower than in garlic and ramsons. Similar to the Amaryllidaceae lectins, the onion, shallot and leek lectins are translated from mRNAs of approximately 800 nucleotides. The cDNA clone encoding the onion lectin, ACA, encodes a pre-proprotein of 17536 Da which contains only a part of the signal peptide. Cotranslational removal of this peptide results in a 16046 Da proprotein with two putative glycosylation sites in its C-terminal sequence. Using the cDNA clone of ACA as a probe, highly homologous (95 per cent) lectin cDNA clones were isolated from A. ascalonicum and A. porrum. The cDNA clones encoding these two lectins yield 18883 Da and 19232 Da precursor polypeptides, respectively, which similar to the onion lectin precursor, contain two possible glycosylation sites in their Cterminal sequence. The cDNA sequences of the lectins from onion, shallot and leek contain the sequence Thr-Gly, which is the processing site for the C-terminal peptide in Amaryllidaceae lectins and also in ramsons.

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An alignment of the onion, shallot and leek lectin sequences with those of ASAI and ASAII revealed an approximately 70 per cent sequence homology in amino acids for the total coding region, whereas they had only 40 per cent homology with the Amaryllidaceae lectins. In contrast to the ramsons lectin, the sequence LECAUA G0, showing only 57 per cent sequence identity with lectins from onion, shallot and leek, the ramsons clones LECAUA G1 and LECAUA G2 had a sequence homology of approximately 70 per cent. Molecular cloning of the Orchidaceae lectin genes Mannose-binding lectins were also reported in the leaves of Listera ovata, a representative of the plant family Orchidaceae (Van Damme et al., 1987b) and other species of this family indicating that lectins may be widespread in the different tribes of Orchidaceae. Indeed, lectins have also been isolated and characterized from Epipactis helleborine and Cymbidium hybrid, species belonging to different tribes. The molecular structure of orchid lectins was similar to that of the Amaryllidaceae, the onion and the leek lectins. Furthermore, the orchid lectins were also potent inhibitors of the infection of target cells by some human and animal retroviruses (Balzarini et al., 1991). Using double immunodiffusion assays, the three orchid lectins reacted with antibodies raised against GNA, indicating that they are all serologically related. In contrast to the Amaryllidaceae and Alliaceae lectins, however, the orchid lectins also strongly interacted with glycoproteins, fetuin and thyroglobulin, suggesting also some differences in their carbohydrate-binding site. To determine the genetic basis of the similarities or differences between the mannose-binding lectins of Amaryllidaceae, Alliaceae and Orchidaceae species, orchid lectins were studied in more detail by molecular cloning. This indicated that, despite some obvious differences, some parts of the amino acid sequences of the different mannose-binding lectins in these three plant families have been conserved during evolution. Molecular cloning of two mannose-binding lectins from Listera ovata Ion-exchange chromatography of an affinity purified lectin preparation from Listera ovata leaves revealed the presence of two lectins. Although the molecular mass of the subunits of both lectins was approximately 12.5 kDa on SDS-PAGE, amino acid sequencing of the lectin polypeptides revealed that the N-terminal part of the sequences were different and had about 65 per cent sequence homology in the N-terminal 20 amino acids. As the N-terminal amino acid sequences of purified LOA showed about 50 per cent homology to the previously sequenced amaryllis lectin, HHA, a cDNA library constructed from a polyA-rich RNA preparation isolated from young leaves of Listera ovata was screened for lectin cDNA clones using a clone of HHA as a probe. As shown in Figure 4.2, two lectin cDNA clones, called LECLOA1 and LECLOA2, with considerable overall sequence similarity (87 per cent) were isolated from the cDNA library. LECLOA1 encodes a pre-proprotein of 176 amino acids with a calculated molecular mass of 18664 Da. Upon transport of this protein through the endoplasmic reticulum a signal peptide of 29 amino acids (2886 Da) is cleaved off resulting in a propeptide of 15778 Da. The cleavage site was determined using the rules of von Heijne (1986) and was found to be in good agreement with the N-terminal sequence of the mature twayblade lectins. Similar to LECLOA1, LECLOA2 also encodes a pre-proprotein with a calculated molecular mass of 18595 Da (175 amino acids) with a signal peptide of 2785 Da. The molecular mass of the propeptides encoded by LECLOA1 and LECLOA2 was approximately 3 kDa larger than that of the mature lectin polypeptides determined by SDS-PAGE. This suggested that a second processing step may take place, similar to that occurring during the cleavage of the C-terminal peptide in Amaryllidaceae lectins after the transport of the propeptide out of the endoplasmic reticulum. The sequence

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Thr-Gly, which is known to be the processing site for the C-terminal peptide in GNA, is also present in the C-terminal part of the deduced amino acid sequence of LECLOA1 and LECLOA2. With a cleavage of the C-terminal peptide in LECLOA1 and LECLOA2 at this site, the molecular size of the resulting lectin polypeptides would be of approximately 11.6 kDa which is in good agreement with the molecular mass of the lectin polypeptides determined by SDS-PAGE. Splitting off the C-terminal peptide at this site also eliminates a possible glycosylation site which is present in the C-terminal parts of the sequences encoded by LECLOA1 and LECLOA2, confirming the fact that the mature tway-blade lectins are not glycosylated. Processing at this site produces lectin polypeptides with pI of 5.8 and 4.6 for LECLOA1 and LECLOA2, respectively, which accords well with the observation that the two mature twayblade lectins can be separated by ion-exchange chromatography on an anion-exchanger column. Despite the obvious differences in N-terminal sequence it is clear that the amino acid sequences coded for by the two lectin cDNA clones showed a high degree of sequence homology; 87 per cent in the total coding region. Although there is only 40 per cent overall sequence homology to mannose-binding lectins from Amaryllidaceae and Alliaceae species, an alignment of the amino acid sequences of LECLOA1 and LECLOA2 to them indicated that some parts of the lectin sequences have been conserved during evolution (Figure 4.2). Similar to the Amaryllidaceae and Alliaceae lectins, except for ASAI, the two isoforms of LOA are also translated from mRNAs of 800 nucleotides. Molecular cloning of the mannose-binding lectins from Epipactis helleborine and Cymbidium hybrid In contrast to Listera ovata, only one lectin component was found in the leaves of these other two orchids. Their cDNA clones were isolated using a cDNA clone LOA as a probe. The cDNA clone, LECEPA, encoding EHA, encodes a precursor of 174 amino acids (18510 Da) with a signal peptide of 2674 Da. Similarly, the cDNA clone LECCHA encoding CHA contains the information for a preproprotein containing 178 amino acids (19109 Da) with a signal peptide of 3264 Da. Both lectin precursors are translated from mRNAs of 800 nucleotides. Similar to LOA, the other two orchid lectin propeptides are larger than the mature lectin polypeptides. This indicates that a second processing step is likely to be involved in the biosynthesis and maturation of the lectins. An alignment of the different Orchidaceae lectin sequences revealed a 60–70 per cent sequence homology for the total coding region. General discussion During the last three decades, several mannose-specific lectins have been isolated and characterized from leguminous species such as Canavalia ensiformis, Pisum sativum, Lens culinaris and Vicia faba. Based on their carbohydrate binding properties these lectins have been classified as mannose-binding lectins (Goldstein and Poretz, 1986). Apart from mannose, however, these lectins also strongly react with glucose and N-acetylglucosamine. In contrast, lectins isolated from Amaryllidaceae, Alliaceae and Orchidaceae species were quite different and had exclusive specificity towards mannose and, therefore, represented a completely new class of lectins. Despite similarities in carbohydrate-binding properties, lectins from Amaryllidaceae, Alliaceae and Orchidaceae were also clearly different with respect to their molecular structure. Although the main subunit in all these lectins has a molecular mass of approximately 12 kDa, the native lectin molecules can be dimers, trimers or tetramers. Moreover, some Alliaceae species contained two different dimeric lectins, one of which was a homodimer whereas the other a heterodimer composed of two subunits of slightly different

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Figure 4.3. Phylogeny of amino acid sequences encoding different mannose binding lectins from Amaryllidaceae, Alliaceae and Orchidaceae species. The dendrogram was constructed using the simultaneous alignment and phylogeny program CLUSTAL. The different sequences are as indicated in Figure 4.2.

molecular mass. A further intriguing finding was that these mannose-specific lectins were of complex mixtures of isolectins. Furthermore, the isolectin composition appeared to be both tissue-specific and developmentally regulated, and could not be explained on the basis of differences in glycosylation or differences in molecular structure. Accordingly, in a major attempt to find a satisfactory explanation, these isolectins were studied at the molecular-genetic levels. Molecular cloning and analysis of genomic DNA have shown that the Amaryllidaceae lectins are encoded by a family of closely related genes and that they encode lectin polypeptides with slightly different amino acid sequences (Van Damme et al., 1991c, 1992a, 1993b). It is possible that differences in the carbohydrate-binding specificities and biological activities of these lectins are dependent on dissimilarities in their sequences. Moreover, these differences in the amino acid sequence resulting in a different charge distribution along the polypeptide chains give an adequate explanation for the finding of lectin polypeptides with different isoelectric points. Although it is not unusual for plant proteins, the concept that lectins are encoded by (extended) multigene families is quite new in lectin research. Earlier findings showing that the concentration of Amaryllidaceae lectins varies in different stages of plant development, suggested that they may function as developmentally regulated storage proteins (Van Damme and Peumans, 1990b). In view of this storage role it is not unexpected that they are encoded by a family of closely related genes. Apart from a storage role, however, Amaryllidaceae lectins can fulfil another function. As these lectins are only present at certain stages of development and then occur in almost all plant tissues, it has been suggested that they may also play an

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active role in plant defence. The recent discovery that GNA is certainly toxic towards insects points in this direction (Hilder et al., 1991). In this light it is possible that by containing multiple isoforms of the same lectin, the plant can broaden its defensive capability against insects. Apart from differences in molecular structure, mannose-specific lectins also show some differences in their biological activities. Thus lectins of Amaryllidaceae, Orchidaceae and the onion and leek lectins have been shown to inhibit the infection of target cells by retroviruses including HIV and cytomegalovirus in vitro and therefore can be used as probes in the study of their surfaces, while ASA and AUA isolectins were completely inactive against these viruses. A sequence comparison of the total coding region of Amaryllidaceae, Alliaceae and Orchidaceae lectins showed that they have about 40 per cent overall sequence homology at the amino acid level. However, this relatively low level of similarity increased to 60 per cent by aligning the sequences of the mature lectin polypeptides of the three families and not the total coding regions. This demonstrated that some parts of the amino acid sequences of the biologically active forms of these lectin polypeptides were highly conserved. Furthermore, the two cysteines, which form an internal disulphide bond, had the same positions in the polypeptide chain of the lectins of all three families. In contrast, the third free cysteine which was present in the Amaryllidaceae lectins was absent in the Alliaceae and Orchidaceae lectins. The similarity between the different lectin sequences was also analysed using the PC Gene program CLUSTAL. The dendrogram (phylogenetic tree) constructed using the pair-wise similarity scores obtained between the different sequences showed that Amaryllidaceae, Alliaceae and Orchidaceae lectin sequences can be separated into three clusters (Figure 4.3). Apparently, the Orchidaceae lectin sequences are more related to those of the Amaryllidaceae lectins than those of the Alliaceae lectins. Within the cluster of the Alliaceae lectins, LECAUAG1 and LECAUAG2 form a subfamily which is located between the sequences of ASA and those of ACA, AAA and APA. The AUA sequence LECAUAG0 forms a separate subfamily distinct from the other Alliaceae lectins. Acknowledgements This work was supported in part by grants from the K.U.Leuven (OT/90/19), the IWONL, the National Bank and the National Fund for Scientific Research (Belgium, FGWO grant 2005989 N). The collaborative work is a part of a European FLAIR Concerted Action Programme (No. 9), coordinated by Dr Pusztai with financial support from the Commission of European Communities. At present E.V.D. is Senior Research Assistant and W.P. Research Director of The National Fund for Scientific Research (Belgium). K.S. acknowledges the receipt of a grant from the IWONL. References Balzarini, J., Schols, D., Neyts, J., Van Damme, E., Peumans, W. and De Clercq, E., 1991, -(1–3)- and -(1–6)-Dmannose-specific plant lectins are markedly inhibitory to human immunodeficiency virus and cytomegalovirus infections in vitro, Antimicrobial Agents and Chemotherapy, 35, 410–16. Balzarini, J., Neyts, J., Schols, D., Hosoya, M., Van Damme, E., Peumans, W. and De Clercq, E., 1992, The mannosespecific plant lectins from Cymbidium hybrid and Epipactis helleborine and the (N-acetylglucosamine)n-specific plant lectin from Urtica dioica are potent and selective inhibitors of human immunodeficiency virus and cytomegalovirus replication in vitro, Antiviral Research, 18, 191–207. Bednarek, S.Y., Wilkins, T.A., Dombrowski, J.E. and Raikhel, N.V., 1990, A carboxy-terminal propeptide is necessary for proper sorting of barley lectin to vacuoles of tobacco, The Plant Cell, 2, 1145–55.

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Cammue, B.P.A., Peeters, B. and Peumans, W.J., 1986, A new lectin from tulip (Tulipa) bulbs, Planta, 169, 583–88. Carrington, D.M., Auffret, A. and Hanke, D.E., 1985, Polypeptide ligation occurs during post-translational modification of concanavalin A, Nature, 313, 64–67. Dahlgren, R.M.T., Clifford, H.T. and Yeo, P.F., 1985, Structure, Evolution and Taxonomy, Berlin: Springer Verlag. Drickamer, K., Dordal, M.S. and Reynolds, L., 1986, Mannose-binding proteins isolated from rat liver contain carbohydrate-recognition domains linked to collagenous tails. Complete primary structure and homology with pulmonary surfactant apoprotein, Journal of Biological Chemistry, 261, 6878–87. Edens, L., Heslinga, L., Klok, R., Ledeboer, A.M., Maat, J., Toonen, M.Y., Visser, C. and Verrips, C.T., 1982, Cloning of cDNA encoding the sweet-tasting plant protein thaumatin and its expression in Escherichia coli, Gene, 18, 1–12. Ericson, M.L., Rodin, J., Lenman, M., Glimelius, K., Josefsson, L.-G. and Rask, L., 1986, Structure of the rapeseed 1.7 S storage protein, napin, and its precursor, Journal of Biological Chemistry, 261, 14576–81. Etzler, M.E., 1986, Distribution and function of plant lectins, in Liener, I.E., Sharon, N. and Goldstein, I.J. (Eds) The Lectins: Properties, Functions and Applications in Biology and Medicine, pp. 371–435, New York: Academic Press. Foriers, A., Lebrun, E., Van Rapenbusch, R., de Neve, R. and Strosberg, A.D., (1981, The structure of the lentil (Lens culinaris) lectin. Amino acid sequence determination and prediction of the secondary structure, Journal of Biological Chemistry, 256, 5550–60. Goldstein, I.J. and Poretz, R.D., 1986, Isolation, physicochemical characterization and carbohydrate-binding specificity of lectins, in Liener, I.E., Sharon, N. and Goldstein, I.J. (Eds) The Lectins: Properties, Functions and Applications in Biology and Medicine, pp. 33–248, New York: Academic Press. Haselbeck, A., Schickaneder, E., von der Eltz, H. and Hösel, W., 1990, Structural characterization of glycoprotein carbohydrate chains by using digoxigenin-labelled lectins on blots, Analytical Biochemistry, 191, 25–30. Higgins, T.J.V., Chandler, P.M., Zurawski, G., Button, S.C. and Spencer, D., 1983, The biosynthesis and primary structure of pea seed lectin, Journal of Biological Chemistry, 258, 9544–49. Hilder, V.A., Gatehouse, A.M.R., Gatehouse, J.A. and Boulter, D., 1991, Beyond Bt toxin; Higher plant genes which enhance insect resistance in transgenic plants, Third International Congress for Plant Molecular Biology, Tucson, AZ, USA, Abstract No 734. Ikeda, K., Sannoh, T., Kawasaki, N., Kawasaki, T. and Yamashina, I., 1987, Serum lectin with known structure activates complement through the classical pathway, Journal of Biological Chemistry, 262, 7451–54. Kaku, H., Van Damme, E.J.M., Peumans, W.J. and Goldstein, I.J., 1990, Carbohydrate-binding specificity of the daffodil (Narcissus pseudonarcissus) and amaryllis (Hippeastrum hybr.) bulb lectins, Archives of Biochemistry and Biophysics, 279, 298–304. Kaku, H., Van Damme, E.J.M., Peumans, W.J. and Goldstein, I.J., 1992, New mannose-specific lectins from garlic (Allium sativum) and ramsons (Allium ursinum) bulbs, Carbohydrate Research, 229, 347–53. Kilpatrick, D.C., Peumans, W.J. and Van Damme, E.J.M., 1990, Mitogenic activity of monocot lectins, in Kocourek, J. and Freed, D.L.J. (Eds) Lectins, Biology, Biochemistry, Clinical Biochemistry, vol. 7, pp. 259–63, St. Louis, USA: Sigma Chemical Co. Klemm, P., 1984, The fim A gene encoding the type-1 fimbrial subunit of Escherichia coli. Nucleotide sequence and primary structure of the protein, European Journal of Biochemistry, 143, 395–99. Krebbers, E., Herdies, L., De Clercq, A., Seurinck, J., Leemans, J., Van Damme, J., Segura, M., Gheysen, G., Van Montagu, M. and Vandekerckhove, J., 1988, Determination of the processing sites of an Arabidopsis 2 S albumin and characterization of the complete gene family, Plant Physiology, 87, 859–66. Lerner, D.R. and Raikhel, N.V., 1989, Cloning and characterization of root-specific barley lectin, Plant Physiology, 91, 124–29. Mahmood, N. and Hay, A.J., 1992, An ELISA using immobilized snowdrop lectin GNA for the detection of envelope glycoproteins of HIV and SIV, Journal of Immunological Methods, 151, 9–13. Oda Y. and Minami, K., 1986, Isolation and characterization of a lectin from tulip bulbs, Tulipa gesneriana, European Journal of Biochemistry, 159, 239–45.

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Peumans, W.J., Allen, A.K. and Cammue, B.P.A., 1986, A new lectin from meadow saffron (Colchicum autumnale), Plant Physiology, 82, 1036–39. Pusztai, A., Ewen, S.W.B., Grant, G., Peumans, W.J., Van Damme, E.J.M., Rubio, L. and Bardocz, S., 1990, Relationship between survival and binding of plant lectins during small intestinal passage and their effectiveness as growth factors, Digestion, 46, 308–16. Pusztai, A., Grant, G., Spencer, R.J., Duguid, T.J., Brown, D.S., Ewen, S.W.B., Peumans, W.J., Van Damme, E.J.M. and Bardocz, S., 1993, Kidney bean lectin-induced Escherichia coli overgrowth in the small intestine is blocked by GNA, a mannose-specific lectin, Journal of Applied Bacteriology, 75, 360–68. Raikhel, N.V. and Wilkins, T.A., 1987, Isolation and characterization of a cDNA clone encoding wheat germ agglutinin, Proceedings of the National Academy of Sciences of the United States of America, 84, 6745–49. Saito, K., Komae, A., Kakuta, M., Van Damme, E.J.M., Peumans, W.J., Goldstein, I.J. and Misaki, A., 1993, Alphamannosyl binding specificity of the lectin from leaves of the orchid twayblade (Listera ovata), European Jonrnal of Biochemistry, 217, 677–81. Sandhu, R.S., Arora, J.S., Chopra, S.K., Pelia, S.S., Kamboj, S.S., Naidu, Y.C. and Nath, I., 1990, New sources of lectins from Araceous indian plants, in Kocourek, J. and Freed, D.L.J. (Eds) Lectins, Biology, Biochemistry, Clinical Biochemistry, vol. 7, pp. 19–26, St. Louis, USA: Sigma Chemical Co. Shibuya, N., Goldstein, I.J., Van Damme, E.J.M. and Peumans, W.J., 1988a, Binding properties of a mannose-specific lectin from the snowdrop (Galanthus nivalis) bulb, Journal of Biological Chemistry, 263, 728–34. Shibuya, N., Berry, J.E. and Goldstein, I.J., 1988b, One-step purification of murine IgM and human a -macroglobulin by affinity chromatography on immobilized snowdrop bulb lectin, Archives of Biochemistry and Biophysics, 267, 676–80. Shinshi, H., Wenzler, H., Neuhaus, J.-M., Felix, G., Hofsteenge, J. and Meins, F. Jr., 1988, Evidence for N- and Cterminal processing of a plant defense-related enzyme: primary structure of tobacco prepro- -1, 3-glucanase, Proceedings of the National Academy of Sciences of the United States of America, 85, 5541–45. Stinissen, H.M. and Peumans, W.J., 1985, Recent advances in biochemistry, cell biology, physiology, biosynthesis and genetics of Gramineae lectins, Biochemie und Physiologie der Pflanzen, 180, 85–106. Sun, C. and Yu, L., 1986, Shengwu Huaxue Yu Shengwu Wuli Xuebao 18, 213 (cited after Chemical Abstracts, 105, 131940r). Van Damme, E.J.M. and Peumans, W.J., 1988, Biosynthesis of the snowdrop (Galanthus nivalis) lectin in ripening ovaries, Plant Physiology, 86, 922–26. Van Damme, E.J.M. and Peumans, W.J., 1990a, Isolectins in Narcissus: complexity, inter-and intraspecies differences and developmental control, Physiologia Plantarum, 79, 1–6. Van Damme, E.J.M. and Peumans, W.J., 1990b, Developmental changes and tissue distribution of lectin in Galanthus nivalis and Narcissus cv Carlton, Planta, 182, 605–9. Van Damme, E.J.M. and Peumans, W.J., 1991, Lectins from monocotyledonae, in Kilpatrick, D.C., van Driessche, E. and Bøg-Hansen, T.C. (Eds) Lectin Reviews, vol. 1, pp. 161–70, St. Louis, USA: Sigma Chemical Co. Van Damme, E.J.M., Allen, A.K. and Peumans, W.J., 1987a, Isolation and characterization of a lectin with exclusive specificity towards mannose from snowdrop (Galanthus nivalis) bulbs, FEBS Letters, 215, 140–44. Van Damme, E.J.M., Allen, A.K. and Peumans, W.J., 1987b, Leaves of the orchid tway blade (Listera ovata) contain a mannose-specific lectin, Plant Physiology, 85, 566–69. Van Damme, E.J.M., Allen, A.K. and Peumans, W.J., 1988, Related mannose-specific lectins from different species of the family Amarylidaceae, Physiologia Plantarum, 73, 52–57. Van Damme, E.J.M., Goldstein, I.J. and Peumans, W.J., 1991a, Comparative study of related mannose-binding lectins from Amaryllidaceae and Alliaceae species, Phytochemistry, 30, 509–14. Van Damme, E.J.M., Kaku, H., Perini, F., Goldstein, I.J., Peeters, B., Yagi, F., Decock, B. and Peumans, W.J., 1991b, Biosynthesis, primary structure and molecular cloning of snowdrop (Galanthus nivalis L.) lectin, European Journal of Biochemistry, 202, 23–30.

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Van Damme, E.J.M., De Clercq, N., Claessens, F., Hemschoote, K., Peeters, B. and Peumans, W.J., 1991c, Molecular cloning and characterization of multiple isoforms of the snowdrop (Galanthus nivalis L.) lectin, Planta, 186, 35–43. Van Damme, E.J.M., Goldstein, I.J., Vercammen, G., Vuylsteke J. and Peumans, W.J., 1992a, Lectins of members of the Amaryllidaceae are encoded by multigene families which show extensive homology, Physiologia Plantarum, 86, 245–52. Van Damme, E.J.M., Smeets, K., Torrekens, S., Van Leuven, F., Goldstein, I.J. and Peumans, W.J., 1992b, The closely related homomeric and heterodimeric mannose-binding lectins from garlic are encoded by one-domain and twodomain lectin genes, respectively, European Journal of Biochemistry, 206, 413–20. Van Damme, E.J.M., Smeets, K., Torrekens, S., Van Leuven, F. and Peumans, W.J., 1993a, The mannose-specific lectins from ramsons (Allium ursinum L.) are encoded by three sets of genes, European Journal of Biochemistry, 217, 123–129. Van Damme, E.J.M., Smeets, K., Engelborghs, I., Aelbers, H., Balzarini, J., Pusztai, A., Van Leuven, F., Goldstein, I.J. and Peumans, W.J., 1993b, Cloning and characterization of the lectin cDNA clones from onion, shallot and leek, Plant Molecular Biology, 23, 365–76. von Heijne, G., 1986, A method for predicting signal sequence cleavage sites, Nucleic Acids Research, 11, 4683–90. Wang, J.L., Cunningham, B.A., Waxdal, M.J. and Edelman, G.M., 1975, The covalent and three-dimensional structure of concanavalin A.I. Amino acid sequence of cyanogen bromide fragments F1 and F2, Journal of Biological Chemistry, 250, 1490–1502. Weiler, B.E., Schröder, H.C., Stefanovich, V., Stewart, D., Forrest, J.M.S., Allen, L.B., Bowden, B.J., Kreuter, M.H., Voth, R. and Müller, W.E.G., 1990, Sulphoevernan, a polyanionic polysaccharide and the Narcissus lectin potently inhibit human immunodeficiency virus infection by binding to viral envelope protein, Journal of General Virology, 71, 1957–63. Wilkins, T.A. and Raikhel, N.V., 1989, Expression of rice lectin is governed by two temporally and spatially regulated mRNAs in developing embryos, The Plant Cell, 1, 541–49.

Chapter 5 Enterocyte-like Caco-2 Cells as a Tool to Study Lectin Interaction J.F.J.G.Koninkx

Introduction The epithelial cells of the small intestine are highly differentiated cells expressing a variety of specific functions. As the immature absorptive cells (the principal cells of the intestinal epithelium), goblet cells, endocrine cells, Paneth cells and other minor cell types migrate out of the crypts towards the tip of the villus, they differentiate progressively. Differentiated enterocytes are characterized by the presence of microvilli at their apical surface firmly anchored to and sustained by a cytoskeleton. The small intestinal cells are polarized and their plasma membrane consists of an apical- and a basolateral domain accompanied by tight junctions between adjoining cells, forming a relatively impermeable barrier from the lumen to the systemic environment. The apical surface of enterocytes has its own characteristic set of proteins and this specialized surface, called the brush-border, contains many enzymes including disaccharidases, aminopeptidases and transport enzymes of glucose and amino acids (Hauri et al., 1985). These proteins are found exclusively in the apical domain of the enterocyte. To study the many aspects of the relationship between lectins and the function, morphology, pathology, differentiation and maturation of epithelial cells and the transepithelial transport of molecules through the small intestinal epithelium both in vivo (Banwell et al., 1988; Bardocz et al., 1989, 1990a; Boldt and Banwell, 1985; Donatucci et al., 1987; Pusztai et al., 1991) and in vitro (Draaijer et al., 1989; Hendriks et al., 1991; Kik et al., 1991b; Koninkx et al., 1992; Lehr et al., 1992; Neeser et al., 1989) mainly animal models have been used. To study the binding of lectins to small intestinal cells, their interference with the cellular metabolism of enterocytes, effect on gut endocrine cells and the gut immune system and their modulation of the microbial ecology of the small intestine, several models have been found convenient. Clearly, the systemic effects of lectins can only be studied in intact animals (Bardocz et al., 1990b; Pusztai et al., 1989a, 1990) or in animals with an isolated self-emptying jejunal loop (Lorenz-Meyer et al., 1985) or a self-emptying blind pouch (Kik et al., 1991a). Organ culture has also been used as an in vitro model to study the binding of lectins to and their interaction with tissues (Danielsen et al., 1982; Quaroni, 1985), but this model has a serious drawback. Following its removal from the animal, the intestinal tissues are particularly rapidly degraded, which restricts the use of this method only to studies of short-term lectin effects in vitro (Kik et al., 1991b). In cultures of enterocyte-like cells it is possible to investigate both short-term and long-term lectin effects at the level of individual cells. Thus, we have been using a cell culture model of small intestinal absorptive cells to study lectin-binding and its influence on cell morphology and cellular metabolism for several years (Draaijer et al., 1989; Hendriks et al., 1991; Koninkx et al., 1992). Two cell lines, Caco-2 (Pinto et al.,

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1983) and HT-29 (Pinto et al., 1982), derived from human colon adenocarcinomas are known, in which the cells can be induced to achieve enterocyte-like differentiation. The Caco-2 cell line, as pointed out by many investigators, is a unique in vitro model for studies on the structural and functional properties of differentiated enterocytes. It also appears to be a suitable model to study the transport of drugs (Hilgers et al., 1990; Lehr et al., 1992), permeability markers across the intestinal mucosa (Cogburn et al., 1991; Hidalgo et al., 1989), the intracellular transport of proteins (Hughson and Hopkins, 1990; Klumpermans et al., 1991), polyamine metabolism (D’Agostino et al., 1989, 1990) and the adherence, penetration and translocation of bacteria (Chauvière et al., 1992; Everest et al., 1992; Finlay and Falkow, 1990; Francis et al., 1992; Kerneis et al., 1991; Konkel et al., 1992; Mounier et al., 1992). In these investigations, filtergrown differentiated Caco-2 cells have been found particularly useful. At late confluency the Caco-2 cells are fully differentiated and exhibit properties of small intesdnal enterocytes both structurally and functionally. Microvilli are present on the apical surface of differentiated Caco-2 cells and polarity is achieved by tight junctions. The high levels of activity of brush borderassociated enzymes including alkaline phosphatase, aminopeptidase and sucrase-isomaltase are characteristic for the functional differentiation of these cells. In this review the binding of different lectins to and their interaction with the fully differentiated enterocytelike Caco-2 cells will be dealt with. The advantage of using this in vitro model for the elucidation of the reaction mechanism of lectins with intestinal cells, to study the effect of lectins upon the translocation of bacteria across the polarized epithelial cell sheet, and for establishing how lectins interfere with transepithelial transport will also be discussed briefly. Growth, structural and functional characteristics of Caco-2 cells The growth characteristics of the Caco-2 cell line are presented in Figure 5.1. The cells start proliferating after a lag time of 2 days during which there is a characteristic and temporary slight decrease in cell number. In the logarithmic phase of growth, which starts on day 3 and continues till day 9, the confluency of the cells is reached on day 6. In the phase of stationary growth, which is entered on day 9, the number of cells remains constant at approximately 0.5×106 cells cm−2. The junctional complexes between Caco-2 cells, which are composed of tight junctions and desmosomes, typical brush-border microvilli oriented perpendicu larly at the surface membrane facing the culture medium and the polarized appearance of the individual cells in the monolayer, are all convincing morphological features of an enterocyte-like differentiation (Pinto et al., 1983). Scanning electron microscopic studies have clearly demonstrated that the microvilli of both the differentiating and the fully differentiated Caco-2 cells are arranged in two different patterns: a carpet-like pattern and flower-like pattern (Figure 5.2). The different patterns of brush-border organization cover areas of equal size. In about half of the cells, the brushborder microvilli form a thick carpet-like pattern, whereas the other half of the cell population is covered by flower-like clusters of microvilli, which appear to join at their apical ends. The specific activity of the brush border-associated enzymes, alkaline phosphatase and sucraseisomaltase, as determined in partially purified brush-border membrane fractions isolated from Caco-2 cells (Pinto et al., 1983) at various time points after cell seeding, is low during the lag phase but increases rapidly at the end of the logarithmic phase of cell growth (Figure 5.3). The highest levels of specific enzyme activities are measured during the stationary phase when the cells have stopped proliferating.

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Figure 5.1. Growth of the human colon carcinoma cell line Caco-2. The results are expressed as the mean cell number per cm2 of four different passages±SD. The day upon which the cells reach confluency, is indicated.

Lectin-binding and lectin-induced effects in differentiated Caco-2 cells Lectin-binding by qualitative and quantitative methods Lectins appear to be resistant to proteolysis during passage through the alimentary tract (Pusztai et al., 1991). Some lectins such as the Phaseolus vulgaris haemagglutinin (PHA), concanavalin A (Con A) and Galanthus nivalis agglutinin (GNA) almost quantitatively survive passage through the rat stomach and small intestine in an immunochemically intact form. Depending on their sugar specificity, lectins which remain biologically active, bind to the terminal sugar or occasionally to longer segments of the oligosaccharide chains of membrane glycoconjugates on the intestinal cells. Thus, it was shown by applying specific immunoreactive antibody-peroxidase-antiperoxidase staining techniques that PHA was extensively bound to the brush-border of the rat small intestinal mucosa. Binding of the lectin from Glycine max (SBA) was less intense, while that of Vicia faba lectin (VFA) and Pisum sativum agglutinin (PSA) was very weak or showed no binding at all.

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Figure 5.2. Microvilli of differentiated Caco-2 cells. The microvilli are arranged in 2 different patterns: a carpet like pattern (A) and a flower-like pattern of brush-border organisation (B). Scale bar is 3 µ m. (From Draaijer et al., 1989; by permission of Biology of the Cell)

As Caco-2 cells are not covered by a mucus layer, binding of lectins to the brush-border membrane can be easily studied with this enterocyte-like cell line. Thus, the binding of PHA-E4, PHA-L4, SBA, VFA, and PSA to intact Caco-2 cells and to purified brush-border membranes of differentiated Caco-2 cells was examined using FITC-labelled lectins (Koninkx et al., 1992) and by an enzyme-linked lectin sorbent assay (ELLSA) (Hendriks et al., 1987), respectively. The most intense FITC-lectin staining of the surface of the cell monolayer was displayed by PHA-E4, PHA-L4, and SBA (Figure 5.4). Staining intensity by VFA was substantially weaker. The fluorescence staining after the binding of FITC-labelled PSA was so weak that no discernible patterns of brush-border organization (Figure 5.2) could be observed. Pre-incubation of the lectins with their specific monosaccharides completely prevented the specific fluorescent labelling, indicating that the binding was receptor-mediated. Dissociation constants which have been reported for the binding of PHA to rat intestinal cells (Donatucci et al., 1987) or to purified rat brush border membranes (Boldt and Banwell, 1985) ranged from 0.67×10−5 mol l−1 to 4.00 ×10−5 mol l−1. Although the dissociation constants of PHA-E4, PHA-L4, SBA, VFA, and PSA with the purified brush-border membranes of differentiated Caco-2 cells were of the same magnitude (10−5; Table 5.1), the number of binding sites or the dissociation constants varied considerably for the different lectins. Thus, the number of lectin-binding sites per mg of brush-border mem

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Figure 5.3. Activity profiles of brush-border associated enzymes during growth of Caco-2 cells. Sucrase-isomaltase activity and alkaline phosphatase activity were determined during growth. The results are expressed as the mean enzyme activity of four different passages±SD. Table 5.1. Maximum binding and dissociation constant of peroxidase-conjugated PHA-E4, PHA-L4, SBA, VFA, and PSA to purified brush-border membranes from differentiated Caco-2 cells. Lectins

Maximum binding (nmol mg−2 bbm protein)

Dissociation constant (mol−1)

PHA-E4 2104±140 (0.11±0.08) 10−5 a PHA-L4 2540±151 (0.43±0.14) 10−5a SBA 4302±149 a,b (0.53±0.12) 10−5a,* a,b,* VFA 3969±66 (1.52±0.17) 10−5 a,b,c a,b,c,d PSA 8600±282 (1.69±0.17) 10−5a,b,c, * The number of lectin-binding sites and dissociation constants in the brush-border membrane of differentiated Caco-2 cells have been established by ELLSA and by means of a computer fit analysis of the data. The results are expressed as the mean maximum binding±SD and the mean dissociation constant±SD of two different passages. a Significant difference between the values displayed by PHA-L , SBA, VFA, PSA and PHA-E 4 4 b between SBA, VFA, PSA and PHA-L 4 c between VFA, PSA and SBA d between VFA and PSA * Not significant

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Maximum binding (nmol mg−2 bbm protein) (From Koninkx et al., 1992; by permission of Gastroenterology) Lectins

Dissociation constant (mol−1)

brane protein for PHA-E4 was about one quarter of that for PSA. However, this was partly compensated for by a pronounced difference in the dissociation constants which was about 15 times less for PHA-E4 than for PSA. In fact, the dissociation constants of lectins could be ranked (Table 5.1). Accordingly, PHA-E4 had the highest binding affinity, while PSA had the lowest binding affinity. Further-more, a comparison of the results of binding studies with FITC- and peroxidase-labelled lectins indicated that a correlation exists between the intensity of fluorescent staining (Figure 5.4) and the dissociation constant of the lectin-cell complexes (Table 5.1). There was no correlation, however, between the staining intensity and the number of lectin-binding sites.

Figure 5.4. Fluorescence labelling of differentiated Caco-2 cells with FITC-conjugated PHA-E4 , PHA-L4, SBA, VFA, or PSA. The monolayer of differentiated Caco-2 cells was incubated at 37°C for 15 minutes with 100 µ g ml−1 of each PHA-E4 (A), PHA-L4 (B), SBA (C), VFA (D), or PSA (E). Scale bar is 50 µ m. (From Koninkx et al., 1992;, by permission of Gastroenterology)

Actin cytoskeletal lesions after exposure to lectins In the brush-border of enterocytes actin cytoskeletal filaments (F-actin) are present in a highly ordered arrangement (Mooseker, 1985; Sager et al., 1986). Each microvillus contains a core consisting of bundles of

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actin filaments which extend into the cytoplasm (Mooseker and Tilney, 1975). Associated with junctional complexes, F-actin is also present in the terminal web (Mooseker, 1985). The F-actin filaments are aggregates of globular subunits (G-actin; Mooseker and Tilney, 1975). The normal balance between polymerization and depolymerization of actin filaments is perturbed by changes in pH or the concentrations of calcium, magnesium and ATP. It is thought that changes in the amount of F-actin in the enterocytes might interfere with the integrity of the intestinal epithelium. Lectins are known to interfere with the cellular processes in different types of cells and this may include their endocytosis (Edelson and Cohn, 1974; Sharon, 1983, 1984). Furthermore, it has been demonstrated that Con A, wheat germ agglutinin (WGA) or PHA can cause destruction of the cytoskeleton of rat enterocytes (King et al., 1980a, 1982, 1993; Lorenzsonn and Olson, 1982; Sjölander et al., 1986). Although the fluorescent patterns of F-actin of differentiated Caco-2 cells incubated with SBA were not different from controls by using 7-nitrobenz-2-oxa-1,3-diazole (NBD) phallacidin as a marker of F-actin (Figure 5.5), a biochemical approach by using the deoxyribonuclease-I inhibition assay (Blikstad et al., 1978) clearly demonstrated that there was a significant and dose-dependent increase in the proportion of G-actin with SBA treatment (Figure 5.6). One of the earliest events to occur after exposure of the cells to SBA is the depolymerization of F-actin. The conversion of F-actin to G-actin, leading to an increase in the percentage of G-actin could be observed as early as 15 min after addition of SBA. Recent experiments have revealed that depolymerization takes place within 2 minutes. The conversion could be fully inhibited in the presence of N-acetyl galactosamine, indicating that the effect was related to the binding of the lectin to the carbohydrate moieties of the brushborder membrane of Caco-2 cells (Table 5.2; Draaijer et al., 1989). Receptor-ligand binding is known to induce rapid cellular changes in pH, calcium concentration and the phosphorylation of cytoskeleton-associated proteins (Mooseker, 1985; Weatherbee, 1981). In turn, these changes disturb the assembly (Mooseker, 1985; Weatherbee, 1981) or organization of actin filaments (Akiyama et al., 1986; Mooseker, 1985). Also, experiments of lectin-binding to cell surface receptors of differentiated Caco-2 cells clearly showed that only a short period of exposure was needed to accomplish the increase in the concentration of G-actin (Table 5.2). Therefore, these experiments suggest that this conversion results from Table 5.2. The percentage of G-actin in differentiated Caco-2 cells after incubation with SBA concentrations for various periods SBA concentration Incubation period (hours)

0 µ g ml−1

20 µ g ml−1

20 µ g ml−1 0.2 M GalNAc

50 µ g ml−1

0.25 58.3±5.8 72.9±5.8 52.7±4.1 78.1±5.2 0.5 49.9±6.6 101.4±6.6 88.9±4.5 1 49.6±3.1 84.9±4.4 — 85.5±2.9 2 51.6±5.4 85.5±4.1 49.3±3.3 99.2±8.0 4 46.4±3.5 89.3±5.7 86.0±5.1 6 43.1±3.1 84.2±3.7 93.4±9.1 24 50.5±3.1 77.6±2.1 — 81.1±2.1 Two different passages were used to determine the effect of SBA concentration and incubation period on the percentage of G-actin. Control Caco-2 cells and SBA-incubated Caco-2 cells differ significantly (P 0.05). Control Caco-2 cells and SBA/inhibitory monosaccharide-incubated Caco-2 cells do not differ significantly.

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Figure 5.5. F-actin distribution in pre-confluent and post-confluent cultures of Caco-2 cells. Fluorescence staining pattern of F-actin filaments of 3 day-old (A; pre-confluent cultures), 7 day-old (B; post-confluent cultures) and 19 day-old Caco-2 cells (C; post-confluent cultures). Caco-2 cells displayed 2 different fluorescent patterns: a diffuse staining pattern (arrow 1) and a patchy staining pattern (arrow 2). Scale bar is 10 µ m. (From Draaijer et al., 1989; by permission of Biology of the Cell). SBA concentration Incubation period 0 µ g ml−1 20 µ g ml−1 20 µ g ml−1 0.2 M 50 µ g ml−1 (hours) GalNAc The results are expressed as the mean percentage of G-actin±SEM. In cach passage the mean percentage of G-actin was determined using duplicate cultures. (From Draaijer et al., 1989; by permission of Biology of the Cell)

cell surface receptor-lectin interactions rather than an increased biosynthesis of G-actin, for which other signals, probably originating from the endocytosis of the lectin or lectin-receptor complex, might be needed. The discovery that the absolute amount of G-actin in the Caco-2 cells did not change, irrespective of lectin concentration and incubation period, supports this view. Similarly, under in vivo conditions, a short-term exposure of the rat intestinal epithelium to Con A or WGA appeared to be sufficient to achieve the disarrangement of the cytoskeleton (Lorenzsonn and Olson, 1982; Sjölander et al., 1986). Thus, at the moment there is increasing evidence to show that membrane receptors of lectins are intimately associated with the underlying actin filaments in the cytoplasm of the cell. Lectin-induced changes in the cellular metabolism. PHA is a potent growth factor for rat small intestine, inducing an increase in its weight which is fully accounted for by the increase in DNA, RNA, protein and carbohydrate concentrations (de Oliveira et al., 1988; Greer et al., 1985; Pusztai et al., 1988a, 1988b). The ratio of protein/DNA did not change suggesting that the growth is mainly by hyperplasia and not by hypertrophy (Pusztai et al., 1989b). In PHA-treated lymphocytes in vitro, the lectin is known to stimulate essentially all metabolic processes though to varying

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Figure 5.6. Dose-dependent changes in the percentage of G-actin in differentiated Caco-2 cells after incubation with SBA. Two different passages have been used to establish this dose-dependent effect. The numbers 6–8 at the top of column 1 indicate that this column representing the percentage of G-actin in control Caco-2 cells differs significantly (P< 0.05) from column 6, 7 and 8 representing the percentage of G-actin in SBA-incubated Gaco-2 cells (10, 20 and 100 µ g ml−1). The results are expressed as the mean percentage of G-actin±SEM. In each passage the mean percentage of G-actin of the SBA-concentrations was determined using quadruplicate cultures. (From Draaijer et al., 1989; by permission of Biology of the Cell)

degrees and at different times after exposure to the mitogen. There are relatively few studies on lectins in relation to cellular metabolism in enterocytes (Banwell et al., 1983; de Oliveira et al., 1988; Donatucci et al., 1987; Greer et al., 1985; Palmer et al., 1987; Pusztai et al., 1988a). Indeed, to the best of our knowledge there are only two studies on the effects of lectins on the synthesis of DNA, RNA, and (glyco)proteins in differentiated Caco-2 cells (Hendriks et al., 1991; Koninkx et al., 1992). Changes in the synthesis of DNA and RNA and glycosylation induced in differentiated Caco-2 cells after exposure to PHA-E4, PHA-L4, or SBA clearly showed that these lectins stimulated cellular metabolism (expressed as relative incorporation of specific radioactive precursors (Figure 5.7)), while VFA principally

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Figure 5.7. Relative incorporation of (2-14C) thymidine, (U-14C) uridine, L-(6-3H) fucose, and D-(6-3H) glucosamine by differentiated Caco-2 cells after incubation with PHA-E4, PHA-L4, SBA, VFA, or PSA. Three different passages have been used to establish the effect of the lectins. The results are expressed as the mean relative incorporation±SEM. The dotted area represents the mean relative incorporation±SKM of cell cultures not exposed to lectins. For each passage of the cells, the relative incorporation of all lectins and lectin concentrations was determined using quadruplicate cultures. (Koninkx et al., 1992; by permission of Gastroenterology)

displayed an inhibitory effect, whereas PSA had little or no effect at all. It is interesting and striking that lectins which were stimulatory on cellular metabolism in differentiated Caco-2 cells (PHA-E4, PHA-L4 and SBA) were specific for complex glycans or N-acetylgalactosamine/galactose, whereas those specific for mannose/glucose either inhibited changes in metabolism or had no effect at all (Pusztai et al., 1991). Recent similar studies with the mannose/glucose-binding Con A or the strictly mannose-specific GNA showed that these lectins did not interfere with cellular metabolism either. Heat inactivation of the lectins was shown to abolish their stimulatory or inhibitory effects, indicating that the changes induced were due to lectin-binding to the sugar-specific sites on the brush-border membrane of differentiated Caco-2 cells. None of the lectins tested so far, appeared to be capable of interfering with RNA-synthesis. It appears that the lectin-induced stimulation of the net rate of initiation of protein synthesis or the translation rate of messenger RNA in Caco-2 cells is due to a modulation of the assembly of functional polysomes controlling

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(glyco)protein synthesis by the lectins. However, it cannot be rigorously excluded at present that the increase in (glyco)protein synthesis is preceded by de novo synthesis of messenger RNA. Binding of lectins through their sugar-reactive sites to the apical membrane of small intestinal cells is thought to be a prerequisite of their toxicity. Indeed, their binding to differentiated Caco-2 cells showed considerable differences both qualitatively (Figure 5.4) and quantitatively (Table 5.1). Quantitatively both the number of lectin-binding sites and the dissociation constants of the various lectins varied considerably. This may indicate a difference in the potential toxicity of lectins for Caco-2 cells because the extent of the lectin-induced changes in cellular metabolism of differentiated Caco-2 cells (Figure 5.7) was clearly correlated with the dissociation constant of the lectin in question. Thus, lectins with relatively low dissociation constants (PHA-E4, PHA-L4 and SBA) strongly interfered with cellular metabolism, whereas lectins with relatively high dissociation constants (VFA and PSA) showed little or no interference at all. In fact, lectins could be graded according to their effects on Caco-2 cells, which diminished in the order of PHA-E4> PHA-L4 SBA>VFA>PSA. In short, there is not only a correlation between the dissociation constants of the lectin-cell interactions (Table 5.1) and the fluorescent staining intensity of the binding of FITC-conjugated lectins (Figure 5.4), but the dissociation constants (Table 5.1) also correlate well with the extent of the changes induced by them of cellular metabolism (Figure 5.7). Morphological alterations in the brush-border membrane It has been shown recently that exposure of differentiated Caco-2 cells to SBA led to cytoskeletal lesions resulting in shortened microvilli (Draaijer et al., 1989). Fur thermore, the in vivo exposure of the rat intestinal epithelium to Con A, WGA and PHA causes a similar disarrangement of the cytoskeleton and shortening of the microvilli of enterocytes (King et al., 1980b, 1982; Lorenzsonn and Olson, 1982; Sjölander et al., 1986). Shortening of the microvilli seems to be accomplished by a shift in the ratio of globular: filamentous actin. As a result of 48 h exposure to 50 µ g ml−1 of each PHA-E4, SBA, and VFA, the lengths of the microvilli of differentiated Caco-2 cells were shortened significantly in comparison with controls (cells incubated with PHA-L4 or PSA; 50 µ g ml−1; Figure 5.8; Table 5.3). Although not yet established, it is most likely that the basis of the PHA-E4- or VFA-mediated shortening of the microvilli is the result of a lectin-induced lesion in the actin-cytoskeleton. After exposure of rat intestinal epithelium to intraluminal dietary lectins clusters of vesicles associated with the brush-border were found (King et al., 1982; Lorenz-sonn and Olson, 1982). An increased number of vesicles was also observed near the brush-borders of PHA-incubated pig small intestinal mucosa in organ culture (Figures 5.9, 5.10). Also, incubation of Caco-2 cells in vitro with lectins led to similar increase in the number of vesicles nearby the brush-borders of these cells. Thus, clusters of vesicles associated with the brush-borders of differentiated Table 5.3. Length of the microvilli of differentiated Caco-2 Cells after exposure to PHA-E4, PHA-L4, SBA, VFA, or PSA Caco-2 cells

Microvillus length (µ m) after incubation with 50 µ g lectin ml−1

PHA-E4-incubated PHA-L4-incubated SBA-incubated VFA-incubated

1.29±0.06a 1.96±0.06c 1.37±0.04a 1.75±0.05b

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Figure 5.8. Microvilli of differentiated Caco-2 cells after incubation with PHA-E4, PHA-L4, SBA VFA, or PSA. Microvilli of Caco-2 cells incubated for 48h with 50 µ g ml−1 of each PHA-E4 (A), PHA-L4 (B), SBA (C), VFA (D), or PSA (E). Microvilli of control Caco-2 cells (F). The arrows point at the presence of the membrane-associated vesicles. Scale bar is 1 µ m. (From Koninkx et al., 1992; by permission of Gastroenterology) Caco-2 cells

Microvillus length (µ m) after incubation with 50 µ g lectin ml−1

PSA-incubated 2.10±0.07c Control 2.04±0.07 Differentiated Caco-2 cells were incubated for 48 h with 50 µ g ml−1 lectin. Two different passages were used to measure the lectin-induced alterations of the microvilli. The results are expressed as the mean±SEM. a P <0.01; b P <0.05; c Not significant (From Koninkx et al., 1992; by permission of Gastroenterology)

Caco-2 cells were observed after exposure to PHA-E4, PHA-L4, SBA, or VFA but not PSA. An increased turnover of the microvillus membrane after exposure of the cells to the lectins may explain the presence of these vesicles. The loss of brush-border membranes results directly in a reduction of the total activity of brush border membrane-associated enzymes. In pig jejunal explants, a clear effect of PHA-E4 on the specific activity of sucrase-isomaltase was evident (Kik et al., 1991b). Indeed, it was shown that the amount of microvillus vesicles tied off and the decrease in the activity of sucrase-isomaltase were well correlated.

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Figure 5.9. Scanning electron micrograph of pig mucosal explants after exposure to PHA-L4. After exposure for 5 h to 50 µ g of PHA-L4 ml−1 Trowell’s T8 medium numerous dots are present on the surface of the microvilli. Scale bar is 100 µ m. (From Kik et al., 1991b; by permission of Gut)

Future studies using the Caco-2 cell line Interference of lectins with transepithelial transport of (macro)molecules The Caco-2 cell line has so far received a great deal of attention as an in vitro model for epithelial cell differentiation. Only recently have there been attempts to utilize this system for transport studies (Cogburn et al., 1991; Hidalgo et al., 1989; Hilgers et al., 1990). Following the growth of Caco-2 cells to form a confluent monolayer on microporous filters, transepithelial transport can be studied by adding compounds to the upper (apical) chamber, which is equivalent to the intestinal lumen, and by monitoring their appearance in the lower basal chamber, equivalent to the serosal side. Transport of low molecular permeability markers, such as mannitol, demonstrated that Caco-2 cell monolayers became less permeable with increasing age in culture (Cogburn et al., 1991). Transepithelial transport of macromolecules through small intestinal cells can generally occur by either passive or endocytotic mechanisms. Passive transport is usually increased in damaged mucosa, while active absorption processes are strongly dependent on the structural integrity of the intestinal barrier and the proper functioning of the intestinal cells.

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Figure 5.10. The microvilli of the absorptive epithelium from pig mucosal explants after exposure to PHA-L4. After exposure for 5 h to 50 µ g of PHA-L4 ml−1 Trowell’s T8 medium the microvilli of the explants are irregularly positioned and shortened and microvillus vesicles are tied off. The microvilli are fragmentary. Scale bar is 50 µ m.

Investigations on transepithelial transport of (macro)molecules have already been carried out in the Caco-2 cells grown as monolayers on microporous filters. Both passive paracellular transport (mannitol, FITC, polyethylene glycol-900, polyethylene glycol-4000), receptor-mediated transcellular transport (glucose, biotin, spermidine, alanine) or passive lipophilic transcellular transport (alprenolol, propanolol, clonidine, diazepam, testosterone, salicylic acid) have been investigated (Cogburn et al., 1991; Hilgers et al., 1990). From the results obtained with glucose (actively transcellularly transported) and passively transported lipophilic solutes of testosterone and salicylic acid it was concluded, that differentiated Caco-2 cells can be used to predict intestinal absorption in vivo (Hilgers et al., 1990). However, it is still unknown, whether lectins can interfere with the in vivo transepithelial transport of (macro)molecules through the intestinal mucosa. All the same, the Caco-2 transport model has given us a means of examining this aspect at the cellular level in the near future. The presence of lectins in the gut lumen appears to enhance the extent of receptor-mediated endocytosis or transporter-mediated transcellular transport (Pusztai, 1989; Pusztai et al., 1991). Based on these findings it is reasonable to suggest that oral drug applications based on lectins either as receptors or ligands might be an effective practical means for drug delivery. In this respect the Caco-2 transport model may be used to predict the intestinal absorption potential of lectin-based drugs.

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Interference of lectins with adherence, penetration and translocation of bacteria To reach the systemic circulation of animals bacteria must cross the intestinal epithelium. Translocation across this barrier is a common mechanism of entry by enteropathogenic bacteria. After translocation pathogens have an immediate access to the underlying tissues, where they can further proliferate. Actually, there are two options for bacteria to cross the intestinal epithelium in vivo and these can be studied using the polarized monolayer of enterocyte-like Caco-2 cells in vitro. Bacteria may enter the intestinal cells through the apical surface or through the tight junctions between the epithelial cells. However in the latter case, entry remains limited to a few bacteria unless a significant potential to disrupt the intercellular spaces exists. Polarized enterocyte-like Caco-2 cells grown on a permeable filter mimic the in vivo animal model very closely and are a suitable in vitro model for studies on the adherence, penetration and translocation of bacteria as these cells provide both a basolateral and an apical surface, separated by tight junctions. It has been demonstrated in this in vitro model that after apical infection with either Salmonella choleraesuis or Salmonella typhimurium, both species of bacteria pen etrated through the monolayer, requiring 2 h before appearing on the serosal side (Finley and Falkow, 1990). Early after infection (30 minutes) both bacteria could be seen adhering to intact microvilli. In contrast to these Salmonella species, Shigella flexneri, does not cross through the apical side of polarized monolayers of differentiated Caco-2 cells. When the tight functions were disrupted artificially (chelation of Ca2+), the entry of the bacteria appeared to occur rapidly through the basolateral membrane (Mounier et al., 1992). Translocation of Campylobacter jejuni across human polarized enterocyte-like Caco-2 cell monolayers occured by passing both through and between the cells (Konkel et al., 1992). Adherence, penetration and translocation of Campylobacter jejuni required both active bacterial (protein synthesis) and target cell metabolic activity. Depending on their carbohydrate specificity, lectins recognize and bind to carbohydrate receptors on the intestinal brush-border membrane. As the attachment of bacteria to carbohydrate receptors on intestinal cells is mediated mainly by lectin-adhesins, it is possible to interfere with their adherence. Dietary supplements of (complex) carbohydrates, which are structurally similar to gut receptors, may competitively inhibit attachment of bacteria. Furthermore, food lectins which possess carbohydrate specificities similar to those of the adhesins may also effectively block the attachment of the bacteria to the intestinal mucosa or the enterocyte-like Caco-2 cells. Thus, adherence of harmful bacteria such as Salmonella choleraesuis, Salmonella typhimurium and Campylobacter jejuni may be blocked by the presence of the appropriate lectins in the cell culture medium. Cytoskeletal lesions in rat enterocytes have been observed after exposure of rat intestinal epithelium to Con A, WGA or PHA (King et al., 1986; Lorenzsonn and Olson, 1982; Sjölander et al., 1986). Recently, we have established that a shift in the ratio of globular to filamentous actin is induced in differentiated Caco-2 cells after exposure to SBA (Draaijer et al., 1989). Lectins, which are able to induce cytoskeletal lesions might well also disrupt the intestinal epithelium or the integrity of the monolayer. As a result of this, bacteria such as Shigella flexneri, which do not normally penetrate the apical membrane (Mounier et al., 1992), may gain free access to the serosal side and then enter the cells through the basolateral membrane. Thus, by applying the principles of chemical probiosis (Pusztai, 1989; Pusztai et al., 1991) using nontoxic lectins, the adherence of harmful bacteria (Salmonella species, Campylobacter jejuni) to the apical membrane can be controlled. However, it must be kept in mind that, if dietary lectins disrupt the integrity of the intestinal epithelium or the polarized monolayer of Caco-2 cells, the otherwise harmless Shigella flexneri can adhere to the basolateral membrane and cause damage. In that case, the remedy might be worse than the disease.

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Working mechanism of lectins Binding of lectins to the brush-border membrane glycoconjugates of differentiated Caco-2 cells (Hendriks et al., 1991; Koninkx et al., 1992) is only the first step in the reaction leading to changes in the cellular metabolism. Interference of lectins with cellular metabolism may result from signals originating from the binding of lectins to their receptors in the brush-border membrane. For the transduction of these signals to the interior of the cells endocytosis of the lectin or the lectin-receptor complex is not required. It is not yet clear, however, whether other signals which originate after the endocytotic uptake of the lectin and/or its receptor (Hendriks et al., 1991; King et al., 1986) by the differentiated Caco-2 cells are involved in signal transduction and what their role might possibly be. Indeed, as the mechanism responsible for the modulation of cellular metabolism after lectin-binding and the internalization of the bound lectin by the cell is still unknown, it is possible that both externally-bound or internally delivered lectins are involved. It has been shown, on the basis of binding studies with radioactively labelled lectins that binding sites are found on both the cytoplasmic and the cisternal surface of the nuclear envelope, the mitochondrial outer membrane, the rough endoplasmic reticulum and on the Golgi apparatus (Lis and Sharon, 1986). Based on these findings and our own results, we suggest as a working hypothesis that the transport of the lectins through the cell and the binding to the various subcellular organelles might be responsible for the changes in cellular metabolism. This hypothesis, however, needs further supporting evidence, possibly from investigations into the localization and transport of labelled lectins within differentiated Caco-2 cells. Acknowledgements The author is indebted to Henriette Eckhardt for typing the manuscript. The micrograph in Figures 5.9 and 5.10 were taken by Dr M.J.L.Kik and her permission to include them in this review is much appreciated. This work is part of a FLAIR Concerted Action Programme (No. 9) supported by the Commission of European Communities. References Akiyama, T., Kadowaki, T., Nishida, E., Kadooka, T., Ogawara, H., Fukami, Y., Sakai, H., Takaku, F. and Kasuga, M., 1986, Substrate specificities of tyrosine-specific protein kinases towards cytoskeletal proteins in vitro, Journal of Biological Chemistry, 261, 14797–803. Banwell, J.G., Boldt, D.H., Meyers, J. and Weber, F.L. Jr., 1983, Phytohaemaglutinin derived from red kidney bean (Phaseolus vulgaris): a cause for intestinal malabsorption associated with bacterial overgrowth in the rat, Gastroenterology, 84, 506–15. Banwell, J.G., Howard, R., Kabir, I. and Costerton, J.W., 1988, Bacterial overgrowth by indigenous microflora in the phytohaemagglutinin fed rats, Canadian Journal of Microbiology, 34, 1009–13. Bardocz, S., Grant, G., Brown, D.S., Ewen, S.W.B. and Pusztai, A., 1989, Involvement of polyamines in Phaseolus vulgaris lectin-induced growth of rat pancreas in vivo, Medical Science Research, 7, 309–11. Bardocz, S., Brown, D.S., Grant G. and Pusztai, A., 1990a, Luminal and basolateral polyamine uptake by rat small intestine stimulated to grow by Phaseolus vulgaris lectin phytohaemagglutinin in vivo, Biochimica et Biophysica Acta, 1034, 46–52. Bardocz, S., Grant, G., Brown, D.S., Ewen, S.W.B., Nevison, I. and Pusztai, A., 1990b, Polyamine metabolism and uptake during Phaseolus vulgaris lectin, PHA-induced growth of rat small intestine, Digestion, 46, 360–6. Blikstad, I., Markey, F., Carlsson, L., Persson, T. and Lindberg, U., 1978, Selective assay of monomeric and filamentous actin in cell extracts, using inhibition of deoxyribonuclease I, Cell, 15, 935–43.

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Boldt, D.H. and Banwell, J.G., 1985, Binding of isolectins from red kidney bean (Phaseolus vulgaris) to purified rat brush-border membranes, Biochimica et Biophysica Acta, 843, 230–37. Chauvière, G., Coconnier, M.-H., Kerneis, S., Fourniat, J. and Servin, A.L., 1992, Adhesion of human Lactobacillus acidophilus strain LB to human enterocyte-like Caco-2 cells, Journal of General Microbiology, 138, 1689–96. Cogburn, J.N., Donovan, M.G. and Schasteen, C.S., 1991, A model of human small intestinal absorptive cells. 1. Transport barrier, Pharmaceutical Research, 8, 210–16. D’Agostino, L., Daniele, B., Pignata, S., Gentile, R., Tagliaferri, P., Contegiacomo, A., Silvestro, G., Polestina, C, Raffaele Bianco, A. and Mazzacca, G., 1989, Ornithine decarboxylase and diamine oxidase in human colon carcinoma cell line Caco-2 in culture, Gastroenterology, 97, 888–94. D’Agostino, L., Pignata, S., Daniele, B., D’Adamo, G., Ferraro, C., Silvestro, G., Tagliaferri, P., Contegiacomo, A., Gentile, R. and Tritto, G., 1990, Polyamine uptake by human colon carcinoma cell line Caco-2, Digestion, 46, 352–59. Danielsen, E.M., Sjöström, H., Norén, O., Bro, B. and Dabelsteen, E., 1982, Biosynthesis of intestinal microvillar proteins: characterization of intestinal explants in organ culture and evidence for the existence of pro-forms of the microvillar enzymes, Biochemical Journal, 202, 647–54. de Oliveira, J.T.A., Pusztai, A. and Grant, G., 1988, Changes in organs and tissues induced by feeding of purified kidney bean (Phaseolus vulgaris) lectins, Nutrition Research, 8, 943–47. Donatucci, D.A., Liener, I.E. and Gross, C.J., 1987, Binding of navy bean (Phaseolus vulgaris) lectin to the intestinal cells of the rat and its effects on the absorption of glucose, Journal of Nutrition, 117, 2154–60. Draaijer, M., Koninkx, J., Hendriks, H., Kik, M., Van Dijk, J. and Mouwen, J., 1989, Actin cytoskeletal lesions in differentiated human colon carcinoma Caco-2 cells after exposure to soybean agglutinin, Biology of the Cell, 65, 29–35. Edelson, P.J. and Cohn, Z.A., 1974, Effect of concanavalin A on mouse peritoneal macrophages. I. Stimulation of endocytic activity and inhibition of phago-lysosome formation, Journal of Experimental Medicine, 140, 1364–86. Everest, P.H., Goossens, H., Butzler, J.-P., Lloyd, D., Knutton, S., Ketley, J.M. and Williams, P.H., 1992, Differentiated Caco-2 cells as a model for enteric invasion by Campylobacter jejuni and C. coli, Journal of Medical Microbiology, 37, 319–25. Finlay, B.B. and Falkow, S., 1990, Salmonella interactions with polarized human intestinal Caco-2 epithelial cells, Journal of Infectious Diseases, 162, 1096–106. Francis, C.L., Starnbach, M. and Falkow, S., 1992, Morphological and cytoskeletal changes in epithelial cells occur immediately upon interaction with Salmonella typhimurium grown under low-oxygen conditions, Molecular Microbiology, 6, 3077–87. Greer, F., Brewer, A.C. and Pusztai, A., 1985, Effect of kidney bean (Phaseolus vulgaris) toxin on tissue weight and composition and some metabolic functions of rats, British Journal of Nutrition, 54, 95–103. Hauri, H.-P., Sterchi, E.E., Bienz, D., Fransen, J.A.M. and Marxer, A., 1985, Expression and transport of microvillus membrane hydrolases in human intestinal epithelial cells, Journal of Cell Biology, 101, 838–51. Hendriks, H.G.C.J.M., Koninkx, J.F.J.G., Draaijer, M., van Dijk, J.E., Raaijmakers, J.A.M. and Mouwen, J.M.V.M., 1987, Quantitative determination of the lectin binding capacity of small intestinal brush border membrane. An enzyme linked lectin sorbent assay (ELLSA ),Biochima et Biophysica Acta, 905, 371–75. Hendriks, H.G.C.J.M., Kik, M.J.L., Koninkx, J.F.J.G., van den Ingh, T.S.G.A.M. and Mouwen, J.M.V.M., 1991, Binding of kidney bean (Phaseolus vulgaris) isolectins to differentiated human colon carcinoma Caco-2 cells and their effect on cellular metabolism, Gut, 32, 196–201. Hidalgo, I.L., Raub, T.J. and Borchardt, R.T., 1989, Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability, Gastroenterology, 96, 736–49. Hilgers, A.R., Conradi, R.A. and Burton, P.S., 1990, Caco-2 cell monolayers as a model for drug transport across the intestinal mucosa, Pharmaceutical Research, 7, 902–10. Hughson, E.J. and Hopkins, C.R., 1990, Endocytic pathways in polarized Caco-2 cells: identification of an endosomal compartment accessible from both apical and basolateral surfaces, Journal of Cell Biology, 110, 337–48.

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Kerneis, S., Bilge, S.S., Fourel, V., Chauvière, G., Coconnier, M.-H. and Servin, A.L., 1991, Use of purified F1845 fimbrial adhesin to study localization and expression of receptors for diffusely adhering Escherichia coli during enterocytic differentiation of human colon carcinoma cell lines HT-29 and Caco-2 in culture, Infection and Immunity, 59, 4013–18. Kik, M.J.L., van den Muysenberg, A. and van Kleef, D., 1991a, Development of a self-emptying blind pouch (SEP) model in the jejunum of piglets, Journal of Animal Physiology and Animal Nutrition, 65, 254–62. Kik, M.J.L., Koninkx, J.F.J.G., van den Muysenberg, A. and Hendriksen, F., 1991b, Pathological effects of Phaseolus vulgaris isolectins on pig small intestinal mucosa in organ culture, Gut, 32, 886–92. King, T.P., Pusztai, A., and Clarke, E.M.W., 1980a, Immunocytochemical localization of ingested kidney bean (Phaseolus vulgaris) lectins in rat gut, Histochemical Journal, 12, 201–8. King, T.P., Pusztai, A. and Clarke, E.M.W., 1980b, Kidney bean (Phaseolus vulgaris) lectin-induced lesions in rat small intestine: light microscope studies, Journal of Comparative Pathology, 90, 585–95. King, T.P., Pusztai, A. and Clarke, E.M.W., 1982, Kidney bean (Phaseolus vulgaris) lectin-induced lesions in rat small intestine. 3. Ultrastructural studies, Journal of Comparative Pathology, 92, 357–73. King, T.P., Begbie, R. and Cadenhead, A., 1983, Nutritional toxicity of raw beans in pigs. Immunocytochemical and cytopathological studies on the gut and the pancreas, Journal of Science of Food and Agriculture, 34, 1404–12. King, T.P., Pusztai, A., Grant, G. and Slater, P., 1986, Immunogold localization of ingested kidney bean (Phaseolus vulgaris) lectins in epithelial cells of the rat small intestine, Histochemical Journal, 18, 413–20. Klumpermans, J., Fransen, J.A.M., Boekestijn, T.C., Oude Elferink, R.P.J., Matter, K., Hauri, H.-P., Tager, J.M. and Ginsel, L.A., 1991, Biosynthesis and transport of lysosomal -glucosidase in the human colon carcinoma cell line Caco-2: secretion from the apical surface, Journal of Cell Science, 100, 339–47. Koninkx, J.F.J.G., Hendriks, H.G.C.J.M., van Rossum, J.M.A., van den Ingh, T.S.G.A.M. and Mouwen, J.M.V.M., 1992, Interaction of legume lectins with the cellular metabolism of differentiated Caco-2 cells, Gastroenterology, 102, 1516–23. Konkel, M.E., Mead, D.J., Hayes, S.F. and Cieplak Jr., W., 1992, Translocation of Campylobacter jejuni across human polarized epithelial cell monolayer cultures, Journal of Infectious Diseases, 166, 308–15. Lehr, C.-M., Bouwstra, J.A., Kok, W., Noach, A.B.J., de Boer, A.G. and Junginger, H.E., 1992, Bioadhesion by means of specific binding of tomato lectin, Pharmaceutical Research, 9, 547–53. Lis, H. and Sharon, N., 1986, Application of lectins, in Liener, I.E., Sharon, N. and Goldstein, I.J. (Eds) Lectins: Properties, Functions and Applications in Biology and Medicine, pp. 293–370, London: Academic Press. Lorenz-Meyer, H., Roth, H., Elsasser, P. and Hahn, U., 1985, Cytotoxicity of lectins on rat intestinal mucosa enhanced by neuraminidase, European Journal of Clinical Investigation, 15, 227–34. Lorenzsonn, V. and Olson, W.A., 1982, In vivo responses of rat intestinal epithelium to intraluminal dietary lectins, Gastroenterology, 82, 838–48. Mooseker, M.S., 1985, Organization, chemistry and assembly of the cytoskeletal apparatus of the intestinal brush border, Annual Review of Cellular Biology, 1, 209–41. Mooseker, M.S. and Tilney, L.G., 1975, Organization of an actin filament-membrane complex, Journal of Cell Biology, 67, 725–43. Mounier, J., Vasselon, T., Hellio, R., Lesourd, M. and Sansonetti, P.J., 1992, Shigella flexneri enters human colonic Caco-2 epithelial cells through the basolateral pole, Infection and Immunity, 60, 237–48. Neeser, J.R., Chambaz, A., Golliard, M., Link-Amster, H., Fryder, V. and Kolodziejczyk, E., 1989, Adhesion of colonization factor antigen II-positive enterotoxigenic Escherichia coli strains to human enterocyte-like HT-29 cells: a basis for host-pathogens interaction in the gut, Infection and Immunity, 57, 3727–34. Palmer, R.M., Pusztai, A., Bain, P. and Grant, G., 1987, Changes in rates of tissue protein synthesis in rats induced in vivo by consumption of kidney bean lectins, Comparative Biochemistry and Physiology, 88C, 179–83. Pinto, M., Appay, M.-D., Simon-Assmann, P., Chevalier, G., Dracopoli, N., Fogh, J. and Zweibaum, A., 1982, Enterocytic differentiation of cultured human colon cancer cells by replacement of glucose by galactose in the medium, Biology of the Cell, 44, 193–96.

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Pinto, M., Robine-Leon, S., Appay, M.-D., Kedinger, M., Triadou, N., Dussaulx, E., Lacroix, B., Simon-Assmann, P., Haffen, K., Fogh, J. and Zweibaum, A., 1983, Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture, Biology of the Cell, 47, 323–30. Pusztai, A., 1989, Transport of proteins through the membranes of the adult gastrointestinal tract—a potential for drug delivery? Advanced Drug Delivery Reviews, 3, 215–28. Pusztai, A., de Oliveira, J.T.A., Bardocz, S., Grant, G. and Wallace, H.M., 1988a, Dietary kidney bean lectin-induced hyperplasia and increased polyamine content of the small intestine, in Bøg-Hansen, T.C. and Freed, D.J.L. (Eds) Lectins: Biology, Biochemistry and Clinical Biochemistry, vol. 6, pp. 117–20, St. Louis: Sigma Library. Pusztai, A., Grant, G., Williams, L.M., Brown, D.S. and Bardocz, S., 1988b, Phaseolus vulgaris lectin induces growth and increases the polyamine content of rat small intestine in vivo, Medical Science Research, 16, 1283–84. Pusztai, A., Grant, G., Williams, L.M., Brown, D.S., Ewen, S.W.B. and Bardocz, S., 1989a, Phaseolus vulgaris lectin induces growth and the uptake of polyamines by the rat small intestine in vivo, Medical Science Research, 17, 143–45. Pusztai, A., Greer, F. and Grant, G., 1989b, Specific uptake of dietary lectins into the systemic circulation of rats, Biochemical Society Transactions, 17, 481–82. Pusztai, A., Ewen, S.W.B., Grant, G., Peumans, W.J., van Damme, E.J.M., Rubio, L. and Bardocz, S., 1990, Relationship between survival and binding of plant lectins during small intestinal passage and their effectiveness as growth factors, Digestion, 46, 308–16. Pusztai, A., Ewen, S.W.B., Grant, G., Peumans, W.J., Van Damme, E.J.M., Rubio, L.A. and Bardocz, S., 1991, Plant (food) lectins as signal molecules: effects on the morphology and bacterial ecology of the small intestine, in Kilpatrick, D.C., Van Driessche, E. and Bøg-Hansen, T.C. (Eds) Lectin Reviews, vol. 1, pp. 1–15, St. Louis: Sigma Library. Quaroni, A., 1985, Development of fetal rat intestine in organ and monolayer culture, Journal of Cell Biology, 100, 1611 22. Sager, P.R., Syversen, T.L.M., Clarkson, T.W., Cavanagh, J.B., Elgsaeter, A., Guldberg, H.C., Lee, S.D., Lichtman, M.A., Mottet, M.A. and Olmsted, J.B., 1986, Structure and funtion of the cytoskeleton, in Clarkson, T.W., Sager, P.R. and Syversen, T.L.M. (Eds) The Cytoskeleton. A Target for Toxic Agents, pp. 3–31, New York, London: Plenum Press. Sharon, N., 1983, Lectin receptors as lymphocyte surface markers, Advances in Immunology, 34, 213–98. Sharon, N., 1984, Carbohydrates as recognition determinants in phagocytosis and in lectin-mediated killing of target cells, Biology of the Cell, 51, 239–45. Sjölander, A., Magnusson, K.E. and Latkovic, S., 1986, Morphological changes of rat small intestine after short-time exposure to concanavalin A or wheat germ agglutinin, Cell Structure and Function, 11, 285–93. Weatherbee, J.A., 1981, Membranes and cell movement: interaction of membranes with the protein of the cytoskeleton, International Review of Cytology, 12, 113–76.

Chapter 6 Lectins as Growth Factors for the Small Intestine and the Gut Susan Bardocz, Stanley W.B.Ewen, George Grant and Arpad Pusztai

Introduction The epithelium of the small bowel is composed of a monolayer of epithelial enterocytes fulfilling the absorptive and digestive functions of the gut. These cells are interspersed with minor cell types: mainly with goblet cells, producing mucins, and enteroendocrine cells, which are responsible for the synthesis of the peptide hormones of the gut. The small intestinal epithelium is organized into two functionally and morphologically distinct compartments: the crypts, where the stem cells proliferate and differentiate, and the villi, where the differentiated cells mature while migrating toward the tip of the villi (Figure 6.1). This is where the absorption and digestion occur (Johnson, 1988). During migration along the crypt-villus axis, there is a continuous change in the cellular membrane; its protein composition, the pattern and activity of the enzymes expressed in it and the state of glycosylation (Figure 6.1) of its components go through distinct phases of development (Johnson, 1988; Cole and Smith, 1989; Gordon, 1989; Shylaja and Seshadri, 1989). Finally, after the fully differentiated and matured cells have reached the apical area of the villus, they are extruded into the lumen of the intestine. As all cell-surface proteins have to be transported from the site of synthesis to the plasma membrane, they have to go through several steps of glycosylation (Cole and Smith, 1989; Shylaja and Seshadri, 1989). The pattern of glycosylation of the membrane glycoconjugates varies in different species (King and Kelly, 1991), but within one species it depends mainly on the stage of differentiation and maturation of the cells, their position along the crypt-villus axis and on the precise location along the gastrointestinal tract. In addition, the pattern will also be influenced by the age and blood group specificity of the animal (Table 6.1). For example, it is believed that the glycosyl side-chains of membrane proteins of the less differentiated crypt cells are usually of the polymannose type, whereas the fully mature cells on the villi express complex glycosyl side-chains. The great variability in the glycosylation of membrane glycoconjugates may help to explain why lectins differ in their ability to interact with the surface of the gut. Unfortunately, at present, quantitative information is limited on the precise carbohydrate structure of the gut surface receptors (see King, Chapter 10), their location along the crypt-villus axis and the changes occurring in them during normal turnover or under different conditions of age and dietary status. However, as the epithelium of the intestinal tract has one of the highest cell turnover rates in the body of mammals (Johnson, 1988) enabling them to react rapidly to any dietary changes, the surface morphology of the gut and changes in its receptors Table 6.1. Factors influencing glycosylation of cells in the gastrointestinal tract •

Species dependent

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• • • • • • • •

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Blood group specificity Age Site in gastrointestinal tract Position along crypt-villus axis Genetic ‘set up’ of stem cell in the crypt (state of differentiation and maturation) Diet Bacterial ecology Pathological alterations

faithfully follow, and can truly reflect, the causes which initially set these changes in motion. Lectin histochemistry and/or the determination of the lectin-binding capacity of brush-border membranes prepared from the small intestine (Hendriks et al., 1987) might provide the tools for mapping the potential receptor sites, give valuable information on the functional state of the gut, and the means to modify and adapt it to our requirement.

Figure 6.1. The structure of the small intestine with carbohydrates projected into the lumen as potential binding sites for plant and bacterial lectins. The different cell types are epithelial cells, stem cells, goblet cells and neuroendocrine cells.

Resistance of plant lectins to proteolytic degradation Dietary proteins are rapidly degraded during passage through the gut by the digestive enzymes present in the small bowel. In the caecum and colon, populations of resident bacteria try to degrade and use all material

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reaching the large intestine as sources of energy. In contrast to dietary proteins, lectins resist degradation by proteolytic enzymes and also by different bacteria. Most lectins survive passage through the entire digestive tract in an immunologically intact form (Table 6.2). Indeed, the secret of lectins as biologically effective molecules lies in their resistance to degradation. Similar to hormones and other growth factors, receptorbound lectins are even more resistant. Although binding to the epithelium may help to stabilize lectins against gut proteolysis, this is not obligatory. The almost complete survival of Galanthus nivalis (GNA), despite the absence of binding to the brush-border in acute exposure, suggests that the molecular structure of some lectins makes them intrinsically resistant to proteolytic breakdown. During the turnover of the gut epithelium, cells on the villus tip with the lectin attached are shed off into the lumen. All cellular material except the lectin is broken down (Pusztai et al., 1990) and the liberated lectin is therefore free to move further down the gut and bind to the next receptor with an appropriate carbohydrate moiety. Furthermore, some carbohydrates are only slightly expressed in the small bowel but are major components in the large intestine. Lectins specific Table 6.2. Binding of lectins to gut mucosa and their survival in the gut Lectin

Binding

Recovery (%)

PHA Phaseolus vulgaris +++ >90 Con A Canavalia ensiformis + >90 GNA Galanthus nivalis − >90 SNA-I Sambuccus nigra + 50–60 SNA-II Sambuccus nigra +++ >60 SBA Glycine max ++ 40–50 LEL Lycopersicon esculentum + 40 50 WGA Triticum vulgare + 50–60 PSL Pisum sativum + 30 40 VFL Vicia faba ± 20 30 DGL Dioclea grandiflora + 18–20 The survival is given as per cent of lectin remaining 1 h after intragastric intubatlon of a solution of 10 mg pure lectin (in physiological saline) to rats fasted for 16 h.

for these rapidly pass through the small intestine and then avidly bind to the surface of the large intestine and affect its metabolism. Binding of lectins to the brush-order membrane Interaction between lectins of plant or bacterial origin and the gut depends on specific recognition by the lectin of membrane glycans projecting into the gut lumen. Therefore biological activity is a direct consequence of lectin function: through recognition and binding to specific carbohydrates on surface membranes, they send signals and deliver messages to cells. The process of recognition between lectins and their receptors is instantaneous. Lectins are potent exogenous growth signals and some can also mimic the action of major metabolic hormones. Most receptors of growth factors and hormones are glycoproteins or glycoconjugates embedded in the cell surface membrane. Receptor proteins are usually composed of more than one subunit, and the subunits exposed on the external side of the membrane are glycosylated. The main external subunit is

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Figure 6.2. Location of the glycosyl-side-chains on the cell-surface proteins in the membrane, and possible ways of lectins interacting with growth factor or hormone receptors.

responsible for binding the signal molecule itself, while the other(s), spanning the membrane, is responsible for the transmission of the signal message and for the activation of second messenger system(s) (Figure 6.2). Depending on their position, the sugar-structures can be present in or near to the active centre of the receptor. They may be on the same subunit as the binding site for the growth factors or hormones is located (Figure 6.2a) or on another subunit (Figure 6.2b). Although the lectin binding site (e.g. the glycosyl side chains) is clearly not the normal functional binding site of the receptor, the resulting conformational change in the receptor subunits embedded in the membrane and the ensuing signal transduction may be similar irrespective of whether the activation was by the physiological ligand or by the lectin. In this case, the lectin can mimic the effect of the natural ligand of the receptor and induce the same (or very similar) physiological reactions. It is also possible, however, that the bound lectin may not induce a conformational change but, by physically blocking the active site of the receptor, attenuate or completely abolish the physiological effect of the natural ligand. Some of the so called non- or anti-mitogenic lectins probably fall into this category. Finally, by an allosteric mechanism, the binding of lectins to the external receptor subunits may also additively or synergistically reinforce the effects of the natural ligand. As glycosylation patterns of brush-border cell surface components are variable and dependent on many factors (Table 6.1), these variations largely determine whether an individual lectin can bind to epithelial membranes and can also explain the differences in their reactivity. However, the strength of lectin binding will also be dependent on the number of free (unoccupied) receptor sites. If there are many carbohydrate sidechains with the ‘right’ sugar structure, the lectin will bind extensively, but if there are just a few binding sites, or the sites are well separated from each other, only weak or no binding occurs. There is evidence that lectins which bind avidly are readily endocytosed or transcytosed. However, the signals necessary for these processes are not well understood at the moment. Metabolic changes induced by the lectins in epithelial cells Lectins which can induce conformational changes in the receptor are also able to send messages via second messengers to the cell. As a result, gene expression in the cell or tissue is altered, leading to changes in the

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amount and composition of proteins and enzymes produced in the cytoplasm or on the surface of the cell. This, in turn alters the cell’s ability to interact with hormones and growth factors. In short, by binding, a lectin may change the entire metabolism of the cell. The most studied example of an exogeneous metabolic signal is the lectin, PHA, from the seeds of kidney bean, Phaseous vulgaris. PHA recognizes and binds to complex carbohydrate structures present on the surface of fully differentiated mammalian cells. Accordingly, as the insulin- and the insulin-like growth factor receptors express complex glycosyl moieties, PHA is an excellent ligand for them. These effects have been observed both in vivo and in vitro (Table 6.3). One of the first effects of exposure of the epithelium of the rat small intestine to dietary PHA is an instant stimulation of protein synthesis in this tissue. This is accomplished by a PHA signal-induced increase in the efficiency of protein synthesis in the cells by converting non-functional ribosomal subunits into fully functional polysomes (Bardocz et al., 1992; Palmer et al., 1987). A second and long lasting increase in protein synthesis is evident after a few hours and this is the result of the synthesis of new RNA (Pusztai et al., 1988). PHA also affects the concentration of the polyamines, putrescine, spermidine and spermine which are essential cell components (Pegg, 1986; Tabor and Tabor, 1984) and are needed for the adaptational growth of the gut (Hosomi et al., 1987; Luk and Baylin, 1983). Table 6.3. Comparison of the effect of insulin and PH A in vitro Signal Seconds binding to receptors activation of receptor tyrosine kinase receptor autophosphorylation Seconds to minutes Stimulation of ionglucoseamino acidnucleic acid-transport alteration of enzyme activities receptor Ser- Thr- phosphorylation changes in gene transcription ligand mediated receptor endocytosis Hours synthesis of nucleic acids proteins receptor down regulation cell growth/proliferation

Insulin

PHA

+ + +

+ + +

+ + + + + + + +

+ + + + + + + +

+ + + +

+ + ? +

Polyamines fulfil many important functions, and amongst others, help to transcribe messages carrying signals for the stimulation of protein synthesis. The growth of the gut induced in conventional rats (rats possessing a normal gut flora) by PHA requires the accumulation of large amounts of polyamines, mostly spermidine, in the tissue (Pusztai et al., 1988). As this is by hyperplasia, the polyamine requirement of the gut under these conditions is highly increased. However, the observation that this accumulation occurs

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without a major increase in the activity of ODC (ornithine decarboxylase, the rate-determining enzyme of polyamine synthesis) of the small intestine (Bardocz et al., 1990a; Pusztai et al., 1988) indicates that, in this instance, ODC may have little to do with polyamine accumulation. In the growing intestine, a part of the polyamine pool originates from the systemic circulation through the basolateral membrane. Indeed, one of the first steps of the PHA-induced growth process is the stimulation of the basolateral uptake of polyamines (Bardocz et al., 1990a, b). Therefore, measurement of the polyamine content of the tissue and the rate of basolateral uptake of polyamines, mostly spermidine, can be used as markers of the metabolic activity of the intestine and to follow the effects of different dietary factors, including the growth factor-like effects of the lectins on gut metabolism. The recognition that lectin-induced changes in cellular metabolism are fully reversible has allowed us to use the PHA-induced rat small intestinal growth model as a convenient tool for magnifying and studying the fundamental metabolic reactions of epithelial cell proliferation, differentiation and maturation and the ensuing changes in gut function and receptor glycosylation. As PHA is strong ly mitogenic, it affects the crypt cell proliferation rate (CCPR), i.e. the number of cells produced. Crypt cell proliferation rate The growth factor activity of lectins is determined mainly by the strength and intensity of their binding to the gut wall (Pusztai et al., 1990). Even when this is relatively weak, the organization of the epithelial membrane is disturbed sufficiently (King et al., 1982; Pusztai et al., 1990) to allow a slight growth of the gut. However, lectins which bind extensively can cause more damage to the cell membrane. As the gut wall is the first line of defence between the individual and the environment, it is essential to keep its integrity. When the structure of the microvillus membrane of epithelial cells is damaged by lectin-binding, the gut becomes leaky and the cells can no longer fulfil their protective and digestive/absorptive functions. Therefore, they have to be replaced by new, healthy cells to maintain the integrity of the intestine and prevent harmful compounds or bacteria entering the body. The result of this is that the CCPR is stimulated, although the exact mechanism by which it occurs is still unknown (Pusztai et al., 1988; 1990). As the lectin first binds to the villus, the growth signal has to be sent to the crypt where the proliferation occurs. Although the mechanism of this is far from clear, it is possible that the signal arises out of the distortion of the epithelial cell membrane after PHA-binding and is transducted by some unknown means to the crypts, or that a stimulatory signal results from the release of a putative endogenous growth factor/hormone by the neuroendocrine cells into the blood circulation and this then exerts its effects on the crypt cells directly. The increase in CCPR might also be a process of detoxification, as the easiest way for the tissue to free itself of the irritant is to shed the affected cells quickly. When CCPR is stimulated, cells which are on the villi in contact with PHA can be eliminated faster. The length of the villus is rarely significantly affected on lectininduced small intestinal growth (Pusztai et al., 1990). In contrast, the crypt size, the number of cells they contain and CCPR are all increased substantially (results not published). These changes correlate well with the effectiveness of the lectins as growth factors. With increased CCPR more cells are produced which then have to migrate up the crypt/ villus axis faster than normal. All newly produced cells, however, need time to differentiate, and as a rule, undifferentiated cells cannot leave the crypt. This can explain why the length of the crypt increases in size and contains more cells than the non-stimulated crypt. Furthermore, the proportion of immature cells on the villi rises because of the pressure exerted by the new cells migrating up the villus column due to the increased CCPR and cell turnover. In an extreme case, most villus tip cells are pushed out to the lumen from the tip of the villi before they have time to mature fully. With continuous exposure to lectins, such as PHA, which bind extensively, the cell turnover time can decrease from 72 to 12

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Figure 6.3. Lectin-induced, second messenger mediated changes in cellular metabolism.

h. Thus, on extended dietary exposure to PHA, the CCPR increases to such an extent that the villi contain an appreciable number of immature cells. Accordingly, by speeding up cell turnover, PHA can and does induce changes in gene expression of gut epithelial cells. As both the composition and quantity of proteins and enzymes of the newly formed cells are typical of the immature cell type, their capacity to digest and absorb

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food components is significantly less than that of the mature enterocytes. Thus, on exposure to PHA, the activity of maturation marker enzymes, such as diamine oxidase (personal communication from Drs Perin and Sessa, Universita Delgi Studi di Milano, Italy) or the specific activity of sucrase isomaltase and alkaline phosphatase (unpublished data) in small intestinal tissues are decreased significantly. Binding of lectins to cells in the crypt Although the biochemical mechanism of the growth stimulation is still not fully understood, some binding of lectins to cells of the jejunal crypts has been detected (Figure 6.3; Pusztai et al., 1990). Therefore, it is possible, that lectin-signals may directly stimulate crypt cell proliferation. Indeed, as PHA binds to crypt cells, its growth factor activity for the small intestine may be the direct result of this binding. However, it is not known how the lectin gains access to the crypt. One suggestion is that, after it has been transported through the epithelium into the systemic circulation, PHA reaches the crypt cells via the bloodstream. However, PHA may also directly access the crypt from the gut lumen. GNA also binds to the crypt (Pusztai et al., 1990), but its effect is the opposite of PHA. This lectin appears to slow down CCPR resulting in a decrease in the length and cell numbers of the crypts. Changes induced in glycosylation of epithelial cells by exposure to lectins Effect of increased CCPR on glycosylation In adult rats there are few free terminal mannose residues on the brush-order membranes. Therefore, binding of the mannose-specific lectin, GNA, to the jejunal epithelium was slight (Pusztai et al., 1990) while the somewhat similar Con A, with specificity for terminal mannose/glucose residues, reacted more strongly, though patchily, with the epithelium (Pusztai et al., 1990). In contrast, the binding of PHA was extensive, particularly on the upper third of jejunal villi (Pusztai et al., 1988, 1990), as most of the glycans on the luminal surface of the small intestine are composed of complex glycosyl side-chains. The Nacetylglucosamine specific wheat germ, Triticum vulgare agglutinin (WGA), also binds to and is endocytosed extensively by epithelial cells. This binding is extensive, particularly at mid villus, but it also reaches down to the lower half of this compartment (Pusztai et al., 1993). Depending on the specificity of the lectin and the strength of its binding, three main types of lectininduced changes were observed in the glycosylation of membrane- and/or cytoplasmic glycoproteins of epithelial cells of the small intestine. After exposure to PHA or to other lectins, which bind avidly to and are endo-cytosed extensively in the small intestine, the pattern of glycosylation of brush-border cells was found to be distinctly different from that of fully mature apical cells in control rats expressing mainly complex glycosyl side chains. Thus, both membrane and cytoplasmic glycoconjugates in these cells of rats fed on lectin-diets contained large amounts of polymannosylated glycans, readily detectable histologically by staining with GNA-digoxigenin (Figure 6.4). As glycans of less differentiated crypt cells are highly polymannosylated, it is possible that with the lectin-induced increase in CCPR and the correspondingly shortened transit time of epithelial cells, these crypt cells penetrate further up the villus than they would do under normal physiological conditions. The more reactive the lectins are, the shorter the transit time becomes, with the result that there are more immature cells on the epithelial surface, and therefore more polymannosyl-type side-chains on cellular glycoproteins. Accordingly, the most effective growth-stimulating lectins are also the most powerful agents for inducing this type of changes in carbohydrate receptor expression of the gut epithelium.

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Figure 6.4. A schematic diagram giving the position of polymannose-type glycoproteins in cells of the small intestine in (a) lactalbumin-diet fed or (b) in PHA-fed rats. The darker the colour, the more polymannose residues can be detected by digoxigenin-labelled GNA.

Reaction of lectins with secreted glycoproteins Lectins, which react with sialic acid in secreted glycoproteins, such as from Sambuccus nigra (SNA-I) and Maackia amurensis (MAA), can induce changes in receptor glycosylation by overstimulating and exhausting the capacity of the goblet cells in the small intestine to synthesise mucin. The action of these lectins results in the partial disappearance of some of the ‘receptor sites’ in mucin containing neuraminyl-2, 6- or neuraminyl-2,3-lactose glycosyl structures respectively and this may leave the luminal surface partially uncovered. Thus, the reactivity of the small intestinal epithelium with SNA-I or MAA disappears almost completely after extended dietary exposure to the respective lectin. As the binding of SBA to glycans with terminal sialic acid is relatively weak, the extent of its binding is increased after the removal of mucins containing terminal sialic acid. Displacement of endogenous ligands by dietary lectins It has been shown that by displacing endogenous ligands bound to glycosyl moieties of luminal receptors, dietary plant lectins can also in effect change the glycosylation of membrane glycans. For example, the binding of strictly mannose-specific GNA by the small intestinal brush-border is essentially absent in rats with a conventional microflora but is significantly increased in germ-free animals (Figures 6.2a and 6.4). This may reflect the fact that limited number of mannosylated glycans in the rat small intestine in conventional rats are blocked by mannose-sensitive fimbriated bacteria (e.g. E. coli) but are free in germ-free rats. When rats are given diets containing GNA, however, the high concentration of the lectin in the lumen competing for

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the same sites allows it to bind to the newly emerging epithelial cells in preference to the bacteria (Pusztai et al., 1990). Nutritional penalty of the stimulation of gut growth Even a small increase in the size of the gut has a slight nutritional penalty for the animal, since the need to renew the gut surface more quickly than normal means that more of the dietary protein and energy are used up for the faster turnover. With lectins, such as PHA, SBA or WGA, which bind avidly to epithelial cells and are more powerful growth factors for the small bowel, the cost in nutritional terms is even more expensive. Indeed, at high dietary intakes of these lectins, most or all of the diet is used by the gut alone with the result that other organs are starved of nutrients (Pusztai, 1989; Pusztai et al., 1991a). However, under most practical conditions where dietary lectin intakes are low, the contribution of the growth stimulating activity of the lectins to nutritional toxicity is relatively slight. It has been shown recently that, despite extensive binding and potent stimulation of the growth of the small intestine, PHA is not toxic for germ-free rats. Accordingly, the striking toxicity of PHA at high dietary intakes in animals with a normal microbial flora is not due to PHA alone. It is likely that one of the major factors, which exacerbates the erosive effect of some lectins on the brush-border, is that they induce a selective bacterial overgrowth in the small intestine and that they, their toxic metabolites or toxins of the bacteria and/or the bacteria themselves are extensively endocytosed by the epithelial cells (see Pusztai et al., Chapter 15). After endocytosis, these toxic substances may enter the blood circulation by transcytosis and exert their deleterious effects by stopping vital cellular functions. They may also interfere with the body’s hormone balance (Pusztai et al., 1991b) and disturb its metabolic equilibrium (Pusztai, 1989; Pusztai et al., 1991b). Future perspectives and practical implications of lectin-gut interaction Changes in glycosylation induced by dietary lectins may lead to either the appearance of new glycans, or the removal of existing glycosyl structures on the surface of the gut and consequently, to changes in the binding potential of the brush-border for dietary lectins. Since a critical step in the bacterial colonisation of the gut is the binding of the bacterium to the gut surface through their fimbrial-and/or surface adhesins, the bacterial ecology of the intestinal tract may be altered by changing the expression of the sugar structures on the luminal surface (see Pusztai et al., Chapter 15). Lectins can also be used to change the physiology and the digestive/absorptive functions of the gut. Lectins with high affinity for binding may be used to induce gut growth or give a kick start to gut growth in cases of total parenteral nutrition or severe cases of gut atrophy. These lectins, almost without exception, may also be used to send messages to the pancreas to stimulate its growth and metabolism and consequently alter the hormonal balance of the body with advantage (see Pusztai et al., Chapter 8). Some lectins may be used to stimulate the secretion of enzymes from the pancreas and of digestive enzymes and mucin from the small intestinal brush-border, when necessary. Anti-mitotic lectins, which are incomplete mitogens, can be used to slow down or stop unwanted cell proliferation in the gastrointestinal tract (see Milton and Rhodes, Chapter 12) although these are yet to be tested. Finally, as the stimulation of reversible gut growth uses up nutrients, lectins such as PHA may be used deliberately to starve the body of nutrients, possibly as slimming agents or, more importantly, to restrict tumour growth by directing nutrients away from the tumour and towards the reversibly growing gut.

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Summary Interaction of lectins with brush-border membranes of the small intestine stimulates the polyaminedependent, reversible growth of the small bowel, the extent of which depends on the strength of the lectin binding. The stronger the binding, the more intensive is the growth of the gut; this leads to more extensive changes in glycosylation patterns of the brush-border membrane glycoconjugates and in the digestive enzyme composition and content of the gut. It is, therefore, possible to alter the gut so that it attains characteristics conducive to increased efficiency in its digestive/absorptive functioning, more advantageous hormonal secretion and more favourable bacterial balance. Thus, the gut may be engineered to suit our requirements and improve our health by the use of natural dietary components, such as lectins. They can stimulate gut growth, change receptor expression, exhaust its capacity to secrete glycoproteins (mucins) and replace endogenous ligands. Acknowledgements This work is part of EEC-FLAIR Concerted Action No 9, EEC AIR Concerted Action 92–569 and was supported in part by the Scottish Office Agriculture and Fisheries Department (Drs Bardocz and Pusztai). References Bardocz, S., Brown, D.S., Grant, G. and Pusztai, A. 1990a, Luminal and basolateral polyamine uptake by rat small intestine stimulated to grow by Phaseolus vulgaris lectin phytohaemagglutinin in vivo, Biochimica et Biophysica Acta, 1034, 46–52. Bardocz, S., Grant, G., Brown, D.S., Ewen, S.W.B., Nevison, I. and Pusztai, A. 1990b, Polyamine metabolism and uptake during Phaseolus vulgaris lectin, PHA-induced growth of rat small intestine, Digestion, 46 (suppl. 2), 360–66. Bardocz S., Brown D.S., Grant G., Pusztai A., Stewart J.C. and Palmer R.M.1992, Effect of the -adrenoreceptor agonist clenbuterol and phytohaemagglutinin on growth, protein synthesis and polyamine metabolism of tissues of the rat, British Journal of Pharmacology, 106, 476–82. Cole, C.R. and Smith, C.A. 1989, Glycoprotein biochemistry (structure and function)—a vehicle for teaching many aspects of biochemistry and molecular biology, Biochemical Education, 17, 179–89. Gordon, J.I. 1989, Intestinal epithelial differentiation: new insights from chimeric and transgenic mice, Journal of Cell Biology, 108, 1187–94. Hendriks, H.G.C.J.M., Koninkx, J.F.J.G., Draaijer, M., van Dijk, J.E., Raaijmakers, J.A.M. and Mouwen, J.M.V.M. 1987, Quantitative determination of the lectin binding capacity of small intestinal brush-border membrane. An enzyme linked lectin sorbent assay (ELLSA), Biochimica et Biophysica Acta, 905, 371–75. Hosomi, M., Stace, N.H., Lirussi, F., Smith, S.M., Murphy, G.M. and Dowling, R.H. 1987, Role of polyamines in intestinal adaptation in the rat, European Journal of Clinical Nutrition, 17, 375–85. Johnson, L.R. 1988, Regulation of gastrointestinal mucosal growth, Physiological Reviews 68, 456–502. King, T.P. and Kelly, D. 1991, Ontogenic expression of histo blood-group antigens in the intestines of suckling pigs: lectins histochemical and immunohistochemical analysis, Histochemical Journal, 23, 43–54. King, T.P., Pusztai, A. and Clarke, E.M.W. 1982, Kidney bean (Phaseolus vulgaris) lectin-induced lesions in rat small intestine. 3. Ultrastructural studies, Journal of Comparative Pathology, 92, 357–73. Luk, G.D. and Baylin, S.B. 1983, Polyamines and intestinal growth—increased polyamine biosynthesis after jejumectomy, American Journal of Physiology, 245, G656–60. Palmer, R.M., Pusztai, A., Bain, P.A. and Grant, G. 1987, Changes in rates of tissue protein synthesis in rats induced in vivo by consumption of kidney bean lectins, Comparative Biochemistry and Physiology, 88C, 179–83.

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Pegg, A.E. 1986, Recent advances in the biochemistry of polyamines in eukaryotes, Biochemical Journal, 234, 249–62. Pusztai, A. 1989, Biological effects of dietary lectins, in Huisman, J., van der Poel, T.F.B. and Liener I.E. (Eds) Recent Advances of Research in Antinutritional Factors in Legume Seeds, pp. 17–29, Wageningen: Pudoc. Pusztai, A., Grant, G., Brown, D.S., Ewen, S.W.B. and Bardocz, S. 1988, Phaseolus vulgaris lectin induces the growth and increases the polyamine content of rat small intestine in vivo, Medical Science Research, 16, 1283–84. Pusztai, A., Begbie, R., Grant, G., Ewen, S.W.B. and Bardocz, S. 1991a, Indirect effect of food antinutrients on protein digestibility and nutritional value of diets, in Fuller M.F. (Ed.) In Vitro Digestion for Pigs and Poultry, pp. 45–61, Wallingford: CAB International. Pusztai, A., Ewen, S.W.B., Grant, G., Peumans, W.J., Van Damme, E.J.M., Rubio, L. and Bardocz, S. 1990, The relationship between survival and binding of plant lectins during small intestinal passage and their effectiveness as growth factors, Digestion, 46 (suppl. 2), 308–16. Pusztai, A., Ewen, S.W.B., Grant, G., Peumans, W.J., Van Damme, E.J.M., Rubio, L. and Bardocz, S. 1991b, Plant (food) lectins as signal molecules: effects on the morphology and bacterial ecology of the small intestine, in Kilpatrick, D.C., van Driessche, E. and Bog-Hansen, T.C. (Eds) Lectin Reviews, Vol. 1, pp. 1–15, St. Louis, MI: Sigma Chemical Company. Pusztai, A., Ewen, S.W.B., Grant, G., Brown, D.S., Stewart, J.C., Peumans, W.J., Van Damme, E.J.M. and Bardocz, S. 1993, Antinutritional effects of wheat germ agglutinin and other N-acetylglucosamine specific lectins, British Journal of Nutrition, 70, 313–21. Shylaja, M. and Seshadri, H.S. 1989, Glycoproteins: an overview, Biochemical Education,17, 170–78. Tabor, C.W. and Tabor, H. 1984, Polyamines, Annual Review of Biochemistry, 53, 747–90.

Chapter 7 The Potential of Bioadhesive Lectins for the Delivery of Peptide and Protein Drugs to the Gastrointestinal Tract Claus-Michael Lehr and Arpad Pusztai

Introduction Due to the rapid progress of molecular biology and biotechnology over the last decade, peptide- and proteinbased reagents have attained considerable relevance for the diagnosis and therapy of diseases, and this trend is still increasing. For the same reason, there is an emerging interest in novel polysaccharides (e.g. heparinderivatives) and polynucleotides (e.g. antisense agents, gene therapy; Anderson, 1992; Davis, 1992). Clearly, the administration of all these new ‘biopharmaceuticals’ require particular formulations which are governed by the structure, physico-chemical properties, stability, pharmacodynamics and pharmacokinetics of these compounds (Lee, 1991). In particular, the new strategies which may be used for their convenient, safe and effective administration to patients in place of injections (parenteral routes) are presently being investigated with great efforts both in industry and academia. Apart from other so-called ‘alternative routes’—e.g. nasal, trans-dermal, pulmonal, buccal, ocular, vaginal or rectal—the peroral route of drug administration is considered as the most convenient. However, from a technical point of view this route is probably the most difficult. The site of actual drug absorption (intestines) is fairly remote from the site of drug administration (mouth). During gastrointestinal transit, numerous unpredictable obstacles (food, mucus, acid, digestive enzymes) are encountered. Apart from these the problem still remains how can such mostly hydrophillic, large and metabolically sensitive compounds be transported across the biological barrier of the intestinal epithelium in sufficiently large amounts for successful therapeutic effects. In order to improve the bioavailability of peptides, proteins and other macromolecular drugs which do not readily pass biological barriers, one of the most promising general strategies is to use bioadhesive drug delivery systems, i.e. pharmaceutical formulations which adhere to the absorbing mucosal epithelium (Harris and Robinson, 1990; Junginger, 1990). In order to achieve durable and strong adhesion between man-made drug delivery devices and the various mucosal tissue surfaces of the human body, different approaches are being pursued. The first approach uses particular synthetic or natural polymers which adhere by some non-specific, general physico-chemical interactions (e.g. interfacial energy effects, hydrogenbonding, electrostatic attraction, etc.) to wet, mucus-covered biological surfaces. This mucus gel layer has to act as a connecting link between the polymers and the actual tissue surface, and hence this special case of bioadhesion is more precisely referred to as mucoadhesion. The second, more sophisticated approach to bioadhesion is to use ligands which bind specifically to the epithelium, such as e.g. plant or bacterial lectins. As these recognize and bind to the sugar moieties of receptors, which are part of the apical surface of the epithelial cells, lectins provide mucus-independent bioadhesion. It is the purpose of this chapter to briefly outline the potential and limitations of bioadhesion technologies generally in oral drug delivery, and

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in particular, to weigh up the possible advantages of the use of specific bioadhesive lectins as gut targeting agents in comparison with non-specific mucoadhesive polymers. Mucoadhesive polymers as a first approach to epithelial adhesion The concept of bioadhesion was first introduced to pharmaceutical sciences more than 10 years ago. The prototypes of such systems were tablets, ointments or powders containing hydrophillic, water-insoluble, high-MW polymers, such as polyacrylic acid or cellulose derivatives, which could adhere to mucosal tissue (Nagai, 1985). Once in contact with the wet, mucous surface the polymers swell and, by absorbing water from the underlying mucus-gel, develop a relatively strong adhesion to the tissue. With this new possibility to prolong and/or to intensify the contact between a drug delivery system and the absorbing biological membrane, some new perspectives for the development of novel dosage forms have risen. First, the formulation could be kept essentially stationary, which—in combination with known controlled-release technologies—allowed to deliver drugs for prolonged periods of time within a well defined area. Second, as the formulation was in close contact with the epithelium, the diffusion pathway for the drug was shorter, its dilution by body liquids reduced and the drug concentration gradient was increased. Therefore, bioadhesive systems could be expected to improve the absorption of polypeptides (>3 amino acids) or proteins crossing the epithelia by passive paracellular diffusion. Possible advantages of oral bioadhesive drug delivery systems While studies with buccal, nasal, rectal or vaginal bioadhesive peptide drug delivery systems have confirmed the feasibility of the concept in animals and man (for references, see Junginger, 1990), the results with oral bioadhesive systems, i.e. which, after swallowing, were supposed to adhere to the mucosa of the gastrointestinal tract, were rather disappointing particularly in man (Ch’ng et al., 1985; Harris et al., 1990; Koshla and Davis, 1987; Longer et al., 1985). In spite of these setbacks the search for a successful oral bioadhesive drug delivery system is still going on for several reasons. First, with an increase in total gastrointestinal transit time it may be possible to keep the oral controlled-release systems within the body for longer periods of time, and hence to reduce the dosage required from, for instance, three times a day to once a day or less. Second, it may be desirable with some drugs to localize the delivery system by means of bioadhesion within a particular area of the gastrointestinal tract, such as e.g. the upper duodenum or the colon, where the drug is more effectively absorbed or exerts a local pharmacological effect on the mucosa. Third, the possibility to enhance the mucosal transport of peptide- and protein-drugs by intensifying their contact with the absorbing biological membrane should in principle also hold for an oral dosage form. Besides, by increasing the premucosal concentration gradient, the drug may effectively be protected from proteolytic degradation by secretory or membrane-bound enzymes before passing through the intercellular junctions between epithelial cells. Physiological problems related to non-specific mucoadhesion within the gastrointestinal tract In our laboratories the potential of, and problems encountered with, oral bioadhesive drug delivery systems have been extensively studied during the last six years, especially the possible use of this approach for the oral delivery of peptideand protein-drugs. Thus, after intraduodenal administration to rats of the octapeptide desglycinamide-arginine-vasopressin (DGAVP) together with a (1%, w/v) mucoadhesive polymer

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Figure 7.1. The adherence of the mucus gel layer to the intestinal epithelium is determined by a dynamic equilibrium (steady state) between secretion of preformed mucus glycoprotein from mucus producing glands or cells and the mechanical and chemical erosion taking place on the luminal surface (modified according to Allen et al., 1984).

polycarbophil, a cross-linked derivative of polyacrylic acid, the plasma levels of the drug were about 3–5 times higher than when it was given dissolved in saline (Lehr et al., 1992b). However, in this instance the higher efficacy of the administration of the drug with a mucoadhesive agent was due not to an increase in penetration but primarily to the inhibitory effect of the polymer on digestive proteolytic enzymes (Lue en et al., 1993). Additionally, the polymer may also have had some direct effect on the tightness of intercellular junctions (not published). In general, experience with the long-term fixation of controlled release systems to the intestinal mucosa using non-specific mucoadhesive polymers has been rather disappointing. There are several reasons for this. First, the adhesion of mucoadhesive polymers or hydrogels to mucus glycans is mediated by non-specific mechanisms, mainly interfacial energy effects or the formation of H-bonds. However, because the intestinal lumen contains not only mucosa-bound mucus, but also soluble or shed-off mucus and other glycans, the chances of the bioadhesive to adhere to the ‘right’ surface are small. In other words, mucoadhesive polymers are likely to become inactivated before they can make contact with the intestinal mucosa, even when the system is protected from premature inactivation in the stomach by enteric coating. Second, in contrast to other (e.g. buccal) mucosa, the intestinal mucosa is covered by a mucus gel layer of appreciable thickness. Although this may not be continuous, the adherent mucus layer is in a steady state that is governed by (1) the rate of the secretion of mucinous glycoproteins from the underlying tissue by specialised glands or cells, and (2) the speed of its mechanical and chemical erosion and degradation on the gut luminal surface (Figure 7.1). Because the mucus gel layer provides the ultimate link between a mucoadhesive system and the actual tissue surface, the maximal duration of potential mucoadhesion is limited by the turnover time of the mucus. The renewal time of the intestinal epithelial cells is of the order of about two days (Lipkin, 1987). In contrast, the turnover time of the mucus gel layer in isolated rat intestinal

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loops is much shorter, between 90 and 240 minutes (Lehr et al., 1991). As this time is about the same as normal small intestinal transit-time in man, mucoadhesive systems cannot be expected to cause a significant delay. Lectins as second-generation bioadhesives How can the problems associated with mucus possibly be overcome in the future? As mucus and other glycoconjugates are ubiquitous in the gastrointestinal tract, a different and more specific approach to bioadhesion will be necessary. These novel bioadhesives will have to bind directly to the cell surface rather than to mucus. Such highly selective binding or ‘targeting’ within the lumen of the gastrointestinal tract may probably only be feasible by involving specific lock-key mechanisms. Most, if not all, known examples for specific binding/bioadhesion in the gastrointestinal tract rely on lectin-sugar interactions. According to the definition of Goldstein et al. (1980), lectins are proteins or glycoproteins of nonimmunological origin which bind to particular sugar structures specifically and with relatively high affinities, similar to those of monoclonal antibodies. However, in contrast to antibodies, lectins are generally stable in the gastrointestinal tract. Their ability to recognize and specifically bind to cellular structures has made lectins valuable tools in histology. It is possible that these same properties may also be exploited for drug delivery purposes in man. While most of the known lectins are of plant origin, lectins are also produced by higher animals (so called ‘endogenous’ or ‘reverse’ lectins), including man, and they may play an important role in the interaction between bacteria or viruses and the host. General bio-medical aspects of exogenous, dietary lectins are discussed in the other chapters of this volume. At present, the investigation of lectin-induced bioadhesion in drug delivery systems is still in its infancy. Once their binding to polarized epithelial cells and other biological properties are sufficiently understood, however, the potential of lectin-targeting for drug delivery purposes may be enormous. This is especially true because the possible applications of specific bioadhesion—as we shall see—are not restricted to the plain fixation of drug delivery systems within the gastrointestinal lumen, but may eventually also be used to induce active trans-cellular transport of the macromolecular drugs or carriers. Lectin-mediated adhesion to the gastrointestinal mucosa By virtue of their specific binding to intestinal mucosal cells and their generally good resistance to digestion within the gastrointestinal tract (Pusztai, 1991; Pusztai et al., 1991), plant lectins would appear to be very attractive carriers for oral drug delivery, were it not for the fact that some plant lectins show acute toxicity or other undesired biological effects. A particularly well studied plant lectin is the Phaseolus vulgaris haemagglutinin (PHA) which has been recognized as the toxic factor of raw bean diet for animals and man, causing severe enteritis and diarrhoea at high dietary levels (Noah et al., 1980; Pusztai and Palmer, 1977). In studies with animals and cell culture systems (Hendriks et al., 1991; King et al., 1980a, 1980b, 1982; Koninkx et al., 1992), high concentrations of PHA and some other plant lectins were found to bind to and disrupt the luminal surface of the small intestine, thereby inducing various morphological and metabolic changes in the enterocytes. It is possible that such effects, though not necessarily fatal, may be prohibitive for the possible use of these compounds as drug delivery adjuvants.

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Tomato lectin For safety reasons, tomato (Lycopersicon esculentum) lectin (TL) appeared to be a particularly interesting candidate. This glycoprotein (Mr of about 70 kDa) was reported by Kilpatrick et al. (1985) to resist digestion, and to bind to rat intestinal villi without any obvious deleterious effects. In view of the widespread consumption of raw tomatoes TL is likely to be safe. The average ingestion of TL has been estimated from the tomato consumption in the USA to range between 100 and 200 mg a year although the exposure of some individuals might be considerably higher (Nachbar et al., 1981). The potential of TL as a drug delivery agent was first shown by Woodley and Naisbett (1988) who reported that in vitro this lectin was bound to everted rat intestinal rings and that this could be specifically inhibited by tetra-(Nacetylglucosamine), (GluNAc)4. However, after intragastric administration to rats, the gastrointestinal transit of the radiolabelled lectin was only slightly different from that of labelled poly(vinylpyrrolidone) or bovine serum albumin which served as controls. In quantitative binding studies of radiolabelled TL with isolated, fixed pig enterocytes or living monolayers of human Caco2-cells (Lehr et al., 1992a), the affinity constant for TL was found to be in the order of 10–6 M for both test models. Fluorescently-labelled microspheres (1 µ m) coated with TL avidly adhered to isolated fixed pig enterocytes in vitro (Figure 7.2a), while the albumin-coated control spheres did not (Figure 7.2b). Moreover, binding appeared to be specific, i.e. could be inhibited by the haptenic receptor oligosaccharide (GluNAc)4. It was also inhibited by relatively small amounts of crude pig gastric mucin. Binding of TL to pig enterocytes was strongest at neutral pH and decreased in acidic milieu. In comparison with mucoadhesive polymers like polycarbophil, this is certainly an advantage when the TL-drug complexes are targeted for the intestine rather than for the stomach. It is also possible that the poor gastric retention of TL observed by Woodley and Naisbett (1988) was due to its lack of binding to tissues at acidic pH values. A major drawback in the use of TL became apparent from in vitro binding studies which showed that the lectin had considerable cross-reactivity with crude mucus. Binding of radiolabelled TL to pig enterocytes was inhibited by crude pig gastric mucin (Sigma, St. Louis) at a IC50% of 2.2 mg ml−1, and adhesion of lectin coated microspheres to the same cells was markedly reduced at a mucin concentration as low as 0.2 mg ml−1. In an in vivo experiment microspheres labelled with different fluorescent dyes and coated with either TL or bovine serum albumin (BSA) were administered to the duodenum of rats (four rats approximately weighing 300 g) to study the transit-time of the lectin-coated in comparison with the control spheres. The rats were killed four hours after administration and the number of microspheres in segments of the small intestine was quantified by fluorescence-activated cell sorting (Ebel, 1990). Although the TL- and BSAcoated microspheres showed clearly different levels of bioadhesion when incubated with isolated enterocytes in vitro, the small intestinal transit-time in the four rats in vivo was the same for all particles (Figure 7.3). In view of the strong cross-reactivity of TL with mucus glycoproteins observed in vitro, it is likely that bioadhesion of the lectin-coated microspheres was inhibited by soluble mucins or shed-off mucus clots ubiquitously present in the gut lumen. In spite of its favourable safety profile, the mucus crossreactivity of TL appears to limit the application of this particular lectin as a gastrointestinal bioadhesive. Lectins specific for carbohydrates others than N-acetylglucosamine Despite their potential antinutrient effects at high dietary concentrations, lectins specific for galactose/Nacetylgalactosamine or the agglutinin from kidney bean, PHA, are useful reagents for exploring the bioadhesive properties of lectin-microsphere conjugates in the rat gastrointestinal tract. Many studies have shown (for references see Pusztai, 1991) that because of the high expression of complex glycans on mature

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Figure 7.2. Adhesion of polystyrene microspheres to pig enterocytes (a) Fluorescently-labelled, TL-coated microspheres (1 µ m) adhering to isolated fixed pig enterocytes resist washing. Insert shows a single enterocyte at higher magnification. (b) Albumin-coated control spheres showing no bioadhesion.

and fully differentiated villus cells these lectins, and particularly PHA, interact extensively with and bind avidly to the gut brush-border epithelium. Indeed, to probe into the possible use of lectins for drug targeting, PHA is unsurpassed as a model lectin.

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Figure 7.3. Effect of TL on the intestinal transit of microspheres. Yellow and red fluorescent microspheres (1 µ m, Polysciences) were coated with TL (black bars) or with BSA (grey bars), respectively. Four hours after mtraduodenal administration, the rats were killed, the intestines removed and cut in segments. After dissolution by treatment with 5N KOH, the microspheres in each segment were counted by fluorescent-activated cell-sorting (Ebel, 1990). Although intestinal transit showed much inter-individual variation, no difference was found in the distribution of BSA- and TLcoated microspheres in each of the four animals.

A systematic study has been carried out to find the most effective method for the chemical coupling of lectins such as PHA (and others) to model fluorescent latex microparticles (‘Fluoresbrite’, Polysciences UK Ltd) of various sizes. A major consideration in these studies was that the lectin had to retain its biological activity after the coupling and that the active lectin coating on the microparticles had to be high enough to confer good adhesive properties to the lectin-particle conjugates. It was shown by fluorescent microscopy in preliminary in vitro testing that qualitatively all lectin-particle conjugates had high reactivity with the appropriate mono-specific anti-lectin antibodies. This indicated that the lectins were at least not denatured by the coupling reaction. Another, more quantitative and specific test was to react the fluorescent lectin-coated microparticles with appropriate affinity matrixes and to estimate the extent of binding by differential centrifugation or other methods. The specificity of the binding of the lectin-coated particle to the affinity matrix (such as Sepharose-4B linked to appropriate carbohydrates) was established by testing the reversibility of the attachment with appropriate carbohydrate haptens and/or change in pH. The most important method which was used to establish the adhesive activity and capacity of lectins in their respective particle conjugates was the testing of their attachment to rat brush-border membranes prepared by a modification of the method of Schmitz et al. (1973). Briefly, rat brush-border scrapings were homogenized in 2 mM Tris—50 mM mannitol buffer, pH 7.1 with an Ultraturrax homogenizer. The

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homogenate was adjusted to 0.01 M with 1 M CaCl2 and centrifuged at 950 g for 10 min after standing for 60 min in the cold. The supernatant was centrifuged again at 33500 g for 30 min at 0°C. The pellet was suspended by stirring in an equal volume of freshly prepared 1.6M Tris and the suspension placed on top of a 0.05 M MgCl2, glycerol gradient of 5.5 ml of 42% (v/v) and 1.5 ml of 37% (v/v) glycerol in tubes for the SW40 rotor of a Beckman ultracentrifuge. After centrifuging for 20 min at 4°C at 63000 g the brush-border membrane (bbm) preparation was collected from the interface, vortexed in water and kept frozen until required for adhesion measurements. After ultracentrifugation in three-step glycerol gradients (28, 36 and 42% v/v glycerol) of small aliquots of bbm suspensions mixed with different concentrations of PHA-coated microparticles (2µ m size; in the range of 100–1000 µ g particle/ tube), no unattached fluorescent PHA-microparticles remained in the 28% glycerol layer even with the highest concentration of particles used. Instead, several new fluorescent bands, intermediate in density between the PHA-microparticles and the original bbm preparation in the 42% (v/v) glycerol layer (Figure 7.4), were observed. This indicated that, as essentially all the lectin remained active in the conjugates, the method of coupling used for the preparation of the lectin-coated microparticles was optimal and highly effective for the retention of lectin activity. The attachment of the PHA-microparticle to bbm was shown to be fully reversible by further glycerol density centrifugation after the isolated bbmparticle complexes were dissociated at low pH values. The in vivo adhesiveness of lectin-microparticles was tested with rats. In a simple preliminary experiment it was shown that 2 h after the rats had been intragastricly intubated with uncoated virgin microparticles (size 2 µ m), about 80% of the fluorescence could be recovered from the distal small intestine. However, after 4 h the recovery was only 23% and an hour later, it was less than 1% of that administered initially. Essentially similar results were obtained with fluorescent microparticles coated with a non-lectin control protein, phaseolin or Glycoprotein II from kidney bean, whose recovery was also less than 1% after 5 h. In contrast, with PHA-conjugated latex particles over 10% of the initial dose was still in the distal small intestine 5 h after the intubation. In further experiments, the dependence of the retardation of lectin-coated microparticles in the small intestine on the carbohydrate-specificity of the lectins was systematically explored. Lectins of different specificities were coupled to fluorescent latex microparticles (size 2 µ m) and tested for small intestinal transit. The rats were intragastrically intubated with the different lectin-coated microparticles, killed 30, 60 or 120min after intubation, and their small intestine and caecum excised. The small intestine was subdivided into 18 pieces of 5 cm each, and these pieces and the caeca were digested with 20% (w/v) KOH at 50°C for 48 h. The fluorescent intensity of the recovered microparticles was measured and recoveries were plotted against the distance from the pylorus (Figure 7.5). As controls, microparticles coated with lactalbumin, human gamma-globulin, or virgin microparticles were used under identical conditions. Generally, control microparticles were reasonably tightly banded and found to be mainly in the small intestinal pieces of 15–18 at the distal end, with about 10–20% of the initial dose in the caecum 2 h after intubation. Microparticles coated with lectins that had specificities for mannose (GNA, Galanthus nivalis agglutinin) or mannose/glucose (con A, concanavalin A) behaved similarly to controls and were not retarded at all. Similarly, wheat germ agglutinin (WGA) with specificity for N-acetylglucosamine, was not retarded, thus confirming the behaviour of tomato lectin, which has a similar carbohydrate specificity. However, even with these lectins the banding was not as tight as with the control particles. In fact, variable amounts of the particles, up to 5–10% of the initial dose, were spread throughout the small intestine. With lectin-particle conjugates composed of lectins such as PHA or those specific for galactose/Nacetylgalactosamine (soya, etc), the spreading was far more extensive and considerable amounts of the particles, 20–30% of the initial dose, was distributed along the entire length of the small intestine as far back

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Figure 7.4. Interaction of PHA-coated latex particles (PHA-Flbr) with brush-border membrane preparations, bbm, in vitro. The fluorescent particles were mixed with a brush border membrane preparation (middle two tubes), layered on a three-step glycerol gradient of 28/36/42% (v/v) glycerol and centrifuged to equilibrium. As a result of binding of PHAFlbr to bbm of different densities, three fluorescent bands were obtained in intermediate positions between the top and the bottom of the glycerol gradient. In the two controls (without bbm, two tubes on either side) the position of PHAFlbr in the absence of bbm was established to be on top of the gradient, as expected.

as the duodenum. In addition, the passage of these particles formed definite wave patterns. Thus, although the main frontal band of the PHA-latex particles progressed to small intestinal pieces 13–16 after 2 h, there was also a definite second wave of the particles in sections 7–12, with a maximum at section 10. Moreover, even the front band was significantly retarded in comparison with controls. Similar findings were made with microparticle conjugates containing lectins with specificity for galactose/N-acetylgalactosamine. One of the main findings of these studies was that the small intestinal transit of lectin-coated microparticles was generally only slightly affected by the size of the particle in the size range of 0.05–2 µ m. Thus, results obtained with particles of 0.05, 0.1 or 2 µ m size were very similar when the experiments were carried out under identical conditions. In contrast, the dietary history of the rats had decisive effects on intestinal transit. The small intestinal transit of microparticles was perceptibly slower in rats which had been fed before intubation than in fasted rats. Even more importantly, the microparticles apparently travelled

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Figure 7.5. Progress of microparticles conjugated to different lectins through the small intestine. Rats, which had been fasted overnight, were intragastrically intubated with ‘Fluoresbrite’ (Flbr; 2 µ m) particles or particles conjugated to (a) PHA (PHA-Flbr) or (b) Aegopodium podagraria lectin (AEG-Flbr), killed 2 h later and their stomach, small intestine (cut into 18 pieces of 5 cm each) and caecum were excised and rinsed with saline. Fluorescent particles were recovered after treatment with 20% (w/v) KOH at 50°C for 48 h, their amounts were determined by reading on a spectrofluorimeter and expressed in arbitrary units of fluorescence per gut section, starting with the stomach (S), then small intestinal sections numbered 1 (proximal) to 18 (distal) and finally the caecum (C).

down the small intestine incorporated in the digesta without any significant differences between lectincoated or control microparticles. Clearly, the results of these preliminary studies have shown that it may be possible to exploit lectins of certain carbohydrate specificities for oral drug delivery and small intestinal targetting. However, a great

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deal of work needs to be done before their use in practice. Most of the experiments described in this section have been carried out with a single and arbitrarily selected dose of the lectin-particle conjugates in young (30-day-old) rats. The effects of varying the age of the rat or the dose of the particles have not been established yet. Neither has there been any testing of the lectin-particles carried out in other animal species including humans. It is also possible that as the surface lectin-density of the microparticles may be a decisive factor in retardation, systematic studies have to be carried out to change the coating concentration of lectins relative to the microparticles. Furthermore, as all this work has been done with a model microparticle (‘Fluoresbrite’), it is not certain whether the findings will have relevance to realistic and pharmacologically tested particles with drugs incorporated. Finally, there have been no attempts to establish whether, on chronic administration, the response of the gut-associated immune system to the lectin would render the complex useless by eliminating the lectin-particles. Thus, final judgement on the possible use of plant lectins in general as second-generation bioadhesives cannot be made yet. Reverse (endogenous) lectins Some bacteria, such as e.g. Shigella, adhere through fucose-, glucose- or mannose-sensitive interactions to the gut. However, the lectin responsible for this adhesion is produced by intestinal cells and not by the bacteria (Izhar et al., 1982; Ashkenazi 1986). As this fucose-binding glycoprotein is host-born, such carbohydrate-binding proteins should be called ‘endogenous’ or ‘reversed’ lectins. Taking advantage of this physiological feature of the intestines, Bridges et al. (1988) synthetized HPMA-copolymers with side chains terminating in the appropriate sugar residues, and found that these ligands had a greater affinity to the rat small intestine in vitro than neutral HPMA-copolymers without sugar moieties. The extent of tissue association increased in the order fucose > mannose > galactose, but the strongest tissue association was displayed by a cationic derivative containing quaternary ammonium groups, presumably mediated by nonspecific electrostatic interactions. In this study, it was unclear whether these polymers were adsorbed directly to the luminal surface of intestinal tissue or bound by the mucus gel layer. Following the same approach, Rathi et al. (1991) prepared HPMA copolymers with side-chains terminating in fucosylamine residues and showed that their adherence to colonic tissue of guinea pigs in vitro was approximately 3–4 times higher than to the small intestine. Indeed, such regional specific binding has a potential for the development of site-specific oral drug delivery systems, especially if the polymeric carriers are not susceptible to degradation by the endogenous enzymes of the host gut but, instead, are degraded by microbial enzymes (azoreductases) in the colon (Brønsted and Kopecek, 1992). Unfortunately, because these polymers bind mainly to the mucus layer rather than to the cell surface, their maximal adhesion time under physiological conditions is probably limited by the turnover rate of the mucus gel layer. According to Ríhová et al. (1992), the binding of radiolabelled HPMA copolymers containing fucosylamine to everted intestinal sacs decreased when the mucus gel layer was removed by treatment with EDTA and dithiothreitol. This may be an indication that although the binding may be caused by a lectin which is secreted together with the mucus, it is still mucoadhesion and not bioadhesion to the epithelial membrane. In the same way as discussed in the context of exogenous lectins, reversed lectins may have the most impact if they were an integral part of the apical membrane of epithelial cells. Bacterial and viral adhesion factors Most bacteria can display a number of adhesins, and at least nine such factors have at present been described for Escherichia coli (Hoepelman and Tuomanen, 1992). Adhesins from this species are also the

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first that have been investigated for drug delivery applications. Caston et al. (1990) isolated Type-1 fimbriae from E. coli and adsorbed the purified protein onto polystyrene microspheres. The presence of functional fimbriae at the surface of the model particulate system was confirmed by mannose-sensitive haemagglutination of guinea pig red blood cells. After intraduodenal administration of radiolabelled microspheres to anaesthetized rats in vivo, the authors reported an increase in the total amount of radioactivity associated with the intestines, from 16.2% to 46.9%, due to the presence of a fimbrial coating. Furthermore, 2 h after administration, the position within the intestines where most radioactivity could be detected in the sections was more proximal with the fimbrial microspheres than with the controls. The reproducibility of this effect, however, particularly after regular oral administration, and the effects of interactions with food, remain to be seen. In comparison with the effects observed with mucoadhesive polymers or tomato lectin, these preliminary data look promising. However, in view of the lack of significant retardation in the small intestine of microspheres coated with mannose-specific plant lectins, it is possible that the increase in the transit-time of fimbrial microspheres is not due to lectin-mediated adhesion. Receptor-mediated attachment systems to colonize and/or invade the host organism are used not only by bacteria, but also by viruses. For instance, Rhinovirus, the mediator of the common cold, binds to the cell adhesion molecule ICAM-1 on the surface of eukaryotic cells, and thereby down-regulates the ability of leucocytes to eliminate virus-infected cells (Greve et al., 1989). Soluble receptor-analogues are therefore under investigation to inhibit viral attachment and infection (Bangham and McMichael, 1990; Marlin et al., 1990). Possibly, such ‘anti-adhesives’ may hence be used as an alternative to vaccination in the future. Vice versa, Rubas et al, (1990) coated liposomes with the reovirus l cell attachment protein, for which binding sites exist on M-cells. Competition studies with reovirus on the mouse L929 cell monolayer demonstrated specific binding, and incubation studies in vitro revealed a tenfold increase in the accumulation of coated liposomes compared with uncoated liposomes in Peyer’s patches. More recently, Ambler and Mackay (1991) demonstrated the in vitro binding and internalization of reovirus 1 and 3 by the apical surface of intestinal cells, using both filter-grown Caco-2 cell monolayers and apical membrane vesicles prepared from rat enterocytes. Specific endo- and trans-cytosis of bioadhesive ligands—A new perspective for the development of macromolecular drug carriers As the above example of viral adhesion factors shows, specific binding to epithelial cells is not necessarily restricted to the plain fixation of a bioadhesive ligand to the outer cell surface, but may also subsequently lead to the internalization of the ligand by the cell. Invagination of the cell membrane, allowing the uptake of tiny droplets of the extracellular liquid (‘fluid-phase endocytosis’ or ‘pinocytosis’) is common to most, if not all types of cells. Usually, this process occurs at a constitutive level, i.e. it occurs continuously and is independent of trigger signals transmitted by ligand binding to the cell (Simionescu et al., 1987). Unfortunately, as the volume of extracellular medium internalized by this process is very small, the socalled ‘endocytic index’ is also small and is in the range of 0.4–2.1 µ l 10–6 cells for various cell types (Williams et al., 1975, Pratten et al., 1980). Thus, the efficacy of this uptake process for macromolecular solutes of the extracellular medium is very small. However, if a particular substrate can be accumulated at the cell surface by means of bioadhesion, the rate and extent at which such membrane bound ligands are internalized along with the normal membrane invagination process may be dramatically increased. Moreover, specific binding to some membrane-bound receptors may additionally transmit trigger signals to the cell, resulting in an increased vesiculation rate and/or a specific intracellular routing/ processing of the internalized material. Various strategies to use endo- and transcytosis for the improved delivery of

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macromolecular drugs have recently been reviewed by Shen et al. (1992) and by Lehr (1993). In this chapter, the discussion of this topic will be restricted to lectins. Receptor-mediated versus adsorptive endo-/trans-cytosis Many physiologically and pathologically relevant macromolecules (e.g. lipoproteins, vitamin B12, transferrin, immunoglobulins, ricin, etc.) are transported into and across mammalian cells by a highly specific membrane invagination processes, called receptor-mediated endocytosis. This important cell biological concept (Goldstein et al., 1985) was formulated first by Goldstein and Brown (1974) for the binding and uptake of plasma low density lipoprotein, and was honoured with the Nobel Prize in 1985. While some studies indicate that these highly specialized transport pathways may also be shuttled along with advanced drug carrier systems, enabling them to cross the gastrointestinal epithelium (Russel-Jones and de Aizpurua, 1988) or the blood-brain barrier (Bickel et al., 1993), it is not a priori clear that this approach may be appropriate for drug delivery purposes. First, the population of high specific affinity receptors (Kd<10–9 M) is relatively small which keeps the total transport capacity of this pathway slight. Second, as the same pathways are already used within the organism for its supply of essential compounds, their additional exploitation to deliver drugs may lead to mutual competition and inhibition. It is possible, therefore, that the binding and subsequent endocytosis of extracellular ligands may be better achieved at a lower level of affinity and specificity, such as by electrostatic (charge-mediated) or sugar-specific (lectin-mediated) interactions. The affinity constants of these interactions are usually in the micromolar rather than in the nanomolar range (Feller et al., 1979, Lehr et al., 1992a), but the number of binding sites available for these interactions is about thousand times higher than for high-affinity receptors. In spite of the relatively low specific affinity, binding to a large number of low-affinity receptors occurs rapidly and is of relatively high strength. To describe such behaviour, the term ‘avidity’ has been suggested (Simmons, 1993). Intermediate (micromolar) affinity but high avidity is typical for lectins and other cell adhesion molecules, whose binding is strong enough to induce rolling or adhesion of lymphocytes on inflamed blood vessel walls (Ley et al., 1991). Lectin-mediated transcytosis Under the difficult conditions of the gastrointestinal tract, charge-mediated binding may not allow the particles to differentiate between cells and other surfaces, such as mucus or food, but sugar-specific (i.e. lectinmediated) interactions may provide a successful outcome. Lectin-mediated transcytosis through the bloodbrain barrier of horse radish peroxidase coupled to WGA, which binds to sialic acid and Nacetylglucosamine residues, has already been successfully demonstrated (Broadwell et al., 1988). The feasibility of this approach for the gastrointestinal epithelium still remains to be demonstrated, though interesting results have been reported for some dietary plant lectins. After intragastric administration of 50 mg kidney bean extract containing 1 µ Ci 125I-labelled PHA to fasted rats (average weight of 100 g), up to 0. 8% of the dose in terms of TCA-precipitable radioactivity was detected in 1 ml blood (Pusztai et al., 1989). From these data it has been estimated that the amount of PHA which crossed the intestinal barrier and was systemically absorbed was 5–10% of the initial dose (Pusztai, 1988). Taking into account the large molecular mass of this glycoprotein (more than 100 kDa), these data would mean a surprisingly high oral bioavailability. Interestingly, under the same experimental conditions in vivo, tomato lectin was barely detectable in blood (less than 0.01%; Pusztai et al., 1989). While these results were obtained after intragastric administration of the lectins to living animals, Naisbett and Woodley (1989) studied the binding

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and transport of radiolabelled tomato lectin with everted rat intestinal sacs in vitro. With this model they found a concentration-dependent uptake of the lectin by the tissue and its transport to the serosal space, which is indicative for lectin-mediated, adsorptive endo-and trans-cytosis. In comparison, a non-adhesive control macromolecule (poly(vinylpyrrolidone)) did not show such saturation, and both its binding and transport were one order of magnitude less. For a direct comparison of the binding and transport of various lectins across epithelial cells under controlled conditions in vitro, experiments were performed with filtergrown Caco-2 cell monolayers (Lehr and Lee, 1993). In comparison with non-adhesive BSA which served as control, tomato lectin (TL) and the two PHA-isolectins L4 and E4 showed appreciable bioadhesion. The affinities of these lectins could be ranked in the order of TL=PHA-L4
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Figure 7.6. Binding/uptake of radiolabelled lectins by Caco-2 cell monolayers (Lehr and Lee, 1993) Bars represent mean ±s.d. (n=6–9). Black bars=reference (43 nM, 37°C), hatched bars=10-fold excess of unlabelled lectin at 37°C, open bars=43 nM, 5°C. Each macromolecule was studied in separate experiments, all treatments were in triplicate using up to 18 monolayers (3 plates) at a time from the same cell passage. Panel (A) shows incubation with the apical side; panel (B) shows basolateral side. Significance is indicated by * and ** at p <0.1 or p <0.05, respectively (non-parametric U-test). For basolateral TL, inhibition by a 10-fold excess of cold lectin failed to reach statistical significance due to large passage-to-passage variations, but within the same experiment the inhibition was always significant.

which specifically binds to a particular class of cell surface receptors (integrins) of mammalian cells. These are functionally connected to the cytoskeleton (Falkow, 1991). As previously reported, latex beads, after coating with purified invasin derivatives, were efficiently internalized by cultured mammalian cells which do not normally show phagocytic activity (Isberg, 1991). Thus, these experiments demonstrate the potential to enhance trans-cellular transport processes by choosing the right bioadhesive ligands. The balance

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Figure 7.7. Transport of radiolabelled lectins across Caco-2 cell monolayers (Lehr and Lee, 1993). Panel (A) shows apical-to-basolateral (A B); panel (B) basolateral-to-apical (B A) direction. For legend see Figure 7.6.

between binding to remote/secreted or strictly membranebound receptors may, as it is for such microorganisms, determine whether or not binding is followed by endocytosis (Karlsson et al., 1991). Concluding remarks Despite the many scientific problems and technical developments which still need to be solved, lectintargeted oral drug delivery is no longer just an interesting idea. Although it is not yet clear which particular type of agglutinins would be the most useful, whether plant-or endogenous-lectins, bacterial fimbrial or

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Figure 7.8. Glycocalyx of an epithelial cell (according to Strous and Dekker, 1992, and Karlsson et al., 1991, modified). The glycocalyx of epithelial cells consists of several functional layers (1) glycolipids, which are immersed in the cell membrane, (2) enzymes and receptors at 10–15 nm distance from the surface, and (3) long-chain mucus glycoproteins, extending up to 150 nm into the intestinal lumen. The extent to which lectins are internalized after binding to extracellular oligosaccharide-moieties depends on the distance of the lectin receptor from the vesicle forming membrane.

surface adhesins, viral cell-attachment proteins, tunnel-forming toxin subunits or invasins should be used for maximum effect, it is certain that because of their high bioadhesive affinity for receptors embedded in the epithelial membrane, lectins appear to be the most promising candidates for drug targeting in the gut. The high avidity of their binding to the gut epithelium ensures that it may be adequate to coat the particle surface with low concentrations of lectins and still obtain good bioadhesive capacity for the microparticles. Accordingly, it may even be possible to use lectins for particle conjugation which are generally regarded as nutritionally toxic at high dietary intakes. Furthermore, as the carbohydrate structure of functional receptors varies along the gastrointestinal tract, the differential carbohydrate reactivity of lectins makes them ideally suited to deliver drugs to different parts and sites in the gut. In addition, lectins are stable reagents and there are already several methods for coupling them to microparticles without compromising their stability or bioadhesive activity. There are also indications that the gut-associated immune system cannot inactivate lectins such as PHA even on chronic administration (Pusztai, 1991). Accordingly as a minimum expectation, delayed transit targeting by lectins may help to increase the efficacy of drug absorption in the gastrointestinal compartment of our choice. With receptor-mediated, lectin-targeted endo-/transcytosis we may also achieve the long-sought after objective of delivering valuable and sensitive polypeptide or other drugs into the systemic circulation by the oral route.

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Harris, D. and Robinson, J.R., 1990, Bioadhesive polymers in peptide drug delivery, Biomaterials, 11, 652–58. Harris, D., Fell, J.T., Sharma, H.L. and Taylor, D.C., 1990, GI transit of potential bioadhesive formulations in man: a scintigraphic study, Journal of Controlled Release, 12, 45–54. Hendriks, H.G.C.J.M, Kik, M.J.L., Koninkx, J.F.J.G., van den Ingh, T.S.G.A.M. and Mouwen, J.M.V.M., 1991, Binding of kidney bean (Phasaeolus vulgaris) isolectins to differentiated human colon carcinoma Caco-2 cells and their effect on cellular metabolism, Gut, 32, 196–201. Hoepelman, A.l.M. and Tuomanen, E.I., 1992, Consequences of microbial attachment: Directing host cell functions with adhesin, Infection and Immununity, 60, 1729–33. Isberg R.R., 1991, Discrimination between intracellular uptake and surface adhesion of bacterial pathogens, Science, 252, 934–38. Isberg, R.R. and Leong, J.M., 1988, Cultured mammalian cells attach to the invasin protein of Yersinia pseudotuberculsis, Proceedings of the National Academy of Sciences of the United States of America, 85, 6682–86. Izhar, M., Nuchamowitz, Y. and Merelman, D., 1982, Adherence of Shigella flexneri to guinea pig intestinal cells is mediated by mucosal adhesin, Infection and Immununity, 35, 1110–18. Junginger, H.E., 1990, Bioadhesive polymer systems for peptide delivery, Acta Pharmaceutica Technologica, 36, 110 26. Karlsson, K.A., Angström, J. and Teneberg, S., 1991, Characteristics of the recognition of host cell carbohydrates by viruses and bacteria, in Wadtström, T., Mäkelä, P.H., Svennerholm, A.M. and Wolf-Watz, H. (Eds.) Molecular Pathogenesis of Gastrointestinal Infections, pp. 9–22, New York: Plenum Press. Kilpatrick, D.C., Pusztai, A., Grant, G., Graham, C. and Ewen, S.W.B., 1985, Tomato lectin resists digestion in the mammalian alimentary canal and binds to intestinal villi without deleterious effects, FEBS letters, 185, 299–305. King, T.P., Pusztai, A. and Clarke, E.M.W., 1980a, Immunocytochemical localization of ingested kidney bean (Phaseolus vulgaris) lectins in rat gut, Histochemical Journal, 12, 201–8. King, T.P., Pusztai, A. and Clarke, E.M.W., 1980b, Kidney bean (Phaseolus vulgaris) induced lesions in rat small intestine: light microscope studies, Journal of Comparative Pathology, 90, 585–95. King, T.P., Pusztai, A. and Clarke, E.M.W., 1982, Kidney bean (Phaseolus vulgaris) induced lesions in rat small intestine. 3. Ultrastructural studies, Journal of Comparative Pathology, 42, 357–73. Koninkx, J.F.J.G., Hendriks, H.G.C.J.M, van Rossum, J.M.A., van den Ingh, T.S.G.A.M. and Mouwen J.M.V.M., 1992, Interaction of legume lectins with the cellular metabolism of differentiated Caco-2 cells, Gastroenterology, 102, 1516–23. Koshla, R. and Davis, S.S., 1987, The effect of polycarbophil on the gastric emptying of pellets, Journal of Pharmacy and Pharmacology, 39, 47–49. Lee, V.H.L., 1991, Changing needs in drug delivery in the era of peptide and protein drugs, in Peptide and Protein Drug Delivery, Lee, V.H.L. (Ed.), pp 1–56, New York: Marcel Dekker. Lehr, C.M., 1993, The transcytosis approach, in de Boer, A.G. (Ed.) Drug Absorption Enhancement, Chur (Switzerland) Harwood Academic Publishers, pp. 325–65. Lehr, C.M. and Lee, V.H.L., 1993, Binding and transport of some bioadhesive plant lectins in the Caco-2 cell model, Pharmaceutical Research, 10, 1796–99. Lehr, C.M., Poelma, F.G.J., Junginger, H.E. and Tukker, J.J., 1991, An estimate of turn-over time of intestinal mucus gel layer in the rat in situ loop, International Journal of Pharmaceutics, 70, 235–40. Lehr, C.-M., Bouwstra, J.A., Kok, W., Noach, A.B.J., de Boer, A.G. and Junginger, H.E., 1992a, Bioadhesion by means of specific binding of tomato lectin, Research, 9, 547–53. Lehr, C.M., Bouwstra, J.A., Kok, W., de Boer, A.G., Tukker, J.J., Verhoef, J.C., Breimer, D.D. and Junginger, H.E., 1992b, Effects of the mucoadhesive polymer polycarbophil on the intestinal absorption of a peptide drug, Journal of Pharmacy and Pharmacology, 44, 402–7. Ley, K., Gaethgens, P., Fennie, C., Singer, M.S., Lasky, L.A., Rosen, S.D., 1991, Lectin-like cell adhesion molecule 1 mediates leukocyte rolling in mesenteric venules in vivo, Blood, 77, 2553–55.

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Chapter 8 Lectins Binding to the Gut Wall are Growth Factors for the Pancreas: Nutritional Implications for Transgenic Plants A.Pusztai, G.Grant, D.S.Brown, S.Bardocz, S.W.B.Ewen, K.Baintner, W.J.Peumans and E.J.M.Van Damme

Introduction Dietary kidney bean (Phaseolus vulgaris) lectin, PHA, is a powerful extraneous growth signal for the entire digestive tract (Pusztai et al., 1988; de Oliveira et al., 1988). It is particularly effective in inducing fully reversible, polyamine-dependent, hyperplastic and hypertrophic growth of the small intestine. However, other lectins which bind to the brush-border epithelium can also induce the growth and stimulate polyamine accretion in small intestinal tissue (Pusztai et al., 1990). PHA also stimulates the hypertrophic growth of the pancreas (Bardocz et al., 1989; de Oliveira et al., 1988; Grant et al., 1990). Similar to that occurring in the small intestine, this growth is accompanied by the accumulation of polyamines in the acinar pancreas, most of which are taken up from the blood circulation without a significant increase in the de novo putrescine biosynthesis by ornithine decarboxylase (ODC) in the tissue. In fact, the uptake of polyamines, mainly spermidine, precedes the actual growth of the pancreas (Bardocz et al., 1989). It is now clear that not only PHA but also a number of other lectins can induce the enlargement of the pancreas and the accumulation of polyamines in it (Pusztai et al., 1990). Thus, although the growth of the pancreas in rats fed on soya bean diets has traditionally been ascribed to the trypsin inhibitor content of soya meal (STI), the seed agglutinin (SBA) is now known to be a more effective growth stimulant than the inhibitors. Although the enlargement induced by either of the two types of soya factor coincides with polyamine accumulation, the mechanism of the stimulation by STI and SBA may not necessarily be the same. Stimulation of the pancreas by dietary trypsin inhibitors is generally thought to be mediated by cholecystokinin (CCK) through a negative feedback mechanism due to reduced trypsin (and other serine protease) concentration in the lumen of the small intestine (Green and Lyman, 1972). In contrast, SBA has no effect on trypsin levels in the gut, therefore, growth stimulation by lectins is not likely to occur through a similar negative feedback mechanism. However, it is possible that following the binding of SBA to the small intestinal epithelium a growth signal is transmitted to the acinar cells of the pancreas by an unknown mechanism and possibly mediated by hormones (CCK) or other growth factors. As polyamines play an important role as second messengers in the signal transduction process, their accretion is obligatory in eukaryotic cells after stimulation by growth factors (Pegg, 1986). Thus, possible differences in the metabolism and accumulation of polyamines during pancreatic enlargement induced by trypsin inhibitors or SBA may show how the pathways of signalling by the two different growth factors differ. Therefore in addition to possible differences in the morphology of the pancreas and its secretion of digestive enzymes, such as -amylase, into the gut lumen after stimulation with similar amounts of various lectins or STI for 10

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days, exploration of changes in polyamine- and ODC levels may give an insight into the biochemical and physiological mechanisms of the lectin-induced pancreatic growth. The possibility of imminent release of transgenic crop plants whose increased insect resistance is due to transfecting them with lectin- and/or trypsin inhibitor genes (see Gatehouse et al., Chapter 3) gives an added urgency to establish the precise mechanism(s) of the antinutritional effects of these factors not only on insects but also, and even more importantly, on mammals who will be the ultimate consumers of these plants. It is now generally accepted that genes that are known to code for highly toxic factors are not suitable for transgenic technology, although they may be very potent insecticides. However, it is not always realised that factors which are not overtly toxic, particularly when expressed at low concentrations in their natural habitat, may become unacceptable health hazards at the highly increased levels that may be needed to improve the insect resistance of transgenic plants in practice. Therefore, diets based on even seemingly innocuous transgenic plants require rigorous testing by appropriate nutritional techniques. This is particularly important because the deleterious effects of some of these factors may be cumulative and are revealed only on extended exposure. Continuous overstimulation of the pancreas is one of the less generally appreciated potential health hazards associated with the consumption of transgenic plant-based foods expressing high levels of lectin and/or trypsin inhibitor genes. This is so despite the recent demonstration that two of the most effective antiinsect gene products, the cowpea trypsin inhibitor (CpTI; Pusztai et al., 1992a) and wheat germ agglutinin (WGA; Pusztai et al., 1993a), respectively, are both potent growth factors for the pancreas. Accordingly, the use of their genes in transgenic crop plants needs to be viewed with considerable caution. It is clear, therefore, that to make use of the high insecticide potential of these and other similar factors further research is needed. One obvious possibility is to find lectins and/or other agents with proven insecticidal activity, which, by nutritional tests, are shown to have no deleterious effects on mammalian species (rats, mice, pigs, etc), and to use their genes for transfer. Unfortunately, as already discussed in this book (Peumans and Van Damme, Chapter 1), because the nutritional evaluation and other physiological tests with animals are timeconsuming and cannot keep pace with work on insect resistance and transgenic research, progress is likely to be slow. However, with a thorough understanding of the mechanism of the lectin-induced growth of the pancreas (and the small intestine; see Bardocz et al., Chapter 6) it may be possible to speed up this process and find physiologically sound strategies of intervention to neutralize the deleterious effects on mammals of transgenic plants expressing high levels of insecticidal proteins without jeopardizing their effectiveness against insects. Thus, in this Chapter the physiological effects of lectins on the pancreas are reviewed and discussed in the light of these considerations. Physiological effects of lectins on the pancreas Lectins with potential insecticidal properties Nutritional testing of rats has been performed with several lectins which may have applications in transgenic technology (see Gatehouse et al., Chapter 3; Peumans and Van Damme, Chapter 1). In most of these tests, PHA, which is specific for complex carbohydrates, has been included as a known positive standard (Pusztai, 1991) because it avidly binds to the brush-border epithelium and is a potent growth factor for both the small intestine and the pancreas (Pusztai et al., 1990). To see whether other lectins specific for complex carbohydrates but different from PHA, were also stimulants of the growth of the small intestine and pancreas, a lectin obtained from tulip bulbs (Cammue et al., 1986) was tested but found to be almost totally inactive. In contrast, most lectins with specificity for N-acetylgalactosamine/galactose (GalNAc/

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Gal), such as SBA purified from de-fatted soya bean on cross-linked guar gum (Pusztai et al., 1991a), Sambucus nigra agglutinin-II (SNA-II) from the bark of elderberry and Robinia pseudoacacia agglutinin (RPA) from tree bark (Peumans et al., 1986) showed strong binding to the small intestinal brush-border and stimulated its growth (Pusztai et al., 1992b). This may rule them out in practice as insecticidal agents. The binding to the gut wall of the different N-acetylglucosamine-specific (GlucNAc) lectins, such as WGA (Peumans et al., 1982), Datura stramonium lectin (DSA; Broekaert et al., 1987) and nettle agglutinin from Urtica dioica rhizomes (UDA; Peumans et al., 1984) was variable, with WGA being the most reactive. Particular attention was given to the animal testing of these (Pusztai et al., 1993a) because GlucNAcspecific lectins have been shown to be particularly effective against a number of economically important plant pests. Similarly, a great deal of research effort has gone into the nutritional evaluation of the group of mannose-specific lectins with high insecticidal activity, such as those from Galanthus nivalis (GNA), Narcissus pseudonarcissus and amaryllis, Hippeastrum hybr. (Van Damme et al., 1987) of the family of Amaryllidaceae and a related mannose-specific lectin from garlic, which all appeared to have only slight binding activity to the gut wall. Furthermore, two sialic acid-binding lectins, MAA, purified from Maackia amurensis bark (Peumans and Van Damme, unpublished) with specificity for neuraminyl- 2,3-galactose and SNA-I, from Sambucus nigra bark (Shibuya et al., 1987), with specificity for neuraminyl- 2,6galactose which may be useful in transgenic technology, were shown to have only slight reactivity with the brush-border epithelium. Accordingly, they are not expected to have major antinutritional effects on mammals. A lectin, so far uncharacterized, isolated from Iris bulbs by chromatography on fetuinSepharose-4B, was also tested but found to be inactive. As positive controls for the stimulation of pancreatic growth, trypsin inhibitors isolated from de-fatted soya seeds (Pusztai et al., 1991a) were also included. This was particularly opportune because the genes of trypsin inhibitors, such as that from cowpeas, have in the past been used with success in plants to improve their resistance against bruchid beetles and other pests. Therefore their nutritional testing with rats or other suitable animals was also of practical necessity. Further-more, because none of the lectins had significant protease inhibitor activity even when tested at ten-fold molecular excess over trypsin, comparing the in vivo effects of lectins and protease (and other enzyme) inhibitors was expected to be particularly revealing. Lectin-induced gut growth As shown previously (Pusztai et al., 1990; 1992b). binding to the epithelium is obligatory for a lectin to be able to induce the growth of and to stimulate the accumulation of polyamines in the small intestine. For example, PHA was not only one of the most potent lectins for inducing the growth and increasing the number of cells and length of crypts of the jejunum of rats, but it was also one of the most effective lectins for inducing polyamine accumulation in this tissue. With lectins which were bound less avidly, the growth of the gut was less prominent. These lectins were also only weak stimulants of polyamine uptake by the mucosa (see Bardocz et al., Chapter 6). As trypsin inhibitors were not bound by the small intestinal mucosa at all (Wilson et al., 1978) they had no significant effects on the growth or crypt length of the small intestine. Lectin-induced growth of the pancreas All lectins which stimulated the growth of the small intestine also induced the enlargement of the pancreas (Table 8.1) and significantly increased its acinar area (Figure 8.1). Generally, lectins which were potent growth factors for the small intestine, such as PHA or RPA, were also highly effective for the pancreas.

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Mannose-specific lectins from Amaryllidaceae, which had only slight effects on the gut, had similarly little effect on the pancreas. However, the relationship between the growth stimulatory effects for the gut and pancreas of lectins was not always clear-cut (Pusztai et al., 1992b). The pancreas enlargement caused by all three Table 8.1 Plant lectin-lnduced changes in the weight, acinar area and polyamine content of the rat pancreas measured after 10 days on 42mg/rat/day pure lectin incorporated into lactalbumin diet. Percentage of lactalbumin control Lectin

Weight

Acinar area

Polyamines

PHA 145 156 140 RPA (Robinia) 143 nd 126 WGA (wheat germ) 118 nd 114 SBA (soya bean) 132 145 153 UDA (nettle) 98 nd 94 DSA (Datura) 111 nd 101 SNA-II (elder) 136 150 140 SNA-I (elder) 132 132 133 MAA (Maackia) 132 132 106 STI (trypsin inhibitor) 135 151 160 Values are expressed relative to those of the lactalbumin control, which is taken as 100 per cent. Lactalbumin control values are: weight of pancreas, 2780±141 mg; acinar area, 118±19µm2; polyamines, 894±76 nmol. Values are mean±SD for 4 rats per treatment group. nd; not determined.

lectins specific for N-acetylglucosamine, WGA, DSA or UDA, was less than that expected from the small intestinal growth which they induced. In contrast, MAA and SNA-I were more effective growth factors for the pancreas than for the small intestine. The polyamine content of the enlarged pancreas in most instances was approximately correlated with the increase of its weight and acinar area. Thus, the PHA-induced growth of the pancreas and its accretion of polyamines were extensive, whereas with the moderately potent GlucNAc-specific lectins, polyamine accumulation was also modest. In contrast, the increase in polyamine levels of the SBA-treated pancreas was high in comparison with the enlargement and, for reasons which are not understood at present, MAA stimulated the pancreas but without a significant increase in its polyamine content. Comparison of the effects of lectins and trypsin inhibitors on the pancreas The effect of diets containing pure trypsin inhibitors on the pancreas was, as expected, appreciable. STI not only induced substantial growth of the pancreas, but it also dramatically increased the polyamine content of the tissue, so much so that, although the enlargement caused by STI was definitely less extensive than that induced by some of the more potent lectins, particularly when expressed on molecular weight basis, the amounts of polyamines in the tissue rose well above that observed with any of the lectins. The reasons for this are not entirely clear at present. In addition to differences in the polyamine content of the pancreas after stimulation with lectins and trypsin inhibitors, secretion of digestive enzymes into the gut lumen was also significantly different. Thus, a single intragastric dose of SBA failed to increase the total -amylase activity in the gut (13.0 units/small intestine)

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Figure 8.1. Pancreatic acini of rats after feeding (a) lactalbumin-or (b) PHA-diets for 10 days. Sections of the pancreas were fixed with 4 per cent buffered (pH 7) paraformaldehyde, embedded in paraffin wax, sectioned at 3 µ m and stained with haematoxylin and eosin. Bar represents 25 µ m.

significantly over that of control values (10.9 units/small intestine), while the same amount of Kunitz trypsin inhibitor (KTI) highly stimulated the secretion of -amylase (80.4 units/small intestine). Similar results were found for the secretion of trypsin and chymotrypsin (results not given). Moreover, different lectins were found to stimulate the secretion of -amylase to different extents and this was not always correlated with the pancreatic growth they induced. Thus, although the enlargement obtained after feeding rats with diets containing PHA or SBA was reasonably similar, SBA did not stimulate the secretion of amylase whereas PHA did (226.2 units/small intestine; Pusztai et al., unpublished). In fact, on acute exposure PHA was a more potent secretagogue than STI. Mechanism of stimulation of pancreatic growth It is generally accepted that the stimulation of pancreatic growth by orally administered trypsin inhibitors is mediated by CCK, a gut peptide hormone with trophic effects on the pancreas (Fölsch et al., 1990; Rosewicz et al., 1988). For the obligatory polyamine accumulation in the tissue, polyamines may possibly be derived from a number of different sources (Bardocz et al., 1989; Fölsch et al., 1990). One of these may be the increased ODC activity in the tissue. Indeed, on infusion of CCK peptides there is an immediate rise in ODC mRNA levels in pancreatic acinar cells suggesting that pancreatic ODC is regulated at pretranslational level (Rosewicz et al., 1988). It would therefore appear that the main initial source of polyamines in the STI-induced pancreatic growth is the de novo synthesized putrescine. However,

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difluoromethylornithine, a specific irreversible inhibitor of ODC did not abolish polyamine accumulation in the pancreas, suggesting that polyamines could also be derived from other sources, possibly via the blood circulation (Bardocz et al., 1989). Although the mechanism of growth-induction after stimulation with lectins is unclear at present, it is apparently distinct from that of the trypsin inhibitors. As lectins have no significant protease inhibitor activity, their stimulation of pancreatic growth could not be due to a fall in protease levels in the lumen of the small intestine. Furthermore, in contrast to the lack of reactivity of protease inhibitors with the mucosa (Wilson et al., 1978), lectins first have to bind to the brush-border to induce its growth. Those which bind avidly to the small intestinal epithelium are potent growth factors for both the gut and pancreas, and stimulate their polyamine accretion. In most instances, the extent of these two processes are proportional, which suggests that polyamine accumulation is obligatory in the lectin-induced growth. However, ODC induction appeared to be minimal in growth-signalling by PHA or SBA (Pusztai et al., 1988; 1989a; Bardocz et al., 1989). With these lectins the substantial increase in the weight of the small intestine or pancreas was attained without significant increase in ODC enzyme activity in the tissue, therefore most of the polyamines must have been derived from the blood circulation. Indeed, in the sequence of events after stimulation with PHA or SBA, first the concentration of blood polyamines was increased followed by an upregulation of the polyamine uptake/transport system of the two tissues before any other growth parameters were increased (Pusztai et al., 1989a; Pusztai, 1991). The main reason why mitogenic lectins are exogenous growth factors for various tissues is that they bind to the receptors of endogenous growth factors or hormones and set in motion the same cascade of events as in the normal signal transduction process (for examples, see Pusztai, 1991). As small amounts of dietary lectins are endocytosed by epithelial cells and transported through the gut wall into the systemic circulation (Pusztai et al., 1989b; Pusztai 1991), the growth of the pancreas may be due possibly to the direct mitogenic effect of the absorbed lectins. Accordingly, the poor efficiency of the N-acetylglucosamine-binding lectins for inducing pancreas growth (Table 8.1) might be adequately explained if, after systemic absorption, they do not circulate freely but are bound by the liver (Pusztai et al., 1989b) or by the endothelial cells of subepithelial venules and lacteals (Pusztai et al., 1993a). It is also suggestive that the best growth factors for the pancreas are those lectins whose binding and endocytosis by small intestinal epithelial cells is the most extensive (Pusztai, 1991). However, no experimental evidence has been obtained so far to show that the mitogenic activity of systemically absorbed lectins is directly responsible for the stimulation of pancreatic growth. It is possible that the effects of lectins on the growth of the pancreas are not direct but are mediated by CCK or other growth factors released from the gut after the binding of lectin to cells, possibly the neuroendocrine cell, of the brush-border epithelium. However, this mediation must be by a mechanism different from that occurring with trypsin inhibitors. The present finding that, in contrast to the potent stimulation of -amylase secretion by dietary KTI, SBA had no effect on the secretion of this digestive enzyme from the pancreas appears to rule out the involvement of CCK in the SBA effect. This is because CCK infusion is known directly to stimulate the discharge of pancreatic enzymes into the lumen of the small intestine and therefore the lack of increased amylase production after treatment with SBA suggests that either CCK is not involved in the pancreatic enlargement or that possibly other hormones are also released by SBA which then can inhibit this effect of CCK. Although this will have to be investigated further, the dramatic and immediate changes in blood insulin and glucagon levels on acute administration of PHA support the view that lectins are capable of triggering the secretion of not only CCK but also of other hormones (Pusztai et al., 1991b; Pusztai, 1993). Thus, failure of a growth factor to stimulate pancreatic enzyme secretion does not necessarily rule out the

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possibility of CCK mediation in the reaction mechanism. Indeed, there is some preliminary experimental evidence to show that, similar to that caused by trypsin inhibitors, lectins such as PHA elevate CCK levels in the blood circulation (Pusztai et al., unpublished). Therefore, CCK-mediation in the lectin-induced growth of the pancreas is now a distinct possibility, particularly, as it was shown recently that CCK-A receptor antagonists of L-364,718 (Merck) and loxiglumide (Rotta) abolished most of the growth factor effects of dietary PHA (Pusztai et al., 1994). In any case, it is possible that the enlargement of the pancreas caused by the diets containing lectins is the result of both direct and indirect stimuli operating simultaneously and this is the reason why lectins are more potent growth factors than trypsin inhibitors. Accordingly, as lectins have no effect on ODC induction but trypsin inhibitors elevate both ODC activity and increase the uptake of polyamines, the accumulation of polyamines in the pancreas after stimulation with trypsin inhibitors is more substantial. Nutritional implications for transgenic plants There are now several well-authenticated examples that lectins and trypsin inhibitors can have a protective role in plants, particularly within the storage tissues of seeds (see Peumans and Van Damme, Chapter 1 and Gatehouse et al., Chapter 3). Thus, the use of genetically engineered crops expressing lectin genes appears to be a promising means of crop production with significantly increased pest resistance. However, increasing the insect resistance of transgenic plants is only one of the most important aspects of this new technology. As these plants will be used in human food or animal feedstuffs, the nutritional safety of the insecticidal products of the transgenes used must be thoroughly tested in experiments with animals. Unfortunately, such animal testings have been rare and even in cases where they have been done this was without coordinating the animal- and insect work. In our laboratory, as a part of the programme of our FLAIR European Concerted Action, we have singled out some of the most promising insecticidal proteins, such as WGA (Pusztai et al., 1993a), CpTI (Pusztai et al., 1992a), GNA (Pusztai et al., 1990), -amylase inhibitor (Pusztai et al., unpublished) and arcelins (Pusztai et al., 1993b), whose genes have been or are going to be transferred to crop plants, and tested them in 10-day rat feeding trials. In these experiments, rats (groups of 4, average weight of 80–90 g; individually housed) were fed 6 g of a fully balanced diet for 10 days, containing 9.3 per cent (w/w) lactalbumin and 0.7 per cent individual pure lectin, except with RPA which was included at 0.3 per cent because the rats refused to eat the diet at higher lectin concentrations. Control rats were fed the same amount of lectin-free diet (10 per cent lactalbumin). The rats were monitored for changes in weight and excretion of faeces and urine. On the morning of day 10, rats were given 2g of their respective diet and killed by ether anaesthesia 2 h later. Their abdomen were cut open and the pancreas and small intestines removed. Sections were cut for morphology and protein, RNA, DNA and polyamine analyses. The results of nutritional tests carried out with rats (Pusztai et al., 1993a) have shown that the use of the WGA gene in transgenic crop plants will have to be viewed with considerable caution. Although WGA is present in staple foods derived from cereals, its concentration is only about 300 mg kg−1 wheat germ, which is far below the level of the 1 per cent shown to be effective against insects. At this low natural level, particularly when diluted with other food ingredients, WGA does not appear to be toxic. However, at 0.7 per cent in the diet this lectin reduces the utilization of dietary proteins, induces wasteful growth of both the small intestine and the pancreas, causes thymus atrophy and depresses the growth of rats (Pusztai et al., 1993a). It is particularly worrying that detectable amounts of functionally and immunochemically intact WGA have been shown to be transported across the intestinal wall into the systemic circulation. The longterm effects of this systemic absorption of WGA on immune function, metabolism and health are unknown.

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Thus, the general use of WGA in edible parts of crop plants as a natural insecticide is not without dangers for potential consumers and its safety may still have to be established. Due to the heat-stability of WGA and its resistance to proteolytic breakdown, this same consideration applies also to the expression of the WGA gene in the leaves of crop plants (e.g. corn) which are used as winter-feed for cattle in many countries. From the limited comparison of the properties of the three GlcNAc-specific lectins in this study and, particularly, from the absence (or the low level) of the transepithelial transport of DSA and UDA, the antinutritional effects of these two lectins appear to be less damaging. However, it may be of particular significance that when tested in vitro, and in line with their diminished toxicity for higher animals, DSA and UDA also appeared to be less active against insects (Czapla and Lang, 1990; Huesing et al., 1991a,b; Murdock et al., 1990). The effect of feeding rats purified CpTI for 10 days (Pusztai et al., 1992a) was a moderate reduction in the weight gain of rats in comparison with control, despite an identical food intake by the two groups. The reduction in the growth rate was about 20 per cent on a live weight basis. However, the corresponding value calculated from the weight of dry carcasses was less, only about 7 per cent, probably because the water content of the body of the two groups of rats was different. Although most of the CpTI was rapidly broken down in the digestive tract, its inclusion in the diet led to a slight, though significant, increase in the nitrogen content of faeces but not of urine. Accordingly, the net protein utilization of rats fed on inhibitorcontaining diets was also slightly depressed while their energy expenditure was elevated. In agreement with results obtained for the protease inhibitors of soya bean, the moderate antinutritional effects of CpTI were probably due mainly to the stimulation of the growth and metabolism of the pancreas. Thus, the nutritional penalty for increased insect-resistance after the transfer of the CpTI gene into food plants is slight in the shortterm. However, the long-term effects on nutrition and health of continuous exposure to the inhibitor are unknown at present and clearly need to be established by further tests. There is firm experimental evidence that -amylase inhibitor is deleterious for a number of insects and therefore the transfer of its gene into crop plants to increase their resistance against these insects has been recommended (see Rousseau et al., Chapter 2). To see what possible effects -amylase inhibitor may have on mammals, we have recently carried out nutritional tests to evaluate the effects on rats of the inclusion of pure kidney bean -amylase inhibitor in their diet at three different dietary concentrations and obtained clear evidence that this inhibitor is deleterious for the health of the animals even at the lowest level (0.2 per cent of the diet; Pusztai et al., unpublished). Accordingly, the use of the -amylase inhibitor gene in transgenic plants intended for consumption by humans and/or animals cannot be recommended for the time being. In contrast, arcelins, the natural insecticidal proteins found in some Phaseolus vulgaris bean cultivars, have been shown to be reasonably safe in short nutritional tests (Pusztai et al., 1993b). However, even with arcelins, further and more thorough nutritional tests may have to be carried out before their safety can be established unequivocally. By far the most extensively tested lectin in both insect and rat trials is GNA. As shown in this book (Gatehouse et al., Chapter 3), GNA has powerful insecticidal activity against a number of insect orders, including the economically so important sap-sucking insects. Furthermore, the gene of this lectin has now been successfully transferred into several plants, some of which have been shown conclusively to express the lectin in most plant tissues and at a high level. As a first step to establish its safety for mammals, the nutritional effects of GNA have been intensively studied with rats in our laboratory (Pusztai et al., 1990; and unpublished). The results show that GNA may be one of the safest lectins to use in transgenic plants. In short-term nutritional experiments it was shown to be essentially non-toxic for rats. Moreover, because GNA substantially reduced the extent of the PHA-induced coliform overgrowth in the small intestine

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(‘Chemical Probiosis’, see Pusztai et al., 1993c and Chapter 15 in this book), the dietary presence of GNA may have considerable health benefits. However, even with such a promising lectin as GNA, it may be prudent to carry out further and longer-term feeding experiments not only with rats but also with pigs and, perhaps even with primates before transgenic crop plants expressing the GNA gene at a high level can be generally released. Conclusion Both lectins and trypsin inhibitors induce pancreatic growth and the accumulation of pancreatic polyamines; however, growth stimulation by these two factors may be fundamentally different. Trypsin inhibitors do not bind to the small intestine, nor induce its growth. They stimulate the enlargement of the pancreas by a negative feedback mechanism mediated by CCK secretion, triggered by falling luminal concentration of serine proteases. Trypsin inhibitors increase polyamine accretion in the pancreas by both ODC enzyme induction and increased uptake from circulation. In contrast, lectins do not reduce the level of proteases in the gut lumen but, due to their avid binding to the brush-border and mitogenicity, lectins induce small intestinal growth by hyperplasia. Lectins which are systemically absorbed may directly exert their mitogenic effects on acinar (and other) cells of the pancreas. However, as there is some evidence to show that following the binding of lectins to gut neuroendocrine cells, circulating CCK levels are increased and that CCK-A receptor antagonists reduce the growth stimulatory effects of lectins on the pancreas, it is possible that this stimulation by lectins is indirect and mediated by CCK and/or other hormones. Possibly both direct and indirect mechanisms operate. However, as lectins are not effective inducers of ODC, polyamine accumulation in the enlarged pancreas and small intestine is mainly by polyamine uptake. The implications of these findings for the safety of transgenic plants transfected with genes of lectins and/ or inhibitors of pancreatic digestive enzymes are obvious. Although the use of genes of overtly toxic proteins in transgenic technology has been ruled out, there is still an urgent need to test not only the insecticidal potential of other apparently non-toxic gene products but, in a concerted fashion, also to carry out thorough nutritional safety testings. From the few such tests completed so far, almost exclusively in our laboratory, the potentially deleterious effects of the insecticidal proteins on the pancreas have emerged as the main problem. Clearly, when lectins such as GNA whose potent insecticidal and anti-bacterial properties are coupled with the lack of short-term antinutritive effects on the pancreas are used, no such problem may arise. Therefore, the gene of GNA appears to be one of the most promising and safest candidates for the transfection of crop plants. However, all other insecticidal proteins tested so far and particularly those which were the most potent in this respect, including even the mildly reactive arcelins, have been shown to be stimulants of pancreatic growth and increased enzyme secretion in rodents. Although it is dangerous to extrapolate to humans from nutritional studies carried out with rats, there is already some evidence that soya meal or trypsin inhibitors instilled into the duodenum of human volunteers stimulate the secretion of pancreatic enzymes in a way which appears to be similar to that occurring in the rat (Calam et al., 1987; Liener et al., 1988). Accordingly, with the possibility that the consumption of transgenic crop plants may lead to a continuous over-stimulation of pancreatic function and its potentially harmful consequences, it may be advisable to exercise considerable caution in accepting transgenic plants without further rigorous testing. Although our insistence that nutritional tests be carried out ought to be one of the preconditions of the general release of transgenic plants, our studies on the mechanism of the lectinand/or trypsin inhibitor-induced pancreatic growth indicate that some of the potentially harmful effects of these transgenes may be alleviated by the use of CCK-A receptor antagonists in the diet. As the use of loxiglumide has already been tried on humans without apparent ill-effects, this and other similar products may

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find a limited use in practice for making transgenic plants more acceptable. However, the real hope is that by intensive and highly focused research we shall be able to identify many more lectins, such as GNA, which have powerful insecticidal activity but whose effects on both the gut and the pancreas are not deleterious for potential consumers. Acknowledgements The collaborative work is part of a European FLAIR Concerted Action Programme (No. 9) and Concerted Action on Polyamines (No. AIRI1-CT92-0569) supported by the Commission of European Communities. This work was supported in part by the Scottish Office Agriculture and Fisheries Department (A. Pusztai, S.Bardocz, G.Grant and D.S.Brown), British-German Academic Research collaboration (A.Pusztai and S.Bardocz) and The National Fund for Scientific Research (Belgium, FGWO grant 20059.89 N; W.J.Peumans and E.J.M.Van Damme). Dr Ewen gratefully acknowledges the generous grant from the Scottish Home and Health Department for the purchase of the Joyce-Loebl ‘magiscan’ image analyser. References Bardocz, S., Grant, G., Brown, D.S., Ewen, S.W.B. and Pusztai, A., 1989, Involvement of polyamines in Phaseolus vulgaris lectin-induced growth of rat pancreas in vivo, Medical Science Research, 17, 309–11. Broekaert, W.F., Allen, A.K. and Peumans, W.J., 1987, Separation and partial characterization of isolectins with different subunit composition from Datura stramonium seeds, FEBS Letters, 220, 116 20. Calam, J., Bojarski, J.C. and Springer, C.J., 1987, Raw soya-bean flour increases cholecystokinin release in man, British Journal of Nutrition, 58, 175–79. Cammue, B.P., Peeters, B. and Peumans, W.J., 1986, A new lectin from tulip bulbs, Planta, 169, 583–88. Czapla, T.H. and Lang, B.A., 1990, Effect of plant lectins on the larval development of European Corn Borer (Lepidoptera: Pyralidae) and Southern Corn Rootworm (Coleoptera: Chrysomelidae), Journal of Economic Entomology, 83, 2480–85. de Oliveira, J.T.A., Pusztai, A. and Grant, G., 1988, Changes in organs and tissues induced by feeding of purified kidney bean (Phaseolus vulgaris) lectins, Nutrition Research, 8, 943–47. Fölsch, U.R., Löser, C. and Alves, F., 1990, Polyamines in pancreatic growth, Digestion, 46 (suppl. 2), 345–51. Grant, G., Bardocz, S., Brown, D.S., Watt, W.B., Stewart, J.C. and Pusztai, A., 1990, Involvement of polyamines in pancreatic growth induced by dietary soyabean, lectin or trypsin inhibitors, Biochemical Society Transactions, 18, 1009–10. Green, G.M. and Lyman, R.L., 1972, Feedback regulation of pancreatic enzyme secretion as a mechanism of trypsin inhibitor-induced hypersecretion in rats, Proceedings of the Society for Experimental Biology and Medicine, 140, 6–12. Huesing, J.E., Murdock, L.L. and Shade, R.E., 1991a, Effect of wheat germ isolectins on development of cowpea weevil, Phytochemistry, 30, 785–88. Huesing, J.E., Murdock, L.L. and Shade, R.E., 1991b, Rice and stinging nettle lectins; insecticidal activity similar to wheat germ agglutinin, Phytochemistry, 30, 3565–68. Liener, I.E., Goodale, R.L., Deshmukh, A., Satterberg, T.L., Ward, G., DiPietro, C.M., Bankey, P.E. and Borner, J.W., 1988, Effect of a trypsin inhibitor from soybeans (Bowman-Birk) on the secretory activity of the human pancreas, Gastroenterology, 94, 419–27. Murdock, L.L., Huesing, J.E., Nielsen, S.S., Pratt, R.C. and Shade, R.E., 1990, Biological effects of plant lectins on the cowpea weevil, Phytochemistry, 29, 85–89. Pegg, A.E., 1986, Recent advances in the biochemistry of polyamines in eukaryotes, Biochemical Journal, 234, 249 62.

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Peumans, W.J., Stinissen, H.M. and Carlier, A.R., 1982, A genetic basis for the origin of six different isolectins in hexaploid wheat, Planta, 154, 562–67. Peumans, W.J., De Ley, M. and Broekaert, W.F., 1984, An unusual lectin from stinging nettle (Urtica dioica) rhizomes, FEBS Letters, 177, 99–103. Peumans, W.J., Nsimba-Lubaki, M., Broekaert, W.F. and Van Damme, E.J.M., 1986, Are bark lectins of elderberry (Sambucus nigra) and black locust (Robinia pseudoacacia) storage proteins? in Shannon, L.M. and Chrispeels, M.J. (Eds) Molecular Biology of Seed Storage Proteins and Lectins, pp. 53–63, Proceedings of the 9th Annual Symposium of Plant Physiology, UCR Riverside. Pusztai, A., 1991, Plant Lectins, Cambridge: Cambridge University Press. Pusztai, A., 1993, Dietary lectins are metabolic signals for the gut and modulate immune and hormone functions, European Journal of Clinical Nutrition, 47, 691–99. Pusztai, A., Grant, G., Brown, D.S., Ewen, S.W.B. and Bardocz, S., 1988, Phaseolus vulgaris lectin induces growth and increases the polyamine content of rat small intestine in vivo, Medical Science Research, 16, 1283–84. Pusztai, A., Grant, G., Williams, L.M., Brown, D.S., Ewen, S.W.B. and Bardocz, S., 1989a, Phaseolus vulgaris lectin induces growth and the uptake of polyamines by the rat small intestine in vivo, Medical Science Research, 17, 215–17. Pusztai, A., Greer, F. and Grant, G., 1989b, Specific uptake of dietary lectins into the systemic circulation of rats, Biochemical Society Transactions, 17, 481–82. Pusztai, A., Ewen, S.W.B., Grant, G., Peumans, W.J., Van Damme, E.J.M., Rubio, L. and Bardocz, S., 1990, Relationship between survival and binding of plant lectins during small intestinal passage and their effectiveness as growth factors, Digestion, 46(suppl. 2), 308–16. Pusztai, A., Watt, W.B. and Stewart, J.C., 1991a, A comprehensive scheme for the isolation of trypsin inhibitors and the agglutinin from soybean seeds, Journal of Agricultural and Food Chemistry, 39, 862–66. Pusztai, A., Ewen, S.W.B., de Carvalho, A.F.F.U., Grant, G., Stewart, J.C. and Bardocz, S., 1991b, Immune and hormonal effects of dietary lectins. Euro Food Tox III, Proceedings of the Interdisciplinary Conference on ‘Effects of Food on the Immune and Hormonal Systems’, Zurich, Switzerland, pp. 20–24. Pusztai, A., Grant, G., Brown, D.J., Stewart, J.C., Bardocz, S., Ewen, S.W.B., Gatehouse, A.M.R. and Hilder, V., 1992a, Nutritional evaluation of the trypsin (EC 3.4.21.4) inhibitor from cowpea (Vigna unguiculata Walp.), British Journal of Nutrition, 68, 783–91. Pusztai, A., Ewen, S.W.B., Grant, G., Brown, D.S., Peumans, W.J., Van Damme, E.J.M. and Bardocz, S., 1992b, Stimulation of growth and polyamine accretion in the small intestine and pancreas by lectins and trypsin inhibitors, in Dowling, H.R., Fölsch, U.R. and Löser, C. (Eds) Falk Symposium 62: Polyamines in the Gastrointestinal Tract, pp. 473–83, Dordrecht: Kluwer Academic Press. Pusztai, A., Ewen, S.W.B., Grant, G., Brown, D.S., Stewart, J.C., Peumans, W.J., Van Damme, E.J.M. and Bardocz, S., 1993a, Antinutritive effects of wheat germ agglutinin and other N-acetylglucosamine specific lectins, British Journal of Nutrition, 70, 313–21. Pusztai, A., Grant, G., Stewart, J.C., Bardocz, S., Ewen, S.W.B. and Gatehouse, A.M.R., 1993b, Nutritional evaluation of RAZ-2, a new Phaseolus vulgaris bean cultiver containing high levels of the natural insecticide protein Arcelin-1, Journal of Agriculture and Food Chemistry, 41, 436–40. Pusztai, A., Grant, G., Spencer, R.J., Duguid, T.J., Brown, D.S., Ewen, S.W.B., Peumans, W.J., Van Damme E.J.M. and Bardocz, S., 1993c, Kidney bean lectin-induced Escherichia coli overgrowth in the small intestine is blocked by GNA, a mannose-specific lectin, Journal of Applied Bacteriology, 75, 360–68. Pusztai, A., Grant, G., Baintner, K. and Bardocz, S., 1994, Lectins are extraneous growth factors inducing CCKmediated pancreatic growth which is blocked by CCK-A receptor antagonists, Digestive Diseases and Sciences, 39, 1751, No. 86. Rosewicz, S., Lewis, L.D., Liddle, R.A. and Logsdon, G.D., 1988, Effects of cholecystokinin on pancreatic ornithine decarboxylase gene expression, American Journal of Physiology, 255, G818–21.

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Shibuya, N., Goldstein, I.J., Broekaert, W.F., Nsimba-lubaki, M., Peeters, B. and Peumans, W.J., 1987, The elderberry (Sambucus nigra L.) bark lectin recognizes the Neu5Ac( 2–6) Gal/GalNAc sequence, The Journal of Biological Chemistry, 262, 1596–601. Van Damme, E.J.M., Allen, A.K. and Peumans, W.J., 1987, Isolation and characterization of a lectin with exclusive specificity towards mannose from snowdrop (Galanthus nivalis) bulbs, FEBS Letters, 215, 140–44. Wilson, P.A., Melmed, R.N., Hampe, M.M.V. and Holt, J.S., 1978, Immunocytochemical study of the interaction of soybean trypsin inhibitor with rat intestinal mucosa, Gut, 19, 260–66.

Chapter 9 Lectins in Immunology D.C.Kilpatrick

Introduction Protein-carbohydrate interactions (including lectin-lectin receptor interactions) occur in association with many biochemical processes. It comes as no surprise, therefore, that lectin-carbohydrate interactions are a common and important feature of molecular events underlying the immune response. Studies with lectins in the field of immunology will be considered under four categories: (1) lectins as polyclonal reagents to investigate the molecular basis of lymphocyte activation and proliferation and their control; (2) lectins as reagents to identify and/or fractionate cells of the immune system; (3) lectins as drugs; and (4) endogenous lectins as first-line agents of defence or as mediators of cellular communication within the immune system. In this review, I will confine myself to the study of human immunology; work involving rodents or other animals will only be referred to when relevant to the human situation. Lectins as polyclonal modulators Mitogenic lectins and their receptors on lymphocytes The value of plant lectins as polyclonal activators is well-known and well-established. The use of lectins including PHA and concanavalin A which transform a majority of resting lymphocytes into lymphoblasts irrespective of antigenic specificity enormously facilitated studies on the biochemistry of this process. With such reagents, the characteristic metabolic changes, Ca2+ and amino acid influxes, acetylation of histones, phosphorylation of nuclear proteins, DNA, RNA and protein synthesis etc, were readily observed and investigated. Although many plant lectins were found to be mitogenic for animal lymphocytes, just as many were not. Of those that were mitogenic, most were largely or exclusively active towards T-lymphocytes. Early reports that immobilised concanavalin A is an exclusively B-cell mitogen (Nicolson, 1974) have not, to my knowledge, been confirmed, and certainly such lectin derivatives do not seem to have been widely used as such. In the mouse, several B-cell specific mitogenic lectins have been reported from various sources (Campbell et al., 1982; Lipsick et al., 1980), but for human B-cells such reagents are rare. It is possible that under some conditions, wheat germ agglutinin (WGA) may preferentially act upon human Bcells (Greene et al., 1981c). Usually, however, any human B-cell activation is dependent on T-cell activation (e.g. as with pokeweed mitogen or lentil lectin) and therefore the discussion which follows refers to T-cell mitogenesis. Readers interested in the molecular events associated with B-cell activation, proliferation and differentiation are referred to the review by Jelinek and Lipsky (1987).

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It is indisputable that mitogenic lectins bind to lymphocytes via saccharide receptors and thereby trigger cell activation, but some non-mitogenic lectins also bind to lymphocytes in a similar saccharide-specific manner. Binding to the lymphocyte cell surface is therefore a necessary requirement of a mitogenic lectin, but simply binding is not enough to guarantee subsequent activation. The heterogeneity of receptors for PHA, concanavalin A and other lectins on lymphocytes (Axelsson et al., 1978; Henkart and Fisher, 1975; Iwata et al., 1977) makes it difficult to relate receptor occupancy to mitogenic activity. Is there a single functional mitogen receptor for a particular lectin and, if so, which one is it ? Are other lectin-receptor interactions irrelevant? Or does mitogenic activity depend on the engagement and perhaps cross-linking of two or more receptors? Suggestions have been made that the essential difference between mitogenic and non-mitogenic lectins in general is that only the former bind to the T3 (CD3) molecule (Fleischer, 1984; Palacios, 1982), or to histocompatibility antigens (Kimura and Ersson, 1981), or to the T-cell receptor (Kanellopoulos et al., 1985). There is also evidence that some lectins can induce a mitogenic response via CD2, the T-lymphocyte receptor for sheep erythrocytes (Leca et al., 1986), via an alternative pathway of activation (Meuer et al., 1984). However, CD2, CD3, HLA and the T-cell receptor (TCR) do not exist as independent molecules on the cell surface; indeed, to some extent they all seem to be linked. Moreover, they may also be linked to CD4, CD8 (Suzuki et al., 1992) and to CD5 (Osman et al., 1992). PHA and concanavalin A bind to both the CD3-T-cell receptor complex and to CD2 (Leca et al., 1986). CD2 is associated to some extent with the CD3-TCR complex (Brown et al., 1989). The latter may serve to regulate the response via CD2 (Holter et al., 1988), while adhesion mediated by CD2 and its interaction with its natural ligand CD58 may enhance TCR mediated activation by stimulating monokine production (Webb et al., 1990). It has now been shown with the use of transfectants that CD2-mediated activation can occur in the absence of the CD3-TCR complex, but that concomitant expression of CD3-TCR markedly reduces the threshold level of CD2 for activation via the alternative pathway (Ohno et al., 1991). Activation of CD2+ CD3−TCR− natural killer cells presumably takes place via this alternative pathway. It seems that once either CD3-TCR, or CD2, or both trigger a mitogenic response, the signals follow a common pathway of protein kinase C activation mediated by diacylglycerol and inositol triphosphate leading to a sequence of phosphosylation reactions (Altman et al., 1990). Perhaps the best generalization that can be made at present is that mitogenic lectins are those which bind to the T3-TCR complex or associated molecules with adequate affinity (Chilson and Kelly-Chilson, 1989) without simultaneously binding to receptors inducing an opposing effect. An affinity requirement could explain why the lima bean lectin tetramer is a weak mitogen while the octameric form of the same lectin is a potent mitogen (Pandolfino et al., 1983). Studies with monoclonal antibodies of precise specificity have demonstrated that a large number of cell surface molecules can apparently influence the mitogenic process in vitro (Table 9.1). The list includes accessory signal receptors like CD28 (Tp 44), CD5 (T1), CD43, CD44 and CD45. These lymphocyte receptors may bind to one or more ligands on macrophages, B-cells or other T-cells, and sometimes the natural ligand is of wide distribution like ICAM-1, a member of the adhesion family. Cellular adhesion seems to be an important function of these interactions (Voss et al., 1991), but for most of the receptors in Table 9.1, monoclonal antibodies have been found which induce mitogenic stimulation either alone or in combination with another agent. There have been suggestions of CD3-TCR and CD2 independent signalling pathways involving some of those receptors, but the situation is far from clear. WGA, for example, appears to stimulate T-cells via such an alternative pathway (Yachie et al., 1987), but the key receptor involved is unknown and hard to identify when WGA may be able to bind 13 different cell surface glycopeptides (Axelsson et al., 1978).

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Receptors for non-mitogenic lectins If lectins can induce a mitogenic response by binding to certain T-cell receptors, it might be expected that other lectins or other lectin receptors may function in the Table 9.1. Receptors on resting T-lymphocytes implicated in activation T-cell receptor

Ligand

Ligand distribution

TCR/CD3 Antigen/HLA APC* CD2(E-R,T11,LFA-2) CD58(LFA-3)CD59 Wide CD4 HLA-class 2 APC CD8 HLA-class 1 Virally infected cells CD5(T1) CD72 B-cells LFA-1(CD11a/18) ICAM-1(CD54) Wide CD26 collagen Wide CD27 unknown CD28(Tp44) B7 Activated B-cells VLA(CD49/CD29) Extracellular matrix Wide CD38(T10) unknown CD43(leukosialin) ICAM-1(CD-54) Wide CD44 hyaluronic acid Wide CD45(LCA) CD22 B-cells IL-1 receptor IL-1 IL-6 receptor IL-6 IL-10 receptor IL-10 Note: For original sources, the reader is referred to the text of Barclay et al. (1993) * Antigen-presenting cell

opposite direction. Indeed, the concept of ‘stimulatory domains’ and ‘inhibitory domains’ on the surface of the same lymphocyte has been proposed (Greene et al., 1981a,b,c). However, an alternative and equally plausible possibility is that occupancy of the same receptor may lead to a positive or negative signal depending on the nature of the ligand and the precise conditions. Therefore a particular lectin might be mitogenic or non-mitogenic depending on the circumstances. Some lectins are certainly anti-mitogenic i.e. they inhibit the action of mitogens in co-culture experiments. Examples include Agaricus bisporus lectin (ABL; Greene et al., 1981a) and the potato and tomato lectins (McCurrach and Kilpatrick, 1988). The tomato lectin binds leukosialin (sialophorin, CD43) and the T-cell form of the leucocyte common antigen (T200, CD45; Kilpatrick et al., 1986). CD43 may function to regulate lymphocyte adhesion (Manjunath et al., 1993), and, if so, its masking by the very basic solanaceous lectins should reduce the net surface charge and promote adhesion. However, as monoclonal antibodies to CD43 can induce T-cell activation or augment the response to specific antigens (Park et al., 1991; Silverman et al., 1989), and its natural absence is associated with immune deficiency (Piller et al., 1989), it is possible that tomato lectin inhibits lymphocyte activation by blocking a physiological signal pathway mediated by CD43. The leucocyte common antigen, CD45, is a tyrosine phosphatase which is an obligatory co-factor for T-cell receptor-mediated signalling (Pingel and Thomas, 1989). It is therefore not surprising that its interaction with tomato lectin influences lymphocyte activation. An unidentified single

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receptor on neuraminidase-treated lymphocytes to which the non-mitogenic Helix pomatia lectin binds possesses characteristics consistent with CD45 (Axelsson et al., 1978). Such lectins interacting with CD45 might act analogously to blocking antibodies rather than analogously to stimulating antibodies. The same lectin can undoubtedly induce opposing effects under different circumstances. Time-honoured examples include the non-mitogenic activity of concanavalin A and PHA at relatively high lectin concentrations, but perhaps the most instructive example is WGA. WGA has been described as simply nonmitogenic (Boldt et al., 1975), actively anti-mitogenic (Barrett et al., 1983), T-cell mitogenic (Brown et al., 1976), B-cell mitogenic (Greene et al., 1981c) and mitogenic only for a subset of T-cells (Gordon et al., 1980). We could certainly confirm that, for T-cells at least, WGA could be either mitogenic or antimitogenic depending on the lectin concentration and other conditions (Kilpatrick and McCurrach, 1987). WGA at low concentrations may activate T-cells via a T3-TCR independent pathway (Yachie et al., 1987) but over a higher concentration range an inhibitory influence may predominate, possibly via binding to interleukin-2 (IL-2) receptors (Reed et al., 1985) as they are synthesized. Another example of how the same lectin can have very different influences upon lymphocytes is provided by Datura stramonium agglutinin (DSA). The receptor(s) for DSA on lymphocytes is not known, but DSA has a rather similar saccharide specificity to WGA, so its dual nature as weak mitogen and anti-mitogen is consistent with its WGA-like qualities. The most remarkable property of DSA is the enormous synergy observed with the phorbol ester, TPA (12–0-tetradecanoyl-phorbol-13-acetate), when the latter was present at a barely mitogenic concentration (McCurrach and Kilpatrick, 1988). A very modest degree of synergy with TPA can be observed with some other lectins and a substantial effect is evident with WGA, but the degree of synergy is an order of magnitude greater with DSA (Kilpatrick et al., 1990). While I myself have used terms like ‘incomplete mitogen’ or ‘co-mitogen’ to describe DSA and WGA, it seems likely that they bind to receptors inducing opposing actions and the influence of TPA is to tilt the balance in the mitogenic direction. It should be noted that antibodies to HLA class 1 or HLA class 2 molecules usually inhibit lymphocyte activation (Akiyama et al., 1985), despite being associated with the T3-TCR complex. These observations rest uncomfortably with the hypothesis that HLA-binding lectins are mitogenic. However, this is entirely consistent with the possibility that the outcome depends not just on the particular ligand-receptor interaction but the biochemical context in which that interaction occurs. Accessoty cells and cytokines Lectin-T lymphocyte interactions cannot be adequately considered without reference to two other factors: accessory cells and cytokines. T-lymphocytes recognize foreign epitopes in the context of histocompatibility antigens. That is to say, foreign epitopes are only recognized as such when associated with self-HLA on the cell surface. For this to happen, the foreign (antigenic) material needs to be ingested by phagocytic cells before being processed and then presented on the cell surface combined with HLA. Antigen presenting cells may be macrophages, dendritic cells or B-lymphocytes. In vitro, mitogenic lectins are also assisted by the presence of accessory cells. When this observation was first made the accessory cells were known to secrete interleukin-1 (IL-1; then called lymphocyte activating factor) while lymphocytes once activated secreted IL-2 (T-cell growth factor). These and other interleukins are pleotropic factors. Although a lengthy list of interleukins, interferons and other cytokines can now be compiled, IL-2 and its receptor on activated lymphocytes are still believed to play a central role in the mitogenic process. While the lymphocyte activation process can be simplified in summary to a series of phosphorylation events leading to the syntheses of IL-2 and its receptor, the precise contributions of accessory cells may be

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as complex as the array of interconnecting receptor-ligand interactions listed in Table 9.1. Representative mitogenic lectins such as concanavalin A are active with a typical mixed population of mononuclear cells at a relatively low concentration range, but are not mitogenic for isolated T-cells under otherwise similar conditions unless the lectin concentration is vastly increased (Roosnek et al., 1985a,b). However, such lectins immobilized on a surface of beads or sheep erythrocytes are mitogenic (at low concentration) for purified T-cells (Gallagher et al., 1986; Warren and Bezos, 1987). Such observations suggest that the benefit of accessory cells is physical (anchoring of the lectin on a cell surface promoting aggregation of lectin receptors on the lymphocyte surface) rather than chemical (IL-1 production). Indeed, as a generalization, IL-1 enhances lymphocyte activation somewhat but does not replace accessory cells (Conti et al., 1991; Palacios, 1985). However, WGA, an entirely accessory cell-dependent mitogen, has its full mitogenic capacity towards isolated T-cells restored by the addition of cell-free conditioned medium (Kilpatrick, 1988) though not by purified IL-1 (Kilpatrick and McCurrach, 1987). PHA is much less dependent on accessory cells for its mitogenic activity towards adult peripheral blood lymphocytes than concanavalin A (Hutchins and Steel, 1983), although the dependence may be qualitatively similar. The accessory cell dependence of WGA and DSA, however, differs radically (Kilpatrick, 1988). WGA requires monocytes for optimal mitogenic activity, but they can be replaced by soluble factors or by TPA; the mitogenic proclivity of DSA is suppressed by monocytes but not by soluble factors secreted into the medium. This cell-mediated suppression can readily be overcome by TPA, which is thought to act by activating protein kinase C in a manner mimicking the action of diacylglycerol derived from plasma membrane phospholipid during physiological lymphocyte activation. TPA or DG can act synergistically with ligands which promote a rise in intracellular [Ca2+], a process mediated physiologically by inositol triphosphate. Accessory cells therefore may exert influence in one or more ways: they can form an attachment surface (analogous to antigen presentation) which may facilitate receptor cross-linking by providing a positive (stimulative) cell-mediated signal (Mueller et al., 1990) or a negative (suppresser) cell-mediated signal; and/ or by the secretion of IL-1 and other cytokines. The relative contributions of, and the outcome of, the interactions between these mechanisms may vary according to the lectin used as reagent and the conditions in which it is used. Conclusion The phenomenology of lectin-lymphocyte interactions has been of major importance in highlighting the complexities of cellular interactions, soluble mediator involvement and receptor signalling mechanisms associated with lymphocyte activation in vitro. These complexities will have to be accounted for if an understanding of the immune response in vivo is to be achieved. Monoclonal antibodies to surface receptors have proved to be more specific tools for this purpose, but it is now becoming fashionable to study immune activation induced by ‘superantigens’. Superantigens are bacterial or viral products which act as polyclonal activators because they have the ability to bind both the TCR and HLA-class 2 antigens (Drake and Kotzin, 1992). Superantigens may be the natural products that plant lectin mitogens mimic, in which case much of what has been learnt about lectins may apply to superantigens and facilitate understanding of their relevance in health and disease. The similarities and differences between mitogenic lectins and superantigens have recently been reviewed elsewhere (Licastro et al., 1993). The differences seem to have been bridged by the unusual lectin from the nettle, Urtica dioica, which—at least for murine T-cells—seems to use the same molecular mechanism as microbial superantigens (Galelli and Truffa-Bachi, 1993).

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Lectins and lymphoid cell identification and separation Alterations in cell surface saccharides are a feature of normal development and differentiation, and also of malignant transformation. Therefore, changes in lectin receptor expression, or lectin binding, might be expected to occur as lymphoid cells differentiate or are transformed to form lymphomas or leukaemias. Indeed, histochemical studies have demonstrated differentiation-related lectin-binding patterns in normal lymphoid tissues (Hsu and Ree, 1983), while malignant cells can be distinguished from their non-malignant counterparts in the same way (Raedler and Raedler, 1985). Lectin-binding ability can be exploited in cell separation techniques which include differential agglutination, lectin affinity chromatography or panning on lectin coated surfaces. Peanut (Arachis hypogaea) agglutinin, PNA, has been much used in this regard. In general terms, it tends to recognize poorly differentiated cells, and so, for example, binds to cortical (immature) thymocytes but not medullary thymocytes. It does not bind appreciably to normal differentiated lymphocytes, but commonly reacts with lymphoid leukaemia cell clones. Amongst peripheral blood cells only monocytes bind PNA, and it may be that those cells which bind most strongly represent a subset at an early stage of maturation (Kilpatrick and Maxwell, 1990). Ironically, PNA also binds very strongly to that most terminally differentiated lymphoid cell, the plasma cell (Rhodes and Flynn, 1989), and this outlying characteristic may turn out to be the most useful in clinical practice. PNA coupled to magnetic beads can be used to purge human bone marrow of plasma cells (Rhodes et al., 1991). This is true of myeloma cells, although the lectin appears to bind to different proteins on the malignant plasma cells of different individuals (Slupsky et al., 1993). A procedure has been developed for removing bone marrow cells of B lineage from the marrow of multiple myeloma patients by a combination of immobilized PNA and immobilized anti-CD19 monoclonal antibodies (Rhodes et al., 1991). This procedure removed all detectable plasma cells, B-cells and pre-B-cells, while allowing a 55 per cent recovery of normal haemopoietic progenitor cells. This procedure may be of value in purging the marrow of myeloma patients undergoing autologous bone marrow transplantation after ablative chemotherapy. If all the malignant cells can be removed before marrow rescue, it seems likely that the high relapse rate associated with this therapy would be reduced. Further developments in this technique are eagerly awaited. The one lectin that has already found therapeutic application in bone marrow modification is soya bean agglutinin, SBA. This lectin was first shown to remove tumour cells from bone marrow (Reisner, 1983), but has been employed in clinical practice as an agent to distinguish T-cells from stem cells and therefore as a means to reduce the risk of graft-versus-host disease. This means of marrow purging which involved the removal of red blood cells, agglutination with SBA and further prolonged incubations of the cells not agglutinated by SBA with sheep erythrocytes for the removal of the remaining T cells, was first used successfully on a baby girl with acute lymphoblastic leukaemia (Reisner et al., 1981). Subsequently a similar procedure has been used on haplo-identical (parental) marrow for transplantation into children with severe combined immune deficiency (Buckley et al., 1986; Cowan et al., 1985), HLA matched or mismatched marrow in a miscellany of leukaemias in adults (Slocombe et al., 1986), and HLA-compatible marrow from adults with chronic myeloid leukaemia (Cunningham et al., 1987). In all of these series, there was a high rate of engraftment and a low rate of graft-versus-host disease. It is currently generally accepted that marrow purging of T-cells does significantly reduce the incidence of graft-versus-host disease, although it is not clear which of the many purging procedures available is the most efficient (Areman and Sacher, 1991; Marmont et al., 1991). It would appear, however, that the SBA agglutination/E-rosetting method is as good as, if not better than, the others. Its major drawback is its relative labour intensiveness. This can be overcome to some extent by substituting incubation with a CD5-ricin immunotoxin for the E-rosetting procedure (Siena et al., 1987). However, it is the necessity for the SBA step which has been called into question by

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Schiff and co-workers (1987), who found that T-cell depletion was just as complete after E-rosetting alone as when done in combination with agglutination by SBA. The omission of the lectin step simplified and therefore shortened the procedure and resulted in a higher yield of cells for transplantation. Of course, SBA might act by removing precursors of the T-cells responsible for graft-versus-host reactions, so the future of SBA applications in the prophylaxis of this complication remains to be clarified. Lymphocytes have also been fractionated into subsets by the use of other lectins including lima bean lectin, WGA and HPA (Sharon, 1983). As mentioned in the previous section, HPA does not bind fresh lymphocytes, but agglutinates them after the cells have been treated with neuraminidase. All desialated Tcells are reactive, but only a minority of B-cells. This was exploited by Schrempf-Decker et al. (1980) to isolate a B-cell preparation for use in routine HLA-DR tissue-typing. We found, however, that neuraminidase-treated lymphocytes were lysed non-specifically in the microcytotoxicity test (Kilpatrick and Darg, 1983) and concluded that caution should be exercised if following the Schrempf-Decker procedure. Indeed, most laboratories chose to use other means of isolating B-cells and, in any case, serological tissuetyping for HLA-class 2 specificities is in the process of being superseded by genotyping technologies (Bidwell, 1992). WGA can be used to fractionate human peripheral blood lymphocytes, although conflicting data on the characteristics of these subsets has been reported (Sharon, 1983). The application of WGA fractionation to bone marrow stem cells might now be warranted in view of the work of Sardina and colleagues (1991) with mice. These workers transplanted WGA-binding stem cells from normal mouse marrow or fetal liver into autoimmune disease-prone mice and observed some inhibition of the genetically derived lesions in the latter. Conversely, the reciprocal transfer resulted in autoimmune lesions in normal mice. These findings indicate that stem cells from autoimmunity-prone animals contain the relevant genetic defects, and also raise the outside possibility that ex-vivo lectin-mediated fractionation of stem cells could alleviate some serious human disorders. Lectins as drugs Mitogenic lectins may act as immunosuppressive agents in vivo. The early animal work on tissue grafting employing PHA and concanavalin A has been reviewed by Nicolson (1974); these experiments demonstrated that lectins or lectin-containing extracts could prolong allograft survival over weak histocompatibility barriers. This line of enquiry was pursued by Hilgert, Horejsi and co-workers who found that lentil lectin had the most potent effect on rodent skin and heart transplants, apparently via the induction of suppresser cells (Hilgert et al., 1980; Hilgert et al., 1983) but it was of limited efficacy over strong histocompatibility barriers. Although lentil lectin was more potent when used in combination with other agents (Hilgert et al., 1984, 1987; Harel and Nelken, 1983), plant lectins are unlikely to be used in human transplantation as they are likely to provoke an immune response and within a short time they would be rendered ineffective by neutralizing antibodies. The same problem has been encountered with murine monoclonal antibodies and the therapeutic solution has been to try to engineer humanized antibodies. As yet, there has been no development of a basically human neomolecule containing a lectin active site, nor is there any real likelihood that any such molecule would not also be immunogenic. In passing, it is of interest that Plouffe et al. (1979) found a geographical association between Hodgkin’s disease and sensitization to PHA from a navy bean storage container; one possible if rather unlikely explanation could be that PHAinduced immunosuppression permitted the causative agent of Hodgkin’s disease (?Epstein-Barr virus) to transform reactive lymphocytes into a malignant lymphoma. In the only well-controlled human experiment

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I am aware of, the anti-mitogenic tomato lectin had no effect whatsoever on delayed hypersensitivity reactions resulting from the intradermal inoculation of recall antigens (Kilpatrick et al., 1985). Lectins have been found to prevent or alleviate autoimmune reactions in non-human animal models. For example, under some experimental circumstances, prior PHA administration can prevent the development of experimental autoimmune thyroiditis in mice (Esquivel et al., 1982). Similarly, electrolectin from Electrophorus can prevent experimental myasthenia gravis in rabbits, and can even alleviate symptoms if administered after the disease has been induced (Levi et al., 1983). Of course, both plant and non-human animal lectins would be limited in their efficacy as therapeutic agents by their immunogenicity, but the same consideration would not apply to endogenous human homologues. Recombinant human placental lectin (injected intravenously into rats) can prevent the symptoms of experimental allergic encephalomyelitis —considered to be an animal model of multiple sclerosis— and therefore, might have beneficial actions on human autoimmune disorders (Offner et al., 1990). . Finally, a brief mention of jacalin ought to be made in this context. Jacalin was the name given to the activity in crude extracts of Artocarpus heterophyllus (integrifolia) which was mitogenic and precipitated IgA. In fact, such extracts contained two lectins, the true jacalin which was specific for D-galactose and could bind IgA1, and a mannose-binding protein which possessed much of the mitogenic activity (Ferreira de Miranda-Santos et al., 1991). Jacalin binds to all human peripheral blood cells, but is selectively mitogenic for CD4-positive (helper) T-cells (Pineau et al., 1990). Moreover, jacalin completely blocks HIV-1 infection of lymphoid cells in vitro (Favero et al., 1993). Unlike some other plant lectins that partially inhibit HIV infection, jacalin does not interact with the viral envelope glycoprotein, gp120. Jacalin does bind to CD4, but does not prevent CD4-gp120 interaction, nor binding of the virus to the cells. However, this is not due to the carbohydrate-binding ability of jacalin but to an active site in its molecular structure comprising amino acids 79–92 on the -chain. This region is homologous with a sequence in the second conserved region of gp120. The 14 amino acid peptide corresponding to this jacalin region has been synthesized and found to possess potent anti-HIV activity itself (Favero et al., 1993). One can only speculate that the peptide acts by interfering with some signalling process that normally follows lymphocyte CD4-HIV gp120 binding. Clearly this peptide can be added to the growing list of potential drugs for HIV prophylaxis. It is unlikely that intact plant lectins will ever be licensed for therapeutic use in humans for the purposes described in the animal models and in vitro systems alluded to above. However, the insights gained from these investigations, and in particular the identification of the precise domains or regions on lectins which can modulate human immunity, may suggest and lead to novel therapeutic strategies which may ultimately prove successful. In contrast, the potential therapeutic applications of endogenous lectins are considerable and will be addressed in the next section. Endogenous human lectins involved in immunity Introduction The original discovery of a galactose-binding agglutinin in rabbit liver was hailed as the first mammalian lectin (Stockert et al., 1974). This claim was not entirely justified (since other lectins such as conglutinin and C-reactive protein were known, but not regarded as lectins), but the discovery prompted a large and fruitful flurry of investigations. Before long, whole families of Ca2+-dependent lectins, sulphydryldependent lectins and others were found in various mammalian tissues, and concomitantly other large phylogenetically conserved families, e.g. the immunoglobulin superfamily, were being described and

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related to one another. Both membrane-bound and soluble mammalian lectins are known to occur in humans, other mammals, and other vertebrates. Although the function(s) of Ca2+-dependent lectins is not known for certain, it is likely that they have a role as Table 9.2. Classification of human lectins involved in immunity Carbohydrate specificity (A) C-TYPE Membrane bound:

Collectins:

Selectins:

Pentraxins (B) S-TYPE

(C) N-TYPE

Asialoglycoprotein receptor Macrophage mannose receptor Hodgkin’s disease lectin Conglutinin Mannan-binding protein (MBP) Surfactant Proteins A and D E-selectin (ELAM-1) L-selectin (LECAM-1) P-selectin (GMP-140) C-reactive protein Serum amyloid P component 14 K group 30–35 K group Others Lymphocyte (and NK cell) surface lectins Lymphocyte soluble lectins Cytokines (IL-1, IL-2, TNF)

GalNAc/galactose mannose GalNAc/galactose GlcNAc mannose mannose/fucose sLex/sLea/heparin sLex/phosphomannan sLex/sLea/heparin galactans, etc. -galactosides

complex rhamnose etc. mannose glycopeptides/ chitobiose

cellular communication and regulation molecules within the immune system as well as acting directly in the first line of defence against infectious organisms. A list of immunologically relevant lectins of human origin is given in Table 9.2. I have classified them within the framework proposed by Drickamer (1988). The two main families are: the C-type lectins, which contain a characteristic carbohydrate-binding domain with a framework sequence of 18 conserved amino acid residues and require Ca2+ for activity; and the S-type lectins, which are thiol-dependent, Ca2+-independent, and -galactoside specific, with their own characteristic highly conserved domain. Other animal lectins are a heterogeneous group with members which are clearly different from either of the afore-mentioned or insufficiently well-characterized to be classified; these are sometimes referred to as ‘N-type’ (not C or S). Membrane-bound C-type lectins The original mammalian hepatic lectin referred to above is now generally known as the asialoglycoprotein receptor and has been extensively studied from rabbit and rat sources. An equivalent lectin has been isolated from human liver (Baenziger and Maynard, 1980), an oligomer with a sub-unit molecular size of 41000. It is found on hepatocytes and has the ability to remove orosomucoid and other desialated glycoproteins from the circulation. This lectin may therefore function to endocytose damaged glycoproteins for degradation or repair, or to remove aged erythocytes and other cells from the circulation. Other functions are also possible. Indeed, a role in cellular interactions has been conjectured. This lectin has also been implicated in the lectinophagocytosis of certain bacteria and protozoa (reviewed by Andersen et al., 1991).

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A separate receptor, found on the Kupffner cells lining the hepatic sinusoids, has the ability to remove damaged circulating glycoproteins in several mammalian species. It differs from the asialoglycoprotein receptor in having specificity for mannose and N-acetylglucosamine rather than galactose and Nacetylgalactosamine, and by its solubilization from tissues at high ionic strengths (Stahl et al., 1980; Sung et al., 1983). It is perhaps most appropriately called the macrophage mannose receptor. It is the cell surface equivalent of the soluble mannan-binding protein found in plasma (Thiel and Reid, 1989), and is capable of mediating the phagocytosis of bacteria, protozoa and fungi (reviewed by Andersen et al., 1991). Of particular interest in these AIDS/HIV disease-conscious days, there is good evidence that the macrophage mannose receptor is important in providing protection against Pneumocystis carinii infection (Ezekowitz et al., 1991). Human alveolar macrophages fail to bind cultured P. carinii in the presence of mannan or after adherence to mannan-coated coverslips. Transfection-experiments with mannose receptor cDNA in COS cells confirmed that the surface expression of the lectin was sufficient for P. carinii uptake. The usual absence of pneumonia in immunocompetent subjects exposed to P. carinii may therefore be attributable, at least in part, to a first-line defensive function of the macrophage lectin. Other membrane-bound C-type lectins have been found in rodents. Cell lines established from human patients with Hodgkin’s disease express a C-type lectin related to the hepatic asialoglycoprotein receptor (Paietta et al., 1986, 1989); this raises the possibility that the abnormal expression of membrane C-type lectins may be linked to malignancy or to the acquired immunodeficiency associated with Hodgkin’s lymphoma. Collectins The collectins constitute a group of soluble C-type lectins with collagen-like sequences analogous to the complement component CIq (Drickamer, 1988; Thiel and Reid, 1989). An approximately 120 amino acid carbohydrate recognizing domain, based on two disulphide bonds and a number of highly conserved hydrophobic residues with binding sites for two calcium ions, is found at the carboxyl terminal end. Much of the remainder of the molecule may consist of collagen-like sequences e.g. repeating units of glycine-prolinehydroxyproline with some glycosylated hydroxylysine. The best-studied human collectin is the mannan-binding protein (MBP), the plasma analogue of the macrophage mannose receptor. It occurs as a large molecule (native molecular weight 600–700×103) made up of identical subunits (Mr=32000). These subunits form collagen-like triplets which cluster to form oligomers with much higher affinity for carbohydrate ligands (Weis et al., 1992). It has a very wide normal concentration range in human plasma and shows a modest increase during an acute phase response (Thiel et al., 1992). A failure to opsonize baker’s yeast in laboratory tests has been associated with a low plasma concentration of MBP and addition of purified MBP can correct the defect (Super et al., 1989). The molecular basis of the low opsonin activity appears to be a single point mutation, causing a switch from a glycine to an aspartic acid residue, and this defect is inherited in an autosomal dominant fashion (Garred et al., 1992; Sumiya et al., 1991). MBP deficiency is found in about 5 per cent of the general population, making it the commonest primary immune deficiency. MBP deficiency has been associated with recurrent infections in a minority of children with the defect (Turner et al., 1991), but whether it contributes to suboptimal immunity in general is not known. MBP (like IgA) deficiency does not appear to predispose to severe life-threatening infections, but may contribute to significant morbidity nonetheless. MBP may act as an opsonin directly (Kuhlman et al., 1989) as well as indirectly by fixing complement via classical (Ikeda et al., 1987) or alternative (Schweinle et al., 1989) pathways. As well as opsonizing

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yeasts/fungi and gram-negative bacteria, MBP is active towards viruses including influenza virus (Hartshorn et al., 1993) and HIV (Ezekowitz et al., 1989). The former property is of potential therapeutic and economic importance, given the lack of vaccines which are strain non-specific and the number of work days lost through influenza infection. The effect of MBP supplementation on AIDS patients is unknown, but the potential of a non-toxic, non-immunogenic factor that can recognize HIV itself and also several of the organisms commonly responsible for AIDS-related opportunistic infections is obvious. Much has been learnt about the structure and the biochemistry of MBP in recent years, but its functional significance in vivo is less well-established. There is now a strong need for well-designed clinical studies to determine the effect of MBP deficiency on health, the value of MBP replacement therapy for deficiency subjects, and the value of MBP supplementation in specific diseases. MBP therapy could take the form of the use of a specific fraction derived from plasma or of somatic gene therapy. Other collectins include conglutinin and the surfactant proteins. Conglutinins from bovidae have been well characterized, but human plasma contains an analogous protein (Thiel et al., 1987). Conglutinins do not seem to activate complement, but can bind to both C3 and to the C1q receptor on phagocytes, and seem to function by augmenting the opsonic process. The human conglutinin-like protein (should it be called ‘conglutinoid’?) binds to iC3b, is calcium-dependent and can be inhibited by N-acetyl-D-glucosamine. Its native molecular weight is 700000 with subunits of 300000 (non-reducing conditions) or 66000 (reducing conditions). Curiously, it is absent from serum. Human lung is the source of two lectins synthesized by epithelial cells (Haagsman, 1994). Surfactant protein A bears a strong structural similarity to MBP, while surfactant protein D bears a closer structural resemblance to conglutinin. It is believed those collectins are important in the first line defence against inhaled pathogens. Selectins Selectins form a family of adhesion molecules characterised by a C-type carbohydrate recognition domain (but at the amino terminus), adjacent epidermal growth factor-like domains and a variable number of domains homologous with complement control protein domains. Additionally, there is a short sequence on the cytoplasmic side of the plasma membrane forming the carboxyl terminal end. This superfamily consisting of L, P and E selectins in humans has recently been reviewed by Bevilacqua and Nelson (1993). These lectins have a complex saccharide specificity which has not been fully clarified, but they recognise fucosylated oligomers related to the sialyl-Lewisx and sialyl-Lewisa structures. L-selectin (LECAM-I) appears to be the ‘homing receptor’ mediating the attachment of lymphocytes to the high endothelial venules of lymph nodes, and is also implicated in the adhesion of monocytes and granulocytes to endothelium at inflammatory loci. It is the only selectin constitutively expressed on the cell surface and, in further contrast to the others, is down regulated on cellular activation. E-selectin (ELAM-I) promotes the adhesion of neutrophils and other granulocytes to cytokine-activated endothelium. E-selectin does not appear to be expressed on the surface of resting endothelial cells, but is synthesized in response to stimuli including IL-I, TNF or endotoxin. P-selectin (GMP-140, CD62), the largest molecule in the selectin family, is present on megakaryocytes as well as activated platelets and activated endothelial cells. It is synthesized constitutively but is stored intracellularly in secretory granules until translocated to the plasma membrane on activation. P-selectin mediates neutrophil-platelet adhesion and the ‘rolling’ attachment of neutrophils to activated endothelium. The primary normal function of selectins appears to be to direct extravasation of leucocytes. Selectin deficiencies have not been described, but the congenital absence of a putative selectin ligand (sialyl-Lewisx)

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was found in association with severe neutrophil adhesion deficiency and recurrent bacterial infection in two unrelated boys (Elziani et al., 1992). Selectins have also been implicated in immunopathological aspects of inflammatory responses; anti-selectin antibodies have been found to be protective in experimentallyinduced neutrophil-dependent lung injury (Mulligan et al., 1992) and myocardial reperfusion injury (Weyrich et al., 1993). E-selectin has even been implicated in the haematogenous spread of colonic cancer (Dejana et al., 1992). Selectins are also found in soluble form in the blood stream and these circulating selectins are sometimes elevated in disease states. The clinical utility of measuring circulating selectins has not yet been established, but any diagnostic or prognostic value in particular disorders should become known within the next decade (Gearing and Newman, 1993). Most knowledge of selectins has come from work reported during the early 1990s, and it may be premature to suggest that the field of study will soon lead to therapeutic applications. However, selectin-carbohydrate interactions are clearly of importance in inflammatory processes and the possibility is raised that greater understanding of these interactions may lead to the development of novel anti-inflammatory compounds. Pentraxins The pentraxins are a conserved family of lectin-like plasma proteins with a characteristic pentameric arrangement of subunits. They do not have a domain equivalent to the carbohydrate recognition domain common to collectins and selectins and were not classified as C-type by Drickamer (1988). Nevertheless, their lectin activity is Ca2+-dependent and therefore it seems justifiable to group them with the C-types rather than the heterogenous mishmash of N-type lectins. The human C-reactive protein and serum amyloid P component can be classified as pentraxin lectins; both can activate complement and may function as part of a primitive host defence mechanism. C-reactive protein, so called because of its ability to bind the C protein of Streptococcus pneumoniae, is the human analogue of the horseshoe crab lectin, limulin. It has a molecular weight of approximately 115000 and is composed of five identical subunits of 23000 (Osmand et al., 1977). It can bind to galactans and DNA as well as phosphoryl choline in the C glycoprotein. C-reactive protein is perhaps the most remarkable of the acute-phase proteins. It has a half-life of a few hours and can change its concentration in the plasma from typically around 0.5 µ g ml−1 to as much as 400 µ g ml−1 or more in less than a day (Whicher and Dieppe, 1985). It is measured as a sensitive marker of inflammation in a wide variety of clinical settings. Sometimes C-reactive protein measurement may provide the only evidence of an inflammatory event; often it is useful for assessing disease activity and response to treatment; it is even a crude means of distinguishing viral (low levels) from bacterial (high levels) infections (Whicher and Dieppe, 1985). Amyloid P component is a tissue and plasma -glycoprotein, which is also a universal constituent of primary and secondary amyloid deposits. Serum amyloid P component (SAP) possesses identical subunits of apparent molecular weight 36000 forming pentagonal units (Benson et al., 1976). Its true native molecular weight appears to be 240000 (Perkins and Pepys, 1986), with subunits of Mr 24000 forming a pair of pentameric rings. SAP displays only a very modest concentration rise during the acute-phase response in humans, despite a 50 per cent amino acid homology with C-reactive protein (Pepys and Baltz, 1983). SAP binds to various carbohydrate and non-carbohydrate structures, but behaves as a lectin with galactan specificity (Li et al., 1984). It is strongly anti-mitogenic towards human mononuclear cells stimulated to proliferate with PPD, PHA or PWM in vitro, raising the possibility of an immunomodulary role in vivo (Li et al., 1984). There is also evidence that the physiological functions of SAP may include opsonization of

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immune complexes (Brown and Anderson, 1993). However, in the absence of a deficiency disease, the precise function(s) of SAP (as for C-reactive protein) is hard to establish. S-type lectins S-type (sulphydryl-dependent) lectins, also called galaptins, S-Lac lectins, and soluble -galactosidebinding lectins, form a large family with highly conserved primary structures and carbohydrate specificity (Harrison, 1991). General features of the family include specificity for N-acetyllactosamine ( -galactosyl 1, 4-N-acetylglucosamine), and inactivation upon oxidation necessitating the presence of reducing agents like 2-mercaptoethanol or dithiothreitol during isolation and storage. Many exhibit developmental regulation within the tissue of origin. By localisation experiments they have been shown to occur both free and membraneattached within the cytoplasm, at the cell surface, and outside the cell. As membrane-bound galaptins can be released with lactose without a need for detergents, they are not integral membrane components. They have been found in a wide variety of tissues, but are particularly associated with muscle and connective tissue. It has been suggested that they may contribute to the organisation of the extracellular matrix and so influence cell adhesion, migration and differentiation (Barondes, 1984; Harrison and Chesterton, 1980). The recent observation that a murine S-type lectin is a growth regulatory and cytostatic factor for embryonic fibroblasts (Wells and Mallucci, 1991) is consistent with such concepts. Significantly, these characteristics were independent of its lectin (haemagglutinating) activity. The best-characterized S-type lectins come from a sub-family containing a single subunit of about 14000 Mr. Human members of this group have been described in lung (Powell, 1980), spleen (Allen et al., 1987), placenta (Hirabayashi and Kasai, 1984; Hirabayashi et al., 1987) and heart and skeletal muscle (Childs and Feizi, 1979). The human placental lectin has been completely sequenced (Hirabayashi and Kasai, 1988) and cloned (Couraud et al., 1989). The recombinant protein can largely prevent the development of experimental autoimmune encephalomyelitis (induced by immunization with myelin basic protein in complete Freund’s adjuvant) in Lewis rats (Offner et al., 1990). This exciting finding is potentially important for two reasons. First, it suggests the possible therapeutic application of human placental lectin to prevent or ameliorate multiple sclerosis and other autoimmune diseases; it is neither immunogenic nor cytotoxic, and so has an advantage over therapeutic options like mouse or hybrid monoclonal antibodies or cytotoxic drugs. Second, this immunosuppressive property suggests a possible natural function in regulating the maternal immune response to fetal antigens. We have studied the localization of the placental lectin within the human placenta by immunoperoxidase staining (Bevan et al., 1994). It is completely absent from the syncytiotrophoblast which is in direct contact with the maternal blood supply, and from the underlying cytotrophoblast (Figure 9.1a). However, some specific staining was observed in columns of extravillous cytotrophoblast obtained early in the first trimester (Figure 9.1b). Villous stromal cells were stained as were the walls of blood vessels. The strongest staining was found in the deciduum, especially in early pregnancy specimens (Figure 9.1c), and similar intensity was observed in endometrial biopsies from non-pregnant patients. The results are consistent with a role for the lectin in the decidual control of trophoblast migration and proliferation, perhaps by interaction with the macrophages and granular lymphocytes of the deciduum as the lectin can induce secretion of cytokines from monocytes (Kajikawa et al., 1986). A second well-conserved sub-family of S-type lectins has molecular sizes in the range 30–35000 (Harrison, 1991). Members have two domains, one similar to the Mr=14000 lectins, the other with a characteristic repeat sequence containing nine amino acids. Human examples include HL-29 from lung

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Figure 9.1. Localisation of human placental lectin. First trimester human placenta was fixed in formaldehyde, embedded in paraffin and sectioned. Sections were treated with rabbit antiserum to placental lectin (or normal rabbit serum) followed by swine anti-rabbit immunoglobulin, rabbit peroxidase-anti-peroxidase and diaminobenzidine as substrate. (a) Villus treated with anti-lectin serum, demonstrating absence of stainmg on trophoblast (×300); (b) extravillous cytotrophoblast columns treated with anti-lectin serum, showing some specific staining (×192); (c) deciduum treated with anti-lectin serum, with intense staining in stromal cells (×120); (d) deciduum treated with normal (non-immune) rabbit serum (×120).

(Sparrow et al., 1987), Mac-2 from macrophages (Cherayil et al., 1990) and BP, an IgE-binding protein on lymphoid cell lines (Robertson et al., 1990). The carbohydrate-binding activity of this subfamily is not

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dependent on the presence of reducing agents (Frigeri et al., 1990) and the amino terminal tandem repeat domain permits self-association and co-operative binding (Massa et al., 1993). These lectins may well function extracellularly and Liu (1993) has proposed a broad spectrum modulatory function in biological responses in addition to a more specific role for BP in amplifying allergic inflammation by binding both IgE and IgE receptors on mast cells. Several S-type lectins not belonging to these major subfamilies have been reported from various mammalian sources and one from human lung (Sparrow et al., 1987). We are at an early stage of understanding the biological significance of S-type lectins, but their ubiquity and highly conserved sequences indicate an important function(s) both within and outside the immune system. Further research will undoubtedly lead to a better understanding of their role in biological processes and concomitantly the opportunity to manipulate the mechanisms concerned for therapeutic benefit either by appropriate exogenous application of recombinant lectin or by exogenous application of its inhibitors. Hopefully the near future will throw more light on the relative importance of the haemagglutinating and growth regulatory active sites, and also on the influence of the oxidative state of the microenvironment in regulating those activities. Non-C, non-S(N)-type lectins The N-type lectins do not constitute a family or superfamily but rather consist of a miscellany of unrelated molecules which share only the feature of being able to bind to some carbohydrate structures. Lymphocytes and other lymphoid cells possess endogenous surface lectins. CD2, the receptor for sheep erythrocytes, seems to bind to saccharide structures on its ligands (Boldt and Armstrong, 1976; Schlesinger et al., 1984). CD8+ human T-cells have non-cytotoxic, rhamnose-binding, antigen non-specific suppressor factors on their surface (Grillon et al., 1991). The role of surface lectins on large granular lymphocytes in natural killer (NK) cell activity has been the subject of recent concise (Bezouska et al., 1991) and full-length (Yokoyama and Seaman, 1993) reviews. The NK cell investigations have even implicated CD45, the leucocyte common antigen discussed earlier as a tyrosine phosphorylase regulator of mitogenesis, as a lectin itself (Bezouska et al., 1993). Soluble suppressor lectins have been described in conditioned media from proliferating mononuclear cell cultures (Fleisher et al., 1981; Greene et al., 1981b). There may be some overlap between these and the secreted forms of the suppressor factors present in CD8+ lymphocytes referred to above (Grillon et al., 1991). Several cytokines appear to possess lectin-like activity independent of their interleukin activities (Muchmore and Decker, 1987; Sherblom et al., 1988, 1989). These observations were based upon the binding of Tamm-Horsfall glycoprotein to IL-1, IL-2 or tumour necrosis factor immobilized on ELISA plates. Doubt has been cast on the validity of these observations, however, by the discoveries that tumour necrosis factor and IL-1 require a conformational change (by binding to a solid phase, or low pH) to bind to Tamm-Horsfall glycoprotein, and that the latter binds non-specifically to proteins under acidic conditions (Moonen et al., 1988). Indeed, Lambert and co-workers (1993) have confirmed that tumour necrosis factor does not bind to the Tamm-Horsfall glycoprotein under physiological conditions. However, a genuine function for tumour necrosis factor that it binds Tamm-Horsfall glycoprotein in an acidic micro-environment cannot be ruled out. Neither can one extrapolate from tumour necrosis factor to the binding properties of IL-2, which incidentally possesses substantial sequence homology with MBP (Sherblom et al., 1989). Understanding the relationship between, and the need for, separate interleukin and carbohydrate-binding activities on the same molecule, as for the separate growth regulating and carbohydrate-binding activities of

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murine S-type lectin (Wells and Mallucci, 1991), should be a great advance in the appreciation of the evolution and function of these molecules. Blood plasma contains a number of immunoglobulins with specificity for carbohydrates (Sela et al., 1975; Lalezari and Jiang, 1984; Summerfield and Taylor, 1986) which, if they were not immunoglobulins could be classified as lectins, and represent an overlap between these two groups of humoral defence molecules. Plasma also may contain other non-immunoglobulin lectins (Taylor and Summer-field, 1987; Hamazaki, 1986) in addition to those listed in the categories above. The fact that there are so many human lectins already characterized, and probably many more still to be described, simply confirms the importance and complexity of lectin-carbohydrate interactions underlying the human immune system. Acknowledgements I thank Dr Bridget Bevan for preparing Figure 9.1, and Mrs Denise Lynch for excellent secretarial assistance. References Akiyama, Y., Zicht, R., Ferone, S., Bonnard, G.D. and Herberman, R.B., 1985, Effect of monoclonal antibodies to class I and class II HLA antigens on lectin and MoAb OKT3-induced lymphocyte proliferation, Cellular Immunology, 91, 477–91. Allen, H.J., Cywinski, M., Palmberg, R. and DiCioccio, R.A., 1987, Comparative analysis of galactoside-binding lectins isolated from mammalian spleens, Archives of Biochemistry and Biophysics, 256, 523–33. Altman, A., Coggeshall, K.M. and Mustelin, T., 1990, Molecular events mediating T cell activation, Advances in Immunology, 48, 227–360. Andersen, O., Laursen, S.B., Svelag, S-E., Holmskov, U. and Thiel, S., 1991, Mammalian lectins in defense mechanisms against microorganisms, in Kilpatrick, D.C., Van Driessche, E. and Bog-Hansen, T-C., (Eds), Lectin Reviews, vol. 1, pp. 41–51, St Louis: Sigma Chemical Company. Areman, E.M. and Sacher, R.A., 1991, Bone marrow processing for transplantation, Transfusion Medicine Reviews, 5, 214–27. Axelsson, B., Kimura, A., Hammarström, S., Wigzell, H., Nilsson, K. and Mellstedt, H., 1978, Helix pomatia A hemagglutinin: selectivity of binding to lymphocyte surface glycopeptides on T cells and certain B cells, European Journal of Immunology, 8, 757–64. Baenziger, J.H. and Maynard, Y., 1980, Human hepatic lectin. Physiochemical properties and specificity, Journal of Biological Chemistry, 255, 4607–13. Barclay, A.N., Birkeland, M.L., Brown, M.H., Reyers, A.D., Dans, S.J., Somoza, C. and Williams, A.F., 1993, The Leukocyte Antigen Facts Book, London: Academic Press. Barondes, S.H., 1984, Soluble lectins: a new class of extracellular proteins, Science, 223, 1259–64. Barrett, D.J., Edwards. J.R., Pietrantuono, B.A. and Ayoub, E.M., 1983, Inhibition of human lymphocyte activation by wheat germ agglutinin: a model for saccharidespecific suppressor factors, Cellular Immunology, 81, 287–97. Benson, M.D., Skinner, M., Shirahama, T. and Cohen, A.S., 1976, P-component of amyloid. Isolation from human serum by affinity chromatography, Arthritis and Rheumatism, 19, 749–54. Bevan, B.H., Kilpatrick, D.C., Liston, W.A., Hirabayashi, J. and Kasai, K., 1994, Immunohistochemical localisation of a -D-galactoside-binding lectin at the human maternofetal interface, Histochemical Journal, 26, 582–86. Bevilacqua, M.P. and Nelson, R.M., 1993, Selectins, Journal of Clinical Investigation, 91, 379–87. Bezouska, K., Pospisil, M., Kubrycht, J., Holan, Z., Lukesova, D. and Kocourek, J., 1991, The role of endogenous lectins in NK cell cytotoxicity, in Kilpatrick, D.C., Van Driessche, E. and Bog-Hansen, T-C. (Eds), Lectin Reviews ,vol . 1, pp. 41–51, St Louis: Sigma Chemical Company.

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Harel, W. and Nelken, D., 1983, The effect of lentil lectin treatment on skin allograft survival in mice and rats , inBogHansen, T.C. and Spengler, G.A. (Eds) Lectins-Biology, Biochemistry, Clinical Biochemistry, vol. 3, pp. 17–25, Berlin: Walter de Gruyter. Harrison, L., 1991, Soluble -galactoside-binding lectins in vertebrates, in Kilpatrick, D.C., Van Driessche, E. and BogHansen, T-C. (Eds) Lectin Reviews, vol. 1, pp. 17–39, St Louis: Sigma Chemical Company. Harrison, F.L. and Chesterton, C.J., 1980, Factors mediating cell-cell recognition and adhesion. Galaptins; a recently discovered class of bridging molecules, FEBS Letters, 122, 157–65. Hartshorn, K.L., Sastry, K., White, M.R., Andes, E.M., Super, M., Ezekowitz, R.A. and Tauber, A.I., 1993, Human mannose-binding protein functions as an opsonin for influenza A viruses, Journal of Clinical Investigation, 91, 1414–20. Henkart, P.A. and Fisher, R.I., 1975, Characterization of the lymphocyte-surface receptors for con A and PHA, Journal of Immunology, 114, 710–14. Hilgert, I., Horejsi, V., Angelisova, P. and Kristofova, H., 1980, Lentil lectin effectively induces allotransplantation tolerance in mice, Nature, 284, 273–75. Hilgert, I., Kristofova, H., Angelisova, P. and Horejsi, V., 1983, Adult transplantation tolerance reduced by lentil lectin IV. Cellular mechanisms involved in adult allotransplantation. Tolerance induced in mice by lentil lectin treatment, Folia Biologica, 29, 307–319. Hilgert, I., Kristofova, H., Kren, V. and Panczak, A., 1984, The effect of lentil lectin treatment on the survival of rat skin allografts in strain combinations with different genetic disparity, Folia Biologica, 30, 104–7. Hilgert, I., Stoyanor, S. and Kristofová, H., 1987, Immunosuppression induced by monoclonal anti-Thy 1,2 antibodies, cyclosporin A, and lentil lectin in mice, Transplantation Proceedings, 19, 1269. Hirabayashi, J. and Kasai, K., 1984, Human placenta -galactoside-binding lectin. Purification and some properties, Biochemical and Biophysical Research Communications, 122, 938–44. Hirabayashi, J. and Kasai, K., 1988, Complete amino acid sequence of a -galactoside binding lectin from human placenta, Journal of Biochemistry, 104, 1–4. Hirabayashi, J., Kawasaki, H., Suzuki, K. and Kasai, K., 1987, Further characterization and structural studies on human placenta lectin, Journal of Biochemistry, 101, 987–95. Holter, W., Majdic, D., Stockinger, H., Howard, B.H. and Knapp, W., 1988, Regulation of the CD2 alternative pathway of T cell activation by CD3, Journal of Immunology, 140, 1043–46. Hsu, S-M. and Ree, H.J., 1983, Histochemical studies on lectin binding in reactive lymphoid tissues, Journal of Histochemistry and Cytochemistry, 31, 538–46. Hutchins, D. and Steel, C.M., 1983, Phytohaemagglutinin induced proliferation of human T lymphocytes: differences between neonate and adults in accessory cell requirements, Clinical and Experimental Immunology, 52, 355–64. Ikeda, K., Sannoh, T., Kawasaki, N., Kawasaki, T. and Yamashiva, I., 1987, Serum lectin with known structure activates complement through the classical pathway, Journal of Biological Chemistry, 262, 7451–54. Iwata, M., Ide H., Terao, T. and Osawa, T., 1977, Membrane receptors of mouse lymphocytes for various lectins, Journal of Biochemistry, 82, 661–69. Jelinek, D.F. and Lipsky, P.E., 1987, Regulation of human B lymphocyte activation, proliferation, and differentiation, Advances in Immunology, 40, 1–59. Kajikawa, T., Nakajima, Y., Hirabayashi, J., Kasai, K. and Yamazaki, M., 1986, Release of cytotoxin by macrophages on treatment with human placenta lectin, Life Sciences, 39, 1177–81. Kanellopoulos, J.M., de Petris, S., Leca, G. and Crumpton, M.J., 1985, The mitogen lectin from Phaseolus vulgaris does not recognise the T3 antigen of human T lymphocytes, European Journal of Immunology, 15, 479–86. Kilpatrick, D.C., 1988, Accessory cell paradox: monocytes enhance or inhibit lectin medi ated human T lymphocyte proliferation depending on the choice of mitogen, Scandinavian journal of Immunology, 28, 247–49. Kilpatrick, D.C. and Darg, C., 1983, Enzymatic modification of the lymphocyte surface. Application to tissue typing and rosetting with sheep erythrocytes, Tissue Antigens, 21, 309–17.

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Chapter 10 Lectin Cytochemistry and Intestinal Epithelial Cell Biology Timothy P.King

Introduction Membrane-associated oligosaccharides fulfil important roles in the development, organization and growth of complex organisms (Feizi, 1988; Feizi and Childs, 1987). In the small intestine these oligosaccharide moieties, which occur on both enzyme and non-enzyme glycoconjugates, provide a wide range of potential binding sites for luminal and circulating biologically active ligands such as growth factors, hormones, bacteria, toxins and lectins (for reviews see Kelly et al., 1992; Pusztai, 1991; Stewart et al., 1993). Because of their high degree of carbohydrate specificity, lectins have been increasingly applied as reliable and discriminating reagents for the cytochemical detection of glycoconjugates (Damjanov, 1987; Pavelka and Ellinger, 1991; Roth, 1993; Spicer and Schulte, 1992). The purpose of this review is to highlight past and recent contributions made by lectin cytochemistry in elucidating vital processes of glycosylation and epithelial differentiation in the mammalian small intestine. In 1974, Etzler and Branstrator published a paper on the use of lectins as cytochemical tools for the differential localization of cell surface and secretory components in the rat intestinal epithelium. Lectins were isolated from Dolichos biflorus, Lotus tetragonolobus, Ricinus communis-1 (RCA-1) and Triticum vulgare (WGA), coupled to fluorescein isothiocyanate (FITC) and applied to sections of rat small intestine. The cytochemical results from this early study demonstrated a clear relationship between intestinal differentiation and cellular glycosylation. During the intervening 20 years, lectin cytochemistry has evolved to become a major investigative tool for gastrointestinal research and is unrivalled for in situ localization of oligosaccharide moieties both within and upon the surfaces of intestinal epithelial cells. The successful evolution of lectin cytochemistry may be attributed to a variety of factors. Vital insights into important interactions between lectins and intestinal glycoconjugates have come from parallel research carried out over the past twenty years on the nutritional effects of dietary lectins (for review see Pusztai, 1991). The expansion of lectin cytochemistry has been fuelled by the work of scientists involved in purifying and characterizing lectins. Many of the most useful lectin probes have been isolated from plant sources and several major advances in blood typing and gastrointestinal research had their origins in botanical science. Lectin cytochemistry also owes much to the skilful development of high quality tissue processing and lectin-labelling procedures. As will become clear in the following sections, a handful of scientists have contributed greatly to this area. It is fortunate that many technical developments, especially those involving lectin-labelling at the electron microscope level, have been undertaken by scientists with an interest in the structure and function of the small intestine.

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Techniques of lectin cytochemistry Many lectins, of plant and animal origin have been purified and their properties defined (Goldstein and Poretz, 1986; Pusztai, 1991; Shibuya et al., 1987; Shibuya et al., 1988; Wang and Cummings, 1988; Wu et al., 1988). Table 10.1 lists several well characterized lectins that have been used to probe intracellular and cell surface glycoconjugates in the mammalian small intestine. The acronyms for lectin names listed in the table are also used in the text. Technical aspects of lectin cytochemistry have been reviewed by several authors (Benhamou, 1989; Horisberger, 1985; Leathem and Atkins, 1983; Schumacher et al., 1991). Early methods used to detect lectin probes were applicable only for light microscopy (LM) but increasingly, electron dense markers have been employed which may be visualized by both LM and electron microscopy (EM). Advances in the design and manufacture of specialized acrylic embedding resins have contributed significantly to the development of cytochemical technique (Ellinger and Pavelka, 1985; Scala et al., 1992). For postembedding lectin cytochemistry, the most popular of many related protocols, it is now possible to chemically or physically fix tissue samples, embed them in resins, cut large area 1 µ m thick sections for LM cytochemistry and then proceed to ultra-thin sectioning and EM labelling on precisely the same cells and tissues. Most lectin cytochemical techniques involve the use of lectin probes which are physically or chemically bound to appropriately sensitive marker systems. The exceptions to this rule are immunocytochemical methods where native lectin probes are detected with anti-lectin antibodies. Cytochemical marker systems have been devised which do not interfere with the sugar-binding characteristics of the lectin probes. Lectins conjugated to FITC, tetramethylrhodamine isothiocyanate (TRITC) or other fluorescent markers are widely used in LM investigations (King and Kelly, 1990; 1991). Modern optical filter systems in fluorescence microscopes and confocal systems are particularly suited for double and triple labelling procedures using lectins labelled with different fluorochromes (Schumacher et al., 1991). Lectin-horseradish peroxidase (HRP) conjugates have been extensively used in both LM and EM labelling protocols with excellent results (Pavelka and Ellinger, 1989, Spicer and Schulte, 1992). Inherent limitations of HRP cytochemical tech Table 10.1. Lectins used in affinity cytochemistry of intestinal glycoconjugates. Lectin (source)

Acronym Nominal carbohydrate specificity

Inhibitory concentrations of sugars used in cytochemistry

Canavalia ensiformis (jack bean) Lens culinaris (lentil) Pisum sativum (pea) Galanthus nivalis (snowdrop) Triticum vulgare (wheat germ)

Con A LCA PSA GNA WGA

Dolichos biflorus (horse gram) Helix pomatia (snail) Arachis hypogaea (peanut) Artocarpus integrifolia Ricinus communis (castor bean) Erythrina cristagalli (coral tree)

DBA HPL PNA Jacalin RCA-1 ECA

200 mM methyl- -mannoside 200–500 mM Man/Glc 50–200 mM Man/Glc 200 mM methyl- -mannoside 10mM triacetylchitotriose 15–150 mM GlcNAc 200 mM GalNAc 10–200 mM GalNAc 50–200 mM Gal 50–300 mM Gal 100–400 mM Gal 10mM Gal 1,4GlcNAc

Man> Glc>GlcNAc Man> Glc>GalNAc Man> Glc=GlcNAc Man>Man 1,3Man GlcNac( 1,4GlcNAc)1–2 > GlcNAc>NeuAc GalNAc 1,3GalNAc» GalNAc GalNAc 1,3GalNAc> GalNAc Gal 1,3GalNAc> and Gal Gal 1,3GalNAc Gal> Gal»GalNAc Gal 1,4GlcNAc

LECTIN CYTOCHEMISTRY AND INTESTINAL EPITHELIAL CELL BIOLOGY

Lectin (source)

Acronym Nominal carbohydrate specificity

Inhibitory concentrations of sugars used in cytochemistry

Lotus tetragonololobus (asparagus pea) Ulex europeus (gorse seed) Sambucus nigra (elderberry) Maackia amurensis

LTA

200 mM Fuc

UEA-1 SNA-1 MAA-2

L-Fuc> L-Fuc1,2Gal 1, 4GlcNAc L-Fuc1,2Gal 1,4GlcNAc NeuAc 2,6Gal/GalNAc NeuAc 2,3Gal 1,4GlcNAc/Glc

157

200 mM Fuc 0.1–1 mM 2,6 sialyllactose 5–50mM 2,3 sialyllactose

niques, often more perceived than actual, include difficulties in suppressing endogenous peroxidase activity and diffusion of the amorphous precipitate produced by the frequently used diaminobenzidine (DAB) reaction. Roth et al. (1992) introduced an anti-HRP antibody-gold complex (anti-HRP-gold) to replace the DAB reaction in the detection of HRP-conjugated lectins. Biotinylated lectins are widely used as cytochemical probes at both the LM and EM levels. Several systems are available for the detection of biotin markers, including FITC-labelled anti-biotin antibodies, anti-biotin: gold complexes, avidin:gold complexes and avidin-biotin: peroxidase complexes followed by the DAB reaction or by the use of anti-HRP: gold (Hsu and Raine, 1982; Roth et al., 1992; Skutelsky et al., 1987). A perceived disadvantage of biotin as a marker is that this hapten may occur naturally in many tissues and organs, often necessitating special treatment of the tissue sections to block endogenous biotin (Wood and Warnke, 1981). The steroid hapten digoxigenin (DIG) has been introduced as a cytochemical marker for lectins (Sata et al., 1990). The primary advantage of DIG is that it does not occur in animal tissues, thus eliminating the need for blocking reactions prior to lectin incubation. DIG may be localized by immunocytochernical techniques employing monospecific anti-DIG antibodies either complexed directly to gold particles or detected by second antibodies linked to HRP. Protocols for the preparation and application of lectin:gold probes have been comprehensively described by Horisberger (1985) and Benhamou (1989). As indicated, gold markers are also widely used in secondary detection systems. At the EM level gold-labelling produces a distinct and permanent signal over subcellular structures with no interference from endogenous enzyme activities. At the LM level this signal may be amplified by the use of simple photochemical silver reactions. The resulting dense black reaction products are precise, clearly localized and may be counterstained with a variety of tissue stains. Silver enhancement has also been introduced as a procedure for enhancing gold-labelling at the EM level where the use of small 1–5 nm gold particles decreases steric hindrance and increases labelling intensity (King et al., 1987). Cytochemical controls in lectin-labelling procedures typically involve incubation of tissue sections with lectins previously incubated with inhibitory levels of competitive sugars (Table 10.1). For differential inhibition of lectin-binding reactions, intestinal tissues have been incubated in the presence of competing sugars at various concentrations (Pavelka and Ellinger, 1991). Non-specific reactions attributable to secondary detection systems are assessed by omitting the initial lectin-labelling steps from cytochemical procedures. Additional tests of specificity involve periodate oxidation or treatment of tissue sections with exoglycosidase enzymes which diminish levels of lectin-reactive sugars.

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Lectin cytochemistry of stem cells and epithelial cell lineages The mammalian small intestine possesses a remarkable capacity to respond, and rapidly adapt, to a diverse array of endogenous and exogenous stimuli. Vital to this adaptive capacity is a complex epithelial surface which is continually undergoing regeneration and differentiation. The intestinal crypt stem cells may be regarded as the vital ‘engines’ that sustain the renewal of the intestinal epithelium (Gordon, 1993). Division of multipotent stem cells located in the crypts of Lieberkuhn gives rise to progeny which undergo amplification and allocation to four principal intestinal epithelial cell lineages (Cheng and LeBlond, 1974). Studies on mice have demonstrated that differentiation programmes of these lineages are expressed during an orderly bipolar migration (reviewed by Gordon, 1993; Gordon et al., 1992). Paneth cells differentiate as they migrate towards the crypt base. Daughter cells representing the three other lineages, the polarized absorptive enterocytes, mucin-secreting goblet cells and enteroendocrine cells, migrate from the crypts to the tips of the intestinal villi where as senescent cells they are extruded into the intestinal lumen. The residence time of these epithelial cells on the villi is only 2–3 days in adult animals, yet during their translocation on the villus surfaces they undergo extensive differentiation. Lectin cytochemical analyses were employed in several simple yet highly innovative investigations into the nature of stem cells and their derived cell lineages on intestinal villi. These analyses were undertaken on selected strains of inbred mice, some of which express intestinal receptors for the lectin Dolichos biflorus agglutinin (DBA) and others which do not express these DBA binding sites (Ponder et al., 1985). DBA recognizes terminal non-reducing GalNac residues in a cell surface receptor encoded by the Dlb-I locus on mouse chromosome 11 (Ponder et al., 1985; Uiterdijk et al., 1986). Chimaeras prepared by aggregation of 4–8 cell embryos from DBA+ and DBA− strains developed into mice producing both DBA+ and DBA− intestinal crypt stem cells (Ponder et al., 1985, Schmidt et al., 1985a,b). The cell lineages from the DBA+ stem cells were visualized on tissue sections and whole mount preparations using peroxidase labelled DBA (Ponder et al., 1986; Schmidt et al., 1984). In neonates it was found that many crypts contained cells of both chimaeric genotypes, indicating the polyclonal origin of the intestinal epithelium at this stage of morphogenesis. The villus surfaces of these animals were characterized by the presence of randomly distributed DBA+ and DBA− cells (Schmidt et al., 1988). In adult chimaeric mice, the epithelium of each intestinal crypt was found to be derived from a single progenitor cell and crypts contained either one or the other DBA phenotype (Schmidt et al., 1985b). Coherent ribbons or bands of cells were observed on the villus surfaces of these animals. Each of the DBA+ and DBA− bands was derived from a single crypt, the width of the bands depended on the number of adjacent crypts with the same genotype. This lectin cytochemical data led to the important conclusion that in adult mice the small intestinal crypts are monoclonal and that their associated villi are polyclonal. The data was also consistent with the unitarian hypothesis of Cheng and LeBlond (1974) which proposed that multipotent crypt stem cells give rise to different epithelial cell lineages. Although lectin cytochemical analysis of DBA associated cellular mosaicism in embryo aggregation chimaeras proved an extremely valuable tool in cell lineage studies, the patch size of the mosaic cell population often proved larger than the affected territory of a single renewing stem cell and its descendants. Winton et al. (1988) devised a novel mutation protocol for marking smaller clones of cells at a chosen time. The intestinal epithelium of the Dlb-Ia/Dlb-Ib heterozygote mouse binds DBA in a uniform fashion. If the DlbIb allele is mutated, either spontaneously or after treatment with a mutagen such as ethylnitrosourea, it can lose its ability to encode a functional DBA receptor. If such a mutation occurs in a crypt stem cell it is recognizable in the small intestinal epithelium as a ribbon of DBA− cells extending up a villus (Figure 10.1). Mutations occurring before crypt formation give rise to clusters or descendent-clones of wholly DBA− crypts in the adult animals (Winton et al., 1988). Gordon et al. (1992) reviewed the potential

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Figure 10.1 Jejunal villus from a C57BL/6J×SWR F1 mouse which had received ethylnitrosourea (50 mg/kg−1) two weeks before killing. The villus was dissected from a whole mount stained with DBA peroxidase. A ribbon of unstained (DBA−) cells is seen (arrowed). Bar=50µ m.

of the DBA marker system for the analysis of spontaneous mutation rates and mutagen sensitivity in developing embryos.

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Epithelial maturation—glycoconjugate expression on the crypt-villus axis Suitability of oligosaccharides as differentiation markers Crypt-villus gradients in expressed glycoconjugates may be modified or diversified by a variety of cellular processes. The oligosaccharide structures of intestinal membrane and secretory glycoconjugates are not themselves primary gene products, but are constructed in a stepwise manner as monosaccharides are added to precursor oligosaccharides via several glycosyltransferases coded for by different genes (for reviews see; Ito and Hirota, 1992; Neutra and Forstner, 1987). The maturing glycoconjugate complexion of each cell type is influenced by many different factors such as the intrinsic composition of glycosyltransferase species defined by the genotype of the individuals, the relative activity or amount of these enzymes (repression, derepression or induction of the enzymes), competition between enzymes with overlapping substrate specificity, the organization of the enzymes in membranes, utilization of precursors and specific substrate sugars, and the activity level of degrading enzymes (Ito and Hirota, 1992). In spite of this considerable potential for diversity, glycosylation processes within the intestinal epithelium frequently proceed in a predictable fashion. As in many epithelia, the oligosaccharide moieties of intestinal membrane and secretory glycoconjugates become more complex during cellular differentiation (Damjanov, 1987; Spicer and Schulte, 1992). With the judicious use of appropriate lectins it is possible to identify carbohydrate sequences which characterize the core, backbone and terminal sequences found on N- and O-linked oligosaccharide chains. Most lectin probes only identify receptor moieties in the terminal or subterminal positions and are therefore of value as in situ markers of the level of completeness of oligosaccharide chains at various positions on the crypt villus axis. However, the absence of lectin reaction does not only signify the incomplete oligosaccharide synthesis; in some cases lectin receptor moieties may be present at a particular villus site but are simply masked by more complex structures. To be of value in defining maturational changes in glycoconjugate composition, lectin cytochemistry must be comprehensive and employ a suitably wide range of lectin probes. For example, using UEA-1 to label intestinal membranes in histo-blood group-A secretor pigs will present a falsely low picture of epithelial fucosylation. This fact only becomes evident when UEA-1 labelling is interpreted alongside lectin or antibody cytochemistry of histo-blood group A-antigen (King and Kelly, 1991). Maturation of absorptive enterocytes Using FITC-labelled lectins, Etzler and Branstrator (1974) demonstrated that rat jejunal epithelial membrane glycoconjugates lost LTA (Fuc) and RCA-1 (Gal) affinity while retaining WGA (GlcNAc) reactivity during cell maturation and migration from the crypts to the upper villi. These authors concluded that as intestinal crypt cells differentiate and move up the villi, their terminal carbohydrate residues are altered. In the small intestine of sucking and weaned pigs, GNA-labelling of high mannose moieties is high in the crypt regions but further along the crypt villus axis the reactivity of this lectin with microvillar membranes is generally much diminished (King, unpublished observations). Conversely, UEA-1 and ECA labelling of histo-blood group-H and precursor Gal 1, 4GlcNAc sequences is consistently stronger on the upper villus surfaces (King and Kelly, 1991 and unpublished observations). In the pig it appears that N-linked and incompletely synthesized O-linked glycoconjugates predominate on the crypt epithelium and fewer Nlinked but more O-linked glycoforms occur on the upper villus surfaces.

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Maturation of goblet cells Oligomucous or immature mucin secreting cells arise by mitosis of crypt stem cells. After 1–3 transit cell divisions, an irreversible differentiation event occurs and discrete goblet cell lineages migrate onto villi (Cheng, 1974; Paulus et al., 1993; Specian and Oliver, 1991). Goblet cells continually synthesise and secrete mucin throughout their life spans (Neutra and Forstner, 1987). In the human small intestine immature goblet cells deep within the crypts produce neutral mucins containing little sialic acid (Specian and Oliver, 1991). As they mature and migrate to the villus tip, the mucins become increasingly sialylated; these sialic acid residues not only increase the acidity of the molecule but are also sites for further modification by N- and O-acylation (Filipe and Fenger, 1979). UEA-1, DBA and WGA labelling of goblet cells was confined to the upper parts of villi in the normal human jejunum (Vecchi et al., 1987) indicating that the expression of histo-blood group-A and O antigens and related oligosaccharide moieties accompanies cellular differentiation. Similar maturation-associated glycosylation changes in goblet cell mucins occur in the porcine small intestine. SNA-1 and MAA-2 labelling of 2,6- and 2,3-linked NeuAc moieties is stronger in cells on the villus surfaces than in the immature crypt cells (King, unpublished observations). Glycosylation mosaicism Although lectin cytochemistry has provided valuable in situ data relating to membrane and mucin glycoconjugate changes associated with the migration of enterocytes along the crypt villus axis, not all the data has been entirely compatible with the model of crypt monoclonality and the orderly migration of cell lineages onto the villus surfaces. In particular, several investigations have revealed situations where adjacent intestinal epithelial cells have differed markedly in their glycoconjugate complexions. In postnatal rats Etzler and Branstrator (1979) observed that RCA-1 and LTA labelling of Gal and Fuc moieties occurred as patches on the villus surfaces. Taatjes and Roth (1990) employed SNA-1 to identify the cells expressing 2,6 NeuAc in the intestinal epithelium of weanling rats. On tissue sections at both the LM and EM levels their cytochemistry revealed the interesting feature of positively and negatively labelled cells interspersed along the crypt villus axis. The patchy or mosaic expression of histo-blood group-A and O antigens and compositionally related oligosaccharide moieties has also been observed in small intestines of pre- and post-weaned pigs (Figure 10.2; Kelly and King, 1991; Kelly et al., 1993; King and Kelly, 1991). The causes of epithelial mosaicism are uncertain. It may reflect subtle differences in the pattern of differentiation between monoclonally derived epithelial cells on the villus surfaces. This hypothesis has been proposed to explain mosaic lactase protein expression in hypolactasic humans (Maiuri et al., 1993a) and histo-blood group-A antigen expression in non-secretor adult human intestines (Maiuri et al., 1993b). As already indicated, mosaic patterns of gene expression may be induced on villus surfaces if the crypt stem cell population is heterogeneous. The assembly of coherent, vertically oriented sheets of clonally derived cells in mice takes about 14 days to become established after birth (Schmidt et al., 1988). The detected variations in epithelial glycosylation may indicate that the ‘purification’ of nascent crypts from polyclonality to monoclonality takes a little longer than was hitherto supposed. Alternatively, as each villus is supplied by several crypts and as a given crypt can supply several adjacent villi it is possible that the cytochemistry is detecting subtle genotypic variations between stem cell lineages. It might be argued that crypt diversity is too simple an explanation for the enormously varied mosaic images obtained. However, it must be remembered that in species such the pig, intestinal villi undergo major changes in shape in the preand post-weaning period (Kelly et al., 1992) and the precise routes of cell lineages from crypt to villus tips have not been determined.

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Figure 10.2. Jejunal villus from a 2 week-old sucking pig. The villus was dissected from a whole mount stained with UEA1-TRITC. A mosaic of UEA1+ and UEA1− cells is present on the villus surface. Bar=50 µ m.

M cell glycosylation markers The transcytosis of bacterial, viral and dietary antigens by specialized intestinal epithelial cells called microfold cells or M cells is one of the major prerequisites for initiation of a secretory immune response in the gut. Research on several species has shown that M cells possess characteristic structural features quite unlike the absorptive cells found on the surfaces of adjacent villi (for review see Kraehenbuhl and Neutra, 1992). In spite of these characteristics, positive identification of M cells, particularly in whole tissues, is not easily performed because of the lack of suitable positive markers. Recently however, Clark et al. (1993) examined the binding of labelled winged bean agglutinin (WBA, from Psophocarpus tetragonolobus) and UEA-1 to the follicle associated epithelium (FAE) overlying fixed mouse small intestinal Peyer’s patches

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and found that the lectins were almost exclusively M-cell specific. The WBA used by these authors consisted, at least in part, of WBA-2 which exhibits an affinity for blood group H structures. The authors concluded that both WBA and UEA-1 were recognizing fucosylated glycoconjugates on the M cell surfaces. Other lectins with affinity for Gal and GalNAc (Soyabean agglutinin and Helix aspersa agglutinin) failed to specifically label mouse M cells. However, similar M cell specific staining was not achieved when UEA-1 and WBA were applied to rat Peyer’s patch FAE or when UEA-1 was applied to that of rabbits (Clark et al., 1993). In the pig small intestine UEA-1 recognizes fucosylated sites on both FAE and normal villi (King and Sansom, unpublished observation). The possibility remains that other speciesspecific M cell glycosylation patterns await to be discovered. Temporal intestinal glycosylation changes Postnatal intestinal development involves extensive epithelial cell proliferation and cytodifferentiation, including changes in the expression of enzymes, receptors and transport systems. Age-related intestinal glycosylation changes play an important role in modifying the properties of intestinal receptors for dietary constituents as well as commensal and pathogenic bacteria (Kelly et al., 1992; Stewart et al., 1993). Intestinal membrane sialylation Sialic acids present on the terminal position of glycoproteins and gangliosides are involved in many aspects of normal and pathological cellular growth and development. These sugars play a key regulatory role in cellular and molecular recognition. In some situations they are the essential structural components of receptors for biological signals, in other circumstances they function as biological masks and via steric hindrance and/or electrostatic repulsion are able to prevent or reduce the accessibility of penultimate recognition sites (Pilatte et al., 1993; Schnaar, 1991). Two forms of lectin cytochemistry have been employed to investigate the expression of membrane sialoglycoconjugates in the developing pig and rat small intestines; indirect cytochemistry, based on PNAlabelling of tissue section Gal 1– 3GalNAc moieties before and after treatment with sialidase, and direct cytochemistry using SNA-1 and MAA-2. In early investigations, using the indirect procedure it was found that intestinal membrane sialylation was a conspicuous feature in newborn, sucking and weaned pigs (Gelberg et al., 1992; Kelly and King, 1991; King and Kelly, 1991). In recent experiments; SNA-1 and MAA-2 have been used to determine the expression of 2,6 and 2,3 sialylated structures in the intestinal membranes of pre- and post-weaned pigs (King, Begbie and Kelly, unpublished observations). SNA-1 labelling of NeuAc 2,6 linked to penultimate Gal or GalNAc residues was strong at birth, declined during sucking and was much diminished during the weaning period. This result is in agreement with similar lectin cytochemical studies showing postnatal decline in sialylation of rat microvillar membranes (Taatjes and Roth, 1990). Moderate and high levels of MAA-2 labelling of microvillar membranes were observed in many of the sucking and weaned pigs, signifying the presence of NeuAc 2,3-linked to Gal 1, 4GlcNAc of complex tri- and tetraantennary asparagine-linked oligisaccharides. 2,3-Linked NeuAc may also be present on N-acetyllactosamine sequences of O-linked oligosaccharides but there is evidence that MAA-2 only binds to such chains with low affinity (Wang and Cummings, 1988). The binding site of the lectin is complex and may recognize, in addition to 2,3-linked NeuAc, aspects of the underlying oligosaccharide sequence (Wang and Cummings, 1988). The presence of 2,6-linked NeuAc in the early postnatal intestine and 2,3-linked NeuAc in sucking and weaned intestines explains why the indirect cytochemical techniques failed to identify marked ontogenic changes in membrane sialylation in the porcine intestine (Kelly and

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King, 1991; King and Kelly, 1991). In the rat, where such changes have been more readily identified, 2,3linked NeuAc is not a common constituent of intestinal membrane glycoconjugates (Biol et al., 1993, Hamr et al., 1993; Taatjes and Roth 1990). Histo-blood group antigens King and Kelly (1990, 1991) investigated changes in the expression of histo-blood group-AO antigens and precursor oligosaccharide sequences in the piglet intestine during an eight week prolonged sucking period. Lectin cytochemistry was undertaken on sections of resin-embedded jejunal tissue and semi-quantitative scoring systems were adopted for categories of FITC- or TRITC-labelled lectins (and some monoclonal antibodies) reactive with carbohydrate moieties present on core, back-bone and terminal oligosaccharide sequences of histo-blood group antigens. Labelling of goblet cell mucins with UEA-1 (and similarly reactive monoclonal antibodies) was low or absent for the first three weeks of the sucking period. However, the mucin granules were strongly labelled with ECA, indicating the presence of Gal 1,4GlcNAc sequences in the mucins of these young animals. These sequences are structural precursors of the UEA-1-reactive Hantigens. By the fifth week of sucking, fucosylation was evident in all animals with the goblet cell contents strongly labelled by UEA-1. By the seventh and eighth weeks of sucking, terminal glycosylation of mucin glycoconjugates varied according to the AO secretor status of the individuals. Mucin granules from Osecretors were strongly labelled with UEA-1 whereas in the A-secretors the majority of the H antigenic sites were masked by terminal GalNAc moieties of histo-blood group A-antigen. The latter was revealed by labelling with HPL and A-specific monoclonal antibodies. In the same investigation similar developmental changes were observed in the glycosylation of membrane glycoconjugates (King and Kelly, 1991). However, unlike the goblet mucin, membrane labelling with UEA-1 was undetectable during the first three weeks of sucking and apparent only during the latter half of the eight week experiment. More complex membrane glycosylation involving increasing levels of fucosylation and/or the expression of histo-blood group A-antigen were detected during the latter part of the sucking period. In other cytochemical investigations on post-weaned pigs, UEA-1 labelling of intestinal membranes has been found to be a conspicuous feature of all O-secretor animals (King, Begbie and Kelly, unpublished observations). Similar pre- and post-weaning changes in the expression of fucosylated glycoconjugates have been observed in the rat intestine. Taatjes and Roth (1990) observed that UEA-1 labelling was restricted to goblet cell mucin in sucking rats, but by postnatal day 23 labelling appeared over the microvillar membranes of some epithelial cells. In adult rats intense staining with UEA-1 was found over goblet cell mucin and microvillar membranes. Postnatal glycosylation `shifts' A progressive change from 2,6 sialylation to 1,2 fucosylation of microvillar glycoconjugates occurs during postnatal development in both pigs and rats (King and Kelly, 1991; King, Begbie and Kelly, unpublished observations; Taatjes and Roth, 1990). In these species the reduction of intestinal 2,6 sialylation and the increase in fucosylation are often separated from one another by a lag phase. Several factors may contribute to the increased expression of fucosylated moieties in the developing intestine. In the rat, fucosyl-, galactosyl- and N-acetylgalactosaminyl-transferase activities are at a low level during the sucking period, are enhanced near weaning and rapidly reach a plateau until adulthood. Conversely, 2,6sialyltransferase activities actively decrease from birth to weaning (Biol et al., 1987, 1993; Chu and Walker, 1986; Ozaki et al., 1989; Ruggiero-Lopez et al., 1991). Specificity of acceptor structure is a key

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determinant of glycosyltransferase activity. For example, if core GalNAc moieties are substituted by 2,6NeuAc, further glycosylation including fucosylation is inhibited (Neutra and Forstner, 1987). As indicated above, ECA-reactive Gal 1, 4GlcNAc membrane glycoconjugates are particularly conspicuous in the pig intestine during the sucking period. Branching of these O-and N-linked backbone sequences is known to affect acceptor structure and influence the shift from sialylation to fucosylation (Clausen and Hakomori, 1989; Dabelsteen et al., 1982). Recent biochemical studies have elegantly demonstrated the effects of endogenous soluble- -galactoside binding lectins and protein inhibitors of fucosyltransferase and other enzymes involved in the intestinal fucosylation process (Biol et al., 1992, 1993; Ruggiero-Lopez et al., 1992). In the same way that lectin cytochemistry has revealed postnatal changes in intestinal 2,6 sialylation and 1,2 fucosylation, there is some evidence that in pigs intestinal membrane oligomannose-type N-linked oligosaccharides may be replaced by complex, or hybrid N-linked structures. GNA-labelling of jejunal membrane in newborn and sucking animals is significantly lower than in weaned individuals. Conversely MAA-2 labelling of 2,3-linked NeuAc is increased after weaning (King and Kelly, unpublished observations). Inter- and intra-species variation in intestinal glycosylation Glycosylation of equivalent cells and cytoplasmic macromolecules frequently differ among species (Damjanov, 1987). Analytical biochemical and lectin cytochemical studies have revealed species-specific differences between glycoconjugates expressed in the small intestines of humans and other mammalian species (Clark et al., 1993; Leffler, 1988; Oriol, 1987; Skutelsky et al., 1989). Using lectin cytochemistry on a wide range of mouse tissues, Spicer et al. (1987) observed extensive glycosylation differences between mouse species and between littermates of outbred species. These differences were not observed between individuals in inbred strains. In humans and many other mammalian species important individual differences in the glycosylation of intestinal membranes and secretions may be correlated with ABO histoblood groups and are thus genetically mediated (Oriol, 1987; Yamamoto et al., 1990). Lectin cytochemical investigations on both the human and pig small intestines have demonstrated that the histo-blood group antigens profoundly influence the glycoconjugate complexions of epithelial surfaces (King and Kelly, 1990, 1991; Mollicone et al., 1986). Mechanisms of intestinal glycosylation Detailed investigations into mechanisms and sub-cellular compartmentalization of cellular glycosylation processes have been relatively slow to develop because of difficulties in preparing and characterizing monoclonal antibodies against glycosyltransferase enzymes. Where such antibodies have been employed, a correspondence between the localization of a glycosyltransferase and the product of its action has frequently been observed (Roth, 1991; Taatjes and Roth, 1991). The recognition of such products by lectin cytochemistry has played a prominent role in elucidating the role of the Golgi apparatus and its associated endomembrane systems in cellular glycosylation processes. Data from several biochemical and cytochemical investigations has led to a general consensus among cell biologists that within the Golgi apparatus and associated endomembrane system the mechanisms of cellular glycosylation are functionally compartmentalized (for reviews see Pavelka and Ellinger, 1991; Roth, 1987, 1991). Glycosyltransferase enzymes, which act early in the glycosylation pathway, are predominantly located in cis-elements of the Golgi apparatus and those acting at terminai steps are located in trans-

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Figure 10.3. Diagrammatic representation of the Golgi apparatus and associated endomembrane system of an intestinal absorptive cell. Lectin cytochemical data from several sources (see text) has been pooled to give an overall impression of the extent of compartmentation of O-linked and N-linked glycosylation.

elements. This functional polarity is not absolute; glycosyltransferase enzymes and their products may be localized in more than one Golgi cisternae (Roth, 1991), or different enzymes may overlap in their distribution in specific Golgi cisternae (Nilsson et al., 1993). Using labelled lectins with specificity for appropriate sequences, several investigators have attempted to characterize the subcellular distribution of core, backbone and terminal sequences found on both N- and Olinked oligosaccharide chains. As already discussed, most lectin probes only identify receptor moieties in the terminal or subterminal positions, a characteristic which can make them particularly valuable as in situ markers of the level of completeness of oligosaccharide chains in different subcellular compartments. A major limitation of lectin cytochemistry is that it cannot discriminate between the different glycoconjugate species which may be traversing the Golgi apparatus at any given time. Carbohydrate groups identified by lectins (and monoclonal antibodies) may not be on the same oligosaccharide chains or even on the same glycoconjugates. Where possible therefore, cytochemical data should be augmented by data from affinity biochemical and/or cell fractionation studies. Absorptive enterocytes In polarized epithelial cells, such as absorptive enterocytes, the Golgi apparatus plays a pivotal role in the organization of protein trafficking pathways (for review see Nelson, 1992). Vital and diverse cellular functions centred on this apparatus include the condensation of secretory proteins, glycosylation and other post-translational processing of proteins and lipids (Roth, 1987). The structure of the Golgi apparatus is complex. In absorptive enterocytes, subunits of the apparatus occur as stacks of flat cisternae, interconnected by tubular-reticular and saccular elements. These cisternal stacks are structurally and functionally polarized (Figure 10.3). The side of a stack which faces transitional elements of the rough endoplasmic reticulum (rER) is termed the cis side; the other side, which often faces secretory granules is named the trans side. High voltage transmission EM on thick sections has revealed systems of tubules bridging the cis, medial and trans cisternae, anastomosing networks of tubules adjacent to the cis cisternae (cis Golgi network) and more extensive networks of tubules (trans-most cisternae or trans Golgi network—TGN) associated with the trans side of the cisternae stack (Noda and Ogawa, 1988; Pavelka and Ellinger, 1991; Roth, 1987).

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Within absorptive enterocytes lectin cytochemistry has revealed predominating lectin-binding patterns with distinct labelling of either cis, medial, trans or trans-most Golgi apparatus regions (Figure 10.3). Many of these patterns have been interpreted in relation to the synthesis or modification of intestinal glycoconjugates. In rat small intestinal absorptive cells Pavelka and Ellinger (1985) found that Con A, which binds preferentially to Man and Gluc residues, intensely labelled dilated cisternae on the cis Golgi side. Labelling with the same lectin was variable in medial and trans cisternae. In the same study, RCA-1, recognizing Gal residues, could only be demonstrated in medial cisternae. In other investigations on rat absorptive cells, ECA labelling of Gal 1–4GlcNAc increased in intensity from medial to trans/trans-most Golgi sections and UEA-1 labelling of fucosylated moieties was particularly prominent in the trans/transmost Golgi regions (Pavelka and Ellinger, 1991). The authors concluded that the predominance of binding sites for Man-binding lectins in cis Golgi cisternae and the preferential localization of reactions for Gal- and Fuc-recognizing lectins in the trans/trans-most regions of the Golgi apparatus, may signal the conversion of high mannose N-glycosidically linked oligosaccharide side chains into complex-type glycans. PSA and LCA are Man- Gluc- and GlcNAc-recognizing lectins that bind with high affinity to fucosylated core regions of N-glycosidically linked glycans. In rat absorptive enterocytes these lectins labelled the rER and were intensely reactive with cis and medial Golgi cisternae (Pavelka and Ellinger, 1989). For inhibition of the intense Golgi labelling, considerably higher concentrations of competitive sugars were necessary than for abolition of the rER label, suggesting that core-fucosylated N-glycosidically linked glycans predominate in the Golgi (Pavelka and Ellinger, 1989). HPL reacts with oligosaccharides containing GalNAc and to a lesser extent GlcNAc moieties. In rat absorptive enterocytes this lectin labelled cis Golgi cisternae, suggesting that these are sites where the initial steps of biosynthesis of O-glycosidically linked saccharides occur (Pavelka and Ellinger, 1985). Reference has already been made to ECA and UEA-1 labelling of medial and trans Golgi cisternae and the suggestion that this reactivity may indicate the sites of complex N-linked glycosylation (Pavelka and Ellinger, 1991). Labelling with these two lectins may also reveal further sites of O-linked glycosylation involving the synthesis of N-acetyllactosamine and the fucosylated histo-blood group O-antigen. Goblet cells The Golgi apparatus of intestinal goblet cells (Figure 10.4) fulfils very similar roles in maintenance of cell polarity as in the absorptive enterocytes. Although the goblet cell Golgi apparatus is involved in multiple Nlinked and O-linked membrane and secretory glycosylation processes it is O-linked mucin glycosylation which dominates the glycosylation machinery. This feature of goblet cell biology has been successfully investigated by lectin cytochemistry. The initial O-glycosylation reaction of mucin involves the transfer of GalNAc residues from UDPGalNAc to the hydroxyl groups of Ser or Thr residues on the polypeptide. Roth (1984) employed HPL-gold conjugates to detect terminal non-reducing GalNAc moieties in intestinal goblet cells. With this probe labelling was absent from the rER but clearly identified over cis and trans Golgi cisternae and mucin droplets. Cisternae in the middle of the Golgi stack were found to be either weakly labelled or not at all. This lectin cytochemical data was interpreted as indicating that core O-glycosylation starts in the cis side of the Golgi apparatus. HPL labelling observed over the trans Golgi cisternae was attributed to the synthesis of histo-blood group A active oligosaccharides (Roth, 1984), a view supported by subsequent immunocytochemical investigations showing the localization of histo-blood group-A 1,3GalNAc transferase in the trans Golgi cisternae (Roth et al., 1988). Similar HPL labelling patterns have been identified in goblet cells in the pig intestine (King and Kelly, 1990).

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Figure 10.4. Diagrammatic representation of the Golgi apparatus, associated endomembrane system and mucin granules of an intestinal goblet cell. Lectin cytochemical data from several sources (see text) has been pooled to give an overall impression of the extent of compartmentation of O-linked glycosylation of mucin proteins.

At least six core structures have been identified in mucin O-linked oligosaccharides (Strous and Dekker, 1992). A given mucin can probably have any combination of cores, although Gal l,3GalNAc-R and Gal 1,3 [GlcNAc 1,6]GalNAc-R cores are the most common in intestinal mucins (Neutra and Forstner, 1987). T antigen (Gal 1,3GalNAc) is recognized by both PNA and jacalin. Sialylation of T antigen inhibits PNA labelling but jacalin continues to react with both the mono-or disialylated forms. In both rat and pig intestinal goblet cells PNA and Jacalin have been found to react with moieties in the medial and/or trans Golgi cisternae (King and Kelly, 1990; Sato and Spicer, 1982). The backbone regions of mucin oligosaccharides consist of series of Gal 1,3 and GlcNAc 1,4 units. The two most common backbones are Gal 1,3GlcNAc (type 1) and Gal 1,4GlcNAc (type 2) structures (Strous and Dekker, 1992). In both rat and pig goblet cells ECA labelling of type 2 structures has been observed in trans Golgi cisternae and also to a lesser degree over newly formed mucin droplets (King, unpublished observations.) The backbone chains of mucin polypeptides are usually terminated with glycosidic linked Gal, GalNAc, Fuc or NeuAc (Neutra and Forstner, 1987). In humans or animals that express histo-blood group-A antigen, HPL reveals the presence of GalNac in the trans cisternae and/or the mucin droplets (King and Kelly, 1990; Roth, 1984). In histo-blood group-O secretors, UEA-1 labelling has identified the 1,2 fucosylated Hantigen in the same locations (Figure 10.4; Ellinger and Pavelka, 1988; King and Kelly, 1990). Although little is known about the distribution of glycosyltransferase enzymes in goblet cells, it clear from the foregoing that at least some compartmentation of O-linked oligosaccharide synthesis occurs in the Golgi apparatus. Cytochemical data from experiments where several lectin probes have been used suggests that this compartmentation is not exclusive and that sequentially occurring terminal glycosylation steps may sometimes take place in the same cisternae (Figure 10.5). Factors other than compartmentation which may play a role in controlling O-linked glycoprotein synthesis include competition of glycosyltransferases for common acceptors and subtle differences in glycosyltransferase substrate specificity. Variations in these

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Figure 10.5. Electron microscope lectin:gold and immunogold labelling of the Golgi apparatus of a rat intestinal goblet cell showing compartmentation of glycosylation. Arrows indicate the cis-trans axis of the Golgi stacks (G). Large (15 nm) HPL-gold probes (GalNAc) are associated with cis and medial cisternae. Small (5nm) PNA-gold probes are associated with medial and trans cisternae. Histo-blood group-H antigen (Fuc-Gal-GlcNAc) in the mucin granules (m) is detected by a monoclonal antibody labelled by anti-mouse IgG:gold probes (10 nm). Bar=250 nm.

controlling influences may account for observed glycosylation heterogeneity within the Golgi apparatus and secretory products (King and Kelly, 1990).

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Future directions in lectin cytochemistry Functional cytochemistry of the Golgi apparatus The compartmentalization model of the Golgi apparatus (Dunphy and Rothman, 1985) proposes that glycosyltransferase enzymes are distributed in sub-compartments according to the order in which they act. As discussed in this review, the universality of this model has been questioned. The strongest evidence for and against the model comes from combined immunocytochemical and lectin cytochemical studies on the distribution of glycosyltransferases and their reaction products. Very few investigations of this kind have been undertaken. In a recent elegant study on the bovine large intestine 1, 4 galactosyltransferase was found to co-localize with RCA-1 reactive products in the trans Golgi cisternae, indicating that this is where galactosylation occurs (Taatjes et al., 1992). Further studies of this kind are necessary to investigate the intracellular mechanisms of glycosylation in the normal and diseased intestine. A major difficulty is that well-characterized monoclonal antibodies against glycosyltransferase enzymes are still relatively scarce. Lectin cytochemistry in the analysis of cloned glycosyltransferase genes In general, the population of surface oligosaccharide molecules displayed by a cell is a reflection of its glycosyltransferase repertoire (Ernst et al., 1989). Cloned glycosyltransferase genes and their cognate cDNAs represent tools to investigate the molecular mechanisms that regulate the expression of oligosaccharide structures during development and differentiation (Larsen et al., 1990). Hitherto, difficulties in isolating mammalian glycosyltransferase enzymes to homogeneity have hindered standard molecular cloning techniques involving antibody screening of expressed cDNA libraries. To circumvent this problem, J.Lowe and colleagues at the Howard Hughes Medical Institute in Michigan have developed new genetic approaches for the isolation of cloned cDNA sequences that determine the expression of cell surface oligosaccharide structures and their cognate glycosyltransferases (Ernst et al., 1989; Larsen et al., 1989; Rajan et al., 1989). These approaches are based on the transfection of glycosyltransferases into COS-1 or mouse L cells which lack the transferase. The very small number of transfected cells expressing the glycosyltransferase of interest are detected using lectins (or monoclonal antibodies) specific for the cell surface-expressed product of the cloned enzyme. For example, Larsen et al. (1989) implemented this gene transfer approach to isolate a cloned murine cDNA that determined surface expression of Gal 1, 3Gal linkages and encoded an 1, 3galactosyltransferase. A cDNA was prepared from a murine cell line known to express the enzyme. Plasmid DNA was prepared and transfected into COS-1 cells that were panned on dishes coated with Griffonia simplicifolia lectin I B4 (GS I-B4), which has a high affinity for Gal1,3Gal moieties. Following several other cloning and subcloning steps, transfected cells were stained with FITC labelled GS I-B4 and isolated by fluorescence activated cell sorting. In future studies subcloning of glycosyltransferase cDNAs into prokaryotic and eukaryotic expression vectors will provide new sources of antigen for the production of monoclonal antibodies. Lectin-labelling and cell sorting procedures, such as those just described, add to the efficiency of these molecular approaches. `Glycosylation engineering' and lectin cytochemistry Stanley (1992) has used the term ‘glycosylation engineering’ to describe new molecular biology techniques developed to experimentally change the expressed carbohydrates of recombinant and non-recombinant glycoproteins. cDNA transfection and lectin-labelling studies also offer new opportunities to investigate

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substrate and enzyme competition within the Golgi apparatus. This principle has already been established by Lee et al. (1989) who purposely altered terminal carbohydrate in vivo by mis-expressing a terminal glycosyltransferase. These authors altered N-linked terminal sequences of Chinese hamster ovary (CHO) cell glycoproteins by transfecting the cells with a 2,6-sialyltransferase cDNA. While wild type CHO cells normally produce sugar chains terminating in the NeuAc 2,3 linkage, the expressed enzyme was found to compete with the endogenous sialyltransferase to attach an alternative terminal sequence, NeuAc 2,6Gal. This structure was revealed on cell surfaces by cytochemistry employing FITC-labelled SNA-1 and SNA-1 :gol d probes. Subcellular localization of the NeuAc 2,6Gal product by SNA-1 :gold-labelling at the EM level revealed localization throughout the Golgi apparatus. O’Hanlon and Lau (1992) have recently used transfection/ SNA-1 labelling experiments to analyse the expression of kidney mRNAs expressed from the rat -galactoside 2,6 sialyltransferase gene. Exposure of cells to differentiation agents such as retinoic acid, butyrate and phorbol esters has produced qualitative changes in levels of several cell surface terminal glycosylation sequences as well as the specific glycosyltransferases that produce them (for reviews see Broquet et al., 1991; Paulson and Colley, 1989). Fluorescence activated sorting of lectin-labelled cells is a highly sensitive method for revealing experimentally induced changes in the expression of surface glycoconjugates (Labarriere et al., 1993). Le Marer et al. (1992) reported a striking increase in Gal 2,6 sialyltransferase activity upon transformation of a rat fibroblast cell line (FR3T3) with the c-Ha-ras oncogene. The cause of the increased Gal 2,6 sialyltransferase activity was demonstrated as the increased steady-state Gal 2,6 sialyltransferase mRNA levels, resulting in increased expression of the enzyme in these transformed cells. Elevated levels of cell surface 2,6NeuAc were cytochemically detected with FITC-labelled SNA-1. Epithelial receptors for enteric micro-organismsÐhost tropism Intestinal glycobiology is at the basis for much important research on malignant transformation and enteric disease. Lectin cytochemistry has a valuable role to play in the spatial analysis of functionally important glycoconjugates within the small intestine. As discussed in this review, the cytochemical approach is at its best when integrated with other investigative approaches. Important advances in the understanding of microbial-epithelial interactions have been facilitated by interaction between microbiologists and epithelial cell biologists (Wick et al., 1991). Many commensal and pathogenic bacteria specifically adhere to small intestinal membrane and mucin glycoconjugates (Stewart et al., 1993). The nature of these receptors plays an important role in host range, tissue tropism, and the triggering of host responses. This is particularly noticeable in neonates where both beneficial and harmful swings in the microbial balance can accompany ontogenic epithelial glycosylation changes (Kelly et al., 1992; Stewart et al., 1993). The relationship may be passive and involve bacterial colonization mediated through binding to expressed glycoconjugates or may involve chemical modification of inhospitable sites through the actions of secreted sialidases and other exoglycosidase enzymes (Corfield, 1992). In vitro adherence assays, employing both lectins and bacterial fimbrial adhesins as cytochemical probes, afford new opportunities for characterizing the nature and distribution of bacterial receptors on intestinal epithelial surfaces (Falk et al., 1993; King et al., 1993). Such approaches, when used alongside more conventional in vivo infection studies, are of particular value in characterizing compositionally related receptors expressed on diverse cell or tissue sites.

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Wu, A.M., Sugii, S. and Herp, A., 1988, A guide for carbohydrate specificities of lectins, in Wu, A.M. and Adams, L.G. (Eds) The Molecular Immunology of Complex Carbohydrates, pp. 205–63, New York: Plenum Press. Yamamoto, F., Clausen, H., White, T., Marken J. and Hakomori, S., 1990, Molecular genetic basis of the histo-blood group ABO system, Nature, 345, 229–33.

Chapter 11 Lectins and CancerÐAn Old Field Revisited Udo Schumacher

Introduction Cancer is the most challenging health-care problem of the developed countries at the end of the twentieth century. This is best illustrated by figures concerning some of the most common neoplasms in these countries, namely breast and colon cancer. Lung cancer is excluded from this discussion, because the main cause of lung cancers—smoking—is well known and the rise in non-smoking policies will, hopefully, result in the decline of this neoplasm. In breast cancer, unfortunately, this is not so: in the United States of America alone about 46000 women are expected to die of it in 1993. The overall mortality rates have, over the years, unfortunately remained almost static because the advances in the detection and prevention of cancer have been offset by the increased incidence of the disease (Anonymous, 1993). A similarly bleak picture must be drawn for cancer of the colon: annually 20000 deaths are reported in the UK. If treated surgically, approximately 50 per cent of the patients survive five years, but if the crude survival rate is taken into account, it drops to approximately 20–25 per cent (Schumacher et al., 1994). An equally pessimistic picture can be drawn for many other solid neoplasms. The reason for the failure of cancer therapy is due mainly to the lack of successful treatment once the tumour has spread. The formation of metastasis is therefore the most important step in determining the fate of cancer patients. Fortunately, some progress has been made in this area of modern cancer research and many of the promising results obtained in this field are related to glycoproteins and lectins. Several excellent reviews are available on the topic of metastasis (Fidler, 1991; Hart et al., 1989) but only recently has the emphasis of cancer research shifted to investigate the cell/cell and cell/matrix interactions (Hart and Saini, 1992). As both the outer surface of the cell membrane and the extracellular matrix consist mainly of glycoconjugates, studies using lectins could create new insights into the processes governing the metastatic cascade. As lectins are such versatile markers, they have been used in a variety of studies using histochemical, biochemical and functional techniques to characterize cancer cells. This review will focus on the results of lectin histochemical studies using clinical material, and results of biochemical and functional studies on these will only be presented if they are of relevance to the histochemical findings. Additionally the role of endogenous lectins will be summarized, and finally a personal and speculative outlook on future developments in the field of lectins and cancer will be given.

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Histochemistry Most of the lectin histochemical studies using clinical material are concerned with tumours derived from epithelial tissues showing glandular differentiation, i.e. tumours of the colon, breast and female genitalia. This is not only due to the fact that these tumours are of the greatest clinical importance but also that lectins are particularly well-suited to investigate these tumours. Taking into account the secretory products of glands, the morphologist distinguishes between serous and mucous glands. Serous glands are negative for the general carbohydrate stain, the periodic acid-Schiff (PAS) reaction, while mucous glands are PAS positive. This is also reflected by their lectin-binding patterns. Thus, lectins which recognize N-linked sugars in the cell membrane glycocalyx, such as Concanavalin A (Con A) or phytohaemagglutinin-L (PHAL), react with serous glandular cells. In contrast, lectins which recognize O-linked glycoproteins bind to mucous cells. These lectins are either specific for N-acetylgalactosamine, such as the Helix pomatia agglutinin (HPA), soya bean agglutinin (SBA) and Dolichos biflorus agglutinin (DBA), or specifically recognize fucose such as the Ulex europaeus agglutinin (UEA-I). These have therefore been widely used as general histochemical reagents to study the mucin-like carbohydrate residues in cancers with glandular differentiation. In comparison, tumours derived from lymphatic tissue, squamous epithelia, and nervous tissue in which mucins and O-linked glycoproteins are less common, have not been extensively studied by lectin histology. Breast cancer Breast cancer is a major clinical problem and milk, the secretory product of the normal lactating breast, is a rich source of a large variety of saccharides. Accordingly, lectins are good markers and well suited to the study of changes in carbohydrate expression in the development of breast cancer. The first studies of breast cancer by lectin histochemical techniques originated from the work of Klein et al. (1981, 1983). These studies indicated that the expression of binding sites for peanut agglutinin (PNA) in breast cancer cells was correlated with the steroid hormone receptor status of the breast cancer and could therefore be used instead of steroid receptor estimation (Klein et al., 1981). However, subsequent studies were more cautious (Dansey et al., 1988; Walker et al., 1985) and although some later studies of breast cancer did reveal differences in lectin-binding between normal and hyperplastic breast tissue and that in breast cancer (Louis et al., 1983; Walker 1984a, 1984b, 1984c, 1985), PNA-binding as a prognostic indicator has never been widely accepted. Most interest in this area was generated by studies using the lectin from the Roman snail (HPA). The initial study reporting HPA-binding to normal breast epithelial cells and breast cancer (Leathem et al., 1983) was further clarified in two subsequent abstracts, in which the correlation between HPA-binding and axillary node metastasis was described (Leathem et al., 1984; 1985). Lectin-binding studies of breast cancer attracted little attention until two further papers by Leathem’s group appeared in the Lancet (Brooks and Leathem, 1991; Leathem and Brooks, 1987) and showed a correlation between HPA-binding to breast cancer cells and the prognosis of the patient. The ability of metastatic breast cancer cells to bind HPA seems to be a constant feature of the tumour because HPA-binding glycoconjugates are similarly expressed in most brain metastases (Schumacher et al., 1992). This seems to be a particular feature of HPA as no constant expression of lectin-binding sites in primary tumours and metastasis could be found with other lectins (Krogerus and Andersson, 1990). Although several groups have corroborated the finding that HPA is of prognostic value in human breast cancer (Alam et al., 1990; Fenlon et al., 1987; Fukutomi et al., 1989) or at least of limited prognostic value (Noguchi et al., 1993; Thomas et al., 1993), this view is by no means accepted by all (Galea et al., 1991;

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Taylor et al., 1991 and an extension of this study by Gusterson et al., 1993). As discussed by Walker (1993), the level of sensitivity in the detection of HPA-binding to tumour cells seems to be critical for it to be of prognostic value. As this depends on tissue processing and the detection methods used which can vary in different laboratories (e.g., Leathem’s group uses an indirect method while Gusterson’s group uses a direct method), differences in the results can be expected. Our own results, using different human breast cancer cell lines, indicate that lectin-binding depends heavily on tissue fixation, processing and methods used for the detection of the lectin-binding sites (unpublished observations). It is possible therefore, that HPA-binding may lead to new discoveries concerning the mechanism by which breast cancer spreads. However, at present it cannot be generally recommended as a routine procedure for risk assessment in patients with breast cancer as the HPA-binding method has not so far been standardized. The most puzzling result of the studies cited above is that receptors for HPA are expressed in both the normal lactating breast and breast cancers that metastasize to the local lymph node but not in those that do not metastasize. In the normal breast, the binding of the lectin HPA was restricted to the apical part of the lactating breast epithelium. SDS-PAGE and Western blotting of the proteins of the human milk fat globule membrane, which is a direct derivative of the apical plasma membrane, revealed that only a limited number of glycoproteins react with HPA (Schumacher, 1990). There is an inter-individual variation in the number of HPA-positive bands in the SDS-PAGE, but, to date, the maximum number is six (Schumacher, 1990). Later studies have shown that in human HPA-positive breast cancer cell lines, which show metastatic abilities in immunosuppressed animals, most if not all membrane glycoproteins are positive for HPA. Therefore, a simple overexpression of one or all of the HPA-positive glycoproteins of the milk fat globule membrane is not the explanation for the fact that HPA-positive breast cancers metastasize (own unpublished results) and that the glycosylation of the cell membrane proteins has changed. So far, unfortunately, it is not known which of the HPA-binding glycoconjugates are responsible for metastasis in breast cancers with a poor prognosis (Walker, 1993). The above cited studies on HPA-binding and breast cancer metastasis were based on long-term survival time (in the studies of Dr Leathem’s group up to 15 years and longer). Indeed, long-term observations are necessary for a meaningful prognosis in breast cancer as the usual five-year survival time appropriate for many other human cancers is not sufficient and often 10- or 15-year survival times have to be considered as more valid in patients with breast cancer. In short-term survival studies peanut agglutinin (PNA), wheat germ agglutinin (WGA), Concanavalin A (Con A), Lotus tetragonolobus agglutinin (LTA) and Ulex europaeus-I agglutinin (UEA-I) gave no further prognostic information than that already provided by histology (Walker, 1990). Colon cancer Impressive progress has been made during the last years in the analysis of the molecular events which lead to colon cancer and a sequence of chromosomal aberrations has been worked out which correlates with the progression normal mucosa >adenoma>carcinoma>metastatic carcinoma (Fearon and Vogelstein, 1990; Fearon and Jones 1992). However, the important step leading to the metastatic phenotype is still not understood. Before referring to the results of a variety of studies using lectins, some methodological problems similar to those described above for breast cancer studies must be discussed. One of the most widely used lectins in colon cancer histochemistry is PNA, the detection of which depends on the sensitivity of the method used. Boland and Roberts (1988) used a quantitative extraction technique to measure the amounts of WGA-and PNA-binding sites in colonic mucins from normal colon and from colon cancer. With this sensitive biochemical assay they could detect a definite, though slight, reaction of PNA with normal

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colonic mucins which would have been difficult to show by fluorescence microscopy. In contrast, PNA positive mucins in colon cancer are more positive for PNA-binding and therefore are more readily detectable by conventional techniques. PNA-binding sites in human colonic mucosa and its neoplasias are more readily detected with an indirect technique such as reaction with biotinylated PNA followed by avidinbiotin-peroxidase, as shown by a study from Boland (1988) who used both FITC-PNA and the indirect technique. However, according to Kellokumpu et al. (1987) FITC-labelled PNA was adequate for the detection of PNA-binding sites of the supranuclear (Golgi) region. After neuraminidase treatment, additional PNA-binding sites were found in the mucin granules at all levels of the crypts, while in colon cancers a strong and direct binding of PNA to the apical cell membranes and to intraluminal secretions occurred. SDSPAGE of normal and tumour samples revealed four distinct carcinoma-associated glycoproteins giving positive reaction with PNA (26, 32, 35 and 50 kD) in addition to the four glycoproteins also positive with PNA which were common to both normal and neoplastic tissues (29, 30, 33 and 36 kD). However, in other studies the results obtained by the use of PNA in diagnostic histopathology of the colon were conflicting. For example, PNA-binding sites in the Golgi region were absent in grade IV adenomas in one study (Orntoft et al., 1991) but present late in the adenoma-to-carcinoma sequence (Boland et al., 1992). Although reaction with PNA was regarded as cancer specific, as this was observed mainly in cancers secreting little or no mucus, its value as a tumour marker is limited (Jass and Smith, 1992). Other studies interpret the changes in lectin-binding with PNA or other lectins during malignant transformation as an indicator of the state of differentiation, not of malignancy (Lee, 1988). Indeed, staining with PNA could not be used for the unambiguous assessment of pre-malignant changes or cancer risk in patients with ulcerative colitis (Fozard et al., 1987). The interpretation of lectin-binding in colonic lesions is further complicated by the fact that the localization (proximal, distal, sigmoid colon or rectum) of the lesion appears to be of importance (McGarrity et al., 1989) and that binding sites for PNA appear to increase with age (Sams et al., 1990). Diet can also influence lectin-binding sites for SBA (Yang et al., 1991) but no similar data are available for PNA. Identification of binding sites for PNA is of special interest in colon cancer, not only because this lectin is used as a tool in histochemistry, but also because PNA is a mitogen for normal human colonic epithelium and for HT29 colorectal cancer cell lines in dosages which can be attained in humans by normal dietary intakes (Ryder et al., 1992). As lectins can be found in many vegetables it is of great scientific and practical importance to evaluate the influence of lectins in our diets as growth signals and even possibly as cocarcinogens. Lectins other than PNA have been used in histochemical investigations of colon cancer, but again with conflicting results. The lectins most often used are UEA-I, DBA and GSA-I (Hohenberger et al., 1990; Kuroki et al., 1991; Lee, 1988; McGarrity et al., 1989; Sams et al., 1990; Watanabe et al., 1992). The study by Hohenberger et al. (1990) is of special interest because it compares the different lectin-binding sites of colorectal cancer, its recurrences and metastases, and has found a heterogeneous marker profile between primary tumours and metastases. There are almost always identical lectin-binding patterns between the primary tumour and local recurrences indicating that these may develop from remnant cells of the primary tumour left after surgery. Summing up histochemical work with lectins on colorectal cancer there seems to be a consensus that PNA and UEA-I are of special interest in colorectal carcinoma. However, the value of these studies is difficult to assess for two main reasons: (1) the conflicting results obtained with PNA or UEA-I may be due to methodological problems which have not been worked out, and (2) larger studies integrating clinical and histochemical data are lacking, with the exception of one study using HPA (Schumacher et al., 1994). Because of the presence of dietary lectins and their role in modulating intestinal proliferation (see above) this field of research will most likely attract more attention in the future.

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Tumours of the hepatopancreatic system Histochemical studies using five different peroxidase-labelled lectins on tissues from normal pancreas, chronic pancreatitis and pancreatic carcinomas have initially indicated no qualitative differences (Ching et al., 1988). However, an increased intensity in PNA-binding of secreted mucins in the pancreatic carcinomata led the authors to suggest the use of PNA for screening purposes. The analysis of PNA-positive mucins was later refined by carbohydrate analysis of the PNA-positive glycoproteins detected in the sera of cancer patients (Ching and Rhodes, 1990). Furthermore, although SBA was not bound by normal ductal epithelium, cancer cell lines showed a positive reaction with this lectin as shown by using cell lines derived from pancreatic cancer (Nishimura et al., 1993). Detection of lectin-binding with Bauhinia purpurea agglutinin (BPA) or Vicia villosa agglutinin (VVA) in addition to PNA on cancer-associated mucins from patients with pancreatic and gastric cancer has been considered a useful approach in the diagnosis of pancreatic cancer (Kawa et al., 1991; Kawa et al., 1992). Changes in the glycosylation of normal pancreas during malignant transformation are not limited to mucins alone but also found in the glycan side chain of gamma-glutamyltranspeptidase (Ohta et al., 1990). A histochemical study of tissues of 25 cases of human hepatocellular carcinoma plus controls using a panel of 12 different lectins revealed no major differences (Zhang et al., 1989). However, changes in the glycosylation of -fetoprotein occurring in hepatocellular carcinoma can be investigated by lectin-binding studies and these, apparently, could be of diagnostic value (Du et al., 1991; Ooi et al., 1990; Tsuchida et al., 1989). Tumours of the genitourinary tract Lectins have been used to characterize the normal urothelium and its tumour, the transitional cell carcinoma. In the normal urothelium, large amounts of WGA, RCA-120 or GSA-II can be bound by all urothelial cells while PNA, SBA, Con A and DBA show only slight binding. Other lectins, such as MPA, UEA-I, GSA-I show an increased binding from basal to superficial cell layers (Ward et al., 1987). Accordingly, as these lectins can be classified as markers of differentiation, they have been used to examine the carbohydrate composition of transitional cell carcinoma. A rationale for using lectins in the analysis of transitional cell carcinoma is that blood group-related carbohydrate antigen expression, which is detectable by several specific lectins, has been used as a predictive parameter in these tumours. Indeed, using cell suspensions from transitional cell carcinoma, aneuploid cells bound PNA more extensively and were less reactive with WGA than diploid cell populations (Orntoft et al., 1988). Using tissue sections and comparing the classical markers of prognosis (aneuploidy and invasion) these changes correlated better with the loss of WGAbinding than with the reduction in PNA-binding (Langkilde et al., 1989a); the latter have been characterized and found to be different to that of the blood group T-antigen (Langkilde et al., 1992). This finding of a decrease in lectin-binding during malignant progression was later extended using a wide range of lectins including PNA, WGA, Vicia faba agglutinin (VFA), Griffonia simplicifolia agglutinin-II (GSA-II), Solanum tuberosum agglutinin (STA), UEA-I, Lens culinaris agglutinin (LCA), DBA and HPA (Langkilde et al., 1989b). With the notable exception of HPA which stained approximately 10 per cent of the cells in invasive tumour cell islands, no binding sites were detected for the other lectins. Other studies could find no systematic difference in lectin-binding between corresponding subpopulations of normal and neoplastic cells (Ward et al., 1992). A similar mixed picture is observed with lectin-binding in prostate cancer. While initial studies indicated that SBA could distinguish between benign and malignant cells by binding only to the latter (Söderström, 1987), these findings were not confirmed in later studies (Loy et al., 1989; McNeal et al., 1991). In addition

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to the primary tumour, bone metastases derived from prostate adenocarcinoma have been studied and the remarkable result of this investigation was that HPA expression was more common in cases with metastases in the bone or other organs (Shiraishi et al., 1992). Several other studies have used lectins to investigate renal tumours, cervical cancer, testicular tumours and ovarian cancer, but these studies are relatively non-discriminatory with the exception of the study by Aoki et al. (1990), who found UEA-I and PNA to be useful indicators of malignancy in endometrial carcinoma. Tumours of other organ systems In addition to the studies above, lectins have been used to characterize the glycoconjugate content of a variety of different tumours. As most of these studies were not correlated with clinical data, their usefulness is limited. However, two groups did highlight a correlation between clinical data and lectin histochemistry: Kakeji et al. (1991), who found an association between HPA-binding and prognosis in gastric cancer and Matsumoto et al. (1992) who found an association between DBA-binding and prognosis in lung cancer. Endogenous lectins In addition to the detection of carbohydrate residues in tumours and their extracts by lectins as reagents, endogenous lectins have also been detected in human and animal cancers. In principle, lectins detected in man and in animals are called endogenous lectins or collectively animal lectins. ‘Tumour-specific’ lectins are a subclass of these and several are probably developmentally regulated. Lotan and Raz (1988) summarized their experiences as follows and their findings serve as a general introduction to the field of tumour lectins: 1. Lectins could be detected, albeit in varying concentrations, on all tumour cell lines so far investigated. 2. Malignant transformation increased the lectin content and was positively correlated with the metastatic potential. 3. Tumour lectins could be involved in cell/cell and cell/matrix interaction. 4. Lectin levels were modulated by inducers of differentiation. As in other fields of lectinology, endogenous lectins in tumours have been identified by a variety of methods. Thus, lectins can be isolated from the tumours or tumour cell lines by affinity chromatography using carbohydrates immobilized on carriers as ligands. Endogenous lectins in the tissues can then be detected using immunohistochemical techniques with antibodies raised to the isolated lectin-proteins. Finally, endogenous tumour lectins can also be demonstrated by labelled neoglycoproteins. Neoglycoproteins are proteins with modified carbohydrate structures derived from existing glycoproteins after the removal of terminal carbohydrate residues (such as neuraminic acid), or they are primary nonglycosylated proteins such as bovine serum albumin to which carbohydrate groups have been covalently linked. After these procedures, a label such as a fluorochrome or biotin is attached for aiding detection in histochemistry (for review, see Gabius and Bardosi, 1991). Several studies have been published using this technique on various carcinomas including breast cancer (Gabius et al., 1988; 1990a), colorectal carcinomas (Gabius et al., 1991), squamous cell carcinoma of the head and neck (Steuer et al., 1991) and cutaneous cancer (Gabius et al., 1990b). However, the results obtained by histochemical techniques seem questionable as normal tissues also reacted with the same neoglycoproteins even in the presence of sugars expected to be

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inhibitory for the reaction. Indeed, as the reactivity of neoglycoproteins could never be inhibited by a simple monosaccharide (Schumacher, 1992), it was not possible to distinguish between low affinity non-specific binding sites and high affinity specific ones. Moreover, this result was not unexpected because during fixation and wax-embedding of tissues the binding activity of the endogenous lectins are not likely to be preserved. A similar situation is known in enzyme histochemistry where the active centre of the enzyme has to be preserved in order to obtain enzyme reactivity and it is well known that, with the probable exception of one example, enzyme histochemistry does not work on routine pathological material. Accordingly, neoglycoprotein histochemistry may have only a limited use in cancer diagnostics. Other more refined and properly controlled studies, however, have demonstrated the presence of endogenous lectins in various tumours and non-neoplastic tissue. Some of the best systems investigated are the galactoside-binding lectins, which were first detected on the surface of various tumour cell lines. These lectins were purified by affinity chromatography on asialofetuin-matrices and shown that the lectin activity was associated with two proteins of Mr 14500 and 34000. Func tional studies indicated that the metastatic potential of cell lines was positively correlated with their lectin content. Indeed, monoclonal anti-lectin antibodies after intravenous injection into the tail vein suppressed the ability of the cells to form lung metastases (Raz and Lotan, 1987). Later the Mr 34000 galactoside-binding lectin was characterized at the molecular level and turned out to be a chimeric gene product formed by an approximately 14000 galactoside-binding lectin and an internal domain of the collagen -gene, the entire sequence showing a greater than 85 per cent homology to a rat low affinity IgE-binding protein (Raz et al., 1989). The level of expression of the 14500 and 34000 galactose-binding lectins varies according to the differentiation of the cell lines in culture: the expression of the 34 kD lectin decreases during differentiation, while the 14.5 kD lectin decreases or increases depending on the cell line used. These results indicate that tumour cell differentiation is accompanied by a distinct modulation of lectin expression and this may recapitulate the developmental regulation of the lectin expression (Lotan et al., 1989b). Similar dependence of lectin expression on differentiation inducers was reported in the mouse melanoma cell line K-1735P (Lotan et al., 1989a). In human colon cancer the expression of two lactose-binding lectins with Mr 31000 and 14500 has been described. The expression of the 31000 lectin, which can be detected cytoplasmically by immunohistochemistry, correlates well with the serum level of carcinoembryonic antigen and the Duke’s stage of the patient while the 14500 lectin shows no correlation with the Duke’s classification and is located apically and in secretory products of the tumour cells (Irimura et al,, 1991; Lotan et al., 1991). The analysis of some of the tumour lectins at a molecular level is quite advanced: the galactose-binding lectin (hL-31) identified in human tumour cells contains a collagen-like sequence and has been cloned into E. coli. Subsequent analysis revealed that the lectin is probably a peripheral membrane protein, whose carboxy- and N-terminal end are both exposed on the outer cell membrane (Ochieng et al., 1993). Similar advances in the molecular analysis of other endogenous lectins have also been reported (Ahmed et al., 1992; Allen et al., 1991; Sharma et al., 1992; Wong et al., 1991; Woo et al., 1991). Despite these advances, the precise role of endogenous lectins in the spread of tumour cells of clinically important cancers is not yet clear. Cell/cell and cell/ matrix interactions obviously play a major role in the spread of tumour cells (see above) and therefore this area of research is promising. However, as the activity of many of the recently discovered cell adhesion molecules are dependent on non-carbohydrate type interactions for their binding, it will take some years for the validity of the different results to be finally evaluated.

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A personal outlook What about the future of lectins and cancer? As can be seen, much data have been acquired concerning lectins and tumours, but most are difficult to evaluate. The changes in glycosylation which occur during the steps of malignant transformation are clear and without any doubt, but what their functional implications are is less clear. The best guess at the moment seems to be linking carbohydrate expression with prognosis and hence with the metastatic potential. This has been carried out in a number of studies, but the problem with these studies is their lack of comparability and the inherent technical problems in the use of formalin-fixed and wax-embedded sections. As the processing of tissues alters their lectin-binding sites and, as the number of these sites in the tissue determines the level of detectability, the problem appears to be difficult to resolve. One way around the technical difficulties may possibly be to isolate the glycoconjugates from the tissues to which the lectins are bound and to raise antibodies to them. However, even this may be difficult as many different tissue glycoproteins are lectin-positive in cancer cells, as described above in the case of HPAbinding sites in breast cancer, and therefore the analysis of lectin-positive bands is of no help. We must define what the altered carbohydrate sequence of glycoconjugates means to the cell in functional terms. If we can begin to understand the functional significance of these changes we will be able to draw a more consistent picture on lectins and cancer. References Ahmed, H., Sharma, A., DiCioccio, R.A. and Allen, H.J., 1992, Lymphoblastoid cell adhesion mediated by a dimeric and polymeric endogenous beta-galactoside-binding lectin (galaptin), Journal of Molecular Recognition, 5, 1–8. Alam, S.M., Whitford, P., Cushley, W., George, W.D. and Campbell, A.M., 1990, Flow cytometric analysis of cell surface carbohydrates in metastatic human breast cancer, British Journal of Cancer 62, 238–42. Allen, H.J., Gottstine, S., Sharma, A., DiCioccio, R.A., Swank, R.T. and Li, H., 1991, Synthesis, isolation and characterization of endogenous beta-galactoside-binding lectins in human leucocytes, Biochemistry, 30, 8904–10. Anonymous, 1993, Strategies for managing the breast cancer program, p. 1, Washington: National Academy Press. Aoki, D., Nozawa, S., Iizuka, R., Kawakami, H. and Hirano, H., 1990, Differences in lectin binding patterns of normal endometrium and endometrial adenocarcinoma, with special reference to staining with Ulex europeus agglutinin-1 and peanut agglutinin, Gynecological Oncology, 37, 338–45. Boland, C.R., 1988, Lectin histochemistry in colorectal polyps, Progress in Clinical Biological Research, 279, 277–87. Boland, C.R. and Roberts, J.A., 1988, Quantitation of lectin binding sites in human colon mucins by use of peanut and wheat germ agglutinins, Journal of Histochemistry and Cytochemistry, 36, 1305–07. Boland, C.R., Martin, M.A. and Goldstein, I.J., 1992, Lectin reactivities as intermediate biomarkers in premalignant colorectal epithelium, Journal of Cellular Biochemistry, Suppl. 16G, 103–9. Brooks, S. and Leathem, A.J.C., 1991, Prediction of lymph node involvement in breast cancer by detection of altered glycosylation in the primary tumour, Lancet, 338, 71 74. Ching, C.K. and Rhodes, J.M., 1990, Purification and characterization of a peanutagglutinin binding pancreatic-cancerrelated serum mucus glycoprotein, International Journal of Cancer, 45, 1022–27. Ching, C.K., Black, R., Helliwell, T., Savage, A., Barr, H. and Rhodes, J.M., 1988, Use of lectin histochemistry in pancreatic cancer, Journal of Clinical Pathology, 41, 324–28. Dansey, R., Murray, J., Ninin, D. and Bezwoda, W.R., 1988, Lectin binding in human breast cancer: clinical and pathologic correlations with fluorescein-conjugated peanut, wheat germ and Concanavalin A binding, Oncology, 45, 300–2. Du, M.Q., Hutchinson, W.L., Johnson, P.J. and Williams, R., 1991, Differential alpha-fetoprotein lectin binding in hepatocellular carcinoma. Diagnostic utility at low serum levels, Cancer, 67, 476–80. Fearon, E.R. and Vogelstein, B., 1990, A genetic model for colorectal tumorigenesis, Cell, 61, 759–67.

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Klein, P.J., Vierbuchen, M., Wurz, H., Schulz, K.D. and Newman, R.A., 1981, Secretion-associated lectin-binding sites as a parameter of hormone dependence in mammary carcinoma, British Journal of Cancer, 44, 476–78. Klein, P.J., Vierbuchen, M., Fischer, J., Schulz, K.D., Farrar, G. and Uhlenbruck, G., 1983, The significance of lectin receptors for the evaluation of hormone dependence in breast cancer, Journal of Steroid Biochemistry, 19, 839–44. Krogerus, L. and Andersson, L.C., 1990, Different lectin-binding patterns in primary breast cancers and their metastases, Cancer, 66, 1802–9. Kuroki, T., Kubota, A., Miki, Y., Yamamura, T. and Utsunomiya, J., 1991, Lectin staining of neoplastic and normal background colorectal mucosa in nonpolyposis and polyposis patients, Diseases of the Colon and Rectum, 34, 679–84. Langkilde, N.C., Wolf, H. and Orntoft, T.F., 1989a, Binding of wheat and peanut lectins to human transitional cell carcinomas. Correlation with histopathologic grade, invasion, and DNA ploidy, Cancer, 64, 849–53. Langkilde, N.C., Wolf, H. and Orntoft, T.F., 1989b, Lectinohistochemistry of human bladder cancer: loss of lectin binding structures in invasive carcinomas, APMIS, 97, 367–73. Langkilde, N.C., Wolf, H., Clausen, H., and Orntoft, T.F., 1992, Human urinary bladder carcinoma glycoconjugates expressing T-(Gal beta(1–3)GalNAc alpha 1-O-R) and T-like antigens: a comparative study using peanut agglutinin and poly- and monoclonal antibodies, Cancer Research, 52, 5030–36. Leathem, A.J. and Brooks, S.A., 1987, Predictive value of lectin binding on breast cancer recurrence and survival, Lancet, 1, 1054–1056. Leathem, A., Dokal, I. and Atkins, N., 1983, Lectin binding to normal and malignant breast tissue, Diagnostic Histopathology, 6, 171–80. Leathem, A., Dokal, I. and Atkins, N., 1984, Carbohydrate expression in breast cancer as an early indicator of metastatic potential, Journal of Pathology, 142, A32. Leathem, A.J., Atkins, N. and Eisen, T., 1985, Breast cancer metastasis, survival and carbohydrate expression associated with lectin binding, Journal of Pathology, 145, 73A. Lee, Y.S., 1988, Lectin expression in neoplastic and non- neoplastic lesions of the rectum, Pathology, 20, 157–65. Lotan, R. and Raz, A., 1988, Lectins in cancer cells, Annals of the New York Academy of Sciences, 551, 385–98. Lotan, R., Carralero, D., Lotan, D. and Raz, A., 1989a, Biochemical and immunological characterization of K-1735P melanoma galactoside-binding lectins and their modulation by differentiation inducers, Cancer Research, 49, 1261–68. Lotan, R., Lotan, D. and Carralero, D.M., 1989b, Modulation of galactose-binding lectins in tumor cells by differentiation-inducing agents, Cancer Letters, 48, 115–22. Lotan, R., Matsushita, Y., Ohannesian, D., Carralero, D., Ota, D.M., Cleary, K.R., Nicolson, G.L. and Irimura, T., 1991, Lactose-binding lectin expression in human colorectal carcinomas. Relation to tumor progression, Carbohydrate Research, 213, 47–57. Louis, C.L., Sztynda, T., Cheng, Z.-H. and Wyllie, R.G., 1983, Lectin-binding affinities of human breast tumors, Cancer, 52, 1244–50. Loy, T.S., Kyle, J. and Bickel, J.T., 1989, Binding of soybean agglutinin lectin to prostatic hyperplasia and adenocarcinoma, Cancer, 63, 1583–86. Matsumoto, H., Muramatsu, H., Muramatsu, T. and Shimazu, H., 1992, Carbohydrate profiles shown by a lectin and a monoclonal antibody correlate with metastatic potential and prognosis of human lung carcinoma, Cancer, 69, 2084–90. McGarrity, T.J., Peiffer, L.P. and Abt, A.B., 1989, Lectin histochemistry of adenomatous polyps. Not a predictor of metachronous lesions, Cancer, 64, 1708–13. McNeal, J.E., Alroy, J., Villers, A., Rewine, E.A., Friha, F.S. and Stamey, T.A., 1991, Mucinous differentiation in prostatic adenocarcinoma, Human Pathology, 22, 979–88. Nishimura, N., Saito, S., Kubota, Y., Moto, o.N., Taguchi, K., Yamazaki, K., Watanabe, A. and Sasaki, H., 1993, Newly established human pancreatic carcinoma cell lines and their lectin binding properties, International Journal of Pancreatology, 13, 31–41.

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Noguchi, M., Thomas, M., Kitagawa, H., Kinoshita, K., Ohta, N., Nagamori, M. and Miyazaki, I., 1993, Further analysis of predicitive value of Helix pomatia lectin binding to primary breast cancer for axillary and internal mammary lymph node metasases, British Journal of Cancer, 67, 1368–71. Ochieng, J., Platt, D., Tait, L., Hogan, V., Raz, T., Carmi, P. and Raz, A., 1993, Structure-function relationship of a recombinant human galactoside-binding protein, Biochemistry, 32, 4455–60. Ohta, H., Sawabu, N., Odani, H., Kawakami, H., Watanabe, H., Toya, D., Ozaki, K. and Hattori, N., 1990, Characterization of gamma-glutamyltranspeptidase from human pancreatic cancer, Pancreas, 5, 82–90. Ooi, A., Nakanishi, I., Sakamoto, N., Tsukada, Y., Takahashi, Y., Minamotor, T. and Mai, M., 1990, Alpha-fetoprotein (AFP)-producing gastric carcinoma. Is it hepatoid differentiation? Cancer, 65, 1741–47. Orntoft, T.F., Petersen, S.E. and Wolf, H., 1988, Dual- parameter flow cytometry of transitional cell carcinomas. Quantitation of DNA content and binding of carbohydrate ligands in cellular subpopulations, Cancer, 61, 963–70. Orntoft, T.F., Langkilde, N.C., Wiener, H. and Ottosen, P.D., 1991, Cellular localization of PNA binding in colorectal adenomas: comparison with differentiation, nuclear:cell height ratio and effect of desialylation, APMIS, 99, 275–81. Raz, A. and Lotan, R., 1987, Endogenous galactoside-binding lectins: a new class of functional tumor cell surface molecules related to metastasis, Cancer Metastasis Review, 6, 433–52. Raz, A., Pazerini, G. and Carmi, P., 1989, Identification of the metastasis-associated, galactoside-binding lectin as a chimeric gene product with homology to an IgE binding protein, Cancer Research, 49, 3489–93. Ryder, S.D., Smith, J.A. and Rhodes, J.M., 1992, Peanut lectin: a mitogen for normal human colonic epithelium and human HT29 colorectal cancer cells, Journal of the National Cancer Institute, 84, 1410–16. Sams, J.S., Lynch, H.T., Burt, R.W., Lanspa, S.J. and Boland, C.R., 1990, Abnormalities of lectin histochemistry in familial polyposis coli and hereditary nonpolyposis colorectal cancer, Cancer, 66, 502–8. Schumacher, U., 1990, Vergleichende histologische, histochemische und ultrastrukturelle Untersuchungen zum Nachweis und zur Bedeutung von kohlenhydrathaltigen Verbindungen in der Mamma: Drüsenepithel, Milchfettkugelmembran, Bindegewebe. Habilitationschrift (Thesis), University of München. Schumacher, U., 1992, A critical evaluation of neoglycoprotein binding sites in vivo and in sections of mouse tissue, Histochemistry, 97, 94–99. Schumacher, U., Kretzschmar, H., Brooks, S. and Leathem, A., 1992, Helix pomatia lectin binding pattern of brain metastases originating from breast cancers, Pathology Research and Practice, 188, 284–86. Schumacher, U., Higgs, D., Loizidou, M., Pickering, R., Leathem, A. and Taylor, I., 1994, The lectin Helix pomatia agglutinin is a good prognostic marker in colon cancer (in press). Sharma, A., DiCioccio, R.A. and Allen, H.J., 1992, Identification and synthesis of a novel 15 kDa beta-galactosied binding lectin in human leucocytes, Glycobiology, 2, 285–92. Shiraishi, T., Atsumi, S., and Yatani, R., 1992, Comparative study of prostatic carcinoma bone metastasis among Japanese in Japan and Japanese Americans and whites in Hawaii, Advances in Experimental Medicine and Biology, 324, 7–16. Söderström, K.O., 1987, Lectin binding to prostatic adenocarcinoma, Cancer, 60, 1823–31. Steuer, M.K., Gabius, H.J., Bardosi, A., Matthias, R., 1991, Histochemische Identifizierung endogener Lektine durch markierte Neoglykoproteine bei humanen Kopf-Hals-Platte-nepithelkarzinomen, Laryngorhinootologie, 70, 243–49. Taylor, C.W., Anbazhagan, R., Jayatilake, H., Adams, A., Gusterson, B.A., Price, K., Gelber, R.D. and Goldhirsch, A., 1991, Helix pomatia in breast cancer, Lancet, 338, 580. Thomas, M., Noguchi, M., Fonseca, L., Kitagawa, H., Kinoshita, K. and Miyazaki, I., 1993, Prognostic significance of Helix pomatia lectin and c-erbB-2 oncoprotein in human breast cancer, British Journal of Cancer, 68, 621–26. Tsuchida, Y., Honna, T., Fukui, M., Sakaguchi, H. and Ishiguro, T., 1989, The ratio of fucosylation of alpha-fetoprotein in hepatoblastoma, Cancer, 63, 2174–76. Walker, R.A., 1984a, The binding of peroxidase-labelled lectins to human breast epithelium I—Normal, hyperplastic and lactating breast, Journal of Pathology, 142, 279–91.

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Chapter 12 Dietary Galactose-binding Lectins and their Effects on Human Colonic Epithelial Cells J.D.Milton and J.M.Rhodes

Introduction The Thomsen-Friedenreich antigen (galactose (1–3) N-acetyl galactosamine- -) behaves as an oncofetal antigen in many epithelial tissues, becoming expressed in hyperplasia or malignancy. There is a considerable literature showing that in the human colon this antigen is increasingly expressed in hyperplastic and adenomatous polyps, inflammatory bowel disease (ulcerative colitis and Crohn’s disease) and in colorectal cancer. The Thomsen-Friedenreich (TF) antigen is recognized by many galactose-binding lectins. Although some of these are toxic or associated with toxins (e.g. Ricinus communis and Abrus precatorius), there is a group of non-toxic dietary lectins which are highly resistant to digestion in the mammalian gastrointestinal tract and avidly interact with TF antigen expressed by the intestinal mucosa. The best characterized of the dietary TF-binding lectins are from the peanut (Arachis hypogaea, PNA), the common edible mushroom (Agaricus bisporus, ABL), jackfruit (Articarpus integrifolia, jacalin or JAC) eaten in India and parts of the Far East and the Amaranthus caudatus agglutinin, ACA, which used to be eaten in South America and is becoming re-established as a food (Vietmeyer, 1986). All these lectins will bind to Gal (1–3)GalNAc -, the TF antigen (Chatterjee et al., 1979; Lotan et al., 1975; Presant and Kornfeld, 1975; Rinderle et al., 1989) but PNA will also bind to Gal (1–3)GalNAc - (Chatterjee et al., 1985) and to a lesser extent poly-Gal (1–3)GlcNAc (Farrer et al., 1980). The other three lectins ABL, JAC and ACA, will bind slightly to Gal (1–3)GalNAc - but can bind to sialyl-Gal-GalNAc (Chatterjee et al., 1985; Rinderle et al., 1989), which PNA cannot. Studies, both in vitro and in vivo, described in this review have shown that these lectins are capable of having marked effects on epithelial proliferation and mucus synthesis. This may have important implications for the increased proliferation that is generally thought to precede carcinogenesis in the colon. Altered lectin-binding to colonic tissue in pre-malignant and malignant disease Many lectin-histochemical studies of gastrointestinal tissues have been conducted over the past decade, particularly with PNA. Although some investigators have reported an absence and some a presence of PNAbinding in the normal adult colon, this confusion seems to be simply a reflection of the technique used. PNA-binding can normally be demonstrated when a sufficiently sensitive technique (such as avidin-biotin amplification) is used (Ryder et al., 1993b). This staining is in the perinuclear region of the goblet cells and not in the theca (goblet) of these cells (Cooper and Reuter, 1983). There is universal agreement that PNA-binding is increased in colon tumour tissue (Cooper, 1982; Rhodes et al., 1986), both within the cells and in secreted material (Boland et al., 1982). Also the location

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within the cells is different in that much of the labelling in tumours is in the apical areas of the goblet cells and in the glycocalyx (Boland et al., 1982). It has also been shown that there is some discrepancy between PNA-binding and the binding of monoclonal antibodies to the TF antigen (Longenecker, 1987; Yuan et al., 1986). This may reflect the fact that PNA can bind to polylactosamine chains and also to Gal (1–3) GalNAc -which occurs on glycolipids rather than glycoproteins. Other investigations have shown that PNA shows increased binding to colonic mucosa from patients with inflammatory bowel disease, both ulcerative colitis (Cooper et al., 1987; Pihl et al., 1985) and Crohn’s disease (Rhodes et al., 1988), which are both premalignant conditions and in patients with either adenomatous or hyperplastic (metaplastic) polyps (Campo et al., 1988; Rhodes et al., 1986). Whilst patterns of staining vary, the general conclusion is that there is more PNA-binding and a different distribution pattern of staining in diseased or malignant tissue. The binding is not in the supranuclear region as in the normal colonic epithelium but in the apical regions and mucus. Recently ACA has also been used to investigate changes in glycoproteins in colonic dysplasia and malignancy. In the normal colon ACA binds to different sites from PNA, binding selectively to cells at the basal (proliferative region) of the colonic crypts, and is localized in the cytoplasm and apical membranes (Boland et al., 1991). A marked increase in labelling of adenocarcinoma of the colon, with particularly intense staining of secreted mucin was found, and similar increases were shown in adenomatous polyps, either sporadic or from patients with familial adenomatous polyposis. The suggestion was made that ACAbinding correlated with areas of increased proliferation. It was also shown that sialidase treatment increased PNA-binding, but did not affect ACA-binding, further evidence for the inability of ACA to distinguish between sialylated and non-sialylated Gal-GalNAc. Sata et al. (1992) confirmed that ACA bound to the lower part of the crypts in the left colon, but found more extensive staining of the full length of crypts, including the goblets, in the right colon. This staining was resistant to galactose oxidase-Schiff treatment indicating that there was another sugar attached to the galactose residue (presumably sialic acid). This finding was supported by a similar staining profile with Maackia amurensis lectin which binds to sialyl(2–3)Gal. In ade nomas and carcinomas ACA was also observed to stain luminal material. Studies with monoclonal antibodies to TF antigen in this investigation and other studies by Longenecker et al. (1987) and Orntoft et al. (1990) surprisingly failed to demonstrate the presence of non-sialylated Gal-GalNAc even in tumour tissue. In contrast, another study using the Dgalactose oxidase/Schiff reaction has confirmed the presence of Gal-GalNAc (Xu et al., 1992) as have recent studies by our own group (Campbell et al., in press). To our knowledge there have been no histological investigations of binding of ABL and JAC to colonic tissue. Effect of lectins on proliferation of colonic tumour cell lines Although most of these studies have used the HT29 colon cancer cell line, some were also performed with Caco-2 cells. The lectin effects can generally be demonstrated more clearly when the cells are grown under suboptimal conditions (low concentrations of fetal calf serum or serum-free medium) so that the stimulatory or inhibitory effects of lectins might be more readily observed. Peanut lectin (PNA) Initial studies (Ryder et al., 1992) showed that PNA at concentrations of 7.5– 100 µ g ml−1 was able to stimulate HT29 proliferation (cells in log phase) by up to 50 per cent as assessed both by 3H-thymidine incorporation after 58 h incubation or by cell counting after 96 h incubation, and that these effects were inhibited by

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preincubation with 0.2M galactose. Similar effects were observed with confluent cell cultures with shorter incubations (Ryder et al., 1993c). No indication of cytotoxicity was found even at the top of this dose range. An optimal concentration (100 pg ml−1) of epidermal growth factor was found to stimulate HT29 cells to a similar extent as PNA, but the two substances together produced a marked synergistic effect (220 per cent increase), which suggests that PNA is not acting via the EGF receptor (which in any case does not contain Gal-GalNAc) (Feizi and Childs, 1985). In contrast to the effect on HT29 cells, PNA had no effect, either stimulatory or inhibitory, on the proliferation of Caco-2 cells. It was found however that Caco-2 cells have a much greater binding ability for PNA than HT29, which will be considered below in relation to the lectin receptors. It should be observed that Caco-2 cells are highly sensitive to other lectins, namely from kidney bean, soya bean, or to broad bean agglutinins (Koninkx et al., 1992). Mushroom lectin (ABL) In contrast to PNA, ABL had an inhibitory effect on the proliferation of HT29 cell grown in serum-free medium, with 80–90 per cent inhibition being observed at 25 µ g ml−1, whether determined by thymidine incorporation or by cell counting (Yu et al., 1993). Cultures in the presence of 2 per cent fetal calf serum showed only 40 per cent inhibition at 50 µ g ml−1, probably due to the presence in fetal calf serum of fetuin which contains sialyl-Gal-GalNAc and would therefore be expected to absorb ABL. This is confirmed by the finding that fetal calf serum can inhibit the agglutination of human red cells by ABL (Yu and Milton, unpublished data). ABL also had a lesser anti-proliferative effect on two other cell lines, Rama-27 and MCF-7, and a modest inhibitory effect on the proliferation of Caco-2 cells (Yu et al., 1993). Again there was no indication of a cytotoxic effect of the lectin as indicated by dye exclusion and the ability of the cells to proliferate after removal of the lectin. The involvement of Gal-GalNAc in the binding of the lectin was demonstrated by the ability of 0.5 mM Gal-GalNAc to reverse the inhibitory effect of the lectin on HT29 cells, whereas 50 mM Gal, GalNac, GalGlc or GlcNAc did not reverse the inhibition. Studies with EGF and PNA confirmed the previous finding of a stimulatory effect, this time in serum free medium rather than 2 per cent FCS, and in both cases the proliferation of the cells was strongly inhibited by ABL, to the level found in ABL treated cells with no added stimulant. ABL was also able to inhibit the proliferation of HT29 cells stimulated by insulin. These studies showed that ABL has a very general inhibitory effect on proliferation of a wide range of epithelial cells. This, combined with its lack of any apparent cytotoxicity, is a very interesting effect which gives it considerable therapeutic potential, possibly as an anti-cancer agent. Jacalin (JAC) JAC was found to inhibit the proliferation of HT29 cells in serum free medium, but even at doses up to 100 µ g ml−1, which did not appear to be toxic, an inhibition of only 60 per cent was found compared with the 90 per cent found with ABL (Zhou et al., 1993). Again proliferation was assessed by both thymidine incorporation and by cell counting. Lack of cytotoxicity was demonstrated by dye exclusion and reversibility of effect after removal of the lectin. The involvement of the Gal-GalNAc binding site of the lectin was demonstrated by the ability of 0.5mM Gal (1–3) GalNAc to reverse the inhibitory effect, but it was also found that 50 mM -methyl Galactose, GalNac and melibiose (Gal-1-6-Gal) could also reverse the inhibitory effect.

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Effect of PNA on proliferation and mucin synthesis in cultured colonic biopsies The only lectin that has been investigated for effects on proliferation in colonic biopsies in vitro is PNA. Initial studies showed that 19 h incubation of normal colonic biopsies with 25 µ g ml−1 PNA caused a 30 per cent increase in crypt cell proliferation rate (CCPR) as assessed by counting of mitotic figures of tissue treated with vincristine for the last 1–3 h of the incubation period (Ryder et al., 1992). It was also found that incorporation of 3H-N-acetylglucosamine into mucin was increased by 77 per cent after 24 h incubation with PNA. Subsequent investigations with biopsies from patients with ulcerative colitis, Crohn’s disease and colonic polyps (Ryder et al., 1993b) showed a similar effect of PNA. Non-stimulated biopsies from patients with ulcerative colitis, particularly those with active disease, showed a higher CCPR than controls (10.9 v 7.2 mitoses crypt−1h−1) whereas biopsies from patients with Crohn’s disease were not significantly increased (7.9). When biopsies were incubated for 19 h with 25 µ g ml−1 PNA, the crypt cell proliferation rate was increased by about 25 per cent regardless of the initial rate. 3H-GlcNAc incorporation into mucin showed an increase in non-stimulated Crohn’s disease biopsies relative to controls whereas reduced incorporation was found in ulcerative colitis, particularly in inactive disease. PNA caused a similar increase in mucin synthesis (about 75 per cent) in tissue from all disease groups and controls. A particularly interesting feature of this investigation was the relationship of the response in biopsies in individual patients to the PNA-binding ability of adjacent biopsies. Thirty five per cent of normal biopsies showed binding to PNA in frozen sections and using either PNA-peroxidase or biotin-PNA/avidinperoxidase. The non-stimulated CCPR was the same regardless of the PNA positivity, but after PNA treatment the CCPR only increased in the biopsies from people who showed positivity for PNA-binding. Similar studies on the response to PNA in tumorous colons were not possible due to the lack of organised crypt structure in tumours. Effect of ingestion of peanuts on colonic cell proliferation PNA has been show to be resistant to digestion in the intestine; at least 50 per cent of its agglutinating activity being recoverable in faeces after ingestion (Ryder et al., 1992). Furthermore, an impressive effect of peanut ingestion on colonic proliferation has recently been demonstrated (Ryder et al., 1993a). Patients had rectal biopsies taken for measurement of mitotic index (number of mitoses per dissected crypt) and then ate 100g raw peanuts per day for one week at the end of which they underwent diagnostic colonoscopy and repeat rectal biopsy. Using avidin-biotin PNA histochemistry on adjacent biopsies it was possible to divide the patients (all of whom had normal colonoscopic appearances and normal routine histology) into patients who were PNA positive (10/36 and those who were negative). The PNA positive patients showed an increase in mitotic index of 41 per cent (p=0.0009) compared with a modest increase (11 per cent) in those who were PNA negative. Binding sites for lectins on colon tumour cells Studies with FITC labelled PNA (Ryder et al., 1993a) have shown that HT29 cells show only about 10 per cent positivity whereas about 30 per cent of Caco-2 cells are labelled. This is in interesting contrast with their respective responses to the lectin. The average number of binding sites for the lectin on each cell is of the same order of magnitude, 2–3×106, but as only a modest percentage of the cells is labelled, the actual number of binding sites per cell on those cells that do react with the lectin is, presumably, considerably higher. Studies of binding of ABL to HT29 using I125 labelled ABL showed about 3×107 binding sites per cell (Yu et al., 1993). Cytochemical studies with peroxidase-ABL on fixed HT29 cells show almost 100 per

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cent labelling of the cells (Milton and Yu unpublished data), though whether this labelling was on the external cell membrane or intracellular structures was not certain. Radio-iodine labelling of the cell membrane of live HT29 or Caco-2 cells by lactoperoxidase and glucose oxidase followed by PNA affinity chromatography, SDS-PAGE and autoradiography (Ryder et al., 1993a) showed three major PNA-binding glycoprotein bands, but the relative mobility of two of these bands was less in the Caco-2 extract than the HT29, suggesting MW of 38 and 32 kD for the smallest pair of bands. Interestingly, a similar extract from colonic tumour biopsy showed bands corresponding to the HT29 but not the Caco-2. Competition studies of binding of radio-iodine-labelled ABL to HT29 cells showed no competition with PNA (Yu et al., 1993). When the cells were treated with sialidase there was a modest increase in the binding of ABL and PNA then could block 15 per cent of the ABL-binding. A possible explanation for this modest competition is that most of the ABL-binding sites were sialylated and sialidase-insensitive, which is most likely to be due to the O-acetylation of the sialic acid making it resistant to sialidase (Schauer, 1985). Possible role of receptors for galactose-binding lectins in the regulation of cellular proliferation The correlation between the proliferative response to peanut ingestion demonstrated in vivo and the PNAreactivity of the adjacent colonic mucosa strongly suggests that this is a lectin-mediated effect rather than some other non-specific effect due to changes in dietary fibre or bacterial content of the colon. The magnitude of this effect is considerable and raises the possibility that epithelial proliferation in the colon may be affected at least as much by the dietary lectin content as by the presence of intraluminal growth factors. It also raises the possibility that the changes in carbohydrate expression detectable by lectin histochemistry in malignant and premalignant disease may have important effects on the proliferative response of the epithelium. How this effect might be produced is difficult to assess as there are no obvious PNA-binding sites seen on the lower crypt cell surfaces in lectin histochemical studies (though this is probably due to the relatively insensitivity of the technique). However, further studies (in preparation) have shown the presence of internalized PNA (identifiable by immunohistochemistry) in the supranuclear region of the surface epithelium in biopsies from patients after peanut ingestion. It is, therefore, possible that at least in part the proliferative response of the epithelium is induced by this endocytosed PNA. The fairly modest stimulatory effect of PNA on HT29 cells might reflect that only a small percentage of cells are responding as it is shown that only a small percentage of cells visibly bind PNA at any one time. In this context, information on PNA-binding site expression and the cell cycle could be of interest here. It must also be remembered that even in a cell line such as HT29, there is a considerable and well-described heterogeneity of the population. There are at least two possible explanations for the finding that ABL and JAC inhibit proliferation of HT29 cells in contrast to PNA. Firstly, this may reflect binding to different cell surface glycoproteins resulting in different transmembrane signalling. Alternatively, although all these lectins may be internalized, their effects may be different because their interaction with intracellular glycoproteins is different. Sialidase treatment of HT29 cells did not, however, result in any very significant change in response to ABL (Yu et al., 1993) suggesting that the latter explanation is more likely. The lectin blots of membrane extracts of HT29 cells (not exclusively cell surface membranes) with the three lectins PNA, ABL and JAC were similar, supporting this hypothesis and also indicating the presence of more than one receptor determinant (e.g., Sial-Gal-GalNAc and Gal-GalNAc) on each lectin-binding glycoprotein. It is, also

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possible therefore, that lectins may have differing effects as a result of binding to different carbohydrate structures on the same glycoprotein. In conclusion, binding of lectins specific for Gal-GalNAc and sialyl-Gal-GalNAc has considerable effect on the proliferation of colonic epithelial cells and cell lines of malignant colonic epithelial origin, but the mechanisms of these effects are unknown. Lectin-binding to cell membranes has been demonstrated and this system could be exploited to investigate changes in enzymes such as protein kinases, which are likely to be involved in control of cellular proliferation. References Boland, C.R., Montgomery, C.K. and Kim, Y.S., 1982, Alterations in human colonic mucin occurring with cellular differentiation and malignant transformation, Proceedings of the National Academy of Sciences of the United States of America, 79, 2051–55. Boland, C.R., Chen, Y-F, Rinderle, S.J., Resau, J.H., Luk, G.D., Lynch, H.T. and Goldstein, I.J., 1991, Use of the lectin from Amaranthus caudatus as a histological probe of proliferating colonic epithelial cells, Cancer Research, 51, 657–65. Campbell, B.J., Finnie, I., Hounsell, E.F. and Rhodes, J.M., Direct demonstration of increased expression of ThomsonFriedenreich (TF) antigen in colonic adenocarcinoma and ulcerative colitis mucin and its concealment in normal mucin, Journal of Clinical Investigation, 95, in press. Campo, E., Condom, R., Palacin, A., Quesada, E. and Cardesa, A., 1988, Lectin binding patterns in normal and neoplastic colonic mucosa. A study of Dolichos biflorus agglutinin, peanut agglutinin, and wheat germ agglutinin, Diseases of the Colon and Rectum, 31, 892–99. Chatterjee, B., Vaith, P., Chatterjee, S., Karduck, D. and Uhlenbruck G., 1979, Compara tive studies of new marker lectins for alkali-labile and alkali-stable carbohydrate chains in glycoproteins, International Journal of Biochemistry, 10, 321–27. Chatterjee, B.P., Ahmed, H., Uhlenbruck, G., Janssen, E., Kolar, C. and Seiler F.R., 1985, Jack fruit (Artocarpus integrifolia) and the Agaricus mushroom lectin fit also to the so-called peanut receptor, Behring Institute Mitteilungen, 78, 148–58. Cooper, H.S., 1982, Peanut lectin binding sites in large bowel carcinoma, Laboratory Investigation, 47, 383–90. Cooper, H.S. and Reuter, V.E., 1983, Peanut lectin-binding sites in polyps of the colon and rectum. Adenomas, hypoplastic polyps, and adenomas with in situ carcinoma, Laboratory Investigation, 49, 655–61. Cooper, H.S., Farano, P. and Coapman R.A., 1987, Peanut lectin binding sites in colon of patients with ulcerative colitis, Archives of Pathology and Laboratory Medicine, 111, 270–75. Farrer, G.H., Uhlenbruck, G. and Kardnok, D., 1980, Biochemical and lectin-serological studies on a glycoprotein derived from edible bird’s nest mucus, Hoppe-Seyler’s Zeitschrift für Physiologische Chemie, 361, 473–76. Feizi, T. and Childs, R.A., 1985, Carbohydrate structures of glycoproteins and glycolipids as differentiation antigens, tumour-associated antigens and components of receptor systems, Trends in Biochemical Sciences, 10, 25–29. Koninkx, J.F., Hendriks, H.G., van Rossum, J.M., van den Ingh, T.S. and Mouwen, J.M., 1992, Interaction of legume lectins with the cellular metabolism of differentiated Caco-2 cells, Gastroenterology, 102, 1516–23. Longenecker, B.M., Willans, D.J., MacLean, G.D., Selvaraj, S., Suresh, M.R. and Noujaim A.A., 1987, Monoclonal antibodies and synthetic tumor-associated glycoconjugates in the study of the expression of Thomsen-Friedenreichlike and Tn-like antigens on human cancers, Journal of the National Cancer Institute, 78, 489–92. Lotan, R., Skutelsky, E., Danon, D. and Sharon, N., 1975, The purification, composition, and specificity of the anti-T lectin from peanut (Arachis hypogaea), Journal of Biological Chemistry, 250, 8518–23. Orntoft, T.F., Harving, N. and Langkilde, N.C., 1990, O-linked mucin-type glycoproteins in normal and malignant colon mucosa: lack of T-antigen expression and accumulation of Tn and sialosyl-Tn antigens in carcinomas, International Journal of Cancer, 45, 666–72.

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Pihl, E., Peura, A., Johnson, W.R., McDermott, F.T. and Hughes, E.S., 1985, T-antigen expression by peanut agglutinin staining relates to mucosal dysplasia in ulcerative colitis, Diseases of the Colon and Rectum, 28, 11–17. Presant, C.A. and Kornfeld, S., 1975,Characterisation of the cell surface receptor for the Agaricus bisporus hemagglutinin, Journal of Biological Chemistry, 247, 6837–45. Rhodes, J.M., Black, R.R. and Savage, A., 1986, Glycoprotein abnormalities in colonic carcinomata, adenomata, and hyperplastic polyps shown by lectin peroxidase histochemistry, Journal of Clinical Pathology, 39, 1331–34. Rhodes, J.M., Black, R.R. and Savage, A., 1988, Altered lectin binding by colonic epithelial glycoconjugates in ulcerative colitis and Crohn’s disease, Digestive Diseases and Sciences, 33, 1359–63. Rinderle, S.J., Goldstein I.J., Matta, K.L. and Ratcliffe, R.M., 1989, Isolation and characterization of amaranthin, a lectin present in the seeds of Amaranthus caudatus, that recognizes the T- (or cryptic T-) antigen, Journal of Biological Chemistry, 264, 16123–31. Ryder, S.D., Smith, J.A. and Rhodes, J.M., 1992, Peanut lectin: a mitogen for normal human colonic epithelium and human HT29 colorectal cancer cells, Journal of the National Cancer Institute, 84, 1410–16. Ryder, S.D., Jacyna, M.R., Levi, A.J. and Rhodes, J.M., 1993a, Peanut eating increases colonic epithelial proliferation, Gut, Abstract 34, 568. Ryder, S.D., Parker, N., Eccleston, D.W., Haqqani, M.T. and Rhodes, J.M., 1994a, Peanut lectin (PNA) stimulates proliferation in colonic explants from patients with ulcerative colitis, Crohn’s disease and colonic polyps, Gastroenterology, 106, 117–24. Ryder, S.D., Smith, J.A., Rhodes, E.G.H., Parker, N. and Rhodes, J.M., 1994b, Proliferative responses of HT29 and Caco2 human colorectal cancer cells to a panel of lectins, Gastroenterology, 106, 85–93. Sata, T., Roth, J., Zuber, C, Stamm, B., Rinderle, S.J., Goldstein, I.J. and Heitz, P.U., 1992, Studies on the ThomsenFriedenreich antigen in human colon with the lectin Amaranthin. Normal and neoplastic epithelium express only cryptic T antigen, Laboratory Investigation, 66, 175–86. Schauer, R., 1985, Sialic acids and their role as biological masks, Trends in Biochemical Sciences 10, 357–60. Vietmeyer, N.D., 1986, Lesser-known plants of potential use in agriculture and forestry, Science, 232, 1379–84. Xu, H., Sakamoto, K. and Shamsuddin, A.M., 1992, Detection of the tumor marker D-Galactose- -(1–3)-N-Acetyl-Dgalactosamine in colonic cancer and precancer, Archives of Pathology and Laboratory Medicine, 116, 1234–38. Yu, L.G., Fernig, D.G., Smith, J.A., Milton, J.D. and Rhodes, J.M., 1993, Reversible inhibition of proliferation of epithelial cell lines by Agaricus bisporus (edible mushroom) lectin, Cancer Research, 53, 4627–32. Yuan, M., Itzkowitz, S.H., Boland, C.R., Kim, Y.D., Tomita, J.T., Palekar, A., Bennington, J.L., Trump B.F. and Kim, Y.S., 1986, Comparison of T-antigen expression in normal, premalignant and malignant human colonic tissue using lectin and antibody immunohistochemistry, Cancer Research, 46, 4841–47. Zhou, Z.Q, Yu, L.G, Milton, J.D., Fernig, D.G. and Rhodes, J.M., 1993, Jacalin causes non-cytotoxic inhibition of proliferation of HT29 colon cancer cells, Clinical Science, 85, 11P (abstract).

Chapter 13 Enterotoxigenic Fimbrial Escherichia coli Lectins and Their Receptors: Targets for Probiotic Treatment of Diarrhoea Edilbert Van Driessche, Rony Sanchez, Ilse Dieussaert, Louis Kanarek, Paul Lintermans and Sonia Beeckmans

Introduction More than a century ago, in 1885, the German paediatrician Theodore Escherich described for the first time a bacterium that could be found in the faeces of healthy individuals and that was called Bacterium coli commune. This facultative anaerobic micro-organism, known today as Escherichia coli, is a normal inhabitant of the large intestine of mammals and birds where it fulfils an important role in the intestinal physiology. It has taken quite some time to realise that some strains of E. coli are the causative agents of diseases as different as diarrhoea, urinary tract infections, cystitis, pyelonephritis, meningitis, peritonitis, septicemia and gram-negative pneumonia. It is surprising that it was 1945 before Bray and Beavan could unequivocally prove that E. coli may be diarrhoegenic in man, especially in children (for a detailed historical review and early references mentioned in this introduction see Robins-Browne, 1987). Outbreaks of severe, often mortal diarrhoea in children have been described both in the USA and Europe since the second half of the seventeenth century. Several investigators had noticed that diarrhoea, especially in childhood, is most prominent during the summer and consequently was also referred to as ‘summer diarrhoea’, and that the disease was most prevalent during the first two years of life at the age of weaning. At the beginning of this century, studies conducted in Britain showed a clear correlation between the socioeconomic status of children and mortality by diarrhoea, and also that breast-feeding was protective against the disease. Although a memorial plaque at the Hillingdon Hospital in Uxbridge (UK) reminds us of the achievements of Bray and Beavan in discovering E. coli as a diarrhoegenic organism, earlier studies had already pointed into this direction. For example, in 1889, Laurelle reported that E. coli can cause peritonitis by perforating the intestine, and Lesage had suggested in 1887 that there are two types of E. coli, i.e. harmless and harmful ones. From a study conducted on Danish children suffering from diarrhoea, Bahr concluded that E. coli is involved in the disease. Similarly, in Germany, in the 1920s, Adam had come to the same conclusion. Today, coligenic diarrhoea in man is of minor importance in developed countries, and when it occurs it can quite easily be kept under control. It should be realised, however, that in developing countries, diarrhoea caused by E. coli or other enteric pathogens remains a life-threatening disease, especially for children. One of the major drawbacks of the intensification of agricultural practice is that animals are kept in conditions which promote the development and fast-spreading of diseases. Newborns and animals, especially, are sensitive to infection by both viruses and bacteria at the age of weaning. In husbandry, coligenic diarrhoea remains a serious problem in young animals not protected by pathogen-specific

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antibodies delivered by the colostrum and milk of the dam. Although antibiotics may be used in an attempt to suppress infections, the intensive use of these compounds has resulted in the selection and fast-spreading of multi-resistant strains. Consequently, alternatives are being developed to treat and/or prevent coligenic diarrhoea in immunologically unprotected animals. Based on their virulent properties, methods of interacting with the intestinal mucosa, clinical syndromes, epidemiology and serotype, diarrhoegenic E. coli are generally classified into four groups, i.e. enterotoxigenic (ETEC), enteroinvasive (EIEC), enteropathogenic (EPEC) and enterohaemorrhagic (EHEC). For a detailed description of the properties of these groups, the reader is referred to the excellent reviews of Robins-Browne (1987), Krogfelt (1991) and Levine (1987). In this chapter we will only consider the enterotoxigenic E. coli responsible for severe diarrhoea in man and his life-stock. The bacteria belonging to this group express at their surface one or several lectins that can be either fimbrial or nonfimbrial in nature, and produce heat-stable (ST) and/or heat-labile (LT) enterotoxins. Actually, it is the toxins that cause diarrhoea by provoking the secretion of water and electrolytes into the lumen of the gut. For recent reviews on the structure of enterotoxins and the way they act at the level of the enterocytes, the reader is referred to papers by Gyles (1992) and Spangler (1992). As a result of the strong interest in ETEC during the last two decades, an overwhelming number of papers have been published on the detection and purification of ETEC lectins and their receptors, on the organization of the genes responsible for fimbriae expression and its regulation, as well as on new strategies in an attempt to prevent colonization of the small intestine by ETEC. Consequently, several reviews have been devoted to these topics which, together with this paper, should give a sound knowledge of the most important issues (Beachey, 1981; Bertels et al., 1991; De Graaf, 1990; De Graaf and Mooi, 1986; Gaastra and De Graaf, 1982; Jann and Hoschützky, 1990; Krogfelt, 1991; Levine, 1987; Moon, 1990; Mouricout, 1991; Nimmich, 1990; Oudega and De Graaf, 1988; Paranchych and Frost, 1988; Pohl, 1993; Sharon, 1987; Van Driessche and Beeckmans, 1993; Wadström, 1993). In this chapter we will focus on the basic mechanisms involved in the binding of E. coli to the mucosa of the gut and in probiotic alternatives to prevent mucosal colonization. Attachment of E. coli to eukaryotic cells is mediated by surface lectins The observation of Guyot in 1908 that some E. coli strains are able to agglutinate erythrocytes of animals and humans, already pointed to the presence of lectins on the surface of the bacteria. Nevertheless, it was only during the mid-1950s that Collier and De Miranda (1955) proved that the observed agglutination can specifically be inhibited by mannose. The finding of Duguid and Gillies (1957) that agglutinating properties of E. coli are correlated to the presence on the bacterial surface of long proteinaceous appendages indicated that the binding of E. coli to eukaryotic cells is mediated by these surface structures, which were originally called pili and are now generally known as fimbriae. These authors also showed that fimbriated E. coli cells attach to intestinal cells. However, conclusive evidence that fimbriae act as bacterial lectins which can recognize glycoconjugates was provided by the elegant studies of Ofek and co-workers (1977). These authors showed that destruction of oligosaccharides on the surface of epithelial cells by sodium metaperiodate, a reagent known to cleave the C—C bond between vicinal hydroxyl groups of sugars, abolishes the capacity of these cells to bind E. coli. Moreover, Ofek et al. (1977, 1978) also found that epithelial cells pre-incubated with a mannose-specific plant lectin such as Concanavalin A no longer bind E. coli that express mannose-specific fimbriae. Similarly, these authors reported that yeast mannan is a strong inhibitor of E. coli attachment, and that yeast is agglutinated by the bacteria. Taken together, these observations clearly showed that mannose-specific lectins, present on the surface of E. coli recognize mannose-

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containing receptors on the epithelial cells. The most convincing evidence to prove that the mannosebinding properties reside in fimbriae was provided by showing that solubilized and highly purified fimbriae prevent E. coli from attaching to the epithelial cells (Ofek et al., 1977). Also Salit and Gotschlich (1977) showed that purified fimbriae agglutinate erythrocytes from several species in a mannose-inhibitable way. Detection of E. coli fimbrial lectins Haemagglutination Based on their haemagglutinating properties, fimbriae are generally classified as mannose-sensitive and mannose-resistant. Mannose-sensitive haemagglutination is ascribed to the presence of type 1 (or ‘common’) fimbriae. These fimbriae (see Table 13.1) are often present on both commensal and enterotoxigenic strains and are rather easily recognized in the electron microscope as 0.2–1 mm long, rigid filaments with a diameter of 7 nm (Figure 13.1A). E. coli strains expressing type 1 fimbriae agglutinate a wide variety of animal and yeast cells, and are expressed in a broad temperature range (18°C−37°C). Mannose-resistant haemagglutination points to the presence of host-specific fimbriae on enterotoxigenic E. coli (Figure 13.1B). These fimbriae confine to E. coli Table 13.1. Characteristics of fimbriae of enterotoxigenic E. coli Fimbriae

Natural host

Morphology

Type 1 (F1)

no host specificity common fimbriae pig

K88 (F4) (ab, ac, ad) 987P K99 (F5) F41

pig pig, lamb, calf pig, lamb, calf

F17 F111 CS31A

calf calf calf

CFA/I (F2)

human

CFA/II (F3)

human

CS 1

Molecular mass major submit (Dalton)

Gene localisation

Erythrocytes agglutinated

rigid, Ø 7 nm 17000

chromosome

guinea pig

MS

mannose, mannosides

flexible, Ø 2. 1 nm rigid, Ø 7 nm flexible, Ø 5 nm flexible, Ø 3. 2 nm

23000– 27000 20000 18500

plasmid

guinea pig, chicken none horse, sheep

MR

galactosides

MR MR

unknown sialic acid

29500

chromosome

MR

GalNac

flexible flexible flexible, Ø 2 nm rigid, Ø 7 nm

19500 17500 30000

chromosome unknown plasmid

human, guinea pig, horse, sheep bovine bovine unknown

MR MR

GlucNac GlucNac unknown

15000

plasmid

human, bovine, chicken bovine, chicken

MR

sialic acid

MR

unknown

plasmid plasmid

plasmid rigid, Ø 7 nm 16800

Inhibiting sugars

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Fimbriae

Natural host

CS 2 CS 3 CFA/III

human

CFA/IV

human

Morphology

Molecular mass major submit (Dalton)

rigid, Ø 7 nm 15300 flexible, 02 15000 nm rigid, Ø 7 nm 18000 flexible

Gene localisation

Erythrocytes agglutinated

plasmid

human, bovine human, bovine

plasmid

Inhibiting sugars

MR

unknown

MR

unknown

CS 4

rigid, Ø 6–7 17000 nm CS 5 rigid Ø 5–6 21000 nm CS 6 fine fibrillar 14500 Abbreviations: MS mannose sensitive; MR mannose resistant; GalNac N-acetylgalactosamine; GlucNac Nacetylglucosamine. For properties of putative colonization factor antigens CS7, CS17, PCFO9, PCFO159 and PCFO166, see text.

their host- and tissue-specific attachment properties and are generally not expressed at temperatures below 18°C. Unlike type 1 fimbriae, host-specific fimbriae will agglutinate or attach to a restricted variety of eukaryotic cells (see Table 13.1). Today several of these fimbriae of enterotoxigenic E. coli have been well characterized with respect to their protein-chemical properties and to the organization of the genes that direct and regulate their synthesis. In vitro attachment to immobilized glycoproteins Although haemagglutination experiments are easy to perform thereby demonstrating the presence of surface lectins, for some strains, although equipped with fimbrial lectins, no agglutinated erythrocytes could be identified. This is the case, for example, for E. coli expressing 987P fimbriae that fail to agglutinate erythrocytes from cow, horse, guinea pig, sheep, pig or rabbit. Similarly, when we started our studies on F17 fimbriae, no erythrocytes were known to be agglutinated by E. coli F17 strains, but more recently we found that cow erythrocytes are agglutinated. It should be emphasized, however, that non-agglutinatable erythrocytes can become agglutinatable after soft enzymatic treatment with, for example, trypsin, papain, neuraminidase, etc., known in some instances to unmask cryptic receptors. Also, temperature is an important parameter in E. coli induced haemagglutination, and whenever new strains are investigated for haemagglutinating properties, it is advisable to perform the tests at 4°C, 25°C and 37°C. For most fimbriae described today, haemagglutination is strongest at 4°C. In order to circumvent the problems met with haemagglutination, we developed an in vitro system based on the recognition of the oligosaccharide chains of glycoproteins covalently attached to a solid support such as Eupergit-C (Van Driessche et al., 1988; see Figure 13.2A). Glycoproteins can easily be covalently linked to this inert polyacrylic matrix, which is substituted with oxirane groups which will react with the free amino groups of proteins under mild conditions, i.e. pH 7.5 at room temperature (see Figure 13.2B). After blocking unreacted oxirane groups with 2-mercaptoethanol, the glycoprotein-derivatized beads can be used

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Figure 13.1. A. E. coli strain expressing type 1 fimbriae. Transmission electron microscopy after negative staining of the bacteria. B. E. coli strain expressing F17 fimbriae. Transmission electron microscopy after negative staining of the bacteria. C. Attachment of E. coli F17 to duodenum of a mouse (scanning electron microscopy). D. Aggregation of purified F17 fimbriae.

for at least three years (Van Driessche et al., unpublished). The main advantage of this in vitro adhesion

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system is that intestinal mucus and/or solubilized brush-border membrane fractions can easily be used to screen for strains that express surface lectins which are of potential interest as mediators of bacterial attachment in vivo. Furthermore, the observation of attachment of bacteria in this system is straightforward using a light microscope. As the oligosaccharides of the covalently linked glycoproteins can readily be oxidized by sodium metaperiodate, the involvement of lectin-sugar interactions in the attachment observed can be demonstrated. Similar to haemagglutination tests, the sugar specificity of the surface lectins can clearly be seen by the addition of potential inhibiting sugars during the incubation of the beads as well as to the washing buffer used to remove non-adherent bacteria. One of the main advantages of the Eupergit-C-glycoprotein system over the widely used in vitro adhesion test developed by Girardeau (1980) is that native receptors or receptor-analogues can be used. Indeed, in the Girardeau-adhesion system, villi recovered from the small intestine have to be fixed by formol or other fixatives, a procedure which can dramatically alter the affinity of the receptors for E. coli surface lectins. For example E. coli F17 strains, which were shown to colo nize the small intestine of mice, failed to react with villi prepared according to Girardeau from the same strain of mice (Sanchez et al., unpublished). Detection of fimbrial lectins by electron microscopy Although either of the in vitro systems described above can demonstrate the presence of E. coli surface lectins, they do not give information on their morphology. Indeed, it is known that E. coli surface lectins can be either fimbrial or non-fimbrial in nature. The presence of fimbrial lectins can quite easily be demonstrated by examination of the bacteria in the electron microscope after negative staining with uranyl acetate (Van Driessche et al., 1993b). Fimbrial lectins are seen as long appendages protruding from the surface of the bacteria. Based on their morphology and diameter, fimbriae are generally described as rigid or flexible (see Table 13.1). Although rigid fimbriae with a diameter of 7nm may point to the expression of type 1 fimbriae, it should be remembered that some host-specific fimbriae also display these morphological properties (see Table 13.1). By electron microscopy, unequivocal identification of fimbriae expressed is only possible by immunological labelling techniques using specific sera directed against known fimbriae. However, this technique is rather cumbersome and the authors prefer to use slide agglutination for the identification of known fimbriae using monospecific antisera directed against highly purified fimbriae. In this assay, agglutination of the bacteria into big clumps will occur nearly instantaneously and can be observed with the naked eye. Purification of E. coli fimbrial lectins By now, several purification schemes have been published for the isolation of fimbrial lectins from E. coli. They essentially consist of two steps, i.e. solubilization and separation of the fimbrial structures from contaminating proteins which may either be released from the surface of the bacteria or from the interior of the cells during the solubilization procedure. Solubilization of fimbriae can be achieved in several ways: (1) by incubating the cells at temperatures around 60°C for 30 min in buffer (De Graaf et al., 1980a); (2) by ultrasonication; (3) by incubation in chaotropic agents such as 3 M KSCN for 1 h at 22°C (Altmann et al., 1982); (4) by mixing the bacterial suspension in a Waring blender or other blending devices such as Ultra Turrax, Virtis homogeniser etc. (Korhonen et al., 1980).

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Figure 13.2. A. Scanning electron microscopy of Eupergit-C-glycoprotein beads with attached E. coli expressing type 1 fimbriae. B. Immobilization of glycoproteins to Eupergit-C beads (Röhm Pharma, Germany). For coupling, glycoproteins, resuspended in 1 M potassium phosphate buffer containing 0.2 per cent (w/v) sodium azide pH 7.5, are mixed with the wet beads at a ratio of 40 mg glycoprotein per gram wet beads. Coupling is allowed to proceed at room temperature for 48 h. After washing the beads with phosphate buffered saline (pH 7.5), unreacted oxirane groups are blocked by shaking the beads in phosphate buffered saline containing 5 per cent (v/v) 2mercaptoethanol. After washing, the derivatized beads can be stored at 4°C in phosphate buffered saline containing 0.02 per cent (w/v) sodium azide until use (Van Driessche et al., 1988).

For example, De Graaf et al. (1980a) used a combination of ammonium sulphate precipitation and gelfiltration in the presence of deoxycholate to purify K99 fimbriae released from E. coli by heat treatment

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(20min, 60°C) in phosphate buffer containing 2M urea. Korhonen et al. (1980) mechanically detached fimbriae and succeeded in purifying them to homogeneity by a procedure consisting of ammonium sulphate precipitation, ultracentrifugation in a sucrose gradient and gelfiltration on Sepharose in the presence of 4M urea. These authors also showed that deoxycholate prevents fimbriae-fimbriae interactions to be formed, which may lead to the formation of huge complexes without affecting the sugar-binding properties or antigenicity. Moreover, deoxycholate was shown to dissociate fimbriae from vesicles. The use of urea in purification procedures is especially useful when fimbriae have to be purified from flagellated E. coli strains. Flagella are known to dissociate in urea, while fimbriae are very resistant to denaturation in urea (Korhonen et al., 1980). A critical evaluation of the procedures generally used to release fimbriae from E. coli revealed that mechanical dissociation is the method of choice for the solubilization of F17 fimbriae. This procedure is a mild one in which fimbriae are sheared off from the bacterial surface and eliminates the risk of protein denaturation by high temperatures or by dissociation when chaotropic solvents are used. Fimbriae released by blending proved to be only slightly contaminated by other proteins and are consequently easy to purify, as will be described below. Sonication on the other hand results in a complete disruption of the cellular structures, yielding a very complex protein mixture as starting material, and making further purification to homogeneity cumbersome. Furthermore, this procedure risks freeing proteolytic enzymes that may seriously reduce the activity or recovery of the proteins to be purified. Recently, investigations conducted by Dieussaert in this laboratory on the release of fimbriae from a new series of enterotoxigenic E. coli strains revealed that the temperature as well as the composition of the growth medium is of critical importance when elevated temperatures may have to be used for fimbriae release. For example, when grown on minimal medium, some strains were completely lysed after incubation at 60°C for 25 min. However, by decreasing the release temperature to 55°C, fimbriae could be successfully and quantitatively solubilized without major contaminating proteins but when the same strains were grown on a rich LB medium, cells readily lysed after incubation at 55°C. This absolutely undesired phenomenon could be avoided by decreasing the release temperature to 47°C. Unfortunately, there also seemed to be a concomitant decrease in the efficiency of fimbriae solubilization. The purification procedure we originally developed for F17 fimbriae (Van Driessche et al., 1993b) consists essentially of two steps, i.e. mechanical solubilization of fimbriae by mixing bacteria in a Waring blender for 2×30–40s, and precipitation of the fimbriae with ammonium sulphate at 20 per cent saturation at 4°C (see Figure 13.3). The homogeneity of these preparations was proven by several procedures. 1. SDS-polyacrylamide gel electrophoresis revealed only one polypeptide with a molecular mass of 20 kDa. No contaminants could be detected after staining the gels by Coomassie brilliant blue. 2. N-terminal sequence analysis (20 amino acids determined) gave one single sequence without any trace of contaminating polypeptides. 3. Electron microscopy of the purified fimbriae could not provide any evidence of contaminating structures such as cell wall fragments or membranes. Purified F17 fimbriae were seen as aggregated, long filaments. When compared with other purification procedures in which gel-filtration or sucrose gradient centrifugation are used to achieve homogeneity, fimbriae prepared by our procedure proved to be of equal quality. Without any doubt, the attraction of this procedure resides in its simplicity and the possibility of scalingup, even at the industrial level if necessary.

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Figure 13.3. Flow sheet describing the purification procedure for enterotoxigenic E. coli fimbriae.

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The procedure originally developed for the purification of F17 fimbriae has also been successfully used for the isolation of type 1 fimbriae and for the K99, F41 and F111 antigens (see Table 13.2). It also proved to be excellent for the purification of fimbriae from Klebsiella and Serratia strains, as well as for the purification of F107 fimbriae from E. coli provoking oedema disease in weaned piglets (De Cupere et al., 1993a,b). It should be emphasized, however, that in cases where different types of fimbriae are expressed on a particular E. coli strain, the procedure may fail to yield pure fimbriae of either type present. However, when the different fimbriae types precipitate quite differently, such as in the case of K99 and F41, differential ammonium sulphate precipitation can still be used (see Table 13.2). Alternatively, separation starting from ammonium sulphate precipitated material can be achieved by ion-exchange chromatography or hydrophobic chromatography. The latter technique, especially, might prove to be a useful one, as different types of fimbriae often differ in their hydrophobic properties. When E. coli strains that also express flagella are under investigation, these structures will also be released by blending and co-precipitate with the fimbriae. However, thanks to the difference in stability between fimbriae and flagella in denaturing agents such as urea, flagella can be dissociated while fimbriae remain intact (Korhonen et al., 1980). The intact fimbriae will still precipitate at low saturation of ammonium sulphate, while the flagella subunits should remain in the supernatant. In view of the high molecular mass of the fimbriae and their strong tendency to aggregate, when necessary, gel-filtration will easily separate fimbriae from contaminating proteins. However, this technique is generally known to be cumbersome, especially when scaling-up for bulk preparations is needed. Table 13.2. Percentage of ammonium sulphate to be used for the purification of different types of enterotoxigenic E. coli fimbriae E. coli strain

Serotype

Type of fimbriae expressed

Ammonium sulphate (per cent saturation)

K514 25KH09 111KH86 B41MC B41 K99

O?:K?:F1 O101:K+:F17 O101:K?:F(Att111) O101:K−:F41 O101:K−:F41,K99 20–40

type 1 (F1) F17 F111 F41 F41

0–20 0–20 0–20 0 20 0–20

An elegant example of a purification procedure that is based on the physicochemical differences between distinct fimbriae expressed on the same E. coli strain has been reported by Karch et al. (1985). The procedure described by these authors essentially relies on the differential depolymerization of different fimbriae by various disrupting agents such as octyl-glucoside, urea, SDS and guanidine-HCl. After exposure of the fimbrial mixture to each depolymerizing agent, intact and depolymerized fimbriae were separated by gel-filtration on a Sepharose-CL4B column. Using this methodology, Karch et al. (1985) succeeded in separating three fimbrial types which are co-expressed on E. coli strain O7:K1:H6. Gene clusters encoding fimbriae biosynthesis: a few well-studied examples From the SDS-electrophoresis patterns of purified fimbriae, it is tempting to suppose that these proteins have a very simple structure built up of one single type of subunit. However, investigations on the organization of gene clusters responsible for fimbriae expression revealed the contrary. Without doubt, no other fimbrial type has been so deeply investigated as the Pap fimbriae, and recent reviews on this topic by

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Hultgren and Normark (1991) and by Hultgren et al. (1993) clearly demonstrate the complexity of fimbriae expression and its regulation. It is now appreciated that in most fimbriae, the sugar-binding subunit is different from the major or structural subunit shown by SDS-polyacrylamide gel electrophoresis, and by molecular biology techniques, fimbriae production and receptor-binding activity can be dissociated. For a detailed description of the gene clusters encoding fimbriae, the reader is referred to Lintermans et al. (Chapter 14), and to De Graaf (1990). Genes encoding fimbriae may be either located on plasmids, on the chromosome, or on both (Table 13.1). Investigations on the expression of fimbriae revealed that the composition and pH of the culture medium and growth temperature are critical parameters. For a better understanding of this paragraph it should be remembered that, in general, gene clusters that encode fimbrial lectins contain at least the following genes (see Figure 13.4 and Note 13.1 at end of chapter). A gene encoding a large outer membrane `pore' protein This molecule has a molecular mass of about 85 kDa (Klemm and Christiansen, 1990; Mooi et al., 1986; Oudega and De Graaf, 1988). It is implicated in the translocation of fimbrial subunits across the outer membrane and serves as a mould upon which fimbriae polymerization occurs. It is believed to form loops in the membrane bilayer. Mutants lacking this protein do not assemble normal fimbriae (Klemm and Christiansen, 1990; Mooi et al., 1982, 1983). It is not yet established whether this pore protein also serves as a permanent anchor after assembly of the fimbriae is terminated. A gene encoding a multifunctional, smaller, periplasmic protein This periplasmic protein (molecular mass about 26 kDa) is involved in stabilizing non-polymerized fimbrial subunits and in transporting fimbrial subunits from the inner to the outer membrane. It obviously acts as a chaperon. In the absence of this protein, none of the fimbrial subunits (i.e. major as well as minor subunits: see below) are detectable in the periplasm (De Graaf and Klaasen, 1986; De Graaf et al., 1984; Klemm, 1992; Mooi et al., 1983; Orndorff and Falkow, 1984). Complexes between the chaperon protein and the major subunit have been detected by De Graaf and co-workers both in K99- and K88-producing E. coli cells. In the K99 system coadsorption was observed of FanE with FanC onto Sepharose-Protein A onto which anti-FanC IgG had been bound (De Graaf et al., 1984). In the K88 system, complexes containing equimolar amounts of FaeE and FaeG were isolated by isoelectric focusing and polyacrylamide gel electrophoresis in non-denaturing conditions (Mooi et al., 1983). Furthermore, FaeE/FaeG complexes were detected by gel-filtration. As their molecular mass was estimated to be 50 kDa, it was concluded that they consisted of one molecule of FaeE associated with one molecule of FaeG (Bakker et al., 1991). It was postulated by Bakker et al. (1991) that a chaperon protein associates with a major subunit through regions of the fimbrial subunit normally involved in subunit-subunit interactions. Three regions in FaeE have indeed been observed to show significant sequence homology with regions in FaeG, at least one of which is normally supposed to be involved in subunit-subunit interaction. Although crystal structures of either FaeE or FanE are not available yet, it was suggested by Holmgren et al. (1992) that these proteins have structures similar to that of the PapD chaperon molecule. The latter protein was shown to have a general topology of an immunoglobulin fold and be built up of two distinct globular lobes (Holmgren and Brändén, 1989).

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Figure 13.4. Biosynthesis of fimbriae from enterotoxigenic E. coli. The fim (type 1), fan (K99) and fae (K88ab) gene clusters are shown and the molecular masses of the respective proteins are indicated (according to data from De Graaf (1990) for fim and fan clusters, and from Bakker et al. (1992b) for the fae cluster). The symbols used in the gene clusters correspond to symbols used for the proteins in the assembly model: the outer membrane pore protein is hatched, the chaperon protein is represented by heavy lines, the fimbrial major subunits are dotted and the minor subunits are shaded either lightly (30 kDa protein which is implicated in initiation and termination of fimbrial growth and which in many cases also is the adhesin), or heavily (smaller proteins implicated in the determination of the number and the length of fimbriae). Other genes (white and with thin lines) are implicated in regulation of gene expression. In the absence of chaperon molecules, fimbriae subunits are degraded by the DegP protease. See text for further details. The model presented here strongly resembles the model for uropathogenic Pap fimbriae biogenesis which has been studied in more detail and recently was reviewed by Hultgren et al. (1993).

A gene coding for the synthesis of the major fimbrial subunit The major subunit builds up the ‘body’ of the fimbriae. This subunit is the most prominent polypeptide

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present on SDS-gels (De Graaf et al., 1984; Gerlach et al., 1989; Minion et al., 1986; Mooi et al., 1981; Pedersen, 1991; Shipley et al., 1981). Genes encoding minor fimbrial subunits These subunits are usually not resolved in SDS-electrophoresis of purified fimbriae. Minor fimbrial subunits have been shown to be involved in initiation and termination of subunit polymerization, in regulation of the extent of fimbrial expression, in determining the length of the fimbriae, etc. These minor fimbrial subunits show substantial amino acid sequence homology with the respective major fimbrial subunits and they are also comparable in molecular mass (i.e. about 17 kDa). In type 1 cells, FimF is thought to be involved in initiation of fimbriae polymerization (see below; Russell and Orndorff, 1992). FimG on the other hand apparently regulates the length of the fimbriae: overproducing strains dispose of shorter fimbriae, whereas E. coli cells having a mutation in FimG produce very extended fimbriae (Maurer and Orndorff, 1987). In K99 E. coli cells, the exact role of minor subunits (FanG and FanH) has not yet been investigated, but these proteins have been hypothesized to fulfil comparable functions as FimF and FimG in type 1 fimbriae (Simons et al., 1991). Both FanG and FanH show significant sequence homology with the major subunit FanC (Roosendaal et al., 1987b). FanG and FanH were shown to be loosely associated in the outer membrane but were found not to be components of the fimbriae themselves (Roosendaal et al., 1987b). In K88ab cells, besides minor subunits of about 17 kDa (i.e. FaeC, which is predominantly located at the tip of the fimbriae (Mooi et al., 1984; Oudega et al., 1989), and FaeF which seems to be required for initiation and/or elongation of the fimbriae), polypeptides of higher molecular mass have also putatively been detected to be minor subunits (FaeI and FaeJ). Amino acid sequence homology was observed between FaeG (major subunit), FaeH, FaeI and FaeJ, particularly at the N- and C-terminal ends (Bakker et al., 1992b). A precise function of the latter minor subunits in the biogenesis and functioning of K88 fimbriae could not be deduced until now. In all clusters a gene is found encoding a protein with a molecular mass around 30 kDa In type 1 E. coli cells, FimH appears to be responsible for the mannose-specific adhesive properties of the fimbriae (Harris et al., 1990; Klemm and Christiansen, 1987; Krogfelt et al., 1990; Maurer and Orndorff, 1987). An E. coli strain over-producing FimH was observed to display an appreciably higher haemagglutination titre and, morphologically, to form so-called fimbriosomes, i.e. 10nm-diameter rounded structures associated with the fimbriae and which were proven to be built up of FimH protein molecules (Abraham et al., 1988a). Moreover, FimH was also found to be involved in fimbriae production. The Cterminal domain of FimH shows substantial sequence homology not only with the major fimbrial subunits (FimA), but also with the other minor subunits FimF and FimG (Klemm and Christiansen, 1987). It was shown by immunoelectron microscopy that FimH is located both laterally and at the tip of the type 1 fimbriae (Abraham et al., 1988a), but from receptor immunoelectron microscopy it was suggested that only the laterally positioned FimH molecules are responsible for sugar-binding (Krogfelt et al., 1990). In K99 cells, FanF was found to be essential for fimbriae biogenesis and it was detected as a minor component in the K99 fimbriae (Simons et al., 1990). FanF was found to exhibit sequence homology with FaeG, the major subunit of K88ab fimbriae. From these results it was suggested by Oudega and De Graaf

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(1988) that a minor component (FanF) through evolution became a major fimbrial subunit in a related species (FaeG). Whereas in type 1 fimbriae, the adhesive properties apparently are confined to FimH molecules, both in K88 and K99 cells adhesion is mediated by the major subunits (Jacobs et al., 1987a,c), although there is some indication that in K88ab cells minor subunits might also be involved (De Graaf, 1990). The 30 kDa polypeptides thus seem to serve several functions, for example, (1) they play a role in fimbriae biogenesis, and (2) in some fimbriae they are responsible for sugar-binding. Genes involved in regulation of fimbriae expression In type 1 E. coli cells expression of fimbriae has been shown to be subjected to phase-regulation i.e. individual cells oscillating between a fimbriated and a non-fimbriated state (Eisenstein, 1981; Nagy et al., 1977). Phase-variation can possibly help E. coli cells to avoid the host’s immune system by regularly eliminating the highly immunogenic fimbrial structures. Phase-variation was shown to result from the inversion of a 314-bp DNA segment (Abraham et al., 1985), the ‘switch’, situated immediately upstream of fimA and presumably containing a promoter. Klemm (1986) demonstrated that the phase-switch is regulated by two proteins, encoded by fimB and fimE genes, located upstream of fimA. FimB and FimE proteins display important sequence homology. They both contain high amounts of positively charged amino acid residues and therefore may interact with DNA. It was shown that FimE switches the invertible segment in the ‘off’ position (i.e. the promoter pointing away from fimA), whereas FimB promotes both ‘on-to-off’ and ‘off-to-on’ inversions with minimal preference to the ‘on’ position (Blomfield et al., 1991; McClain et al., 1991). Whereas it is generally accepted that the switch regulation by FimB/FimE is unresponsive to growth conditions, it was recently shown that additional proteins, encoded by genes not belonging to the fim gene cluster, are involved as well in the inversion (Blomfield et al., 1993). The possibility of the latter proteins being subjected to environmental regulation was put forward by Blomfield et al. (1993). In K99 cells fimbrial biosynthesis was shown to be controlled by two regulatory, remarkably homologous proteins, FanA and FanB (De Graaf, 1988; Roosendaal et al., 1987a). These proteins are synthesized without signal peptide, indicating that they are not exported. They also show sequence homology with FaeB, a regulatory protein of K88ab E. coli cells. Mutations in either FanA or FanB cause a 10-fold decrease in fimbriae synthesis. A model for transcriptional regulation of K99 gene cluster expression was put forward by De Graaf (1988). FanA and FanB are thought to form a dimer attaching to RNA polymerase within the fanA gene, thereby preventing termination of transcription at two terminator sequences present upstream of FanC. Based on the above data, a model for fimbriae biogenesis was put forward by De Graaf (1990). It is schematically represented in Figure 13.4 (see also Hultgren et al., 1993, for a review on the assembly and functioning of fimbriae in uropathogenic [Pap] E. coli). All fimbrial major and minor subunits are synthesized as larger precursors having an N-terminal signal sequence. In the periplasm a pool of subunits is built up. They are stored complexed with chaperon proteins which are supposed to prevent the subunits from precocious polymerization and protect them against proteolytic degradation. A cell-envelope protease (DegP) is indeed known to be present and to play an important role in the degradation of unstable proteins to be exported beyond the cytoplasm (Strauch et al., 1989). In the outer membrane, 85 kDa pore proteins are available as assembly platforms onto which fimbrial biogenesis will start. First, a 30 kDa minor subunit is bound onto the pore protein. This subunit is thought to block the pore unless a number of distinct minor subunits are adsorbed, thereby pushing the 30 kDa subunit

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outside the cell and detaching the initiation complex from the pore protein. Subsequently, major fimbrial subunits are bound. Upon integration of fimbrial subunits, the chaperon molecules are set free and can be recycled. Growth of a fimbrial superstructure is supposed to stop when another 30 kDa protein is incorporated in such a way that it again blocks the pore. It is hypothesized that further translocation of such a 30 kDa molecule is prevented because the subunit is inserted in opposite orientation (i.e. using its C-terminal domain for interaction with the last major subunit). According to this model, the length of the fimbriae will depend on the relative concentrations of major versus minor subunits. It should be emphasized that variations on this general model most probably have been developed by several E. coli strains during evolution. In this respect it should also be remembered that E. coli strains easily loose, acquire and exchange genes, as will be described below. Structure-function relationships in fimbrial E. coli lectins and their receptors Type 1 fimbriae As mentioned in the introduction, mannose-specific fimbrial lectins, known as type 1 fimbriae, were the first to be investigated and shown to be implicated in the attachment of E. coli to eukaryotic cells. Type 1 fimbriae are rod-like filaments with a diameter of 7 nm and an average length of 1 µ m. These proteinaceous filaments are built up of some 1000 major, structural subunits (FimA) in combination with several minor ones such as FimF, FimG and FimH (as discussed above). Today, FimH has been unequivocally identified as the adhesin. Tewari et al. (1993) succeeded in purifying FimH in quantities that allowed the study of this adhesin in homogeneous form. These authors showed that the purified adhesin displays the same mannoseinhibitable binding to human neutrophils and provokes the same metabolic effects as type 1-fimbriated E. coli, and that microspheres coated with FimH are phagocytosed by neutrophils. As well as binding and affecting the metabolism of neutrophils, purified type 1 fimbriae have also been shown to stimulate T cellindependent proliferation and secretion of immunoglobulins of the IgM isotype by B lymphocytes (Ponniah et al., 1992). These two examples clearly show that, like plant lectins, bacterial fimbrial lectins are not just mediating attachment, but that binding to eukaryotic cell recep tors may also elicit intracellular responses within the cells. In type 1 fimbriae, the adhesin (FimH) has been shown to be localized both at the tip (Hanson and Brinton, 1988) and laterally, positioned at intervals along the fimbriae (Abraham et al., 1987; Krogfelt et al., 1990). This latter localization is in agreement with the findings of Ponniah et al. (1991) i.e. that fragmentation of type 1 fimbriae by freezing and thawing results in an increased haemagglutinating activity, increased stimulation of human lymphocyte proliferation and increased binding of the mannose-containing enzyme horseradish peroxidase. Studies on the quantitative inhibition of the agglutination of mannancontaining yeast cells revealed that the combining site of type 1 fimbriae best fits structures found on short oligomannosidic chains of N-glycosidically linked glycoproteins (Firon et al., 1982). From these studies it was concluded that the sugar-binding site of type 1 fimbriae is an extended one corresponding to the size of a trisaccharide. Most probably this site consists of three subsites each of which accommodates a monosaccharide. As p-nitrophenyl- , D-mannoside is a much stronger inhibitor than methyl- , Dmannoside, it might be possible that a hydrophobic region is present adjacent to one of the subsites. The latter conclusion was confirmed by Firon et al. (1987) when investigating the effects of aromatic glycosides of mannose on the agglutination of mannan-containing yeast and on the adherence of type 1fimbriated E. coli to guinea pig ileal epithelial cells. Similarly, Falkowski et al. (1986) came to the same

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conclusion upon studying the inhibitory affect of p-nitrophenol and related compounds on the adherence of type 1-fimbriated E. coli to vaginal epithelial cells. Hydrophobic sites in the vicinity of the carbohydratebinding site have also been found in several plant lectins such as ConA (Van Driessche, 1988). In view of the specific recognition of common parts of oligosaccharides of N-linked glycoproteins, it is not surprising that type 1-fimbriated E. coli attach to a wide variety of eukaryotic cells. The implication of type 1 fimbriae in pathogenesis is still a matter of debate, although it is known that, in addition to host-specific fimbriae, many strains causing, for instance diarrhoea or pyelonephritis, also express type 1 fimbriae. Nevertheless, in several cases, type 1 fimbriae mediated adhesion has been noticed. For example, Jayappa et al. (1985) showed that porcine small intestines are successfully colonized with E. coli known to express exclusively type 1 fimbriae. These authors also found that the E. coli only attach on the brush-border and, using immunofluorescent staining, that type 1 fimbriae are also expressed on in vivo attaching E. coli cells. Furthermore, when newborn pigs were fed anti-type 1 fimbriae serum prior to challenge, the number of the gut-associated bacteria was reduced by six-fold and vaccination of gilts with type 1 fimbriae resulted in protection of the newborn pigs. These findings thus clearly show that type 1 fimbriae can be considered as a virulence factor and that purified type 1 fimbriae act as an effective vaccine antigen. In calves, Dieussaert et al. (unpublished results) could show that receptors for type 1 fimbriae are present, both in the mucus layer and the brush-border membranes of epithelial cells all along the small intestine of animals up to four months. Similarly, Sajjan and Forstner (1990) showed that rat intestinal mucin contains receptors for type 1-fimbriated enterotoxigenic E. coli. These receptors were found to be located on N-linked oligosaccharides of the 118-kDa linker glycopeptide region of mucin and to be partly covered by non-covalent bound lipid. In view of the high similarity of the sugar-binding properties of type 1 fimbriae expressed by different members of the Enterobacteriaceae (Abraham et al., 1988b), mucin as well as the brush-border membranes of enterocytes might contain potential receptors for type 1 fimbriae and consequently these fimbriae may be of importance in the intestinal colonization by E. coli and other members of the Enterobacteriaceae. Moreover, mannose-sensitive fimbriae have been shown to be implicated in gram-negative bacillary bacteraemia and meningitis in neonatals (Cox and Taylor, 1990; Guerina et al., 1983), and in urinary tract infections (Aronson et al., 1979; Svanborg-Eden et al., 1990). Recently, Kukkonen et al. (1993) reported that type 1-fimbriated E. coli bind to laminin and reconstituted basement membranes. This finding is of special interest because it might indicate that type 1 fimbriae are implicated in penetration through the basement membrane to cause septicemia. This might also be true for other members of the Enterobacteriaceae, as investigations by Gerlach et al. (1989) have shown that the mechanism of synthesizing type 1 fimbriae within the members of this family appears to be highly conserved, as is demonstrated by complementation studies leading to the production of functional fimbriae. Although type 1 fimbriae are often present on intestinal strains of E. coli, Bloch et al. (1992) considered them to be at most only indirectly implicated in intestinal colonization. This statement is based on the observation that not all E. coli strains colonizing the intestine produce type 1 fimbriae and that, as a result of phase-variation, E. coli type 1 fimbriae positive cells may be present in the fimbriae negative phase in the intestine. Moreover, mutants obtained by site-directed mutagenesis and prevented from producing type 1 fimbriae are as efficient in colonizing the intestine as the wild type cells. Instead of being directly implicated in intestinal colonization, type 1 fimbriae might play a key role in the individual to individual transmission of E. coli as a consequence of oropharyngeal colonization. Unlike in experimental in vivo conditions, where high amounts of bacteria are given to the animals, natural infection occurs with only a few or, in any case, with a small number of bacteria. By first colonizing the oropharyngeal mucosa, E. coli would be able to multiply before moving down the gastrointestinal tract, thereby ensuring that at least part of the inoculum survives the inactivation and killing by the stomach. It should be kept in mind however that

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salivary glycoproteins can bind to type 1-fimbriated E. coli and cause these cells to agglutinate (Babu et al., 1986). This interaction might cause an early removal of type 1-fimbriated bacteria and prevent colonization of the oropharyngeal mucosa. Consequently, both the saliva and the ecological conditions in the stomach may strongly reduce the number of type 1 fimbriae, able to interact with the mucosa of more distal parts of the gastrointestinal tract. The receptor for type 1 fimbriae present on guinea pig erythrocytes has been purified and characterized by Giampapa et al. (1988). These authors succeeded in purifying the receptor by affinity chromatography on immobilized type 1 fimbriae. The receptor was shown to be a glycoprotein of 65 kDa molecular mass with a pI of 8.5–8.7, containing N-linked oligosaccharides that are also recognized by ConA. An association constant of 6×106 M−1 has been determined for the interaction of type 1 fimbriae with the receptor. On human leukocytes, the integrins CD11 and CD18 were identified as receptors (Gbarah et al., 1991). Type 1-fimbriated E. coli were shown to mediate phagocytosis by bacteria in the absence of opsonins, a process known as lectino-phagocytosis (Ofek and Sharon, 1988; Sharon, 1987). In vivo, lectinophagocytosis might be important in tissues with low serum activity. Rodriguez-Ortega et al. (1987) succeeded in purifying the type 1 fimbriae receptors of human polymorphonuclear leukocytes, which were shown to be glycoproteins with a molecular mass of respectively 150, 100 and 70–80 kDa. The gp150 and gp100 were suggested to be identical to the - and -subunits of leukocyte complement receptors and adhesion glycoproteins involved in complement-mediated opsonophagocytosis. More recent results reported by Keith et al. (1990) however demonstrated that type 1 fimbriation might impede killing of E. coli by macrophages. These authors used different E. coli K12 mutants with defined lesions affecting either type 1 fimbriae expression, receptor-binding activity or length of the fimbriae produced. It was found that fimbriated cells are three times more resistant than non-fimbriated mutants, and that resistance to killing is not related to the length of the fimbriae but, contrarily, depends on receptorbinding activity of the fimbriae expressed. Thus, at the moment, the implication of type 1 fimbriae in nonopsonic killing of E. coli by macrophages remains unclear. Important E. coli fimbrial lectins and their receptors in cattle and piglets K99 fimbriae The K99 antigen was identified by Burrows et al. (1976) as a virulence factor in the pathogenesis of neonatal diarrhoea in calves. These authors reported that the K99 antigen causes mannose-resistant haemagglutination of sheep erythrocytes and it was shown to be responsible for the attachment of E. coli K99 cells to brush-borders prepared from 1–2-day-old calves. Several procedures are now available for the purification of K99 fimbriae. Isaacson (1977) used ammonium sulphate precipitation and column chromatography on dimethylaminoethyl-Sephadex, and noticed the strong tendency of purified fimbriae for self-aggregation. The isoelectric point of these fimbriae was determined by Isaacson to be higher than 10, which is very unusual for E. coli fimbriae. Most remarkably, whereas the fimbriated E. coli strain B41 was found to agglutinate guinea pig erythrocytes, these red blood cells were not agglutinated by the purified fimbriae. However, the guinea pig haemagglutinating activity was recovered from the DEAE-Sephadex column in a fraction that contained no K99 activity. This agglutinin was later determined to be the F41 fimbrial lectin that is co-expressed with K99 on the B41 strain (Morris et al., 1978, 1980). F41 fimbriae agglutinate guinea pig erythrocytes and have a pI of 4.2 (Isaacson, 1978), whereas K99 fimbriae agglutinate horse erythrocytes. The expression of K99 fimbriae is strongly dependent on the composition and temperature of the culture medium. De Graaf et al. (1980b) reported that K99 fimbriae production is optimal in minimal

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salt medium with glucose, and in semisynthetic Minca medium. In complex media, the expression of K99 fimbriae was found to be strongly reduced. Similarly, growth temperatures below 30°C repress the expression of these fimbriae (Roosendaal et al., 1986). Similarly, the pH of the medium was found to influence the production of K99 as well as of F41 fimbriae (Van Verseveld et al., 1985). According to De Graaf et al. (1980b), the temperature-dependent expression of fimbriae is relevant, as expression is only necessary at 37°C in the intestine of infected animals. Analysis of the gene cluster encoding K99 fimbriae expression revealed that 6 genes, designated C-H, are necessary (see discussion above). Unlike many other fimbrial E. coli systems, in K99 fimbriae sugarbinding activity is confined to the major subunit. Chemical modification studies (Jacobs et al., 1985) revealed that the integrity of the S—S bond in the K99 fimbriae subunits is essential for receptor-binding activity. Whereas modification of carboxyl groups or tyrosine residues has no effect on the sugar-binding properties of K99 fimbriae, modification of one arginine per subunit inactivates the fimbriae. Similarly, modification of lysine residues with 4-chloro-3,5 dinitrobenzoate results in loss of binding capacity of K99 fimbriae to horse erythrocytes. Lys-132 and lys-133 were suggested to be part of the receptorbinding domain of K99 fimbriae, because, in the presence of the glycolipid receptor, these two lysine residues were protected from modification. Furthermore, these two positively charged residues may be of special importance for the interaction with the negatively charged sialic acid residues of the glycolipid erythrocyte receptor (Jacobs et al., 1986). Using site-directed mutagenesis, Jacobs et al. (1987b) confirmed the importance of lys-132 and arg-136 for receptor-binding activity of K99 fimbriae. Indeed, replacement of these residues by threonine and serine respectively abolishes the adhesive capacity of the fimbriae. Jacobs et al. (1987b) also reported that a tryptophan residue, trp-67, plays an essential structural role, as replacement of this residue by leucine results in unstable polypeptides which were found to be undetectable in these mutants. The equine erythrocyte receptor for K99 fimbriae was characterized by Smit et al. (1984) as a glycolipid with the structure Neu5Gc- (2 3)-Gal- (1 4)-Glc- (1 1)ceramide. This glycolipid was unequivocally identified as the K99 receptor by several approaches: (1) pre-incubation of E. coli K99 cells or purified fimbriae with the receptor was found to inhibit equine erythrocyte haemagglutination; (2) the receptor inhibits attachment of E. coli K99 to porcine intestinal cells; (3) guinea pig erythrocytes, which are normally not agglutinated by E. coli K99 cells or purified fimbriae, can be rendered agglutinatable by preincubating the cells with the purified receptor; and (4) the receptor can be isolated by affinity chromatography using CNBr-activated Sepharose to which K99 fimbriae are covalently linked. Similarly, the K99 receptor in piglet small intestine has been shown by Ono et al. (1989) and Teneberg et al. (1990, 1993) to be the gangliosides NeuGc-GM3, NeuGc-GM2, NeuGc-CD1a and NeuAc-5PG. Non-acid glycolipids do not display receptor activity. When investigating the post-natal changes of GM3 Neu Gc, Yuyama et al. (1993) found a good correlation between the GM3 Neu Gc content and the susceptibility of piglets to E. coli K99 infection. This receptor is maximally expressed at birth and gradually decreases to of its initial quantity in 5month-old piglets. In agreement with the findings of Ono et al. (1989), the composition of the ceramide part of the receptor was also shown by Yuyama et al. (1993) to be important for E. coli K99 binding. Investigations on the binding of K99 fimbriae to cryostat sections of pig small intestine (Lindahl and Carlstedt, 1990) revealed binding sites to be located in the mucus but not in submucosal connective tissue. Receptor activity was shown to reside in high molecular mass mucin glycopeptides from the pig small intestine. Desialylation of the mucin glycopeptides resulted in a concomitant significant reduction in binding and confirms the sialic acid specificity of K99 fimbriae. In piglets, two phenotypes exist that differ from each other by the susceptibility of their intestinal brush-borders to adhesion of K99-fimbriated E. coli (Seignole et al., 1991). These differences were shown to be due to differences in the glycolipid composition

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of the membrane. Enterocyte membranes of receptive piglets have a higher content of monosialoglycolipids and oligosialogangliosides, whereas non-receptive piglets have mainly monosialogangliosides. Moreover, enterocyte membranes of receptive piglets were found to be richer in glycolipids containing K99 receptor structures than those of non-receptive animals. The calf intestinal mucus layer has been shown by Mouricout and Julien (1987) to contain receptors for K99 fimbriae which proved to be high molecular mass glycoproteins of 2000–4000 kDa. Desialylation of the mucus reduced the attachment of K99+ E. coli confirming again the specificity of K99 fimbriae for sialic acid. Moreover, these authors reported that the binding of E. coli K99 to mucus shows positive cooperativity. 987P fimbriae E. coli strains expressing 987P fimbriae are known to be causative agents of neonatal diarrhoea in piglets. Schifferli et al. (1990) could show that the genes encoding 987P expression, as well as the genes that encode the toxin STIa, are localized on a plasmid of 35–40 megaDa. Genetic analysis revealed that eight genes are involved, designated fasA to fasH, all of which are necessary for 987P expression and adhesion. Moreover, these authors suggested that the fimbrial subunit itself is the adhesin (Schifferli et al., 1991a,b). More recently, Casey et al. (1993) found that E. coli 987 strains contain plasmid as well as chromosomal copies of 987P genes, and that interaction between both types of genes may influence 987P fimbriae expression. De Graaf and Klaasen (1986) already described the presence of chromosomal 987P genes encoding five polypeptides with a molecular mass between 16.5 and 81 kDa. These authors identified the 25 kDa protein as the fimbrial subunit, and the others as accessory proteins involved in fimbriae production. Subsequent investigations of Klaasen et al. (1990) revealed that the 987P gene cluster contains a transposon that encodes the heat-stable enterotoxin STa. More detailed analysis by Klaasen and De Graaf (1990) showed that a protein designated FapR activates the promoter of the 987P gene cluster, and that the expression of the fapR gene is dependent on an adjacent IS1 element that flanks the enterotoxin gene (Klaasen et al., 1990). As with type 1 fimbriae, 987P fimbriae undergo phase-variation and are very resistant to depolymerization, which can only be achieved by heat or by 6 M guanidine-HCl. Van der Woude et al. (1989) showed that the composition of the growth medium influences both phase-variation and overall production of 987P fimbriae. For example, a shift from minimal to complex medium induces a rapid reduction in the amount of fimbriae per P+ cell, while a shift from complex to minimal medium results in an increase in the percentage of P+ cells and a constant amount of fimbriae per cell. More recently, Carroll et al. (1991) reported that 987P fimbriae expression was enhanced and in some cases restored by passing the organisms through Craigie’s tubes. Until now, no erythrocytes could be identified that are agglutinated by 987P fimbriae. Enterotoxigenic E. coli 987P colonize the small intestine of <6-day-old neonatal piglets. Type 987P-fimbriated E. coli were shown by Dean and Isaacson (1985) to attach in vitro to small intestinal epithelial cells of adult rabbits, but not to cells from infant animals. The receptor from adult rabbit brush-border membranes was shown to be released upon storage and was characterized as a low molecular mass glycoprotein containing 81 per cent carbohydrate and 19 per cent amino acids by weight. The isolated receptor agglutinates 987P fimbriated E. coli. It was shown that the agglutination can be inhibited by amino sugars and their derivatives, by high concentrations of salt and by D-galactose or L-fucose specific lectins (Dean and Isaacson, 1985). Localization studies using fluorescein-labelled antibodies against the 987P receptor revealed the receptor to be present in adult rabbits in the jejunum and ileum along the entire villous surface and in the goblet cells.

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In infant rabbits, receptors could only be localized in goblet cells. In the ileum of the neonatal piglet, material antigenically similar to the rabbit receptor was demonstrated in goblet cells (Dean and Isaacson, 1985). The colonization of the small intestine of pigs by 987P E. coli was shown by Dean et al. (1989) to be agedependent. Both the colonization and the incidence and severity of diarrhoea were found to be highest in neonatal piglets, while older pigs are not colonized and do not develop diarrhoea upon infection. Nevertheless, fimbriated bacteria adhere in vitro to intestinal epithelial brush-borders from both sensitive (neonatal) and older (resistant) pigs, indicating that resistance or sensitivity is not directly related to the lack or presence of 987P receptors. Resistance was shown not to be due to the inability of E. coli 987P to grow in the intestine of older pigs. Dean and co-workers (1989) hypothesized that resistance is due to the release of 987P receptors in the intestinal lumen and that blocking the 987P fimbriae prevents attachment. More detailed investigations by Dean (1990) showed the brush-border receptors from both susceptible and resistant animals to be glycoproteins with a molecular mass between 33 and 40 kDa. The mucus receptor proved to be a low molecular mass glycoprotein in older pigs, only trace amounts of which could be detected in susceptible neonates. In SDS-electrophoresis, the pig mucus receptor co-migrates with the receptor isolated from adult rabbits. These results show conclusively that resistance to infection by E. coli 987P is due to the presence of attachment blockers secreted in the small intestinal mucus layer of older pigs. F41 fimbriae As was mentioned earlier in this chapter, F41 fimbriae were originally detected as an unexpected ‘contaminant’ on E. coli strains expressing K99 fimbriae (Morris et al., 1978, 1980). The same authors could show that some enterotoxigenic E. coli produce F41 fimbriae but lack K88, K99 and 987P fimbriae (Morris et al., 1983). The investigations of Moseley et al. (1986) had revealed that the genes encoding F41 expression are localized on the chromosome and that a genetic relationship exists between the F41 and K88 determinants. Using K88 and F41 gene probes, these authors found that some strains hybridize with these probes but do not produce K88 or F41 fimbriae. As these strains display mannose-resistant haemagglutination of human erythrocytes, Moseley et al. (1986) concluded that additional types of adhesins might exist whose genetic determinants are related to those of F41 and K88. Anderson and Moseley (1988) identified and characterized the genes necessary for F41 fimbriae production. Four polypeptides with molecular masses of 29, 30, 32 and 36 kDa were shown to be encoded by the F41 determinant. The 29 kDa polypeptide was identified as the F41 fimbrial subunit. These authors further showed that a high degree of homology exists between the genetic determinants of K88 and F41 fimbriae. The homologies were shown to include sequences encoding accessory proteins implicated in fimbriae biosynthesis. However, the K88 and F41 fimbrial subunits are non-homologous. Anderson and Moseley (1988) hypothesized that, in view of homology of the genes that flank the subunit genes, in the evolution of K88 and F41, a fimbrial subunit gene from one type of fimbriae has been replaced by another. Recent studies of Cox and Houvenaghel (1993) on the in vitro adhesion properties of E. coli strains expressing either F41, K99, K88 or 987P fimbriae revealed that F41+ and 987P+ strains adhere all along the small intestine of 4–5-week-old piglets, whereas K99 strains preferentially attach to the caudal half of the small intestine. K99/F41+ strains were found mainly to adhere to the jejunum and ileum, and K88+ strains to jejunal villi.

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F17 fimbriae In calves, coligenic neonatal diarrhoea has mostly been correlated to the colonization of the small intestine by E. coli expressing K99 and/or F41 fimbriae. Another important fimbrial antigen, originally described as Att25 in Belgium by Pohl et al. (1982) and as F(Y) in France (Contrepois and Girardeau, 1985) proved to be expressed by several E. coli strains (Morris et al., 1985; Pohl, 1993). A comparison of both fimbriae by Lintermans et al. (1988a, b) revealed they are identical and since then, these fimbriae have been rebaptized as F17. Epidemiological research has shown that F17 fimbriae are commonly expressed by enterotoxigenic E. coli infectious for calves in the whole EC, Japan (Shimizu et al., 1987) and even India (Gulati et al., 1992). Genetic analysis revealed that at least four genes are required for the expression of adhesive F17 fimbriae (Lintermans et al., 1991). The F17-G protein was identified as the adhesin and is responsible for the haemagglutinating properties and the attachment of F17 fimbriae to the small intestinal mucosa of calves (Lintermans et al., 1991). Although no erythrocytes agglutinated by E. coli F17 strains or purified F17 fimbriae could initially be identified, the authors found that cow erythrocytes are readily agglutinated in an N-acetylglucosamineinhibitable way. However, upon investigating the adhesion properties of several E. coli strains, known to express exclusively F17 fimbriae, to glycoproteins immobilized on Eupergit-C beads, it was found that F17 fimbriae display carbohydrate-binding heterogeneity. From Table 13.3 it is clear that all strains investigated recognize the oligosaccharide chains of bovine submaxillary gland mucin. On the other hand, only some strains bind to the oligosaccharides present on ovomucoid or fetuin. In all cases, adhesion to the glycoprotein-derivatized beads was shown to be mediated by lectin-carbohydrate interactions, as oxidation of the sugar residues of the glycoproteins by sodium metaperiodate results in complete abolishment of bacterial adhesion. Inhibition studies with simple sugars revealed that, whereas some strains are prevented from binding to the beads by N-acetylglucosamine, others were insensitive to N-acetylglucosamine but binding can be inhibited by either mannose or N-acetylglucosamine. For some E. coli strains, attachmentinhibition could only be achieved when both N-acetylglucosamine and mannose are present during the incubation of the bacteria with the beads and in the washing buffer. Based on these observations, Van Driessche and co-workers (Van Driessche et al., 1988, 1993a) hypothesized that at least three types of carbohydrate-binding sites of the F17 adhesin can be distinguished (see Figure 13.5), in other words: 1. Strains solely inhibited by N-acetylglucosamine contain a small binding site that can accommodate this sugar. 2. Strains that can be prevented from binding to Eupergit-C-glycoproteins by either mannose or Nacetylglucosamine have a more extended carbohydrate-binding site, possibly consisting of two subsites which are partially overlapping. Saturation of either of the two subsites might prevent binding. 3. For strains that require a mixture of N-acetylglucosamine and mannose to prevent being bound to Eupergit-C-mucin beads, two hypotheses can be put forward. It might be possible that two types of adhesins are expressed, i.e. an N-acetylglucosamine specific fimbrial one, and a second mannosespecific one. The latter should be outer-membrane bound as there was no evidence of type 1 fimbriae, either by type 1 fimbriae antiserum, by SDS-electrophoresis of the purified fimbriae present, or by immuno-electron microscopy. Alternatively, the adhesin of this subtype of F17 fimbriae might contain an extended carbohydrate-binding site consisting of two subsites, i.e. one specific for Nacetylglucosamine and one specific for mannose. In this case, occupation of both subsites would be necessary to prevent attachment. However, the observation that the agglutination of yeast can be inhibited by either N-acetylglucosamine or mannose excludes the possibility that two types of adhesins are involved.

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Table 13.3. Attachment of E. coli F17 strains and E. coli K514 to Eupergit-C-glycoprotein beads E. coli strain

Ovomucoid

PBS

Man

Fetuin NAG

PBS

Man

Mucin NAG

PBS

Man

NAG

43KH85 – – – – – – + + _ 32KH85 – _ – – – – + + – 33KH85 – – – – – – + – – Att25 + + + + – + + + + 233S85* 25KH09 + – + – – – + – – K514 + – + + – + + – + (−) no attachment; (+) attachment PBS: phosphate buffered saline; Man: mannose; NAG: N-acetylglucosamine * attachment of E. coli Att25 233S85 to Eupergit-C-mucin beads can be inhibited by a mixture of mannose and Nacetylglucosamine

F17 fimbriae purified from strains corresponding to each of the three subgroups cannot be distinguished on the basis of their amino acid composition, indicating that at least the same major subunit is expressed on each subtype (Van Driessche, unpublished). These observations are of special interest, both from a fundamental and from a practical point of view. Indeed, the results show that fimbriae which are considered to be identical by cross-reacting with the same antiserum or when comparing the molecular mass of their major subunit or their overall amino acid composition, may nevertheless display different carbohydrate binding properties. This finding is of crucial importance when adhesin-blockers may be used in vivo to prevent colonization of the small intestine. Although haemagglutination-inhibition studies and investigations on the attachment and attachmentinhibition using immobilized glycoproteins provide information on the sugar-binding properties of fimbriae, they do not inform us of the nature and distribution of the receptors present in the small intestinal mucosa. Similarly, these studies give at the very best semi-quantitative information on the inhibitory potency of potential attachment-inhibitors. In order to study the intestinal receptors for F17 fimbriae, an ELISA (enzyme-linked inmunosorbant assay) was developed by Sanchez et al. (1993b, 1993c). In this technique, the wells of microtiter plates are coated with purified brush-border membranes isolated from enterocytes or mucus and incubated with E. coli. After the non-attached bacteria have been washed away, adherent cells are quantified using antibodies directed against purified fimbriae and Protein A-phosphatase. By using this technique, Sanchez et al. (1993a, b, c) could show that receptors for F17 fimbriae are present in both the intestinal mucus and brush-border membranes all along the small intestine of an 11-day-old calf. Attachment of F17 fimbriated E. coli was shown to be due to lectin-sugar interactions as pre-treatment of either brushborder membranes or mucus with sodium metaperiodate strongly reduces or completely prevents bacteria from binding. Similarly, the inhibition of attachment caused by pre incubating the bacteria with monosaccharides and glycoproteins points in the same direction. These effects will be discussed in more detail later in this chapter. Detailed investigations by Dieussaert (1992) on the age-dependent expression of F17 receptors revealed them to be present in both the brush-border membranes and mucus of neonatal calves and animals of at least four-months-old (see Figure 13.6).

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Figure 13.5. Model to explain the observed carbohydrate-binding heterogeneity of F17 fimbriae. (1) Presumed carbohydrate-binding site of F17 fimbriae exclusively inhibited by N-acetylglucosamine (NAG); (2) carbohydratebinding site of F17 fimbriae whose sugar-binding activity is inhibited by either mannose or N-acetylglucosamine; (3) carbohydrate-binding site of F17 fimbriae that can only be prevented from attachment by a mixture of mannose and Nacetylglucosamine.

F111 fimbriae Unlike F17 receptors, those of F111 fimbriae are hardly detectable in intestinal mucus preparations of calves of different age. Moreover, they were shown by Dieussaert et al. (1993) to be expressed mainly in

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Figure 13.6. Binding of E. coli strain Att25 233S85 expressing F17 fimbriae (shaded), and E. coli strain 111KH86 expressing F111 fimbriae (hatched) to mucus (A) and brush-border membranes (B) of calves of different age. Wells of microtitre plates were coated with mucus or brush-border membranes, and subsequently incubated with fimbriated E. coli. After non-adherent bacteria had been washed away, the wells were incubated with monospecific anti-fimbriae antiserum, and with protein A-phosphatase. Phosphatase activity stain was recorded at 405 nm. Age 1: four days; age 2: six weeks; age 3: four months. D: duodenum; JP: jejunum proximale; JD: jejunum distale; I: ileum.

the brush-border membranes of the duodenum, and their presence was found to decrease in older animals. These results clearly show that receptors recognized by F17 and F111 fimbriae are different, at least at the

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level of their oligosaccharide chains. It was a rather unexpected observation indeed, as F17 and F111 fimbriae are highly homologous (Bertels et al., 1989). Both fimbriae agglutinate cow erythrocytes in an Nacetylglucosamine-inhibitable way, show cross-reactivity when tested with antiserum raised against either of them, and the N-terminal amino acid sequence of their major subunits is identical up to residue 30. These results confirm the statement mentioned above that adhesins, differing in their fine sugar specificity, can be combined to identical or at least very similar major subunits in the fimbrial structure. Although it has not yet been proven, it is tempting to speculate that the genomes encoding F17 and F111 are highly homologous and that the F111 adhesin evolved from the F17 one. Similarly, the F111 major subunit may have evolved from the F17 one by deletion of part of the C-terminus. Further studies conducted by Dieussaert (1992) on the effect of monosaccharides on the attachment of E. coli expressing F17 or F111 fimbriae revealed that, while some sugars inhibit attachment, some particular monosaccharides on the contrary stimulate attachment, especially at relatively low concentrations (Table 13.4). For example, for E. coli F17 strain Att25 233S85, even 0.05M mannose or methyl- , Dmannopyranoside was found to inhibit adhesion by up to 60 per cent. At the same concentration, glucose, Nacetylglucosamine, D-glucosamine-hydrochloride and fructose were observed to stimulate attachment with a factor of respectively 1.57, 1.35, 1.26 and 1.52. At high concentrations, i.e. 250 mM, all the monosaccharides tested have some inhibitory effect. At this concentration, both mannose and , Dmethylmannopyranoside inhibit the attachment at both the brush-border membranes and mucus completely. On the other hand, N-acetylglucosamine for example inhibits the attachment to mucus by 60 per cent, and has no effect on the adhesion to brush-border membranes. As mentioned above, from haemagglutinationinhibition studies it had previously been concluded that F17 fimbriae can be classified as Nacetylglucosamine-specific lectins. In combination, these results conclusively show that the identification of a haptenic sugar for lectins depends to a major extent on the experimental system used. Our results also clearly show that the generally accepted statement that mannoseTable 13.4. Stimulating effect of particular sugars (0.05 M) on the attachment to mucus of the small intestine E. coli strain Sugar

Att25 233S85

25KHo9

FIII

None 1 1 1 Glucose 1.57 1.53 ++ N-Acetylglucosamine 1.35 1.59 +++ Glucosamine-HCl 1.26 1.53 4.07 Fructose 1.52 1.54 2.57 Mannose 0.40 0.40 ++ Glucosamine 1 1 1.76 Arabinose 1 1 ++ Galactose 1 1 1.43 -Methylgalactopyranoside 1 1 1 ++ and +++ : strong, and very strong stimulation respectively, but not reproducibly quantifiable

Type I 1 1 1 1 1 0.10 1 1 1.38 1.33

sensitive binding is due to the presence of type 1 fimbriae does not hold. The same conclusion was formulated earlier by Van Driessche et al. (1993a) based on attachment-inhibition studies using Eupergit-Cglycoprotein beads.

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Figure 13.7. Competitor-activator model explaining the carbohydrate-specific increase in attachment of fimbriated E. coli F17 and E. coli F111 to mucus or brush-border membranes. The adhesins are supposed to have two carbohydratebinding sites, i.e. a receptor-binding site and (an) activator site(s). The activator site(s) display(s) a high affinity for particular monosaccharides. Upon saturation of the activator site(s), a conformational change in the receptor-binding site is induced with a concomitant increase in its affinity for the receptor. At high concentrations, activating sugars may become inhibitors.

The adhesion-stimulatory effect provoked by N-acetylglucosamine, glucose and fructose has been observed for different E. coli strains, producing exclusively F17 fimbriae. Also for E. coli type 1 strains, concentration-dependent stimulation of attachment has been observed (Table 13.4). It is interesting to note that similar effects have been observed by De Cupere et al. (1993a, b) with an E. coli 107 strain that provokes oedema disease in weaned piglets. The physiological significance of the attachment-stimulation observed by low concentrations of sugars is not clear at this moment. In order to attempt to explain the effects observed, a model was developed as depicted in Figure 13.7. In this competitor-activator model it is supposed that two types of sugar-binding sites exist at the level of the adhesin, i.e. a receptor-binding site and an activator site. Some sugars display high affinity-binding for the activator site which will consequently be saturated at low sugar concentrations. Saturation of this site is supposed to induce a transconformation in the receptor-binding site with a concomitant increase in affinity for the receptor. Consequently, a higher signal will be recorded in the attachment assay. Above a certain sugar concentration, the sugar can competitively compete with the mucosal receptors and behave as an inhibitor. CS31A fimbriae Some time ago, Girardeau et al. (1988) isolated from bovine enterotoxigenic and septicemic E. coli strains a new fimbrial antigen which was designated CS31A. Electron microscopy revealed that CS31A are very thin, fibrillar organelles of 2nm diameter that form capsule-like structures. The expression of CS31A fimbriae was shown by Girardeau et al. (1988) to be strongly dependent on the composition of the medium: fimbriae production is weaker in liquid medium and repressed by L-alanine. CS31A fimbriae consist of 30 kDa

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subunits and were shown to be immunologically related to K88 fimbriae. Also the N-terminal amino acid sequence shows a high degree of homology with that of K88 subunits. Until now, no erythrocytes could be identified that are agglutinated by CS31A expressing transconjugants harbouring the 105-megaDa plasmid containing the genes which encode these fimbriae. These cells also fail to adhere to intestinal cells. Recent investigations of Contrepois et al. (1993) revealed that CS31A producing strains have a common genetic background and are representatives of an important part of bovine pathogenic E. coli. The investigations of Korth et al. (1991) unequivocally identified CS31A fimbriae as a colonization factor. These authors showed that E. coli HB101, harbouring the cloned CS31A determinant, attach to cultured epithelial cells. The CS31A subunit gene shares extensive sequence homology in its signal sequence to the subunit genes encoding F41 and K88 fimbriae. Whereas no homology between the mature F41 and CS31A could be detected, substantial correlation to the K88 subunit was found. Based on the genetic homology between CS31A, F41 and K88, Korth et al. (1991) concluded that these fimbriae are encoded by a group of related genetic determinants differing in their structural subunit genes but having a related mechanism of adhesin expression. Using a CS31A-specific probe, the same authors confirmed that these fimbriae are expressed in bovine E. coli isolates in France, Canada, India and Japan, and are often present together with K99 fimbriae. K88 fimbriae K88 fimbriae are often expressed by E. coli strains causing neonatal diarrhoea in piglets. Three serological variants designated K88ab, K88ac and K88ad have been identified (Dykes et al., 1985; Foged et al., 1986) and at least six genes have been found to be involved in K88 fimbriae expression (De Graaf, 1990; see also discussion above). Similar to K99 fimbriae, the adhesive properties of K88 fimbriae reside in the major fimbrial subunit (Bakker et al., 1992a). This statement is in agreement with the observations of Jacobs et al. (1987c), that tripeptides ser-148-leu-phe, and ala-156-ile-phe, isolated from the major K88 subunit, inhibit the adherence of K88 fimbriae to erythrocytes and intestinal cells. Moreover, these tripeptides were found to be able to elute attached bacteria from agglutinated erythrocytes. Previous investigations had revealed that arginine residues are also implicated in the receptor-binding activity of K88 fimbriae (Jacobs et al. 1985). Using site-directed mutagenesis, Jacobs et al. (1987a) showed that, while replacement of phe-85 or phe-158 by serine has no effect on the biosynthesis or adhesive capacities of K88 fimbriae, substitution of phe-150 by serine results in a dramatic decrease in the adhesive capacity of K88 fimbriae. Receptors for K88 fimbriae were shown by Sellwood (1980) to be present in brush-borders of intestinal epithelial cells from pigs which are sensitive to infection by E. coli K88, while animals that are resistant to infection lack the K88 receptors. The K88ab-specific receptor in the porcine small intestine mucus was identified as a glycoprotein of 40–42 kDa molecular mass (Metcalfe et al., 1991) and the K88ac-specific receptors were shown to be glycoproteins with a molecular mass of 210 and 240 kDa (Erickson et al., 1992). These receptors were found to be present in adhesive brush-borders but absent from non-adhesive ones. From these results, in combination with those of Sellwood (1980), it can be concluded that the sensitivity or resistance to E. coli K88 infection is directly related to the presence or absence of fimbrial-specific receptors. Studies on the age and serotype-dependent binding of K88 to porcine intestinal receptors (Willemsen and De Graaf, 1992) showed that both mucus and brush-border membranes contain receptors which are glycoproteins in nature. In mucus, three glycoproteins displaying receptor activity were identified with a respective molecular mass of 25, 35 and 60 kDa, while in brush-border membranes, receptors were found to be glycoproteins of 16 kDa and a set of glycoproteins with a molecular mass between 40 and 70 kDa. Some

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differences were detected when the binding of the three K88 serotypes to mucus and brush-border preparations were compared (Willemsen and De Graaf, 1992). For example, K88ad fimbriae bind with low affinity to mucus receptors while K88ab fimbriae recognize an additional mucus glycoprotein of 16 kDa that does neither bind K88ac nor K88ad fimbriae. Furthermore, the presence of the mucus receptors was reported to be age-dependent. They were indeed shown to be expressed until at least day 35 after birth. Expression of the brush-border receptors on the other hand is age-independent. Pigs resistant to E. coli K88 infection do not contain receptors in their brush-border membranes. Investigations of Conway et al. (1990) clearly demonstrated the importance of intestinal mucus in pathogenesis. Upon studying the age-dependent presence of K88-specific receptors in porcine ileal mucus, these investigators found that K88-specific receptors in mucus are protective when present in high concentrations and that the incidence of infection is inversely proportional to the amount of receptors present. According to Conway et al. (1990), the low concentration of K88-specific mucus receptors in neonatal piglets would allow bacteria to adhere to the epithelial cells, while high concentrations of mucus receptors as those found in for example 35-day-old unweaned piglets would prevent epithelial colonization. This view is in full agreement with the findings of Dean and co-workers (1989) on 987P fimbriae. E. coli strains expressing K88 fimbriae but not producing enterotoxins were shown to colonize the small intestine of sensitive piglets (Duval-Iflah et al., 1990). Colonized animals died between 2 and 7 days postinfection and had developed gastric stasis and enteric congestions, but diarrhoea was never observed. Some sensitive piglets were not colonized although they were also shown to express K88-specific receptors at the level of the intestinal mucosa. From these observations it was concluded that some as yet unknown factors may counteract the bacterial colonization of the small intestine of adhesive piglets by E. coli K88. Receptors for K88ab fimbriae have been identified in both the mucus and brush-border membranes of 5– 8-week-old CD-1 mice (Laux et al., 1986). The mucus receptors were shown to be glycoproteins with a molecular mass of 57 and 64 kDa and are also present in the brush-border membranes which contain an additional receptor protein of 91 kDa. Attachment of E. coli K88ab to both mucus receptors and brushborder membranes can be inhibited by D-glucosamine, proteolytic digestion of the receptors and sodium metaperiodate oxidation. The studies of Laux and Cohen (1989) revealed that E. coli K12 (K88ab) attach to both mouse small intestinal epithelial cells and mouse small intestine mucus immobilised on polystyrene. However, in an in vitro system where epithelial cells had been covered with mucus prior to the application of bacteria, it was found that the E. coli K12 (K88ab) cells do not attach to the underlying cells, but remain attached to the mucus. When the mucus receptor sites are first blocked with purified fimbriae, adhesion to epithelial cells was readily observed. These results thus clearly demonstrate that the mucus layer may prevent E. coli adhesion to epithelial cells by entrapping the bacteria or by saturating the fimbrial receptorbinding sites by soluble receptors. In vivo studies confirmed this principle, as it was shown that E. coli K12 and E. coli K12 (K88ab) are cleared from the mouse intestine at equal rates. Although the fimbrial antigens described above have been most thoroughly investigated in animals suffering from diarrhoea, it does not imply that no other surface fimbrial adhesins are involved in attaching enterotoxigenic E. coli to the gut. For example, Salajka et al. (1992) detected a new colonization factor on enterotoxigenic E. coli isolated from the intestinal content of piglets suffering from postweaning diarrhoea. This factor was shown to be different from K88, K99, F41, 987P, CFA/I, CFA/II and F17 fimbriae. E. coli strains expressing this new adhesin, which was called antigen ‘8813’, cause diarrhoea and colonize the intestinal epithelium in pigs, and attach in vitro to pig intestinal brush-borders. The adhesin was shown to be produced at 37°C, but not at 18°C. No erythrocytes agglutinated by E. coli expressing exclusively the 8813 surface antigen could be identified. Similarly, Nagy et al. (1992) identified two enterotoxigenic E. coli strains from piglets suffering from fatal postweaning diarrhoea that express fimbriae which are different

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from K88, K99, F41 and 987P. These strains do not attach to isolated brush-borders of newborn, 1-day-old animals in the presence of mannose, but display mannose-resistant attachment to brush-borders of older animals. Consequently it was suggested that the attachment is dependent on receptors that progressively develop with age during the first three weeks after birth. The presence of new fimbriae was also suggested by Woodward et al. (1993) to be implicated in the development of neonatal diarrhoea in Australian pigs. Similarly, in this laboratory, Dieussaert et al. (unpublished) could identify at least five enterotoxigenic E. coli strains, isolated from diarrhoeic calves, that express fimbriae which were not described before. Human colonization factor antigens (CFA's) As mentioned in the introduction, enterotoxigenic E. coli are important causative agents of diarrhoea in people visiting developing countries, and especially in children in these parts of the world. Often, as a result of very fast dehydration, this diarrhoea is fatal. By now, several important and widely spread fimbrial antigens have been identified and some have been investigated in great detail (see also Table 13.1). CFA/I fimbriae are composed of 15 kDa subunits, CFA/II consists of a complex of three distinct antigens designated as CS1, CS2, CS3. While CS1 and CS2 are rigid fimbriae with a diameter of 6–7 nm, CS3 consists of thin, flexible fimbriae of 2–3 nm diameter. The subunits of CS1 and CS2 have a molecular mass of 16.8 kDa and 17 kDa respectively, while CS3 fimbriae are built up of fimbriae of 14.5 and 15.5 kDa subunits. CFA/I and CFA/II fimbriae have unequivocally been shown to be responsible for binding of E. coli to human enterocyte brush-borders (Knutton et al., 1984a). The receptor for CFA/I on human erythrocytes has been identified as a glycoprotein containing important sialic acid moieties (Pieroni et al., 1988) and has an apparent molecular mass of 26 kDa. Furthermore, as the receptor binds to wheat germ agglutinin-Sepharose, it was concluded that it is a sialoglycoprotein. Investigations of Wennerås et al. (1990) on the receptors for CFA/I and CFA/II fimbriae revealed that both fimbriae recognize different receptors, both in rabbit intestinal brush-border membranes and human intestinal cell lines. As the binding pattern of chloroform-methanol treated membranes was found to be identical to that of non-treated membranes, these authors concluded that the CFA/I and CFA/II receptors are glycoproteins rather than glycolipids. The human intestinal cell line receptors migrate in SDSelectrophoresis with a molecular mass in the range of 30–35 kDa but are absent from non-intestinal cell lines. Neuraminidase treatment of the nitrocellulose blots prior to incubation with fimbriae completely abolishes binding, confirming the importance of sialic acid residues for binding. All three subcomponents of CFA/II, i.e. CS1, CS2 and CS3 bind to the same receptors as does CFA/I. The elegant investigations of Bühler et al. (1991) revealed that the receptor-binding activity of CFA/I fimbriae resides in the tip subunit and succeeded in dissociating CFA/I fimbriae by heating at 100°C. The dissociated subunits were found to retain their receptor-binding properties as they were able to inhibit CFA/ I induced haemagglutination. Monoclonal antibodies raised against the CFA/I subunits were shown to be strong haemagglutination inhibitors and in immunoelectron microscopy studies these MAb only labelled the tips of the fimbriae, indicating that only the tip subunit displays receptor-binding activity. As CFA/I fimbriae were shown to be built up of only one type of subunit, it is thought that the potential receptorbinding sites of the internal subunits are masked in the fimbrial structure. According to Bühler et al. (1991), this property might be of special interest as breakage of fimbriae by shearing forces might unmask new receptor-binding sites and enable the bacteria to bind again to the intestinal mucosa. In order to determine the role of each subcomponent of the CFA/II complex in colonization and in eliciting protective antibodies, Svennerholm et al. (1990) investigated the colonization capacity of E. coli strains expressing either CS1, CS2 or CS3 in a nonligated rabbit intestine model (RITARD). It was found

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that all three antigens act as colonization factors and provoke anticolonization immunity. Similarly, Knutton et al. (1984b) had previously shown by electron microscopy that CFA/I and CFA/II mediate attachment to isolated human duodenal enterocytes and that bacterial adhesion involves many interactions between the tips of the fimbriae and receptors on erythrocytes or the enterocyte brush-border membrane. E. coli expressing type 1 fimbriae on the other hand attach to the basolateral membranes. Similar to CFA/II, colonization factor CFA/IV (previously designated as PCF 8775) also consists of three antigenic compounds, CS4, CS5 and CS6 (Wolf et al., 1989), which were investigated ultrastructurally as well as with respect to their attachment properties to human small intestine enterocytes and cultured human intestinal mucosa (Knutton et al., 1989). Whereas CS4 and CS5 fimbriae, which have a diameter of 6–7 nm and 6 nm respectively, were unequivocally shown by immunoelectron microscopy to promote adhesion, the role of CS6 as an adhesin involved in attachment is not clear. As CS6 was found to be difficult to characterize morphologically, it is possible that this antigen has a very fine fibrillar structure. Although E. coli strains lacking CS6 are non-adherent, strains expressing CS6 do adhere. However, it should be mentioned that CS6+ strains used in these studies also produce fimbrial or fibrillar structures which have not yet been characterized, and which might be responsible for the adhesion observed for CS6+ strains. Knutton et al. (1989) argued that non-adherence of CS6-only strains can be explained in several ways. First, the specific human duodenal enterocytes used in these studies may lack receptors in general, or some individuals may be devoid of these receptors. However, the authors considered the latter possibility as improbable as the same observation was made with 12 donors. Second, previous studies by Svennerholm and co-workers (1992) revealed that CS6-only E. coli readily attach to the ileum of rabbits, therefore it might be possible that CS6-specific receptors are only expressed at particular sites along the small intestine. Third, the possibility cannot be excluded that the non-adherent phenotype of CS6-only cells results from an insufficient expression of CS6. It is well-known that the expression of fimbriae is strongly dependent on the growth conditions and consequently it cannot be excluded that the micro-environment of the rabbit ileum favours the expression of CS6 to such an extent that adhesion and subsequent colonization is feasible. The subunit molecular mass of CS4 and CS5 is 17 and 21 kDa respectively, while that of CS6 subunits has been reported to be either 14.5 kDa, or 14.5 and 16 kDa, depending on the strain used (McConnell et al., 1988). Recently, a new type of fimbriae related to CS5 has been found to be present on the human enterotoxigenic E. coli strain 334 (Hibberd et al., 1990). Electron microscopy revealed that these fimbriae have a helical structure similar to CS5. However, 334 fimbriae are immunologically different from CS5 as well as from other known colonization factor antigens such as CFA/I, CFA/III and CFA/IV. SDSpolyacrylamide gel electrophoresis of 334 fimbriae gave subunits of about 21.5 kDa, and on western blotting the subunits of 334 and CS5A fimbriae shared common epitopes. 334 fimbriae were shown to be encoded by genes located on a plasmid that also contains the genes encoding the enterotoxin. The fimbriae of strain 334 have been designated by Hibberd et al. (1990) as CS7. CFA/III fimbriae were characterized by Honda et al. (1984) and shown to be responsible for the high hydrophobicity of strains expressing them. These fimbriae were purified to homogeneity by chromatography on Sepharose-4B and phenyl-Sepharose-CL4B, and proved to be built up of subunits of about 18 kDa molecular mass. Enterotoxigenic E. coli expressing CFA/III fimbriae were shown to colonize suckling mice and infant rabbits (Honda et al. 1984). The enterotoxigenic E. coli strain O144:H21 displayed mannose-resistant haemagglutination of bovine erythrocytes and expressed fimbriae with a diameter of about 7nm (McConnell et al. 1990). SDSelectrophoresis of the purified fimbriae revealed two types of subunits with a molecular mass 17.5 and 15 kDa respectively. Genes encoding these fimbriae, designated as CS17, were shown to be located on a 100 megaDa plasmid. This plasmid also contains the information for LT production.

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Another new putative fimbrial colonization factor has been described by Tacket et al. (1987). These fimbriae are expressed by the enterotoxigenic E. coli O159:H4 strain and are built up of 19 kDa subunits. This putative colonization factor has been designated as PCFO159:H4 and was shown to be plasmidencoded. Investigations of McConnell and Rowe (1989) on the prevalence of CFA/III and PCFO159:H4 revealed that both antigens occur infrequently in ETEC. Recent investigations by Svennerholm et al. (1992) on the role of different putative colonization factor antigens of human enterotoxigenic E. coli in rabbits revealed that PCFO159, CS7, CS17 and CFA/III are all colonizing factors as well as strong immunogens. Using the non-ligated rabbit intestine model (RITARD), these authors reported that E. coli expressing these adhesion antigens are present significantly longer in the stool than PCF/CS-negative strains, and that the infected animals develop a loose stool for a few days. As E. coli expressing PCFO166 were not present in the faeces for longer than normal flora E. coli, it was suggested by Svennerholm et al. (1992) that these fimbriae are not (at least in rabbits) involved in colonization. These authors argued that in humans, PCFO166 may play a role in colonization of the intestine as it was observed earlier that E. coli strains expressing these fimbriae attach to human enterocytes from duodenal biopsy tissues. Investigations of Sommerfelt et al. (1992) on the genetic relationship of PCFO166, CFA/I and CS4 revealed that the N-terminal amino acid sequence of PCFO166 shows a high degree of homology with that of CFA/I and CS4, suggesting that these three fimbrial systems are encoded by operons that are genetically related. This suggestion is further supported by the finding of Sommerfelt et al. (1992) that transformants harbouring the CfaD gene that codes for a positive regulator of the CFA/I and CS4 genes, produce high amounts of PCFO166 both in the presence or absence of bile salts which are normally necessary for PCFO166 expression. When screening human ETEC strains that attach to human proximal small intestinal mucosa but lack mannose-resistant haemagglutinating properties, Knutton et al. (1987) detected on E. coli strain O148:H28 a new type of fimbriae which was shown to be morphologically different from CFA/I, CFA/II and CFA/IV. These fimbriae consist of curly fibrils with a diameter of about 3nm. Heuzenroeder et al. (1990) described a new fimbrial antigen designated PCFO9 which was shown to be present on LT+ enterotoxigenic E. coli O9:H-LT+ strains isolated from infants in central Australia. These fimbriae were shown to be built up of major subunits of 27 kDa molecular mass, and encoded by genes located on the chromosome. Although some E. coli strains displaying mannose-resistant haemagglutination were originally considered not to express fimbriae, more detailed analysis revealed them to produce extremely fine fimbriae which hardly can be resolved by electron microscopy. For example, Ørskov et al. (1985) reinvestigated three E. coli strains originally described to be devoid of fimbriae. Immunoelectron microscopy of embedded and sectioned bacteria, that had first been treated with specific antisera raised against the haemagglutinins and with ferritin-labelled anti-rabbit IgG, revealed that these bacteria are surrounded by a capsule which was shown by negative staining to consist of very fine fimbriae. Recent studies by Kernéis et al. (1992) revealed that Caco-2 and HT-29 cells are excellent cell lines to be used in investigations on the molecular interactions between CFA’s with the brush-borders. These authors showed that CFA receptors are expressed during the differentiation process of these cells in culture. However, as these cell lines still differ from normal human enterocytes, a comparison of the structural features of receptors expressed by both cell types should be made before results obtained in vitro can be extrapolated to the situation found in the human intestine.

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Strategies to prevent colonization of the intestine by E. coli The studies mentioned above do not only contribute to our understanding of how E. coli manage to colonize mucosal surfaces, but they also open new avenues to interfere with this process. As already mentioned, attachment of E. coli to mucosae is a first, but essential step in pathogenesis. Consequently, different approaches have been used and/or are in full development to prevent E. coli adhesion. They have been described by Beachey in 1981 in a review paper on bacterial adhesion: (1) application of purified adhesin or receptor materials or their analogues as competitive inhibitors; (2) administration of sublethal concentrations of antibiotics that suppress the formation and/or expression of functionally active adhesins; and (3) development of vaccines against bacterial surface components involved in adhesion to mucosal surfaces. The potential of using sublethal concentrations of antibiotics to interfere with fimbriae production has been reviewed (Chopra and Linton, 1986; Zhanel and Nicolle, 1992). However, as antibiotics are known to contribute considerably to the selection of antibiotic-resistant variants, it is hoped that in the future, the use of these drugs can be further reduced and replaced whenever possible by alternative treatments. Prevention by antibodies The development of vaccines against E. coli fimbriae has proven to be successful, especially because fimbriae are known to be strong immunogens. In domestic animals especially, treating neonatal diarrhoea by vaccination of pregnant dams has in many cases resulted in the development of high titres of antibodies in the colostrum that will protect the offspring. For example, in the 1970s, Rutter et al. (1976) described the antibacterial activity of colostrum and milk of sows vaccinated with K88 fimbriae and showed that this activity was due to the presence of anti-adhesive K88 antibodies. These authors had already suggested that glycoproteins present in colostrum and milk might prevent K88-positive E. coli from attaching to mucosal receptors by binding to the K88 fimbriae. When E. coli are expressing different types of fimbriae, colostrumcontaining antibodies to each of them may be necessary to prevent mucosal colonization. Contrepois and Girardeau (1985) noticed that calves were not protected against E. coli expressing K99, F41 and FY fimbriae by either colostrum of cows vaccinated against K99+F41 or against FY, but a mixture of both colostra was found to be effective. Similarly, Runnels et al. (1987) found that piglets suckling sows which were vaccinated with pilus antigen, are only protected against infection by E. coli strains expressing the same fimbriae. For example, F41+ vaccine gave protection against challenge with E. coli F41 but not against challenge with E. coli K99 or against strains expressing both K99 and F41 fimbriae. These results clearly demonstrate the urgent need to search for new types of adhesins present in the E. coli population, so that they can be included to extend existing vaccines. Successful protection of newborn lambs (Sojka et al., 1978), piglets (Nagy et al., 1978) and calves (Acres et al., 1979) by colostrum antibodies raised against K99 fimbriae in dams have been reported. Similarly, colostral antibodies against 987P fimbriae (Lösch et at., 1986) passively protected piglets against infection by 987P positive E. coli. The use of colostrum to protect the newborn from intestinal colonization by enterotoxigenic E. coli has, at this time, several drawbacks. First, it implies that dams should be vaccinated during pregnancy, which is not always the case; second, colostrum is only available in limited amounts and, as a consequence, expensive to use. An attractive alternative might be immunization of chickens which are known to produce antibodies in the egg yolk (Heller et al., 1990; Kühlmann et al., 1988; Lösch et al., 1986; Shimizu et al., 1989). For example, Yokoyama et al. (1992) could passively protect neonatal colostrumdeprived piglets against fatal enteric colibaccillosis with powder preparations of specific antibodies obtained by spray-drying the water-soluble fraction of egg yolks from hens immunized against K88, K99 and 987P

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fimbriae. Similarly, rabbits could be prevented from developing diarrhoea when they were fed egg yolks from hens previously immunized with E. coli that produced heat-labile enterotoxin and colonization factor antigen-I (O’Farrelly et al., 1992). All information available until now shows convincingly that antibodies against E. coli fimbriae can passively protect animals from developing diarrhoea resulting from the colonization of the intestine by enterotoxigenic E. coli. This statement is further confirmed by many other investigations which cannot all be discussed here. However, there is still debate on the mechanism by which anti-fimbriae anti-bodies prevent colonization. Antibodies could block the fimbrial receptor binding site and thus directly prevent attachment to epithelial cells. Antibodies could also act in a less specific way such as by agglutination, by changing the surface charge properties, or by entrapping bacteria in the mucus so that they cannot reach the epithelial cells. Interestingly, Duchet-Suchaux et al. (1992) showed that suckling mice from dams intravenously injected with monoclonal antibodies raised against different epitope clusters of F41 fimbriae were protected against challenge with F41-positive E. coli. These results clearly show that protection of the suckling infants by the monoclonals that passed from the serum to the colostrum and to the milk is not necessarily attributed to antibodies raised against the receptor-binding site of the fimbriae. Investigations by Nagy et al. (1986) revealed that serial passage of K88-positive E. coli through medium containing immune colostrum resulted in the disappearance of K88 positive cells from the culture. Loss of the potency to express K88 fimbriae was not observed with colostrum from animals that had not been immunized with the K88 antigen. Although it had previously been suggested that immune serum contained a heat-stable factor responsible for curing plasmids encoding K88 fimbriae, the results of Nagy et al. (1986) show that immune colostrum favours the accumulation of viable cells that no longer produce K88 fimbriae. These cells are known to be present at low frequency in normal cultures of K88 producing cells. These authors further showed that loss of K88 plasmids was not due to non-specific plasmid curing effects, as other plasmids present in the bacteria were not lost upon passage through immune colostrum. Nagy et al. (1986) argued that spontaneous loss of K88 plasmids in combination with serological variation might be how E. coli had developed to cope with the immunological pressure in immunized animals. More recent studies of Casey and Moon (1990) revealed that loss of K99 and STaP genes during infection occurs both by plasmid curing and deletion of K99 genes. Prevention by receptor analogues Blocking the receptor-binding site of fimbrial lectins by receptor analogues such as glycoproteins constitutes an alternative and/or complementary way to prevent colonization. The potential of this strategy has already been indicated by the pioneering work of Aronson et al. (1979) who reported that methyl- , Dmannopyrano-side, a sugar previously found to inhibit in vitro the binding of two E. coli strains to human buccal epithelial cells, prevented colonization of the urinary tract of mice. The investigations of Neeser et al. (1986) showed that short oligomannoside-type glycoasparagine glycopeptides from ovalbumin, as well as oligomannoside-type glycopeptides obtained from legume storage proteins, are able to inhibit agglutination of erythrocytes by type 1-positive E. coli as well as binding of the bacteria to human buccal cells. Similarly, Mouricout and Julien (1986) reported that the agglutination of sheep erythrocytes by enterotoxigenic E. coli expressing K99 and F41 fimbriae can be inhibited by bovine plasma glycoprotein glycans. Further investigations of Mouricout and Julien (1987) revealed that receptors for K99, F41 and FY fimbriae of enterotoxigenic E. coli are present in intestinal mucus of calves. In in vitro studies, Mouricout et al. (1990) demonstrated that glycoprotein glycans of bovine plasma inhibit K99 mediated E. coli adhesion to erythrocytes and protect colostrum-deprived newborn calves against lethal doses of K99-positive E. coli. As

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mentioned earlier in this chapter, the attachment of E. coli F17 to mucus was confirmed by Sanchez et al. (1993a, b, c). These authors also showed that receptors for F17 fimbriae are present all along the small intestine of an 11-day-old calf. Although earlier investigations on the inhibition of cow erythrocyte agglutination revealed that N-acetylglucosamine is a strong haemagglutination inhibitor, this sugar hardly affected the binding of E. coli F17 strains to either mucus or brush border membranes. Except at very high concentrations (0.25 M), N-acetylglucosamine displayed no inhibitory activity. Similarly, other monosaccharides such as mannose and glucose were only inhibitory at very high concentrations and, consequently, the inhibiting effect observed was considered to be non-specific. These results clearly indicate that the receptors present at the surface of cow erythrocytes are different from the ones expressed in the intestinal brush border membranes or mucus. From these observations, it is also evident that inhibition studies using erythrocytes as a probe are of very limited value in screening potential attachment blockers and justify the use of brush border membranes and mucus components isolated from susceptible animals in in vitro systems such as ELISA. On the other hand, glycoproteins such as fetuin, ovomucoid, submaxillary gland mucin, hen egg white glycoproteins, as well as plasma glyco proteins from cows strongly inhibited the attachment of E. coli F17 cells to mucus and brush border membranes all along the small intestine. Plasma glycoproteins, especially from the cow, were very efficient inhibitors. In the ELISA test used, a 50 per cent reduction of binding had already been measured at a concentration of 0.1 mg ml−1. Similarly, hen egg white glycoproteins gave 50 per cent inhibition at 0.5 mg ml−1. In view of the sugar-binding heterogeneity displayed by F17 fimbriae, it is interesting to note that both plasma glycoproteins from cow as well as hen egg white glycoproteins inhibit the attachment of all F17-producing E. coli strains tested. To confirm that the attachment-inhibition observed in vitro is due to glycoproteins and not to specific anti-F17 fimbriae antibodies, different treatments of the preparations were performed. It was found that neither heat denaturation, nor complete proteolytic degradation of plasma proteins or hen egg white had any effect on the inhibitory properties of these preparations. Also, removal of the antibodies from the cow plasma preparations had no effect on the inhibition caused by the preparations. Moreover, the absence of specific anti-F17 fimbriae antibodies in the plasma preparations was demonstrated by ELISA, and plasma preparations prepared from cows never in contact with E. coli F17 cells and shown to be devoid of F17 antibodies also displayed strong inhibitory activity. The results described by Mouricout and co-workers (Mouricout and Julien, 1986; Mouricout et al., 1990) on E. coli K99 in combination with those of the authors on E. coli F17 demonstrate that cow plasma is an excellent source of adhesion blockers. These preparations also fulfil the requirements requested for their applicability in practice. First, they should bind with high affinity to the bacterial adhesins so that they can be used at reasonable concentrations. Second, when glycoproteins are to be used as inhibitors, their inhibitory power should not be lost upon partial or complete digestion of their polypeptide part in the stomach or intestine. Third, and most important, attachment inhibitors should be abundantly available at low cost. Fourth, the inhibitors should be easy to deliver to the animals, preferentially as a feed additive. The potential of cow plasma and hen egg white preparations to inhibit in vivo attachment of E. coli F17 has been investigated by Sanchez et al. (1993c). Previously it had been observed by Van Driessche et al. (unpublished) that the small intestine of young mice is readily colonized by E. coli F17 without provoking diarrhoea. Sanchez et al. in the authors’ laboratory showed that cow plasma glycoproteins and hen egg white glycoproteins strongly interfere with the colonization of the intestine of young mice by E. coli F17. When the animals received these inhibitors 24 h prior to infection, as well as during the whole period of the experiment, colonization was completely prevented. Although the results mentioned justify optimism on the further development of alternatives for antibiotics, it is possible that the successful application of receptor analogues might not be as

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straightforward as originally supposed. For example, although De Cupere et al. (1993a, b) in this laboratory were able to show lectin-mediated attachment of edema disease provoking E. coli to both mucus components and brush-border membranes of unweaned as well as of weaned piglets, all attempts to inhibit this attachment by glycoproteins expected to act as receptor analogues were unsuccessful (De Cupere et al., 1993a, b). After all, this is not so surprising as it is generally appreciated that high affinity binding of lectins to glycoconjugates does not only critically depend on the overall composition, but also depends on the finestructural features of the covalently linked oligosaccharide chains, and in some instances even on the nature and properties of the polypeptide backbone of glycoproteins. Thus, the potency of glycoproteins or their derived glycans to act as receptor analogues and compete with the native receptor for high affinity binding to fimbrial lectins, will depend on the structural properties of the competing glycans. There is no doubt that further research on the structure of intestinal receptors, present both in mucus and in the brush-border membranes of epithelial cells, will substantially contribute to the further development of receptor analogue therapy. As long as the sugar sequences of intestinal receptors are unknown, screening for receptor analogues remains a matter of trial and error, and it might be possible that glycans which are at present considered to be devoid of attachment-inhibiting activity can easily be changed, just by some simple enzymatic modification, into high-affinity competitive inhibitors. Also, receptor analog therapy may be complicated by the sugar-binding heterogeneity of E. coli fimbrial lectins. For example, Van Driessche et al. (1993a) reported that different E. coli solely expressing F17 fimbriae displayed different binding and binding-inhibition patterns when tested in vitro on Eupergit-C-glycoprotein beads. Obviously, only these receptor analogues that can adequately prevent all known strains from binding to intestinal mucosae are of any use in receptor analogue therapy. Moreover, in many instances E. coli express more than one type of fimbrial lectin, therefore successful receptor analogues will be those that block the receptor-binding sites of all fimbriae expressed. Recently, Schroten et al. (1992) reported on the importance of milk, more particularly the milk fat globule, as natural protective agent against enteric infections. These authors found that S-fimbriated E. coli could be prevented from binding to human buccal cells by human milk fat globule membranes. This protective effect was not found in adapted infant formula, or in artificial lipid emulsions. The protective effect of milk fat globules was shown to reside in the mucus glycoproteins of the membrane. As milk fat globule membranes, isolated from stools, are still agglutinated by S-fimbriated bacteria, the authors supposed that milk fat globules or their derived membranes might be protective all along the intestinal tract. The fact that human milk, but not formula milk fat globule membranes are able to bind enteric pathogens, clearly underlies the validity of the ‘old-fashioned’ principle of the beneficial effect of breast milk. Moreover, as well as fat globules, milk contains several other components such as lysozyme, the lactoperoxidase system, macrophages and lymphocytes as well as secretory IgA that display anti-bacterial activity. Wold et al. (1990) reported that the protective action of IgA is not solely due to its specific antigenbinding properties, but also to its covalently linked oligosaccharide chains that bind to the mannose-specific lectins from type 1 fimbriated E. coli. As such, s-IgA does not only display antibacterial activity by acting as a specific antibody, but also as a glycoprotein that can prevent mucosal colonization by saturating the carbohydrate binding sites of the surface lectins from E. coli and other pathogens. The possible protective role of sialic acid-specific lectin receptor analogues present in milk to intestinal infections has been discussed by Wadström (1993) and by Wadström et al. (1983). Obviously, there is an urgent need in the revaluation of milk as an excellent source of antibacterial compounds.

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Prevention by blocking the intestinal receptors Instead of blocking the receptor-binding site of bacterial fimbrial lectins, colonization of the intestine might be prevented by blocking the receptors with, for example, plant lectins or isolated bacterial lectins. Indeed, most plant lectins are known to be rather resistant to proteolytic breakdown in the gastrointestinal tract and are able to bind to mucus cells and epithelial cells lining the intestine (Pusztai et al., 1993a, b). Consequently, lectins may occupy the fimbrial intestinal receptor and as such prevent colonization. Although this approach is of high potential, much more research will be necessary in order to investigate the possible adverse effects lectins may have, once they interact with epithelial cell receptors. For a detailed overview of the application of plant lectins as attachment inhibitors, the interested reader is referred to Chapter 15 by Pusztai et al., and to Pusztai et al. (1993a, b) and references mentioned therein. Prevention by receptor modification Another approach to prevent intestinal colonization might be the temporal modification of intestinal receptors. For example, intestinal colonization in rabbits by a diarrhoeogenic E. coli strain expressing the human colonization factor CFAI could be prevented by the administration of an enteric-coated protease preparation, 18h prior to infection (Mynott et al., 1991). This new approach could prove to be extremely promising for the future, because its applicability does not depend on the availability of antibodies or attachment inhibitors. Especially when previously unknown new E. coli adhesins are involved, receptor modification therapy might be a ready to use approach for treating diarrhoea. However, this approach has only been tested on a limited number of animals, and the protease treatment was only given in one dose. The possibility that protease treatment during a longer period of time might seriously interfere with the normal physiology of the intestine cannot be excluded for, as well as receptors used by pathogens for colonization, other receptors or structural components of the mucosa may also become proteolyzed, or modified in such a way that their function is seriously impaired. Epilogue In this chapter we have attempted to summarize our current knowledge on the structure and function of fimbrial lectins that enable enterotoxigenic E. coli to colonize the small intestine of humans and animals. It is now generally appreciated that the specific recognition of oligosaccharides from the host by lectins expressed at the surface of E. coli cells is responsible for the species-, tissue- and genetic tropism displayed by E. coli and other micro-organisms. Protein-sugar interactions are now known to be of general importance in specific recognition phenomena between different organisms, as well as between cells within one organism. For example, lectin-carbohydrate interactions are known to be involved in as diverse phenomena as metastasis of tumour cells, cell-cell recognition and communication between cells of the immune system, differentiation, vector-parasite relations, symbiosis, etc. The recognition of lectin-carbohydrate interactions as the basic mechanisms governing the attachment of enterotoxigenic E. coli to the gut has opened new avenues to interfere with this process, which is now considered to be an initial but essential step in pathogenesis. The massive application of antibiotics over the last decade in treating microbial infections has resulted in the selection for highly resistant, often multiresistant E. coli strains that cannot be kept under control. This is why alternatives to antibiotics have to be developed to cope with microbial infections. As is summarized in this chapter, different approaches are now under intensive investigation to interfere probiotically with the attachment process (see Figure 13.8). It is evident that the success and application of these new strategies for the benefit of mankind critically depends

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on the basic knowledge of the structural properties of the two partners involved in the attachment, i.e. the E. coli lectins and their receptors expressed by the host. It is clear that our understanding of how E. coli lectins are synthesized and expressed at the bacterial surface, as well as on their diversity, has considerably increased during the last decade. At the same time, as more information becomes available on E. coli lectins, we realize that only a small fraction of these lectins has been identified and characterized to some extent. Consequently, it can reasonably be expected that in the years to come, many new, both fimbrial and nonfimbrial E. coli lectins will be described. Apart from extending our fundamental knowledge on these proteins, it is clear that the results of these investigations are of utmost importance in vaccine development and in modifying already available vaccines. When compared to our knowledge of E. coli lectins, our understanding of the mucosal receptors is negligible. Today, in most instances we can only talk about them as being glycoconjugates, either glycoproteins or glycolipids present in the mucus layer and/or the brush borders of the enterocytes. As described in this chapter, only in a few cases have receptors been isolated and purified to such an extent that the elucidation of their structural properties is feasible in the near future. The unravelling of the sugar sequence of the receptor’s oligosaccharides, and the determination of their spatial conformation is of paramount importance to develop and tailor probiotics that can be used to block either the intestinal receptors or the carbohydrate binding sites of the E. coli lectins. From these considerations it is evident that in the near future the joint effort of epidemiologists, microbiologists, protein and carbohydrate chemists, immunologists, geneticists, nutritionalists and many others will be necessary to further unravel the basic mechanisms that govern the intimate, sophisticated and complex relationships and interactions between pathogens and their hosts. This tremendous task and challenge sometimes seems to be a utopia. In this context it is stimulating and refreshing to remember the rhetoric questions formulated recently by Dr C. de Lannoye (1993): ‘Is there any important project in the history of human civilization that did not develop from utopic enthusiasm? Has pure pragmatism not always cumulated in mediocrity, massification, and even in the cultivation of triviality?’ Acknowledgements The Belgian Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw (IWONL), n.v. VEOS Belgium, and Innogenetics are kindly acknowledged for financially supporting the research on E. coli lectins performed in the laboratories of the authors. S.Beeckmans is Senior Research Associate of the Belgian National Fund for Scientific Research. R.Sanchez is post-doctoral fellow, and I.Dieussaert holds a fellowship of the IWONL. Our colleague G.Charlier is kindly acknowledged for providing the electron microscopy pictures. E.Van Driessche is deeply indebted to Mrs Josée Haenegreefs for her devoted and meticulous assistance in literature surveying, and to Mrs Greta Devuyst for her appreciated secretarial help.

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Figure 13.8. A–B Attachment of fimbriated E. coli to the intestinal mucosal surface. Fimbrial lectins recognize glycoconjugates, i.e. glycoproteins or glycolipids, expressed at the luminal site of enterocyte brush-borders (A), or glycoconjugates present in the mucus layer covering the epithelium (B). Receptors present in mucus and brush-border membranes can either be identical or different. Enterotoxigenic E. coli attached to the mucosa will rapidly multiply to colonize the small intestine. Enterotoxins are liberated from the cells in close vicinity to toxin receptors present at the surface of the enterocytes. C–D Saturation of the fimbriae receptors in mucus and brush-border membranes by fimbrial lectins (C) or plant lectins (D) may prevent fimbriated E. coli from binding to the mucosa. Consequently, the pathogens will rapidly be removed from the gut without causing damage to the host. E–F Saturation of the carbohydrate-binding sites of the fimbriae by receptor analogues that will compete with endogenous receptors prevents attachment (E). Antibodies directed against fimbriae, present in colostrum or sIgA secreted by the mucosa will prevent attachment by agglutinating the bacteria, or by blocking the carbohydrate-binding site of the fimbriae. The latter can be anti-(carbohydrate-binding site) antibodies, or glycosylated antibodies whose covalently attached oligosaccharides are recognized by the adhesin (F). G–H Sub-lethal concentrations of antibiotics may result in the expression of non-functional, non-adhesive fimbriae that fail to recognize mucosal receptors (G). Specific proteases or glycosidases may temporarily modify mucosal receptors that are not recognized by the fimbrial lectins (H).

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Sojka, W.J., Wray, C. and Morris, J.A., 1978, Passive protection of lambs against experimental enteric colibacillosis by colostral transfer of antibodies from K99 vaccinated ewes, Journal of Medical Microbiology, 11, 493–99. Sommerfelt, H., Grewal, H.M.S., Svennerholm, A.M., Gaastra, W., Flood, P.R., Viboud, G. and Bhan, M.K., 1992, Genetic relationship of putative colonization factor O166 to colonization factor antigen I and coli surface antigen 4 of enterotoxigenic Escherichia coli, Infection and Immunity, 60, 3799–806. Spangler, B.D., 1992, Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin, Microbiological Reviews, 56, 622–47. Strauch, K.L., Johnson, K. and Beckwith, J., 1989, Characterization of degP, a gene required for proteolysis in the cell envelope and essential for growth of Escherichia coli at high temperature, Journal of Bacteriology, 171, 2689–96. Svanborg-Edén, C., Andersson, B., Aniansson, G., Lindstedt, R., De Man, P., Nielsen, A., Leffler, H. and Wold, A., 1990, Inhibition of bacterial attachment: examples from the urinary and respiratory tracts, Current Topics in Microbiology and Immunology, 151, 167–84. Svennerholm, A.M., Wennerås, C, Holmgren, J., McConnell, M.M. and Rowe, B., 1990, Roles of different coli surface antigens of colonization factor antigen II in colonization by and protective immunogenicity of enterotoxigenic Escherichia coli in rabbits, Infection and Immunity, 58, 341–46. Svennerholm, A.M., McConnell, M.M. and Wiklund, G., 1992, Roles of different putative colonization factor antigens in colonization of human enterotoxigenic Escherichia coli in rabbits, Microbial Pathogenesis, 13, 381–89. Tacket, C.O., Maneval, D.R. and Levine, M.M., 1987, Purification, morphology, and genetics of a new fimbrial putative colonization factor of enterotoxigenic Escherichia coli O159:H4, Infection and Immunity, 55, 1063–69. Teneberg, S., Willemsen, P.T.J., De Graaf, F.K. and Karlsson, K.A., 1990, Receptor-active glycolipids of epithelial cells of the small intestine of young and adult pigs in relation to susceptibility to infection with Escherichia coli K99, FEBS Letters, 263, 10–14. Teneberg, S., Willemsen, P.T.J., De Graaf, F.K. and Karlsson, K.A., 1993, Calf small intestine receptors for K99 fimbriated enterotoxigenic Escherichia coli, FEMS Microbiology Letters, 109, 107–12. Tewari, R., MacGregor, J.I., Ikeda, T., Little, J.R., Hultgren, S.J. and Abraham, S.N., 1993, Neutrophil activation by nascent FimH subunits of type 1 fimbriae purified from the periplasm of Escherichia coli, Journal of Biological Chemistry, 268, 3009–15. Van der Woude, M.W., De Graaf, F.K. and Van Verseveld, H.W., 1989, Production of the fimbrial adhesin 987P by enterotoxigenic Escherichia coli during growth under controlled conditions in a chemostat, Journal of General Microbiology, 135, 3421–29. Van Driessche, E., 1988, Structure and function of leguminosae lectins, in Franz, H. (Ed.) Advances in Lectin Research, vol. I, pp. 73–134, Berlin: Springer-Verlag, Heidelberg. Van Driessche, E. and Beeckmans, S., 1993, Fimbrial lectins from enterotoxigenic E. coli: a mini-overview, in Van Driessche, E., Franz, H., Beeckmans, S., Pfüller, U., Kallikorm, A. and Bøg-Hansen, T.C. (Eds) Lectins: Biology, Biochemistry, Clinical Biochemistry, vol. 8, pp. 207–16, Helletup, DK: Textop. Van Driessche, E., Schoup, J., Charlier, G., Lintermans, P., Beeckmans, S., Zeeuws, R., Pohl, P. and Kanarek, L., 1988, The attachment of E. coli to intestinal calf villi and Eupergit-C-glycoprotein beads, in Bøg-Hansen, T.C. and Freed, D.L.J. (Eds) Lectins: Biology, Biochemistry, Clinical Biochemistry, vol. 6, pp. 55–62, St. Louis, MO: Sigma Chemical Company. Van Driessche, E., Beeckmans, S., Furrazola, G., Charlier, G., Pohl, P., Sanchez, R., Lintermans, P. and Kanarek, L., 1993a, Studies on the carbohydrate-binding properties of E. coli F17 strains, in Van Driessche, E., Franz, H., Beeckmans, S., Pfüller, U., Kallikorm, A. and Bøg-Hansen, T.C. (Eds) Lectins: Biology, Biochemistry, Clinical Biochemistry, vol. 8, pp. 217–22, Hellerup, DK: Textop. Van Driessche, E., Sanchez, R., Beeckmans, S., De Cupere, F., Charlier, G., Pohl, P., Lintermans, P. and Kanarek, L., 1993b, A general procedure for the purification of fimbrial lectins from Escherichia coli, in Gabius, H.J. and Gabius S. (Eds) Lectins and Glycobiology, pp. 47–54, Springer-Verlag: Heidelberg. Van Verseveld, H.W., Bakker, P., Van der Woude, T., Terleth, C. and De Graaf, F.K., 1985, Production of fimbrial adhesins K99 and F41 by enterotoxigenic Escherichia coli as a function of growth-rate domain, Infection and Immunity, 49, 159–63.

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Wadström, T., 1993, Sialic-acid specific bacterial lectins of enterotoxigenic Escherichia coli (ETEC) and Campilobacter pylori, in Van Driessche, E., Franz, H., Beeckmans, S., Pfüller, U., Kallikorm, A. and Bøg-Hansen, T.C. (Eds) Lectins: Biology, Biochemistry, Clinical Biochemistry, vol. 8, pp. 229–34, Hellerup, DK: Textop. Wadström, T., Faris, A., Lindahl, M., Lönnerdahl, B. and Ersson, B. 1983, Nonmannosespecific lectins on enterotoxigenic E. coli recognizing different glycoconjugates, in Bøg-Hansen, T.C. and Spengler, G.A. (Eds), Lectins: Biology, Biochemistry, Clinical Biochemistry, vol. 3, pp. 503–10, New York: W.deGruyter. Wennerås, C., Holmgren, J. and Svennerholm, A.M., 1990, The binding of colonization factor antigens of enterotoxigenic Escherichia coli to intestinal cell membrane proteins, FEMS Microbiology Letters, 66, 107–12. Willemsen, P.T.J. and De Graaf, F.K., 1992, Age and serotype dependent binding of K88 fimbriae to porcine intestinal receptors, Microbial Pathogenesis, 12, 367–75. Wold, A.E., Mestecky, J., Tomana, M., Kobata, A., Ohbayashi, H., Endo, T. and Svanborg Eden, C., 1990, Secretory immunoglobulin A carries oligosaccharide receptors for Escherichia coli type 1 fimbrial lectin, Infection and Immunity, 58, 3073–77. Wolf, M.A., Andrews, G.P., Tall, B.D., McConnell, M.M., Levine, M.M. and Boedeker, E.C., 1989, Characterization of CS4 and CS6 antigenic components of PCF8775, a putative colonization factor complex from enterotoxigenic Escherichia coli E8775, Infection and Immunity, 57, 164–73. Woodward, J.M., Connaughton, I.D., Fahy, V.A., Lymbery, A.J. and Hampson, D.J., 1993, Clonal analysis of Escherichia coli of serogroups O9, O20, and O101 isolated from Australian pigs with neonatal diarrhea, Journal of Clinical Microbiology, 31, 1185 88. Yokoyama, H., Peralta, R.C., Diaz, R., Sendo, S., Ikemori, Y. and Kodama, Y., 1992, Passive protective effect of chicken egg yolk immunoglobulins against experimental enterotoxigenic Escherichia coli infection in neonatal piglets, Infection and Immunity, 60, 998–1007. Yuyama, Y., Yoshimatsu, K., Ono, E., Saito, M. and Naiki, M., 1993, Postnatal change of pig intestinal ganglioside bound by Escherichia coli with K99 fimbriae, Journal of Biochemistry, 113, 488–92. Zhanel, G.G. and Nicolle, L.E., 1992, Effect of subinhibitory antimicrobial concentrations (sub-MICs) on in-vitro bacterial adherence to uroepithelial cells, Journal of Antimicrobial Chemotherapy, 29, 617–27. Note 13.1

FimX, FanX, FaeX, etc., are used to define the protein, while fimX, fanX, faeX refer to the gene.

Chapter 14 Identification of the F17 Gene Cluster and Development of Adhesion Blockers and Vaccine Components P.Lintermans, A.Bertels, E.Van Driessche and H.De Greve

Introduction This work describes the characterization of the F17 fimbriae, the analysis of the F17 fimbrial gene cluster and the identification of putative F17-specific adhesion blockers and vaccine components. In 1979, the Escherichia coli strain 25KH9 was isolated from faeces of a diarrhoeal calf at the National Institute for Veterinary Research, Brussels (Pohl P. et al., 1982). This strain adhered in vitro to isolated intestinal calf villi. Electron microscopy revealed the presence of wiry flexible appendages or fimbriae on the surface of the bacteria. Serological analysis indicated that the strain 25KH9 produced no K99 (F5), K88 (F4), 987P (F6), nor F41 fimbrial antigens. The fimbriae were provisionally designated as ‘Att25’ (i.e. attachment of strain KH9). Fimbriae homologous to the Att25 fimbriae were also isolated in France at the same time; they were designated F(Y) (Girardeau et al., 1979). When the authors demonstrated that the Att25 and F(Y) fimbriae were identical, both antigens were designated ‘F17’ (see below). Epidemiological studies indicated that the F17 fimbriae were found on the surface of bovine enterotoxigenic E. coli (ETEC) and that F17 piliation was correlated with the production of CNF2 (Cytotoxic Necrotizing Factor 2) in E. coli strains (El Mazouari K., manuscript in preparation). F17 fimbriae could also be isolated from non-enterotoxigenic E. coli strains; F17 piliation and production of aerobactin in septicaemic E. coli strains was positively correlated (Pohl et al., 1984, 1986). The frequency of isolation of the F17 antigen was significantly higher in diseased calves compared with healthy animals. A recent survey carried out on strains isolated from diarrhoeal bovines in Canada, the USA and Europe indicated an overall presence of F17 fimbriae in 21.6 per cent of the strains against 8.8 per cent for the K99 fimbriae. That F17 fimbriae are produced in vivo was shown when fresh faeces samples were analysed by immunofluorescence. Calves infected with an F17-positive strain excreted many fluorescent organisms which could be identified as F17-positive bacteria in bacterial cultures. These data indicated the importance of the F17 antigen in veterinary medicine and encouraged the authors to analyse in some detail the F17 fimbriae. This research consisted of the characterization of the F17 fimbriae and the cloning of the F17 gene cluster. The main purpose of this approach was the identification of the F17 fimbrial adhesin. The F17 adhesin has three potential applications which can be used in the prevention or therapy of neonatal diarrhoea. The F17 adhesin can be purified and used to isolate the intestinal F17 receptor for studies of the interaction between the F17 fimbriae and the receptor. It is expected that these may lead to the development of adhesion-blockers. A second application of the F17 fimbrial adhesin is to use the adhesin protein as an oral adhesion blocker. The efficacy of this oral treatment to protect new-born calves from non-vaccinated

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cows can then be tested. Finally, the adhesin protein can be incorporated into a new generation of vaccines which should provide a more efficient and specific protection for the new-born calves in comparison with existing generation of diarrhoeal vaccines. Many bacterial vaccines against neonatal calf diarrhoea that are currently available contain killed bacteria. Adhesins: a review Fimbrial Adhesins In order to understand the interactions between bacteria and eukaryotic cells, a short description of the gramnegative bacterial surface structures is given. E. coli possess the following main surface structures: the Oantigen or somatic antigen, the K-antigen or capsular antigen, and the H- or flagellar antigen (Stanier et al., 1979). Determination of these antigens results in the O-, K-, H-types of a given E. coli strain (Ørskov and Ørskov, 1984). Later, a fourth surface antigen known as pili or fimbriae was also detected. Fimbriae and pili are morphologic terms used to denote these nonflagellar and filamentous appendages radiating from the bacterial cell surface (Brinton, 1959; Duguid et al., 1955). The term pili is often used when one describes filamentous structures involved directly in bacterial conjugation while the term fimbriae is reserved for filamentous appendages involved in bacterial adhesion and colonization. The fimbrial antigens of E. coli are designated as F-antigens even though other classification schemes are also used (Ørskov and Ørskov, 1983). Fimbriae may vary in number from a few to several hundred, they are 2–7 nm in diameter and can extend to 4 mm from the bacterial surface. They are polymers consisting of subunits, called pilins (Clegg and Gerlach, 1987; Gaastra and de Graaf, 1982; Klemm, 1985) which have a molecular mass of about 20 kD. Morphologically, fimbriae can be subdivided into two groups; type 1 (F1) and 987P (F6) fimbriae are rigid and have a diameter of around 7 nm while other fimbriae are characterized by a diameter of 2–4 nm and are more flexible. Phosphate, carbohydrate and phospholipids can be associated with fimbriae; these can be important in receptor recognition (for review, see also: Arp, 1988; Mooi and de Graaf, 1985). The ETEC strains produce at least two virulence factors: fimbriae and enterotoxins (Dean et al., 1972; Gyles et al., 1974; Smith and Gyles, 1970; Smith and Linggood, 1971; So et al., 1976). In one case (K99 or F5) the linkage of fimbrial and toxin genes on one plasmid could explain the close linkage between K99 expression and ST production in bovine ETEC strains (Mainil, 1988). The mechanisms by which bacteria interact with the mucosal surface can be categorized as association, adhesion or invasion (Arp, 1988; Lewin, 1984; Reed and Williams, 1978; Takeuchi, 1967). Association describes a loose contact between bacteria and mucosal surface (Marshall, 1984) with enhancement by chemotaxis and flagellar motility (Allweiss et al., 1977). Adhesion or adherence describes an interaction based on a ligand-receptor model (Jones and Rutter, 1974). Bacterial surface structures with small radii such as fimbriae decrease the repulsive forces between bacteria and mucosal cells. These organelles are able to extend through repulsive regions and establish stable ligand-receptor interactions (Gubish et al., 1982; Rutter and Vincent, 1980). Detection of fimbrial antigens is carried out by slideagglutination, immunofluorescence and ELISA. The classical agglutination assay is limited due to the suppression of in vitro fimbrial production. This is welldocumented for the F5 (K99) fimbriae which are grown on semi-synthetic Minca medium (de Graaf et al., 1980b). The use of ELISA in epidemiological studies is hampered by the presence of copro-antibodies directed against fimbriae (Van Zijderveld and Overdijk, 1983). In contrast, the immunofluorescence technique developed by the authors has many advantages such as its insensitivity against copro-antibodies and antibiotics, it is well-adapted to the detection of fimbrial expression in vivo, and it has high sensitivity

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(Lintermans and Pohl, 1983a, 1983b, 1984). The most recent development in fimbrial detection is based on DNA probes which contain only the pilin gene sequence or the complete fimbrial gene cluster. Fimbriae are prepared by forceful mixing of the bacterial culture or by incubation at 60°C. The fimbriae are then separated from bacterial debris by differential centrifugation and further purified by ammonium sulphate precipitation or by sucrose gradient centrifugation (Korhonen et al., 1980; Lintermans, 1986). Attachment of the bacteria to erythrocytes leads to haemagglutination which facilitated the identification and characterization of many fimbrial antigens. However, analysis of haemagglutination data is difficult if one species expresses two or more different fimbrial antigens (Burrows et al., 1976; Old, 1972). The expression of fimbriae is affected by phase variation (F1, F6) and by quantitative fimbrial variation (F5) (Abraham et al., 1985; Contrepois et al., 1983; Eisenstein, 1981; Orndorff et al., 1985; Pallesen et al., 1989). Phase variation is the result of DNA sequence inversion. The DNA rearrangements consist of the inversion of a 314 bp region, capable of inverting upstream of the F1 pilin gene, pilA. The quantitative variation was best studied for the F5 (K99) fimbriae as, apparently, the F5 genes are subject to glucosemediated catabolite repression (Contrepois et al., 1981, 1983; Isaacson, 1980). Type 1 (F1) fimbriae can be isolated from about 80 per cent of E. coli strains (Gaastra and de Graaf, 1982). The role of F1 fimbriae in the pathogenicity of F1-positive strains is not clear (Blumenstock and Jann, 1982; Jayappa et al., 1985; Silverblatt et al., 1979). The genetic analysis of the F1 fimbrial gene cluster revealed that the pilE gene was responsible for the attachment of F1 fimbriae to guinea pig erythrocytes. PilE mutants expressed normal F1 fimbriae but they did not adhere to these erythrocytes. Thus, the pilE gene product appeared to play some role in the adhesion of F1 fimbriae. As a general conclusion, strains carrying the F1 gene cluster adhere to the F1 receptor if there is normal piliation (i.e. pilA) and if the pilA, B, C, and E genes are intact (Hanson and Brinton, 1988; Maurer and Orndorff, 1985, 1987; Minion et al., 1986). F4 (K88) fimbriae are present on the surface of porcine enterotoxigenic E. coli strains (Jones and Rutter, 1972; Ørskov et al., 1961). Three serological variants of the F4 antigen could be identified: K88ab, K88ac and K88ad, the a epitope being a common antigen (Ørskov et al., 1964, 1969). This antigenic variability can be the result of the vaccination strategy but could also be the bacterial response to changes in the intestinal receptors (Bijlsma et al., 1982). The fact that the K88ab antigens are becoming less important and are replaced by the recent K88ac and K88ad variants supports this hypothesis. The genes encoding the subunits of the different F4 variants were cloned and sequenced (Josephsen et al., 1984; Klemm, 1981; Shipley et al., 1981), revealing that amino acid changes occurred only in the protein domains oriented to the outside of the fimbriae. The K88ac gene cluster was characterized and found to encode for six polypeptides. This gene cluster consists of two operons; one operon encodes the subunit protein of 23.5 kD, the second operon encodes the 17-, 29-, and 70 kD proteins. As mutants in the 70 kD protein secrete K88 fimbriae into the medium, it is possible that the 70 kD protein functions as an anchoring protein. F5 (K99) fimbriae and F41 fimbriae are located on the surface of bovine ETEC strains (Ørskov et al., 1975) but are rarely found on porcine ETEC strains. Biosynthesis of F5 fimbriae is repressed by alanine (de Graaf et al., 1980a). Pap fimbriae (pili associated with pyelonephritis) are produced on the surface of E. coli isolates associated with human pyelonephritis (Normark et al., 1986). Several serotypes have been described and designated F7 to F13. Genetic analyses of Pap fimbriae were carried out in detail. The major conclusion from these analyses were: 1. The papA subunit gene is dispensable for Pap-specific adhesion; when a synthetic DNA linker was introduced in papA in such a way that translation after the insertion will go out of frame, the obtained

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mutants produced no detectable fimbriae but mediated digalactoside-specific haemagglutination (Bäga et al., 1984; Uhlin et al., 1985). 2. Some mutations in the Pap gene cluster result in piliated but non-adhesive bacteria; the papF and papG gene products are essential for expression of digalactoside-binding adhesin but not for the biogenesis of Pap fimbriae (Lindberg et al., 1987; Lund et al., 1985, 1988; Norgren et al., 1984). 3. Other genes of the pap gene cluster are necessary to obtain a functional adhesin; the papC and papD genes are required for the export and assembly of the Pap fimbrial adhesin (Lindberg et al., 1984). 4. The pap gene cluster also encodes the so-called minor pilins; the papH, papE and papF genes encode proteins having the characteristics of pilins. These proteins have two cysteines and a penultimate tyrosine residue. The hydrophilicity profile is similar to other pilins. As the papE, papH and papF are not normally seen on SDS-PAGE gels, they are denoted ‘minor’ pilins (Normark et al., 1986). Pili also occur on other bacterial species (see Arp, 1988 for review); fimbrial antigens are described on Bordetella, Moraxella, Bacteroides and Neisseria. The adhesion of Bordetella pertussis to respiratory cilia is mediated by both a filamentous haemagglutinin and the pertussis toxin (An der Lan et al., 1986; Cowell et al., 1986). At least two virulence factors are required for the pathogenicity of Moraxella bovis: fimbriae for adhesion and a pitting factor for penetration of the cornea (Chandler et al., 1985; Frank and Gerber, 1981; Pedersen et al., 1972). The virulence of Bacteroides nodosus requires the production of fimbriae and extracellular proteases (Anderson et al., 1984; Lee et al., 1983). The antigenic variation in Neisseria pilins and opacity-associated proteins (OPAs) can possibly be another strategy of the bacteria to escape host defence and enhance virulence (Hermodson et al., 1978; King and Swanson, 1978; Swanson et al., 1971). Non-fimbrial adhesins In the first part of this review, the focus was on the correlation between fimbrial expression and the adhesive properties of E. coli. However, some E. coli may exhibit mannose-resistant adhesion without expressing fimbriae and this is ascribed to the presence of non-fimbrial adhesins (NFA or AFA; see Bertels et al., 1991 for review). The non-fimbrial adhesive structures are subject to temperature regulation and phase variation as has been described for fimbrial adhesins. The major difference between fimbriated and non-fimbriated strains is the lack of a transcribed gene encoding for the major fimbrial subunit. Two new haemagglutinins of E. coli have been described, an N-acetyl-D-glucosamine specific G fimbriae and a nonfimbrial blood group M specific agglutinin (21000 d). NFA-1 and NFA-2 non-fimbrial haemagglutinins from E. coli have been purified; both proteins tend to form aggregates and have a subunit molecular weight of 21000 (NFA1) and 19000 (NFA2). Electron microscopic examinations showed the presence of an extracellular capsule-like layer in adhering E. coli but not in non-adhering bacteria. Both proteins are antigenically distinct but recognize a common receptor. NFA-1 adhesin from the uropathogenic E. coli strain 827 was cloned using the cos 4 cosmid vector. A clone was isolated which promoted haemagglutination and showed the same biological properties as the adhesin produced by the wild type strain. Both expressed the adhesin at 37°C, but not at 18°C. Similarly, the presence of 1 per cent glucose in the culture medium inhibited adhesin production. The adhesin purified from the clone formed high molecular weight aggregates which, upon denaturation, dissociated into 21 kDa subunits identical to those produced by the wild type strain. The genes for the adhesin were isolated and the NFA-1 operon was localized to a 6.5kb region (Hales et al., 1988). A non-fimbrial bacterial surface protein of 16000 (AFA-I) was identified on the surface of an ETEC strain isolated from a patient with acute infantile diarrhoea. A detailed analysis of the AFA-I a-fimbrial adhesin from human pyelonephritic E. coli

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strains was carried out. The AFA-I adhesin on the bacterial surface is composed of a single repeating 16000 polypeptide subunit. Its amino acid composition is remarkable as there are 22 per cent non-polar residues and 2–3 cysteines per subunit. The N-terminal sequence of AFA-I is unrelated to other known E. coli fimbrial sequences. The AFA-I gene was cloned and the recombinant strain agglutinated only human and gorilla erythrocytes indicating a preference for receptor molecules which are present only on the red cells of human or anthropoid apes. The recombinant clone expressing AFA-I carries five genes belonging to the same transcriptional unit; the afaE gene was identified as the structural gene encoding AFA-I. This protein has a typical 21-residue prokaryotic signal peptide and a 131-residue-long mature polypeptide. The gene organization of fimbriae consisting of adhesive subunits is analogous to that of NFA. Hoschutzky et al. (1989) characterized the non-fimbrial adhesin NFA-4 from uropathogenic E. coli O7:K98. The adhesin consists of non-covalently linked 28 kDa subunits which form aggregates of a molecular weight of more than 106 Da. The N-terminal sequence showed 70% homology to the corresponding region of the F4 subunit. A monoclonal antibody against NFA-4 inhibited the haemagglutinating activity of both bacteria and purified NFA-4. Some of the isolation procedures were similar to those described for fimbriaassociated adhesins. Essentially, this technique is based on the release of non-fimbrial adhesins by heating the bacteria at 50–70° C, removal of the bacteria by centrifugation, differential ammonium sulphate precipitation in the presence of EDTA and purification by high resolution ion exchange chromatography. With this method, NFAs were isolated from six different E. coli strains, with yields ranging from 1–10 mg NFA protein per 10 g wet weight of bacteria. In conclusion, from results of SDS-PAGE it is clear that NFA adhesins consist of noncovalently linked subunits with molecular weight of 15–30000, which are comparable to those of fimbrial adhesins. The NFAs are large polymers consisting of more than 20 subunits. Electron microscopic analysis of thin sections of E. coli expressing NFA-1, NFA-2 and NFA-4 revealed that these adhesins surround the bacterial cell like a capsule. Kroncke et al. (1990) performed electron microscopic studies of co-expression of adhesive protein capsules and polysaccharides and found that the bacteria expressed composite capsules with the adhesin as a recognition peptide at the cell-distal outer region and the K antigen at the cell-proximal inner region indicating that the adhesin part of the capsule was necessary for interaction with the eukaryotic receptor. F17 Fimbrae Characterization (Lintermans et al., 1988a) The strain 25KH9 was serotyped as O101: K+: H−. This strain was isolated from a faeces sample of a twoday-old calf suffering from diarrhoea. A derivative strain lacking toxin production was designated 25KH9st and used in further studies. The strain 25KH9st did not haemagglutinate unmodified human, bovine, chicken or guinea pig erythrocytes. The fimbriae produced by E. coli 25KH9st were isolated and purified. These fimbriae were composed of subunit proteins of 20 kD. Immuno-electron microscopy identified the Att25 antigen as fimbriae with a diameter of 3–4 nm. Fingerprints of the tryptic peptides from Att25 (Belgian isolates) and F(Y) (French isolates) were almost identical (Hewick et al., 1981; Hirs, 1956, 1967). Therefore, F(Y) and the Att25 fimbriae were designated as ‘F17’. Both Fab fragments of F17 antibodies and purified F17 protein blocked the binding of the strain 25KH9st to calf villi. Immunogold-labelling of calf villi coated with F17 fimbriae revealed the accumulation of electron-dense material near the border of the villi. Binding of F17 fimbriae could be inhibited with 50 mM N-acetyl-glucosamine (NAG) (Girardeau, 1980).

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Strain 25KH9st produced fimbriae which were almost identical to the F(Y) fimbriae (see also Morris et al., 1985); and therefore also designated as ‘F17’. These fimbriae were shown to be the adhesive factor on the surface of the strain 25KH9st. This was proved by using Fab fragments of F17 and the F17 protein in adhesion blocking studies. Furthermore, the binding of F17 fimbriae to the calf microvilli and mucus could be visualized with immunogold electron microscopy. Cloning of the F17 gene cluster and identification of the F17-A subunit gene (Lintermans et al., 1988b) Total bacterial DNA prepared from the strain 25KH9st was partially digested with Sau3A and fractionated. DNA fragments of 15–20 kb were cloned in the cloning vector pUC8. The obtained transformants were blotted onto nitro-cellulose membranes and incubated with F17 antibodies. One clone out of the 2000 tested, reacted with the F17 antiserum. This clone contained a 20-kilobase plasmid which was designated pPLHD1. E. coli (pPLHD1) produced fimbriae on the surface of the bacteria and these were identified as F17 fimbriae by immuno-electron microscopy. The fimbriae encoded by pPLHD1 were functional as the strain HB101(pPLHD1), could adhere to intestinal calf villi, and could also be inhibited with NAG. Internal deletions were made in pPLHD1 with several restriction enzymes. The EcoRI deletion resulted in an 8.7kilobase DNA fragment. This construction was designated pPLHD2. E. coli (pPLHD2) adhered to calf villi, and its binding was blocked with NAG. A fimbrial preparation of E. coli K514(pPLHD2) migrated in SDSPAGE in the same position as F17 fimbriae isolated from wild type strain 25KH9st. In Western blots, both bands reacted with F17 antibodies. Two selected tryptic peptides of the F17-A protein served as templates for the synthesis of appropriate DNA probes (Southern, 1975). The Asp-Gln-Thr-Cys tetrapeptide was the base for the synthesis of the GAT/C CAA/G ACA/G TGT/C oligonucleotide mixture (Holm, 1986; Mahoney and Hermodson, 1980; Suggs et al., 1981). The mixture of these 16 DNA oligonucleotides was used to localize the F17-A gene (subunit gene) in plasmid pPLHD2. The sequence analysis (Maxam and Gilbert, 1977; Sanger et al., 1977) of the region that hybridized with the labelled oligonucleotide revealed the presence of an open reading frame (ORF) encoding 180 amino acids. The F17-A pilin is transcribed from its natural promoter in the plasmid pPLHD2 because restriction analysis and sequencing of the vector borders indicated that the F17-A pilin gene is not under control of the lacZ promoter. Plasmid pPLHD50 is a pPLHD2 derivative deleted in the pilin gene (F17-A−). E. coli (pPLHD50) became negative for fimbrial production and adhered no longer to calf villi. F17-A pilin shares the common characteristics of most E. coli pilins: a hydrophobic N-terminus (signal sequence), a cysteine loop in the Nterminal half of the protein and a penultimate tyrosine at the C-terminus. The two cysteine residues of the mature peptide may be involved in the formation of an intra-chain disulphide linkage similar to that inferred for Pap fimbriae, K99 fimbriae, Type 1 fimbriae and gonococcal fimbriae. The F17-A pilin is characterized by a hydrophobic COOH-terminal part which has been proposed to be involved in subunit-subunit interaction and/or membrane embedding (Hopp and Woods, 1981). Amino acid sequence comparison of F17-A with F5, F4 (small subunit), PapA and F1 indicated a closer relationship of F17-A with PapA and F1 than with F5 and F4. The amino acid residues conserved seemed to be located at both the carboxy- and amino-termini. These regions are probably involved in functions common to this group of proteins such as anchorage to the outer membrane, transport and fimbriae subunit interaction (Inouye and Halegoua, 1980). A new blood-group-specific agglutinin was recently identified on human pyelonephritogenic E. coli strains. This haemagglutinin recognized terminal N-acetyl-D-glucosamine and was associated with a new

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type of fimbriae: G fimbriae. A high homology was found between the N-terminal regions of the F17-A and G pilins; furthermore the findings that the molecular weight of both fimbriae was 19.5 kD on SDS-PAGE and that they both recognized N-acetyl-D-glucosamine indicated that these fimbriae are closely related. They are however not identical as amino acid analyses of the purified G and F17-A fimbriae indicated differences in the methionine (none for F17, two for G) and arginine content (one for F17, five for G; Rhen et al., 1986). Analysis of the organization of the F17 gene cluster (Lintermans, 1990) The experiments described in this chapter were aimed at the identification of other genes involved in the F17 piliation. In a first approach, deletions and subclones of the pPLHD2 plasmid were made. These experiments indicated that large fractions of the pPLHD2 plasmid were necessary to obtain normal F17 piliation. The pPLHD62 plasmid was the smallest construction that could encode for functional F17 fimbriae. This plasmid contained a 6 kb DNA insert. Minicell analysis of the derivatives of pPLHD2 allowed the partial mapping of the F17 gene cluster. From this it was concluded that at least four genes are necessary to encode functional F17 fimbriae: F17-A, F17-C, F17-D and F17-G. The F17-A gene encodes the F17 subunits. Tn5 transposon mutagenesis was carried out to obtain F17-C, F17-D and F17-G mutants. However, it was observed that the Tn5 transposon could not be inserted at random, Therefore site-specific linker mutagenesis experiments were initiated. A nonsense-linker containing a XbaI site was chosen for this purpose. The introduction of the XbaI linker in the pPLHD2 and pPLHD52 plasmids enabled the authors to mutate separately the F17-A, F17-D, F17-C and F17-G genes. These mutants were analysed with electron microscopy, ELISA and in vitro adhesion assays using calf villi or Eupergit-C particles coated with bovine mucin. The phenotypic analysis indicated that the F17-A, F17-C, F17-D and F17-G genes are indispensible in order to obtain adhesive F17 fimbriae. The F17-A, F17-C and F17-D mutants were not piliated and lost their adhesive properties to calf villi. The F17-G mutant produced fimbriae which were morphologically identical to the F17 fimbriae purified from strains containing the intact F17 gene cluster but this mutant could no longer adhere to the Eupergit-C spheres. Furthermore, the adhesion of this F17-G mutant to calf villi was weak in comparison with strains containing the intact F17-G gene. Function and nucleotide sequence of the F17-G gene (Lintermans et al., 1990) Transformants containing the pPLHD53 (F17-G−) construction produced F17 fimbriae that could not adhere to Eupergit-C spheres. The K514(pPLHD52) and K514(pPLHD53) strains, which were respectively F17-G+ and F17G− , produced identical fimbriae when they were analysed with immunogold electron microscopy. Furthermore, ELISA and SDS-PAGE could not discriminate between fimbriae produced by the two strains, indicating that the fimbriae produced by adhesive and non-adhesive clones were similar. Complementation studies revealed that the F17-G protein acted in trans. As the purified fimbriae obtained from E. coli (pPLHD2) blocked the adhesion of F17-positive strains, the authors concluded that the adhesin component must have been present in this fimbrial fraction and that the F17-G protein was probably the adhesin responsible for the direct interaction with the intestinal receptor or for a non-defined modification of the F17-A subunits leading to the same effect. Immunogold studies with F17-G antibodies indicated the presence of the F17-G protein on fimbriae of the strain K514 (pPLHD52) while no significant labelling was seen on fimbriae of the strain K514(pPLHD53), therefore the F17-G adhesin is a minor component of the F17 fimbriae. Lindberg et al. (1984) also located the Pap-

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adhesin protein at the tip of the fimbriae by immunogold techniques. All these results do not exclude the possibility that the Pap or F17 adhesin proteins act by pilin modification as it has not yet been shown if one could block adhesion of these fimbriae to receptor cells by antibodies directed against the Pap or F17 adhesin proteins. The following hypothesis is proposed for F17-mediated binding. Because the F17-A subunit proteins are characterized by a high degree of hydrophobicity and bind to calf mucus, F17 fimbriae could first bind to calf mucus. This binding is based on hydrophobic interactions and followed by a more specific NAG-dependent binding to the receptors which are located on the epithelial surface of the intestine. Function and nucleotide sequence of the F17-C gene (Lintermans, 1990) The F17-C gene encodes a protein of 90 kD and the nucleotide sequence of the F17-C gene and minicell studies indicated the presence of a signal sequence. All fimbrial gene clusters of E. coli contained a gene encoding a 70–90 kD protein. Studies of the 80 kD protein of the F4 fimbriae showed that it probably functions as a base protein on which the fimbrial subunits are polymerized. Function and nucleotide sequence of the F17-D gene (Lintermans, 1990) The nucleotide sequence of the F17-D gene indicated a close homology with Pap-D and the F17-D protein had a molecular weight of about 28 kD. Mutation of F17-D resulted in nude non-adhesive strains (as is the case with Pap-D mutations). The function of F17-D is unknown but its homology with Pap-D indicated a possible role as a subunit stabilization protein. Effect of incubation temperature on the expression of F17 fimbriae (Lintermans, 1990) The expression of F17 fimbriae is temperature-dependent and there have been attempts to identify regulatory sequences in the F17 gene cluster responsible for this effect mainly because these regulatory sequences could also eventually regulate the expression of other (non-fimbrial) virulence factors. To study the effect of temperature on F17 expression, bacteria were first incubated at 18°C; the incubation temperature was then increased to 37°C for 30, 60, 90 or 120 min. Bacterial surface structures of these cultures were analysed with immunogold electron microscopy. Protein expression of the cultures grown at 18°C and 37°C were compared with the in vitro transcription-translation system. The results showed that the influence of temperature on F17 fimbrial expression was post-translational; F17-positive bacteria grown at 18°C produced a low level of F17 subunits which were rarely polymerized into fimbriae. The fimbrial subunits of cultures incubated at 18°C were localized on the cellular membrane of the bacteria; after incubation of these cultures at 37°C for 60 min fimbrial subunits were polymerized into fimbrial structures and the bacteria became positive in adhesion assays. In vitro transcription-translation experiments carried out at 18°C could not demonstrate the presence of F17-A protein. Moreover, no proteins could be visualized at this temperature by using pUC8 plasmid DNA, indicating a low background activity which was not detected with autoradiography. Reduced membrane fluidity or an insufficient concentration of F17 pilin on the bacterial membrane can explain the reduced piliation at low incubation temperature. Furthermore, the expression of F17 fimbriae was not affected by the addition of various sugars and amino acids to the incubation media (M. Contrepois, personal communication).

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Characterization of F17 variants (Bertels et al., 1989) In 1986, the authors isolated an E. coli strain from the faeces of a diarrhoeal calf adhering to calf villi in vitro and weakly cross-reacting with F17 antiserum. The strain was designated 111KH86 and its fimbriae ‘F111’ It was of considerable interest to investigate the F111 fimbriae in order to evaluate the necessity to incorporate the strain (or fimbriae) in colibacillosis vaccines. The F111 fimbriae were purified and its N-terminal amino acids determined. Furthermore, the serological relationship of the F111 fimbriae with CS31A, F17, F1, F2, F3, F4, F5, F6, F41 and F165 was studied using immunogold electron microscopy, agglutination, immunofluorescence and immunoelectrophoresis. The results indicated that the F111 fimbriae are composed of subunits of 18 kD and were shown by immunogold electron microscopy to react with F111 antibodies and to a lesser degree with F17 antibodies. No crossreaction was observed with other fimbrial antigens. Furthermore, the F111 fimbriae could block the adhesion of F111-and F17-positive strains. The N-terminal amino acid sequence of F111 and F17 fimbriae was almost identical. As both F111 fimbriae and F111 antibodies could inhibit the binding of F17-positive strains, and both were characterized as NAG-specific, we concluded that the F111 and F17 fimbriae probably recognize the same or a homologous intestinal receptor (Van Driesche et al., 1988). Incorporation of F111 fimbriae in colibacillosis vaccines is not necessary if these vaccines already contain a F17-positive strain. Recently, fimbriae were described on the surface of CNF2-producing E. coli strains, which similar to F111 strains, hybridized with the F17-A probe but were not agglutinated by F17 antiserum. Both the fimbrial subunit gene and adhesin gene were cloned and sequenced. They showed homology with the structural subunit (F17-A) and adhesin (F17-G) genes of the F17 gene cluster. In view of the homology with F17 fimbrial gene cluster, the fimbriae encoded by the CNF2-producing E. coli strains were termed F17b (El Mazouari, personal communication). Hybridization experiments (Lintermans, 1990) The purpose of these hybridization experiments of F1, F2, F3, F4, F5, F6, F111 F41 and F167 fimbrial gene clusters with the F17-A and F17-G probes was to study the prevalence of the F17 subunit and F17 adhesin DNA sequences in other fimbrial gene clusters. It was shown that the F17-A probe and the F17-G probe reacted only with DNA obtained from F17-, CNF2- and F111-positive strains. This confirmed earlier results which indicated a high degree of homology between the F17 and F111 fimbrial systems based on serological studies. Furthermore, the F17-A probe was used to determine the number of subunit gene sequences in the strain 25KH9st. First, total DNA from the strain 25KH9st was digested with various restriction enzymes, separated by electrophoresis and blotted to nylon membranes. The resulting hybridization data allowed the authors, together with a detailed restriction map of pPLHD2, to conclude that the F17-A gene was present as a single copy gene in the strain 25KH9st. F17-Specific adhesion blockers and vaccines The F17-G gene was cloned in the pGEMEX expression vector; this vector allowed the expression of fusion proteins with the T7 gene 10 capsid. After induction of the transformants with IPTG, 50 per cent of the total protein consisted of the F17-G fusion product which had a molecular weight of 62 kD. The fusion protein was purified and solubilized. After induction, bacteria were centrifuged and treated with lysozyme. Inclusion bodies were obtained and washed; denaturation took place in 8 M urea and partial renaturation was done by

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dialysis against phosphate buffer. Cleavage of the F17-G protein from the leader protein was impossible as no known protease sites were available in the fusion region. The purified F17-G fusion’s were used to immunize rabbits and chicken. As negative controls, rabbits and chicken were also immunized with the T7 gene 10 leader protein. Live bacteria expressing the F17-G fusion were also used as immunizing antigen. Antibody titres were determined by ELISA using plates coated with the fusion and leader proteins. High titres were obtained using both live bacteria and purified inclusion bodies; pre-immune sera and eggs remained negative in the ELISA test. Both rabbit antibodies and egg-derived antibodies recognized the F17-G fusion and the leader protein in a Western blot analysis. Evaluation of the adhesion blocking activities of the F17-G fusion was carried out using isolated calf villi, brush-border membranes (BBM), Caco-2 cell lines and haemagglutination (Girardeau, 1980). Antibodies directed against the F17-G fusion obtained from rabbits could block the adhesion of two different F17 strains but the adhesion of a K99 strain was not blocked; the pre-immune serum could not block the adherence of the F17-positive strains. Antibodies obtained after injection of live bacteria or after injection of purified inclusion bodies were able to block the binding of F17 strains. Antibodies obtained from chicken could also block their adherence but the results were less clear-cut. Using BBM, the purified and solubilized F17 fusion-protein could bind to membrane receptors and block the subsequent adherence of F17-positive bacteria to the BBM. In contrast, the Caco-2 cells were not suitable as no binding could be obtained using F17-positive strains as positive controls. Preliminary data obtained indicated that rumen epithe lial cells could also function as an adhesion model for F17 bacteria (Neogrady S., personal communication). All inhibition studies done with the anti-leader anti-bodies were negative in the blocking assays. Haemagglutination assays using fresh bovine red blood cells and NAG as the blocking sugar failed to differentiate between agglutination obtained with the purified F17 product and the leader protein; both F17 fusion product and the purified T7 capsid product agglutinated bovine erythrocytes in a NAG-specific manner. It is possible that the leader protein recognized NAG-specific receptors on bovine erythrocytes or reacted non-specifically. Therefore, further cloning work was initiated to express the F17-G protein without a major leading peptide. For this an industrial expression vector using specific protease cleavage sites and a poly-histidine based purification system has been used. Future studies will have to address the crucial problem of testing the blocking activity of F17-G antibodies in new-born calves; purified F17-G protein will also have to be evaluated as a vaccine component for gestating cows. General conclusions The F17 gene cluster was cloned and sequenced. The smallest construction which encoded normal F17 fimbriae was designated pPLHD62 and contained a 6kb DNA insert in the pUC8 cloning vector. It has been shown that at least four genes are necessary to obtain functional F17 fimbriae. The F17-A gene encodes a subunit peptide of 20 kD. Amino acid sequence comparison of F17 with other fimbrial proteins indicated a much closer relationship of F17-A with PapA and PilA than with F4 and F5 subunit peptides. The F17-G protein has a molecular weight of 36 kD, functions as the adhesin and is a minor fimbrial component. Deletion of F17-G gene resulted in normal but non-adhesive F17 fimbriae. The other fimbrial genes were designated F17-C and F17-D and were found to be indispensible for piliation. Expression of F17 fimbriae is dependent on incubation temperature. Using immunogold electron microscopy it was shown that this effect was post-translational. No regulatory gene sequences were detected in the F17 gene cluster.

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The F111 and F17b fimbriae, which are serologic variants of the F17 fimbriae, have been purified. Apparently the F17 and F111 fimbriae recognize analogous intestinal receptors. While sequence variation has been observed between the F17-A, F111-A and F17b-A subunit genes, the corresponding adhesin genes are very similar. This report describes in extenso the dissociation between piliation and adhesion in the F17 system. The experiments described in this work are not only relevant for the veterinary medicine in general but are important for the development of new strategies for the prevention and therapy of neonatal calf diarrhoea. Indeed, the protection of new-born calves from cows not vaccinated against colibacillosis remains one of the main problems in veterinary medicine. Oral rehydration and antibiotic treatment are not efficient once diarrhoea is observed. The identification of the F17 adhesin could therefore be a first step to find a rational treatment for this disease. The adhesin component has three possible and important applications. First, to study the protective capacity of colibacillosis vaccines based on adhesin components. Secondly, the purified adhesin could be used for the purification of the F17 intestinal receptor and also for studying the interactions between the receptor and the adhesin. The results of these studies may then stimulate the development of drugs for the blockage of the adherence of pathogenic E. coli strains in the small intestine. Thirdly, this adhesin protein is also a possible candidate as an oral blocker to be used to protect calves of non-vaccinated cows after birth. It is important to note that this last approach may find other applications in the prevention or therapy of bacterial diseases where adhesion is a critical step in the pathogenicity of the bacterial agent. Acknowledgements We thank the Instituut voor Aanmoediging van het Onderzoek in Nijverheid en Landbouw (IWONL) for the financial support for the cloning of the F17 gene cluster (grant no. 5398A). References Abraham, J.M., Freitag, C.S., Clements, J.R. and Eisenstein, B.I., 1985, An invertible element of DNA controls phase variation of type 1 fimbriae of E. coli, Proceedings of the National Academy of Sciencs of the United States of America, 82, 5724–27. Allweiss, B., Dostal, J., Carey, K.E., Edwards, T.F. and Freter, R., 1977, The role of chemotaxis in the ecology of bacterial pathogens of mucosal surfaces, Nature, 266, 448–50. An der Lan, B., Cowell, J.L., Burstyn, D.G., Manclark, C.R. and Chrambach, A., 1986, Characterization of the filamentous hemagglutinin from Bordetella pertussis by gel electrophoresis, Molecular and Cellular Biochemistry, 70, 31–55. Anderson, B.J., Bills, M.M., Egerton, J.R. and Mattick, J.S., 1984, Cloning and expression in E. coli of the gene encoding the subunit of Bacteroides nodosus fimbriae, Journal of Bacteriology, 160, 748–54. Arp, L.H., 1988, Bacterial infection of mucosal surfaces: an overview of cellular and molecular mechanisms, in Roth, J.A. (Ed.) Virulence Mechanisms of Bacterial Pathogens, pp. 3–27 American Society for Microbiology, Washington, USA. Bäga, M., Normark, S., Hardy, J., O’Hanley, P., Lark, D., Olsson, O., Schoolnik, G. and Falkow, S., 1984, Nucleotide sequence of the papA gene encoding the Pap pilus subunit of human uropathogenic Escherichia coli, Journal of Bacteriology, 157, 330–33. Bertels, A., Pohl, P., Schlicker, C., Van Driessche, E., Charlier, G., De Greve, H. and Lintermans, P., 1989, Isolation of the F111 fimbrial antigen on the surface of a bovine Escherichia coli strain isolated out of calf diarrhea:

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characterization and discussion of the need to adapt recent vaccines against neonatal calf diarrhea, Vlaams Diergeneeskundig Tijdschrift, 58, 118–22. Bertels, A., De Greve, H. and Lintermans, P., 1991, Function and genetics of fimbrial and nonfimbrial lectins from Escherichia coli, in Kilpatrick, D.C., Van Driessche, E. and Bøg-Hansen, T.C. (Eds), Lectin Reviews, vol. I, pp. 53–67, St. Louis, MO, USA: Sigma Chemical Company. Bijlsma, I.G.W., De Nijs, A., Van Der Meer, C. and Frik, J.F., 1982, Different pig phenotypes affect adherence of E. coli to jejunal brush borders by K88ab, K88ac, or K88ad antigen, Infection and Immunity, 37, 891–94. Blumenstock, E. and Jann, K., 1982, Adhesion of piliated E. coli strains to phagocytes: differences between bacteria with mannose-sensitive pili and those with mannoseresistant pili, Infection and Immunity, 35, 264–69. Brinton Jr., C.C., 1959, Non-flagellar appendages of bacteria, Nature, 183, 782–86. Burrows, M.R., Sellwood, R. and Gibbons, R.A., 1976, Haemagglutinating and adhesive properties associated with the K99 antigen of bovine strains of Escherichia coli, Journal of General Microbiology, 96, 269–75. Chandler, R.L., Smith, K. and Turfrey, B.A., 1985, Exposure of bovine cornea to different strains of Moraxella bovis and to other bacterial species in vitro, Journal of Comparative Pathology, 95, 415–23. Clegg, S. and Gerlach, G.F., 1987, Enterobacterial fimbriae, Journal of Bacteriology, 169, 934–38. Contrepois, M.G., Girardeau, J.P., Dubourguier, H.C. and Gouet, P., 1981, Mise en evidence de quelques facteurs intervenant dans la biosynthese de l’ag K99, in Pohl P. and Leunen J. (Eds) Resistance and Pathogenic Plasmids, pp. 206–27, Brussels:CEC seminar. Contrepois, M.G., Girardeau, J.P., Gouet, P. and Der Vartanian, M., 1983, Expression of K99 pilus of E. coli. Annales de Recherches Vétérinaires, 14, 400–7. Cowell, J.L., Urisu, A., Zhang, M., Steven, A.C. and Manclark, C.R., 1986, Filamentous hemagglutinin and fimbriae of Bordetella pertussis: properties and roles in attachment, in Leive L. (Ed.) Microbiology, pp. 55–58, Washington, DC: American Society for Microbiology. Dean, A.G., Ching, Y., Williams, R.G. and Harden, L.B., 1972, Test for E. coli enterotoxin using infant mice: application in a study of diarrhea in children in Honolulu, Journal of Infectious Diseases, 125, 407–11. de Graaf, F.K., Klaasen-Boor, P. and Van Hees, J.E., 1980a, Biosynthesis of the K99 surface antigen is repressed by alanine, Infection and Immunity, 30, 125–28. de Graaf, F.K., Wientjes, F.B. and Klaasen-Boor, P., 1980b, Production of K99 antigen by enterotoxigenic Escherichia coli strains of antigen groups O8, O9, O20, and O101 grown at different conditions, Infection and Immunity, 27, 216–21. Duguid, J.P., Smith, I.W., Dempster, G. and Edmunds, P.N., 1955, Non-flagellar filamentous appendages (‘fimbriae’) and haemagglutinating activity in Bacterium coli, Journal of Pathology and Bacteriology, 70, 335–48. Eisenstein, B.I, 1981, Phase variation of type 1 fimbriae in E. coli is under transcriptional control, Science, 214, 337–39. Frank, S.K. and Gerber, J.D., 1981, Hydrolytic enzymes of Moraxella bovis, Journal of Clinical Microbiology, 13, 269–71. Gaastra, W. and De Graaf, F.K., 1982, Hostspecific fimbrial adhesins of noninvasive enterotoxigenic Escherichia coli strains, Microbiological Reviews, 46, 129–61. Girardeau, J.P., 1980, A new in vitro technique for attachment to intestinal villi using enteropathogenic E. coli, Annali di Microbiologia, 131B, 31–37. Girardeau, J.P., Dubourguier, H.C. and Contrepois, M., 1979, Attachement des E. coli entéropathogènes a la muqueuse intestinale, in Société Francaise de Buiatrie (Ed.) Gastro-entérites Néonatales du Veau, Vichy, 25–26 October (53–66). Gubish, E.R., Chen, K.D.S. and Buchanan, T.M., 1982, Attachment of gonococcal lectin-resistant clones of Chinese hamster ovary cells, Infection and Immunity, 37, 189–94. Gyles, C.L., So, M. and Falkow, S., 1974, The enterotoxin plasmids of E. coli, Journal of Infectious Diseases, 130, 40–49. Hales, B.A., Beverly-Clarke, H., High, N.J., Jann, K., Perry, R., Goldhar, J. and Boulnois, G.J., 1988, Molecular cloning and characterisation of the genes for a non-fimbrial adhesin from E. coli, Microbial Pathogenesis, 5, 9–17.

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Hanson, M.S. and Brinton, C.C., 1988, Identification and characterization of E. coli type-1 pilus tip adhesion protein, Nature, 332, 265–68. Hermodson, M.A., Chen, K.C.S. and Buchanan, T.M., 1978, Neisseria pili proteins: aminoterminal amino acid sequences and identification of an unusual amino acid, Biochemistry, 17, 442–45. Hewick, R.M., Hunkapiller, M.W., Hood, L.E. and Dreyer, W.J., 1981, A gas-liquid solid phase peptide and protein sequenator, Journal of Biological Chemistry, 256, 7990–97. Hirs, C.H.W., 1956, The oxidation of ribonuclease with performic acid, Journal of Biological Chemistry, 219 611–21. Hirs, C.H.W., 1967, The determination of cysteine as cysteic acid, Methods in Enzymology, 11, 59–62. Holm, L., 1986, Codon usage and gene expression, Nucleic Acids Research, 14, 3075–87. Hopp, T.P. and Woods, K.R., 1981, Prediction of protein antigenic determinants from amino acid sequences, Proceedings of the National Academy of Sciences of the United States of America, 78, 3824–28. Hoschutzky, H., Nimmich, W., Lottspeich, F. and Jann, K., 1989, Isolation and characterisation of the non-fimbrial adhesin NFA-4 from uropathogenic E. coli O7:K98:H6, Microbial Pathogenesis, 6, 351–59. Inouye, M. and Halegoua, S., 1980 Secretion and membrane localization of proteins in Escherichia coli, Critical Reviews in Biochemistry, 7, 339–71. Isaacson, R.E., 1980, Factors affecting expression of the E. coli pilus K99, Infection and Immunity, 28, 190–94. Jayappa, H.G., Goodnow, R.A. and Geary, S.J., 1985, Role of Escherichia coli type 1 pilus in colonization of porcine ileum and its protective nature as a vaccine antigen in controlling colibacillosis, Infection and Immunity, 48, 350–54. Jones, G.W. and Rutter, J.M., 1972, Role of the K88 antigen in the pathogenesis of neonatal diarrhea caused by E. coli in piglets, Infection and Immunity, 6, 918–27. Jones, G.W. and Rutter, J.M., 1974, The association of K88 antigen with haemagglutinating activity in porcine strains of E. coli, Journal of General Microbiology, 84, 135–44. Josephsen J., Hansen, F., de Graaf, F.K. and Gaastra, W., 1984, The nucleotide sequence of the protein subunit of the K88ac fimbriae of porcine enterotoxigenic E. coli, FEMS Microbiology Letters, 25, 301–6. King, G.J. and Swanson, J., 1978, Studies on gonococcus infection. XV. Identification of surface proteins of Neisseria gonorrhoeae correlated with leukocyte association, Infection and Immunity, 21, 575–84. Klemm, P., 1981, The complete amino acid sequence of the K88 antigen, a fimbrial protein from E. coli, European Journal of Biochemistry, 117, 617–27. Klemm, P., 1985, Fimbrial adhesins of E. coli, Reviews of Infectious Diseases, 7, 321–40. Korhonen, T.K., Nurmiaho, E.L., Ranta, H. and Edén, C.S., 1980, New method for isolation of immunologically pure pili from Escherichia coli, Infection and Immunity, 27, 569–75. Kroncke, K.D., Ørskov, I., Ørskov, F., Jann, B. and Jann, K., 1990, Electron microscopic study of coexpression of adhesive protein capsules and polysaccharide capsules in E. coli, Infection and Immunity, 58, 2710–14. Lee, S.W., Alexander, B. and McGowan, B., 1983, Purification, characterization, and serologic characteristics of Bacteroides nodosus pili and use of a purified vaccine in sheep, American Journal of Veterinary Research, 44, 1676–81. Lewin, R., 1984, Microbial adhesion is a sticky problem, Science, 224, 375–77. Lindberg, F.P., Lund, B. and Normark, S., 1984, Genes of pyelonephritogenic E. coli required for digalactoside-specific agglutination of human cells, EMBO Journal, 3, 1167–73. Lindberg, F., Lund, B., Johansson, L. and Normark, S., 1987, Localization of the receptorbinding protein adhesin at the tip of the bacterial pilus, Nature, 328, 84–87. Lintermans, P., 1986, Aanhechtingsantigenen van boviene enterotoxigene E. coli: detektie, zuivering en de genetische karakterizatie van het Att25 operon. Thesis verdediging voor het bekomen van de graad van eerst-aanwezend assistent bij het Min. van Landbouw. Lintermans, P., 1990, The genetische analyse van de F17 gencluster, Aggregaatsthesis, UG. Lintermans, P. and Pohl, P., 1983a, ‘Detection of bovine enterotoxigenic E. coli’, Proceedings of the Third International Symposium of the World Association of Veterinary Laboratory Diagnosticians, June 13–15. Iowa, USA (pp. 511–517).

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Lintermans, P. and Pohl, P., 1983b, Detection of bovine enterotoxigenic E. coli possessing a K99 or Att25 adherence antigen by a radial immunodiffusion tecnique, Veterinary Record, 113, 376. Lintermans, P. and Pohl, P., 1984, Detection of bovine enterotoxigenic E. coli: a comparative study of a direct fluorescent antibody technique and conventional culturing methods, British Veterinary Journal, 140, 44–53. Lintermans, P.F., Pohl, P., Bertels, A., Charlier, G., Vandekerckhove, J., Van Damme, J., Schoup, J., Schlicker, C., Korhonen, T., De Greve, H. and Van Montagu, M., 1988a, Characterization and purification of the F17 adhesin on the surface of bovine enteropathogenic and septicemic Escherichia coli, American Journal of Veterinary Research, 49, 1794–99. Lintermans, P., Pohl, P., Deboeck, F., Bertels, A., Schlicker, C., Vandekerckhove, J., Van Damme, J., Van Montagu, M. and De Greve, H., 1988b, Isolation and nucleotide sequence of the F17-A gene encoding the structural protein of the F17 fimbriae in bovine enterotoxigenic Escherichia coli, Infection and Immunity, 56, 1475–84. Lintermans, P.F., Bertels, A., Schlicker, C, Deboeck, F., Charlier, G., Pohl, P., Norgren, M., Normark, S., Van Montagu, M. and De Greve, H., 1990, Identification, charaterization, and nucleotide sequence of the F17-G gene which determines receptor binding of E. coli F17 fimbrae, Journal of Bacteriology, 137, 3366–72. Lund, B., Lindberg, F., Baga, M. and Normark, S., 1985, Globoside-specific adhesins of uropathogenic E. coli are encoded by similar trans-complementable gene clusters, Journal of Bacteriology, 162, 1293–301. Lund, B., Lindberg, F. and Normark, S., 1988, Structure and antigenic properties of the Tip-located P pilus proteins of uropathogenic E. coli, Journal of Bacteriology, 170, 1887– 94. Mahoney, W.C. and Hermodson, M.A., 1980, Separation of large denatured peptides by reverse phase high performance liquid chromatography. Trifluoroacetic acid as a peptide solvent, Journal of Biological Chemistry, 255, 11199–203. Mainil, J., 1988, Escherichia coli entérotoxigènes bovins: identification des facteurs et des plasmides de virulence par hybridation ADN-ADN, These de Doctorat. Marshall, K.C. (Ed.), 1984, Glossary, in Microbial Adhesion and Aggregation, pp. 397–99, New York: SpringerVerlag. Maurer, L. and Orndorff, P., 1985, A new locus, pilE, required for the binding of type 1 piliated Escherichia coli to erythrocytes, FEMS Microbiology Letters, 30, 59–66. Maurer, L. and Orndorff, P.E., 1987, Identification and characterization of genes determining receptor binding and pilus length of Escherichia coli type 1 pili, Journal of Bacteriology, 169, 640–45. Maxam, A.M. and Gilbert, W., 1977, A new method for sequencing DNA, Proceedings of the National Academy of Sciences of the United States of America, 74, 560–64. Minion, F.C., Abraham, S.N., Beachey, E.H. and Goguen, J.D., 1986, The genetic determinant of adhesive function in type 1 fimbriae of Escherichia coli is distinct from the gene encoding the fimbrial subunit, Journal of Bacteriology, 165, 1033–36. Mooi, F.R. and de Graaf, F.K., 1985, Molecular biology of fimbriae of enterotoxigenic E. coli, Current Topics in Microbiology and Immunology, 118, 119–35. Morris, J.A., Sojka, W.J. and Ready, R.A., 1985, Serological comparison of the E. coli prototype strains for the F(Y) and Att25 adhesins implicated in neonatal diarrhoea in calves, Research in Veterinary Science, 38, 246–47. Norgren, M., Normark, S., Lark, D., O’Hanley, P., Schoolnik, G., Falkow, S., Svanborg Eden, C., Bäga, M. and Uhlin, B.E., 1984, Mutations in E. coli cistrons affection adhesion to human cells do not abolish Pap pili fiber formation, EMBO Journal, 3, 1159–65. Normark, S., Bäga, M., Goransson, M., Lindberg, F.P., Lund, B., Norgren, M. and Uhlin, B.-E., 1986, Genetics and biogenesis of E. coli adhesins, in Mirelman, D. (Ed.) Microbial Lectins and Agglutinins, pp. 113–43, New York: Wiley Interscience. Old, D.C., 1972, Inhibition of the interaction between fimbrial haemagglutinins and erythrocytes by D-mannose and other carbohydrates, Journal of General Microbiology, 71, 149–57. Orndorff, P.E., Spears, P.A., Schauer, D. and Falkow, S., 1985, Two modes of control of pilA, the gene encoding type 1 pilin in E. coli, Journal of Bacteriology, 164, 321–30. Ørskov, I. and Ørskov, F., 1983, Serology of E. coli fimbriae, Progress in Allergy, 33, 80–105.

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Ørskov, F. and Ørskov, I., 1984, Serotyping of E. coli, Methods in Microbiology, 14, 43–112. Ørskov, I., Ørskov, F., Sojka, W.J. and Leach, J.M., 1961, Simultaneous occurrence of E. coli B and L antigens in strains from diseased swine, Acta Patholologica et Microbiologica Scandinavica, 53B, 404–22. Ørskov, I., Ørskov, F., Sojka, W.J. and Wittig, W., 1964, K. antigens K88ab (L) and K88ac (L) in E. coli. A new Oantigen: O147 and a new K-antigen: K89(B), Acta Pathologica et Microbiologica Scandavica, 626, 439–47. Ørskov, I., Ørskov, F., Wittig, W. and Sweeney, J.E., 1969, A new E. coli serotype O149:K91 (B), K88ac (L):H10 isolated from diseased swine, Acta Pathologica et Microbiologica Scandavica, 75:491–98. Ørskov, 1., Ørskov, F., Smith, H.W. and Sojka, W.J., 1975 The establishment of K99, a thermolabile, transmissible E. coli K antigen, previously called ‘Kco’, possessed by calf and lamb enteropathogenic strains, Acta Pathologica et Microbiologica Scandavica, 83B, 31–36. Pallesen, L., Madsen, O. and Klemm, P., 1989, Regulation of the phase switch controlling expression of type 1 fimbriae in E. coli, Molecular Microbiology, 3, 925–31. Pedersen, K.B., Frøholm, L.O. and Bøvre, K., 1972, Fimbriation and colony type of Moraxella bovis in relation to conjunctival colonization and development of keratoconjunctivitis in cattle, Acta Pathologica et Microbiologica Scandavica, 80B, 911–18. Pohl, P., Lintermans, P., Van Muylem, K. and Schotte, M., 1982, Colibacilles entérotoxigènes du veau possédant un antigène d’attachement different de l’antigène K99, Annales de Médicine Vétérinaire, 126, 569–71. Pohl, P., Lintermans, P. and Van Muylem, K., 1984, Fréquence des adhésines K99 et Att25 chez les E. coli du veau, Annales de Médicine Vétérinaire, 128, 555–58. Pohl, P., Lintermans, P., Moury, J., Van Muylem, K. and Marin, M., 1986, Facteurs de virulence chez les E. coli septicemiqaes et saprophytes du veau, Annales de Médicine Vétérinaire, 130, 515–20. Reed, W.P. and Williams Jr., R.C., 1978, Bacterial adherence: first step in pathogenesis of certain infections, Journal of Chronic Diseases, 9, 470–87. Rhen, M., Klemm, P. and Korhonen, T.K., 1986, Identification of two new hemagglutinins of E. coli, N-acetyl-Dglucosamine-specific fimbriae and a blood group M-specific agglutinin, by cloning the corresponding genes in E. coli K 12, Journal of Bacteriology, 168, 12134–42. Rutter, P.R. and Vincent, B., 1980, The adherence of microorganisms to surfaces: physicochemical aspects, in Berkeley, R.C.W., Lynch, J.M., Melling, J., Rutter, P.R. and Vincent B. (Eds), Microbial Adhesion to Surfaces, pp. 79–92, Chichester, England: Ellis Horwood. Sanger, F., Nicklen, S. and Coulson, A.R., 1977, DNA sequencing with chain-terminating inhibitors, Proceedings of the National Academy of Sciences of the United States of America, 74, 5463–67. Shipley, P.L., Dougan, G. and Falkow, S., 1981, Identification and cloning of the genetic determinant that encodes for the K88ac adherence antigen, Journal of Bacteriology, 145, 920–25. Silverblatt, F.J., Dreyer, J.S. and Schauer, S., 1979, Effect of pili on susceptibility of E. coli to phagocytosis, Infection and Immunity, 24, 218–23. Smith, H.W. and Gyles, C.L., 1970, The relationship between two apparently different enterotoxins produced by enteropathogenic strains of E. coli of porcine origin, Journal of Medical Microbiology, 3, 387–401. Smith, H.W. and Linggood, M.A., 1971, Observations on the pathogenic properties of the K88, Hly and Ent plasmids of E. coli with particular reference to porcine diarrhea, Journal of Medical Microbiology, 4, 467–85. So, M., Boyer, H.W., Betlach, M. and Falkow, S., 1976, Molecular cloning of an E. coli plasmid determinant that encodes for the production of heat-stable enterotoxin, Journal of Bacteriiology, 128, 463–72. Southern, E.M., 1975, Detection of specific sequences among DNA-fragments separated by gel electrophoresis, Journal of Molecular Biology, 98, 503–17. Stanier, R.Y., Adelberg, E.A. and Ingraham, J.L., 1979, General Microbiology, pp. 314–363, New York: Prentice-Hall. Suggs, S.V., Wallace, R.B., Hirose, T., Kawashima, E.H. and Itakura, K., 1981, Use of synthetic oligonucleotides as hybridization probes: Isolation of cloned cDNA sequences for human 2-microglobulin, Proceedings of the National Academy of Sciences of the United States of America, 78, 6613–17. Swanson, J., Kraus, S.J. and Gotschlich, E.C., 1971, Studies on gonococcus infection. I. Pili and zones of adhesion: their relation to gonococcal growth patterns, Journal of Experimental Medicine, 134, 886–906.

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Takeuchi, A., 1967, Electron microscope studies of experimental salmonella infection. I. Penetration into the intestinal epithelium by Salmonella typhimurium, American Journal of Pathology, 50, 109–36. Uhlin, B.E., Norgren, M., Baga, M. and Normark, S., 1985, Adhesion to human cells by E. coli lacking the major subunit of a digalactoside-specific pilus-adhesin, Proceedings of the National Academy of Sciences of the United States of America, 82, 1800–4. Van Driessche, E., Schoup, J., Charlier, G., Lintermans, P., Beeckmans, S., Zeeuws, R., Pohl, P. and Kanarek, L., 1988, The attachment of E. coli to intestinal calf villi and Eupergit-C-glycoprotein beads, in Bøg-Hansen, T.C. and Freed, D.L.J. (Eds) Lectins: Biology, Biochemistry, Clinical Biochemistry, vol. 6, pp. 55–62, St. Louis, MO: Sigma Chemical Company. Van Zijderveld, F.G. and Overdijk, E., 1983, Experiences with the ELISA for detection of the E. coli K99 antigen in calf faeces, Annales de Recherches Vétérinaires, 14, 395–99.

Chapter 15 Infection of the Gut by Pathogenic Bacteria is Inhibited by Dietary Lectins: Chemical Probiosis A.Pusztai, G.Grant, S.W.B.Ewen, W.J.Peumans, E.J.M.Van Damme and S.Bardocz

Introduction Growth and health are ultimately dependent on the efficient assimilation of nutrients from the small intestine. To achieve maximum efficiency in food conversion it is not only necessary for the diet to be of high nutritional quality, but the optimal functioning of the digestive tract is also essential. However, as food is digested and absorbed in the presence of bacteria, this is seldom attained. Although the main digestive/ absorptive compartment of the gastrointestinal tract, the small intestine, in healthy individuals is usually regarded as essentially free from coliforms and other potentially harmful bacteria, it is far from being germfree. Moreover, it is clear that damage to the epithelial surface or simply even a change of diet may lead to speedy proliferative changes in the bacterial content of the small intestine and, therefore, gastrointestinal upsets can occur under conditions of strict hygiene (Savage, 1987; Shimizu and Terashima, 1982). Lectin-induced coliform overgrowth in the small intestine The stability of the bacterial flora in the small intestine of healthy, well-fed rats was clearly demonstrated by our inability to orally infect them with Type-1, mannose-sensitive fimbriated E. coli isolated from their gut without also changing their diet. Even after six days of oral exposure of the rats to broth containing 108 and 109 viable E. coli ml−1, the counts of this bacteria remained at control levels, 103–104 coliform bacteria of the whole small intestine (Pusztai et al., 1993). In contrast, one of the most clear-cut examples of a damaging overgrowth of coliforms induced by dietary changes was the demonstration that when rats were given good-quality diets into which increasing amounts of kidney bean (Phaseolus vulgaris) lectin (PHA) were included, a highly significant, dose-dependent and fully reversible increase of E. coli occurred (Figure 15.1). This selective overgrowth of the rat small intestine by E. coli was not caused by administration of the bacteria but originated from the commensal flora of the animals. Although at high doses of PHA there was also a slight increase in some non-lactosefermenting coliforms (mainly Proteus spp.), counts of Lactobacillus spp. and Bacteroides were essentially unchanged, even at the highest PHA concentrations. The proliferating E. coli was shown to bind avidly to the small intestinal glycocalix and they could not be removed by simple washing of small intestinal sections without the presence of mannose in the washing buffer. This provides convincing experimental evidence for the involvement of bacterial lectins in the adhesion of bacteria already implicit in the sugar-specific inhibition of bacterial haemagglutination (Old, 1972; Salit and Gotschlich, 1977). In contrast, the intestinal washings contained high counts of Lactobacilli, bacteria which are not regarded as strictly adhering species in the proximal small intestine (Pusztai et al., 1993).

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Figure 15.1. Log10 counts of Escherichia coli in jejunum of rats given different doses of pure kidney bean lectin, PHA, for three days.

The reversibility of the E. coli overgrowth was demonstrated by feeding the rats control diets without PHA for three days because the coliform counts then reverted to the original control levels of 103–104 cfu g −1 wet tissue. The overgrowth of the small intestine by commensal, Type-1, mannose-sensitive fimbriated E. coli after dietary lectin treatment is not confined to PHA alone. Similar overgrowth can be induced by most avidly binding lectins, such as soya bean agglutinin, SBA, wheat germ agglutinin or Robinia lectin, although to achieve the same effect their concentrations may have to be increased in comparison with PHA. Recent morphological studies have now provided convincing experimental evidence that E. coli overgrowth in the small intestine occurred only with those lectins which were also powerful growth factors for the small intestine. As seen elsewhere in this book (see Bardocz et al., Chapter 6), by binding avidly to the brush-border epithelium these lectins induce dose- and polyamine-dependent, fully reversible and hyperplastic growth of the small intestine with stimulation of the rate of crypt cell proliferation and speeding up the turnover of villus cells. In healthy rats the villus epithelium is populated mainly by highly differentiated, mature enterocytes whose membrane- and cytoplasmic glycoconjugates contain complex saccharide side-chains and very few terminal -linked mannose residues. Consequently, in the normal small intestine the residual population of Type-1, mannose-sensitive fimbriated E. coli is low. The scarcity of undifferentiated, immature epithelial cells on the villi with high levels of polymannosylated cellular glycans is clearly shown by the slight reactivity of intestinal sections of these rats with mannose-specific lectins such as GNA, the agglutinin from snowdrop (Galanthus nivalis) bulbs (Figure 15.2a). However, after the PHA-induced overgrowth of E. coli

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Figure 15.2. Binding of GNA (a mannose-specific lectin from snowdrop, Galanthus nivalis, bulbs) to jejunal brushborder of rats (a) given diets containing GNA for six days; or (b) given GNA for six days with PHA for the last three days; and (c) which were returned to control diets for three days after exposure to both GNA and PHA for three days. Binding of GNA was demonstrated on paraformaldehyde-fixed sections which were first reacted with anti-GNA antibodies followed by secondary peroxidase-antiperoxidase treatment. Bars represent 25 µ m.

in the stimulated small intestine, there was an abundance of -linked terminal mannosyl glycans as shown by their extensive reaction with GNA (Figure 15.2b). Consequently, the brush-border could now provide the bacteria with the opportunity for adherence and proliferation. The reversibility of the overgrowth was demonstrated by the disappearance of the mannosyl residues within three days of the removal of the mitogenic stimulus of PHA from the diet (Figure 15.2c). The biological significance of the lectin-induced coliform overgrowth is likely to be great and not confined to laboratory conditions. It is commonly accepted that injury of the small intestinal epithelium automatically stimulates its hyperplastic growth to make good the damage regardless of whether it is caused by dietary or bacterial lectins, or by other erosive factors or diseases. Under these conditions the proportion of immature cells expressing polymannosylated membrane- and/or cytoplasmic glycans will increase with the direct consequence that coliform bacterial overgrowth will inevitably occur. The resulting diarrhoea and other digestive/absorptive problems usually exacerbate the initial gut damage and, when combined with poor nutritional status, such as in children in Third World countries, may lead to serious and wide-ranging health problems. Prevention of bacterial diseases by probiosis It is a commonly held belief that a stable bacterial flora in the gut is beneficial for the health of humans or animals. The most convincing evidence for this is the frequent observation that orally administered antibiotics enhance the proliferation of pathogenic species by suppressing the normal protective gut flora, resulting in diarrhoea and other intestinal problems. There is also some evidence that resident bacteria may contribute to the resistance of the host to infection by pathogens by competitive exclusion (Fuller, 1989, 1992). Accordingly, there have been many attempts, albeit not very successful, to use live bacterial

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supplements as probiotic agents to improve the resistance of humans or animals to bacterial infection (Fuller, 1989, 1992). A more rational approach to the protection of the mammalian gastrointestinal tract against pathogenic infection is likely to come from an understanding of the scientific principles involved in the mechanism of bacterial adhesion to gut surface structures. The clear host specificity of most pathogens argues strongly in favour of the existence of a general ‘lock-key’ mechanism which predetermines whether adhesion and/or infection can occur. Accordingly, if a particular pathogen has the proper adhesion molecules (keys) which specifically fit into epithelial structures (locks), interaction between the bacteria and the surface of the gut wall can occur. As a result, the bacteria can withstand the peristaltic downward pressure operating in the gut and can successfully colonize it. A corollary of the key-lock hypothesis is that substances (or keys) which mimic the adhesin-lectin responsible for the interaction between the bacteria and the tissue surface will be able to competitively block the adherence of that particular species (chemical probiosis). This blocking may be achieved by different types of inhibitory substances. By definition, the oral administration of adhesin-lectins isolated and purified from bacteria can effectively block the infection of the intestines by this bacterial species or those which adhere to these tissues by the same or similar adhesins. Unfortunately, the large-scale production of most adhesins is not easy to accomplish at present. A second and possibly effective way of blocking bacterial adhesion is the oral administration of specific antibodies produced against the adhesin. Although antibodies are obvious and potent inhibitors of adhesion in vitro, they are rapidly broken down in the intestines by proteases, and are consequently poor inhibitors in vivo. A third and more practical solution to the problem of preventing infection is to administer in the diet a large excess of saccharides, glycans or glycoconjugates whose terminal carbohydrate structures closely mimic the carbohydrate side chains of the bacterial receptors on the gut wall. This method is cost-effective particularly if such glycans are cheap and readily available. Finally, one of the most promising and highly effective ways to interfere with bacterial infection is to include in the diet plant lectins whose binding specificity is similar or the same as that of the infecting bacterial species. The probiotic plant lectin actually occupies the same site as the bacteria, whereas the complementary saccharides only compete with it. Thus, although the probiotic sugar has, according to mass law, to be given in high excess, the lectin needs to be administered only at slightly over equimolar amounts. In the following section, the power of this approach is demonstrated by a few examples. Probiosis by inhibition of bacterial adhesion in the intestines Blocking by saccharides As described above and also reviewed elsewhere in this book (see Van Driessche et al., Chapter 13 and Lintermans et al., Chapter 14), there is a substantial evidence that bacterial adhesion to cells of the gut epithelium or other epithelial tissues both in vivo and in vitro is a specific process which is in most instances mediated in a strictly carbohydrate-specific mannerby bacterial adhesins, lectins. Thus, from the definition of lectins it follows that bacterial adhesion can be specifically abolished or reduced substantially in the presence of sufficiently high concentrations of saccharides appropriate for the carbohydrate-specificity of the bacterial adhesin. This was, for example, most elegantly demonstrated in studies with Pseudomonas aeruginosa in Balb/c mice (Ko et al., 1987) where its in vitro attachment to tissue sections was completely inhibited in the presence of N-acetyl-neuraminic acid. Similarly, the extent of adherence of this bacteria to various organs in vivo was dramatically reduced in the presence of this sugar, whereas non-related

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carbohydrates (such as D-galactose) had no significant effect. Indeed, the administration of N-acetyl-neuraminic acid protected the animals from septicaemia and death (Ko et al., 1987). Similar results were obtained with the sugar-specific blocking of other bacterial species, such as the inhibition by high dietary intakes of -D-methylmannoside of PHA-induced E. coli overgrowth of the rat small intestine (Table 15.1). The inhibition was even more effective when instead of a simple sugar, glycopeptides containing high-mannose side chains were given orally to rats before they were given PHAdiets (Table 15.2; Pusztai et al., unpublished). It is now realised that probiosis can be achieved not only by the rather unpredictable supplementation of the diet with live ‘probiotic’ bacteria but also by the use of appropriate sugars to Table 15.1. Number of Escherichia coli in the jejunum of rats treated with PHA in the presence or absence of methylmannoside Control group (n=7) E. coli Median Log10 SED Significance

8.2×107 7.9 1.2 p<0.05

-mannoside group (n=7) 7.7×105 5.9 1.1

Table 15.2. Number of Escherichia coli in the jejunum of rats treated with PHA in the presence or absence of an egg albumin glycopeptide preparation

E. coli Median Log10 SED Significance

Control group (n=7)

Egg gg albumin group (n=7)

1.7×108 8.2 1.0 p <0.05

1.1×106 6.0 1.1

reduce the number of unwanted bacteria in the gut lumen. It is, therefore, not surprising that most food/feed manufacturers now market a range of carbohydrate-based probiotic supplements (Fuller, 1992). Blocking by plant lectins As shown above in the PHA-induced E. coli overgrowth rat model, the small intestinal epithelium is increasingly populated by immature cells displaying glycan side-chains of the polymannosyl type due to the accelerated turnover of the brush-border (Figure 15.2b). High concentrations of saccharides containing terminal mannose residues were therefore effective in reducing the adherence of Type-1, mannose-sensitive fimbriated E. coli to the polymannosylated glycans of these juvenile villus cells of the small intestine (Tables 15.1 and 15.2); however mannose-specific lectins, such as GNA were even more effective as a significant reduction in E. coli was achieved at dietary intakes of lectin which were smaller by an order of magnitude than those of the sugars (Figure 15.3; Pusztai et al., 1993). As GNA is a strictly mannose-specific lectin which does not directly interact with PHA, its effectiveness as an adhesionblocker of E. coli in vivo would appear to be the result of its competitive binding to the newly emerging terminal mannose residues of gut receptors. This was shown conclusively by the finding that,

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Figure 15.3. Log10 of Escherichia coli counts in the jejunum of rats which were given diets containing GNA, PHA, a combination of both lectins or control.

although GNA reduced by 103 the number of E. coli in the small intestine of PHA-treated rats it did not prevent the PHA-induced growth and other morphological changes of the brush-border epithelium such as the lengthening of small intestinal crypts and erosive surface damage (Pusztai et al., 1993). Neither did GNA reverse the PHA-induced changes in brush-border membrane glycans, as polymannosyl structures were still present on the glycocalix, at concentrations that were actually greater than those found with PHA alone (Figure 15.2b); GNA by itself had only a slight effect on the composition and histology of the small intestine (Figure 15.2a). These results therefore suggest that the specific and increased binding of GNA to terminal mannosyl residues was responsible for the reduction in adherence of mannose-sensitive fimbriated E. coli, as both were competing for the same adhesion sites. Unlike other lectins, GNA does not react with E. coli under in vitro conditions (Baintner et al., 1993) and it is unlikely that the in vivo reduction in bacterial numbers was caused by the precipitation of E. coli by GNA in the small intestine and its removal by peristalsis. These observations therefore provide the most convincing experimental evidence for chemical probiosis (Pusztai et al., 1990), i.e. that sugar-specific binding of bacteria in the gut can be blocked by dietary lectins of the same specificity. The use of plant lectins for the blockage of pathogenic infections is now protected by an International Patent Application (No. PCT/GB91/02236)

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Future perspectives Type-1, mannose-sensitive fimbriated E. coli is an important pathogen which is responsible for a number of serious gut-related health problems (diarrhoea in children, non-specific colitis in humans and pigs, etc.), and there is considerable benefit in being able to block its proliferation by the use of lectins. However, lectins have potential applications as blockers of many other pathogenic bacterial infections. Extensive work is now in progress in several laboratories, including the authors’ at the Rowett Research Institute, on the application of the principle of chemical probiosis to inhibit bacterial species other than Type-1 E. coli. For example, one such important aim of these studies is to inhibit the infection of mammals with different strains of Salmonella typhimurium or S.enteritides by the use of appropriate lectin-blockers in the diet. The rat model has been found particularly useful for studies of infection by Salmonellae and its inhibition because the physiological effects of these bacteria in rats are well-understood and highly reproducible. The use of the rodent model is also very pertinent because of emerging evidence that the spread of salmonellosis in nature to livestock occurs mainly through field mice and rats which can carry the disease under normal conditions. Similar to Type-1, mannose-sensitive fimbriated E. coli, most strains of Salmonellae are also fimbriated and may in fact use Type-1 fimbriae for adhesion in the gut. However, the evidence for the involvement of Type-1 fimbriae in colonization by Salmonellae is controversial. As most Salmonella species, in addition to Type-1 fimbriae, can elaborate at least two other and different fimbriae that are not specific for terminal mannose residues, it is likely that blockage of these pathogens will need the combined effects of several lectins or other blockers, each inhibiting the adhesion of one particular fimbrial species. Thus, the situation with Salmonellae is similar to that found with F17 E. coli (see Van Driessche et al., and Lintermans et al., Chapters 13 and 14 respectively) whose adhesion to calf brush-border membranes could not be inhibited by a simple sugar but required a combination of both N-acetyl-D-glucosamine and D-mannose. In the same manner, it is now thought that the binding of Helicobacter pylori to the stomach epithelium is dependent on glycans containing both N-acetyl-neuraminic acid and fucose in terminal positions. Accordingly, it is likely that at least two lectins will be needed to block the adhesion of this bacterial species, one specific for sialic acid and the other for fucose. It seems, therefore, that the successful blockage by a single lectin, GNA, of the specific overgrowth of Type-1, mannose-sensitive fimbriated E. coli in the mammalian small intestine is most likely to be an exception to the rule and that future developments will be dependent on improved understanding of the complexity of the carbohydrate-specificity of bacterial adhesion. This will undoubtedly increase the difficulties and the effort necessary for alleviating the effects of pathogenic infections on human and animal health without resorting to the use of undesirable antibiotics. As plant lectin-based antibacterial agents can be regarded as natural food components, it is likely that the pharmaceutical use of lectins will increase in future. Their use to improve both the quality and safety of our diet should reduce the need to use expensive antibacterial medical formulations for a healthier life. Acknowledgements This work was supported by The Scottish Office Agriculture and Fisheries Department and in part by a grant of the National Fund for Scientific Research (Belgium; FGWO grant 20059.89 N) to Dr Peumans who is the Research Director, and Dr Van Damme, who is a Research Assistant of this Fund. Dr Ewen gratefully acknowledges the generous grant from the Scottish Home and Health Department for the purchase of the Joyce-Loebl ‘magiscan’ image analyser. The work was also a part of EEC-FLAIR Concerted Action No 9 and No AIRI1-CT92-0569.

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References Baintner, K., Duncan, S.H., Stewart, C.S. and Pusztai, A., 1993, Binding and degradation of lectins by components of rumen liquor, Journal of Applied Bacteriology, 74, 29–35. Fuller, R., 1989, Probiotics in man and animals, Journal of Applied Bacteriology, 66, 365–78. Fuller, R., 1992, Probiotics. The Scientific Basis, London: Chapman and Hall. Ko, H.L., Beuth, J., Sotter, J., Schroten, H., Uhlenbruck, G. and Pulverer, G., 1987, In vitro and in vivo inhibition of lectin mediated adhesion of Pseudomonas aeruginosa by receptor blocking carbohydrates, Infection, 15, 237–40. Old, D.C., 1972, Inhibition of the interaction between fimbrial haemagglutinins and erythrocytes by D-mannose and other carbohydrates, Journal of General Microbiology, 71, 149–57. Pusztai, A., Grant, G., King, T.P. and Clarke, E.M.W., 1990, Chemical Probiosis, in Haresign, W. and Cole, D.J.A. (Eds) Recent Advances in Animal Nutrition, pp. 47–60, London: Butterworths. Pusztai, A., Grant, G., Spencer, R.J., Duguid, T.J., Brown, D.S., Ewen, S.W.B., Peumans, W.J., Van Damme, E.J.M. and Bardocz, S., 1993, Kidney bean lectin-induced Escherichia coli overgrowth in the small intestine is blocked by GNA, a mannose-specific lectin, Journal of Applied Bacteriology, 75, 360–68. Salit, I.E. and Gotschlich, E.C., 1977, Type I Escherichia coli pili: Characterization of binding to monkey kidney cells, Journal of Experimental Medicine, 146, 1182–94. Savage, D.C., 1987, Microorganisms associated with epithelial surfaces and stability of the indigenous microflora, Die Nahrung, 31, 383–95. Shimizu, M. and Terashima, T., 1982, Appearance of enterotoxigenic Escherichia coli in piglets with diarrhoea in connection with feed changes, Microbiology and Immunology, 26, 467–77.

Index

987P fimbriae 256–8, 294 antibodies 273 8813 antigen 268 -amylase inhibitor ( -AI) bean 24–31, 35, 50–2 pancreatic growth 146, 150 Streptococcus tendae 24–5 Acanthoscelides obtectus 46, 47 accessory cells 159–60 actin cytoskeletal lesions 87–9, 92, 96 Acyrthosiphon pisum 45 adenomatous polyps 226 adhesins fimbrial 251–2, 294–7 non-fimbrial 297–8 adhesion 295 adsorptive ento-/trans-cytosis 131–6 Aegopodium podagraria 10, 128 AFA-I 298 Agaricus bisporus lectin (ABL) 158 colonic epithelial cells 225, 227–8, 230, 231 alkaline phosphatase 111 allergic encephalomyelitis 163 Alliaceae lectins 75–7 anti-insect properties 18 biomedical/biophysical properties, and biological activities 60 amino acid analyses 62 applications 63–4 purification and molecular structure 61–2 biosynthesis and molecular cloning 69–73 carbohydrate-binding specificity 59–60, 62–3 Allium 60 Allium ascalonicum agglutinin (AAA) 61,62, 63 biosynthesis and cloning 67, 72–3 Allium cepa agglutinin (ACA) 61, 62, 63 biosynthesis and cloning 67, 72–3

Allium porrum agglutinin (APA) 61, 62, 63 biosynthesis and cloning 67, 72–3 Allium sativum agglutinin (ASA) 61–2, 63 biosynthesis and cloning 67, 69–71 Allium ursinum agglutinin (AUA) 62, 63 biosynthesis and cloning 67, 69, 71–2 Amaranthus caudatus agglutinin (ACA) 225, 226–7 Amaryllidaceae lectins 75–7 anti-insect properties 18 biochemical/biophysical properties, and biological activities 60 amino acid analyses 62 applications 63–4 purification and molecular structure 61–2 biosynthesis and molecular cloning 64–9 carbohydrate-binding specificity 59–60, 62–3 amaryllis (Hippeastrum) 61, 62 amino acid composition bean -amylase inhibitor 25, 26–8 mannose-binding monocot lectins 62, 67, 76–7 ASA 70–1 AUA 71, 72 CHA 75 EHA 74 GNA 65–8 LOA 73–4 plant storage proteins 5, 8 amyloid P component 169–70 anti-insect properties see insecticidal lectins anti-mitogenic lectins 107 antibodies, to prevent E. coli colonization 272–4 antinutrients 13 Arachis hypogaea see peanut agglutinin arcelin pancreatic growth 150 potential 40 structure 25, 27 270

INDEX

Zabrotes subfasciatus 36 asialoglycoprotein receptor 165–6 association 295 autoimmune thyroiditis 163, 170 avidity 132, 136 B-cell mitogens 155–6 bacteria adherence penetration and translocation 95–6 bioadhesion 130, 135 gut infection 313–16 future perspectives 320–1 probiosis 316–20 Bacteroides nodosus 297 bark 4, 7, 10, 11–12 Bauhinia purpurea 42 Bauhinia purpurea agglutinin (BPA) 42, 216 bioadhesive lectins 117–18, 120–37 biotinylated lectins 186 blowfly (Lucilia cuprina) 45, 48 bone marrow 161–2 Bordetella pertussis 297 breast cancer 211, 212–14 brush-border 81 binding of lectins to 106–7, 114 Caco-2 cells 83, 85–7 morphological alterations 91–4 drug delivery systems 125–6 E.coli 252, 257, 261–3, 266–7 bulbs 4, 7–9, 10 C-reactive protein 169 C-type lectins 165–70 Caco-2 cells 82 differentiated, lectin-binding and lectin-induced effects 84–94 future studies 94–7 growth, structural and functional characteristics 82–4 Callosobruchus maculatus see cowpea weevil Camel’s foot tree 42 Campylobacter jejuni 96 Canavalia brasiliensis 42 Canavalia ensiformis 45 cancer 211–12 breast 212–14 colon 214–16 endogenous lectins 217–19 future 219–20 gastric 217

histochemistry 212 lung 211, 217 prostate 217 tumours 217 of genitourinary tract 216–17 of hepatopancreatic system 216 carbohydrate-binding activity, plant lectins 13–15 CFA/I fimbriae 268–9, 271, 277 CFA/II fimbriae 268–9 CFA/III fimbriae 270, 271 CFA/IV fimbriae 269 chaperon proteins 248, 250–1 children, diarrhoea 235, 236 chitin-binding lectins 41 anti-insect properties 16–17 fungitoxic properties 16 cholecystokinin (CKK) 141–2, 146, 147–8 Clivia 60 Clivia miniata 9 Clivia miniata agglutinin (CMA) cloning 67, 68, 69 molecular structure 61, 62 cloned glycosyltransferase genes 202–3 CNF2 293 Coleoptera 39–42 coliform overgrowth 313–16 colitis, ulcerative 226, 229 collectins 165, 166–8 colon cancer 211, 214–16, 219 colonic epithelial cells 225–31 colonization factor antigens (CFA’s) 268–71 Colorado beetle 37 colostra 272–3 common bean (Phaseolus vulgaris) -amylase inhibitor 24–31 Acanthoscelides obtectus 46 arcelin 36 expression of lectin 50–2 PHA toxicity 17, 35, 40 complex specificity 3, 14 Concanavalin A (Con A) 45, 46–8 actin cytoskeletal lesions 87, 89, 92 cancer 212, 214 cellular metabolism, changes in 91 drug delivery systems 127 hydrophobic sites 252 immunology 155, 156, 158 lectin-binding 84 epithelial cells 111

271

272

INDEX

conglutinins 167 continuous cropping 37 corms 4, 12 corn earworm 37 cotton bollworm (Heliothis virescens) 37, 46, 49, 50–1 cotyledons 4, 6, 11 cowpea trypsin inhibitor (CpTI) 142, 149–50 cowpea weevil (Callosobruchus maculatus) 35, 36, 40–2, 46–7 -amylase inhibitor, bean 24 Cratylia floribunda 42 Crohn’s disease 226, 229 crop protection 23–4, 35, 36–9 crypt cells binding to 111 proliferation rate (CCPR) 109–11 effect on glycosylation 111–12 PNA 228, 229 crypts 103 CS1 fimbriae 268–9 CS2 fimbriae 268–9 CS3 fimbriae 268–9 CS4 fimbriae 269, 270, 271 CS5 fimbriae 269, 270 CS6 fimbriae 269–70 CS7 fimbriae 270, 271 CS17 fimbriae 270, 271 CS31 Afimbriae 264–6 Cymbidium hybrid agglutinin (CHA) biosynthesis and cloning 67, 74–5 molecular structure 61, 62 cytochemistry 183–4 future directions 202–4 stem cells and epithelial cell lineages 186–8 techniques 184–6 cytokines 159–60, 173 cytomegalovirus in vitro 63, 76 cytoskeletal lesions 87–9, 92, 96 cytotoxic lectins 15 daffodil 9, 42, 61 Datura stamonium agglutinin (DSA) immunology 158–9, 160 pancreatic growth 143, 145, 149 defence, plant carbohydrate-binding activity of lectins 13–15 crop protection 36–9 experimental evidence 15–18 insecticidal properties 3, 16–17, 18

-amylase inhibitor 24–31 mechanism of toxicity 45–54 phytophagous insects 39–45 problems and solutions 12–13 role of lectins 35–6 desglycinamide-arginine-vasopressin 119 Diabrotica undecimpunctata (Southern corn rootworm) 41, 46 diamine oxidase 110–11 diaminobenzidine (DAB) reaction 186 diarrhoea 235–6 digoxigenin 186 Dioclea paraguariensis 42 Dioclea rostrata 42 Diptera 45 Dolichos biflorus agglutinin (DBA) 187–8 cancer 212, 215, 217 drugs delivery systems 117–18, 136–7 bioadhesive lectins 120–35 mucoadhesive polymers 118–20 lectins as 163–4 E-selectin 168 edema disease 245, 264 elderberry (Sambucus nigra) 10, 12, 14, 41 electrolectin 163 Electrophorus 163 Empoasca fabae 45 encephalomyelitis 163, 170 endocytic index 131 endocytosis 131–2 endogenous lectins 121 cancer 217–19 drug delivery systems 129–30 human, immunology 164–73 endogenous ligands 113 endometrial carcinoma 217 endosperm 4, 6 Enterobacteriaceae 253 enterocytes, absorptive 196–8 maturation 189–90 enteroendocrine cells 103 enterotoxigenic fimbrial E. coli lectins and receptors 236– 7, 277–81 detection of 237–42 gene clusters encoding fimbriae biosynthesis 246–51 important 254–72 prevention of colonization 272–7

INDEX

purification of 242–6 structure-function relationships 251–4 surface lectins 237 enterotoxins 295 Epipactis helleborine agglutinin (EHA) biosynthesis and cloning 67, 74–5 molecular structure 61, 62 epithelial cells 46, 48, 81, 103 bacteria 95 cytochemistry 183, 187–8 dietary galactose-binding lectins 225–31 drug delivery systems 135, 136 E. coli 237, 252, 267 glycosylation of 111–13 maturation 189–92 metabolic changes induced by lectins in 107–11 receptors for enteric micro-organisms 203–4 temporal intestinal glycosylation changes 192 Escherich, Theodore 235 Escherichia coli bioadhesion 130 coliform overgrowth 313–16, 318–20 fimbrial lectins and receptors 235–7, 277–81 detection of 237–42 F17 fimbriae 293–306 gene clusters encoding fimbriae biosynthesis 246–51 important 254–72 prevention of colonization 272–7 purification of 242–6 structure-function relationships 251–4 surface lectins 237 Galanthus nivalis agglutinin 64 and glycosylation 113 eukaryotic cells, attachment of E. coli to 237 Eupergit-C-glycoprotein system 240–1, 259, 261 European cornborer 42 F17-A gene 299–300, 301, 304 F17-C gene 301, 302 F17-D gene 301, 302 F17 fimbriae 258–63, 264, 305–6 adhesion-blockers and vaccines 304–5 analysis 300–1 characterization 299 of variants 303 cloning 299–300 function and nucleotide sequence 301–2 hybridization experiments 303–4 identification 293–4, 299–300

273

incubation temperature effects 302–3 purification 243–5 receptor analogs 274–5, 276 F17-G gene 301–2, 304–5 F41 fimbriae 254, 258, 296 antibodies 272, 273 purification 245 F111 fimbriae 263–4, 303–4 F-actin 87–8 F-antigens 294 familial adenomatous polyposis 226 fimbrial E. coli lectins and receptors 236–7, 277–81 detection of 237–42 F17 fimbriae 293–306 gene clusters encoding fimbriae biosynthesis 246–51 important 254–72 prevention of colonization 272–7 purification of 242–6 structure-function relationships 251–4 surface lectins 237 fimbriosomes 249 flagellae, E. coli 243, 245 fluid-phase endocytosis 131 follicle associated epithelium 192 fucosylation 194, 195 fungitoxic lectins 13, 16 G-actin 87–9 G fimbriae 300 galactoside-binding lectins 218–19, 230–1 Galanthus 60 Galanthus nivalis (snowdrop) 9, 17, 41, 42 Galanthus nivalis agglutinin (GNA) 41, 44–5, 48–9 applications 63–4 biosynthesis and cloning 65–8 carbohydrate-binding specificity 62–3 cellular metabolism, changes in 91 coliform overgrowth 315, 316, 319–20 crypt cells, binding to 111 drug delivery systems 127 endogenous ligands, displacement of 113 epithelial cells, binding to 111–12 expression 52–4 isolation and characterization 60 lectin-binding 84 molecular structure 61, 62, 64 pancreatic growth 143, 150–1 proteolytic degradation, resistance to 105 garlic (Allium sativum) 42, 61–2, 69

274

INDEX

gastric cancer 217 gene clusters encoding fimbriae biosynthesis 246–51 genitourinary tract, tumours 216–17 Glycine max 84 glycocalyx 135, 136 glycoproteins in vitro attachment of E. coli fimbrial lectins 240–2 receptor analogs 274–6 secreted 112–13 glycosylation 103–4, 107, 111 13, 114 cancer 219–20 cytochemistry 183, 189 ‘engineering’ 203 inter- and intra-species variation 195 M cell markers 192 mechanisms 195–201 mosaicism 190–2 temporal changes 192–5 goblet cells 103, 198–201 E. coli receptors 257 maturation 190 Golgi apparatus cancer 214, 215 glycosylation 196–202 Griffonia simplicifolia agglutinin 217 ground elder (Aegopodium podagraria) 9–11 gut, lectins as growth factors 103–5, 114–15, 144 brush-border membrane, binding to 106–7 epithelial cells glycosylation, changes 111–13 metabolic changes 107–11 future perspectives and practical implications 114 nutritional penalty 113–14 proteolytic degradation, resistance to 105–6 H-antigens 294 haemagglutination 237–40 Helicobacter pylori 321 Heliothis virescens 37, 46, 49, 50–1 Heliothis zea 37 Helix pomatia agglutinin (HPA) 158, 162, 198 cancer 212, 213–14, 217 heparin 135 hepatopancreatic system, tumours 216 heterogeneity, plant lectins 2–4 storage proteins 5 hevein 16 Hippeastrum 60 Hippeastrum hybrid agglutinin (HHA)

applications 63, 64 carbohydrate-binding specificity 63 cloning 67, 68, 69 molecular structure 61, 62, 64 pancreatic growth 143 histo-blood group antigens 193–4 histochemistry, cancer 212 HIV 63, 64, 76 jacalin 164 mannan-binding protein 167 Hodgkin’s disease 163, 166 Homoptera 42–5 horseradish peroxidase (HRP) 184–6 HPMA copolymers 129–30 HT-29 cells 82 human colonization factor antigens (CFA’s) 268–71 human lectins 164–73 human placental lectin 170–2 hyperplastic polyps 226 immunology 155 drugs 163–4 endogenous human lectins 164–73 lymphoid cell identification and separation 161–3 polyclonal modulators 155–61 inflammatory bowel disease 226 influenza virus 167 inhibitory domains 158 insecticidal lectins 13, 16–17, 18 -amylase inhibitor, common bean 24 pancreatic growth 143–4, 149–51 insecticides 37 integrated pest management (IPM) programme 37–9 integrins 135 interleukin-1 (IL-1) 159–60, 173 interleukin-2 (IL-2) 159, 173 interleukin-6 (IL-6) 63 invasin 135 Iris lectin 144 jacalin (JAC) 164,200 colonic epithelial cells 225, 228, 231 K88 fimbriae 258, 266–8, 296 antibodies 272, 273–4 and CS31A fimbriae 265 gene clusters 248, 249–50 K99 fimbriae 254–6, 258, 295, 296 antibodies 272–3, 274

INDEX

gene clusters 248, 249–50 purification 245 receptor analogs 274, 275 K5145 fimbriae 259 K-antigens 294 Klebsiella 245 Kunitz trypsin inhibitor (KTI) 146 L-selectin 168 Lacanobia oleraceae 54 Lactobacillus 314 leaves 4 lectin-like protein (LLP) 24, 52 lectino-phagocytosis 254 leek (Allium porrum) 61, 62, 69 Lens culinaris agglutinin (LCA) 217 lentil lectin (LLA) 46–8, 163, 198 Lepidoptera 42 Leucojum 60 lima bean lectin 157, 162 limulin 169 Listera ovata agglutinin (LOA) 60 applications 63 biosynthesis and cloning 67, 73–4 carbohydrate-binding specificity 63 molecular structure 61, 62 Lotus tetragonolobus agglutinin (LTA) 214 loxiglumide 152 Lucilia cuprina (blowfly) 45, 48 lung cancer 211, 217 Lycopersicon esculentum see tomato lectin lymphocytes 63, 161– 2 mitogenic lectins 155–7 lymphoid cell identification and separation 161–3 Maackia 42 Maackia amurensis 14 Maackia amurensis agglutinin (MAA) 112–13 colonic epithelial cells 226 pancreatic growth 144, 145, 146 macrophage mannose receptor 166 Manduca sexta 42 mannan-binding protein (MBP) 166–7, 173 mannose-binding monocot lectins 59–64, 75–7 biosynthesis and molecular cloning Alliaceae lectins 69–73 Amaryllidaceae lectins 64–9 Orchidaceae lectins 73–5 mealworm 52

275

membrane-bound C-type lectins 165–6 meningitis 253 metabolism, cellular 90–1 metastases 211, 213–14, 215, 217 microfold (M) cells 192 milk 276 mitogenic lectins 155–7 monocot lectins, mannose-binding 59–64, 75–7 biosynthesis and molecular cloning Alliaceae lectins 69–73 Amaryllidaceae lectins 64–9 Orchidaceae lectins 73–5 monoculture 37 Morazella bovis 297 mosaicism, glycosylation 190–2 mucin 112–13 mucoadhesive polymers 118–20 multiple sclerosis 163, 170 myasthenia gravis 163 myeloma cells 161 Myzus persicae see peach/potato aphid N-type lectins 165, 172–3 Narcissus 60 Narcissus pseudonarcissus 10, 67, 68, 69 Narcissus pseudonarcissus agglutinin (NPA) 61, 62,64 natural killer (NK) cells 173 Neisseria 297 neoglycoproteins 218 Nephotettix cinciteps 45 nettle (Urtica dionica) 9, 16 NFA-1 adhesin 297–8 NFA-2 adhesin 297 NFA-4 adhesin 298 Nilaparvata lugens see rice brown planthopper non-fimbrial adhesins (NFAs) 297–8 non-mitogenic lectins brush-border membrane 107 immunology 156 receptors 157–9 O-antigens 294 oligosaccharides as differentiation markers 189 goblet cells 200 onion (Allium cepa) 61, 62, 69 opacity-associated proteins (OPAs) 297 oral bioadhesive drug delivery systems 118–19 Orchidaceae lectins 2, 75–7

276

INDEX

bimedical/biophysical properties, and biological activities 60–1 amino acid analyses 62 applications 63–4 purification and molecular structure 61–2 carbohydrate-binding specificity 62–3 molecular cloning 73–5 ornithine decarboxylase (ODC) 108 pancreatic growth 141, 142, 146–7, 148 Ostrinia nubilalis 42 P-selectin 168 pancreatic growth 141–6, 151–2 nutritional implications for transgenic plants 148–51 stimulation mechanism 146–8 Pap fimbriae 246, 296–7, 301–2 parenchyma 4, 5, 6, 11–12 PCFO9 fimbriae 271 PCFO15:H4 fimbriae 270–1 PCFO166 fimbriae 271 pea (Pisum sativum) 41, 46 expression of lectin 49–50 pea aphid 45 peach/potato aphid (Myzus persicae) 45, 53–4 peanut agglutinin (PNA) 161, 200 cancer 212–13, 214–15, 216–17 colonic epithelial cells 225–6, 227, 228–31 pentraxins 165, 169–70 peptide drugs 117–18, 136–7 bioadhesve lectins 120–35 mucoadhesive polymers 118–20 periodic acid-Schiff (PAS) reaction 212 periplasmic proteins 248 peritonitis 235 peritophic membrane 46, 48 pesticides 23, 37–8 phagocytosis 254 phase-variation 250, 295 phaseolin 40 Phaseolus vulgaris see bean Phaseolus vulgaris agglutinin (PHA) 36, 40 actin cytoskeletal lesions 87, 92 brush-border membrane, morphological alterations in 92–4 cancer 212 coliform overgrowth 313–16, 318–20 drug delivery systems 121, 125–7, 128, 132, 133–5 immunology drugs 163

polyclonal modulators 156, 158, 160 lectin-binding 84, 85–7 epithelial cells 111–12 metabolic changes induced by 90–1, 107–11 pancreatic growth 141, 143, 144–6, 147, 148 PHA-E 25, 27 PHA-L 25, 26, 27 stimulation of gut growth 144 nutritional penalty 113 toleration by Acanthoscelides obtectus 46, 47 toxicity 17, 35 phosphatase, alkaline 111 phytohaemagglutinins carbohydrate-binding activity 13–14 heterogeneity 3 as recognition molecules 1 phytophagous insects 39–45 pili 294, 296–7 pilins 294 pinocytosis 131 Pisum sativum see pea Pisum sativum agglutinin (PSA) 84, 85–7 brush-border membrane, morphological changes in 92– 3 cellular metabolism, changes in 90–1 cytochemistry 198 placental lectin, human 170–2 plasma cells 161 Pneumocystis carinii 166 pneumonia 166 polyamines pancreatic growth 141, 142, 146–7, 148, 151 PHA, effects of 107–8 polycarbophil 119 polyclonal modulators 155–61 polynucleotide drugs 117 polyposis, familial adenomatous 226 polyps 226, 229 polysaccharide drugs 117 ‘pore’ proteins 246 postnatal glycosylation ‘shifts’ 194–5 potato leafhopper 45 probiosis 316–20 Proteus 314 prostate cancer 217 protein drugs 117–18, 136–7 bioadhesive lectins 120–35 mucoadhesive polymers 118–20 protein storage vacuoles 5, 6

INDEX

proteolytic degradation 105–6 Pseudomonas aeruginosa 318 Psophocarpus tetragonolobus see winged bean putrescine 147 pyelonephritis 296 ramsons (Allium ursinum) 62, 69 receptor analogues 274–7 receptor-mediated endo-/trans-cytosis 131–6 receptor modification therapy 277 recognition molecules 1 retroviruses 63 reverse lectins see endogenous lectins Rhinovirus 130 rhizomes 4, 7, 9–11 rice brown planthopper (Nilaparvata lugens) 37–8, 42, 43, 44–5, 48–9 rice green leafhopper 45 Ricinus communis agglutinin (RCA) 42 Robinia 314 Robinia pseudoacacia agglutinin (RPA) 143, 144, 149 root stocks 4, 7, 12 S-type lectins 165, 170–2 saccharides 317–19 Salmonella 320–1 Salmonella choleraesuis 95–6 Salmonella enteritides 320 Salmonella typhimurium 95–6, 320 salmonellosis 320 Sambucus nigra see elderberry Sambucus nigra agglutinin (SNA) 112–13 pancreatic growth 143, 144, 145 seed lectins 5–6 heterogeneity 3 location 4 selectins 165, 168–9 septicemia 253 Serratia 245 serum amyloid P component (SAP) 169–70 shallot (Allium ascalonicum) 61, 62, 69 Shigella flexneri 96 sialic acid 112–13 sialylation, intestinal membrane 193 small intestine, lectins as growth factors 103–5, 114–15 brush-border membrane, binding to 106–7 coliform overgrowth 313–16 epithelial cells glycosylation changes 111–13

277

metabolic changes 107–11 future perspectives and practical implications 114 nutritional penalty 113–14 proteolytic degradation, resistance to 105–6 snowdrop (Galanthus nivalis) 9, 17, 41, 42 Solanum tuberosum agglutinin (STA) 217 Sophora japonica 12 Southern corn rootworm 41, 46 soya bean agglutinin (SBA) 42, 84, 85–6 actin cytoskeletal lesions 87–9, 91–2 bacteria adherence, penetration and translocation 96 brush-border membrane, morphological alterations in 91–3 cancer 212, 215, 216, 217 cellular metabolism, changes in 90–1 coliform overgrowth 314 glycoproteins, reaction with 113 immunology 161–2 pancreatic growth 141–2, 143, 146, 147–8 stimulation of gut growth, nutritional penalty 113 soya trypsin inhibitor (STI) 141, 142, 146–7 spermidine 108, 141 stem cells 187–8 stems 4 stimulatory domains 158 stinging nettle 9, 16 storage proteins, plants 4–8 bark 11–12 bulbs 8–9 heterogeneity 3 rhizomes 9–11 Streptococcus pneumoniae 169 Streptomyces tendae 24–5 sucrase-isomaltase 110–11 superantigens 160–1 surfactant proteins 167–8 T-cell receptor (TCR) 156–7 T-lymphocytes 63, 155–9 Tamm-Horsfall glycoprotein 173 tendamistat 24–5, 26–8, 29–31 Tenebrio molitor 52 Thomsen-Friedenreich (TF) antigen 225, 226, 227 thyroiditis, autoimmune 163 tobacco budworm see Heliothis virescens tomato hornworm 42 tomato lectin (TL) drug delivery 121–4, 127, 132, 133, 135 immunology 158, 163

278

INDEX

transcytosis 131–6 transepithelial transport of (macro)molecules 94–5 Triticum vulgare 42 trypsin inhibitors 141, 142, 144, 146, 148, 151–2 tubers 4, 7, 12 tulip 9, 10 tumour necrosis factor 173 tumour-specific lectins 217–18 tumours 217 colonic 226, 227–8, 229–30 endogenous lectins 217–19 of genitourinary tract 216–17 of hepatopancreatic system 216 type 1 fimbriae 294, 296 detection 237–9, 242 gene clusters 248–9, 250 structure-function relationships 251–4 ulcerative colitis 226, 229 Ulex europeus agglutinin (UEA) 192, 194 cancer 212, 214, 215, 217 Urtica dioica 9,16 Urtica dioica agglutinin (UDA) 16 immunology 160–1 pancreatic growth 41, 143, 145, 149 vaccines, F17 304–5 vegetative storage tissues 4, 6–12 Vicia faba agglutinin (VFA) 84, 85–6, 217 brush-border membrane, morphological alterations in 92–3 cellular metabolism, changes in 90–1 Vicia villosa agglutinin (VVA) 216 villi 103 viruses bioadhesion 130–1 mannan-binding protein 167 wheat germ (Triticum vulgarae) 42 wheat germ agglutinin (WGA) 16, 18, 46–8 actin cytoskeletal lesions 87, 89, 92 cancer 214, 216–17 coliform overgrowth 314 drug delivery systems 127, 132 epithelial binding 111 immunology lymphoid cell identification and separation 162 polyclonal modulators 156, 157, 158, 159, 160 pancreatic growth 142, 143, 145, 149

potency 41, 42, 44, 45 stimulation of gut growth, nutritional penalty 113 winged bean (Psophocarpus tetragonolobus) 35–6, 40 winged bean agglutinin (WBA) 192 working mechanism of lectins 96–7 Xenophum leavis 65 Zabrotes subfasciatus 40