Lectin-Microorganism Interactions

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LectinMicroorganism Interactions exmcil by R. doDoyo@

University of Louisville Louisville,. Kentucky

Allegheny General Hospital and Medical Collegeof Penns yhtania Pittsburgh, Pennsylvania

Marcel Dekker, Inc. New York.Basel*Hong Kong

Library of Congress Cataloging-in-PublicationData Lectin-microorganism interactiondedited by R. J. Doyle, Malcolm Slifkin. p. cm. Includes bibliographical references and index. ISBN 0-8247-91 13-4 (alk. paper) 1. Lectins. 2. Microbialpolysaccharides. I. Doyle,Ronald J. 11. Slifkin, Malcolm. [DNLM: 1. Lectins--physiology. 2. Latins--diagnosticuse. 3. Microbiology.] QP552.IA2IA15 1994 574.19’245--d~20 CIP The publisher offers discounts on this book when ordered in bulk quantities. For moreinformation,writetoSpecialSales/ProfessionalMarketingatthe address below. This book is printed on acid-free paper.

Copyright @ 1994 by MARCEL DEKKER, INC.

All Rights Reserved.

Neither this book nor any part may be reproducedor transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.. MARCEL DEKKER, INC. 270 Madison Avenue, New York, New York

10016

Current printing (last digit): l 0 9 8 7 6 5 4 3 2 1 PRINTED IN THE UNITED STATESOF AMERICA

Preface

Lectins have become irreplaceable tools for modern microbiologists, molecular biologists, and biochemists. These carbohydrate-binding proteins have been employed as diagnostic reagentsfor viruses, bacteria, fungi,and protozoa. They have also been used in epidemiological investigations in infectious diseases. The use of lectins to isolate microbial toxins, microbial mutants, cell surface glycoconjugates, and viral coat glycoproteins is now well established. This is the first book devoted solely to lectin-microorganism interactions. The text contains an introduction to lectins and their interactions with microorganisms aswell as chapters on lectins as probesfor viral, bacterial, fungaland protozoal surfaces. Two chapters were contributed by Russian scientists who have reviewed much the lectin-microorganism of literature from Eastern Europe. Another chapter is concerned about advances in the use of lectinsin studying blood groups. Although blooddocells not qualify as microorganisms, the traditional associationbetween blood bank laboratories and diagnostic microbiology laboratories is strong enough to justify a chapter on lectin-red cell interactions. A proposal to provide a uniform method to abbreviate lectins is also presented. The book’s abundant references encompass most of the literature on lectin interactions with microorganisms. The text provides a thorough overview of lectin-microorganism interactions including applications and fundamental aspects of the interactions. A list of the most common applications of lectins in microbiologyand serology would include the following: Diagnostic microbiology Epidemiological characterization of microorganisms iii

iv

Preface

Research on protozoa Applications to fungi and yeasts Routine grouping of erythrocytes Automated blood-grouping apparatuses Assembly of cell surfaceBacillus in subtilis Study of bacteriophage receptors Use in microbialultrastructure Purification of teichoic acids Purification of viral components Characterization of glycoproteins in tissue culturecells Purification of microbial enzymesimportant in biotechnology Mechanism ofbacterial adhesion Solution structure of teichoic acids Mechanism ofroot nodulation Structural determination of microbial polysaccharides Characterization of lipopolysaccharidestructure of Neisseria gonorrhoeae This book is intended for all scientists who employ lectins as tools. Although the book does not provide detailed methods or physicochemical descriptionsoflectins,microbiologists,biochemists,bio-techengineers, physicians, epidemiologists, serologists,and other health care workerswill find this volume an invaluable resource.The book can also serve as a text for a one-semester course in lectins and their applicationsto microbiology. The versatility of lectins as reagents and tools in microbiology and serology is emphasizedthroughout the book. The availabilityof new lectins with new specificities will make it possible to identify even more applications for lectins in microbiologyand serology. R . 3. Doyle Malcolm Sliflin

Contents

Preface Contributors 1. Introduction to Lectins and Their Interactions with R . J. Doyle 2.

Use of Lectins General in and Diagnostic Virology Sigvard Olofsson, Stig Jeansson, and John-Erik Stig Hansen

3. Epidemiological Applicationsof Lectins to Agents of Transmitted Sexually William 0.Schalla and StephenA. Morse 4. Application ofClinical Lectins Bacteriology in Malcolm Sliflin 5.

iii vii

l 67

111 143

Lectin Specificities Relevant to the Medically Important Yeast Candida albicans Hans C. Korting and Markus W. Ollert

Interaction6. Lectin-Leishmania R. L.Jacobson Interactions 7. Trypanosome-Lectin Justus Schottelius and Martins S. 0.Aisien

191 225 V

vi

Contents

8. Lectin Sorbents in Microbiology V. M . Lakhtin

249

9. MicrobialLectins for theInvestigation of Glycoconjugates K. L. Shakhanina, N. L. Kalinin, and V. M . Lakhtin

299

10. Lectin-Blood Interactions Group C. Levene, Nechama Gilboa-Garber, and Nachman C. Garber

327

Index

393

Contributors

Martins S. 0 . Aisien Department of Zoology, University of Benin, Benin City, Nigeria

R. J. Doyle Professor of Microbiology,DepartmentofMicrobiology, School of Medicine; Associate Dean for Research, School of Dentistry, University of Louisville, Louisville, Kentucky Nachman C. Garber Professor of Microbiology, Department of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel Nechama Gilboa-Garber Professor of Biochemistry, Department of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel John-Erik Stig Hansen Head of Research, Laboratory of Infectious Diseases, Hvidovre Hospital, Hvidovre, Denmark

R. L. Jacobson Medical Parasitologist, Department of Parasitology, Hebrew University-Hadassah Medical School, Jerusalem, Israel Stig Jeansson Associate Professor, Department of Clinical Virology, University of Gothenburg, Gothenburg, Sweden N. L. Kalinin Department of Biological Sciences (Immunology),'Gamaleya Institute of Epidemiologyand Microbiology, Russian Academy of Medical Sciences, MOSCOW, Russia vii

viii

Contributors

Hans C. Korting Department of Dermatology, University of Munich, Munich, Germany

V. M. Lakhtin Head, Laboratory of Lectinology, Institute for Applied ScienceofMoscowUniversity, andInstituteofFoodSubstances,Russian Academy of Medical Sciences, Moscow, Russia

C. Levene Director, Reference Laboratory

for Immunohematology and Blood Groups, Ministryof Health, Jerusalem, Israel

Stephen A. Morse Director, Division of Sexually Transmitted Diseases Laboratory Research, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, Atlanta, Georgia Markus W. Ollert Department of Biochemistry and Molecular Biology, University of Hamburg, Hamburg, Germany Sigvard Olofsson Associate Professor, Department of Clinical Virology, University of Gothenburg, Gothenburg, Sweden William 0. Schalla Chief, Model Performance Evaluation Program, Division of Laboratory Systems, Centers for Disease Control and Prevention, Public HealthService, U.S. Department of HealthandHumanServices,Atlanta, Georgia Justus Schottelius Privatdozent, Department of Protozoology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany

K.L.Shakhanina Head, Department of Biological Sciences (Immunology), Gamaleya Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences, Moscow, Russia Malcolm Slifkin Head, Section of Microbiology, Department of Laboratory Medicine, Allegheny General Hospital; Professor of Microbiology and Immunology, and Professor of Pathology and Laboratory Medicine, Medical Collegeof Pennsylvania, Allegheny Campus, Pittsburgh, Pennsylvania

LectinMicroorganism Interactions


1 Introduction to Lectins and Their Interactions with Microorganisms R. J.DOYLE University of Louisville, Louisville, Kentucky

1. INTRODUCTION AND THE DEFINITION OFA LECTIN

Lectin research is now more than 100 years old. Most lectinologists acknowledge the valuable contribution of Stillmark [l]as the beginning ofthe centennial on lectin identification, purification, characterization, biological properties, and functions. Stillmark, for his Ph.D. thesis at the University of Dorpat (now Tartu, in Estonia), recorded the hemagglutinating properties of extracts of Ricinus communis seeds and of members of the family Euphorbaceae. He observedthat red cells of some species were refractory to hemagglutination, giving rise to the concept of lectin specificity. Since the 1960s,lectin research has seemedto gain an exponential strength. This has necessitated a critical examination of the word “lectin,” followed by new attempts to define a lectin. The original definition of a lectin was proposed by Boyd and Shapleigh lectin to account for the blood group specificity of plant extracts. The word itself is taken from the Latin legere, meaning to select or choose. As a tangential comment,Boyd and Shapleigh [2]made the prophetic statement “They [the lectins] promise to have practical and theoretical importance.” Some researchers simply called the extracts possessing blood group specificity as agglutinins, hemagglutinins,or phytohemagglutinins. As pointed out by Boyd and Shapleigh [2], some immunologists didnot seem very happy by applying the word “agglutinin” to a material from a plant tissue. The word lectin is now in much more common use, no doubt because red cellagglutinating substances are found in almost all living tissues examined. Becauseof the genesisoflectinresearch,Goldstein et al. [3] were 1

Doyle

2

prompted to redefine a lectin. They proposed a lectin as “a sugar-binding protein or glycoprotein of non-immune origin which agglutinates cells and or precipitates glycoconjugates.” This definition assumes that all lectinsare multivalent. It assumes that the specificity ofthe lectin is largely dependent on monosaccharide terminii. The definition takes into account the fact that lectins may be soluble or tissue-bound. The definition ascribes lectinlike properties to certain enzymes, such as amylases or phosphorylases, which may precipitate polysaccharides. Kocourek and Horejsi [4] contested the definition of lectinof Goldstein et al. [3]. They proposed that “lectins are sugar-binding proteins or glycoproteins of non-immune origin which are devoid of enzymatic activity towards sugarsto which they bind anddo not require free glycosidic hydroxyl groups on these sugars for their binding.” This definition, therefore, dispenses with enzymes as lectins, and it also dispenses with the requirement of multivalency. Kocourek and Horejsi[4] agree that lectins are nonimmune proteins. Dixon [5], on behalf of the Nomenclature Committee ofthe International Union of Biochemistry, accepted the definition of Goldstein et al. [3]for a lectin. Dixon arguedthat the definition proposed by Kocourek and Horejsi [4] was “ . . . too broad to be useful, since it includes substances such as sugar-transport proteins, chemotaxis receptors, certain bacterial toxins, hormones and interferons.” Dixon further argued that some “ . certain proteins hitherto known as lectinspossessglycosidaseactivity.”Dixon and the committee chose to remove the word “glycoprotein” from the Goldstein et al. definition because glycoproteins are a class of proteins. It seems clear that a single, simple definition of a lectin may be impossible. Both the Goldstein et al. and the Kocourek and Horejsi definitions have merit and the criticisms of Dixon are reasonable. The now-known multiple functions of lectins may compromise any of the foregoing definitions. Barondes [6] has now made a convincing attempt to establish a new definition for lectins.Barondeshas pointed out that many of the well-characterized lectins have binding sites for noncarbohydrate ligands. For example, discoidin I, a multivalent protein from Dictyostelium discoideum, bindsN-acetylgalactosamine(GalNAc) and galactose (Gal) and contains an kg-Gly-Asp (RGD) sequence. The RGD sequence isimportant to cell-substratum adhesive events in animal cells, but it is also required for the developmental cycle of D. discoideum. Small peptides containing the RGD sequence interfere with developmental processes ofthe slime mold. Therefore, it is clear that discoidin I is bifunctional, of which one function or property is dependenton carbohydrate and the other on a noncarbohydrate-binding amino acid sequence. Similarly, the asialoglycoproteinreceptor(Gal,-GalNAc-specificlectin) also containsan amino acid sequencethat tethers itto cellular membranes. Barondes [6], in an effort to take into account the known properties of

..

Lectin-Microorganism Complexes

3

carbohydrate-binding proteins, has defined a lectin as “a carbohydratebinding protein other than an enzyme or an antibody.” This is the most satisfying,least-restrictivedefinitionofalectinyetproposed.Butthis definition is not perfect, as it must include periplasmic (nonmembraneanchored) carbohydratetransport proteins of bacteria, such asthe arabinose- and galactose-binding proteins ofEscherichia coli. It also maybe that certain proteins, such as limulin, a sialic acid-binding protein from the horseshoe crab Limuluspolyphemus is a type of “immune” protein, exhibiting antibodylike properties. Nevertheless, the definition of a lectin given by Barondes will be adhered to in this book. A lucid and detailed history of [7]. the development of researchon lectins has been given by Kocourek This book is concerned with the interactions between lectins and microorganisms, including bacteria, fungi and yeasts, protozoa, metazoa, and viruses. Only a brief review will be presented on the properties of lectins. Lectins of microorganisms will not be discussed in terms of their functional roles as adhesins. A comprehensive review of the chemical and biological properties of lectins and their functions and applications was published in 1986 [8]. A readable account of lectinology has also been published by Sharon and Lis [9]. No single comprehensive review on lectin-microorganism interactions is available, although reviews by Pistole [lo], Doyle and Keller [l l], Slifkin and Doyle [l21 and Doyle and Slifkin [l31 outline selected areas of the lectin-microbe literature. Table 1 provides a brief description of selected paperson lectins and lectin-microorganism complexes. The table is designedto provide an overview of how lectins have been used to study microbial surfacesand glycoconjugates. Some of the experiments cited in Table1 will be discussed morethoroughly in thisand other chapters of the book. II. SOURCES AND FUNCTION OF LECTINS

Lectins seem ubiquitous in nature. They occur in the simplest life forms (viruses) to the most complex (mammalian tissues). In plants, more than loo0 species have been reported to possess lectins. In fact, most plants examined yield lectins or lectinlike activities. There are no rapid-screening methods for all lectins. Because most lectins tend to be multivalent, they generally havethe ability to aggregate cells, suchas erythrocytes. In examining biological specimensor their extractsfor lectins, it must be considered that frequently lectins exhibit a narrow specificity. One kind of red cell may be agglutinatedby a lectin,or only the red cells ofone or afew species may be susceptibleto aggregation. This is because different red cells have unique glycoconjugate compositions and unique distributions of lectin receptors.Nevertheless,hemagglutinationis the mostreliable and direct

Table 1 Selected Major Experiments in Lectin Researchand Lectin-Microorganism Interactions

Year

Contributor(s)

1888

Stillmark (1)

1936

Sumner and Howell (14)

1948-52

Renkonen (15); Bird (16-

1957

1960

Makela (19) (also work of Morgan and Watkins; Boyd, Reguera, and others; further reviewed by Levene et al. (Chapter 10 of this book), Bird(20) and Crookston (21) Nowell (22)

1960s

Goldstein et al.(23)

1960s

Kohler et al. (24-27); Wagner (28) Doyle etal. (29); Goldstein and Staub(30)

1968-70

18)

1971

Tkacz et al. (31)

1972-73 1973

Archibald and Coapes (32); Birdsell and Doyle (33) Doyle et al.(34)

1973

Martinez-Palomo et al.(35)

1977

Ebisu et al.(36)

1978

Stoddart et al. (37)

1979

Schaefer et al.(38)

1984

Graham etal(39)

4

Observations Plant extracts could specifically agglutinate erythrocytes of various animals. ConA aggregated membersof the genera Mycobacterium and Actinomyces. Lipid extracts ofM. paratuberculosk were aggregated byC o d . Developed use of lectinsas blood group reagents. Further studies on blood group antigen interactions with lectins.

Discovery of lectin-induced mitogenesis of lymphocytes. Specificity of ConAfor nonreducing sugar termini shown. Demonstrated lectin specificity for microorganisms. ConA was shown to specifically bind lipopolysaccharides of certain gramnegative bacteria. Identification of budding sites Sacin charomyces. ConA blocked binding of bacteriophage to Bacillus subtilis. First affinity purification of a teichoic acid employingCod-agarose columns. Lectins employedas probes for pathogenic protozoa. Lectins were usedas structural probes for a streptococcal group-specific polysaccharide. Identification of fungi in paraffin sections of tissues. First use of lectins in diagnostic microbiology. Enzyme-linked lectinosorbent assay (ELLA) developedfor bacteria and bacterial spores.


5

Table 1 (Continued)

Observations Year

Contributor(s)

1984

Mobley et al. (40)

1985

Schalla et al. (41)

1988

Karayannopoulou et al.

1989

Slifkin and Cumbie (43)

(42)

ConA was used monitor to the insertion of and subsequent fate of teichoic acids of Bacillus subtilis. Lectins were first employed as reagents in the epidemiologyof bacterial infec-

tious agents. In situ identificationof fungi in tissue sections. Use of lectins in diagnostic virology with infected tissue cultures.

means of screening for lectins. Furthermore, once a lectin has been shown to clump a particular cell (red cells, fungi, bacteria, or other), the specificity of the lectin can be determined by hapten-inhibition experiments. Knowledge ofthe specificity then frequently leads to affinity purification methods for the isolation of the glycoconjugate-binding proteins. One reason monovalent lectins have not been discovered may be that there are no rapid means for their detection. It may be possible for monovalent lectins to compete with polyvalent lectins and, thereby, renderthe latter incapable of causing cellular aggregation, but as far as is known, no systematic search for monovalent lectins has been undertaken. Monovalent lectins would also be expectedto be retarded, but not retained, by affinity columns. Table 2 outlines the major sources of lectins. In plants, lectins have been found in the roots, sap, fruit, seeds, flowers,barks, stem, and leaves. Some plants have more than one lectin,and some lectinsare synthesized as allelic variants or as isolectins. Some lectins are glycoproteins, but in many instances, the carbohydrate is not required for lectin activity. In bacteria, lectins may O C C U ~on the cell surface or may be found in the periplasm (transport proteins) or cytoplasm.Thebacterium Pseudomonas ueruginosu is the onlyknown prokaryote to express internal lectins [M].The spectrum of lectin sources is impressive. For some years, it was a theme of some lectin researchers to find a universalfunction for the proteins.Now, it seems that the function is related more to the origin of the lectin (Table 3). For example, surface lectins of bacteria are thought to be important in adhesive events. Mutants lacking surface lectins tendto be avirulent [45]. Furthermore, inhibitorsof bacterial lectins have been reported to reduce the incidence of experimental infections [46]. Similarly, viral lectins (the spikes of influenza viruses are

6

Doyle

Table 2 Sources of Lectins inNature

Avian Invertebrates Eggs, serums, tissues Crustaceans, insects, slugs, snails Bacteria Cell wall Eggs, lymphocytes, serum, sperm, various tissues Cytoplasm Plants Cytoplasmic membrane Flowers Fimbriae (pili) Fruit Outer membrane Leaves Periplasm akes eels, Fish, Roots Serums Venoms yeasts, Fungi, protozoa Stems Surface structures Viruses Bacteriophages Spikes of some animal viruses

the best example)and fungal lectins may have roles in adhesion to glycoconjugates [47]. The influenza virus binds to receptors containing terminal sialic acids. Sialidase treatment receptorcontaining of cells rendersthe cells resistant to the influenza virus. In bacteria, many bacteriophage particles [48]. require a-glycosylated teichoic acids as receptors Etzler 1491 has reviewed many of the proposed functional roles for lectins in plants. In one interesting experiment, Marsh[50] grew Dolichos Table 3 Some Proposed Functional Roles for Lectins ~~

Source(s)

~

Function@)

Bacteria, viruses Adhesion to glycoconjugates Fungi, molds Adhesion; matingfactors; differentiation Adhesion; trapping of potential nutrients Nematodes, protozoa Eel and fish serum, Primitive antibodies; agglutininsfor bacteria crustacean’tissues Mammalian tissues Lectinophagocytosis; removal of desialyated glycoproteins Plants Anti-insect; anti-fungal; primitive immune proteins; symbiosis;storage protein Immune Insects factors against protozoa Eggs glycoconjugates spermRecognition of

s

Ledin-Microorganism

7

bifronrs in the presence and absence of blood group A antigen and then analyzed the seeds for the anti-A lectin. Both groups of seeds gave riseto the anti-A lectin, suggesting that the lectin was not synthesized by virtue of antigenic stimulation.It seems likelythat lectins of plantsare not analogous to antibodies in animals.Plants, however, may not have a needto respond a more direct role against immeto potential antigens. The lectin may play diate challenges such as from fungi or viruses. Wheat germ agglutinin can inhibit the growth of the plant pathogen Trichoderma viride [51]. Furthermore, the lectin can bindto hyphal tipsand septa of the fungus. Antifungal properties of the lectin of Solanum tuberosurn have been reported. The lectin, which binds oligomers of N-acetylglucosamine, inhibited hyphal extension ofBotrytis cinera [52] and caused the release of cytoplasmic constituents of Phytophthora infestans [53]. Other studies have appeared that describe antifungal properties of lectins. Lectin from barley is known to reduce the infectivity of barley stripe mosaic virus 1541. In work in the author's laboratory, several lectins capable of binding to bacteria were incapable of inhibiting cell division, so it appears unlikely that lectins possess general antibacterial properties. Specific lectins, however, may inhibit selected bacteria. If, indeed, lectins do play a general role in reducing the infectiveness of plant pathogens, it must bethrough an as yet to be determined mechanism. Lectins of some plantsmay be toxicto insect predators.The larvae of bruchid beetles are killed by the lectin ofPhaseolus vulgaris[S]. For some years, there has been a controversy surrounding the role of lectins in symbiosis between nitrogen-fixing bacteriaand legumes. It is clear that the root lectins of some sprouts of plants can specifically bind nitrogen-fixing bacteria. But it is also clear that these same lectins can bind other kinds of microorganisms as well. Furthermore, occasionally, the absence of lectin in mutants of the plants has not led to loss of the ability of the bacteria to adhere to root tips [reviewed in 491. In animal tissues, some macrophages possess cell surface lectins capable of recognizingbacterial (and possiblyother microbial) glycoconjugates. These lectins may be required to achieve nonopsonic phagocytosis. This process has been termed lectinophagocytosis by Ofek and Sharon [56]. Livercellspossesslectinscapableofbindingasialoglycoproteins.These lectins presumably functionin the removal ofthe glycoproteins from circulation. The hepatocyte lectins are sometimes called C-lectins as they are Ca*+-dependent. The C-lectins bind galactose residues in mammals and are involvedinendocytosisofasialoglycoproteins.Anotherclassofanimal lectins is called S-lectins. These lectins are soluble in the absence of detergents and are usually specific for @-galactosides. Neither C- nor S-type lectins have been employed in microbiology, as far as is known. Animal

8

Doyle

cell lectins [reviewedin 571 are also consideredto be involved in egg-sperm recognition, cellular differentiation, metastasis, lymphocyte migration, and hormonal function [a summaryof these proposed functions is given in91. Lectins have now been firmly established in numerous biological processes. The increasing availability of lectins from many sources provides more probes for the studyof microbial and viral glycoconjugates. 111. SPECIFICITIES OF LECTINS

Traditionally, the specificities of lectins have been defined based on the simplest monosaccharidesto inhibit hemagglutinationor to bind directlyto the protein. Some lectins, however, cannot be inhibited by monosaccharides (or disaccharides). For example, the ar-1,6-glucan-binding lectin of Streptococcus Cricetus cannot be inhibited by high concentrations of isomaltotriose, but can be inhibited by relatively low concentrations of isomaltooctaose or higher oligomers [58]. Moreover, peptides contribute to the specificity of some lectins capable of complexing with complex saccharides. Figure 1 shows the structures of most of the sugars and monosaccharides that have been reportedto bind with lectins.The figure shows the Haworth structures and, for some, the stable chair conformations. Symbols are included for the monosaccharides commonly found in polysaccharides or glycoconjugates of bacteria, plants,and animals (all possible monosaccharides with symbols cannot be described in this brief overview of lectin specificities, but the figure contains the best-represented ones the in literature). Microorganisms, including viruses for purposes of this book, are known to contain glycoconjugates possessing all the structures shown in that interact with microbial Figure 1. In addition, lectins have been reported and viral glycoconjugates containing the structures shown in the figure. Figure 2 shows ways of presenting some of the oligosaccharide structures known to interactwithlectins.Someof the structures are animalcellderived, but many of the structures are found on viral coats or on cells transformed by viruses (seeChapter 2for the complete descriptionof viral glycoconjugates capableof interacting with lectins). Concanavalin A (ConA), the first lectin for which the specificity was studied in detail, binds to unsubstituted nonreducing a-D-glucose (Glc)or cm-mannose (Man) residues [23]. The basic requirement is that hydroxyls at C-3, C-4, and C-6 must be available. Microbial polymers, such as dextrans (a-1,6-glucan), which have only one nonreducing D-glucose per chain, bind to ConA,but the protein cannot precipitate with them. Introduction of branches into the linear dextran may result in increased availability of nonreducing termini, leading to precipitation with the lectin. Linear teichoic acids may precipitate with Con4 because of the substitution (in ef-

C1l1011

D-GLUCOSE

0 OH

cu1a

0

D-GALACTOSE

D-MANNOSE

L-FUCOSE

N-ACETYL-D-GLUCOSAMINE

N-ACETYL-D-GALACTOSAMINE

N-ACENLNEURAMINIC ACID (SIALIC ACID)

0-

OH

H Z

D-GLUCOSAMINE L O

O

N-ACETYLMURAMICACID

H

O

H

a-L-RHAMNOSE

'

b

D-GALACTURONIC ACID

Figure 1 Lectin-reactive carbohydrate structures. The figure shows the most common structure known to complex with lectins. Symbols for some of the structures are shown to the very right. The figure also shows Haworth (pyranose) and chair conformations for many of the carbohydrates. (For carbohydrate structures frequently found on glycoproteins; see Fig. 2. Modified from Ref. 9.)

9

10

Doyle

v\ t v/

Manu 3 (Mana6) Man Manu 6 Manu 3'

'Man

Manu 2 Manu 3 [Manu 3 (Manu 6) Manu 6jManp4GkNAcp4GlcNAc Manu ,6

Manu6 Manu 3° Mana2Mana3/

Galp4GlcNAcp2Manu6

Manp4GlcNAcp4GlcNAc

\

Galp4GlcNAcp2Mana3

GlcNAcpG \ GlcNAcp2Mana6

v-t-

L-Fuc~~ I ManplGlcNAcfMGlcNAc

>

Manp4GkNAcp4GlcNAc

GlcNAcp2Mana3

GlcNAcp2Mana6 GlcNAcpl Manp4GlcNAcp4GlcNAc GlcNAcpPManaii

v\

~l~~~~p2Mana3[Mana3(Mana6)Mana6]Man~4G~C~~C~~~~~~~ Mana6, ManaJ/ >aM GlcNAcp2Mana3

Manp4GlcNAcplGlcNAc

W.\

W-.

e-4

Figure 2 Representation of lectin-reactive sites commonlyfound on glycoproteins. The saccharides may be derived from animal cells, viral-transformed cells, or micro,organisms, suchas fungi. Lectins frequently complex with oligosaccharidesglycoin proteins. Usually, the most importantresiduesare the nonreducingterminiand their accompanying penultimate residues. (Modified from Ref. 9.)

fect, a typeof branching) of a-D-glucose residueson the glycerol or ribitol moiety (a later section describesthe microbial structuresthat may interact with lectins). Mannansand glycoproteins may containa-l ,Zmannose linkCod. ages that readily interact with The Appendix liststhe most common lectins studied to date. Specificities are given in terms of monosaccharides or oligosaccharides that best complex with the lectins. For many of the lectins, only monosaccharides have been studied as inhibitors, whereas for others, inhibitors have yet to

. Lectin-Microorganism Complexes

11

be discovered. The table also lists the common name of the lectin source and, when known,the blood group specificity.A new means for the abbreviation of lectins is proposed as well. Some commonabbreviations, such as WGA, for wheat germ agglutinin,are too well entrenched in the literature to propose a change. When possible, abbreviations should begin with the first two letters of the genus, followed by the first letter of species. When abbreviations overlap, multiple letters both of the genus and species should be employed. Throughout this book, the abbreviations shown in the Appendix will be employed. An inspection of the specificities of the lectins listed inthe Appendix reveals that numerous lectins bind Gal or GalNAc residues. A few of the lectins are specific for anomeric linkages, although most Gal or GalNAcbinding lectins can complex with eithera-or 0-linked saccharides. Importantly, although a particular lectinmay bind a particular saccharide, there is no certainty that the lectin will bind those residues on a microbial surface. Frequently, hydrophobic residues enhance the interaction between a saccharide and a lectin. Probablythe most unimportant hydroxyl group recognized by lectins is C-2. For example, ConA can bind mannose or glucose, monosaccharides that differ only in the spatial orientation of the C-2 hydroxyl. In general, galactose-specific lectins have no affinity for glucose, and vice versa, showing the role of C-4 in lectin recognition. For many lectins, the penultimate saccharide has a large influence on binding. For Eranthishyemalis @RH) canbindGal& example, the lectinfrom 1,4GlcNAc somewhat betterthan Gala-l ,4Glc. Similarly,the Japanese pagodatreelectin(SOJ, from Sophora japonica) bindsGalp-l,3GalNAc somewhat betterthan Gal&1,3GlcNAc. Most GlcNAc-binding lectins combine best with oligomers of GlcNAc, although as far as is known, all of these lectins can complex with GlcNAc alone. In the Appendix, it should be noted that there is a paucity of 0-glucose-binding lectins.A lectin from the fungus Sclerotium rorfsii(SCR) has been reportedto bind Glcp-l,3Glc. In addition, Cytisus sessifolius (CSS) has a weak affinity for cellobiose (Glc@-1,4Glc). Lectins capable of binding @-glucosides would be welcome in diagnostic microbiology, as many bacteria and fungi produce &linked glucosidic polymers. The so-called 0-lectins [69] are known to interact with hydrophobic 0-glucosides, but these have not yet provedto be of value in microbiology. There are also only a few lectins specific for uronic acids. The Aplysia depilans ( M D ) lectin is inhibited by galacturonic acid, but Abramis brama (ABD) has this lectin also binds galactose. The lectin from been reportedto complex with rhamnose (Rha) residues, but this lectin also binds galactose. The very few lectins reportedto be specificfor 0-D-glucose, uronic acids, or rhamnose may reflect that researchers havenot looked for such specificities. The ubiquitous occurrence of 0-D-glucose, a-L-rhamnose, and uronicacidssuggests that lectins are available that canbindthese

Doyle

12

monosaccharides. Lectins specific for sialic acids frequently complex with N-acetylneuraminic acid (NeuAc), whereasothers may complex with 9-0acetylneuraminic acid or N-glycolylneuraminic acid. Only a few bacteria make sialic acid-containing polymers,but many viral protein coats contain sialoglycoproteins that can interact with lectins. Some lectins interact primarily with mucin-type or 0-linked glycoconjugates, including the lectins from Agaricus bisporus and Bauhiniapurpurea. Other lectins seemto have Ricinus high affinities for N-linked glycoconjugates, such as the lectin from communis. The now widespread availability of numerous lectins increases the probability that lectins will be applied more extensivelyto the study of microbial surfaces.

W.

THE HYDROPHOBIC EFFECT AND LECTINS

Several lectins are known to possess multiple-combining sites. In addition to saccharide-specificsites,lectinsmaybindmetalions and hydrophoa hydrophobicbic ligands. A common occurrence in legume lectins is combining site spatially separated from the saccharide-combining site. In addition, there are hydrophobic sites very near saccharide-specific sites. Concanavalin A can bind p-nitrophenyl-a-D-mannoside better than it can bind methyl-a-D-mannoside which, inturn, is complexed better than Q-Dmannose. Also, Con4 has a site specific for hydrophobic groups, such as inositol, -adenine, and various fluorescent dyes. Frequently, the affinity constant for the bindingof a hydrophobic groupis greater than the affinity constant between the lectin and its specific monosaccharide. Microorganisms have numerous hydrophobins [70] on their surfaces, including proteins,glycolipids,lipoteichoicacids, and others.Thesehydrophobins, which may or may not be associated with glycoconjugates, nodoubt contribute to the ability of a lectin to bind to a microbial surface.It is interesting that, among the legumes, the hydrophobic cleft has been largely conserved throughout evolution,suggesting a functionalrole for thesite. Hydrophobic sites adjacent to carbohydrate-specific sites also extend to unrelated species. Pseudomonas aeruginosa lectins bind hydrophobic saccharidesbetter than unsubstitutedsaccharides.Similarly, the mannosespecific lectinof Escherichia coli has a much higher affinity for hydrophobic mannosidesthan for hydroxylated mannosides[71]. V. ISOLATIONANDPURIFICATIONOFLECTINS

There are no general strategies for the isolation of lectins. The isolation procedure@)are usually dictated by the source (seed, serum, bacteria, or other) and may involve classic protein purification schema. Some extracts

lectin-Microorganism Complexes

13

maybeinitiallyfractionated by ammoniumsulfateandthenbyionexchange chromatography.If the specificity of a lectin is known, the lectin may be purified to homogeneity on affinity sorbents. Elution of the lectin can then be realized by use of solutions of saccharidesor chaotropes, orby lowering the pH. It is essential that eluting agents be removed from the purified lectin(@and that the agents do not irreversibly alter the saccharidebinding site@). Affinity methods rarely separate isolectins, although a combination of affinity and ion-exchange techniques may afford reasonable separations. For studieson microbial surfaces, it is best that pure lectins be employed when possible. This is because many sources of lectins yield two or more carbohydrate-binding proteins with distinct specificities. The book by Liener et al. [8] provides extensive discussionson the methods involved in lectin isolationand purifications. VI.LECTINDERIVATIVES

IN MICROBIOLOGY

The microbiological applications of lectins frequently require that the lectin possess a sensitive tag or reporter. Lectins, as proteins, can be derivatized with any of the same reagents that have been employed for antibodies or enzymes [72]. Fluorescein isothiocyanate (FITC) derivatives of lectins have been used in microbiology to detect microorganismsand spores [73,74]and to study the distribution of wall polymers [75]. Figure 3 shows an FITC derivative of soybean agglutinin bindingto spores of Bacillus anthracis, a gram-positive organism possessing a cell wall polysaccharide with terminal D-galactose residues. The obvious goal of introducing a marker onto the lectin is to be able to monitor glycoconjugates on microbial surfaces or in solution. It must be rememberedthat the chemical modification of a lectin may led to a partial loss of its activity. Consequently, chemical modifications are normally performed in the presence of specific saccharides in an effort to prevent inactivationof the combining sites. Many lectins bind directly onto latex particles, resulting in passively sensitized spheres that can be used in aggregation reactions (Table 4). Latex-sensitized particles also have been used in establishing specificities of lectins. Such sensitized particles may be susceptible to aggregation by microbial glycoconjugates. Lectins may be coupled with enzymes, thereby permittingenzyme-linkedlectinsorbentassays(ELLA).TheELLAtechniques have been used successfully to detect low densities of bacteria and bacterial spores[39]. Salt-enhanced ELLA assays (SELLA)take advantage of the fact that many substrata for lectins can be salted-out onto plastics. The SELLA techniques make .i’t.,possible to detect very low concentrations of microbial glycoconjugates.The acronym FELLA is reservedfor the use of fluorescent derivatives of lectins, whereas GELLA is proposed to refer

14

Doyle

Figure 3 Binding cIf fluorescein-labeled soybean agglutinin (SBA) with Bacillus unthruck spores. Vegetative cells also bind SBA. The SBA-reactive materialon the spore surfaces may or may not be similar in structure to that on vegetative cell walls. (From Ref. 76.)

to lectin-gold mixtures as probes for glycoconjugates. A main advantage for using dot-blot-like assays with lectin-colloidal gold is that glycoconjugates can be detected on membrane filters and very low concentrations of gold can be detected visually. The lectin-colloidal gold (GELLA) assays are convenient for the screening of large numbers of samples for glycoconjugates. VII. MICROBIAL SUBSTRATA FOR LECTINS

There are numerous microbial structuresthat serve as receptors for lectins. 5 . In bacteria, Most ofthe lectin-reactive glycoconjugatesare listed in Table cell wall-or outer membrane-associated componentsare the most common lectin receptors. Teichoic acids, covalently linkedto peptidoglycans, occur in many gram-positive bacteria. Polymersof glycerol phosphate or ribitol phosphate may contain a- or &linked carbohydrate substitutions on the carbons not attachedto phosphates. InB. subtilis W23, a p-D-glucosyl unit is attached to the C-2, 3, or 4 groups of the ribitol, whereas for B. subtilis


15

Table 4 Lectins, Lectin Derivatives,and Procedures Involving Lectinsin Micro-

biology Methods

Description

Agglutination (direct) Soluble lectins bind to microbial surface. Latex spheresare passively sensitized with lecAgglutination (indirect) tins. Biotin-conjugated lectinmay be detected by an BELLA avidin-enzyme conjugate. used Lectin may bindto plastic, or lectin may be Enzyme-linked lectinsorbent assay (ELLA) to complex with plastic-bound microbe or antigen. FELLA Fluorescent usedis lectin to detect glycoconjugates or microbes. GELLA Lectin-colloidal mixture detects gold low densities of microbes or low concentrationsof glycoconjugates. derivatized RELLA be Lectin may to contain radioactive 3H, 14C, '"I, or '=I. SELLA lectinenzyme-linked Salt-enhanced sorbent assay; ammoniumsulfate promotes binding of lectin or protein antigen to polystyrene. WELLA Western blot modified so lectin bind can a to a macromolecule ina gel. Enzyme assays Lectin coupled is to enzyme, such as &galactosidase or a peroxidase. Lectin in combination withflu- Lectin may detectone kind of bacterium, but conventional fluorogenicor chromogenic suborogenic or chromogenic substrate strate may detect closely related bacteria. Lectin issubstituted for antibody in rocketelecLectinophoresis trophoresis.

168, the C-2 of the glycerol contains a-D-glucosyl substitutions. As indicated in the foregoing, ConA and other a-D-Glc-specific lectins can combine with the teichoic acid. Figure 4 shows an example of a teichoic acid structure, along with someother microbial polymers knownto be reactive with lectins. Some strains of Staphylococcus aureus possess ribitol phosphate teichoic acidsthat are substituted with botha-and 8-GlcNAc. These ConA and WGA, respectively. teichoic acids interact with Muramic acid (usually in the N-acetylated form) will react weakly with some GlcNAc- and sialic acid-binding proteins. Most muramic acid residues possess amino acid substitutions on the lactyl groups on C-3, so in pepti-

16

Doyle

Table 5 Lectin-Reactive Sites on Microorganisms and Viruses

Bacteria Capsules Glycolipids Chitin(infrequent) Glycoproteins Group-specific polysaccharides Galactans ucans (polyfructans) Levans Lipomannans nans Lipooiigosaccharides rotozoa Lipopolysaccharides Lipoteichoic acids Peptidoglycans Surface array layers Teichoic acids Teichuronic acids Type-specific polysaccharides’

Fungi Arabinan Capsule

Galactomannans Glycolipids Glycoproteins Lipophosphoglycans Phosphoglycans Viruses Envelope glycoproteins

‘Types of polysaccharides that occur in oral streptococci are not to be confused with type-specific “protein of pyogenic cocci.

doglycans, muramic acids generally form poor receptors. Peptidoglycans, however, are able to complex with WGA, presumably through interaction with nonreducing GlcNAc termini[77]. The lipopolysaccharide shown in Figure4 would be expectedto interact with WGA and galactose-binding lectins. Connelly and Allen[78] and Allen et al. [79] showed that a battery of lectins could be used in structural determinations of lipolysaccharides from Neisseria gonorrhoeue. Doyle et al. [29] found that Shigella flexneri lipopolysaccharides could precipitate with ConA. Several other lipopolysaccharides formed weak complexes with the lectin. Goldstein and Staub [30] observed that although some lipopolysaccharidespossessedtherequisiteterminala-D-glucoseresidues,they would not precipitate with ConA. These findings suggested that penultimate or nearest-neighbor residues may influence the interaction with the lectin. Mutants in lipopolysaccharide oligosaccharide structures may result in exposedor lost lectin reactive sites. Hammarstrom et[80] al. have studied the reactivity of lipopolysaccharides froma Salmonella sp. and found that the extent of mutation may leadto various lectin reactivities. For example, Helixpothe wild-type salmonellar lipopolysaccharide was unreactive with matia agglutinin (HPA; see Appendix), but mutations leading to “rough” a colonies gave riseto a product capable of interaction with the lectin, and final “deep rough” mutant was again unreactive. The lipopolysaccharides

l7


ElhN-P

I

KDO

A lipopolysaccharide

IUpldAI- $..-oal-Gp

A peptidoglycan -(MurNAcp-l

I

HepH,ep.KWKDO ( Mac-Rhm-Gal),

GkNAc P-P-ElhN Gal

I

, 4 GlcNAcp);

peptide@) Chitin

(GICNACP-~,~),

A capsular polysaccharide (GlcU

p - 1,4Gk),

Figure 4 Potential lectin receptors derived from microorganisms. Teichoic acids are covalently boundto peptidoglycan of many gram-positive bacteria. Lipoteichoic acids @TA) are anchored in cell membranes and not associated with the walls of most (group A streptococci excepted) gram-positive cells.The LTAs, consisting of a chain of poly(glycero1 phosphate) may be glycosylated, similar to wall teichoic acids. Lipopolysaccharide (LPS) structures are dependent on the genus or species of the organism producing them. Capsular polysaccharides also exhibit diversities of structures, either from gram-positive or gram-negative microorganisms. The simplified structures shown in the figure do not represent all potential lectin-reactive materials produced by microorganisms. A more complete list given is in Table5.

18

Doyle

of many gram-negative bacteria possess limulin-reactive 2-keto-3-deoxyoctonate (KDO; see Fig. 4) [81]. Gilbride and Pistole [82] have suggested that limulin may serve as a type of immune factor in the horseshoe crab, because of the ability of the lectin to bind lipopolysaccharides. Branched dextrans (a-glucans) precipitate with ConA and other aglucose-binding lectins. Increased branching, leading to a greater proportion of nonreducing glucose termini, enhances reactivity with ConA [23]. Therefore, ConA is a tool for studying a-glucan structures. Mannans, levans, galactans, arabinogalactans, galactomannans, various group-specific polysaccharides of streptococci, and teichuronic acids havebeen reported to bind with oneor more lectins. An acidic lipomannanfrom Micrococcus luteus has been detected by ConA in lectinophoresis [83] (see Table 4), a type of rocket electrophoresis substituting lectin for antibody. Several reports describe the interaction between bacterial teichoic acids and lectins [84-861. Precipitin reactions in gelshavebeenemployed to detect complexformation between ConA and teichoic acidsfrom members of the genus Bacillus 1871. In addition, soluble teichoic acids are precipitated by ConA. Cell wall-bound teichoic acids can be detected on the surfaces of the bacilli by FITC-ConA. Lectins from Triticum vulgaris (WGA) and Helixpomatia (HPA) have been used to study cell wall teichoic acids fromstaphylococci.Reeder and Ekstedt [85] found that an a-D-glucosylated teichoic acidfrom a strain of Staph. epidermidis was precipitated by ConA. Thiswas unusual because most teichoic acids from Staph. epidermidis strains are not a-D-glucosylated. Also, ConA can be used as a probe for the surface teichoic acid ofStreptococcus faecalis (Enterococcus hirae) [87]. When the teichoic acid is removedby extraction with acids, the reactivity of Strep.faecalis with ConA is abolished. Individual chapters in this book will provide details on interactions between lectinsand various specific surface glycoconjugates of microorganisms and fungi. The reactivity of a lectin with a particular glycoconjugate depends on several factors (Table 6). For example, lectin molecular weights vary from a few thousand to over one-half million. Therefore, some lectins can penetrate to sites inaccessible to antibodies or other higher molecular weight lectins. Furthermore, some glycoconjugates may be masked, rendering them inaccessible to any lectin. An example is the peptidoglycan of certain bacteriathat is not available to lectins because ofouter membranes or capsules. Dextran-covered streptococci could be made ConA-reactive by incubation of the cells with dextranase [88; see also 891. In Mycoplasma, lectin-reactive sitesare exposed by treating the bacteria with proteases[W]. In addition, salts may decrease the interaction between lectins and glycoconjugates by causing an unfavorable conformation in the glycoconjugate [91J. Work in my laboratory has shown that some bacteria become lectin-

near


19

Table 6 Some Factors Governing the Reactivity of Microbial Glycoconju-

gates with Lectins Factors

Comment

High MW lectins may be excluded from carbohydrate receptors. Adjacenthydrophobicresi-Somelectinspreferentiallybind to sites dues Presence salts ofSalts induce rigid-rod a random to coil conformation in teichoic acids, thereby diminishing the rate of binding with lectins. Cell wall turnover some Inbacteria, lectin-reactive sites are shed into medium (“turned over”). Time of interaction In microorganisms with low adensity of lectin receptor, .considerable time an interacmay be required to detect tion. Locationoflectin -reactiveSomeglycoconjugatesmaybemasked membrane, peptidbyouter capsule, site@) oglycan, or other factor, thereby preventing their accessibility to lectins. Proteases abolish) lecProteases expose (ormay tin-reactive siteson microorganisms. Lectin molecular weight (MW)

reactive onlyafter prolonged incubation times with the protein. Hydrophobic interactions may playa role in complex formation between lectins and glycoconjugates. For example, a-glucosylated lipoteichoic acids of B. subtilis cannot be readily eluted from ConA-Sepharose columns unless mild chaotropes are presentalongwithmethyl-a-D-mannoside.Finally,some microorganisms shed their cell surface components during growth. These shed or turned over materials may not be replaced, resulting in loss of lectin-reactive sites [92]. VIII. APPLICATIONS OF LECTINS IN MICROBIOLOGY

Lectins have now become essential toolsfor thestudy of microbial glycoconjugates. A few of the uses of lectins in microbiology were givenin Table 1, where someof the historical aspects of lectin-microorganism interactions were listed. The purpose of this section is to provide an overview of the applications of lectins in various practical and research problems relatedto

20

Doyle

microbiology. Table 7 lists some of the most common applications of lectins in microbiology reported to date. In diagnosticmicrobiology,lectinshavenowbecome standard reagents. In a typical application, a suspension of a microorganism may be mixed with a solutionof lectin on a glass slide. An agglutination can often be takenas a confirmationof an organism, providingthat some knowledge of the history of the organism is available. For example, N. gonorrhoeae can be confirmed by agglutinating with WGA, assuming the isolate came from a urethral exudate and was a gram-negative diplococcus. the If isolate originated from a spinal tap, a throat swab, or the skin, WGA may be expected to aggregate a few strains of N. menigitidis or N. laetamica. The agglutination of N. gonorrhoeae by WGA requires a microgramor less of the lectin, whereas the other bacteria are usually agglutinated by higher concentrations. In fact, one of the advantages of employing lectinsas diagnostic reagents isthat they are generally activeat very low concentrations. Moreover, agglutination reactions are usually rapid. Th? use of lectin derivatives (see Table 4) potentiates the possibilities for lectin applications in diagnostic procedures.The detection of herpesviruses in tissue cultures [43] and the detectionof infectious agents in tissue sections [42] are good examples of how derivatized lectins can be used in diagnostic microbiology procedures. Table 8 offers an outline of some of the major uses of lectins in diagnostic procedures. Slifkin, Chapter4 in this book, provides a comprehensive review ofthe uses of lectinsin clinical diagnostic microbiology. Table 7 General Applications of Lectins in Microbiology

Affinity sorbents for microbial polymersand microbial products Detection of intracellular viruses and microorganisms Detection of microorganisms in situ Detection of mutants in surface glycoconjugates Probes for monitoring insertionand fate of cell surface glycoconjugates Probes for solution properties of polyelectrolytes Purification of immunoglobulins Reagents for diagnostic microbiology Reagents to be employed in establishing structures of glycoconjugates Reagents to establish epidemiologicalpatterns of infectious agents Study of symbiosis between bacteria and sponges Reagents that can be employedto determine receptor identitiesfor bacteriophages Selectiveinhibitors of enzymes Studies on adhesion mechanisms of microorganisms Use in identification of antigens, including blood group antigens Source: Details may be foundin Refs. 93-111.

Table 8 Summary of Some Specific Applications of Lectins in Diagnostic Microbi-

ology and Epidemiology from other bacilli byits The pathogen Bacillus anthraciscan be distinguished growth at 37OC and aggregation withthe Glycine maw lectin. Neisseria gonorrhoeaeand nonencapsulated N. meningtidis are selectively agglutinated by low concentrations of wheat germ agglutinin. Serogroup A Campylobacterfetus could be correctly identified with lectins. Group B streptococci can be specifically agglutinated by lectins bound to polystyrene particles. Cross-reactivity with streptococcal groups A,C,D,F, and G was not observed. A lectinfrom Cepaea hortensisis specific for group B streptococci. Group C streptoccal antigen is selectively agglutinated with a lectin from Dolichos biflorus bound to polystyrene particles. Enzyme-linked lectinsorbent (ELLA) assays can be usedto detect low numbers of Bacillus anthracis. Surface antigen of Streptococcusfaecalis isolates from endocarditis patients could be identified byblotting techniques using lectins. Most common isolates Listeria of monocytogenescould be grouped with a battery of lectins. Fluorescein-conjugated lectins selectively bound to microbial isolatesfrom the human cornea. Fungi could be directly observed in tissue sectionsby use of lectins. from plant tissue by use of three lecRhabdoviruses of plants could be distinguished tins. Subtypes of Marek disease virus could be discriminated by a battery of lectins. Lectins could discriminate between pathogenic and nonpathogenic South American trypanosomes Smooth and rough strains of Brucella sp. displayed unique agglutinationpatterns with lectins. Fluorescein-labeled Helixpomatia lectin could rapidly distinguish herpes simplex types 1 and 2 in cell culture. Thermophilic Campylobacter sp. demonstrated unique reactivities witha battery of lectins and plant agglutinins. A battery of lectinswas used to distinguish betweenNeisseria gonorrhoeaeisolates from various geographic locations. Campylobacterjejuni and C. coli were grouped accordingto their reactivities with several lectins. Isolates ofHemophilus ducreyifrom different geographic areas gave riseto unique agglutination patterns with lectins Wheat germ agglutinin has been proposed as a reagentto discriminate between gram-positive and gram-negative bacteria. Source: Refs. 11-13,110-128. 131, 132. Chapter 10 describesthe use of individual lectins in blood banking.

22

Doyle

Levy [l331 showed that 2-10 pg/ml of WGA blocked attachment of Chlamydia psittaci to mouse fibroblasts. The blockage could be inhibited byGlcNAc, but not other saccharides or sugars. Moreover, ConA and RCA-1,II were unableto block the chlamydial attachment.A strain of C. trachornatis, isolated from a lymphogranuloma venereum lesion,was also prevented from adheringto the fibroblasts by WGA. Identification of carbohydrates involved in bacterium-animal cell interaction may leadto new means of preventing certain infections.An agglutinin (lectinlike substance of unknown composition) from Persea americana prevents the adhesion of Strep. mutans to saliva-coated hydroxylapatite [98]. The saliva-coated hydroxylapatite (S-HA) serves as a model for tooth surfaces, so considerable efforts have been made to prevent attachment of cariogenic streptococci to the surfaces of the S-HA beads. Streptococcus downei (formerly Strep. mutans serotype h) adhesionto the S-HA was not inhibitedby various carbohydrates, but the binding was abolished bya protease. The bacteria had to be pretreated with P. arnericuna agglutinin to achieve significant inhibition of adhesion. Halverson and Stacey [95] have employed various lectins as mediators of adhesion betweenRhizobium (Bradyrhizobium)japonicum and soybean root. A mutant of R. japonicum, incapable of causing rood modulation, could be made phenotypically wild-type (modulationpositive) by very low concentrations ofsoybeanagglutinin(SBA). Concentrations of SBA as low as ten molecules per bacterium were effective in restoring the wild-type characteristics. The results suggestedthat modulation was dependent on adhesion of the bacterium to the root surface. Efforts are underway in several laboratories to study the role of lectins in mediating attachment of bacteria to plant tissues. Electron microscopic techniques, employing SBA-ferritin, were used to localize the lectin binding site on Rhizobium japonicum. The SBA-ferritin binds to a capsular polysaccharide at one end of the cell [134].The capsular polysaccharide was not contaminated with lipopolysaccharide, nor did outer membrane bind with the lectin. The polysaccharidea good is candidatefor bridging between nitrogen-fixing bacteriaand legume root tip lectins. Furthermore, the localization of capsule at one end ofthe bacterium raisesan important question in bacteria physiologyabout sites of secretion of exopolymers. The capsular polysaccharides of pneumococci vary considerably in composition and linkages. The pneumococcal S-l4polysaccharide studied by Lindberg et al.[l351was proposed to have the followingstructure

- GlcNAc~-1,3Gal~-l,3Glc~-1,6-Gal~-1,3 Ebisu et al. [36]found that WGA and RCA-1,II.could precipitate the S-l4 polysaccharide, but the a-D-Gal-specific lectinfrom Griffonia(Bandeiraea) simplicifolia could not. The lack of reactivity with the griffonial lectin

Lectin-Microorganism

23

confirmed the existenceofthe8-D-galactoseterminallinkage. A linear polymer ofS-14 could be derived by periodate oxidation, followed by Smith degradation, which removesthe 8-D-galactosyl residues. The linear polymer retained its abilityto bind with WGA,but its reaction with RCAwas lost, as expected. These results showthe versatility of lectins in structural work on bacterial glycoconjugates. Wheat germ agglutinin has an affinity for sialic acid residues, aswell as S-linked GlcNAc residues. Gray et al. [l361 took advantage of the reactivity of WGA for sialic acids to aid in the purification of streptococcal group B, type-specificpolysaccharides. The type-specific,but not the group-specific, polysaccharide, couldbe eluted from WGA-Sepharose columns in reasonably high yield and free of contaminants. Lectin bound to magnetic microspheres has also been employed in detecting and concentrating bacteria in dilute suspensions. Patchett et al. [l091 coated spheres with the H. pomatia lectin and observed that strains of Listeria monocytogenes would bind avidly to the lectin-sphere conjugate. They further showed that L. monocytogenes would adhere to HPAagarose columns, only to be eluted by GalNAc. The column and sphere techniques made it possible to concentrate L. monocytogenes from low densities of cells. Such techniques may have value in the food and dairy industries for which L. rnonocytogenes is a frequent contaminant. Interestingly, of several GalNAc-specific lectins, only HPA interacted with most of the strains of L. rnonocytogenes. Most other food-borne bacteria, such as members of the genera Salmonella, Bacillus, and Staphylococcus, did not complex withHPA. It has been suggestedthat lectins can be employed to enumerate yeasts in a suspension [137]. Lectin-conjugates were loaded into plastic syringes, then suspensions of yeastswere poured over the columns. The yeasts were assayed bytheir abilityto produce metabolites,the concentrations of which were proportional to the densities of yeasts bound onto the columns. The method is clever,but there are several items of concern. Some yeasts simply do not interact with any known lectins. Some yeasts, although capableof interacting witha lectin, maynot respire or ferment. The method has promise for distinguishingbetweenlimitednumbers of metabolicallyactive yeasts, but for a pure culture, direct-counting methods wouldseem superior. The detection of human immunodeficiency virus(HIV) envelope antigens has become important in clinical and hospital laboratories. The antigens are generally glycosylatedand, therefore, are potentially ableto interact with lectins. Robinson al. et [l381 coated microtiter plates with solutions of ConA, then used the coatedwells to trap soluble HIV envelope antigens. The antigens were obtained from detergent-solubilized glycoproteins re-

24

Doyle

leased into the culture medium of HIV-l-infected cells grown in serum-free medium. The HIV antigens, trapped on the solid surface, could then be quantitated by use of enzyme-linked immunosorbent assay @LISA) techand niques. No false-positive results occurred among 16 HIV-negative sera, no false-negative results occurred among 14 HIV-positive sera.

1x0

BACTERIAL CELL WALLS, BACTERIOPHAGES, AND LECTINS

Bacterial viruses frequently bind to carbohydrates as a first step in the infective process.In B. subtilis, glucosylated teichoic acidsare required for the binding of many bacteriophage particles[48]. The teichoic acid, however, must be covalently boundto peptidoglycan to serve as phage receptor sites. Concanavalin Aand bacteriophage 425 competed for the same site@) on the cell wall ofB. subtilis 168 [33],a bacterium containing a glucosylated poly(glycero1 phosphate) teichoic acid. Table 9 shows that phage 425 absorbs to glucosylatedwalls, but, when ConA is present, no adsorption occurs. Removal of the teichoic acid with 100 mM sodium hydroxide resulted in a wall incapable of binding phage 425. Soluble glucosylated teichoic acid didnot compete with cell walls, but neutralized the effect of the lectin. Figure S shows that ConA, when added to a mixture of walls and phage 425, interrupted the adsorption of the virus. When the ConA inhibitor, methyl-a-D-glucopyranoside,was added, adsorption commenced. The ConA may be directly competing with the virus for receptors, or the lectin may cover a composite site consisting of teichoic acidand peptidoglycan. A soluble autolysateor lysozyme digest ofthe cell wall will not bind to the virus, showing that an intact cell wall containing glucosylated teichoic acid is the actual phage receptor [33]. Concanavalin A not only blocked phage B. subtilis, but also blocked sites on Staph. auras, providreceptor sites on a-D-N-acetylglucosaminylresing the staphylococcus possessed nonreducing idues in its teichoic acid[32]. As a control, it was shown that ConA would not alter phage binding to B. subtilis W23, a bacillus possessing 0-linked D-glucose residues in its teichoic acids. Lactobacillus casei,another gram-positive rod, possesses a cell surface polysaccharide outsidethe peptidoglycan that serves asa receptor for bacteriophage PL-1. L-Rhamnose, a component of the wall-associated polysaccharide, inhibits phage adsorption to the cells [139]. Slight inhibition was observed for D-mannose and L-fucose. Astreptomyceshemagglutinin (crude lectin) of anti-B activity (specific for L-rhamnose and D-galactose; see Appendix) inhibits phage binding to cellwallsof L. casei [140]. In addition, small reductions in phage binding were noted for D-glucose (or


0 0 0 0 0 0

25

26

Doyle

I

A

A

A

107

za

106

I

105 I

0

I

2

I

I

4 6 TIME (MIN)

I

0 I

8

10

Figure 5 Effect of C o d on the kinetics of bacteriophage 425 adsorption to cell walls of B. subtilis 168. The reaction mixtures contained (in atotal volume of 1.0 ml) 0.2 mgofcellwalls, 2.0 mg of c o d , 1.5 x 10’ plaque-forming units of 425, and 0.1 M methyl-a-D-glucopyranoside.At the times indicated, sampleswere removed, diluted 1 : 100 in cold medium, further diluted, and plated by the agaroverlay procedure. Symbols:0, untreated cell walls; 0 ,cell walls plus methy1-a-Dglucopyranoside (a-MG); A, ConA added at zero time; W, ConA added at first arrow andthe glucoside addedat second arrow. (From Ref. 33.)

D-GlcNAc), but not for lectins that bound only GlcNAc. It was suggested that L-rhamnose wasthe primary receptorfor the phage [140]. X. INTERACTION OF TElCHOlC ACIDS WITH LECTINS

Teichoic acidsare frequently substituted with lectin-reactive carbohydrates. In B. subtilis 168, the teichoic acid is a-D-glucosylatedat the glycerol C-2, whereasin B. subtilis W23,fl-D-glucosyl substitutions occur on carbon positions 2, 3, or 4, but these glucose residuesare not receptorsfor readily CY-Davailablelectins. In Staph.aureus, the presenceofteichoicacid glucosaminyl residues (mostly N-acetylated) renders the cells agglutinable by ConA. Doyle and Birdsell [86] found that double diffusion in agargels

Ledin-Microorganism

27

was a goodway to monitor lectin-teichoic acid interactions. They observed that teichoic acids of B. subtilis 168would form precipitin bands with (DConA in agar gels. When the gels were soaked in ConA inhibitors mannose or methyl-a-D-mannopyranoside),the precipitin lines dissolved. Reeder and Ekstedt [85]used a similar method to study ConA-teichoic acid complexes in staphylococci. When solubleB. subtilis 168 teichoic acid was allowed to interact with ConA, typical precipitinlike profileswere oban equivalence zone, tained, characterized bya zone of teichoic acid excess, and a zone of ConA excess [91]. Inhibition of precipitation was brought about by the same inhibitors of ConA-neutral polysaccharide complexes. Results suggest that teichoic acids may exist in two conformations. One conformation is random-coil, found in reasonably high salt solutions. The other is rigid-rod, found in dilute salts and buffers. The teichoic acid in the rigid-rod conformation is readily precipitated by ConA, whereas the random-cell teichoic acid is less readily able to interact with the lectin (Fig. 6)[91]. Interaction ofteichoicacidswithlectins,therefore, ,is saltdependent, whereas neutral polysaccharide structure is largely unaffected by salts [91] (Table 10). The rigid-rod conformation of teichoic acids in low-ionic-strengthmediumisprobablydue to electrostaticrepulsion groups inthe teichoic acid backbone structure. Ions would tend to neutralize the phosphate groups, resulting in a random-coil conformation. Teichoic acid conformation may be important in interactions with specific antibodies or autolysin bindingto walls. Concanavalin A has provedto be a good probe to distinguish between random-coiland rigid-rod conformations of teichoic acids. Peptidoglycans of Staph. auracs are receptors of WGA, if the peptidoglycans possess terminal nonreducing GlcNAc residues [77]. Similarly, staphylococci possessing teichoic acids substituted with 0-GlcNAc residues were good receptors for WGA. As far as is known, there have been no attempts to purify soluble peptidoglycans using WGA affinity sorbents(see also Chapter 8). Classic preparative schema for cell wall teichoic acids involve extraction of walls with acids or bases. These methods yield polydisperse and impure preparations. Doyle et al. [34] were able to isolate an undegraded (based on physical properties) teichoic acid of B. subtilis 168byuseof ConA-agarose column (Fig. 7). They showed that when autolysates were poured over the ConA column, most of the peptidoglycan and protein emerged nearthe void volume. The teichoic acidwas eluted only by ConA inhibitors. The teichoic acid isolated by ConA affinity chromatography gave a narrow, single band in the analytical ultracentrifuge. In contrast, teichoic acids preparedby conventional extraction procedures were polydisperse, as revealed by analytical ultracentrifugation. Interestingly, the ConA

28

Doyle

140

a

2.8

I

h

1 E

h

120

2.4

100

2.0

'

h

E C

1.6

E (U

v

W

60

1.2 0

40

0.8

20

0.4

z

c3)

=L

t

0

S

(r:

0

EFFLUENT VOLUME (ml) Figure 6 Affinity chromatography of a bacterial teichoic acid on Cod-agarose. An autolysate of cell walls of B. subtilis 168 was poured over a Cod-agarose column. The glucosylated teichoic acid was eluted with the addition of methyl-a-D-

glucopyranoside to the column (elution with (From Ref. 34.)

the glucoside began at the arrow).

column has proved useful inthe isolation of mutants deficient in cell wall glucosylation. Teichoic acid, prepared by the affinity method, is a good antigen when mixed witha polymer of opposite charge. Several other reports document the use of lectinaffinity chromatography for the isolation of teichoic acids. Ndule et al. [l411 observed that a small fraction of GlcNAc-containing teichoic acid from Staph. aureus is retained on WGA-Ultrogel. The teichoic acid fraction seemed to partially bind to the column by ionic phenomena.Later, Ndule and Flandrois 11421 showed that the wall fraction contained ribitol phosphate, GlcNAc, and alanine. A GlcNAc-containing ribitol-teichoic acid of B. subtilis W23 can be resolved on WGA-Sepharose [143]. Lectin chromatography was useful in the separation of glycerol teichoic acids and mannitol teichoic acids in members of the genus Brevibacteriurn [ l a ] . The streptococcal group N antigen could be purified as a galactosyllipoteichoic acid [145]. Recently, Leopold and Fischer [l461 were able to purify lipoteichoic acids from En-

29

lectin-Microorganism Complexes Table 10 Solubilities of Concanavalin A-TeichoicAcid and Conca-

navalin A-Glycogen Complexes Complex

Teichoic acid' Tris (50 mM, pH 7.5) NaCl(100 mM) NaCl(1 .O M) KC1 (1.0 M) Galactose (100 mM) Methyl-a-D-mannopyranoside (100 mM)

Glycogen

Tris (50 m M , pH 7.5) NaCl(1 .O M) Galactose (100 mM) Methyl-a-D-mannopyranoside(100 mM)

49

390 645 580

55

827

33 50 53 761

Complexes werefrom reaction mixtures containing 1.O mg ConA, 1.O mg B. subtilis 168 teichoic acid, or 2.0 mg rabbit liver glycogen, in 50 mM Tris @H 7.5) in 2.0 m1 volumes. Followingincubation for 2 hr at 3OC, the precipitates were collected by centrigugation. washed twice with Tris, and finally, suspended in 4 m1 of the indicated solvents. Soluble protein was measured following an additional 2 hr incubation at room temperature. 'For teichoic acid-ConA complexes, radioactive ConA was employed, the soluble contents of which were determined by scintillation counting. For ConA-glycogen complexes the soluble ConA was assayed by a colorimetric method. Source: Ref. 91.

terococcus faecalis, E. hirae, and Leuconostoc mesenteroides on columns containing ConA. Interestingly, the LTAs were shownto be heterogeneous in the extent of their glucosylation and chain length. The amounts of alanine ester inthe LTA were uniform. Furthermore, the ester-linked alanine and the glucose moieties werefound on the same poly(glycero1 phosphate) chains. Various papers in the 1960s and early 1970s suggested that cell wall teichoic acids were arrangedon the outer surface of the gram-positive cell wall. The appearance of electron-denseouter regions of the walls gave rise to the notion that the teichoic acids were distributed asymmetrically. Doyle et al. [75] found that when walls were partially autolyzed,the walls bound more ConA (Fig. 8). The amount of ConA bound to a partially autolyzed wall wasgreater than the lectin boundto native walls. However,the relative amount of hexose remaining in the wall was virtually constant, a finding

30

Doyle

Y

I

0.125

0.25

-I Y

0.5 TElCHOlC ACID (mg)

0.75

1.5

3.0

Figure 7 Precipitin profile between ConA and the teichoic acid of B. subtilk 168. ConA (1.0 mg), with the indicated concentration of teichoic acid (2.0 ml final volume), was incubated for 2 hr at 25OC. The precipitates were removed by centrifugation, washedoncewith 5 m1 of the indicated solvent, and analyzed for protein content. (A), 50 mM tris(hydroxylmethy1amino)methane (Tris), plus 1 mM Mg2+, pH 7.5; (B), 50 mM Tris; (C), 50 mM Tris plus 1 M sodium chloride. (From Ref.91.)

that suggested that walls were "loosened" by autolysis, making masked receptors available for interaction with the lectin. Calculations suggested that at least one-half ofthe teichoic acid ofB. subtilis 168 was intercalated within the wall matrix and available for interaction with the lectin only after autolysis (or lysozyme digestion). Anderson et al. [147], Mobley et al. [40] Kirchner et al. [1481, and Kemper et al. [l491 appliedF1-ConA to the study of surface expansion in bacterial gram-positive rods, particularly B. subtilis. For several years, it was a mystery howB. subtilis expanded its surface during division. Several authors assumed that surface expansion in bacilli was analogous to that of streptococci, where a single growth zone defined the boundary of expansion. Mobley et al. [M]were able to make use of several known obserB. subtilis. First, ConA vations on the cellwallsandteichoicacidsof specificallyand reversibly bindsto cm-glucosylated teichoic acids. Second, teichoic acids and peptidoglycan are coordinately assembled inB. subtilis.


31

Finally, phosphoglucomutase mutants ( g t s ) cannot glucosylate their teichoic acids. Mobleyet al. used fluorescein-labeled ConAto study the insertion and fate of cell wallin temperature-sensitiveg t f c mutants of B. subtilis. They found (Fig. 9) that whenConA-reactivecells,grown at the permissive temperature, were shifted to the nonpermissive temperature,the lectin-reactive sites disappeared randomly over the cell cylinder surfaces, but were retained in the polar areas. In contrast, when cells were shifted from nonpermissive to permissiveconditions,ConA-reactivesites(new wall) were found very early in cell septa, but appeared diffusely in cell cylinders(Fig. 10). Oldpolesdid not bind the-lectin at all for several generations. The results were taken as evidencefor the diffuse intercalation of wall in the cell side walls during division process. Poles (matured septa) were consideredto be assembled ina manner analogousto that forstreptococci. Side walls seemed to elongate because of the random (or diffuse) addition of new wall polymers on the face of the wall near the plasma

0.24

0.20

0.12

0.04

0.00 0

30

60

90

120

150

AUTOLYSIS TIME (MIN)

Figure 8 Release of Cod-reactive teichoic acid from cell walls of B. subtilis 168. Autolysis was carried out at room temperature in 40 mM Tris-HC1 (pH 7.4). As autolysis proceeded, samples were withdrawn, heat inactivatedat 100°C for 15 min, and mixed with radioactive C o d (final volumes were 2.0 m1 containing 2.0 mg ConA of 1940 cpm/mg). After an incubation, Cod-teichoic acid or autolysate complexes were removed by centrifugation, washed, and radioactivity determined on supernatant fractions. Wallconcentration was 0.5 mg/ml. (From Ref.75.)

32

Doyle

Figure 9 Binding of fluorescein-ConA to B. subtilis gra strain C33 after a shift from permissive(35OC) to nonpermissive(45OC) conditions. Intervals (generations) after the shift: 0 (A), 1.6 (B), 2.8 (C), 3.2 (D) and 5.0 Growth rates were 33 min per generation atthe nonpermissive temperature. (FromRef. 40.)

(E).


33

Figure 10 Interactionbetweenfluorescein-ConAand B. subtilis gtaC33 after a shift from nonpermissive to permissive conditions. Cells were prepared for photography 0.8 generations afterthe temperature shift. Note that the cell poles are not as fluorescent as the cell cylinders or the septa. Growth rate in the minimal medium was 40 min per generation at the permissive temperature. Cells grown at the nonpermissive temperature were unable to bind the lectin.Bar, 15 pm. (From Ref.40.)

membrane, followed by even more addition of wall.New wall pushed old wall awayfrom the cytoplasmic sideto the cell surface. More recent refinements of the mechanisms of surface expansion of bacilli have been presentedbyKirchneret al. [l481 and byKemperet al. [149].The results provide a good descriptionof how a lectin (ConA)has been usedto study a major problem in bacterial physiology. Figure 11 shows that asthe temperature-sensitive bacilli are shifted from permissive to nonpermissive conditions, nearly three generations are required before most ConA-reactive sites are turned overor diluted. Presumably, these ConA-binding sites are residual side wall materialsand old poles. In contrast, when the cells are shifted from nonpermissive conditions, only one generation is needed for ConAreactive sitesto appear. The newly inserted sitesare from new poles (septa) and side wall material "pushed"to the outer surface.

34 LT

9

Doyle

260

180

120

40

1

2

3

4 5

1

2

3

4

GENERATIONS Figure 11 Agglutination of B. subtilis gtaC33 by C o d . The panel on the left

shows the amount of ConA required to aggregate the cells whenthe growth temperature was shifted from a permissive to a nonpermissive condition. Cells were removed, mixed with ConA and examined for aggregation under a dissecting microscope. The panel on the right shows the aggregation results when the cells were shifted from anonpermissive to permissive temperature. (Some ofthe results courtesy ofHLT Mobley.)

XI. MICROBIAL ULTRASTRUCTURE AS PROBEDBY LECTINS

Concanavalin A has also been employed to study the organization of teichoic acid in the wall ofB. subtilis 168 [150]. Thin sections ofthe walls of the bacterium were relatively uniform,but Cod-bound walls exhibitedan asymmetry (Figs. 12 and 13). The Cod-treated walls exhibited irregular fuzzy external surfaces, whereas untreated walls were relatively smooth. It was speculated that the lectin condensed the peripheral teichoic acids, resulting inthe observed asymmetry of the walls (Fig. 14). The current view is that the outer surface of the walls are serrated because of cellularturgor and autolysins [1491. In Staph. aureus, walls also seem to be serrated or rough on the outer surface. Morioka et al. [l481 observed that colloidal gold-WGA labeled partially autolyzed cells very densely (Fig.15). Wall material could be observed sloughing off fromthe cell surface. The sloughedoff wall material


35

... ,

"

Figure 12 Thin sections of B. subtilis 168. (a) Untreated; (b) treated with ConA. (From Ref. 150.)

is, no doubt, due to turnover of wall during normalcell surface expansion [92]. Figure 15 shows the labeling pattern ofWGA-gold on S. auras. Morioka et al. [l511 considered that the WGA was binding to cell wall teichoic acids. They did not show micrographs of isolated walls, but because of staining of thin sections postembedding, were able to show the binding pattern of the lectin on the inner- and outer-wall faces. Interestingly, the WGA seemed to bind to three distinct layers in the septum or

36

Doyle

Figure 13 Thin sections of cell walls of B. subtilis 168. (a,b) untreated controls; treated with C o d . (From Ref. 150.)

(c,d)

cross-wall. When staining with WGA-gold was done in the presence of GlcNAc there was no evidence of labeling. Electron microscopic techniques with lectin probes have been used to study the surface localizationof lipoteichoic acids in group A streptococci [152]. Concanavalin A labeled with ferritin was employedto show that the lipoteichoic acid (LTA) could be found on the outer surface of the cell. Pepsin digestionof the cells resulted in increased binding of ConA-ferritin. Furthermore, isolated walls were also capable of binding the lectin, but

37


only on the periphery ofthe walls. Strains exhibiting high hydrophobicities bound more lectin than the hydrophilic strains. The results are consistent with the view that LTA molecules are surface-exposed ingroup A streptococci and may serve in interacting with mucosal cells. The specificity of ConA binding to cellular (and wall) LTA was confirmed by first mixing the lectinwithpurifiedLTA. The purifiedLTAcaused a reduction in the binding of ConA to cell surfaces. The resultsare interesting from the viewpoint that a membrane-anchored LTA molecule can become exteriorized and bind stronglyto cell walls. The walls must have complementary receptors for the LTA molecule. The addition of LTA to LTA-depleted walls was not attempted, although such experiments may be useful in establishing the identity of LTA receptors on cell walls. The ConA-ferritin (and gold) probes employed [l521 demonstrate the value of lectins in characterizing microbial surface adhesins. Maruyama etal. [l011 isolated twomutants of E. coli that were highly sensitive to sodium dodecyl sulfate (SDS). Both of these mutants were of U n t r e a t e dC e l lW a l l s

/ Cell Wallsfollowing FixationandDehydration

Surfacr Teichoic Acid

Stainable Cell Wall

CellWalls plus Concanavalin A Concanavalin A eeeeeeeeeeeeee*eee

Figure 14 Schematic representation of the organization of the cell surface of B. subtifis 168. (From Ref. 150.)

38

Doyle

Figure 15 Binding of wheat germ agglutininto cell wallsof Stuphylococcusuureus.

(A) Thin section of Stuph. uureus incubated with WGA-gold. Label is found on both the inner and outer wall faces. (B) Same as A, but the label is shown to bind strongly to aseptum.(C)Interaction of WGA-goldwithapartiallyautolyzed Stuph. uureus. (From Ref. 151.)

readily aggregated by ConA, but the parent isogenic strain was relatively refractory to the lectin. Presumably, the mutants contained defective outer membrane components,leading to exposure of glycoconjugates on the plasma membrane. Maruyama [153-1551 employed the agglutinability by ConA to assay for spheroplast formation in Escherichia coli. The identity


39

the glycoconjugate(s)on the spheroplast surface capable of interacting with ConA hasnot been defined, although lipopolysaccharide structures may be involved. Lectins have been employed to study the surface carbohydrates in various morphological forms of rumen fungi [156]. For example, the fucoseand specificLaccaria amesthystinalectin bound spores, flagella, sporangia, rhizoids of a Neocaiiimastix strain, but in another strain, the flagella were unable to bind the lectin. Similarly,the same lectin boundto the rhizoids of some Piromonas strains, but not to others. Lectins not only distinguished between various morphological structures, but also distinguishedthe genera Neocaiiimastix, Piromonas, and Sphaeromonas [156]. The lectin-binding sites were located on the fungal structures by fluorescence. Lectins thus serve as powerful probes to study the surface differentiation of life cycles of fungi. Bonfante-Fasolo et al. [l571 were able to use WGA-gold to localize chitin in the spore wallsof the fungus Glomus versiforme. Chitin was localized in the fibrillar wall components. Labeling was not observed in the cytoplasm or the areas separating primary and secondary wall. The ultrastructural results are in agreement withthe notion that chitin synthesis occurs at the plasmalemmalevel.Laterstudiesbythese authors [158], employing lectin-gold markers, were directed to determining the surface location of carbohydrates inthe fungal symbiont,Hymenoscyphus ericae. They showed that WGA-gold bound exclusively to septa and to an inner electron transparent layer on longitudinal walls. The ConA-reactive material was seen to be radiating from the wall of infective strains, suggesting that this material was involved in adhesion to host cells. In fact, when the infective strain was in contact with the host, there wasevidence for an abundance of ConA-gold sites. The results may be useful in establishing the molecular basisfor fungi-plant interactions. Miragall et al. [l591 and Rico et al. [l601 have studied the surface expansion in Candida aibicans, an opportunistic fungal pathogen. They observed that protoplasts of C. aibicans lacked WGA-gold-reactive sites (or WGA-peroxidase sites), but when the protoplasts were allowed to regenerate their wall materials, the WGA sites were synthesized slowly. The initial WGA-reactive sites were found on or near the plasmalemma, but later sites were marked rather uniformly overthe entire surface. TheConAferritin sites appeared on the very outermost wall layer as the protoplasts were converted into vegetative cells. Althoughthe ConA sites were considered to besynthesizedonly during later stages ofdivision, there is no obvious explanationof how the sites are assembled on the surface. Rico et al. [l601 established that a lytic process in localized areas of the wall was required for surface expansion. This process may result in cell wall turnover [92], but this is not known with certainty.It is thought that yeasts add their

40

Doyle

newly synthesized materialsat sites of septationand at diffuse sites covering the growing bud. Concanavalin A is a marker for the mannoproteins of yeasts, including C.albicans [31,37,94]. From the results of Miragall et al. [l591and Rico et al. [160], it seems that the assembly of yeast cell wall occurs in distinct steps. The initial steps involve the synthesis of chitin (WGA-reactive), whereas the latter steps incorporate mannoproteins (ConA-reactive). This is one example of the application of lectinstheinstudy of microbial growth processes. XII. INFLUENCE O F LECTINS ON MICROBIAL PHYSIOLOGY In an interesting applicationof lectins in microbial physiology, itwas observed that ConA preventedthe uptake of DNA byB. subtilis [ 1611. Compethe lectin and transforming DNA tent cultures of B. subtilis were mixed with added. TheConA prevented the expression ofnew genetic markers, but the effect of ConA was negated by methyl-a-D-glucoside. In control experiments, it was established that the lectin had no effect on cell viability. It appears that ConA sequestered potential DNA receptors (probably teichoic acids) on the cell surface, rendering the cells incapableof bindingthe DNA. Although ConA is unable to aggregate B. cereus 14579, the lectin can induce several physiological responses the in organism. For example, ConA stimulated growth rates and culture yields of the bacterium [162-1641. In addition, it increased oxygenuptake, and RNA, DNA, and protein synthesis in cultures of B. cereus. The enhanced physiological responses were unrelated to the lectin serving asa source of amino acids, because methyl-aD-mannoside reversed the effects of ConA and, in control experiments, it was shown that the lectin was not internalized. Cell fractionationsrevealed that ConA was capable of binding the cytoplasmic membrane,but not the cell wall. These experiments are analogous to the now familiar studies on ConA-induced mitogenesis of lymphocytes. There is no obvious explanation for the effects of ConA on B. cereus. The authors speculate the cyclic guanosine 3’3 ’-monophosphate (cGMP), also increased in the presence of the lectin, may regulate macromolecular synthesis. As far as is known, these studies have not been followed up. Additional studies are needed to define the mechanism of how ConA triggers physiological responses in bacilli. Lectin-enhanced metabolism couldbe of significant value in industrial microbiology. X111. LECTIN-BINDING PROTEINS OFBACTERIA

Only a few bacteria are able to glycosylate their proteins [165]. The most widely studied glycoproteins of bacteria belong to the Archaeobacteria, although a few eubacteria have been reported to synthesize carbohydrate-

lectin-Microorganism

41

conjugated proteins. Bose et al. [l661 reported that purified elementary bodies of the pathogen, Chlamydia trachomatis, were unable to bind to HeLa cultures inthe presence of WGA. Later, Swanson and Kuo [167,168] were able to show that C. trachomatis proteins could bindto ConA, DBA (Dolichos biflorus), Ulexeuropaeus (Ulex-I), SBA (soybean agglutinin), and PNA (peanut agglutinin). The results were consistent with the bacteriumhavinglectinreceptorsconsisting of mannose,fucose, and Nacetylgalactosamine (or galactose). Two proteins of 18 and 32 kDa were isolated, and when treated with periodate, were unableto bind the lectins. Furthermore, the two polypeptides were able to bind to the surfaces of HeLacells,suggesting that theywereadhesins.Theseproteinsmaybe candidates for a chlamydial vaccine. Mycoplasma pneumoniae,a wall-free bacterium, has been reported to [169]. These glycoproteins contain glycoprotein in its plasma membrane may bethe receptors for lectins knownto selectively interact with mycoplasmal membranes [90]. Kahane and Tully [90] found that WGA, RCA, and ConA would bind to cells of Mycoplasma and to plasma membranes, although the bindingof WGA was low. Proteolysis of the membranes led to an increase in lectin-reactive sites, suggesting that the carbohydrates may be partially masked in vegetative cells. In addition, the lectins tended to complex only the outer surface, showingthat carbohydrate distribution in the membranes was asymmetric. Later, Kahane and Brunner [l691 isolated a glycoprotein with a relative molecular mass (M,) of 60,000 from M. pneumoniae membranes. The glycoprotein contained about 7% of weight of carbohydrate (Glc, Gal, GlcN). No studies on the linkage of carbohydrate to peptide or carbohydrate to carbohydrate were performed, so it is not proved that the lectin receptors were indeed the 60,000 M, protein. Carbohydrates on bacteria may be recognized by macrophage surface lectins, leading to lectinophagocytosis [56]. Whether the binding of carbohydrates by macrophage lectins contributesto the pathogenesis of the organism isas yet unknown. A WELLA techniquewas used to detect a glycoprotein in the plasma membrane of the mollicute, Spiroplasma citra [170]. Membranes were extracted with chloroform/methanol to remove lipids and then fractionated on sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDSPAGE). The proteins were blotted onto nitrocellulose membrane filters, which were then overlaid with [3H]ConA. Autoradiography revealed the presence of a ConA-binding protein of about 84,000 M,. Affinity chromatography on ConA-agarose was then used to isolate the glycoprotein. Currently, no function has been ascribed to the glycoprotein. Furthermore, the nature of the linkage(s) between the polypeptide and its glycan substituent@)remains to be defined.

42

Doyle

A larvacidal toxin from spores of Bacillus sphaericus contains 12% carbohydrate, but cannot be purified by ConA chromatography [1711. Lectin probes have been employed to rule out the presence of carbohydrate on a surface array protein of Campylobacterfetus [172]. Many surface array proteins studiedto date have been glycoproteins. The C.fetus surface array protein, although highly hydrophobic, would not bind to ConA [172]. An interesting glycoprotein autolysin was isolated by Kawamura and Shockman [l731 with the aid of ConA-Sepharose chromatography. The autolysin was glucosylated, a rare finding for glycoproteins. It seems unlikely that glucosylation was by a nonenzymatic reaction, because subfractions ofthe autolysin contained oligomeric glucose residues. Further studies on possible glycoproteins in prokaryotes have been provided by Aitchison et al. [93]. They studied surface antigens of Strep. faecalis by electrophoresing whole-cell extracts, cell walls, and other cell fractions. Following SDS-PAGE, the gels were blotted onto nitrocellulose papers and mixed with lectin-peroxidase conjugates (WELLA).It was observed that lectins specific for L-fucose (UEA), D-glucose to D-mannose (ConA) and N-acetylglucosamine (WGA) boundto two prominent protein bands. Proper controls were run to eliminate the contributions of growth medium constituents. The results suggest that Strep. faecalis elaborates glycoprotein antigens.The attachment of the carbohydrates to polypeptide has not been studied, however. From the reports of Kawamura and Shockman [l731 and of Aitchison et al. [93], it seems that glycoproteins may be more common in prokaryotes than heretofore recognized. XIV. THE GRAM STAIN AND LECTINS

Sizemore et al. [l321 proposed that WGA could be used to selectively bind gram-positive cells, whereasthe gram-negative bacterium would failto interact with the lectin. They found that all gram-positive cells tested bound fluorescein-labeled WGA. Ody some Pseudornonassp.of the gram-negative bacteria yielded binding reactions with WGA. This study, although detailed in its testing of a variety of gram-positive and gram-negative bacteria, must account for some important exceptions. For example, many members of the genus Bacillus do not bind the WGA [76]. Furthermore, the gramnegative N. gonorrhoeae readily complexes with WGA [104]. Members of the genera Brucella and Yersinia have also been reported to interact with WGA [174,175]. Gram-negative bacteria with defective outer membrane, or with lipopolysaccharides containing 8-1,Clinked GlcNAc may be expected to interact with WGA. It seems unlikely that the proposed test will be adopted; however,it is possiblethat newly discovered lectins will be able to discriminate between gram-positive and gram-negative bacteria.


43

APPENDIX Sources and Specificities of Common Lectins Systematic Common name name

or Specificities source

Aaptos papillata Abramis brama

Sponge (M) (Isolectins) Fish (ABD)

Abrus precatorius Achatinafulica Achatina granulata Adenia digitata Adenia volkensii Aegopodium podagraria Agaricus bisporus

Jequirty bean (ABP) Snail (ACF) Snail (ACG) Shrub (ADD) Shrub (ADV) Ground elder (AEP) Common mushroom (AGP)

Agardhiella tenera Agaricus campestris Agrocybe aegerita Agropyrum repens

Red alga (AGT) Fungus (AGC) Mushroom (AGEE) Couch or quack grass (AGR)

Albizzia julibrissin Alcyonium digitatum Aleuria aurantia

Mimosa tree seed (ALJ) Marine cnidarium (ALD) Orange peel fungus (ALA)

Allium cepa Allium porrum Allium sativum AIIomyrina dichotoma Aloe arborescens Amaranthus caudatus Amaranthus leucocarpus

Onion (ALCE) Leek (ALPO) Garlic (ALS) Japanese beetle (AlloA) Aloe plant (Aloe) Inca wheat (AMA) Tropical herb (AML)

Amaranthus retrofexus Amaroucium stellatum Amphicarpaea bracteata

Pigweed (AMR) Tunicate (AMs) Hog peanut (AMB)

Androctonus australis Angelica archangelica Anguilla anguilla

Scorpion (ANA) Medicinal plant (ANA) Eel (AAA)

(GlCNAc/3-1,4), L-Rha >C Y - D - G ~ (Anti-B) B - D - G> ~~ GlcNAc D-G~ Sialic acid D-Gal b-~-Gal D-G~NAc GalD-l,3GalNAc; Gal Complex Complex Complex Gal@-1,3GalNAc; GalNAc (Anti-A) Complex CY- or B-D-G~ CY-L-FUC; CY-L-FUC CY1,2Gal D - M ~D-G~c ; D - M ~D-G~c ; Complex D-Gal& 1,4GlcNAc Complex D-Gal Galp-l,3GalNAc [Anti-M(T)] Glc, Man Complex GalNAca1,3GalNAc (Anti-A,) Complex (Anti A>H) Sialic acid Glycoprotein L - F u c c Y - ~ .[Anti~G~~

Anmilla rostrata

Eel (ANR)

L-FUC

Amphicarpeae edgeworthy Hog Peanut (AME)

ow11

44

Doyle

Systematic Common name name


Anopheles albopictus Antheraea pemyi Anthocidaris crassispina Antirrhinum majus Aplysia californica Aplysia depilans

Mosquito (ANAL) Chinese oak silk moth (AMP) Sea urchin (eichinoidin) Snapdragon (ANM) Sea hare (APC) Sea mollusc (APD)

Arachis hypogaea

Peanut (PNA)

Arion empiricorum Aristolachia galeata Artocarpus altilis

Snail (ARE) Medicinal herb (ARG) Breadfruit (ARA)

Artocarpus heterophyllus (integrifolia) Artocarpus lakoocha

Jackfruit (jacalin) (JCA)

Ascidia malaca Asteriasforbesi Astragalus onobrychis (distortus) Avena sativa Axinella polypoides

Sea squirt (ASM) Sea star (ASF) Milk vetch (ASO)

Complex a@)~-Gal Gala-l ,3GalNAc Glycoprotein Complex D-Galacturonic acid; D-Gal Gala-l,3GalNAc; Gal (Anti-TITk, Ths

Azolla caroliniana Balea perversa Bauhinia candicans Bauhinia carronii Bauhinia purpurea

Jackfruit (ATL)

Oat (OAT) Sponge (MP-I, 11) (Isolectins) Water fern (AZC) Snail (BAPE) Shrub (BAC) Shrub (BACA) Camel’s foot tree (BAP)

Complex D-Gal (Anti-B>A) Gala-l,3GalNAc~~ (Anti-T) GalB-l,3GalNAc (Anti-T) Gala-1,fGalNAc; CY-D-G~~ Gala-l ,6Glc >Gal Complex D - G ~ N A(AntiA) c O-D-G~C GalS-l,6Gal

D-G~ D - G (Anti-B,A) ~ D-Gal; GalNAc O-D-Gd GalS-l,3GalNAc; Gala-l,3Gal (Anti-T) .Bauhinia tomentosa Complex Shrub (BAT) Beta vulgaris Complex Beet root (BEV) Biomphalaria glabrata Water snail (BIG) (Isolectins) Fru >L-Gal >D - M ~ (Anti-AI>A2>B) Birgus latro Sialic acid Coconut crab (BIL) Sialic acid Boltenia ovipera Tunicate (BOO) Boodlea coacta Green alga (BOC) (Isolectins) Glycoprotein Botylloides leachii D-Gal Colonial asidian (BOL) (Isolectins) Bowringia milbraedii D - M ~ Shrub (BOM) Brachypodium sylvaticum Brome grass (BRS) (GlCNAC~-l,4),, > >GlcNAc


Systematic name

Common name

45

or source

Brussica oleracea Bryonica dioica Bryopsis hypnoides

Red cabbage (BRO) White bryony(BRD) Algae (BRH)

Butea frondosa

Bastard teak(BUF)

Calendula officinalis

Artichoke or pot marigold (CAQ Callinectes sapidus Blue crab (CAS) Calliphora erythrocephala(CAE) Blowfly Culpurinaaurea D-Ga1;GalNAc (CAA)

Specificities Complex GalNAc;Gala-l,6Gal D - G [Anti~ B >Awl1 Fucal,2Gal; Gala-l ,3GlcNAc D-G~c;Man

Complex D - M ~D-G~c ; (AntiA,B) Canavalia ensiformis JackA) (Con bean Mana-1;Glca-l; GlCNACa-l Canavalia gladiata Japanese jack bean (Con G) Mana;Glca Cancer antennarius California or blue crab (CAA) Sialic acid Canna indica Indian herb (CAI) Hydrophobic 8glucosides Capsicum annuum Hot herb (CAA) (GlcNAc&I ,4)" Garagana arborexens Siberian pea tree (CAAR) D-GalNAc (Isolectins) Caragnafrutex Siberianpea shrub (CAFR) D-GA [AntiB>A(H)I Carcinoscorpius rotunda Horseshoe crab (CarcinoSialic acid cauda (NeuAca-2,6Gal) scorpin) Carpopeltisflabellata alga Marine ( C M Glycoproteins Caucasotachea astro- (CAAS) Snail D-G~~NA (Anti c labiata A>H) Caulerpa paspaloides Marine alga (CAPA) Complex Cepaea hortensis acid Sialic(CEH)Snail Cepaea nemoralis (CEN)Snail D-GalNAc (AntiA>H) Cerastium tomentosum Snow in summer (CET) Complex Cercis siliquastrum Red bud or Judas tree (CES)Complex Chamaespartium sagittale Winged broom (CHS) Complex Channa leucopunctatus Fish(CHL)(Isolectins 1-111)GalNAccu1,3GalNAc; GalNAc Charybdisjaponica Crab (CHJ) D-Gal (Anti-B >A) Chelidonium majus Greater celandine (CHM) D-GlcNAc Cicer arietinum Complex (CIA)peaChick Citrullus vulgaris Watermelon Man;Glc;GlcNAc (CIV) Cladoniapyxidata Marine lichen (CLP) G1c;Man (Anti A > AB)

46


Doyle


Clavelinapicta Clerodendron inerme Clerodendron trichotomum Clitocybe geotropa Clitocybe nebularis Clupea harengus

Tunicate (CLPI) Asian shrub (CLI) Asian shrub (CLT) Fleshy mushroom(CLG) Nebelkappe (CLN) Herring egg (CLHO)

scoparius Cytisus sessilifolius

Shrub (CYSE) (Isolectins)

Complex Complex D-Ga1NAc;Gal U-L-FUC ~-GalNAc;Gal D-Rha,D-Gal (AntiB >A,) Codium fragile Sponge seaweed (COF) D-G~~NA (Anti-A) c Colchicum autumnale Meadow saffron (COA) a,P-D-Gal;GalNAc Colinus coggygria Cashew likeshrub (COC) Glycoprotein Colocasia esculenta Taro (COE) (Isolectins) Complex Coronilla varia Crown vetch (COW D-Ga1;GalNAc (AntiA>B) Cotinus ceggyaria Smoketree (COCE) Complex . Crassostrea virginica Oyster (cRv) Complex Crenomytilus grayanus Mussel (CRG) D-Gal Crotolaria aegypteaca Middle Eastshrub (CRA) D-GalNac (Anti A>H) Crotolaria juncea Sunhemp (CRJ) D-Gal >GdNAc Crotolaria striata Shrub (crotalarin) D - G ~ ~ N> AD c -G~ (Anti-A) Croton tiglium (Euphorbiaceae)(CRT) Complex Cucumis sativa Cucumber (CUS) Glycoprotein Cucurbita maxima Winter squash (CUM) (GlcNAc&l,4),, Cucurbita pepo Squash (CUP) (GlcNAcj3-1,4),; GlcNAc Cuscuta europea(gronovii) Dodder (CUE) Complex Cycad siamensis Primitive gymnosperm (CYS) Complex Cynara scolymus Globe artichoke (CYSC) Glycoprotein Cystocloniumpurpureum Marine alga (CYP) Complex Cytisus (Sarothamnus) Scotch broom(CYSCO) D-Ga1;D-GalNAc

Datura innoxia Datura stramonium Daucus carota Dicentrarchuq labrax Dictyostelium discoideum

Harmless jimsonweed@AI) Thorn apple @AS) (or Jimsonweed) Carrot (DAC) Sea bass (DIL) Slime mold (discoidin)(Isolectins)

(GlCNAC/3-1,4),,; cellobiose; L-FUCU-~ ,2Gal [Anti O(H), AJ (GlCNAC&1,4), GalP-I ,3(4)GlcNAc (GlCNAC&l,4), D-FUC(Anti-H) D-Ga1NAc;D-Fuc

s


47


Dictyota dichotoma Didemnum candidum


Dolichos biflorus

Brown alga (DID) Ascidian (DIC-1,II) (Isolectins) (similar to jack bean) (DIG) Horse gram @BA)

Drosera rotundifolia Echinocereus engelmanii Electrophorus electricus Elettaria cardamomun Eobania vermiculata

Sun dew (DRR) Cactus (ECE) Electric eel (EEL) Cardomon (ELC) Snail (EOV)

Eranthis hyemalis

Winter aconite root @RH)

Dioclea grandiflora

Erythrina corrallodendron Coral tree (ECor) Erythrina cristagalli

Coral tree (ECA)

Erythrina variegata Euhadra callizona (amaline) Euhadra periomphala

Coral tree (EVA) Snail (EUC)

Euphorbia antiquorum Euphorbia cyparissias Euphorbia heterophylla Evonymus europaeus Fagopyrum esculentum Falcata japonica

Evergreen (EUA) Cypress spurge @VC) Evergreen (EUH) Spindle tree (EUE) Buckwheat (FAE) Shrub (FAJ)

Fomesfomentarius

Sapwood rot (FOF')

Fucus vesiculosus Galanthus nivalis Galega officinalis Ganoderma lucidum (applanatum) Geodia cydonium

Brown alga (FUV) Snow drop (GAN) Goat's rue (GAO) Fairies teaspoon (GAL)

Genista tinctoria

Greenweed (GET)

Snail (EUP)

Sponge (GEC)

Complex Gal@-l,4)Fru; Gala-1,4GlC;D-Gal D-Man >D-G~c GalNAca1.3GalNAc (AntiA, > A, > Cad) Complex Complex D-Gal Complex D-G~~NAc (AntiA>H) Gala-l ,4GlcNAc; Gala- 1,4Glc (AntiOW)>A'B) Gala- 1.4GlcNAc; GlcNAc>D - G ~ GalP-l,4GlcNAc; Galoll,6Gal;Gal Gala-l '4GlcNAc Complex D-G~~NAc (AntiA > H) D-G~ a-~-Gal D - G ~ ~ N> ALac c Gala-l,fGal (Anti-B) Complex D-Ga1NAc;Gal (AntiAI > A 3 a-~-Gal;GalNAc (Anti-B>0) Complex Mana-l,3Glc ~-Glc;Man 8-D-Gal Gala-l,4GlcNA~; Gal/3-1,3GlcNAc; Ga1;GalNAc Complex

4a

Systematic name Geum urbanum Glossinafuscipes

Doyle

Common nameor source

Town herb (GEU) Tsetse fly (TSF)(numerous

Specificities Glycoprotein Complex

other Glossinaspp also produce lectins) Glycine max (soja) Soybean (SBA) (Isolectins) I GalNAca(or/3)1,3 Gal;D-Gal 4-0-Methyl-DI1 glucuronic acid Gracilaria tikvahiae Ceylon moss (GRT) Sialic Acid (Anti(confervoides) &B) Grvfithsiaflocculosa Red(GRF) Complex alga(activity enhanced by GalNAc or GlcNAc) Griffoniasimplicifolia African legume CS-I a-D-Ga1;D-GalNAc (Anti-A,B) (Isolectins) CS-IB., a - ~ - G a(Anti-B) l CS-IAB, a-D-Gal; D-G~~NAc (Anti-A,B) GS-IA,B, a-D-Ga1;D-GalNAc (Anti-A,B) GS-IA3BI a-D-Ga1;D-GalNAc (Anti-A,B) CS-I& a-~-GalNAc (Anti-A) CS-I1 D-G~cNAc(AntiB,Tk,T) CS-IV WL-FUC-I ,2Gal (Anti-Leband Y) (I) D-Gal >GalNAc Grifolafrondosa Mushroom (GRFR) (Isolectins) D-GalNAc (11) Gypsophila elegans Complex Maiden’s breath (GYE) L-FUC; D-GlCu; Halichondricpanicea Marine sponge(&W) D-GalU Halocynthia roretzi Solitary ascidian (HAR) DGal; Me1 Hardenbergia comptoniana ShrubRaf (HAC) annus Helianthus Complex (HEA) Sunflower aspersa Helix snail Garden WEAS) a-D-GalNAc (Anti-A) hortensis Helix Snail (HEH) acid Sialic Helix Iucorum Snail (HEL) a,@-GalNAc Helixpomatia Garden snail (HEP) GalNAca1,3GalNAc (Anti-A)


49



Helleborus purpurascens Hellebore (purplebear's foot) (HW Hevea brasiliensis Rubber tree (HEB) Hippeastrum spp Amaryllis (HIA) Homarus americanus Lobster (HOA)(L-Agl) (Isolectins) (L-Ag2) Hordeum vulgare Barley (HOV) Hura crepitans Sand boxtree (HUC) Hypnea japonica Marine alga(HYJ) (Isolectins) Hyptis suaveolens Tropical tree (HYS) Iberis amara

Shrub (IBA)

Iberis umbellata Iphygena plicatula

Shrub (IBU) Snail (IPP)

Ipomoea rubrocoerulea (purpurea) Ischnoderma resinosum Laburnum alpinum

Morning glory(IPR) Mushroom (ISR) Golden chain (LAA)

Complex GlcNAc@-l,4),, Ma11~~-1,3Gal;Mana Sialic acid D-G~~NAc GlcNAc D - G ~ ~ N> AG cal Glycoproteins Ga1;GalNAc (Anti-A) Complex [AntiM(M +N)1 D-Gd D-Gal;Rha [AntiB,A(H)I D-G1u;Man

8-D-Gal (Anti-A) L-Fuccu-l,2Gal [Anti-O(H)] Laburnum anagyroides Bark (LBA) L-FUC [Anti-OW)] Laccaria amethystina Mushroom (Isolectins) 8-D-Gal;D-GalNAc L-FUC [Anti-O(H)] (LAAM) Lactarius deliciosus Mushroom (milkycap) (LAD) GdD-l,3GalNAc Lactariusperlatum Red pepper (LAP) P-D-Gd Laelia autumnalis Orchid (LAAU) a,P-D-Gal (AntiA,>Az>Le"> Le? Lathyrus ochrus Vetchling (Isolectins) (LoL I, D-M~II;D-G~c 11) Lathyrus odoratus Sweet pea (LAO) D-M~II >D-Gk > GlcNAc Lathyrus sphaericus Pea (LAS) D-Man;Glc Lathyrus sylvestrus Purple sweet pea (LASY) D-GalNAc (AntiA>H) Laurencia undulata Algae (LAU) D-Gd (AntiB>A>H) Laurus nobilis Bay leaf (LAN) Complex Leciniaria biplicata Snail (LEB) D-Ga1,Rha [AntiB,A(H)I Lens culinaris (esculenta) Lentil (LCA) Mana-1;Glca-1;NAc Leonurus cardiaca Motherwort (LEC) 8-D-GalNAc (AntiCad, but not Tn)

50

Systematic name

Doyle

Common name or Specificities source

Lepidium sativum Complex (LES) Garden cress Leucojum vernum White snowflower (LEV) D - M ~ Levisticum officinale Aromatic herb (LEO) Complex Limaxjlavus acid Sialic (LFA) Slug Limuluspolyphemus Horseshoe crab (limulin) (LIP) Sialic acid Listera ovata (LIO) Twayblade D - M ~ Litchi chinensis Lychee nut Complex (LIC) Lonchocarpus capassa Complex (LOC) leaf Apple Lophocereus shotti(LOS) Cactus Complex Lotononis bainesii Perennial pasture legume Complex (LOB) Lotus tetragonolobus Asparagus (LOTUS) (winged ~ - F u ~ ~ - l , 2 G [Antial Pea) O(H)1 Luffa actangula Gourd (LUA) (GlCNACO-l ,4), Lumbricus terrestris Earthworm (LUT) Complex Lycoperdon perlatum Puff ball mushroom (LYP) Complex Lycopersicon esculentum Tomato (LYE) (GlCNACP-l ,4), Lygos monosperma Willow-like reedtree (LW)Complex (Anti-Cad, but not T,Tk,T,,, Lygos sphaerocarpa

TJ

Reed tree Complex (LYS) (Anti-T,

T,, but not Cad) Algae (LW) Complex Baja cactus (MAE) (Isolectins) a-L-Fuc;D-GalNAc Osage orange (MAP) GalO-l,3GalNAc (Anti-T) Macrotyloma axillare Shrub (MAAX) D-GalNAc (AntiA, > A 3 Mangifera indica Mango (MA11 Complex Marasrnius oreades Fairy-ring mushroom (MAO) D-Gal (Anti-B>0) Medicago lupulina Black medic (MEL) Glycoproteins Medicago sativa (truncu- Alfalfa (MES) D-Man;Glc lata) Megabalanus rosa Barnacle (MER) (Isolectins) D - G ~ Megapetaria squalida D-G~NAc Clam (MES) Microciona porifera Marine sponge (MIP) Complex Moluccella laevis Irish bell (MOL) Complex (Anti-A,N) Momordica charantia D-Gal >GalNAc Bitter pear melon(MOC) Moringia olifera Moringin Complex Myrica gale Sweet gale (MYG) Complex Narcissus pseudonarcissus Daffodil ( N A P ) Man~~-1,3;Mana-1,6 Ocimum basilicum Basil (OCB) Complex Octopus vulgaris (bairdi) octopus (OCV) 8-D-Gal

Lyngbya majusarla Machaerocerus eruca Maclura pomifera


51


or source Specificities

Oncorhynchusspp Onobrychis viciifolia Ononis hircinia Ononis spinosa Origanum vulgare Oryza sativa Otala lactea

Salmon eggs Sanfoin (ONV) Restharrow (ONH) Spiny restharrow (ONS) Oregano (ORV) Rice (ORS) Snail (OTL)

Pachycereus pringlei

Cactus (PAP)

Palmariapalmata Papaver dubium Papaver somniferum

Marine alga(PAP) Doubtful poppy (PAD) Sleep-bringing or opium POPPY (PAS) Parsnip (PASA) Lichen (PEC) Brown alga (PEC) Perch (PEF) Cockroach (PEAM) Avocado fruit seed (PAA) Lamprey (PEM) Parsley (PEC) Stinkhorn mushroom (PHI) Fungus (PHC)

Pastinaca sativa Peltigera canina Pelvetia canaliculata Percaflaviatilis Periplaneta americana Persea americana Petromyzon marinus Petroselinum crispum Phallus impudicus Phanerochaete chrysosporium Phaseolus acutiofolius Phaseolus coccineus Phaseolus lunatus(I, 11) Phaseolus vulgaris

Phlebotomuspapatasi Phlomisfruticosa Phlox drumondii Phoradendrom californicum Photoliota squarrosa

a,B-~-Gal CY-M ~lC >C~Y-G CY,S-D-G~~;D-G~NAC cw-D-Giil Complex D-GlcNAc D-GalNAc (AntiA>H) Complex (AntiA>B) Complex (GlCNACS-l,4)n (GlcNAc(3-l,4)n Complex Complex Unknown D-G~c;D-M~~;L-Fuc Complex Complex Complex Complex Complex (GW1,4)n

Terpary bean (PHA) (Isolec- Complex tins) Scarlet runner bean (PHC) Unknown Lima bean (LBA) (Isolectins) GalNAca-l,3Gal (Anti-A) Red kidney bean(PHA) (Iso- Complex lectins) GalP-1,4 GlcNAc(3-1, Z M ~ ~ C Y (Anti-A) Sandfly (PHP) (numerous Complex other Phlebotomus spp also produce lectins) Jerusalem sage(PHF) Me1;GlcNAc; 2-deoxy-~-Gal Ornamental thread (PHD) D-G1c;Man D-G~ Desert mistletoe (PHCA) Broad-leaf tree (PHS) CY-L-FUC

Doyle

52



Pichia anomala Pila globosa Pisum sativum Plecoglossus altivelis Pleurotus ostreatus

Common reed(PHAU) (Iso- D-G~NAc lectins) Pokeweed (PWM) (Isolectins) Gal&1,4(3)GlcNA~; (GlCNAC,/3-1,4),; Manar-l,2Man Yeast (PIA) (GW-1A n Sialic acid Snail (PIG) Manu-1;GlW-l Pea (PEA) L-Rha (Anti-B) Fish egg (PLA) Fucosyllactose [AntiMushroom (PLO)

Pleurotus spodoleucus

Mushroom (PLS)

Plumaria elegans Polyandrocarpa misakiensis Polygonatum multiforum

Marine alga(PLE) Tunicate (POM)

Phragmites australis Phytolacca americana

Polygonumpersicaria Psathyrella velutina Pseudomonas aeruginosa

Perennial flower, USSR (POMU) Buckwheat or lady’s thumb smartweed (POP) Mushroom (PSBacterium (PA-I, PA-11)

om11

Fucosyllactose [AntiO(W1 Complex D-Gal Complex D-G~ D-G~cNAc Thiogalactosides

>

Psophocarpus tetragono- Winged bean (PST) lobus South Afr. kiatt tree (PTA) Pterocarpus angolensis Red marine algae(PTP) Ptilotaplumosa English or red oak (QUR) Quercus rubra Frog eggs (RAC) Rana catesbaiena Frog (RAJ) Rana japonica Radish ( M S ) Raphanus sativus Garden rhubard (RHR) Rheum rhabarbarum Reduviid bug(RHP) Rhodnius prolixus Ricinus communis

Castor bean (Isolectins) (RCA-1,II)

Robinia pseudoacacia Rumex obtrisfolia

Black locust (ROP) Bitterdock (RUD)

gal;

L-Fuc;L-Gal; D-Man D-Gal >D-G~NAc (Anti-A,B) D-G~c,D-Man D - G (Anti-B) ~ (GlcNAC/3-1,4), Complex, Sialic acid Complex Glycoproteins Glycoprotein D-ManNAc; D-G~NAc; D-Gal Gal&1,3(4)GlcNAc Gal&1,3GalNAc; GalNAc Complex Hydrophobic 8glycosides

Ledin-Microorganism


53


Rumexpatientia Shrub ( R U P ) Rutilis rutilus Fish egg Saccharomyces cerevisiae Yeast (SAC) Salk alba White willow ( S a ) Salmo solar Salmon (SAS) Salvia horminum Bluebeard shrub ( S A H ) Salvia sclarea Sambucus edulus Sambucus nigra Sambucus racemosa Sarcophaga peregrina Satureja hortensia Saxidomus giganteus Saxidomuspurpuratus Sclerotium rolfsii Secale cereale Sesamum indicum Simulium ornatum Soja hipspida Solanum alatum Solanum dulcamara Solanum melongena Solanum tuberosum Solieria chordalis Sophora japonica

Glycoprotein a-L-Rha >@-D-Gal D-Man (GlcNAc@-l ,4)n D-Gal (Anti-B>A) Complex (AntiT. Cad) GalNAccu-l ,ser Clary shrub (SAC) (or thr) (Anti-Tn) Daneworth (SAE) D - G (Anti-A>B,O) ~ Elderberry (SNA) (Isolectins D-Gal;GalNAc; Sialic acid 1-11) Complex Shrub (SAR) Complex Flesh fly (SAP) Savory herb ( S A H ) Complex Butter clam(SAG) D-GalNAc (Anti-A,) Shellfish (SAPU) (Isolectins) IX-D-G~CNAC GlC/3-1,3Glc Fungus (SCR) (Isolectins) Rye (SCL) D-GlcNAc Sesame (SEI) D-G~cNAc Blackfly (SIO) Complex Bean (SOH) ~-GlcNAc;Gal Winged nightshade (SOA) (GlcNAc@-1,4),, Woody nightshade (SOD) Complex Eggplant (SOM) (GlcNAc@-1,4), Potato (STA) (GlcNAc@-l,4)ps Marine alga(SOC) Glycoprotein Japanese pagoda tree (SOJ) Gal@-1,3GalNAc; Gal@-1,3GlcNAc [Anti-B> A >

+

ow11

Sorbus aucuparia

Complex

Streptomyces spp Styela plicata Synadenium grantii Tachypleus tridentatus Tams bactata

Bacterium (STR) Tunicate (STP) Community seed tree (SYG) Asian horseshoecrab (TAT) Gymnosperm or Yew (TAB)

Tetracarpidium conophorum

Nigerian walnut (TEC)

Complex (Anti B>H) a-L-Fuc;D-Man Sialic acid ~t-~-Gal Sialic acid Hydrophobic @-glucosides Gal@-l,rlGlcNAc

European mountain ash (SDA) Spondyliosoma cantharus Sea bream (SPC)

54

Doyle


Tetragonolobus maritimus Thymus vulgaris Tichocarpus crinitus Tilia cordata Trichosanthes anguina Trichosanthes kirilowii Tridacna crocea Tridacna maxima Trifolium repens Trigonellaprocumbens Trimeresums mucrosquamatus venum Triticum vulgaris (aestivum) Tropaeolum majus Tulipa gesneriana UIex europaeus UImus glabria Ulva arasakei

Ulva arasakii Ulva lactuca Urtica dioica Vaejovisspinigerus Velesunio ambigus Verbascum blattaria Viburnum lantana Viburnum opulus Vicia cracca Vicia cretica Viciafaba Vicia graminea Vicia hyrcania

Vicia villosa

or Specificities source Winged pea(TEM) Glycoprotein Complex Thyme (TW Red alga (TIC) Complex Basswood (TICD) (GlcNAcp-1,4), Gourd (hair flower)(TU) Complex Chinese gourd (TRK) D-Gal Mollusc (TRC) D-Gal Clam (TRM) Gala-l,4GlcNAc White clover (Trifoliin) 2-Deoxyglucose Fenugreek (TRP) Complex Poison scaly fungus(TRMU) Complex Wheat (WGA)

(GlcNAcp-l,4),, Sialic acid Nasturtium (TR") D-GANAc (Anti-A) Tulip (TUG) DMan Gorse (or furze) (Isolectins) ~-F~ca-l,2Gal[Anti(UEA-l, UEA-11) Om)];(GlCNACa1,412 [Anti-O(H)I Smooth elm (ULG) (GlCNACa-1,4), Algae (ULA) Complex [AntiA,H(O)I Algae (UAR) D-GalNAc (AntiA+H) Green marine algae (ULL) LY-L-FUC [Anti-O(H)] (GlCNAC&1,4), Stinging nettle (URD) Complex; sialic acids Scorpion (VAS) Complex Murray mussel (WA) Smooth moth plant (VEB) Complex Wayfaring tree (VIL) Glycoprotein Guelder rose(VIO) Glycoprotein D-GalNAc (Anti-A) Common vetch (VIC) Complex (AntiVetch (VICRE) TITh,A) Fava (broad) bean (favin) Maria-1;GlW-l Herb (VIG) Gala-l ,3GalNAc (Anti-N 0) Shrub (VIH) (Isolectins) Gala-l ;3GalNAc (Anti-T) GlcNAc,Glc (Anti-T,) Hairy vetch (WA) (Isolectins) GalNAca-l ,ser (Anti-TJ (or thr)



55

or source

Wisteriafloribunda

Mung bean (VIR) Cowpea (VILJ) Mistletoe (Isolectins) (ML-I, 11,111) Japanese wisteria (WIF')

Wisteria sinensis Xenopus laevis Xylaria polymorpha Zea mays Zingiber officinale

Flower (WIS) Frog OCEL) Mushroom (XYP) Maize (ZEM) Chinese ginger(ZIO)

Vigna radiata Vigna ungiuculata Viscum album

Specificities D-G~ D-Gal gal; D-G~WH, GalNAcar-l,6Gal; GalNAc;Gal DGal ar,B-~-Gal Complex D-Man;Gal;GalNAc Glycoprotein

Specificitiesdeterminedbyhemagglutination inhibition, equilibriumdialysis,fluorescence quenching, other. Specificities may reflectcontributions of penultimate residues, or peptides. In some cases, specificities reported in the literature do not reveal anomeric preferences or blood group reactivities. In other cases, reports may disagree. When di- or tri-saccharide specificities are shown, usually the lectin has an affinity for the nonreducing carbohydrate residue. The table does not list hydrophobic derivatives of saccharides, although frequently the hydrophobic derivatives are capable of binding with higher affinities than unmodified saccharides. Many lectins have a higher affinity for di-, tri-,or multi-, antennary complex carbohydrates than for linear carbohydrate sequences, but the table has been simplifed to include only the latter. Vertebrate lectins, although now recognized to be numerous, are not thoroughly reviewed inthe table. Finally, bacterial lectins, with the exception of Pseudomonas aeruginosa and Streptomyces spp, have not been purified in significant quantities and arenot described in the table. Fru = fructose; Fuc = fucose; Gal = galactose; GalNAc = N-acetylgalactosamine; GalNHz = galactosamine; Glc = glucose.GlcNAc = N-acetylglucosamine; Lac = lactose; Man = mannose, Me1 = melibiose; NeuAc = neuraminic acid, Raf = raffinose. Major commercial sources of lectins are E-Y Laboratories, San Mateo, CA (USA); Lectinola, Charles University, Prague, Czech Republic; Lectinotest, Lvov Medical Institute, Lvov, Ukraine and Sigma Chemical Company, St. Louis, MO (USA). Abbreviationsare based on common usage and on first two letters of the genus and first letter of the species. In some cases, it is necessary to employ the first two letters of both the genus and species. Table was derived from the reviews of Mogoset al. (61), Antonjuk et al. (62), Sharon and Lis (g), Liener et al. (8). B i d (20), Wu et al. (60).Etzler (M),Strosberg et al. (63), Goldstein and Poretz (M), Goldstein and Hayes (59). Garber et al. (66.67). Gilboa-Garber et al. (65), and Mandal and Mandal(68), commercial pamphletsand original papers of numerous investigators.

REFERENCES 1. Stillmark H. Uber Ricin, ein giftiges Ferment aus den Samen von Ricinus communis und einigen anderen Euphorbiaceen. (Ph.D. Thesis), University of Dorpat (Tartu), Estonia, 1888. 2. Boyd WC, Shapleigh E. Specific precipitating activity of plant agglutinins (lectins). Science 1954; 119:419.

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3. Goldstein IJ, Hughes RC, Monsigny M, Osawa T, Sharon N. What should be called a lectin? Nature 1980; 285:66. 4. Kocourek J, Horejsi V. Defining a lectin.Nature 1981; 290:188. 5. Dixon HBF. Defining a lectin.Nature 1981; 292:192. 6. Barondes S. Bifunctional properties of lectins: lectins redefined. Trends Biochem Sci 1988; 13:480-482. 7. Kocourek J. Historical background. In: Liener IE, Sharon N, Goldstein IJ, eds. The lectins:properties, functions, and applications in biologyand medicine. Orlando: Academic Press, 1986:l-32. 8. Liener IE, Sharon N, Goldstein IJ, eds. The lectins: properties, functions and applications in biology and medicine. Orlando: AcademicPress, 1986. 9. Sharon N, Lis H. Lectins. London: Chapman & Hall, 199O:l-27. 10. Pistole TG. Interaction of bacteria and fungi with lectins and lectin-like substances. AnnuRev Microbiol 1981; 35:85-112. 11. Doyle R, Keller K. Lectins in diagnostic microbiology.Eur J Clin Microbiol 1983; 3:4-9. 12. Slifkin M, Doyle RJ. Lectins and their application to clinical microbiology. Clin MicrobiolRev 1990; 3:197-218. 13. Doyle RJ, Slifkin M. Applications of lectins in microbiology. ASM News 1989; 55:655-658. 14. Sumner JB, Howell SF. The identification of the hemagglutinin of the jack bean with concanavalinA. J Bacterioll936; 32:227-237. 15. Renkonen KD. Studies on haemagglutinins present in seeds of some representatives ofthe family Leguminosae. Ann Med Exp Fenn 1948; 26:66-72. 16. Bird GWG. Specific agglutinating activity for human red blood corpusclesin extracts of Dolichos biflorus. Curr Sci 1951; 20:298-299. 17. Bird GWG. Relationship of the blood subgroups A,, A,, and A,B, A2B to haemagglutinins present in the seed of Dolichos biforus. Nature 1952; 170: 674. 18. Bird GWG. George Bird’s reminisces. In: BBg-Hanson TC, Freed DLJ, eds. Lectins, biology, biochemistry, clinical biochemistry, vol. 6. St. Louis: Sigma Chemical CO, 1988. 19. Makela D. Studies in hemagglutinins of leguminosae seeds. Ann Med Exp Fenn 1957; 35(suppl 11):l-130. 20. Bird GWG. Lectins in haematology and blood banking.Adv Haematol 1988; 17~125-148. 21. Crookston MC. Blood typing and cross-matching procedures. In: Rose NR, Friedman H, eds. Manual of clinical immunology, 2nd ed. Washington, DC: American Societyfor Microbiology, 1980:716-725. 22. Nowell PC. Phytohemagglutinin: aninitiator of mitosisin cultures ofnormal human leukocytes. Cancer Res 1960; 20:462-466. 23. Goldstein IJ, Hollerman CE, Smith EE. Protein-carbohydrate interaction. 11. Inhibition studies on the interaction of concanavalin A with polysaccharides. Biochemistry 1965; 4:876-883. 24. Kohler W, Prokop 0. Agglutination von streptokokken ver gruppe C durch ein agglutininaus Helixpomutiu. Z ImmunitaetsforschAllerg Klin Immunol 1967; 133~30-53.


57

25. Kohler WD, Prokop 0. Agglutinationsversuche am streptokokken mit dem phytogslutinin aus Dolichos biflorus. Z Immunitaetsforsch Allerg Klin Immunoll967; 133:171-175. 26. Prokop 0,Kohler W. Agglutinations reactions en von mikroorganiumen mit Helix pomatia eiweiss drusnextract. Z Immunitaetsforsch Allerg Klin Immunoll967; 133:176-179. 27. KohlerW, Prokop 0, Kuhnemund 0. Routine identification of group C streptococci by means an agglutinin (protectin) from the albumen gland of the edible snail,Helixpomatia. J Med Microbiol 1973; 6:127-130. and strep28. Wagner M. Interaction of wheat-germ agglutinin with streptococci tococcal cell wall polymers. Immunobiology1979; 15657-64. 29. Doyle RJ, Woodside EE, Fishel CW. Protein-polyelectrolyte interactions. The concanavalin A precipitin reaction with polyelectrolytesand polysaccharide derivatives.Biochem J 1968; 106:35-40. 30. Goldstein IJ, Staub AM. Interaction of concanavalin Awith polysaccharides of salmonellae. Immunochemistry1970; 7:315-319. 31. Tkacz JS, Cybolska EB, Lampen JO. Specific staining of wall mannan in yeast cells with fluorescein-conjugated concanavalin A. J Bacteriol 1971; 105: 1-5. 32. Archibald AR, Coapes HE. Blocking of bacteriophage receptor sitesby concanavalin A. J Gen Microbiol1972; 73581-585. 33. Birdsell DC, Doyle RJ. Modification of bacteriophage 425 adsorption to Bacillus subtilisby concanavalin A. J Bacterioll973; 113:198-202. 34. Doyle RJ, Birdsell DC, Young FE. Isolation of teichoic acid of Bacillus subtilis 168 by affinity chromatography. Prep Biochem 1973; 3: 13-17. 35. Martinez-Palomo A, Gonzales-Robles A, de la Torre M. Selective agglutination of pathogenicstrains of Entamoeba histolytica induced by concanavalin A. Nature 1973; 245:186-187. 36. Ebisu S, Lonngren J, Goldstein IJ. Interaction of pneumococcal S-l4 polysaccharide with lectinsfrom Ricinus communis, Triticum vulgaris,and Bandeiraeasimplicifolia. Carbohydr Res 1977; 58:187-191. 37. Stoddart RW, Herbertson BM. The use of fluorescein-labelled lectins in the detection and identification offungipathogenic for man: a preliminary study. J Med Microbiol 1978; 11:315-324. 38. Schaefer RL, Keller KF, Doyle RJ. Lectins in diagnostic microbiology: use of wheat germ agglutinin for laboratory identification of Neisseria gonorrhoeae. J Clin Microbioll979; 10:669-672. 39. Graham K,Keller K, Ezzell J, Doyle R. Enzyme-linked lectinosorbent assay (ELLA) for detecting Bacillus anthracis. Eur J Clin Microbiol 1984; 3:210212. 40. Mobley HLT, Koch AL, Doyle RJ, Streips UN. Insertion and fate of cell wall in Bacillussubtilis. J Bacteriol 1984; 158:169-179. 41. Schalla WD, Whittington WL, Rice RJ, Larsen SA. Epidemiological characterization of Neisseria gonorrhoeae by lectins. J Clin Microbiol 1985; 22: 379-382. 42. Karayannopoulou G, Weiss J, DamjanovI.Detection of fungi in tissue sections by lectin histochemistry. ArchPathol Lab Med 1988; 112:746-748.

58

Doyle

43. Slifkin M, Cumbie R. Rapid detection of herpes simplexvirus with fluorescein-labeled Helixpomatia lectin. J Clin Microbiol1989; 27:1036-1039. 44. Gilboa-Garber N. Lectins of Pseudomonas aeruginosa:Properties, biological effects and applications. In: Mirelman D, ed. Microbial lectins and agglutinins. New York: John Wiley & Sons, 1986:255-269. 45. Sharon N. Bacterial lectins,cell-cell recognition and infectious disease. FEBS Lett 1987; 217:145-157. 46. Aronson M, Medalia 0, Schori L, Mirelman D, Sharon N, Ofek I. Prevention of colonization of the urinary tract of mice with Escherichia coli by blocking of bacterial adherence withmethyl-a-D-mannopyranoside. J Infect Dis 1979; 139:329-332. 47. Wiley DC, Skehel JJ. The structure and function of the hemagglutinin membrane glycoprotein of influenzavirus. Annu Rev Biochem 1987; 56:365-394. acid adsorption of phage 48. Young FE. Requirement of glucosylated teichoicfor in Bacillussubtilis 168. Proc Natl Acad Sci USA 1967; 58:2377-2383. 49. Etzler ME. Distribution and function of plant lectins. In: LienerIE, Sharon N, Goldstein IJ, eds. The lectins: properties, functions, and applications in biology and medicine. Orlando: Academic Press, 1986:371-435. 50. Marsh WL. The effect ofplant root environment on the activity of Dolichos biflorus lectin. Vox Sang 1967; 12:68-70. 51. Mirelman D, Galan E, Sharon N, Lotan R. Inhibition of fungal growth by wheat germ agglutinin.Nature 1975; 256:414-416. 52. Callow JA. Recognition, resistanceand role of plant lectins in host-parasite interactions. Adv Bot Res 1977; 4:1-49. 53. Garas NE, Kuc J. Potato lectin lyses zoospores of Phytophthora infkstans and precipitates elicitors of terpenoid accumulation produced by the fungus. Physiol Plant Pathol 1981; 18:227-237. 54. Partridge J, Shannon L, Gumpf D. A barley lectin that binds free amino sugars. I. Purification and characterization. BiochimBiophys Acta 1976; 451:470-483. 55. Janzen DH, Juster HB, LienerIE. Insecticidal activityof the phytohemagglutinin in black beanson a bruchid beetle. Science 1976; 192:795-796. 56. Ofek I, Sharon N. Lectinophagocytosis: a molecular mechanism of recognition between cell surface sugars and lectins in the phagocytosis of bacteria. Infect Immun 1988; 56539-547, 57. Drickamer K. Two distinct classes of carbohydrate-recognition domains in animal lectins. J Biol Chem 1988; 263:9557-9560. 58. Drake D, Taylor KG,BleiweisAS,Doyle RJ. Specificity of the glucanbinding lectin ofStreptococcus Cricetus. Infect Immun 1988; 56:1864-1872. 59. Goldstein IJ, Hayes CE. The lectins: carbohydrate-binding proteins of plants and animals. AdvCarbohydr Chem Biochem1978; 35:127-340. 60. Wu A M , Sugii S, Herp A. A guide for carbohydrate specificities of lectins. Adv Exp MedBioll988; 288:819-847. 61. MogosSM, Stefan E, Hulea SA, Andras M, Trif M, Pop A, Buzila L, Dihoru G, Roman N, Motas C. Screening for new lectins in the Romania flora. Rev Rom Biochim 1989; 26:139-144.


59

62. Antonyuk VA, Formagyuk V, Levashou V. Use of lectins in microbiology.Z Mikrobiol EpidemiolImmunobioll987;6:97-104. 63. Strosberg A, Buffard D, Lauwereys M, Foriers A. Legume lectins: a large family of homologous proteins. In: LienerIE, Sharon N, Goldstein IJ, eds. The lectins: properties, functions, and applications in biology and medicine. Orlando: Academic Press, 1986949-264. D. Isolation, physicochemical characterization, and 64. Goldstein J, Poretz R carbohydrate-binding specificity of lectins. In: Liener E, Sharon N, Goldstein J, eds. The lectins: properties, functions, and applications in biology and medicine. Orlando: Academic Press,1986:33-247. 65. Gilboa-Garber N, SussweinA, Mizrahi L, Avichezer D. Purification and characterization of the gonad lectin of Apl'ysia depilans. FEBS Lett 1985; 181~267-270. 66. Garber N, Guempel U, Gilboa-Garber N, Doyle RJ. Specificity ofthe fucosebinding lectin of Pseudomonas aeruginosa.FEMS Microbiol Lett 1987; 48: 331-334. 67. Garber N, Guempei U, Belz A, Gilboa-Garber N, Doyle RJ. On the specificity of the D-galactose-binding lectin (PA-I)of Pseudomonas aeruginosaand its strong binding to hydrophobic derivatives of D-galactose and thiogalactose. Biochim Biophys Acta 1992; 1116:331-333. 68. Mandal C, Mandal C. Sialic acid binding lectins. Experientia 1990, 46:433441.

69. Jermyn MA. Flavonoid glycosides, beta-lectins and plant cell membranes. Proc Aust Biochem SOC1977; 10:89-104. 70. Rosenberg M, DoyleRJ. Microbial cell surface hydrophobicity: history, measurement and significance. In: Doyle RJ, Rosenberg M, eds. Microbial cell surface hydrophobicity. Washington, DC: American Society for Microbiology, 1990:1-37. 71. Firon N, Ashkenazi S, Mirelman D, Ofek I, Sharon N. Aromatic alphaglycosides of mannose are powerful inhibitors of the adherence of type 1 fimbriated Escherichia coli to yeast and intestinal epithelial cells. Infect Immun 1987; 55:472-476. 72. Bayer EA, Skutelsky E, Wilchek M The ultrastructural visualization of cell surface glycoconjugates. MethodsEnzymoll982; 83:195-215. 73. Damjanov I. Biology of disease. Lectin cytochemistry and histochemistry. Lab Invest 1987; 575-20. 74. Jackson M, Chan R, Matoba AY, RobinJB. The use of fluoresceinconjugated lectins for visualizing atypical mycobacteria. Arch Ophthalmol 1989; 107:1206-1209. 75. Doyle RJ, McDannel ML, Helman JR, Streips UN. Distribution of teichoic acid in the cell wall ofBacillussubtilis. J Bacteriol1975; 122:152-158. 76. Cole HB, Ezzell JW Jr, Keller KF, Doyle RJ. Differentiation of Bacillus anthracis and other Bacillus species by lectins. J Clin Microbiol1984; 19:4853. 77. Lotan R, Sharon N, Mirelman D. Interaction of wheat-germ agglutinin with bacterial cells and cell-wall polymers.Eur J Biochem 1975; 55:257-262.

60

Doyle

78. Connelly MC, AllenPZ. Chemical and immunochemical studies lipopolysaccharides from pyocin 103-sensitive and resistant Neisseria gonorrhoeae.Carbohydr Res 1983; 120:171-186. 79. Allen PZ, Connelly MC, ApicellaMA. Interactions of lectins withNeisseria gonorrhoeae. Can J Microbiol 1980; 26:468-474. 80. Hammarstrom S, Lindberg AA, Robertsson ES. Precipitation of lipopolysaccharides from rough mutants of Salmonella typhimurium by an A-hemagglutinin from Helixpomatia. Eur J Biochem 1972; 25:274-282. 81. Pistole TG. Bacterial agglutinins from Limulus polyphemus-an overview. Prog Clin Biol Res 1979;29547-553. 82. Gilbride KJ, Pistole TG. Isolation and characterization of a bacterial agglutinin in the serum of Limuluspolyphemus. Prog Clin Biol Res 1979; 29525535. 83. Owen P, Salton MRJ. Membrane asymmetry and expression of cell surface antigens of Micrococcus lysodeikticusestablished by crossed immunoelectrophoresis. J Bacterioll977; 132:974-985. 84. Archibald AR, Coapes HE. The interaction of concanavalin A with teichoic acids and bacterial walls. Biochem J 1971; 123:665-667. 85. Reeder WJ, Ekstedt RD. Study of the interaction of concanavalin A with staphylococcal teichoic acids. Immunoll971; J 106:334-340. 86. Doyle RJ, Birdsell DC. Interaction of concanavalin A with the cell wall of Bacillus subtilis. J Bacterioll972; 109:652-658. 87. Garland JM, Archibald AR, Baddiley J. An electron microscopic study of the location of teichoic acidand its contribution to staining reactions in walls of Streptococcusfaecalis 8191. J GenMicrobioll975; 89:73-86. 88. Hamada S, Gill K, Slade HD. Binding of lectins to Streptococcus mutans cells and type-specific polysaccharides, and effect on adherence. Infect Immun 1977; 18:708-716. 89. Kashket S, Guilmette KM. Aggregation of oral streptococci in the presence of concanavalinA. Arch Oral Bioll975; 20:375-379. 90. Kahane I, Tully JG. Binding of plant lectins to Mycoplasma cells and membranes. J Bacteriol 1970; 128:l-7. 91. Doyle RJ, McDannel ML, Streips UN, Birdsell DC, YoungFE. Polyelectrolyte nature of bacterial teichoic acids. J Bacterioll974; 118:606-615. 92. Doyle RJ, Chaloupka J, Vinter V. Turnover of cell walls in microorganisms. Microbiol Rev 1988; 52554-567. 93. Aitchison EJ, Lambert PA, Farrell ID. Antigenic composition an ofendocarditis associated isolate of Streptococcusfaecalis and identification of its glycoprotein antigensby ligand blotting with lectins. JMed Microbiol 1986; 21: 161-167. 94. Bauer H, Horisberger M, Bush D, Sigarlakie E.Mannan as a major component of bud scarsof Saccharomyces cerevisiae.Arch Microbioll972; 85:202272. 95. Halverson LJ, Stacey G. Effect of lectinon nodulation by wild-typeBradyhizobium japonicum and nodulation-defective mutant. Appl Environ Microbiol 1986; 51:753-760.


96. 97. 98. 99. 100. 101. 102. 103. 104.

61

Fogg GC, Yang L, Wang E, Blaser MJ. Surface array proteins of Campylobacter fetus block lectin-mediated bindingto type A lipopolysaccharide. Infect Immun 1990; 58:2738-2744. Holm SE, Bersholm A, Wagner B, Wagner M. A sialic-acid-specific lectin from Cepaea hortensis that promotes phagocytosis of a group-B, type Ia, streptococcal strain. J Med Microbiol 1985; 19:317-323. Staat RH, Langley SD, Doyle RJ. Streptococcusmutans adherence: presumptiveevidence for protein-mediated attachment followedbyglucandependent cellular accumulation. Infect Immun 1980; 27:675-681. Meade NA, Staat RH, Langley SD, DoyleRJ. Lectin-like activity from Persea americana.Carbohydr Res 1980; 78:349-363. Goldstein IJ, Misaki A. Interaction of concanavalin A with an arabinogalactan from the cell wall of Mycobacterium bovis. J Bacteriol 1970; 103:422425. Maruyama HB, Arisawa M, Ono-Onitsuka M. Simplified assay of concanavalin A-dependent bacterialagglutination by using cellsurface mutants. Infect Immun 1975; 11:1320-1324. Doyle RJ, KellerKF,Ezzell JW. Bacillus. In:Lennette EH, Balows A, Hausler HJ Jr,Shadomy HD, eds. Manual of clinical microbiology, 4th ed. Washington, DC: American Society Microbiology,1985:211-215. Delucca AJ 2nd. Lectin grouping of Bacillus thuringiensis serovars. Can J Microbiol1984;30:1100-1104. Doyle RJ, Nedjat-Haiem F, Keller KF, Frasch CE. Diagnostic value of interactions between members of the family Neisseriaceae and lectins. J Clin Microbiol 1984; 19:383-387. Senne JE. Lectin agglutination of Neisseriagonorrhoeae. Clin Microbiol Newslett 1981; 3:lO. Frasch EC. Role of lipopolysaccharide in wheat germ agglutinin mediated agglutination of Neisseria meningitidis and Neisseria gonorrhoeae. J Clin Microbiol 1980; 12:498-501. Yajko DM,Chu A, Hadley W K.Rapid confirmatory identificationof Neisseria gonorrhoeae with lectins and chromogenic substrates. J Clin Microbiol 1984; 19~380-382. Vazquez JA, Berron S. Lectin agglutinationtest as an epidemiological marker for Neisseria gonorrhoeae.Genitourin Med 1990; 66:302. Patchett R A , Kelley AF, Kroll RG. The adsorption of bacteria to immobilized lectins. JAppl Bacterioll991; 71:277-284. Hemming C. The kinetics of lectin-mediated cell agglutination. Adv Lectin Res 1991; 4 9 - 1 2 1 . Wong KH, Skelton SK, FeeleyJC. Interaction of Campylobacter jejuni and Campylobacter coliwith lectinsand blood group antibodies. J Clin Microbiol 1985; 22:134-135. Corbel JJ, Gill KPW. Lectin agglutination of thermophilic Campylobacter species. Vet Microbiol1987; 15:163-173. Gill KPW, CorbelMJ. Lectin agglutinationfor thedifferentiation of Campylobacterfetus serotypes. Vet Rec 1986; 118:158. '

105. 106. 107. 108. 109. 110. 111. 112. 113.

62

Doyle

114. Slifkin M, Gil GM. Rapid biochemicaltests for the identification of groups A,B,C,F, and G streptococci from throat cultures. J Clin Microbiol 1983; 18~29-32. 115. Slifkin M, Gil GM. Identification of group C streptococcal antigen extracts with lectin-bound polystyrene particles. J Clin Microbioll984; 19:83-84. 116. Pistole, T. Naturally occurring bacterial agglutininin the serum of the horseshoe crab Limuluspolyphemus.J Invert Path 1976; 28:153-154. 117. Wagner M. Agglutination of bacteria by a sialic acid-specific lectin of the snail Cepueu hortensis.Acta Histochem 1982; 71:35-39. 118. Slifkin M, Cumbie R. Identification of group-B streptococcal antigen with lectin-bound polystyrene particles. J Clin Microbiol 1987; 25:1172-1175. 119. Davidson SK, Keller KF, Doyle RJ. Differentiation of coagulase-positive and coagulase-negative staphylococci by lectins and plant agglutinins. J Clin Microbiol 1982; 15547-553. 120. Adam G , Heegard P, Bog-Hansen TC, Mundry KW. Lectins as probes for the assay of rhabdovirus infections in plants. J Virol Methods 1987; 17:263275. 121. Svennerholm B, Olofsson S, Jeansson S, Vahlne A, Lycke E.Herpes simplex virus type selective enzyme-linkedimmunosorbent assay with Helix pomutia lectin-purified antigens. J ClinMicrobioll984; 19:235-239. 122. Corbel MJ, Cockren DS, Brewer RA. Differentiation of smooth and rough Brucella strains by lectins. Vet Rec 1983; 113:261-262. 123. Chatterjee BP, Guha AK, Pal P, Bhattachayya M. Lectin typingof Pseudomonus ueruginosu strains of different serotypes, Habs and Fisher types. Zentralbl Bakteriol Hyg1989; A271:362-371. 124. Eaves DJ, Doyle RJ. Surface characteristics of Pseudomonas cepuciu. Microbios 1988; 53:119-128. 125. AvN I. Affra RC, RobinJB, Rao NA. Lectins for theidentification of ocular bacterial pathogens. MetabPediatr Syst Ophthalmoll987; 19:45-47. 126. Malkinson M, Orgad U, Becker Y. Use of lectins to detect and differentiate subtypes of Marek’s disease virus and turkey herpesvirus glycoproteins in tissue culture. J Virol Methods1986; 13:129-133. 127. deMiranda Santos IKF, Pereira MEA. Lectins discriminate between pathogenic and non-pathogenic South American trypanosomes. Am J Trop Med Hyg 1984; 33:839-844. 128. Korting HC, AbeckD, Johnson A P , BallardRC,Taylor-RobinsonD, Braun-Falco 0. Lectin typingof Huemophilus ducreyi. Eur J Clin Microbiol Infect Dis 1988; 7:678-680. 129. LeMinor L, Tournier P, Chalon AM. Agglutinabilite par la concanavaline a de divers bacilles a gram negatif. Etude chez les Sulmonellu de la correlation avec la structure antigenique 0. Ann Microbiol (Paris)1973; 124A:467-476. 130. Doyle RJ, Nedjat-Haiem F, Miller RD, Keller KF. Interaction between plant agglutinins and Legionellu species. J ClinMicrobioll982; 19973-975. 131. Nutsubidze NN, Lakhtin VM, Demina VA, Obraztsova IN, Marinova DN. Possibility of identifying microorganisms by the rate of their agglutination by lectins. Drikladnaya Bioklim Mikrobioll991; 27:274-278.


63

132. Sizemore RK, Caldwell JJ, Kendrick AS. Alternate Gram staining technique using a fluorescent lectin. Appl Environ Microbiol1990,562245-2247. 133. Levy NJ. Wheat germ agglutinin blockage of chlamydial attachment sites: antagonism by N-acetyl-D-glucosamine. Infect Immun 1979; 25:946-953. 134. Tsien HC, Schmidt EL. Localizationand partial characterization of soybean lectin-bindingpolysaccharideof Rhizobium japonicum. JBacteriol 1981; 145~1063-1074. 135. Lindberg B, Lonngren J, Powell DA. Structural studies on the specific type14 pneumococcal polysaccharide. Carbohydr Res1977; 58:177-186. 136. Gray BM, Dillon HC Jr,Pritchard DC. Interaction of group B streptococcal type-specific polysaccharides with wheat germ agglutinin and other lectins. J Immunol Methods 1984; 72:269-277. 137. Mattiasson B, Johansson P-A. A simple rapid methodfor quantifying microorganisms by their metabolic activitywhen bound to a specific adsorbent. J Immunol Methods 1982; 52:233-240. 138. Robinson JE, Holton D, Liu J, McMurdo H, Murciano A, GohdD. A novel enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to HIV-l envelope glycoproteins basedon immobilization of viral glycoproteins in microtiter wells coated with concanavalin A. J Immunol Methods 1990; 132:63-71. 139. Watanabe K, Takesue S, Ishibashi K. Reversibility ofthe adsorptionof bacteriophage PL-1to the cell walls isolatedfrom Lactobacillus casei.J Gen Virol 1977; 34:189-194. Ishibashi K, Takesue S, Watanabe K, Oishi K. Use of lectins to characterize l#. the receptor sitesfor bacteriophage PL-1 of Lactobacillus casei.J Gen Microbiol 1982; 128:2251-2259. 141. Ndulue A, Flandrois JP, Marmet D. Abnormal affinity of Staphylococcus aureus N-acetylglucosamine ribitol teichoic acidfor wheat-germ agglutinin. J Chromatogr 1981; 209:323-328. 142. Ndulue A, FlandroisJ-P. Immunochemical studies ofStaphylococcus aureus Oeding-Haukenesantigen a,: aphosphorus-containingpolysaccharide. J Gen Microbiol 1983; 129:3603-3610. 143. Hancock IC. The biosynthesis of wall teichoic acid by toluenised cells of BacillussubtilkW23. Eur J Biochem1981; 119:85-90. in the 144. Fiedler F, Bude A. Occurrence and chemistry of cell wall teichoic acids genus Brevibacterium. J Gen Microbiol 198; 1392837-2846. 145. Wicken AJ, Knox KW. Characterization of group N Streptococcus lipoteichoic acid. Infect Immun 1975; 11:973-981. 146. Leopold K, Fischer W. Separation of the poly(g1ycerophosphate) lipoteichoic acids of Enterococcus faecalis Kiel 27738,Enterococcus hirae ATCC 9790 and Leuconostoc mesenteroidesDSM 20343 into molecular speciesby affinity chromatography on concanavalin A. Eur J Biochem 1991; 196:475-482. 147. Anderson AJ, Green RS, Sturman AJ,Archibald AR. Cell wall assembly in Bacillus subtilis:location of wall material incorporated during pulsed release of phosphate limitation, its accessibility to bacteriophages and concanavalin A, and its susceptibilityto turnover. J Bacteriol1978; 136:886-899.

64

Doyle

148. Kirchner G, Kemper MA, Koch AL, Doyle RJ. Zonal turnover of cell poles of Bacillussubtilis. Ann Inst Pasteur (Microbiology) 1988; 139545-654. 149. Kemper MA, MobleyHLT, Doyle RJ. How do bacilli elongate?In: Actor P,

Daneo-Moore L, Higgins ML, Salton MRJ, Shockman GD, eds. Antibiotic inhibition of bacterial cell surface assembly and function, Washington, DC: American Societyfor Microbiology, 1988:98-108. 150. Birdsell DC, DoyleRJ, Morgenstern M. Organization of teichoic acid inthe cell wall ofBacillus subtilis. J Bacteriol 1975; 121 :726-734. 151. Morioka H, Tachibana M, Suganuma A. Ultrastructural localization of carbohydrates on thin sections of Staphylococcus aureus with silver methenamine and wheat germ agglutinin-gold complex. J Bacteriol 1987; 169:13581362. 152. Ryc M, Wagner B, Wagner M, Bicova R. Electron microscopic localization of lipoteichoic acid on group A streptococci. Zentralbl Bakteriol Hyg 1988; A269:168-178. 153. Maruyama HB. Agglutination of bacterial spheroplast. 1. Effect of concanavalin A. Biochim BiophysActa 1972; 2743499-504. 11. Agglutination154. Maruyama HB. Agglutination of bacterial spheroplast: 155. 156. 157. 158. 159. 160.

dependent degradation of Escherichia coliribosomal ribonucleic acid. BacJ teriol 1973; 115:47-51. Maruyama HB. Agglutination of bacterial spheroplast. 111. Relationship of labelled concanavalin Ato the agglutinability. J Biochem 1974; 75:165-170. Guillot J, Breton A, Damez M, Dusser M, Gaillard-Martinie B, Millet. Use of lectinsfor a comparative study of cell wall composition of different anaerobic rumenfungal strains. FEMS MicrobiolLett 1990; 67:151-156. Bonfante-Fasolo P, Vian B, Testa B. Ultrastructural localization of chitinin the cell wall of a fungal spore. Biol Cell 1987; 57:265-270. Bonfante-Fasolo P, Perotto S, Testa B, Faccio A. Ultrastructural localization of cell surface sugar residues in ericoid mycorrhizal fungi by gold-labeled lectins. Protoplasma 1987; 139:25-35. Miragall F, Rico H, Sentandreu R. Regeneration of the cell wall in protoplasts ofCandida albicans.A cytochemicalstudy using wheat germ agglutinin and concanavalin A. ArchMicrobioll988; 149:286-290. Rico H, Herrero E, Miragall F, Sentandreu R. An electron microscopystudy of wall expansion during Candida albicansyeast and mycelial growth using concanavalin A-ferritin labelling of mannoproteins. Arch Microbiol 1991;

156~111-114. 161. Chu C-Y, Chen K-C. Inhibition of DNA-induced transformation by concanavalin A inBacillus subtilis. Biochem Biophys Res Commun 1979; 91:170176. Bacillus species to concanav162. Chan K-Y, Lau T-M. Physiological responses of alin A. 1. The binding of concanavalin A to B. cereus ATCC 14579 and B. lichenifoms I F 0 12107. Microbios 1984; 39:121-128. of species to concanav163. Chan K-Y,Lau T-M. Physiological responses Bacillus alin A. 3. Effect on RNA, DNA and protein synthesis, and galactose uptake of B. cereus ATCC 14579. Microbios 1984; 40:195-204.


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164. Lau T-M,Chan K-Y.Physiological responsesof Bacillus species to concanavalin A. 2. Effect on growth, oxygen uptake, enzyme activitiesand intracellular cyclic guanosine 3',5 '-monophosphate level of B. cereus ATCC 14579. Microbios 1984; 39:137-150. 165. Lechner J, Wieland F. Structure and biosynthesis of prokaryotic glycoproteins. Annu Rev Biochem 1989; 58:173-194. 166. Bose SK, Smith GB,Paul RG. Influence of lectins, hexoses, and neuraminidase on the association of purified elementary bodiesof Chlamydia trachomatis W - 3 1 with HeLa cells. Infect Immun 1983; 40:1060-1067. 167. Swanson A F , Kuo C-C. Identification of lectin-binding proteins in Chlamydia species. Infect Immun 1990; 58:502-507. 168. Swanson AF, Kuo C-C. The characterization of lectin-binding proteins of Chlamydiatrachornatis as glycoproteins.Microb Pathogen 1991;10:465473. 169. Kahane I, Brunner H.Isolation of a glycoprotein from Mycoplasma pneumoniae membranes. InfectImmun 1977; 18:273-277. 170. Simoneau P, Labarere J. Detection of a concanavalin A binding protein in the mollicute Spiroplasma citriand purification from the plasma membrane. Arch Microbioll989; 152488-491. 171. Narasu ML, Gopinathan KP. Purification of larvicidalprotein from Bacillus sphaericus 1593. Biochem Biophys ResCommun 1986; 141:756-761. 172. Durbreuil JD, Logan SM, Cubbage S, Eidhin DN, McCubbinW D , Kay CM, Beveridge TJ, Ferris FG, Trust TJ. Structural and biochemical analysesof a surface array protein of Campylobacter.fetus. J Bacteriol 1988; 170:41654173. 173. Kawamura T, Shockman GD. Purification and some propertiesof the endogenous, autolytic N-acetylmuramoylhydrolaseof Streptococcus faecium, a bacterial glycoenzyme. J Biol Chem 1983; 258:9514-9521. 174. Corbel MJ, Cockrem DS,Brewer RA. The interaction of Brucella cell surface components withplant agglutinins. Dev Biol Stand 1984; 56:169-175. 175. Cavalcanti MSM, Almeida AMP, Coelho LCBB. Interaction of lectins with Yersiniapestis strains. Appl Biochem Biotechnol1990; 26:125-131.


2 Use of Lectins in General and Diagnostic Virology SICVARD OLOFSSON and STlC JEANSSON University of Gothenburg,

Gothenburg, Sweden

JOHN-ERIKSTlC HANSEN Hvidovre Hospital, Hvidovre, Denmark

1.

INTRODUCTION

One obvious explanation for the success of lectins as tools in the microbiology of cellular organisms is the huge amount of different carbohydrate structures associated with various microorganisms. The structures of these glycans are strictly under control of the microorganism through the concerted actions of a variety of glycosyltransferases. As demonstrated in other chaptersinthisvolume,manyspecies are associatedwith unique carbohydrate structures, whichcouldbe identified by individual for lectins or by selected panels of lectins. These lectins could be used identification of certain species as well as for purification of structural components. Enveloped animal viruses contain large amounts of glycoproteins, but in contrast with cellular microorganisms,the structural information for the carbohydrates of viral glycoproteins is not specified by the virus genome, but by its host cell. This means that the oligosaccharides of virus-specific glycoproteins should differ very little from the oligosaccharides of uninfected cells, suggestingthat lectins could be of limited value for general and clinical virology. However, the prominent virus-induced changes in the host cell metabolism may have consequences also for the glycosylation process, usually resulting in sufficiently unique carbohydrates to be distinguished from uninfected cell structures by the aid of lectins. Accordingly, lectins have provedto be useful for several purposes both in general and diagnostic virology. 67

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II. VIRUSES AND VIRAL GLYCOPROTEINS A. Maturation of Enveloped Viruses

Lectins have most frequently been used for studies on enveloped viruses. The infectious cycle of a model enveloped virus is depicted in Figure 1. Only the steps of interest for understanding the interactions between lectins the infectious and viruses are considered. For a comprehensive treatment of cycle of enveloped viruses, the reader is referred to a recent textbook [l]. The first phase in the infection of a permissive cell byan enveloped virus is specific attachment between the virus adsorption complex (VAC), usually consisting of oneor more virus-specific glycoproteins,and a cellular receptor, exposed from the cell surface (see process 1, Fig. 1). The next step is penetration of the viral nucleocapsid throughthe plasma membrane ofthe infected cell (process2) by fusion between the viral envelope and the lipid bilayer of the plasma membrane. The penetration is carried out either by or may not be identical with the action of a viral fusion protein (which may

Figure 1 The replication of a prototype enveloped virus. The diagram, which in essence describes the multiplicationof a togavirus [148], is simplified to emphasize the biosynthetic stepsof relevance for lectinstudies. Details of the figure are commented on in the text.

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proteins of VAC) or catalyzed by the low pH of endocytosis vesicles after engulfment ofthe virus particle. These processes may be inhibited by addition of several lectins (see Section 1II.F). Process 3 is uncoating of the viral nucleic acidfrom core and nucleocapsid proteins. The uncoated nucleic acid is expressed by virus-encoded and host cell-codedfactors (process 4 of Fig.1 refers to the replication of a small negative-stranded RNA virus). For simplicity, the processes by which mRNAs are generated willnot be reviewed. Relativeto their ultrastructural localization, two types of mRNAs are discerned. Viral glycoproteins are synthesized from mRNAs associated with ribosomes attached to the vesicles of the rough endoplasmic reticulum (RER; see Fig. 1, process 4b), whereas soluble and nonglycosylated structural viral proteins are synthesized from polyribosomes localized in the cytoplasmic sap (see process 4a). Multiple copies of genomic viral nucleic acid are synthesized by virus-coded polymerases (see process Sa) and, subsequently, combined withnewly formed viral capsomers to form progeny nucleocapsids (see process 6a). The viral glycoproteinsare handled by the same mechanismthat operates for cellular membrane proteinsand, during the translation of a glycoprotein mRNA, a 15- 30-amino acid long sequence of hydrophobic amino acids at the NH,-terminal portion of the growing polypeptide signals for membrane insertion (see process Sb). This signal is the passport for the glycoprotein to gain accessto the glycosyltransferases situated inthe lumen of RER and the Golgi apparatus. A tight association betweenthe growing polypeptide and RER force the polysomes to stick to the RER vesicles. The polypeptide moves farther into RER vesicles until a special stop-transfer peptide sequence appears, which permanently anchors the glycoprotein in the membrane. This membrane-associated glycoprotein travels out through the vesicles of the RER and the Golgi apparatus and is continuously processed by the various enzymes, including glycosyltransferases and glycosidases in these organelles (see process 6b). Finally, the glycoprotein reaches the cell surface, usually exposingthe NH,-terminal portion with its carbohydrates from the exterior of the cell. The enveloped virus is usually formed by a budding process, involving interactions between the COOH-terminal part of cell surface-associated viral glycoproteinsand proteins of an adjacent nucleocapsid (see process 7 Fig. 1). The glycoprotein spikes of these virus particles contain mature oligosaccharides with potential to bind to different lectins, whereasthe intracellular processing intermediates of viral glycoproteins will be recognized mainly by lectins, reacting with immature oligosaccharides. The taxonomy and a brief morphological characterization of the viruses discussed in the present chapter are found in Table 1. Even if glycoproteins are attributes of enveloped viruses, some nonenveloped viruses,

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including adenovirusesand papovaviruses, may encode glycoproteins [2,3]. Rotaviruses constitute icosahedral nonenveloped virus particles, but one maturation step includes buddingthrough intracellular vesicles [4]. Some of the capsid proteins are glycosylated [5], probably because the intracellular budding process presents the capsid proteinsto the glycosylation machinery of the cell. B. Clycosylation of Viral Envelope Proteins 1. Structures of Oligosaccharides

The oligosaccharide chains of viral glycoproteins can be classified into two groups by the nature of the covalent linkage between oligosaccharideand polypeptide backbone [reviewed in 61. The first class is called asparagineor N-linked oligosaccharides, because they are N-glycosidically linked from an N-acetylglucosamine (GlcNAc) to the amide nitrogen of asparagine. The second class is called0-linked oligosaccharides, as theyare O-glycosidically linked from an N-acetylgalactosamine (GalNAc)to the hydroxyl oxygen of serine or threonine. These oligosaccharides are sometimes referredto as mucin-type oligosaccharides,as they are important constituents of different mucins. Some examples of N- and 0-linked oligosaccharides are given in Figure 2. In general, 0-linked oligosaccharides of viral glycoproteins do not contain mannose,and N-linked oligosaccharides usually do not contain GalNAc [reviewed in 61. Recently, 0-linked glycans, containing the GlcNAc-Ser(Thr), have been describedfor viral glycoproteins, associated with the nucleus [7]. The N-linked oligosaccharides are derived from a common biosynthetic precursor (further outlined in Section II.B.2). They can be further divided into three subclasses: (1) high-mannose oligosaccharides (structure A); (2) complex-type oligosaccharides, containing peripheral GlcNAc, Gal, and sialic acid residues,as indicated instructure B. In addition, fucose may be attachedto the innermost GlcNAcand at other positions. Complex-type oligosaccharides may be further substituted to form tri-, tetra-, and pentaantennary structures (Fig.3). The structures of such oligosaccharidesand the implications for the lectin-binding properties are discussed later. (3) Hybrid-type oligosaccharides, which have both high-mannose and complextype characteristics(see Fig. 2, structure C). The 0-linked glycans are very heterogeneous in structure and size. Usually, theyare relatively small, comprising only one to five monosaccharides, but also larger structures have been identified, although not for viral glycoproteins. StructuresD-G (see Fig. 2)are examples of0-linked glycans found in viral glycoproteins.The 0-linked oligosaccharides are clustered in


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Figure 2 Examples of structures of N-linked (structures A-C) and 0-linked @-G) glycans described for viral glycoproteins. The N-linked glycansare designated: (A) high-mannose, (B) complex-type, and (C) hybrid-type glycans.0-linked glycans DF were detected in the HSV-l-specified glycoprotein gC-l [62] and structure G was found in the mouse hepatitisvirus glycoprotein [149]. (Structural data fromRef. 9, 52.)

pronase-resistant arrays in several viral glycoproteins (discussed in more detail in Section III.B.2). 2. Biosynthesis of N-Linked Glycansof Viral Glycoproteins

The synthesis of N-linked oligosaccharides, which is summarized in Figure 4, is a complex process, involving a multitude of intermediates. All early steps in this process (see phases A and B in Fig. 4) are common for all N-linked oligosaccharides from mammalian cells, whether or not cells are infected with viruses. A large lipid-linked oligosaccharide intermediate is built up, and this precursor oligosaccharide is transferred en bloc to the growing polypeptide chain (see Fig.1, process 5b). The N-linked oligosaccharides are added to asparagine residues ofthe polypeptide, participating in the sequence Asn-X-Ser(Thr) [X not Pro] [&lo]. This largecarbohydrate

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Figure 3 Structures of multiantennarycomplex-typeoligosaccharides.Thecomplete glycosidic linkages are indicated for all branching positions. The roman numerals indicate the designation of the individual GlcNAc transferases responsible for initiation of each branch. (A) The structure of a tetraantennary oligosaccharide and (B) a triantennary structure with a bisecting GlcNAc are depicted. (Data from Ref. 150.)

structure is trimmed by specific glucosidases and mannosidases, situated in the RER or in the &Golgi vesicles (see Fig. 4, process B) to form a small oligosaccharide. The oligosaccharide resultingfrom process B is a substrate for subsequently acting, and partly competing glycosyltransferases, localized inthe medial and trans-regions of the Golgi (see Fig. 4, process C) to form a variety of hybrid and complex type oligosaccharides. Thisstructural variability is generated by the dual specificities of glycosyltransferases: (1) Each glycosyltransferase has a specificity for the sugar nucleotide and for the oligosaccharide, and (2) each enzyme will establish only one type of glyco[l l]. Consequently, the informasidic bondout of half a dozen possibilities tion for structural arrangements of monosaccharides in the final oligosaccharides is transferredfrom the genomeby the consecutivelyacting glycosyltransferases, for which each newly added monosaccharide generates a suitablesubstrate for one or more subsequently acting transferases. The complex-type oligosaccharides differ in degree of branching, glycosidic bond configurations, degree of sialylation, and fucosylation. Variations in branching degree and configuration of complex-type oligosaccharides (compare Fig. 2, structure D, and Fig. 3) are especially important for their lectin-binding properties. The initiation step in the formation of a given

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branch is catalyzedby a specific GlcNActransferase, as indicated in Figure 3. These differences in the peripheral structures of complex-type oligosaccharides reflect cell-specific differencesquantitative in and qualitative properties of the Golgi-associated glycosyltransferases and differences in the intraluminal concentrations of sugar nucleotides. 3. Biosynthesis of 0-Linked Glycans in Viral Glycoproteins

The O-linked oligosaccharidesare synthesized without any involvement of lipid-linked intermediates.After addition of the first GalNAc to the polypeptide, elongationof O-linked oligosaccharides proceeds by the successive actions of individual glycosyltransferases directly on the peptide-associated, growing oligosaccharide. As withthe peripheral structuresof N-linked oligosaccharides, there also is significant variation in the structures of 0linked oligosaccharides. The peptide signal requirementsfor addition of O-linked oligosaccharides are not completely understood. Biosynthesis of O-linked oligosaccharides takes place when the protein has acquired a tertiary structure in the Golgi apparatus. In contrast with the linear peptide stretches, signaling N-glycosylation, it is assumed that three-dimensional determinants, probably rich in threonine, serine, and proline residues, constitute signals for O-glycosylation [12,13]. Addition of the firstGalNAcresidueprobably takes place in the cis-compartment of the Golgi region [14], whereas later glycosylation stepsare carried out in the medial and trans-compartmentsof the Golgi apparatus concomitantly with the later steps of N-glycosylation [15,16]. 111. INTERACTIONS BETWEENVIRAL CLYCOCONJUCATESAND LECTINS A. Possible Mechanisms for Generation of Virus-Specific lectin-Binding Determinants

7.

Virus-Specified Glycosyltransferases, Do They Exist?

The structures of glycoprotein oligosaccharides are specified through the concerted action of a variety of glycosyltranferases, for which each such enzyme is responsiblefor addition to the growing oligosaccharide chain of one monosaccharide in a highly specific manner. Undoubtedly, the virus could change the peripheral composition, by specifying only one single glycosyltransferase, acting at a strategic point in the biosynthesis of glycoproteinoligosaccharides.Virus-specifiedglycosyltransferasesweredescribed for bacteriophages byLosick and Robbins in 1967[17]. In that report, the authors demonstrated an altered bacterial surface after phage


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conversion and showed that this phenomenon was dependent on a viruscoded galactosyltransferase, with specificities other than transferases encoded by the cellular genome, resulting formation in of new terminal carbohydrate determinantsat the bacterial surface. However, available data now favor the hypothesis that virus-encoded glycosyltransferases, if they exist at all, do not play any role in the infectious strategy of animal viruses (6,14,18-22). 2. Other Virus-Coded Factors, lnfluencing the Glycosylation Status of the lnfected Cell

Although animal virusesdo not specify any glycosyltransferases, it is possible that some viruses may encode regulatoryfactors, which may influence the transcription of individual cellular glycosyltransferases.If certain strategic enzymesare affected, this phenomenon would have crucial effects on the structural composition of the oligosaccharides synthesized by the infected cells. Alterations in the glycosylation pattern as a consequence of infection withan animal viruswere initially reportedby Onodera et al.[23], demonstrating an increase in sialic acid content of surface glycoproteins correlated to increased levels of sialyltransferase of cells transformed by a temperature-sensitive mutant ofsimianvirus 40 (SV40). More recently, Adachi et al. [24] found that infection of a variety of CD4' lymphoid cell lines with human immunodeficiency virus (HIV) resulted in a dramatically increasedexpressionofLey-active carbohydrate determinants. Increased amounts of Ley-active oligosaccharides were also detected on T lymphocytesisolated from patientswithacquiredimmunodeficiencysyndrome (AIDS) and those with AIDS-related complex (ARC), indicating that this phenomenon also exists in vivo. Hansen et al. [25] demonstrated that not only LeY,but also twoother newly expressed carbohydrate determinants of HIV-infectedcellswereassociatedwith the HIV-specified glycoprotein, designated gp120. These determinants are blood group A,, and sialyl T,. Fossum et al. [26] found that bovine B lymphocytes, infected with bovine leukemia virus (BLV), containedHelix pomatia agglutinin (HPA)-binding surface glycans, normally a characteristic for T-cells. This change in the glycosylation of h e l l s took place without any detectable synthesis of viral proteins [MoreinB, personal communication]. The mechanismof induction of these carbohydratestructures is largely unknown. The situation for HIV, as just related, seems to be relatively complex inasmuch as both primitive carbohydrates [27], such as sialylT,, or more complexstructures, such as AI substance, are induced. Recentdata have given some insight into the mechanisms of changes in glycosylation. ras,asmay inducealterThus, certain oncogenes and protooncogenes, such ations after transfection into mammalian cells [28]. Moreover, H-ras was

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correlated to altered glycosylation in Madin-Darby canine kidney (MDCK) cells after transformation by murine sarcoma virus[29]. Since the ras gene contains a region homologous with genes encoding the family of G-binding that one branch of glycosylationcontrol proteins [30], it may be speculated is carried out by the activities of signal transducing G-proteins [29,30]. If this principle is applicable also for the complex HIV-induced changes in glycosylation, this phenomenon might be explained by modulation of the activity of several signal transducing G-proteins. It is clear that such induced changes in carbohydrate structures are relevant when discussing only cells infected with viruses that do not immediately give riseto the classic shutdown of host cell macromolecular synthesis. The transformation process implies continued expression of host cell genes, but, even infection by a lytic retrovirus permits coexpression of host cell and viral genes for a considerable amount of time. Althoughnot positively demonstrated, the same phenomenon could also take place during the latent phases of human herpesvirus infection. Alterations theinexpression of host cell glycosyltranferases, induced by virus-coded factors, therefore, could be expected to result in corresponding changes carbohydrate in structures that are detectable with lectins. Thereare numerous viruses, characterized by a delayed or an absent shutdown of host cell macromolecular synthesis,for which lectins could be of use to detect glycoproteins with altered lectinbinding patterns. 3. Virus-Induced Physical Modulation of Cellular Glycosyltransferases

Changes inthe lipid compositionmay modulate the conformation of membrane-associated glycosyltransferases, resulting in changes in the kinetic properties of such enzymes [31,32]. The late stages in the replicative cycle of an enveloped virus are associated with major changes in the membrane system ofthe infected cell, suggesting that this phenomenon could also take place in virus-infected cells. Small, but significant, changes in the kinetic properties, for both glycon and aglycon specificity, have been reported for sialyl- and galactosyltransferases late in the herpes simplex virus (HSV) infectious cycle [33]. Most viral glycoproteins are late gene products and, therefore, are produced in large amounts. If critical glycosyltransferases or certain nucleotide sugar precursorsare not available at sufficiently high concentrations in the infected cells, oligosaccharide structures of viral glycoproteins might differ from those expressed in uninfected cells owingto exhaustion of the cellular capacity to add a particular monosaccharide to the growing oligosaccharide chain. Probably the high content of terminal galactose inHSV

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glycoproteins, when expressed in certain cells [34], is due to exhaustion of the sialylation capacity. 4. Unusual Glycans, Acquired by Specific Peptide Stretches of Viral Glycoproteins

The addition of N- and 0-linked oligosaccharides to proteins require the presence of specific acceptor peptide stretches characteristicfor each type of oligosaccharide. One of these signals, for example, the sequence Asn-XSer(Thr), signaling the addition of an N-linked glycan, is more or less ubiquitous in membrane sequences, whereas certain patterns of O-glycosylation occur only rarely in such glycoproteins. One such of structural type arrangement isthe occurrence of peptide stretches harboring multiple small 0-glycans in protease-resistantclusters,resultingincharacteristiclectin receptors. For example, such clustered 0-linked units of GalNAc bind excellently to Vicia villosa (VVA) B., isolectin [35]. Although such clustered 0-glycans are characteristic for mucins, most cells usedfor propagation of animal viruses are more or less devoid of these structures [6]. The signal requirement for this type of glycosylation is not known in detail, but most likely it is dependent on higher-order structural features of the protein [131. If a viral glycoprotein contains this latter type of rare signal sequence, it will be heavily 0-glycosylated, provided the appropriate glycosyltransferases are present in the infected cell. It cannot be excluded that exhaustion of the capacity to add p-1,3galactose to GalNAc contributes to the increased levels of GalNAc-Ser(Thr) units (see Section III.A.3). B. lectin-Binding Determinants of Carbohydrates in Viral Glycoproteins

7.

General

Viral glycoproteins contain oligosaccharide substructures that are accessible for a variety of lectins. Several of these lectins have been especially useful for characterization and isolation of viral glycoproteins. The specificities of such lectins, with special reference to their bindingto viral glycoproteins are discussed in the following section. The reader is referred to Chapter 1 and to other reviews for more general aspectson lectin specificities [36,37]. 2. Lectins Binding to N-Linked Glycans

Mannose-Binding and ggBranch-SeIective” Lectins. With only a few exceptions, all viral glycoproteins contain N-linked oligosaccharides,which are accessible for one or more of the mannose-binding lectinsand lectins from Phaseolus vulgaris. The most frequently used mannose-binding lectinsare

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isolated from Canavaliaensifonnis (concanavalin A; ConA),Lens culinaris (lentil; LCA), and Pisum sativum (PEA), all having the monosaccharide specificity mannoseand glucose. One ofthe first lectins usedfor characterization of viruseswas ConA [38-40]. The oligosaccharide specificity of ConA differs significantly from that of LCA and PEA [41]. Both lectins bind to moderately branched N-linked oligosaccharides, but LCA and PEA have a strict requirement for fucose (Fuc), situated as depicted in Figure 5. Thismeans that LCA and PEA will bindonly to fucose-containing complex-type oligosaccharides, whereas ConA, in addition, will bind to high-mannose and hybrid oligosaccharides (see structures A and C, Fig. 2). Consequently, the use of these lectins makes it possible to distinguish between matureand precursor formsof viral glycoproteins.A wide variety of enveloped viruses contain oligosaccharides capable of binding to these lectins [6]. Viral glycoproteins with more extensively branched oligosaccharides Phaseolus vulgarisleukoagglutinating (L-PHA)and may be investigated by erythroagglutinating (E-PHA) lectins.The L-PHA lectin binds to tri- and tetraantennary oligosaccharides containinga GlcNAco-l,6Man branch(see Fig. 3, structure A), whereas E-PHA binds to a triantennary oligosaccharide, witha bisecting GlcNAc (see Fig. 3,structure B) [42,43]. Immobilized L-PHA retards, rather than arrests, the passage of oligosaccharides through an affinity column [42,44]. Chromatography with L-PHA has been used for isolation of multiantennary N-linked glycans of herpes simplex virus

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glycoprotein C [45]. A lectin chromatography strategy that uses serial chromatographic procedureson the aforementioned lectins has been developed to structurally characterize N-linked glycans released from glycoproteins by pronase digestion [42]. This strategy has been successfully used to characterize N-glycans of an influenza virus hemagglutinin [46]. The results just reviewed [42,43] wereobtained with pronase-released oligosaccharides. Many more overlapping-binding patterns were reported for these lectins when N-glycanase-released oligosaccharides were analyzed [44]. Fucose-Binding Lectins. Theglycoproteinsofmostenvelopedviruses contain complex-type oligosaccharides, with fucose attached to GlcNAc in an a-l,6 linkage (see Fig. 2B). Several lectinsthat bind to fucose have been reported [for a review,see361, but not all fucose-binding lectins accept this configuration. Thus, the Ulex europeaus lectin I (UEA-l), which is a frequently used fucose-binding lectin in glycobiology, binds Fuca-l,6Gal the determinant of the blood groupH substance, but not the Fuca-l,6GlcNAc determinant described earlier. This lectin failed to bind both HSV (unpublished) and HIV [47] glycoproteins inour laboratory, although theseglycoproteins contain several fucose-containing glycans [48,49]. A more suitable lectin for work with fucose-containing viral glycoproteins should be the lectin ofAleuria aurantia,which wasreported to bind to fucose ina variety of linkages, includingthe a-1,6 configuration [50,51]. Galactose-Binding Lectins. Galactose is present as a terminal or penultimate sugar in complex-type oligosaccharides of viral glycoproteins (see Fig. 2). The most frequently occurring linkage is Gal&l,4GlcNAc [9,52], to which bindsto the Ricinus communislectin [RCA] (53). The RCA binds branched complex glycans that contain two or more terminal galactose residues [53]. A single galactose residue on a diantennary oligosaccharide may bind to RCA and other galactose-binding lectins, if the galactose is associated withthe penultimate GlcNAc, attachedto the a-1,3- and not the a-1,6-mannose of the oligosaccharide [53; see Fig. 5, structure B). For an explanation of this phenomenon, the reader is referred to the review by Montreuil [37]. Terminal sialic acid usually abolishes RCA binding, but tetraantennary oligosaccharides, containing a-2,6-linked sialic acid are reported to bind to RCA [37,53,54]. The results indicate that removal of terminal sialic acid from HSV glycoproteins isa prerequisite for RCA binding [48,55]. 3. Ledins Preferentially Binding to 0-Linked Glycans

N-Acetylgalactosamine-BindingLectins. Several GalNAc-binding lectins, such as the Helixpomatia lectin (HPA), the Vicia villosaB4isolectin (VVA B4),the Glycine Max (SBA) lectin, and the Arachis hypogaea lectin (PNA), with main specificity for the disaccharide sequence Gal/3-1,3GalNAcYare

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useful for experimental and clinical virology (discussedin detail in Section 1V.C). Although both GalNAc and the Gal/3-1,3 linkage may occurin certain mammalian N-linked oligosaccharides, such structures are very rare, if occurring at all, in the N-linked glycans of enveloped viruses [6]. Therefore, a specific, elutable, binding of a viral glycoprotein withofone these lectins could be provisionally considered an indication of O-linked oligosaccharides. The use of HPA and SBA affinity chromatography and subsequent chemical confirmation first demonstrated the O-linked glycans in viral glycoproteins [56,571. Thus, of more than seven antigenically distinct viral glycoproteins, only one species, designatedgC-l, bound to SBA and HPA ~81. The GalNAc-binding lectins have different “next-best-eluting monosaccharides.” For example, the affinity of HPA could bedescribed as GalNAc > GlcNAccomparedwithGalNAc > Gal for SBA[36]. The GalNAc-binding lectins also display a considerable heterogeneity of the oligosaccharide specificity [36,59]. It has been reported that HPA binds severalrelativelylargeoligosaccharideswithterminalGalNAc,including blood group A substance, which contains the terminal disaccharide GalNAca-l,3Gal [36]; the Forssmanantigen, which containsterminal GalNAca-l ,3GalNac [36];and the desialylated derivative of the ganglioside GM2, which contains terminal &linked GalNAc, as mentioned earlier [60]. Surprisingly, the HPA-binding activity of HSV-l-specified gC-l (see foregoing) was not associated withan O-linked oligosaccharide containing any of these terminal carbohydrate determinants. In contrast, the structures responsible for HPA-binding constituted clustered unitsof single GalNAc molecules, each O-glycosidically linked to a serine or threonine residueof the polypeptide [61,62]. A model of such an HPA-binding cluster is depicted in Figure6. Here, the specificity ofHPA resembles that of the W A B4 lectin, which has a pronounced affinity for clustered O-linked GalNAc residues [35]. Accordingly, it was shown that HPA and VVA B4 bound to the same GalNAc-containing stretches of gC-l [62,22]. No lectin-binding structure of a viral glycoprotein has yet been correlated with a complex GalNAc-containing oligosaccharide, such as blood group A substance or the Forssman antigen. For instance, the HIV-specified glycoprotein gp120, for which blood group A reactivity was demonstrated with a highly specific monoclonal antibody [25], failed to react with GalNAc-binding lectins [47]. One reason for this discrepancy in the reactivity between lectins and highly specific antibodies could be that the affinity constants for lectins binding to blood group A substance are too low to permit lectin bindingto a single oligosaccharide of a viral glycoprotein. Indeed, the blood group specificities of GalNAc-binding lectins were established under multivalent hemagglutination conditions[for review, see 361. Moreover, GalNAc-binding lectins, such as DBA and VVA, failed to


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Serllhr

HPA

Figure 6 The peptide-carbohydratestructural arrangement responsiblefor binding of viral glycoproteinsto PNA and HPA.(A)Peptide organization of HSV-specified glycoproteins gC-l and gC-2. The lollipops denote positions of N-linked glycans. The peptide is represented by a thick continuous line.A region in gC-l which either is nonhomologous withthe corresponding region inHSV-2 or is missingin the gC-2 sequence is indicated by a hatched square. The thin continuous line in the graphic representation of gC-2 denotes a stretch of low homology with gC-l, whereas the thin broken line denotes that the corresponding gC-l sequence has no counterpart at all in gC-2. [Peptide sequence data from Ref. 151-153, and carbohydratestructures and organizationfrom Ref. 61,62,99.]

detect single 0-linked GalNAc residues ingp120 [47], although such structures were detected with a monoclonal antibody with high specificity for the T,, antigen (GalNAc-Ser(Thr)) [Hansen et al., submitted]. This is in contrast with HSV glycoproteins, in which GalNAc-Ser(Thr) units were readily detectable with HPA and W A . This difference between HSV-

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specified gC-l and HIV-specified gp120 may be explained by differences in GalNAc-Ser(Thr) density, as outlined in Figure 7. These data emphasize that great care must be takentheininterpretation of data from experiments with GalNAc-binding lectins and viral glycoproteins. Galactosefl-I,3-BindingLectins. Afamilyoflectins,comprising PNA, BPA, and AGP bind to the Galfl-l,3GalNAc sequence [reviewed in 631, which isa frequentcore structure of 0-linked glycans in viral glycoproteins [6]. The specificity of AGP is complex, and the immobilized lectin is also reported to retain sialylated 0-linked oligosaccharides [63]. Affinity chromatography with PNA has been usedfor studies on HSV glycoproteins. If the peptide stretchesof gC-l depicted in Figure 6 are equipped with larger 0-linked glycans, the HPA binding will be replaced by affinity for PNA W]. 4. Lectins Binding to Carbohydrate Determinants of N-and 0-Linked Oligosaccharides

WheatGerm Lectin. Thewheatgermlectin (Triticum vulgaris; WGA) has been widely used for studies of viral glycoproteins [7,38,48,55,64,65]. WGA has a wide potential for glycoconjugate research: (1) a high affinity

Binding to:

HPAMIA

a -Tn

HIVgp120

-

t

HSVgC-1

t

t

0 Gal

0 GalNAc

Figure 7 Reactivity of gp120 and gC-l, specified by HIV-l and HSV-l, respectively, with GalNAc-binding lectins and a monoclonal antibody, directed against T. [GalNAc-Ser(Thr)]. The different densitiesof N- and 0-linked glycans between the proteins is illustrated in the diagram.

in

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for GlcNAc residues of preferentially N-linked glycans [66],an(2) intermediate affinity for sialic acid [mainly (a2-3)N-acetylneuraminic acid; 67,681 situated inN- and O-linked glycans [69],and (3) high affinity for GlcNAc, O-glycosidically linkedto certain viral glycoproteins destined for export to the cellular nucleus [7]. Sialic Acid-Binding Lectins. Sialic acid-binding lectins are, with the exception of WGA, not regularly found in plants. Three lectins from invertebrates, Carcinoscorpius rotunda caudalectin, L i m a flaws, and Limulus polyphemus (LPA) bind sialic acid with slight,but significant, differences in their specificities [36].The LPA lectin has been usedfor purification of glycoproteins specifiedby a bovine herpesvirus[70]. IV. USE OF LECTINS I N GENERAL VIROLOGY

A. General Guidelines for Use of Lectins in Virological Work

If results from experiments with lectins are to be interpreted in terms of carbohydrate structures, twoimportant points must be considered: First, it must be borne in mind that several lectins, such as ConA, contain hydrophobic domainsthat may interact with noncarbohydrate determinants of glycoconjugates. This is especially important during lectin blot experiments with electrophoretically separated proteins and after r i a tion for lectin cytochemistry [71], in which denatured glycoproteins are studied. Therefore, great care should be taken to include control experiments that show that the interactions studiedare blocked by addition of a relevant monosaccharide. In general, a lectin-glycoconjugate binding should be considered specific onlyif the interaction is inhibited by adequate concentrations of a relevantlow molecular weight saccharide. Second, specific binding itself is not a chemical proof for a particular carbohydrate structure. The published specificities are often based on hemagglutination-inhibition, or on data from other experimental systems, not always compatible withthe virological experimental situation [36]. An example is the HPA- and SBA-binding glycans associated with HSV-1 specified 9C-1 [58]. Given the current literature on lectin specificitiesat the time of these experiments,it was initially believedthat the lectin-binding pattern revealed the presence of larger O-linked glycans with blood group A or Forssmanspecificity[72,73].In contrast, subsequent structural analyses demonstrated a previously unknown specificity of HPA: the repeated units of O-linked GalNAcas depicted in Figure 6 [62]. It should also be rememberedthat released oligosaccharides,may have a different lectin-bindingthan corresponding protein-associated structures; even the choice of method for release could influence the lectin-binding

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pattern of a particular oligosaccharide [a].In conclusion, results from lectin-binding experiments should be considered hints for further structural work. If structural conclusions are to be drawnfrom lectin-binding experiments, it is strongly recommendedthat they are confirmed by other tools, including use of specific inhibitors and the growing panel of specific glycosidases available. B. Detection of Specific Carbohydrate Substructures 7.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Lectin-Blotting

The classic SDS-PAGE according to Laemmli [74] allows separation of complex mixtures of proteins from virus-infected cells, by relative molecular mass. The separated protein bands are available for analysis of their lectin-binding properties accordingto several experimental protocols. Lectin-binding of such separated glycoproteins may even be assayed directlyon the acrylamide gels.By using fluorescein isothiocyanate (F1TC)-conjugated ConA for incubation of an acrylamide gel and visualization, by ultraviolet light, the presence of ConA-binding oligosaccharideson feline herpes virus type-l glycoproteins was demonstrated [75]. A similar procedure has been used for direct staining glycoproteins in lysates of cells infected with Friend leukemia virus [a], in which the acrylamide gel was incubated with 1251labeled lectins (ConA, WGA, RCA-1). The lectin blot technique (WELLBA) is an adoption of the original immunoblot technique [76] in which antibodies are replacedbylectins. Thus, electrophoretically separated glycoproteinsare transferred to nitrocellulose sheetsand subsequently incubated with lectin solutions. Unbound lectins are washed away,and bound lectinsare detected by autoradiography (radioiodinated lectins [77,78]) or by an enzyme-linked assay (biotinylated lectin and enzyme-conjugated avidin [71]). This technique offers unique possibilities to extract relatively detailedinformation about individual viral glycoproteins, even if these glycoproteins are not available in pure form. The technique has been mainly used for characterization of glycoproteins of plant [71] or insect [77] viruses, but also for certain mammalian viruses, such as primate cytomegalovirus(CMV) [78] and HIV [47,79],for which it is difficult to obtain pure glycoprotein preparations. Compared with direct lectin-staining of acrylamide gels used for glycoprotein separation, transfer to nitrocellulose or other membranous supports allows enzymatic digestion of the separated glycoproteins before lectin staining. In a case such as ConA in which the lectin has more than one binding specificity, digestion with a glycosidase may allowa more precise characterizationof the ligand 1471. Proteins, denatured by SDS during electrophoresis, expose hydropho-


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bic domainsthat may bind nonspecificallyto lectins [71], and it is strongly recommended to check whether lectin binding can be inhibited by addition of relevant low molecular weight sugarsor saccharides, even if all incubations take place inthe presence of detergents. Using a lectin blot assay, it was possible to demonstrate that nucleocapsid proteins of granulosis virus (baculoviruses), infecting Indian meal moth, contained glycans, by binding to DBA (GalNAc), UEA (fucose), and LPA (sialic acid). All these lectin-virus interactions were inhibited by addition of relevant monosaccharides.The result with LPA is remarkable, since N-linked glycans of insect cells have been reported not to contain sialic acid [80,81]. In similar assays the major HIV glycoprotein gp120 binds to ConA, LCA, PSA, WIF, WGA, and PHA-E (47,791. 2. LectinCytochemistry

BothFITC-basedlectinfluorescence [82-841 and lectinperoxidase [85] techniques have been used for cytochemical detection of viral glycoproteins. These techniques permit resolution of subcellular components [84, 851. Lectin fluorescence has been used to study the dynamics of viral glycoprotein appearance on cells infected with Newcastle disease virus [83] and for monitoring the intracellular transport of viral glycoproteins through the Golgi vesicles [84]. Lectin cytochemistry parallels the high-resolution power witha great potentialto handle multiple samples. The method, therefore, is suitable for diagnostic purposes both in human[82] and in veterinary [85] clinical virology. Moreover,the method is applicable in situations during which onlya small proportion (1 070 or less) ofa cell culture expresses viral glycoproteins. In our laboratory, we have found double fluorescence (rhodamine-avidin biotinylatedlectin and FITC-labeledantibodies against viral glycoproteins) useful to correlate lectinaffinity to glycoprotein expression in such cases. Simultaneous staining of EBV-specified glycoproteins(greenfluorescence) and SBA-bindingcarbohydrates(redfluorescence) of EBV-immortalizedP3HRl cells is shown asan example in Figure 8 (Olofsson S, unpublished). It is evident that the same two cellsthat stain for viral glycoprotein also stain for SBA-binding activity, indicating that expression of late virus gene productsis essential for induction of the new carbohydratestructures,

+

C. Purificationof Virus andViral Components

1. GeneralComments

Lectin affinity chromatography has become a powerful tool for purification of both envelope glycoproteinsand complete infectious enveloped virus particles. The major advantage of this technique is that specifically

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Figure 8 Double fluorescence cytochemistry of EBV-transformed P3HRl. Green fluorescence biotinylatedHPA (light grey), visualized by use of FITC-conjugated avidin; red fluorescence a-EBV glycoprotein serum (dark grey), visualized by the use of a second antibody, conjugated with Texas red. The preparate was repositioned aboutten cell diameters between exposure of film for FITC and rhodamine fluorescence, to demonstrate thatthe same two cells were stained by both lectin and

antibody.

bound material may be eluted by relatively low concentrations of simple mono- or disaccharides and not by high concentrations of chaotropic ions or extreme pH values, which often occurs with immunosorbent techniques. The almost physiological elution conditions of lectin chromatography permit isolation of purified glycoproteins, with preserved three-dimensional conformation and biological activity. In addition, the lectin affinity chromatography provides useful structural information about the glycans of the viral glycoprotein. The procedure can be usedboth for purification of infectious virusesand for purification of viral strupural components. 2. Purification of lnfectious Enveloped Viruses

There are relatively few reports on purification of enveloped virus particles. In some situations, however, lectin chromatography can be a complement to physical methods for purificationof virus. Thus,Crotolariajuncea lectin chromatography was used after density-gradient centrifugation to purify bovine diarrhea virus(Flaviviridae),which is notoriously difficultto purify


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with maintained infectivity. In this study, the gel was eluted with 0.2 M lactose, and recoveries ofabout 65% of viral infectivity were obtained [86]. Electronmicroscopydemonstrated a monodispersesuspensionofenveloped particles,but the low incorporation of radiolabeled metabolites into bovine diarrhea virus particles made it impossible to measure punty [86]. Neukirch et al. [87] used affinity chromatography with another galactosebinding lectin, RCA, of polyethylene glycol (PEG)-concentrated infected cell supernatants and reported a 40% recovery of viral infectivity and an almost 1000-fold purification of protein. Early attempts to purify retroviruses by lectin coagglutination resulted in recovery of infectivity, but only a two- to threefold purification[ M ] . 3. Purificationof Enve/ope Glycoproteins

Lectins perhaps have been most important for virological research inaffinity chromatography for purification of viral glycoproteins. Such purified glycoproteins have been usedfor several purposes, including serodiagnosis of viral infections, production of subcomponent vaccine candidates, and pioneering experimentsto explore the early interactionsof viruses and their target cells. Several techniques for purification of viral glycoproteins are presented in Table 2. Several problems must be addressed duringthe optimizationof a purification protocol. As viralglycoproteins,with a few exceptions, are anchored by a hydrophobic peptide stretch through the viral envelope or the cell membranes, it is essential to include a detergent solubilizationstep to release viral glycoproteins.It is important to choose a detergent that preserves a biologically activeconformation of both the lectin used and the viral glycoprotein studied. In most studies deoxycholate (DOC) and nonionic detergents have been used. The principles for detergent solubilizationof biological membranes have been reviewed [108], and detailed analysis ofthe detergent compatibilityof several lectins have been published [1091. During the last years, the nonionic detergent octylglucoside has been used for solubilization of viral glycoproteins as well as cellular receptors for viruses [l lo]. This detergentis dialyzableand, therefore, may be removed fromthe solubilized glycoproteins. Another problem is that glycoproteins of enveloped viruses usually contain the same oligosaccharidestheasnumeroushost cell-specified glycoproteins, which may coelute with and contaminate the viral proteins. Therefore, precautions must be taken to select virus glycoproteinsfrom host cell proteins. Four principal ways have been usedto solve this problem: (1) The host cell glycoproteins are present only in the cell membrane and usually not in the envelopes of virus particles.A rate zonal gradient centrifugation in dialyzable gradient medium to isolate enveloped particles is a relatively simple procedure to enrich viral glycoproteins from most of the cellular

n

V 0

a

Olofsson et al.


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proteins. (2) If metabolically radiolabeled glycoproteinsare to be studied, it is often unnecessary to eliminate host cell glycoproteins before chromatography, asthe infection of susceptible cells results in a rapid shutdown of host cell macromolecular synthesis; hence, no incorporation of radiolabel into these products. (3) Viruses might introducecarbohydrate neoantigens that are associated with viral, and not host, cell glycoproteins. Here, it is possible to select for lectins that could be used to selectively purify a viral glycoprotein. (4) Some glycoproteinsare soluble and secreted into the culture medium at high concentrations, eliminating the need for an extra separation step. Table 2 summarizes published procedures for purification of some viral glycoproteins and how the problem of selectivity has been dealt with in each study. The most frequently used principle to establish selectionfor viral glycoproteins is the inclusion of a gradient centrifugation step for isolation of these glycoproteins. For most viruses it is appropriate to use a dialyzable medium, such as sucrose, but for especially vulnerable viruses, such as herpesviruses, it is essential to avoid osmotic stress; therefore, a high-molecular gradient medium, such as dextran [l111 or colloidal silica [l 121, must be used to ensure preservation of intact particles. The use of lectins for purification of equine infectious anemia virus (EIAV) gp90 is especially interesting, because the gene encoding this glycoprotein is very variable [89]. Therefore, it is a great advantageto use a lectin for purification, since all gp90 from all virus variants express complex-type N-linked glycans, despite considerable changes in the primary sequence [89]. It is also possible to separate two viral glycoproteins from the same virus by changing the detergent duringthe elution. Thus, the two Sendai virus glycoproteins will be separated if the detergent Empigen is substitutedfor DOC after elution of one glycoprotein [92]. A high degree of preservation of biological activity is associated with lectinaffinity chromatography of detergent extracts from purified virions indicated by the suggested use of the purified glycoproteins in immunoassays [95,96] and as subcomponent vaccine candidates [90,91]. The use of lectin affinity chromatography for isolation of glycoproteinsextracted from [3H]GlcN-labeledvirus-infectedcellsis an efficient way to explore the oligosaccharide processing of viral glycoproteins. the As entire infected cell is used for extraction, itis possible to isolate the various processing intermediates of viral glycoproteins by affinity chromatography by usingboth lectins bindingto immature oligosaccharidesand lectins binding to completely glycosylated proteins [89]. Viral glycoproteins may contain specific peptide stretches that signal for acquisition of carbohydrate units with unusual lectin-binding properties. One such example is the HPA- and VVAB,-binding structures of

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HSV-glycoproteins gC-l and gG-2, as discussed in Sections III.A.4 and 6. This type of B.3. The organizationof such a cluster is depicted in Figure clustered 0-linked glycans with HPA or VVA B4 affinity is rare, not only in glycoproteins of cell lines used for virus production, but also in most viral glycoproteins. This meansthat HPA chromatography of HSV offers not only selectivity for viral glycoproteins, but also a potential to pick out one single viral glycoprotein, despitethe presence of at least seven others. As discussed later in Section IV.C, the HSV-specified HPA-binding glycoproteinshavebeenusefulasantigensin the serodiagnosisof HSV infections. There are reasons to believe that chromatography with lectins, binding to 0-linked glycans, is applicable for purification of other viral glycoproteins. The equine herpesvirus glycoprotein gp300 has 0-linked oligosaccharides withthe same arrangement,and it binds to HPA [1131. Although no HPA affinity chromatography data are yet available, the structural arrangements of 0-glycans in single glycoproteins specified by human cytomegalovirus [1141, certain retroviruses[1151, and respiratory syncytial virus [l161 suggest that isolation of these glycoproteins by HPA or PNA affinity chromatography is possible. Someviralglycoproteins are secreted into the cultivationmedium wheretheybecomethe dominant glycoprotein species [117]. Therefore, secreted glycoproteins, whichare completely glycosylated, may be purified to reasonable purity from host cell contaminants by only one chromatography step [65]. The increased amount ofexpressionvectorsencoding truncated viral glycoproteinsfor which the membrane anchor sequence is missing will probably increase the use of culture media as a source for affinity purification of viral glycoproteins. Because lectin chromatography is usually nondestructive and often quantitative, this method has been combined with other techniques when a high degree of separation from host cell material or other viral glycoproteins has been required. References to such protocols, including one for purification of a glycoprotein encodedby an nonenveloped virus,are given in lowerpart of Table 2. 4. Purificationof Oligosaccharides from Viral Glycoproteins

Lectin affinity chromatography has been useful for purification of individual oligosaccharides releasedfrom viral glycoproteins by pronaseor endoglycosidase treatment. These procedures have been efficient, especially’for N-linked glycans.The main advantageof lectin chromatography is the combination of purification power with structural characterization. Chromatography with a relatively restricted panel of lectins, usually comprising LCA, ConA, and RCA, combined with gel filtration, often results in surprisingly well-defined preparations of individual oligosaccharides [63]. A lectin chromatography strategy, witha special protocol for serial chroma-


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tography using gel-bound C o d , PEA (or LCA), L-PHA, and E-PHA, has been developedto isolate and to structurally characterize many possible N-linked glycans released from glycoproteins by pronase digestion [42]. This strategy has been successfully used to characterize N-glycans of an influenza virus hemagglutinin [46]. Lectins have been less frequently used to purify individual O-linked oligosaccharides, probably because of the low-binding constants between the appropriate lectins and monovalent 0-glycans. The lectins HPA and VVAB4 have been used to purify small glycopeptide stretches of viral glycoproteinscontainingpronase-resistantclusters ofO-linkedGalNAcSer(Thr) [22,61,62], whereasPNA chromatography maybe used for isolation of aggregates containing Gal&1,3GalNAc. D. Lectins as Tools to Study Immunological Properties of Viral Glycoproteins

7.

General

The carbohydrate complement of many viral glycoproteins contributesto their immunological properties [l 181. In some, the carbohydrate determinants themselves constitute the antigenic epitopes [25], but in most, the carbohydrates act to modulate the antigenic activityof protein-derived epitopes [55,119]. Lectins have been of great value in experimental strategies, two of which are reviewed in the following, to determine the significance of carbohydrates inthe antigenicity of viral glycoproteins. 2. Solid-Phase Method to Identify Carbohydrate-Dependent Epitopes

By combining the enzyme-linked lectin-binding assay, developed by van der Schaal et al. [l201 and a sialidase-periodate-based method for sequential degradation of peripheral carbohydrate determinants ofglycoproteins, coated onto microplates,Sjoblometal.developedamethodbywhich modulation of antigenic activity can be correlated to specific changes in the peripheral glycosylation[55]. The method is presented in FigureBy 9. using defined monoclonal antibodies,it is possibleto map the precise locationsof peptide epitopes,which are inducible by removalof masking carbohydrate epitopes, in parallel withthe identification of the particular carbohydrate determinants responsiblefor masking antigenic activity.The use of periodate treatment to remove peripheral carbohydratesis convenientand especially usefulif large amountsof antibodies are to be screened. Elimination by use of specific glycosidases is more specific,but also more laboriousto perform in the solid-phase assay and, therefore, could be used to confirm the results demonstrating periodate-induced alterations of particular epi-

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Sialidase and

Monoclwral Lectins antibodies

/

\

NeuAc Ne Ac ACA

+

I

P

GP1

GlcNAc GlcNAc

I

I

lo00

Man

I

GlcNAc

I GlcNAc I

500

- Asn

Figure 9 Principle for the sialidase-periodate assay for demonstration of carbohydratedependent epitopes. The cavities ofa microplate are coated with purified virus glycoprotein andthe eight rowsof the plate are treated with sialidase and increasing concentrations of periodate, as indicated in the figure. After the sialic acid is removed, the penultimate sugarsare sequentially removed, rowby row. The columns may now beincubated with reporter biotinylated lectins and antibodies at a constant dilution, and the bindingisvisualizedin an enzyme-linkedsystem.Changes in absorbance reflect changes in epitope activity, caused by alterations in the peripheral structures of oligosaccharides. The absorbance profiles of the reporter lectin RCA (specificity Gal; see figure)and two antibodies are depicted; one antibody reacting with a galactose-dependent epitope and the other reacting with an epitope, partially masked by sialic acid. (Redrawnfrom Ref. 55.)

topes. Experiments with specific exoglycosidases demonstrated that both &linked galactoseand a-linked fucose promote antigenicity of a cluster of peptide epitopes of an HSV-specified glycoprotein [49,55]. By adding a highly specific galactosyltransferase and an appropriate donor sugar nucleotide to microplate wells, in which the activity of a galactose-dependent epitope was destroyed by &galactosidase treatment, it was possible to restore the antigenic activity in parallel with the restoration of RCA-binding activity. Because of the extremely high specificity of glycosyltransferases [l l], it was possible to conclusively demonstratethat the determinant GalP1,4GlcNAc was necessaryfor maintenance of antigenic activity[49].


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3. Lectin-Mediated hhibition of Virus lnfeaion

As the glycoproteins of enveloped virusesare the mediators of viral adhesion and penetration, some interest has focusedon the carbohydrate parts of these glycoproteins for their possible role in the early events of viral infection (see Fig. 1). Several lectins (ConA, LCA, WGA, E-PHA) bind to the major envelope glycoprotein, gp120, of HIV [47,79], and these lectins also block infection of HIV, during which the initial event is binding of gp120 to cellular CD4 [121]. The lectins also block the fusion of gp120bearing infected cellsand uninfected CD4-bearing cells[47], even when the a infected cells are solely expressing the viral glycoproteins mediated by vaccinia vector [122]. These data suggest that both steps 1 and 2 (see Fig. 1) in the HIV infection may be blocked by lectins. However, penetration may be more affected than initial adhesion, as shown with ConA inhibitionof HSV infection [40]. The carbohydrate part of a viral glycoprotein can mediate infection through binding to an endogenous lectin in the cell membrane [123]. But an observation that a lectin blocks infection invitro after binding to viral envelope glycoproteinscannot in itself be taken as evidence that viral glycans constitutea binding site. Aggregation of virions, as well as conformational changes or steric hindrance, should be taken into consideration. In several cases, lectins can also block infection by binding to a cellular glycoconjugate involved in viral binding [124,1251. Whereas Cow4 inhibits CMV infection of human fibroblasts by binding to the CMV envelope, PHA inhibits CMV infection by binding to the fibroblasts [126]. In some situations witha broad specificity ofthe lectin, asfor ConA, inhibitionof virus infection can occur bothby binding to the virus aswell as by bindingto the that lectins can also interfere cell [124,125]. Finally, it should be mentioned with the proper assembly and budding of enveloped viruses. Thus ConA 7, Fig. 1) by can prevent release of virions from VSV infected cells (see step cross-linking of glycoconjugates inthe infected cell-membrane[127]. Although ConAand PHA have been reportedto inhibit viral infection in vivo [128], and endogenous lectin mannose-binding protein in human plasma blocks HIV infection in vitro [129], lectins have primarily been employed in vitrofor studies of the functional roleof viral glycoproteins in virus infection. Thismay generate not onlyinformation on mechanisms of entry, but may also point out viral carbohydrate structures of interest in anticarbohydrate immune responses [25]. E. Viruses as Probes in Studies on General Cell Biology

Viruses have been widely used in cell biologyto explore a variety of complex subcellular phenomena, from RNA processing to intracellular traffic of

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proteins. The usefulness of viruses as probes in such studies hinges on the facts that (1) viruses are totally dependent on the cellular machinery for energy supply, biosynthetic processes, and assembly; and (2) most viruses shut off biosynthesis of macromolecular synthesis early in the infectious cycle. The virus-infected cell offers a possibility to follow the biosynthesis and intracellular traffic of only a few viral gene products, instead of monitoring the synthesis and breakdown of a myriad of various gene products in a normal cell. The availability of numerous defined conditionally lethal mutant virus strainsadds further potential to this technique [84]. So far as biosynthesis and maturation of glycoproteins are concerned, the combination of viral probes and the use of lectins have been a very successful combination, and much of the knowledge about the complex metabolism of cell surface glycoproteins is derived from such experiments. Thus, the intracellulartransport and processing of glycoproteins have been mapped in detail by the use of organelle-specific lectins and temperaturesensitive virus mutants [84,130]. Intracellular localizations for several of the various steps of protein glycosylation involving both RER vesicles as well as all three classes of Golgi cisternae have been determined. As an example, one could mention the mapping of galactosylation of complextype N-linked glycansto the tram-Golgi cisternae by the use of RCA and SFV-infected BHK cells in colloidal gold-aided electron microscopy[l3l]. The combinationof virus-infected cellsand lectins has also been useful for detection of missing glycosyltransferases in mutant or tumor cells[22], restricting the use of time-consuming glycosyltransferase assays only for confirmational purposes. V.

LECTINS IN CLINICAL VIROLOGY

A. General

Major goals in clinical virology are to diagnose and treat viral infections. The methods usedare based on (1) detection and identification of the viral pathogens; (2) serological methods for detection of virus-specific antibodies; and (3) antiviral treatment, when targeting the drug to the virus-infected cells would be of advantage. In these fields,. lectins have proved useful or may have a potential to be used.

B. Detection and Identification of the Viral Agents Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are antigenically similar, and differentiation between these two virus types is, for clinical reasons in many cases,important. HSV type-specific monoclonalantibodies are generally used for discrimination between HSV-1and HSV-2 [132,


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1331. Slifkin and Cumbie [82] have used the selective binding of HPA for HSV glycoproteins gC-l and gG-2 [58,99] and employed FITC-conjugated HPA for typingofHSV-1 and HSV-2. The HSV-l-infected cellsshow a homogeneous fluorescence, whereas cells infected with HSV-2 display fluorescent spots. This principle is an interesting new approach for typing HSV, but it needs to be confirmedand tested on a larger number of HSV-1 and HSV-2 strains. An advantage of the lectin method of typing HSV, in comparison withthe method basedon the use of monoclonal antibodies, is that only one FITC-HPA conjugate is needed. The cost for a HPA conjugate also may be considerably lower than that for reagents basedon monoclonal antibodies. A similar method has been used in veterinary virology for differentiation of Marek disease virus strains[S]. Influenza A virusoften occurs in epidemic or pandemic form, and the epidemics are a public health problem. Amajor antigenic change of influenza A is called antigenic shift and the new virus type may cause a pandemic often or anepidemic [for review, see 1341. Epidemics of lesser intensity are associated with a more gradual change called antigenic drift. Important antigens subject to antigenic change are the hemagglutinin (HA) and the neuraminidase (NA). The antigenic drift ofNAof influenza A can be analyzed by a lectin neuraminidase test (LPN) as described by Luther et al. [135,1361. The principle of LPN is that erythrocytes incubated with virus NA are specifically agglutinated by the lectins HPA and PNA. The viral NA splits off sialic acid from the erythrocytes, andthe galactose or GalNAc residues inthe terminal positionof the sugar side chain will be exposedand accessible for the reporter lectins[137]. The LPN test is sensitivefor detection of neuraminidase activity, and the test also can be used asa sensitive method for detection of anti-NA activity in human sera. This test hasalso been described for analysis of NA and NA antibodies against influenzaB and mumps viruses [135,136]. C. Serological Methods for Detection of Virus-Specific Antibodies

Purified virus glycoproteins will increase sensitivity and specificity when used as antigens in serodiagnosis of virus infections,and they can also be used for immunization of animals and production of hyperimmune seraor monoclonal antibodies. Several of the purification protocols, reviewed in Table 2, may be used for production of viral glycoproteins of adequate quality for use as immunogensor antigens in serological tests. Some examples of preparations that have been used in routine diagnosis will be reviewed in the following. From HSV-infected cells, soluble viral glycoproteinsare secreted into

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the culture medium. Jeansson et al.[65] used ammonium sulfate precipitation for concentration, and wheat germ lectin chromatographyfor separation, of soluble viral glycoproteins. When tested against HSV-1 glycoprotein-specific monoclonal antibodies, onlygD-l and gE-l antigens couldbe detected in the purified material [unpublished observation]. The purified HSV glycoproteins were used in a thin-layer immunoassay for determination of antibodies to HSV. Results fromthe thin-layer immunoassay correlated well with assaysfor neutralizing antibodies [65]. It is also possible to .use lectin chromatography for purification of antigens for type-selective serodiagnosis of infections with closely related viruses. The HSV-1 glycoprotein gC-l and the HSV-2 glycoprotein gG-2, which may be purified by HPA affinity chromatography (see Table 2), are immunodominant antigens [58,138,139]. Glycoprotein gG-2, and to a certain degree alsogC-l, express a high degree of serological type-specificity and, especially,gG-2 has beenuseful for detection oftype-specific HSV-2 antibodies, diagnosis of HSV-2 infection, and seroepidemiological [140,1411. The purified HSV-2 gG-2antiinvestigations of HSV-2 infection gen can be used in high dilution in ELISA for detection of type-specific 1gG antibodies [138]. The HPA-affinity-purified gC-l antigen will generally react with both HSV-l and HSV-2 antibodies whenused in ELISA, in accordance with findings reported by Suchankova et al. [139], although single batches of gC-l with a high degree of type-specificity have been isolated. Probably this reflects effects of differences in glycosylation between different batches [Olofsson S, Jeansson S, unpublished]. Recently, Wasmuth et al. [l421 used lentil lectin affinity chromatography for purification of glycoprotein antigens from a Triton X-100 lysate of varicella-zoster virus-infected cells. An ELISA based on this purified glycoprotein antigen was highly sensitive and specific and correlated well with neutralizing antibodyand cell-mediated immunity. This ELISA method has been useful for evaluation of the humoral immune response to live attenuated varicella vaccine [143]. Cross-reactions with other human herpesviruses, such as HSV-l and HSV-2, which were reported previously [144], are not detected with this ELISA. Robinson et al. [l451 described a method of coupling HIV-1 envelope glycoproteins to the solidphase of microtiter plates using ConA. First ConA was adsorbed to the wells of a microplate and, in a second step, detergent-solubilized HIV glycoproteins were bound to insolubilized ConA. This ELISAwas highly sensitivefor detection of antibodiesto native a 1 2 0 when tested on a panel of 30 sera. This approach to serodiagnosis of HIV is interesting and should be evaluated in detail to assess the sensitivity and specificity ofthe assay.

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D. Antiviral Treatment: Targeting of Antiviral Drugs to Virus-Infected Cells

Membrane lectins, which bind and assist internalizing mannosylated and 6-phosphomannosylated ligands, have been demonstratedat the surface of normal macrophages. Synthetic neutral, glycosylated, biodegradable, and nonimmunogenic polymers, have been conjugated to antiviral drugs, and the conjugateswere more activethan the free drug inhibitingthe multiplication of herpes virus in human macrophages in vitro [146,147]. For viruses, the primary targetsof which are macrophages and other lymphocytes carrying endogenous membrane lectins, this may be a worthwhile approach to antiviral therapy. VI. CONCLUDING REMARKS

It is clear that experimental techniques basedon lectins have playeda fundamental role for the understanding of the biology of enveloped viruses. Lectin-based techniques have also been important tools in diagnostic virology. Recently, lectin-based techniques have met a challenge from experimental systems based on monoclonal antibodyand DNA technology. There are, however, sufficient reasons to believe that lectins will continue to be important both for general and diagnostic virology. Whereas lectin bindingto small O-linked glycans probablywill be replaced by monoclonal antibodies with more appropriate specificities and association constants(see Section III.B.3), it may be assumedthat the lectins, described in Section III.B.2, bindingto N-glycans, will be competitive in the future. One important example isthe continuous search for new human pathogenic retroviruses,for which the high genetic variability, even within a single virus type, constitutes major a obstacle for general and clinical studies. Here, lectins are extremely helpful, because it is possible to select lectins recognizing N-linked glycans of in essence all retroviral glycoproteins, independent of strain-specific differences in the polypeptide part [see 891. Consequently, lectin affinity chromatography has the potential to be the key procedure for making the variant glycoprotein molecules of retroviruses available to other techniques in generaland diagnostic virology. ACKNOWLEDGMENTS

Work in the author’s laboratory was supported by grants to SO from the Swedish Cancer Society(Grant 2962-B89-01XA), the Swedish Medical Research Council (Grant 9083), the National Swedish Board for Technical Development (Project 87-0256P), and Bristol-Myers Squibb, Inc.

100

Olofsson et al.

REFERENCES 1. Fields BN, Knipe D, eds. Field's virology, vol. 1,2. New York: Raven Press, 1990. 2. Schmitt MK, Mann K. Glycosylation of simian virus40 T antigen and localization of glycosylated T antigen in the nuclear matrix. Virology 1987; 156: 268-281. 3. Ishibasi M, Maize1 JV. The polypeptides of adenovirus. VI. Early and late glycopolypeptides. Virology1974; 58:345-361. 4. Estes MK. Rotaviruses and their replication. In: Fields BN, Knipe D, eds. Fields virology, 2nd ed. New York: Raven Press, 1990:1329-1352. 5. Caust J, Dyall-Smith ML, Lazdins I, Holmes IH. Glycosylation, an important modifier ofrotavirus antigenicity. ArchViroll987; 96:123-134. 6. Datema R, Olofsson S, Romero P. Inhibitors of protein glycosylation and glycoprotein processingin viral systems.Pharrnacol Ther 1987; 33:221-286. 7. Hanover JAYCohen CK, Willingham MC, Park MK. 0-Linked N-acetylglucosamine is attached to proteins of the nuclear pore. Evidence for cytoplasmic and nucleoplasmic glycoproteins. J BiolChem 1987;262:98879894. 8. Marshall RD. Glycoproteins. Annu Rev Biochem 1972; 41:673-702. 9. KornfeldR,Kornfeld S. Comparative aspectsofglycoprotein structure. Annu Rev Biochem 1976; 49217-234. 10. Montreuil J. Primary structure of glycoprotein glycans. Basis for the molecular biology of glycoproteins. Adv Carbohydr Chem Biochem 1980; 37:157223. 11. Roseman S. Complex carbohydrates and intercellular adhesion In:Lee EYC, Smith EE, eds. Biology and chemistry of eucaryoticcell surfaces. New York: Academic Press, 1974:317-354. 12. Kessler MJ, Mise T, Ghai RD, Bahl OP. Structure and location of the 0glycosidiccarbohydrate units of human chorionic gonadotropin. J Biol Chem 1979; 254:7909-7914. 13. Mellis SJ, Baenziger N.Structures of the 0-glycosidically linked oligosaccharides of humanIgD. J Biol Chem 1983; 256:11557-11563. 14. Serafini-CessiF, Dall'Olio F, Scannavini M, Costanzo F, Campadelli-Fiume G. N-Acetylgalactosaminyltransferaseactivity involvedin 0-glycosylation of herpes simplex virus type1 glycoproteins. J Viroll983; 48:325-329. 15. Kim YS, Perdoma J, Nordberg J. Glycoprotein biosynthesisin small intestinal mucosa. I. Astudy of glycosyltransferasesin microsomal subfractions. J Biol Chem 1971; 2465466-5476. 16. Hanover JA, Lennarz WJ, Young JD. Synthesis of N- and 0-linked glycopeptides in oviduct membrane preparations. J Biol Chem 1980; 255:67136716. 17. Losick R, RobbinsPW.Mechanism of€15 conversion studiedwith a bacterial mutant. J Mol Bioll967; 39:445-455. 18. Etchison JR, Summers DF, Georgopoulos G. Variations inthe size and structure of radiolabelled glycopeptidesfrom the glycoprotein of vesicularstoma-


19.

20. 21.

22.

23.

24.

25.

26. 27. 28. 29. 30.

101

titis virus grown in four mouse teratocarcinoma cell lines. J Biol Chem 1981; 256:3366-3369. Campadelli-FiumeG, Poletti L, Dall’Olio F, Serafini-CessiF. Infectivity and glycoprotein processing of herpessimplex virus type 1 grown in a ricinresistant cell line deficient in N-acetylglucosaminyl transferase I. JViroll982; 43~1061-1071. Campadelli-Fiume G, Serafini-Cessi F. Processing of the oligosaccharide chains of herpes simplex virus type1 glycoproteins. In: Roizman B, ed. The herpeseviruses. New York: Plenum Press, 1985:357-382. Serafini-Cessi F, Dall’Olio F, Scannavini M, Campadelli-Fiume G. Processing of herpes simplex virus glycans in cells defective in glycosyl transferases of the Golgi system: relation to cell fusion and virion egress. Virology 1983; 131~59-70. Lundstrom M, Jeansson S, Olofsson S. Host cell-induced differences in the 0-glycosylation of herpessimplex virus gC-l. 11. Demonstration ofcellspecific galactosyltransferase essential for formation of 0-linked oligosaccharides. Virology 1987; 161:395-402. Onodera K, Yamaguchi N, Kuchino T, Aoi Y. Alterations in surface glycoproteins and levels of sialyltransferase of cells transformed by a temperaturesensitive mutant of simian virus40. Proc Natl Acad Sci USA 1976; 73:40904094. Adachi M, Hayami M, Kashiwagi N, Mizuta T, Ohta Y, Gill MJ, Matheson DS, Tamaoki T, Shiozawa C, Hakomori S. Expression of Ley antigen in human immunodeficiency virus-infected human T cell linesand in peripheral lymphocytes of patients with acquired immune deficiency syndrome (AIDS) and AIDS-related complex (ARC). J Exp Med 1988; 167:323-331. Hansen JE, Clausen H, Nielsen C, Teglbjaerg LS, Hansen LL, Nielsen CM, Dabelsteen E, Mathiesen L, Hakomori SI, Nielsen JO. Inhibition of human immunodeficiency virus (HIV) infection in vitro by anticarbohydrate monoclonalantibodies: peripheral glycosylationofHIVenvelopeglycoprotein gp120 may be atarget for virus neutralization. JViroll990; 64:2833-2840. Fossum C, Burny A, Portetelle D, Mammerickx M, Morein B. Detection of B and T cellswithlectins or antibodies in healthy and bovine leukemia virus-infected cattle.Vet Immunol Immunopathol 1988; 18:269-278. Clausen H, Hakomori S. ABH and related histo-blood group antigens; immunochemical differencesin carrier isotypesand their distribution. Vox Sang 1989; 56:1-20. Bolscher JG, van der Bijl MM, Neefjes JJ, Hall A, Smets LA, Ploegh HL. rus (proto)oncogene induces N-linked carbohydrate modification: temporal relationshipwith induction of invasivepotential. EMBO J 1988; 7:3361-3368. Bruyneel EA, Debray H, De Mets M, Mareel MM, Montreuil J. Altered glycosylation in Madin-Darby canine kidney(MDCK) cellsafter transformation by murine sarcoma virus. Clin Exp Metastasis 1990; 8:241-253. Hurley JB, Simon MI, Teplow DB, Robishaw JD, Gilman AG. Homologies between signal transducing G proteins and rus gene products. Science 1984; 226960-862.

102

Olofsson et al.

31. Beadling L, Rothfield L. Modulation of the conformation of a membrane glycosyl transferase by specific lipids. Proc Natl Acad Sci USA 1978; 75: 3669-3672. 32. Mookerjea S, Yung JWM. Studieson uridine-galactosepyrophosphatase and uridine diphosphate-galactose :glycoprotein galactosyl transferase activities in microsomal membranes. Arch Biochem Biophys 1975; 166:223-236. 33. Olofsson S, Khanna B, Lycke E. Altered kinetic properties of sialyl and galactosyl transferases associated with herpes simplex virus infection GMK of and BHK cells. J Gen Virol1980; 47:l-9. 34. Kumarasamy R, Blough HA. Characterization of oligosaccharides of highly purified glycoprotein gC of herpes simplex virus type 1. Biochem Biophys Res Commun 1982; 109:1108-1115. 35. Tollefsen SE, Kornfeld R. The B., lectin from Vicia villosa seeds interacts with N-acetylgalactosamine residuesa-linked to serine or threonine residues in cell surface glycoproteins. JBiol Chem 1983; 2585172-5176. 36. Goldstein IJ, Poretz RD. Isolation, physicochemical characterization, and carbohydrate-binding specificity of lectins. In: Liener IE, Sharon N, Goldstein IJ, eds. The lectins: properties, functions, and applications in biology and medicine. Orlando: Academic Press, 1986:233-245. 37. Montreuil J. Structure and conformation of glycoprotein glycansIn: Olden K, Parent JB, eds. Vertebrate lectins.New York: Van Nostrand, 1987:l-26. 38. Ozanne B, Sambrook J. Binding of radioactively labelled concanavalin A and wheat germ agglutinin to normal and virus-transformed cells. Nature 1971; 232:156-160. 39. Zarling JM, Tevethia SS. Expression of concanavalin A binding sites in rabbit kidney cells infected with vaccinia virus. Virology 1971; 45:313-316. 40. Ito M, Barron AL. Inactivation of herpes simplex virusby concanavalin A. J Viroll974; 13:1312-1318. 41. Kornfeld K, Reitman ML, Kornfeld R. The carbohydrate-binding specificity of pea and lentil lectins. Fucose is an important determinant. J Biol Chem 1981; 256~6633-6640. 42. Cummings RD, Kornfeld S. Fractionation of asparagine-linked oligosaccharides by serial lectin-agarose affinity chromatography. A rapid, sensitive technique. J Biol Chem 1982; 257:11235-11240. 43. Cummings RD, Kornfeld S. Characterization of the structural determinants required for the high affinity interaction of asparagine-linked oligosaccharides with immobilizedPhaseolus vulgarisleukoagglutinating and erythroagglutinating lectins. J Biol Chem 1982; 257:11230-11234. 44. Green ED,Baenziger JU. Oligosaccharide specificities ofPhaseolus vulgaris leukoagglutinating and erythroagglutinating phytohemagglutinins. Interactions withN-glycanase-releasedoligosaccharides.JBiolChem 1987;262: 12018-12029. 45. Olofsson S, Datema R.New virus-selectiveinhibitor of terminal glycosylation increasing immunological reactivityof a viral glycoprotein. Antiviral Chem Chemother 1990: 1:17-24. 46. Deom CM, Schulze IT. Oligosaccharide composition of an influenza virus


47.

48.

49.

50.

51. 52. 53.

54. 55.

56. 57. 58. 59.

60.

103

hemagglutinin with host-determined binding properties. J Biol Chem 1985; 260:14771-14774. Hansen JE, Nielsen CM, Nielsen C, Heegaard P, Mathiesen LR,Nielsen JO. Correlation between carbohydrate structures on the envelope glycoprotein gp120 of HIV-1 and HIV-2 and syncytium inhibition withlectins. AIDS 1989; 3:635-641. Olofsson S, Lundstrom M, Datema R. The antiherpes drug (E)-5-(2bromovinyl)2"deoxyuridine (BVdU) interferes with the formation of Nlinked and O-linked oligosaccharides of the herpes simplex virus type 1 glycoprotein C. Virology1985; 147:201-205. Olofsson S, Sjoblom I, Jeansson S. Activity of herpes simplex virus type l-specified glycoprotein C antigen site I1 epitopes modulated by peripheral fucose or galactose units of glycoprotein oligosaccharides. J Gen Virol 1990; 71:889-895. Ohlson C, Karlsson JO. Glycoproteins of axonal transport: polypeptides interacting with the lectin from Aleuria aurantia. Brain Res 1983; 264:99104. Gustavsson S, Ohlson C, Karlsson JL. Glycoproteins of axonal transport: affinity on fucose-specific lectins.J Neurochem 1982; 38:852-855. Kornfeld R, KornfeldS. Structure of glycoproteinsand their oligosaccharide units. In: Lennarz WJ, ed. The biochemistry of glycoproteins and proteoglycans. New York: Plenum Press,1981:l-34. Green ED, Brodbeck RM, Baenziger JU. Lectin affinity high-performance chromatography liquid chromatography. Interactions with Ricinus communis agglutinin 1 and Ricinus communis 11. J BiolChem1987;262:1203012039. Pierce-Crktiel A, Izhar Y, Nuchmanowitz Y, Strecker J, Montreuil J, Spik G, Mirelman D. Oliogosaccharidestructure of the soluble agglutinin released from guinea pig colonic epithelial cells. FEMS Lett 1983; 20:237-242. Sjoblom I, Lundstrom M, Sjogren-Jansson E, Glorioso JC, Jeansson S, Olofsson S. Demonstration and mapping of highly carbohydrate-dependent epitopes in the herpes simplex virus type l-specified glycoprotein C. J Gen Virol 1987; 68545-554. Olofsson S. Studies on herpes simplex virus glycoproteins. Dissertations of the Medical Faculty, University of Goteborg, 1980. Olofsson S, Blomberg J, Lycke E. 0-Glycosidic carbohydrate-peptide linkages of herpes simplexvirus glycoproteins. Arch Virol1981; 70:321-329. Olofsson S, Jeansson S, Lycke E. Unusual lectin-binding properties of a herpes simplex virus type l-specific glycoprotein. J Virol 1981; 38:564-570. Hammarstrom S, Murphy LA, Goldstein IJ, Etzler M. Carbohydrate binding specificity offour N-acetyl-D-galactosamine-"specific"lectins: Helixpomatia A hemagglutinin, soy bean agglutinin, lima bean lectin and Dolichos bifrorus lectin. Biochemistry 1977; 16:2750-2755. Mansson JE, Olofsson S. Binding specificities of the lectins from Helkpomatia, soybean and peanut against different glycosphingolipids in liposome membranes. FEBS Lett 1983; 156:249-252.

Olofsson et al.

104

61. Olofsson S, Sjoblom I, Lundstrom M, Jeansson S, Lycke E. Glycoprotein C of herpes simplex virus: characterization of 0-linked oligosaccharides. J Gen Viroll983; 64:2735-2747. 62. Lundstrom M, Olofsson S, Jeansson S, Lycke E, Datema R, Mansson J-E. Host cell induced differences in 0-glycosylation of the herpes simplex virus gC-l. I. Structures of nonsialylated HPA- and PNA-binding carbohydrates. Virology 1987; 161:385-394. 63. Osawa T, Tsuji T. Fractionation and structural assessment of oligosaccharides and glycopeptides by use of immobilized lectins. Annu Rev Biochem 1987; 56:21-42. 64. Elia G, Ferrantini M, Belardelli F, Proietti E, GresserI,Amici C, Bendetto A. Wheat germ agglutinin-binding protein changes in highly malignant Friend leukemia cells metastasizing to the liver. Clin ExplMelastatis 1988; 6:347-362. 65. Jeansson S, Elwing H, Nilsson L-A. Thin-layer immunoassay for determination of antibodiesto herpes simplex virus. J Clin Microbiol1979; 9:317-322. 66. Allen A K , Neuberger A, SharonN. The purification, composition, and specificity of wheat germ agglutinin. Biochem 1973; J 131:155-162. 67. Peters BP, Ebisu S, Goldstein IJ, Flashner M. Interaction ofwheatgerm lectin with sialic acid. Biochemistry 1979; 275505-5511. 68. Kronis K A , Carver JP. Wheat germ agglutinin dimers bind siayloligosaccharides at four sites in solution: proton magnetic resonance temperature studies at 360 MHz. Biochemistry 1985; 24:826-833. 69. Wright CS. Localization of the N-acetyl-D-neuraminic acid binding site in wheat germ agglutinin: a crystallographic study at 2-8 resolution. J Mol Bioll980; 141:267-291. 70. Badia M, QuerolE. Detection and purification of bovine herpesvirus1 glycoproteins by lectin affinity. J Virol Methods1988; 22:23-29. 71. Adam G, Heegardy P, [email protected] as probes for the assay of rhabdovirus infectionsin plants. J Virol Methods1987; 17:263-275. 72. Hammarstrom S, KabatEA. Purification and characterization of a blood group A-reactive hemagglutinin from the snail Helixpomatia and a study of its combining site. Biochemistry1969; 8:2696-2705. 73. Lis H, Sharon N. Lectins: their chemistry and application to immunology In: Sela M, ed. The antigens, v01 4. New York: Academic Press, 1977:429529. 74. Laemmli UK. Cleavageof structural proteins during the assemblyof the head of bacteriophageT4. Nature 1970; 227:680-685. 75. Fargeaud D, JeanninCB,Kat0 F, Chappuis G.Biochemical study of the feline herpesvirus 1. Identification of glycoproteins by affinity. Arch Virol 1984; 80:69-82. 76. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA1979; 76:4350-4354. 77. Russell DL, Consigli RA. Glycosylation of purified enveloped nucleocapsids of the granulosis virus infecting PIodia interpunctellaas determined by lectin binding. Virus Res1985; 4:83-91.

A


105

Primate cytomegalovirusglycoproteins:lectin78. BenkoDM,GibsonWA. binding propertiesand sensitivitiesto glycosidases. J Virol 1986; 59:703-715. 79. Hammar L, ErikssonS, Morein B. Human immunodeficiency virus glycoproteins: lectin binding properties. AIDS Res Hum Retroviruses 5:495-506. 1989; 80. Kuroda K, Geyer H, Geyer R, Doerfler W, Klenk HD. The oligosaccharides of influenza virus hemagglutinin expressed in insect cells by a baculovirus vector. Virology 1990; 174:418-429. 81. Butters TD, Hughes RC, Vischer.P. Steps in the biosynthesis of mosquito cell membrane glycoproteins and the effects of tunicamycin. Biochim Biophys Acta 1981; 640:672-686. 82. Slifkin M, CumbieR. Rapid detectionof herpes simplex virus with fluorescein-labeled Helixpomatia lectin. J ClinMicrobioll989; 27:1036-1039. 83. Poste G, Reeve P. Increased mobility and redistribution of concanavalin A receptors on cells infected with Newcastle disease virus. Nature 1984; 247: 469-47 1. 84. Kaiiriiriiiinen L, Virtanen I, Saraste J, Keriinen S. Transport of virus membrane glycoproteins, useoftemperature-sensitive mutants and organelle-specific lectins. MethodsEnzymoll983; %:453-456. 85. Malkinson M, Orgad U, Becker Y. Use of lectins to detect and differentiate subtypes of Marek’s disease virus and turkey herpesvirus glycoproteins in tissue culture. J Virol Methods1986; 13:129-133. 86. Moreno-Lopez J, Kristiansen T, Kbsnas P. Bovine viral diarrhea virus: affinity chromatography on Crotalariajuncea lectin. J Virol Methods 1981; 2: 293-300. 87. Neukirch M, Moennig V, Liess B. A simpleprocedure for the concentration and purification of hog cholera virus (HCV) using the lectin of Ricinus communis. Arch Virol 1981; 69:287-290. 88. Stewart ML, Summers DF, Soeiro R, Fields BN, Maize1 JV. Purification of oncornaviruses by agglutination with concanavalin A. Proc Natl Acad Sci USA 1973; 79~1308-1312. 89. Montelaro RC, West M, IsselCJ. Isolation of equine infectious anemia virus glycoproteins. Lectin affinity chromatography procedures for high avidity glycoproteins. J Virol Methods1983; 6:337-346. 90. Hayman MJ, Skehel JJ, Crumpton MJ. Purification of virus glycoproteins by affinity chromatography using Lens culinaris phytohemagglutinin. FEBS Lett 1973; 29:185-188. 91. Kristiansen T, Sparrman M, Heller L. Towards a subunit influenza vaccine prepared by affinity chromatography on immobilized lectin. J Biosci 1983; 5~149-155. 92. Gething MJ, White JM, Waterfield MD. Purification of the fusion protein of Sendaivirus: analysis ofthe NH,-terminal sequence generatedduring precursor activation. Proc Natl Acad Sci USA 1978; 75:2737-2740. 93. Lund GA, Salmi AA. Purification and characterization of measlesvirus haemagglutinin protein G. J Gen Virol 1981; 56:185-193. I 94. Christie M, Endresen C, Haukenes G. Purification of measles virus H polypeptide and of F polypeptide. Arch Virol 1981; 69:177-187.

106

Olofsson et al.

95. Devare SG, Stephenson JP. Biochemical and immunological characterization Of the major envelope glycoprotein of bovine leukemia virus.J Vir01 1977; 23M3-437. 96. Westenbrink F, Koornstra W, Bentvelzen P. The major polypeptides of the murine-mammary-tumor virus isolated by plant-lectin affinity chromatography. Eur J Biochem1977; 76:85-90. 97. Zwaagstra JC, Armstrong GD, Leung WC,The use of lectin affinity columns for selection of precursor or fully glycosylatedforms of glycoprotein gD1 of herpes simplex virus type1. J Virol Methods1988; 20:21-32. 98. Respess RA, Pancake BA, Edson CM, Schaffer PA. A rapid procedure for the enrichment of undenaturated. antigenically active herpes simplex virus glycoproteins. J Virol Methods1984; 8:27-45. 99. Olofsson S, Lundstrom M, Marsden H, Jeansson S, Vahlne A. Characterization of a herpes simplexvirus type Zspecified glycoprotein withaffinity for N-acetylgalactosamine-specificlectins and its identificationas g92K or gG. J Gen Virol 1986; 67:737-744. 100. Friedrichs WE, Grose C. Glycoprotein gpl18 of varicella-zoster virus: purification by serial affinity chromatography. J Viol 1984; 49:992-996. 101. Thorley-Lawson DA, Poodry CA. Identification and isolation of the main component (gp350-gp220) of Epstein-Barr virus responsible for generating neutralizing antibodiesin vivo. J Virol 1982; 43:730-736. 102. Qualtiere LF, Chase R, Pearson GR. Purification and biologic characterization of a major Epstein Barr virus-induced membrane glycoprotein. J Immuno1 1982; 129814-818. 103. Pachl C, Burke RL, Stuve LL, Sanchez-PescadorL, VanNest G, Masiary F, Dina D. Expression of cell-associated and secreted forms of herpes simplex virus type 1 glycoprotein Bin mammalian cells. JViroll987; 61:315-325. 104. Persson H, KatzeMG,PhilipsonL. Purification of a nativemembraneassociated adenovirus tumor antigen. J Virol 1982; 4295-917. 105. Neurath AR, Prince AM, Lippin A. Affinity chromatography of hepatitis B antigen on concanavalin A linked to Sepharose. J Gen Virol 1973; 19:391395. 106. Ponce de Leon M, Hessle H, Cohen GH. Separationof herpes simplex virus antigens by concanavalin A affinity chromatography. J Virol 1973; 12766774. 107. Keller PM, Lonergan K, Neff BJ, Morton DA,EllisRW. Purification of individual varicella-zoster virus (VZV)glycoproteins gpI, gpII, and gpIII and their use in ELISA for detection of VZV glycoprotein-specific antibodies. J Virol Methods 1986; 14:177-188. 108. Helenius A, Simons K. Solubilization of membranes by detergents. Biochim Biophys Acta 1975; 415:29-79. 109. Lotan R, Beattie G, Hubbell W, Nicolson GL. Activities of lectins and their immobilized derivatives in detergent solutions. Implications on the use of lectin affinity chromatography for the purification of membrane glycoproteins. Biochemistry 1977; 16:1787-1794.


l07

110. Krah DL. Characterization of octyl glucoside-solubilized cell membrane receptors for binding measles virus. Virology1989; 172:386-390. 111. Spear PG, Roiman B. Proteins specified by herpes simplex virus.V. Purification and structural proteins by the herpesvirion. J Virol1972; 9:143-159. 112. Svennerholm B, Vahlne A, Jeansson S, Lunden R, Olofsson S, Svantesson 113.

114. 115. 116.

G, Lycke E. Separation of herpes simplex virusand nucleocapsids on Percoll gradients. J Virol Methods1980; 1:303-309. Whittaker GR, Wheldon LA, Giles LE, StocksJ-M, Halliburton IW, Killington RA, MeredithDM. Characterization of the high MW glycoprotein (gp3OO) of equine herpesvirus type 1 as a novel glycoprotein with extensive 0-linked carbohydrate. J Gen Virol1990;71:2407-2416. Kari B, Gehrz R. Isolation and characterization of a human cytomegalovirus glycoprotein containing a high content of 0-linked oligosaccharides. Arch Virol 1988; 98:171-188. Pinter A, Honnen WJ. 0-Glycosylation of retroviral envelope gene products. J Virol 1988; 62:1016-1021. Wertz GW, CollinsPL, Huang Y,Gruber C, Levine S, Ball LA. Nucleotide sequence of the G protein gene of human respiratory syncytial virus reveals an unusual type of viral membrane protein. Proc Natl Acad Sci USA 1985;

82:4075-4079. 117. Kaplan A, Erickson JS, Ben-Porat T. Synthesis of proteins in cells infected 118. 119.

120. 121. 122. 123.

124.

with herpesvirus. X. Proteins excreted by cells infected with herpes simplex virus type 1and 2. Virology 1975; 64:132-143. Alexander S, Elder JH. Carbohydrate dramatically influences immune reactivity ofantisera to viral glycoprotein antigens. Science 1984; 226:1328-1330. Skehel JJ, Stevens DJ, Daniels RS, Douglas HR, Knossow M, Wilson IA, Wiley DC. Acarbohydrate side chainon hemagglutinin of Hong Kong influenza viruses inhibits recognition by a monoclonal antibody. Proc Natl Acad Sci USA 1984; 81:1779-1783. van der Schaal I A M , Logman TJJ, Dim CL, Kijne JW. An enzyme-linked lectin binding assayfor quantitative determination of lectin receptors. Anal Biochem 1984; 140:48-55. Robinson WE Jr, Montefiori DC, Mitchell WM. Evidence that mannosy1 residues are involved inhuman immunodeficiency virustype 1(HIV-l) pathogenesis. AIDS Res Hum Retroviruses 1987; 3:265-282. Lifson J, Coutrt S, Huang E, Engleman E. Role of envelope glycoprotein carbohydrate in human immunodeficiencyvirus (HIV) infectivityand virusinduced fusion. J Exp Med 1986; 164:2101-2106. Larkin M, Childs RA, Matthews TJ, Thiel S, Miziochi T, Lawson AM, Savi11 JS, Haslett C, Dim R, Feizi T. Oligosaccharide-mediatedinteractions of the envelope glycoproteingp120 of HIV-1that are independent of CD4 recognition. AIDS 1989; 3:793-798. Mastromarino P, Conti C, Orsi N. Effect of concanavalin Aon early interactions of Sindbis virus with goose erythrocytesand BHK 21 cells. Microbialogica 1986; 9:295-303.

108

Olofsson et al.

125. Bowen DL, Isaak DD, CernyJ. Inhibition of in vitro Friend murine leukemia virus infection of lipopolysaccharide-activated B-cells with concanavalin A. JNCI 1979; 62:1497-1502. 126. Ito M, Girvin L, Barron AL. Inactivation of human cytomegalovirus by phytohemagglutinin. Arch Virol 1978; 57:97-105. 127. Cartwright B. Effect on concanavalin A on vesicular stomatitis virus maturation. J Gen Virol 1977; 34249-256. 128. Dent PB.Inhibition of mortality and induction of immunityto Friend disease by lectin-treated virus.JNCI 1973; 50511-513. 129. Ezekowitz RAB, Kuhlman M, Groopman JE, Byrn RA. A human serum mannose-binding protein inhibits in vitro infection by the human immunodeficiency virus. J Exp Med 1989; 169:185-196. 130. Griffiths G, Quinn P, Warren G. Dissection ofthe Golgi complex. I. Monensin inhibits the transport of viral membrane proteins from medial to truns Golgi cisternae in baby hamster kidney cells infected with Semliki Forest virus. J Cell Bioll983; 969335-850. 131. Griffiths G, Brands R, Burke B, Louvard D, Warren G. Viral membrane proteins acquire galactose in truns Golgi cisternaeduring intracellular transport. J Cell Biol 1982; 95:781-792. 132. Nilheden E, Jeansson S, Vahlne A. Typing of herpes simplex virus by an enzyme linkedimmunosorbent assay with monoclonalantibodies. J Clin Microbioll983; 17:677-680. 133. Frame B, Mahony JB, Balachandran N, Rawls WE, Chernensky MA. Identification and typing of herpes simplex virus by enzyme immunoassay with monoconal antibodies. J Clin Microbiol1984; 20:162-166. 134. Murphy BR,WebsterRG.Orthomyxoviruses.In:FieldsBN,KnipeDM, eds. Fields virology, 2nd ed.New York: Raven Press, 1990:1091-1144. 135. Luther P, AdamczykB,BergmanKC.Simple test for detection of virus neuraminidase and antineuraminidase using lectins. Zentralbl Bakteriol [A] 1980; 248:281-285. 136. Luther P, Bergman KC, Oxford JS. An investigation of antigenic drift of neuraminidase of influenza A (HlN1) viruses. J Hyg (Lond) 1984; 92:223229. 137. Bird GWG.Anti-T in peanuts. Vox Sang 1964; 9:748-752. 138. Svennerholm B, Olofsson S, Jeansson S, Vahlne A, Lycke E. Herpes simplex virus type-selective enzyme-linkedimmunosorbent assay withHelix pomatiu lectin-purified antigens.J Clin Microbioll984; 19:235-239. 139. Suchankova A, Hirsch I, Kremar M, Vonka V. Determination of herpes simplex virus type-specific antibodies by solid-phase RIA on Helixpomutiu lectin-purified antigens.J Infect Dis 1984; 149:964-972. 140. Ades A E , Peckham CS, Dale GE, Best JM, Jeansson S. Prevalence of antibodies to herpes simplex virus types 1 and 2 in pregnant women, and estimated rates of infection. J Epidemiol CommunityHealth 1989; 4353-60. 141. Lowhagen GB, Jansen E, Nordenfelt E, Lycke E. Epidemiology of genital herpes infection inSweden. Acta Derm Venereol (Stockh)1990; 70330-334. 142. Wasmuth EH, Miller WJ. Sensitive enzyme-linked immunosorbent assayfor


143. 144.

145.

146.

147. 148. 149. 150. 151. 152. 153.

109

antibody to varicella-zoster virus using purifiedVZV glycoprotein antigen. J Med Virol1990; 32:189-193. Provost PJ, Krah DL, Kuter BJ, Morton DH, Schofield TL, Wasmuth EH, White CJ, Miller WJ, Ellis RW. Antibody assays suitable for assessing immune responsesto live varicella vaccine. Vaccine 1991; 9:111-116. Kitamura K, Namazue J, Campo-Vera H, Ogino T, Yamanishi K. Induction of neutralizing antibody against varicella-zoster virus (VZV) by VZV gp3 and cross reactivity betweenVZV gp3 and herpes simplexgB. Virology 1986; 149~74-82. Robinson JE, Holton D, Liu J, McMurdo H, Murciano A, Gohd R. A novel enzyme-linked immunosorbent assay (ELISA)for thedetection ofantibodies to HIV-1 envelope glycoproteins basedon immobilization of viral glycoproteins in microtiter wells coated with concanavalin A. J Immunol Methods 1990,132:63-71. Midoux P, Negre E, Roche AC, Mayer R, Monsigny M, Balzarini J, De Clercq E, Mayer E, Ghaffar A, Gangemi JD. Drug targeting: anti-HIV-l activityofmannosylatedpolymer-bound 9-(2-phosphonylmethoxyethyl)adenine. Biochem BiophysRes Commun 1990; 167:1044-1049. Roche AC, Midoux P, Pimpaneau V, Negre E, Mayer R, Monsigny M. Endocytosis mediatedby monocyte and macrophage membrane lectins-applications to antiviral drug targeting. Res Virol 1990; 141243-249. Schlesinger S, Schlesinger MJ. Replication of Togaviridaeand Flaviviridae. In: Fields BN, Knipe DM, eds. Fields virology, 2nd ed. New York: Raven Press, 1990: 697-712. Niemann H, Geyer R, Klenk H-D, Stirm S, Wirth M. The carbohydrates of mouse hepatitis virus (MHV) A59: structures of the 0-glycosidically linked oligosaccharides of glycoproteinEl. EMBO J 1984; 3565-670. Schachter H. Biosynthetic controls that determine the branching and microheterogeneity of protein-bound oligosaccharides. BiochemCellBiol1986; 64:163-181. Frink RJ, Eisenberg R, Cohen G, Wagner EK. Detailed analysis ofthe portion of herpes simplex virus genome encoding glycoprotein C. J Virol 1983; 45~634-647. Swain MA, Peet RW, Gallaway DA. Characterization of the gene encoding herpes simplex virus type 2 glycoprotein and comparison with the type 1 counterpart. J Virol 1985; 53561-569. Dowbenko DJ, Laskey LA. Extensive homology betweenthe herpes simplex virus type 2 glycoprotein F gene and the herpes simplex virus type 1 glycoprotein C gene. J Viroll984; 52:154-163.


3 Epidemiological Applicationsof Lectins to Agents of Sexually Transmitted Diseases WILLIAM 0. SCHALLA and STEPHEN A. MORSE Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, Atlanta, Georgia

1. INTRODUCTION

The first reported use of lectins to agglutinate bacteria was published in 1936 and involved mycobacteria [l]; however, it was not until the 1960s that lectins were used to study the cell surface carbohydrate moieties of bacteria and fungi [2-51. Since that time, the amount of published information on lectin characterization of pathogenic and nonpathogenic microorganisms has dramatically increased.A review of the interaction of bacteria [6]. The and fungi with lectinsand lectinlike substances has been published interested reader is referred to this as well as other reviews [7-1 l] for further information. Many studies on lectin-microorganism interactions have focused on lectin binding to whole organisms. Virtually any carbohydrate-containing cell surface structure has the potential of binding one or more lectins. Some of these components have been identified and include fungal cell wall constituents, such as mannans [6,7] and chitin [6,7], and bacterial components, such as capsular polysaccharides [6,7,12-141, lipopolysaccharides [6,7,12,13,15], teichoic acids[6,7,16,17], and noncapsular polysaccharides [18,191 (see Chapter 1). Although it has been reported[20] that lectin-binding maydifferentiate between microbial genera, it is evidentthat lectin-binding can be used as a typing method to study intraspecies differences [8,21-321. Because lectins Use of tradenames is for identification only and does not constitute endorsement by the Public Health Service or by theU.S. Department of Health and Human Services. 111

l

112

Schalla and Morse

have the ability to bind to a wide variety of microbial components that contain either simpleor complex carbohydrates, they can be used to detect changes in the bacterial cell envelope, identify cell surface carbohydrates, or differentiate between strains of bacteria on the basis of variations in cell surface carbohydrates. The latter use has epidemiological implications in that it can provide information that can be used either directly or in conjunction with other typing systems to study the population dynamics of pathogenic microorganisms.This chapter will focus onthe use of lectinsto study sexually transmitted microorganisms such as Neisseria gonorrhoeae, Haernophilus ducreyi, Treponema pallidurn subsp pallidurn, and Treponema pallidurn subsp pertenue. II. NHSS€R/AGONORRHOEA€

A. TheOrganism

Neisseria gonorrhoeae is a gram-negative diplococcus that colonizes human mucosal surfaces. Gonococci cause symptomatic or asymptomatic localized infections including urethritis, cervicitis, proctitis, pharyngitis, and conjunctivitis. Disseminated infections occur either by direct extension to adjacent organs [pelvic inflammatory disease (PID), epididymitis], or by bacteremic spread (skin lesions, tenosynovitis, septic arthritis, endocarditis, and meningitis).

B. Cell Envelope The outer membrane is composed of proteins, phospholipids, and lipopolysaccharide. Neisseria gonorrhoeae and other mucosal pathogens, such as N. meningitidis and Haemophilus influenzae, produce highly branched and relatively short lipopolysaccharides [33]. These lipopolysaccharides lack the repeating and variable 0-antigens that are characteristic of the lipopolysaccharide of the Enterobacteriaceae. To reflect these structural differences, gonococcal lipopolysaccharide andother bacterial lipopolysaccharidesthat share similar featuresare referred to as lipooligosaccharides(LOS). l . Properties of Gonococcal lipooligosaccharide Gonococcal LOS consists of several components with molecular weights

ranging from 3200 to 7100 [34]. Immunochemical analysis of gonococcal LOS using monoclonal antibodies has revealed the presence of discrete individual LOS components [35,36]. GonococcalLOS share structures and epitopes with human glycosphingolipids that are precursors to blood group antigens [37]. TheseLOS are sialylated in vivo by host cytidine monophospho-N-acetylneuraminic acid in a manner similar to host glycosphingolipids

Applications Agents of to Lectins

of STD

113

[38,39]. The sialic acid is readily lost during in vitro cultivation [M]. The structures of the oligosaccharide component ofthe two major LOS components of N. gonorrhoeae strain F62 have recently been determined [41] and are illustrated in Figure 1. These are the only LOS structures that have 1B) been completely determined. The terminal Galp-l,4GlcNAcPl (see Fig. is the structure that is sialylated in vivo [38]. Gonococci are apparently able to vary the expression of LOS determinants [42]. The basis for this variation is incompletely understood. C. Interaction with Lectins

The LOS is primarily responsible for the interaction of N. gonorrhoeae with lectins, suchas wheat germ agglutinin (WGA) [13]. Schaefer al. et [26] observed that WGA reacted with 165 of 165strains of N. gonorrhoeae and as an aid in identifying proposed that agglutination by WGA could be used this organism. Yajko et al. [27] observed that rare strains (4 of 126) were not agglutinated byWGA and proposed that a combination ofWGA, soybean agglutinin (SBA), and chromogenic substrates could be used for the confirmatory identificationof N. gonorrhoeae. Lectin-binding studies have been useful in determining the structure of the gonococcal LOS. Allen et al. [l51 observed that some lectins [WGA, SBA, Phaseolus vulgaris (PHA), and ricin] agglutinated all strains, suggesting that P-linked D-N-acetylglucose-(GlcNAc)and P-D-galactose (Gal) units werecommon structural features of the LOS. Otherlectins[Dolichos biforis (DBA),limulin, and Sophora japonica] agglutinated only some strains, suggesting that cy-D-GalNAc units and p-~-Gallinked to GalNAc

GalNAc~l+3Gal~l+4GlcNAc~l+4-Glc~1+4Hepa+KDO

(A)

3

t

GlcNAcal+2Hepal

Gal~l+4GlcNAc~l+3Gal~l+4-Glc~l+4Hepa+KDO 3

(B)

T

GlcNAcal+2Hepal Figure 1 Structure of the two major dioligosaccharides derived from the lipooligosaccharides of N. gonorrhoeae strain F62. (Data fromRef. 41

.)

Schalla and Morse

114

occurred as structural features of the LOS of some strains. In retrospect, these resultsare consistent withthe recently determined structures shown in Figure 1. The differentialbinding of lectins to N . gonorrhoeae that was observed in previous studies [15,24] suggested the presence or exposure of specific carbohydrates may vary between strains, and these differences may be used to provide useful epidemiological information. 1. Methodology

The following protocol has been used successfully for lectin-typing of N . gonorrhoeae. Fourteen lectins with specificities for the carbohydrates found in gonococcal LOS, including many of those used by Allen et al. [l51 to obtain structural information about the gonococcal cell envelope and by Doyle et al. [24] to distinguish between members of the family Neisseriaceae, were obtained as commercially prepared lectin agglutination panels from E-YLaboratories (San Mateo, California). The 14 lectins and their carbohydrate specificitiesare shown in Table1. Gonococcal (GC) agar base (Difco Laboratories, Detroit, Michigan) containing1 Vo (vol/vol) IsoVitaTable 1 Lectins Used in the Commercially Prepared Panelsand their Carbohydrate Specificities'

Lectin Concanavalin A Lens culinaris Trichosanthes kirilowii Griffoniasimplicifolia I Arachis hypogeae

Con A LCA TRK

Glycine m a Dolichos bifrorus Griffoniasimplicifolia I1 Solanum tuberosum Triticum vulgaris

SBA DBA GS-I1 STA WGA

L i m aflavus Phaseolus vulgaris Ulex europaeus I Lotus tetragonolobus

LFA PHA UEA-I LOTUS

GS-I PNA

a-~-Man> CY-D-G~C CY-D-G~CNAC a-D-Man > CY-D-G~C D-G~ a-~-Gal> ~x-D-G~NAc D-Gal-B-(1+3) > B-D-GdNH, = a-~-Gal

CY-D-G~NAC > p - ~ G a l N A ca - ~ - G a l a-~-GalNAc> CY-D-G~ a-~-GlcNAc= 8-D-GlcNAc @-D-GkNAc)ZJ > U-D-G~CNAC) (B-D-GlcNAc), > @-D-GkNAc), > W-D-G~CNAC) N-Acetylneuraminic acid D-G~NAc IX-L-FUC CY-L-FUC

'Lectin specificities accordingto Goldstein and Hayes[7] and E-Y Laboratories. bMan. mannose; Glc, glucose; Fuc, fucose; Gal, galactose. Source: Ref. 47.

Applications of Lectins to Agents of STD

115

lex enrichment (BBL, Cockeysville, Maryland) and 2% (vol/vol) fetal bovine serum (Hyclone Laboratories, Ogden, Utah), pH 7.2, is the medium of choice for cultivatinggonococci for lectintyping. The inclusion of Hyclone fetal bovine serum had no effecton the agglutination patterns of control strains of N. gonorrhoeae (unpublished data). Likewise, varying the initial pH of the medium from 6.5 to 7.8 did not affectthe agglutination patterns of control strains. Culturesof N. gonorrhoeae grown on chocolate agar for more than 18 hr often exhibited cell lysis and autoagglutination, causing difficulty in interpreting lectin agglutination patterns [15]. This can be prevented by using a commercially available antiautoagglutination reagent [27] or by incorporating deoxyribonuclease (DNase) the in suspending buffer [15]. To reduce the effects of cell lysis and autoagglutination, cultures of N. gonorrhoeae are incubated for 16-18 hr at 35OC under aerowood bic conditions with5% COz.For agglutination studies, cotton-tipped applicator sticks are used to transfer cell growth to tubes containing Trisbuffered saline (TBS), pH 7.5. For standardization, the cell suspensions are adjusted spectrophotometrically (535 nm) to the optical density of a McFarland No. 4 standard. This results in a suspension containing about lo9colony-forming units (cfu)/ml, determined to be optimum for providing agglutination reactions that could be visually read without requiring the use of a magnifying device. A 50- to 100-p1volume of cell suspension is addedto each well in the panel of dried lectins. The panels are placed on a gyratory platform, rotated at room temperaturefor 5-10 min, read for agglutination activity,and the reactions recorded. Although each panel contains a cover to prevent drying of the lectin-cell suspension, it is still very important to use onlythe number of panels that can be easily read and interpreted within a 10- to 15-min period. Additionally, gonococcal strains with known agglutination patterns should be included as controls with each set of panel runsto determine any lot-to-lot variation in manufactured lectin panels. To ensure the specificity of the reactions, samplesof carbohydrate solutions specificfor each lectin are added to the wells to demonstrate inhibitionof the agglutination reactions. Also, gonococcal strains are blindly repeated on different days to ensure that agglutination patterns do not change over time. 2. EpidemiologicApplications

Vasquez and B e r m 1431 suggested that auxotyping [M]and serotyping 145,461 were not an efficient means of discriminating among many gonococcal isolates and proposed that adding the lectin-binding pattern would markedly increasethe discriminating power.We have used lectin agglutination to study the characteristics of 150 strains of N. gonorrhoeae that were isolated during epidemiological investigationsCalifornia, in Hawaii, &or-

116

Schalla and Morse

gia, and Pennsylvania [47]. Twenty-four different agglutination patterns were observed (Table 2). All of the strains were agglutinated by lectins (TRK, SBA, and GS-I) that exhibitspecificities for C Y - D - G or~ ~CY-DGalNAc. Conversely, none ofthe strains was agglutinated by LFA, which hasaspecificity for N-acetylneuraminicacid(sialicacid).Interestingly, not all of the strains were agglutinated by lectins sharing a specificity for D-galactose.Forexample,PNAagglutinated 149/150 isolates;whereas, DBA agglutinated only57/150, and PHA agglutinated 7/150 isolates. It is possible that the specific carbohydrateis either in the wrong conformation, is inaccessible to the lectin, or that it is not present on every strain. Six isolates were not agglutinated withWGA; five of these strains were isolated from patients with gonococcal meningitis and will be discussed in detail later. The agglutination patterns observed with the 150 isolates were designated as lectin groups. Table3 shows that most strains (67%) belonged to lectin groups 6 and 7. These lectin groups accounted for 70.6% of the isolates from California and Hawaii, 56.8% of the isolates from Georgia, and 72.7% of the isolates from Pennsylvania. In this limited study, we were unable to observe any correlation between a specific lectin group, the sexof the patient, or geographic area. Fortunately, paired isolates were available for five couples from Georgia (Table 4). The auxotypeand serovar agreed for all of the sexual partner isolates; however, the lectin group agreed in only three of five isolate pairs. Among the disparate pairs, the difference in lectin group represented the loss and gain of reactivity to just a single lectin. Interestingly, one male sexual partner (patient A) had four female partners,and the lectin group was the same in three offour female partners. In light of the variability of gonococcal LOS, the role of host factors (e.g., antibodies) in LOS variation (and lectin group variability) should be examined. Twelve pairs of isolates from sexual partners werealsoavailable among the isolates from California and Hawaii (Table 5). Three of four paired isolates belonging to different lectin groups also differed in either serovar or auxotype, suggesting that these may represent infections with multiple strains. Approximately 15% of patients have been infected with more than one strain of N. gonorrhoeae [48]. The lectin group, serovar, and auxotype agreedfor 5 of 12 paired isolates; in only one pair werethe auxotype and serovar the same and the lectin group different. Previous studies have shown that the auxotype [49] and serovar [46] are relatively stable characteristicsof a particular strain of N. gonorrhoeae;therefore, it is likelythat the recently described heterogeneityof gonococcal LOS[42] is responsible for the variability of lectin agglutinationpatterns among partner isolates with identical auxotypes and serovars. When the two prepon-

Table 2 Lectin Agglutination Patterns of the California, Hawaii, Georgia, and PennsylvaniaGonococcal Isolates

Lectin group

3 4 5 6

7 8

I0 11

+

+ + +

12 13 14

15 16 17 18 19 20

21 22 23 24

B-. 5a -.

ConA LCA

1 2

9

P

Lectin'

+

+

TRK

+ + + + + + + + + + + + + + + + + + + + + + + +

GS-I PNA

SBA

DBA GS-I1 STA WGA LFA

+ + + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + + + + + +

+ + + +

+ + + + + + + + + + + + + + + + + + + + + + +

+ + +

+ + +

+

+

+ + + +

+ +

+ + +

+; +

+ +

+ + + + + + + + + + +

+ + + + +

PHA UEA-I LOTUS

+ +

+ +

+ +

+ +

+ + + +

v)

0, F

2

I ,

c . 3 v)

cc

0

>

op

(D

+ +

+ + + + + +

n

a

& Ln

4

Y

+

U

+ + + +

+

+

'Con A, concanavalin A; LCA, Lens culinaris; TRK. Trichosanthes kirilowii; GS-I, Griffonia simplicifoliaI; PNA, Arachk hypogeae; SBA, Gfwinemax; DBA, Dolichos biflonrs; GS-11. Griffonia simplicifolia II; STA, Solanum tuberosum;WGA. Triticum vulgar& LFA, Limaxflavus;PHA, Phaseolus vulgoris; UEA-I, Ulex europaeus I; LOTUS, Lotus tetragonolobus. Source: Ref. 47.

-L

d

U

Schalla and Mom

118

Table 3 Geographic Distribution of Lectin Agglutination Groups

No. of isolates from Lectin group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

California and Hawaii Georgia Pennsylvania

31

0 0 0 2 0 13 23 1 0 0 0 1 3 0 1 0 1 0 1 1 0 1. 2 1

1 2 1 1 1 12 13 1 1 1 1 1 4 1 1 1 0 1 0 0 0 0 0 0

0 0 0 0 0 9

0 0 0 0 9 0 0 0 0 0 0 0 0 4 1 0 1

derant lectin groups recorded for this study (Table 6) were examined, it was observed that lectin group 6 and lectin group7 could not be differentiated basedon IA or IB servovars. AlthoughIA and IB serovars were found in both groups, IB serovars were more frequently present inboth groups. Auxotropic requirements showedthat most of these isolates required proline or were prototrophic. Plasmid analysis revealedthat all of the isolates contained the 2.6-MDa cryptic plasmid, and most contained either no plactamase plasmid (3.2- and 4.4-MDa plasmids), either a combination of the 2.6-MDa cryptic plasmid, and the 4.4- or 3.2-MDa 0-lactamase plasmid, or the combination of the 4.4 0-lactamase plasmidand the 24.5-MDa

of STD

Applications Agents of to Lectins

119

conjugative plasmid. No differentiation of these lectin groups could be based on plasmid content. Lectins have also been used to retrospectively examine chromosomally mediated penicillin-resistant (Pen')strains isolated duringan epidemiological investigation in New Mexico [50]. Nineteen Pen' isolates of N. gonorrhoeae were obtained from 8 heterosexual and 11 homosexualmen; 21 penicillin-susceptible (Pen') isolateswereobtainedfrom 15 heterosexual and 6 homosexual men. All of the patients resided in the same area of Albuquerque, New Mexico. Strains were characterized by serotype [45,46], auxotype [M],plasmid content [51,52], and lectin group. The results are presented in Table 7 . All of the heterosexual isolates belonged to lectin groups 7 and 12, with the exception of one isolate that belonged to lectin group 25. Both the Pen' and Pen' heterosexual isolates were distributed between lectin groups7 and 12. All of the Pens and Pen' isolates obtained from homosexual men belonged to lectin group 7, however, indicatingthat isolates infecting these homosexual men shared a common characteristic. Among these gonococcal isolates, eight were from homosexual partners and two from heterosexual partners (Table 8). All of the sexual partner isolates belonged to lectin group 7 , and all had 2.6-MDa cryptic plasmids; two isolates contained 24.5-MDa conjugative plasmids. Eight of the ten sexual partners were the same serovar, whereas only four of ten had similar auxotrophic requirements. The two heterosexual partners isolates that belonged to lectin group 7 had the same serovar and auxotrophic requirements. When all ofthe isolates from this studywere compared, the heteroTable 4 Relationships Among Sexual Pairs, Serovars, Lectin

Groups, and Auxotypesfor Georgia Isolates ~~

Patient A B A C D E A E A F

Sex'

Serovar

Lectin group

Auxotype

M F M F M F M F M F

IB-5 IB-5 IB-5 IB-5 IB-5 IB-5 IB-5 IB-5 IB-5 IB-5

6 8 6 6 5 6

Prototrophic Prototrophic Prototrophic Prototrophic Prototrophic Prototrophic Prototrophic Prototrophic Prototrophic Prototrophic

'M, male; F, female. Source: Ref. 47.

6 6 6 6

Schalla and Morse

120

Table 5 Relationships Among Sexual Pairs, Serovars, Lectin Groups, and Auxotypes for Californiaand Hawaii Isolates

Patient No. Lectingroup Serovar Auxotype Sex" 1 2 3 4 5

6

7

8 9 10 11 12 13 14 15

16 17 18 19 20 21 22

23 24

M F M F F M F M F M

IB-5 IB-5 IB-13

6 6 8

IB-5 IB-1

7

F

IBd IB-5

M F M F M M F M F F M F M

IB-l IA-3 IA-3 !BC IBc IB-l

IB-l IB-5 IB-13 IB-5 IA-6

IA-3

6 6 22 6 6 6 18 6 7 7 7 7 7 7

IA-3 IB-5

6 22 6

IB-5 IA-4

6 7

IA-4

7

Prototrophic Prototrophic Prototrophic Prototrophic prob prob Prototrophic prob Prototrophic Prototrophic Prototrophic Prototrophic Prototrophic prob Prototrophic Prototrophic Prototrophic prob prob Prototrophic Prototrophic Prototrophic Prototrophic Prototrophic

"M, male; F, female.

bpro, proline requiring. Wnclassified in the present nomenclature for serological classsification. Source: Ref. 47.

sexual Pen' isolates had proline growth requirements, whereas the homosexual Pen' and Pens isolates and the heterosexual Pens isolates required either proline or were prototrophic. Homosexual and heterosexual Pen' isolates belongedto protein IB serovars, whereas the homosexual and heteroxexual Pen' isolates belongedto protein IA and IB serovars. This was the first reported study in which gonococcal isolates from the same geographic area showed the same lectin agglutination group for the sexual partners and that isolates obtained from different patient anatomical sites agreed with lectin groupand serovar.

)

Applications of Ledins to Agents of STD

121

Table 6

Serovars, Auxotypes, and Plasmid Contents Associated with the Two Preponderant Lectin Groups Plasmid content

IA

Lectin group

serovar

Auxotype"

6 (34)'

W141

Arg/Pro (5), Pro (14)

IB(20)

Arg (1)

( M m

Proto (14) Pro (24) Proto (34) Arg/Pro (6) Ser (1) Arg/Pro/Hyx (1) Pro/Met (1)

7 (67) (42) IB

2.6 (18) 2.6,3.2 (1) 2.6,4.4 (5) 2.6,4.4,24.5 (10) 2.6 (42) 2.6,3.2 (1) 2.6,4.4 (9) 2.6,4.4,24.5 (15)

'kg. arginine; Pro, proline; Ser. serine;Met,methionine; Hyx, hypoxanthine; Proto, proto-

trohic. bMDa, megadaltons. %umber of isolates listed in parenthesis.

3. Lack of Wheat Germ Agglutinin Reactivity as a Virulence Marker Various studies have reported that between 3 and 5% of gonococcal isolates do not react withWGA [27,43]. The lack of reactivity withWGA has also

been noted among strains ofN. gonorrhoeae associated with meningitis,a relatively rare complication of disseminated gonococcal infection (DGI) [53-571. In 1984, three cases of meningitis associated with disseminated gonococcal infections were reported in Pennsylvania; one of the patients died of complications[58,59]. Neisseria gonorrhoeaewas isolated fromthe Table 7

Lectin Agglutination Patterns of New Mexico Penicillin-Sensitive (Pen? and Penicillin-Resistant (Pen') Gonococcal Isolates by Sexual Preference Sexual preference

Homosexual Lectin group 7 12 25

Heterosexual

6

6 9 0

4 3 1

0 0

11 0 0

122

and

Schalla

Morse

Table 8 Relationship of Lectin Group, Serovar, Plasmid Content, and Auxotype of Isolates forthe New Mexico Sexual Partners*

Sexual partners A

B C

D E F G

H I J

Sexb

M M M M M M

M M F M

Culture site'Auxotype Serovar

R U U R

U R U U C

U

IB-l IB-1 IB-l IB-l IB-4 IB-4 IB-4 IB-1 IB-l IB-1

Prototrophic Proline Proline Prototrophic

Prototrophic Proline Prototrophic Prototrophic Proline Proline

'All isolates were in lectin group 7, and all had 2.6 MDa plasmids.

bM,male; F, female. a,rectum; U, urethra; C, cervix. Source: Data from Ref. 50.

blood and cervix of the deceased patient, from the cerebrospinal fluidand cervix ofthe second patient, and from the blood of the third patient. When examined with lectin panels, all five isolates failed to react with WGA. Fifty urogenital and twoDGIarthritis-associatedisolatesof N. gonorrhoeae were then obtained from the same Pennsylvania community and tested for agglutination by WGA; 49 of 50 urogenital and the 2 arthritisassociated isolates were agglutinated by WGA. Results theof lectin agglutination of the 5 meningitis-associated, 50 urogenital, and 2 DGI arthritisassociated isolates obtainedfrom this geographic area, and 13PID isolates obtained from the Centers for Disease Control and Prevention (CDC)culture collection are shown in Table 9. One isolate obtained from a case of uncomplicated or urogenital gonorrhoea did not agglutinate WGA. The failure of WGA to agglutinate the strains isolatedfrom patients with gonococcal meningitis may be a marker for a virulence factor. A comparison of serovar and lectin reactivity with WGAbetween the 5 Pennsylvania meningitis-associated isolates,10 DGI isolatesnot associated with meningitis and obtained fromthe CDC culture collection,and the 50 Pennsylvania urogenital isolates causing uncomplicated gonorrhoea is summarized inTable 10. The meningitis-associated isolates did not agglutinate WGA and were preponderantly IA serovars. The DGI isolates not associated with meningitis were also preponderantly IA serovars; however, were they agglu-


123

Table 9 Various Lectin Reactions with the Pennsylvania N. gonorrhoeae Isolates

from Different Anatomical Sites No.of reactive isolates/total

Lectin Concanavalin A Arachis hypogeae Dolichos bifrorus Solanum tuberosum Triticum vulgaris

DGI,'

DGI,

0/2 2/2 2/2 2/2 2/2

0/5 5/5 5/5 5/5 0/5

meningitis Symbol arthritisPID' Urogenital Cod PNA

0/13 13/13 13/13 12/13 12/13

0/50 50/50 13/50 46/50 49/50

DBA

STA WGA

'PID, pelvic inflammatory disease; DGI, disseminated gonococcal infection. Source:Data from Ref.58.

tinated by WGA. The isolates from uncomplicated infections were preponnot agglutinate WGA. Frasch derantly IB serovars, and only one isolate did [131observed that encapsulated strains of N. meningitidis were not agglutinatedby WGA, whereasnonencapsulatedstrainswereagglutinatedby WGA. No capsule could be demonstrated when the five gonococcal isolates from meningitis patients were examined further. A subsequent study revealed that a gonococcal isolate associated with meningitis, obtained from a different geographic areaof the United States, was agglutinated by WGA [m].These findings suggestedthat thestrains isolated in Pennsylvania may represent the spread of a single clone; however, this is unlikely, since serotype analysis indicatedthat these strains belonged to both IA and IB serovars. Table 10 Comparison of Serogroupand Wheat Germ Agglutination Among Patient Isolates ofN. gonorrhoeae

patients Diagnosis DGI"/meningitis DGI/without meningitis Uncomplicated gonorrhoea

Protein 'Wheat germ I-serogroup agglutination No. of no yes IA IB 5 10 50

'DGI, disseminated gonococcal infection. Source: Data from Ref.59.

4 8 6

1 2

44

-

10 49

5

-

1

124

Schalla and Morse

4. Use of Lectins to Assess Treatment Failure

Another application of lectin typing has been in drug treatment trials to characterize pre-and posttreatment isolatesof N. gonorrhoeae. Two studies have been reported that characterized pre- and posttreatment isolates following enoxacin [61] or cefodizime versus cefotaxime [62] therapy for gonorrhea. Lectin typing has been used in conjunction with auxotyping, serovar analysis,and minimum inhibitory concentration (MIC) to the investigational drug to ascertain whether the pre- and posttreatment isolates were identical, thus representing either treatment failure, reinfection from an untreated partner, or emergence of an antibiotic-resistant strain. Nonidentical pre-and posttreatment isolates suggest reinfection with a different strain. Twelve pairs of pre- and posttreatment isolateswere available from the enoxacin trial. The results of this study are shown in Table 11. Eleven of twelve pairs of pre- and posttreatment isolates had identical auxotypes and serovars; two isolate pairs differed in lectin group. In one pair, the difference represented the loss of reactivity with GS-I1 and the gain of reactivity with DBA. In the other pair, the posttreatment isolate lost reactivity with GS-11. In addition, 11of12 pairs of pre- and posttreatment strains had the same MIC to enoxacin. One of the posttreatment strains exhibited a 67-fold increase inthe MIC value and probably represents the selection of a resistant strain. In the other study involving treatment of uncomplicated gonorrhea in men and womenwith either cefodizime or cefotaxime [62], all of the preand posttreatment isolateswere identical for auxotype, serovar, and lectin group. The results of these studies suggest that, with one exception, the posttreatment isolates represent either treatment failureor reinfection from an untreated partner. 111. HAFMOPHILUS DUCREYI

A. The Organism

Haemophilus ducreyiis a gram-negative, nonmotile rodthat is the etiological agent of chancroid. Chancroid is one of the five classic venereal diseases (gonorrhea, syphilis, lymphogranuloma venereum, donovanosis, and chancroid) and is characterizedby painful genital ulcersand frequent lymphadenopathy. Chancroid was considered to be a relatively uncommon sexually transmitted disease in the United States; however, the number of reported cases have increased more than sixfold since 1984 [63]. Haemophilus ducreyi is a major cause of genital ulcers in Asia and Africa, where the prevalence of chancroidoften exceeds that of syphilis. This bacterium is difficultto isolate directly from genital lesions [63]. Sensitivity of culture is dependent on the medium, the specimen, and the

Applications of Lectins to Agents of STD 125

Schalla and Morse

126

expertise ofthe laboratorian. Relatively little is known about the epidemiology of chancroid because thereare relatively few phenotypic characteristics that can be usedto differentiate among isolatesof H. ducreyi. Outer membrane protein profiles [64,65] and amino peptidase profiles [66,67] have been used, butdo not provide suitable discrimination. Recently,the taxonomy of H. ducreyi has been calledinto question [68,69]. Sequence analysis of ribosomal RNA genes indicates that H. ducreyi belongs in the family Pasteurellaceae, but is not a member of the genus Haemophilus [70]. It has been suggested that H. ducreyi may comprise a single-species genus and, thus, may be ofclonal origin. Therefore,it is not surprising that it has been difficult to differentiate among strains ofH. ducreyi. The identification of (RFLPs) amongribosomal restrictionfragmentlengthpolymorphisms RNA genes (ribotyping) has recently been useful in typing isolates of H. ducreyi [71]. B. Cell Envelope

In electron micrographs,the outer membrane of H. ducreyi appears morphologically similar to those of other gram-negative bacteria [63,72]. The outer membrane profile is also typical of gram-negative bacteria, with one to several proteins predominating [64,73,74]. Changes in growth medium composition, atmospheric conditions, and temperature do not appear to affect the outer membrane protein profile; however, the electrophoretic pattern of the LPS is apparently affectedby conditions of growth [75]. The presence of a capsule has not been adequately demonstrated. Bertram [76] observed the presence of antibody-stabilized extracellular capsular material by electron microscopy. In contrast, Johnson et al. [77] failed to observe extracellular acidic polysaccharideon thin sections of H. ducreyi stained with ruthenium red. 7.

Properties of Haernophilus ducreyi Lipooligosaccharides

Haemophilus sp. have been reported to produce both smooth and rough LPS[78]. Several investigators have recently examined the type of LPS produced by H. ducreyi. Haemophilus ducreyi possesses a low molecular weight LPS that contains heptose, ketodeoxyoctonate, glucose, galactose, glucosamine, and galactosamine [79]. It is similar in size to that of other mucosal pathogens and therefore, is referred to as LOS. The H. ducreyi LOS is more toxic than the LOS from H. influenzae or the LPS from Escherichia coli, and has produced skin abscesses in animal models [80]. Two patterns of reactivity with monoclonal antibodies have been observed among isolates ofH. ducreyi [81]. The LOS from 24 of 25 strains examined 3F11, whichrecognizes a terminal Gal& boundmonoclonalantibody


127

1,4GlcNAc epitopethat has a structure similar to human blood group antigens [37,81]. This epitope is also presenton some gonococcal LOSSand is apparently sialylated in vivo [39]. WhetherH. ducreyi LOS is sialylated in vivo remainsto be determined. Onestrain has been identifiedthat does not react with monoclonal 3Fll. This strain produces a smaller LOS than the other strains that have been examined [81]. C. Interaction of Haemophilus ducreyi with Lectins 7.

Methodology

Laboratory cultivation of H. ducreyi can be accomplished with heartinfusion agar (Difco-Laboratories)supplemented with 5%rabbit erythrocytes, 10% fetal bovine sera (Hyclone Laboratories), and 1% IsoVitaleX (BBL). Various media have been described for cultivating H. ducreyi [63,82-911. After growth for 18 hr at 35OC in 5% COz, cells were suspended in TBS, pH 7.5, as described for N. gonorrhoeae. Unlike gonococci, H. ducreyi cells form clumps or chains when in suspension that interfere in interpreting lectin agglutination reactions. By allowing suspensions of H. ducreyi to stand for 1-2 min, cell clumps and chains of cells settle to the bottom of the tube. The liquid at the top of the tube, containing single or pairs of cells, is removed and transferred to another tube. This suspension can now be adjusted spectrophotometrically (535 nm) to an optical density of a McFarland No. 4 standard. Samples (50~1)of the cell suspension are added to the lectin panel wells, panels are placedon a gyratory platform, rotated for 5-10 min, and agglutination read. Additionally, plastic panels containing no lectins were used to place a suspension of each isolate. Withthe aid of a stereomicroscope (Bausch and Lomb) at 5 x or 10 x magnification, the granulation between wells containing organisms and lectins could be compared with the wells containing only organisms. In those instances when there was difficulty determining if agglutination was occurring, or granulation was causing a false-positive reaction, specific carbohydrates were usedto show reversibilityof agglutination. 2. EpidemiologicalApplications A total of 63 isolates ofH. ducreyi obtained from outbreaks in California, Florida, Georgia, New York, and Massachusetts were examined. The results of the H. ducreyi lectin agglutination activity are shown in Table 12.0f the 63 isolates examined, two preponderant lectin groups were observed. Differences in lectin agglutination focused on the reactivity of isolates with G!”. Among these isolates, 27(43070) did not agglutinate with GS-11, whereas 36 (57%) isolates did agglutinate with this lectin. What appeared to be agglutination activity with LCA and PHA could not be reversed

++ + ++++

++ + ++++

+ + + +

++ + ++++

++ + ++++

++ + ++++

++

++

++

+

++

++

++

Schalla and Morse

++ + ++++

++

wt-

++ + ++++

"rncl)

++ VI

++ + ++++ VIW

.5 U

+

+

+

+

++++ ++

++++ ++

+

++++ ++

++++ ++

++++ ++

+ +

+ +

+ +

+ +

+ +

+ +

+ +

-Applications of Lectins to Agents of STD

+

++++ ++ + +

++

+

++++ ++ + +

+

+ ++++ ++ m

+ m

l""

d.-

d

n VI

129

Schalla and Morse

130

with specific carbohydrates. It was concluded that this activity was due to nonspecific clumping that resulted in false-positive agglutination. The results presented in Table 12 are different from those presented by Korting et al. [92]. In that study, all of the isolates were agglutinated by LCA and PHA, with some agglutination activity reported for DBA,UEA-I, and Lotus. In contrast, we were unable to show agglutination activity by LCA, DBA, PHA, UEA-I or Lotus. However, all of the isolates were agglutinated by TRK, GS-I, PNA, SBA, and STA. Similarto the results reported by Korting et al. [92], some isolates were agglutinated by GS-11. Nodifference was found in agglutination activity of isolates relative to geographic location inthe United States.The similarity in lectin agglutinationpatterns for these isolates may suggest that these outbreaks were caused by a few strains; however,the inability of lectins to differentiate many agglutination types among the H. ducreyi strains examined is consistent with data suggesting that few phenotypic properties exist that can beused for strain typing [63].

W.

TREPONEMA PALLIDUMSUBSPECIES PALLIDUM AND TREPONEMA PALLIDUMSUBSPECIES PERTENUE

A. The Organism

Treponema pallidumsubsp. pallidum (T.pallidurn) and T. pallidurn subsp. pertenue (T. pertenue) are nonculturable (pathogenic) spirochetesthat are the causal agents of syphilis and yaws, respectively. They belong to the family Spirochaetaceae, genus Treponema. Syphilis is a chronic disease that occurs throughout the world. Infection within the host is characterized by a primary lesionor soft chancre that usually develops within a few weeks. Treponemes canoften be observed by direct examination of lesion fluid by darkfield microscopy. Treponema pertenueis the causative agent of yaws, transmission occurs through skin contact, and the disease occurs primarily in the tropics. BothT. pallidurn and T. pertenue are gram-negative organism and are considered to be microaerophilic [93,94]. Treponemapallidum takes up oxygen and possesses an electron transport system [95,96]. Both microorganisms exhibit corkscrew motility that is accompaniedby rotation around the longitudinal axis and a bending or flexing. The ends of these spirochetes are somewhat pointed or tapered [97,98]. Electron microscopy has shownthat these treponemes possessan outer membrane, also referred to as an outer envelope, which covers the axial filaments located on the surface [97,98]. Antigenically, these treponemes are considered identical, and a species-specific antigen has not yet been identified. Pathogenic and nonpathogenic treponemes can be differentiated by DNA homology, but

Applications Lectins of

to Agents of STD

131

this method does not differentiate between T. pallidurn and T. pertenue [99,100]. B. The Outer Envelope and Cell Wall

The pathogenic treponemesare nonculturable, although limited success has been obtained with tissue cell cultures [101-1031.Because it is difficult to grow pathogenic treponemes in vitro, very little information is available concerning their biochemical reactions, metabolism, and chemical composition, although the lipid composition of T. pallidurn was reported by Mathews et al. [W].Some reports have indicated that the pathogenic treponemes possess a surface acidic polysaccharide[105-1071,but other reports question whetherthis polysaccharide is synthesized by the treponeme, or is derived from host tissue [108-11 l] The . polysaccharide possesses glucose and galactose residues[104,107,110]. C. Interaction with Lectins

Very little information is available about the interaction of T. pallidurn or T. pertenue with lectins. Baseman et al. [l121 used lectins noncovalently bound to Leighton tube coverslips to bind freshly extracted treponemes and facilitate the removal of cellular debris and fluids. They observedthat T. pallidurn bound to lectin film coverslips containing ConA,WGA, PHA, and pokeweed mitogen (PWM). Binding ofT. pallidurn to ConA occurred with greater affinitythan to WGA and P m . Fitzgerald and Johnson [l091 reported that T. pallidurn interacted with WGAand SBA, and they postulated that the ligand was the acidic polysaccharide. The surface polysaccharide of T. pallidurn is reported to be composed of hyaluronic acid and chondroitin sulfate or related acidic glycosaminoglycans[107].Hyaluronic acid, will interact acid, consisting of N-acetyl-D-glucosamine-D-glucuronic withWGA.Similarly,chondroitin sulfate consistsofN-acetyl-wgalactosamine-D-glucuronicacid,whichwillinteractwithSBA.Theseacidic polysaccharides are present in syphilitic lesions [108-11l], and it is unclear whether the polysaccharide material ispart of the treponemal cell surface, or results from host tissue damage during the infection process[108-1 1 l]. 1. Methodology

Since T. pallidurn and T. pertenue cannot be easily subcultured in vitro, growth of the organismsisaccomplishedbyintratesticularpassagein healthy rabbits. Rabbit testes are excised 11-14 days postinfection. The treponemes are harvested by slicing the testes, placing thema solution in of 0.02 M phosphate-buffered saline (pH 7.2), containing 0.075 M sodium citrate, and teasing the treponemes out of the testicular tissue with gentle

132

Schalla and Morse

agitation. The pathogenic treponemes are separated from other cellular debris by density gradient centrifugation in 20% Percoll (Pharmacia Fine Chemicals, Piscataway, New Jersey). Treponemes purified by density gradient centrifugation are washed three times with TBS, pH 7.5, to remove contaminating Percoll. Treponemaphagedenis biotype Reiter was cultivated in NIH thioglycolate broth (Difco Laboratories) containing10% inactivated normalrabbit sera. Growthof this nonpathogenic treponeme was accomplished at 35OC for 4-7 days. Purified pathogenic treponemes and the nonpathogenic T. phagedenis were suspended in TBS,pH 7.5, adjusted spectrophotometrically to yield a suspension equivalent to a McFarland No. 4 standard, and examined for agglutination activity with the panel was prepared by of 14 lectins. Normal rabbit testicular extract (NRTE) pulverizing nitrogen-frozen testicular tissue in a stainless steel mill. The powdered testicular tissuewas extracted three times with TBS, pH 7.5, and layered onto 20% Percoll for density gradient centrifugation as described earlier. The NRTE and 20% Percoll were also examined for agglutination activity withthe 14 panel lectins. 2. EpidemiologicalApplications

Three strains of T. pallidurn and two strains of T. pertenue, all isolated from different geographic areas,and one nonpathogenicstrain were examined for lectin reactivity.The results of lectin agglutination withthe treponemal strains are shown in Table13. The same lectin agglutinationpattern was observed for all T. pallidurn and T. pertenue strains. The pathogenic treponemes reacted with ConA, GS-I, SBA, and WGA. The NRTE also showed reactivity with these same lectins. It is uncertain whether the treponemal reactivity with these lectins istodue treponemal cell surface carbohydrate, or to contaminating testicular tissue present on the surface of the treponemes. The pathogenic strains could not be differentiated from the T. phagedenis biotype Reiter owing to the uncertainty of testicular tissue contamination. Agglutination of lectins with the pathogenic treponemal suspensions was reversed with specific carbohydrates, indicating specific reactivity; however, with the uncertainty of testicular tissue contamination, the specificity of these reactions may reside in contaminating testicular material. The 20% Percoll preparation did not show lectin agglutination. V. CONCLUSIONS

The information reported in this chapter represents only preliminary information concerningthe applications of lectins to some organisms associated with sexually transmitted diseases. All agglutination characteristics of these microorganisms were determined using prepared panels of 14 lectins. Fur-

Table 13 Lectin' Agglutination Activity of Pathogenic Treponemal Strains,Compared with One Nonpathogenic Treponemal Strain

ConA LCA TRK T. phagedenb biotype Reiter T. pallidurn subsp. pallidum Strain 1 Strain 2 Strain 3 T. pallidum subsp. pertenue Strain 1 Strain 2 20% Percoll NRTE~

-L

PNA

SBA

+

+

DBA GS-I1 STA WGA LFA

+

+

* * *

+ + +

+ + +

* *

+ +

+

+

+ + +

+ + +

+ + +

+ + +

+ + +

+ + +

+ + +

+ +

+ +

+ +

+ +

+ +

+ + +

+ +

+ +

+ + +

+ + +

+

f

+

PHA UEA-I LOTUS

+

+

See Table 1 for identification of lectins. bNRTE, normal rabbit testicular extract.

W W

GS-I

+

+

134

Schalla and Morse

ther characterization of cell surface carbohydrates and their role in cell surface structure may require the use of more-specific lectin reagentsthan those contained in panels used for studies reported in this chapter. The agglutination reactions of N. gonorrhoeue, based on structural components of the LOS, were carbohydrate-specificand could be inhibited. The agglutination of N. gonorrhoeae appeared to be due to the availability of exposed terminal structures of the carbohydrate residues. Masking of some carbohydrate structures or influenceofaneighboring structural charge phenomenon mayoffer an explanation.why lectinsof similar specificity do not agglutinatethe same gonococcal strains. Twenty-four different lectin groups were observed in these studies. It has been suggested that gonococcal LOS epitopes may undergo phase variation, which may, in turn, affect lectin agglutinability[42]. However, subculture didnot appear to affect lectin agglutination ofN. gonorrhoeae strains used in these studies. Strains of N. gonorrhoeae were repeatedly subcultured and used as blind controls. The original agglutination pattern of each strain did not change after subculture. The observationthat differences in lectin agglutination with GS-I1 and DBA may be suggestive of structural differences in CY-D-G~NAC or u-D-G~cNAc residues, lectin accessibilityto residues, absence of these residues in some strains, or the population of residues presented to lectin reagents. Inthe studies presented in this chapter, two strains of N. gonorrhoeae agglutinated with ConA, which represented an uncommon lectin agglutinationpattern among the 150 strains examined. In contrast, Vazquez and Berron 1431observed a more frequent agglutinationof N. gonorrhoeae strains by ConA.Perhaps these strains, which wereisolated in different geographic areasof the world, exhibited more LOS structural differences than the 150 strains examined from the United States. The strains isolated in the United States showed infrequent agglutination with UEA-I, LCA, and PHA, when compared with the agglutination patterns reported by Vazquezand Berron [43]. The data presented in this chapter concerning lectin agglutination differences amongstrains of N. gonorrhoeae represent a different typingsystem that can be used in conjunction with serotypingand auxotyping. The use of lectin agglutination, serotyping, and auxotyping can be useful in clinicalstudies to determine antibiotic treatmentfailures,emergence of antibiotic-resistant strains, and reinfection from an untreated sexual partner. Strains of H. ducreyi reported in this chapter did not show a high degree of variability in lectin agglutination patterns. Repeated subculture of theseH. ducreyi strains did not affect the reproducibilityof lectin agglutination patterns, suggesting that subculturing did notaffect LOS epitopes present on the cell surface. That only two lectin agglutinationpatterns for


135

the isolates of H.ducreyi were available for examination in this study is consistent with results of studies indicating few phenotypic characteristics are available for differentiating strains of this organism. Studies of lectin agglutination with H. ducreyi reported by Korting et al. [92] with strains isolated from other world geographic areas did show differences in lectin agglutination. At least 20 different lectin agglutination patterns were recorded, suggesting that there may be methodological differences or differences in phenotypic characteristics among these strains. The lectin agglutination patterns of the pathogenic treponemes were identical. No differences in lectin agglutinationwere observed betweenthe strains examined.A previous report indicated that lectin agglutination with T. pallidurn was due to acidic polysaccharides[107]. However, whetherthis polysaccharide is of treponemal origin or host tissue origin is still unclear [108-11 l]. Difficulty in obtaining high yields of treponemesfor lectin agglutination or in preparing pathogenic treponemes to ensure absence of host tissue fluid or debris will be a continual problem to produce reliable and accurate lectin agglutination results. Other preliminary studies have been initiatedto examine other agents of sexually transmitted diseases. Withthe same panel of 14 lectins, strains of Chlamydia trachornatishave been examinedfor agglutination by lectins. Strains of C. trachornatis were agglutinated by lectins specificfor u-D-GIc, a-~-Gal,and a-~-GalNAc.McCoy tissue culture cells usedfor passage of C. trachornatis were also agglutinatedby the same lectins, however, raising concern that the agglutination may be due to contamination by McCoy tissue culture cell epitopes. Additionally, the panel of 14 lectins has been used to determine lectin agglutination patterns of Mycoplasma horninis. Only after proteolytic treatment were M. horninis strains agglutinated by lectins specific for Q-D-G~c,a-D-Gal, and a-~-GalNAc,in accord with results reported by Kahane and Tully [l 131. Schiefer et al. [l 141, however, did not observe agglutinationof M . horninis strains by lectins, and treating of these strains with pronase failed to enhance agglutination reactivity. Further examination of these microorganisms is necessary to determine the suitability of lectin applications and to determine the stability and reproducibility of lectin agglutination activity. The applicationof lectin agglutinationto agents of sexually transmitted diseases may facilitate the detection of inter-and intrastrain variations. ACKNOWLEDGMENTS

The authors thank R. E. George, E. F. Hunter, and S. A. Larsen for their technical assistanceand guidance in growth of pathogenic treponemes, and G. M. Mast for assistance inpreparation of this chapter.

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REFERENCES 1. Sumner JB, Howell SF. The identification of the hemagglutinin of the jack bean with concanavalin A. Bacterioll936; J 32:227-237. 2. Kohler W,Prokop 0. Agglutinationversuchean streptokokkenmit dem phytagglutinin aus Dolichos biflorus. Z Immunitaetsforch Allerg Klin Immunol 1967; 133~171-175. 3. Prokop 0 , Kohler W. Agglutinations reactions en von mikroorganisamen mit Helix pomatia eiweiss drusenextrakt. Z Immunitaetsforsch Allerg Klin Immunoll967; 133:176-179. 4. Goldstein IJ, Misaki A. Interaction of concanavalinA with an arabinogalactan from the cell wall of Mycobacterium bovis. J. Bacteriol 1970; 103:422425. 5. Goldstein IJ, Staub AM. Interaction of concanavalinA with polysaccharides of salmonellae. Immunochemistry 1970;7:315-319. 6. Pistole TG. Interaction of bacteria and fungi with lectins and lectin-like substance. AnnuRev Microbioll981; 35:85-112. 7. Goldstein IJ, Hayes CE. Thelectins: carbohydrate-binding proteins of plants and animals. Adv Carbohydr Chem Biochem 1978; 35:127-340. 8. Slifkin M, Doyle RJ. Lectins and their application to clinical microbiology. Clin.Microbio1Rev 1990; 3:197-218. 9. Doyle RJ, Keller K. Lectins in diagnostic microbiology.Eur J Clin Microbiol 1984; 3:4-9. 10. Lis H, Sharon N. Lectins as molecules and tools. Annu Rev Biochem 1986; 55135-67. 11. Liener IE, Sharon N, Goldstein IJ, eds. The lectins: properties, functions, and applications in biology and medicine. Orlando: Academic Press, 1986. 12. Lotan R, Sharon N, Mirelman D. Interaction of wheat germ agglutinin with bacterial cellsand cell wall polymers.Eur J Biochem 1975; 50257-262. 13. Frasch CE. Role of lipopolysaccharide in wheat germ agglutinin-mediated agglutination of Neisseria meningitidis and Neisseria gonorrhoeae. J Clin Microbioll980; 12:498-501. 14. McSweegan EF, Pistole TG. Interaction of the lectin limulin with capsular polysaccharides from Neisseria meningitidis and Escherichia coli. Biochem Biophys ResCommun 1982; 4:1390-1397. 15. Allen PZ, Connelly MC, Apicella MA. Interaction of lectins with Neisseria gonorrhoeae. Can J Microbioll980; 26:468474. 16. Doyle RJ, Birdsell DC. Interaction of concanavalin A with the cell wall of Bacillussubtilk. J Bacteriol 1982; 109:652-658. 17. Reeder NJ, Ekstedt RD. Study of the interaction of concanavalin A with staphylococcal teichoic acids. J Immunol 1971; 106:334-340. 18. Archibald AR, Coapes HE.The interaction of concanavalinA with teichoic acids and bacterial walls. Biochem J 1971; 123665-667. 19. Cifonelli JA, Rebers P, Perry MB, Jones JKF. The capsular polysaccharide of pneumonococcustype XII, SXII. Biochemistry 1966;5:3066-3072. 20. Stoddart RW, Herbertson BM. The use of fluorescein-labelled lectinsin the


21. 22. 23. 24.

25. 26. 27. 28. 29. 30. 31. 32. 33. 34.

35.

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detection and identification offungipathogenic for man:apreliminary study. J Med Microbioll978; 11:315-324. Cole HB, Ezzell JW Jr, Keller KF, Doyle RJ. Differentiation of Bacillus anthracis and other Bacillus species by lectins. J Clin Microbiol 1984; 19:4853. Curtis .GDW, Slack MPE. Wheat-germ agglutination in Neisseria gonorrhoeae. Br J Vener Dis 1981; 57:253-255. Davidson SK,Keller KF, Doyle RJ. Differentiation ofcoagulase-positive and coagulase-negative staphylococci by lectinsand plant agglutinins. J Clin Microbiol 1982; 15547-553. Doyle RJ, Nedjat-Haiem F, Keller KF, Frach CE. Diagnostic valueof interactions betweenmembers of the family Neisseriaceae and lectins.JClin Microbioll984; 19:383-387. Senne JE. Lectin agglutination of Neisseriagonorrhoeae. Clin Microbiol News1 1981; 3:lO. Schafer RL, Keller KF, Doyle RJ. Lectins in diagnostic microbiology:use of wheat germ agglutinin for laboratory identification of Neisseria gonorrhoeae. J Clin Microbiol1979; 10:669-672. Yajko DM, Chu A, Hadley KW. Rapid confirmatory identification of Neisseria gonorrhoeae with lectins and chromogenic substrates. J Clin Microbiol 1984; 19~380-382. Doyle RJ, Nedjat-Haiem F, Miller RD, Keller KF. Interaction between plant agglutinins and Legionella species. J ClinMicrobioll982; 15:973-975. Ottensooser F, Nakamizo Y, Sat0 M, Miyamoto Y, Takmwa K. Lectins detecting group C streptococci. Infect Immun1974; 9:971-973. Slifkin M, Cumbie R. Identification of group B streptococcal antigen with lectin-bound polystyrene particles. J Clin Microbioll987; 25:1172-1175. Wong KH, Skelton SK, FeeleyJC. Interaction of Campylobacter jejuni and Campylobacter coliwith lectinsand blood group antibodies. J Clin Microbiol 1985; 22~134-135. Meade NA, Staat RH, Langley SD, DoyleRJ. Lectin-like activity from Persea americana.Carbohydr Res 1980; 78:349-363. Mintz CS, Apicella MA, Morse SA.Electrophoretic and serological characterization of the lipopolysaccharide produced by Neisseria gonorrhoeae. J Infect Dis 1984; 149544-552. Griffiss JM, O’Brien JP, Yamasaki R, Williams GD, Rice PA, Schneider H. Physical heterogeneity of neisserial lipooligosaccharides reflects oligosaccharides that differ in apparent molecular weight, chemical composition, and antigenic expression. Infect Immun 1987; 55:1792-1800. Dudas KC, Apicella MA. Selection and immunochemical analysis of lipooligosaccharide mutants of Neisseria gonorrhoeae.Infect Immun 1988; 56:499504.

36. Griffiss JM, Schneider H, Mandrel1 RE, Yamasaki R, Jarvis GA, Kim JJ, Gibson BW, Hamadeh R, Apicella MA. Lipooligosaccharides: the principal glycolipids of the neisserial outer membrane. Rev Infect Dis 1988; lO(supp1 2):S287-S295.

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37. Mandrell RE, Griffiss JM, Macher BA. Lipooligosaccharides(LOS) of Neisseria gonorrhoeae and Neisseria meningitidis have components that are immunochemically similarto precursors of human bloodgroup antigens. J Exp Med 1988; 168:107-126. 38. Parsons NJ, Pate1 PV, Tan EL, Andrade JRC, Naim CA, Goldner M, Cole JA, Smith H. Cytidine 5'-monophospho-N-acetylneuraminicacid or a related compound is the low molecular weight factor from human red blood cells which induce gonococcal resistance to killing by human serum. J Gen Microbiol 1988; 134:3295-3306. 39. Mandrell RE, Lesse AJ, Sugai JV, Shero M, Griffiss JM, Cole JA, Parsons NJ, Smith H, Morse SA, Apicella MA. In vitro and in vivo modification of Neisseria gonorrhoeaelipooligosaccharide epitopestructure by sialylation. J Exp Med 1990; 171:1649-1664. 40. Ward ME, Watt PJ, Glynn AA. Gonococci in urethral exudate possess a virulence factor lost on subculture. Nature 1970; 227:382-384. 41. Yamasaki R, Bacon B, Nasholds W, Schneider H, Griffiss JM. Structural determination of the oligosaccharides derivedfrom lipooligosaccharide (LOS) of Neisseria gonorrhoeaeF62 by chemicaland 2D NMR (500 MHz) methods: MAbs 1-1" and 3F1 l-defined LOS epitopes have a N-acetylgalactosaminylneolactotetraose and neolactotetraoseat their non-reducing terminus, respectively. Biochemistry 1991; 30:10566-10575. 42. Schneider H, Hammack CA, Apicella MA, Griffiss JM. Instability of expression of lipooligosaccharides andtheir epitopes in Neisseria gonorrhoeae.Infect Immun 1988; 56:942-946. as an epidemiological 43. Vasquez JA, Berron S. Lectinsagglutinationtest marker for Neiserria gonorrhoeae.Genitourin Med 1990; 66:302-305. 44. Catlin BW. Nutritional profiles of Neisseria gonorrhoeae, Neisseria meningitidis, and Neisseria lactamica in chemically defined media and the use of growth requirementsfor gonococcal typing. J Infect Dis 1975;128:178-194. EG, Holmes KK, Knapp JS, Siadak 45. Tam MR, Buchanan TM, Sandstrom AW, Nowinski RC. Serological classification of Neisseria gonorrhoeae with monoclonal antibodies. Infect Immun 1982; 361042-1053. 46. Knapp JS, Tam MR, Nowinski RC, HolmesKK, Sandstrom EG. Serological classification of Neisseria gonorrhoeaewith use of monoclonal antibodies to gonococcal outer membrane proteinI. J Infect Dis 1984; 150:44-48. RJ, Larsen SA. Epidemiological charac47. Schalla WO, Whittington WL, Rice terization of Neisseria gonorrhoeae by lectins. J Clin Microbiol 1985; 2 2 379-382. 48. Short HB, Ploscoe VB, Weiss JA, Young FE. Rapid method for auxotyping multiple strains of Neisseria gonorrhoeae.J Clin Microbiol 1977;6:244-248. 49. Catlin BW. Characteristics and auxotyping of Neisseriagonorrhoeae. In: Bergan T, Norris JR, eds. Methods Microbiol1978;10:345-380. 50. Schalla WO, Rice RJ, Biddle J W , JeanLouis Y, Larsen SA, Whittington WL. Lectin characterization of gonococci from an outbreak caused by penicillin-resistant Neisseria gonorrhoeae.J Clin Microbiol1985; 22:481-483. 51. Meyers JA, Sanchez D, Elwell LP, Falkow SL. Simple agarose gel electro-


52. 53. 54. 55.

56. 57.

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phoresis methodfor the identification and characterization of plasmid DNA. J Bacteriol 1976; 127:1629-1633. Perine PL, Thornsberry C, Schalla WO, Biddle J, Seigel MS, Wong KH, Thompson SE. Evidence for two distinct types of penicillinase-producing Neisseria gonorrhoeae.Lancet 1977; 2:993-995. Holmes KK, Counts GW, Beaty HN. Disseminated gonocococal infection. Ann Intern Med 1971; 74:979-993. Sayeed Z A , Bharudi U, Howell E, Meyers HL Jr. Gonococcal meningitis: a review. J Am Med Assoc 1972; 1730-1731. Swierczewski JA, Mason EJ, Cabrera PB, Liber M. Fulminating meningitis with Waterhouse-Friderichesen syndrome due to Neisseria gonorrhoeae.Am J Clin Patholl970; 54:202-204. Pasquariello CA, Plotkin SA, Rice RJ, Hackney JR. Fatal gonococcal septicemia. Pediatr Infect Dis 1985; 4204-206. Granato PA, Howard R, Wilkinson B, Laser J. Meningitis caused by maltose-negative variant of Neisseria meningitidis. J Clin Microbiol 1980;11:

270-273. 58. Rice RJ, Schalla WO, Whittington WL, Biddle JW, JeanLouis Y, DeWitt

WE, Thompson SE. Investigation ofNeisseria gonorrhoeaecausing disseminated infection, endotoxemia,and meningitis and identification of a possible virulencemarker.In:SchoolnikGK,Brooks GF, Falkow S. FraschCE, Knapp JS, McCutchan JA, Morse SA, eds. Pathogenic neisseriae. Washington, DC: American Society for Microbiology, 1985:61-65. 59. Rice RJ, Schalla WO, Whittington WL, JeanLouis Y, Biddle J W , Goldberg M, Dewitt W, PasquarielloA, Abrutyn E,Swenson R. Phenotypic characterization of Neisseria gonorrhoeae isolated from three cases of meningitis. J Infect Dis 1986; 153:362-365. 60. Del Rio C, Stephens DS, Knapp JS, Rice RJ, Schalla WO. Comparison of isolates ofNeisseria gonorrhoeaecausing meningitis and report of gonococcal meningitis in a patient with C8 deficiency: J Clin Microbiol 1989; 27:10451049. 61. Van Der Willigen

AH, Van Der Hoek JCS, Wagenvoort JHT, Van Vliet HJA, Van Klingeren B, Schalla WO, Knapp JS, Van Jost T, Michael MF, Stolz E. Comparative double-blind study of 200- and 400-mg enoxacin given orally in the treatment of acute uncomplicated urethral gonorrhea in males. Antimicrob Agents Chemother1987; 31535-538. 62. Van Der Willigen AH, Wagenvoort JHT, Schalla WO, KnappJS, Boot JM, Heeres-Weststrate PL, Michel MF, Van Klingeren B, Stolz E. Randomized comparative study of 0.5 and 1 g of cefodizime (HR221) versus 1 g of cefotaxime for acute uncomplicated urogenital gonorrhea. Antimicrob Agents Chemother 1988; 32:426-429. 63. Morse SA. Chancroid and Haemophilus ducreyi. Clin Microbial Rev 1989; 2~137-157. 64. Odumeru JA, Ronald AR, Albritton WL. Characterization of cell proteins of Haemophilus ducreyi by polyacrylamide gel electrophoresis. J Infect Dis 1983; 148:710-714.

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65. Taylor DN, Echevema P, Hanchalay S, Pitarangsi C, Slootmans L, Piot P. Antimicrobial susceptibilityand characterization of outer membrane proteins of Haemophilus ducreyiin Thailand. J Clin Microbiol1985; 21:442-444. 66. CasinIM,Sanson-Le Pors MJ, Gorce MF, Ortenberg M, Perol Y. The enzymatic profile of Haemophilus ducreyi. Ann Microbiol (Paris) 1982; 133B3379-383. 67. Van Dyck E, PiotP. Enzyme profiie of Haemophilus ducreyistrains isolated on different continents. Eur J Clin Microbiol1987; 6:40-43. 68. Casin IM, Grimont F, Grimont PAD, Sanson-Le Pors MJ. Lack of deoxyribonucleic acid relatedness betweenHaemophilus ducreyiand other Haemophilus species. Int J Syst Bacteriol1985; 35:23-25. Fast DM, Holler JS, 69. Carlone GM, Schalla WO, Moss CW, Ashley DL, Plikaytis BD. Haemophilus ducreyiisoprenoid quinone content and structure determination. Int J Syst Bacterioll988; 38949-253. 70. Rossau R, Duhamel M, Jannes G, Decourt JL, Van Heuverswyn H. The development of specific rRNA-derived oligonucleotide probes for HaemophiIus ducreyi, the causative agent of chancroid. J Gen Microbiol 1991;137: 277-285. 71. Sarafian SK, Woods TC, Knapp JS, SwamhathanCB,Morse SA. Molecular characterization of Haemophilus ducreyi by rRNA fingerprinting. J Clin Microbiol 1991; 29:1949-1954. 72. Kilian M, Theilade J. Cell wall ultrastructure of strains of Haemophilus ducreyi and Haemophiluspisicum. Int J Syst Bacteriol1975; 22351-356. 73. Abeck D, Johnson AP. Identification of surface-exposedproteins in Haemophilus ducreyi. FEMS Microbiol Lett 1987; 44:49-51. 74. Saunders JM, Folds JD. Immunoblot analysis of antigens associated with Haemophilus ducreyiusing serum from immunized rabbits. Genitourin Med 1986; 62~321-328. Y, Fontaine EA, Taylor75. AbeckD, Johnson A P , BallardRC,Dangor Robinson D. Effect of cultural conditions on the protein and lipopolysaccharide profiles ofHaemophilus ducreyianalyzed by SDS-PAGE. FEMS Microbiol Lett 1987; 48:397-399. 76. Bertram PD. Studies on Haemophilus ducreyi. MSc. thesis, University of Manitoba, Winnipeg, May 1980. 77. Johnson A P , Abeck D, Davies HA. The structure, pathogenicity and genetics of Haemophilus ducreyi. J Infect Dis 1988; 17:99-106. Characterization of Haemophilusparain78. Roberts MC, Mintz CS, Morse SA. fluenzae strains with low-M, or ladder-like lipopolysaccharides. J Gen Microbiol 1986; 132:611-616. 79. Odumeru JA, Wiseman GM, Ronald AR. Relationship between lipopolysaccharide composition and virulence ofHaemophilus ducreyi. J Med Microbiol 1987; 23~155-162. 80. Campagnari AA, Wild LM, Griffiths GE, Karalus RJ, Wirth MA, Spinola SM. Role of lipooligosaccharide in experimental dermal lesions caused by Haemophilus ducreyi. Infect Immun 1991; 59:2601-2608.

Applications of Lectins to Agents of STD 81. Campagnari AA, Spinola SM, Lese AJ, Kwaik YA, Mandrel1

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RE, Apicella

MA. Lipooligosaccharide epitopes shared among gram-negativenonenteric

mucosal pathogens. Microb Pathogen1990; 8:353-362. 82. Hammond GW, Slutchuk M, Scatliff J, Sherman E, Wilt JC, Ronald AR. 83. 84. 85. 86. 87.

Epidemiologic, clinical,laboratory andtherapeutic features ofan urbanoutbreak of chancroid inNorth America. Rev. Infect Dis1980; 2:867-879. Plummer FA, Nsanze H, Karasira P, D’Costa LJ, Dylewski J, Ronald AR. Epidemiology of chancroid and Haemophilus ducreyi in Nairobi, Kenya. Lancet 1983: 2:1293-1295. Rajan VS, Sng EH, Lim AL. The isolation ofHaemophilus ducreyiin Singapore. Ann Acad Med (Singapore) 1983; 1257-60. Taylor DN, DuangmaniC, Suvonge C, O’Connor R, Pitarangsi C, Panikabutra K, Echeverria P. The role of Haemophilus ducreyi in penile ulcers in Bangkok, Thailand. Sex Transm Dis 1984; 11:148-151. Sng EH, Lim AL, Rajan VS, Goh AJ. Characteristics of Haemophilus ducreyi. Br J Vener Dis 1982; 58:239-242. Sottnek FO, Biddle JW, Kraus SJ, Weaver RE!, Stewart JA. Isolation and identification of Haemophilus ducreyi in a clinical study. J Clin Microbiol

1980; 12170-174. 88. Hannah P, Greenwood JR. Isolation and rapid identification ofHaemophilus ducreyi. J Clin Microbioll982; 16:861-864. 89. Dylewski J, Nsanze H,Maitha G , Ronald A. Laboratory diagnosisof

Haemophilus ducreyi: sensitivity of culture media. Diagn Microbiol Infect Dis 1986; 4241-245. 90. Lubwama SW, Plummer FA, Ndinya-Achola H, Nsanze W, Namaara LJ. Isolation and identification of Haemophilus ducreyi in a clinicallaboratory. J Med Microbiol 1986; 22:175-178. of dot91. Schalla WO, Sanders LL, Schmid GP, Tam MR, Morse SA. Use immunobinding and immunofluorescence assaysto investigate clinically suspected cases of chancroid.J Infect Dis 1986; 153:879-887. JohnsonAP, BallardRC,Taylor-Robinson D, 92. KortingHC,AbeckD, Braun-Falco 0. Lectin typing ofHaemophilus ducreyi. Eur J Clin Microbiol Infect Dis 1988; 7:678-680. 93. Norris SJ, Miller JN, Sykes JA, Fitzgerald TJ. Influence of oxygen tension, sulfhydryl compounds; and serum on the motility and virulence of Treponema pallidum (Nichols strain) in a cell-free system. Infect Immun 1978; 22: 689-697. 94. Fieldsteel AH,StoutJG,

BeckerFA.Comparativebehaviorofvirulent strains of Treponemapallidum and Treponema pertenuein gradient cultures of various mammalian cells. Infect Immun1979; 24:337-345. 95. Cox CD, BarberMK. Oxygen uptake by Treponemapallidum.Infect Immun

1974; 10:123-127. 96. Lysko PG, Cox DC. Terminal electron transport in Treponema pallidurn. Infect Immun 1977; 16:885-890. 97. Hovind-Hougen K. Determination by means of electron microscopy of mor-

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98.

99. 100. 101. 102. 103. 104. 105. 106.

107. 108. 109. 110. 111. 112. 113. 114.

Schalla and Morse phological criteria of value for classification of some spirochetes, in particular treponemes. Acta Pathol Microbiol ScandSuppll976; 255:1-41. Fitzgerald TJ, Cleveland P, Johnson RC, Miller JN, Sykes JA. Scanning electron microscopy ofTreponemapallidum(Nichols strain) attached to cultured mammalian cells. J Bacterioll977; 130:1333-1344. Mia0 RM, Fieldsteel AH.Genetics ofTreponema:relationshipbetween Treponemapallidum and five cultivable treponemes.Bacterioll978; J 133:lOl-107. Mia0 RM, Fieldsteel AH. Genetic relationship between Treponema pallidum and Treponema pertenue, two noncultivable human pathogens. J Bacteriol 1980; 141:427-429. Fieldsteel AH, Cox DL, Moeckli RA. Cultivation of virulent Treponema pallidum in tissue culture.Infect Immun 1981; 3298-915. Fieldsteel AH, Cox DL, Moeckli RA. Further studies on replication of virulent Treponema pallidum in tissue cultures of SflEp cells. Infect Immun 1982; 35:449-455. Noms SJ. In vitro cultivation of Treponema pallidum: independent confirmation. Infect Immun 1982; 36:437-439. Mathews HM, Yang "K, Jenkin HM. Unique lipid composition of Treponemapallidum (Nichols virulentstrain). Infect Immun 1979; 24:713-719. Fitzgerald TJ, Johnson RC, Wolfe ET. Mucopolysaccharide material resulting from the interaction of Treponema pallidum (Nichols strain) with cultured mammalian cells. Infect Immun 1978; 22575-584. Van Der Sluis JJ, Ten Kate FJ, Vuzevski VD, Stolz E. Light and electron microscopy of rabbit testesinfectedwith Treponemapallidum (Nichols strain): nature of deposited mucopolysaccharides and localisation of treponemes. Genitourin Med 1987; 63:297-304. Fitzgerald TJ, Johnson RC. Surface mucopolysaccharidesof Treponemapallidum. Infect Immun 1979; 24:244-251. Fitzgerald TJ, Johnson RC, Ritzi DM. Relationship of Treponemapallidum to acidic mucopolysaccharides. Infect Immun1979; 24:252-260. Fitzgerald TJ, Johnson RC. Mucopolysaccharidase ofTreponemapallidum. Infect Immun 1979; 24:261-268. Wos SM, WicherK. Antigenic evidencefor host originof exudative fluidsin lesions of Treponemapallidum-infectedrabbits. Infect Immun 1985; 47:228233. Fitzgerald TJ, Repesh LA. The hyaluronidase associated with Treponema pallidum facilitates treponemal dissemination. Infect Immun 1987; 55:10231028. Baseman JB, Zachar Z, Hayes NS. Concanavalin A-mediated affinity film for Treponemapallidum.Infect Immun 1980; 27:260-263. Kahane I, Tully JG. Binding of plant lectins to mycoplasma cells and membranes. J Bacterioll976; 128:l-7. Schiefer H-G, Gerhardt U, Brunner H, Krupe M. Studies with lectinson the surface carbohydrate structures of mycoplasma membranes.Bacterioll974; J 120~81-88.

4 Application of Lectins in Clinical Bacteriology MALCOLM SLlFKlN Allegheny General Hospital and Medical College of

Pennsylvania, Pittsburgh, Pennsylvania

1. INTRODUCTION

The application of lectins for the identification 'of various microorganisms was first broughtto the attention of the microbiologist through the investigations of Sumner and Howell, in 1936 [l]. These investigators observed that various lectins capable of agglutinating agglutinated erythrocytes could be inhibitedby simple sugars[2]. These observationswere the first to present evidence that the lectin from the jackbean, concanavalin A (Cod), agglutinated certain species of Mycobacterium and Actinomyces. The lipids and ether of Mycobacterium paratuberculosis,when extracted with acetone and incorporated in a salt suspension, were reported to agglutinate in the presence of C o d . This latter report of Sumner and Howell, therefore, may be viewed as one that brought forth the initial momentum of lectin application into the realm of clinical microbiology as well as into many other facets of microbiology. Bacterial lectins have been demonstrated to be associated with a role in infectious disease[3]. The bacterial surface lectins serve as molecules of recognition in cell-cell interactions [2,4]. The reader is thus referred to references on bacterial lectins that function as adhesins in host-pathogen interactions p]. There are many reviews on lectins and applications of these unique proteins [6-171. At present, there are several reviews on the application of lectins as diagnostic reagentsfor use in clinical microbiology[18-231. This chapter will (l) review the pertinent applicationsof nonbacterial lectinsfor the identification or classificationofvariousgram-positive and gram143

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negative bacteriaand (2) present examples ofthe use of lectins as diagnostic probes. Specialattention is devotedto the presentation of methods applicable to the detection of various bacteria,by lectin interaction, for the clinical bacteriology laboratory. II. USE O F LECTINS FOR GRAM-POSITIVE BACTERIA A. Bacillus

Concanavalin A reacts with a wall polymer of B. subtilis, represented by [M]. Variouslectinshave polyglucosylglycerolphosphateteichoicacid made it possible to eliminate the relatively expensiveand time-consuming culturing or serological testing. For example, the laboratory identification of B. anthracis has traditionally been a time-consuming and inexact exercise for several reasons. These problems include overlapof common antigens, an overlap for y phage receptor sites, and the need for special media and reagents. Some lectins bind to plastic surfaces allowinga procedure to be developed known as an enzyme-linked lectinosorbent assay (ELLA) (see Chapter 1). This assay was demonstratedto detect as few as 26,000 cells of B. anthracis employing horseradish peroxidase-conjugated soybean lectin 1251. Soybean, Abrus precatorius and Griffonia simplicifolia (GSA-l) agglutinins were reported to agglutinate B. anthracis and B. mycoides [25]. Bacillus anthraciswill bind with fluorescein-conjugated soybean agglutinin [27]; however, B. mycoides does not divide at 37OC, so a Bacillus culture incubated at 37OC and agglutinated by soybean agglutinin may be considered to be B. anthracis [181. Confirmation of B. anthracis depends on the de[27]. termination of exotoxinsor on the cell wall-associated polysaccharide A procedure usinga snail lectin,Helixpomatia, and the soybean lectin for the differentiation of B. anthracis can befound in the fourth edition of the Manual of Clinical Microbiology, published in 1985 1271. This agglutination procedure, reported byColeetal. [26], can be performedwith minimal reagentsand can be completed in only minutes.A Bacillus isolate mixed in a solution of soybean lectin will agglutinate within1-2 min if the bacterium is B. anthracis or B. mycoides. A second lectin,Helixpomatia, is positive for only B. mycoides. Lectins have also been used to classify many strainsof the insect pathogenB. thuringiensis [28]. B. Staphylococcus and Related Bacteria

A relatively wide spectrum of gram-positive bacteria have been examined for their respective affinity for plant- or animal-derived lectins. Various lectins have proved useful reagentsfor probing structural features of poly-

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saccharides.Lectinscanrecognize N-acetyl-D-galactosamine(GalNAc) structures. These lectinsappear to respond to differences of terminala-or 8-galactose or a- or 8-GalNAc [29]. Some a-GalNAc-specific lectins are Dolichos biflorus, Griffonia simplicifolia, and Salvia sclarea. The lectins derived from Helix pomatia, Vicia villosa, Wistariafloribunda, and Glycine max react witha-as wellas &linked GalNAc residues. Concanavalin A was reported to precipitate with a-glucosylated, but not with 0-glucosylated teichoic acids, from Staph. epidennidis by agar diffusion [30]. This lectin reacted with only a-linked N-acetylglucosamine teichoic acidsfrom strains of Staph. aureus. Concanavalin A is reported to precipitate the teichoic acid associated with a strain of Staph. aureus that residues, but does not precipitate with contains a-N-acetyl-D-glucosaminyl teichoic acids ofother strains containing8-N-acetyl-D-glucosaminylgroups P11. Staphylococcus aureus and the related bacterium Micrococcus lysodeikticus were reported to react with concanavalin A [32]. Staphylococcus aureus was reported to be agglutinated by a fraction of horseshoe crab distinct from limulin [33]. Purified lectinfrom the horseshoe crab Limulus polyphemus agglutinatesStaph. aureuscells [33]. Generally, most laboratories employ the catalase and coagulase tests, along with Gram stain reaction, for the differentiation of Staph. aureus from the coagulase-negative staphylococci [34]. Coagulase production is reported to be associatedwithfalse-negative [35] andfalse-positiveresponses [35,36]. A battery of lectins have been demonstrated to be useful for the differentiationof coagulase-negativeand coagulase-positive staphyto differentiatethesetwo lococci [37]. Thismethodprovidesameans groups of staphylococci in only 5 mins. Staphylococcus aureus will not agglutinate in the presence of the lectin from cells of the horseshoe crab L. polyphemus nor the combination of lectins derived from the mango, Mangifera indica, and from wheat germ, Triticum vulgaris. Agglutination with these lectins occurs with the coagulase-negative staphylococcal strains tested. This slide agglutination test correctly differentiated 29 strains of Staph. aureusand 30 strains of coagulase-negative staphylococci.

c.

streptococcus

Most strains of Strep. mutans tested were reported to be agglutinated 'by concanavalin A [38-401. Enhancement of this agglutination response was achieved by addition of sucrose or addition of dextranase to the bacterial cells grown in the presence of sucrose. Inhibition studies indicated that different binding siteson the bacterial cells from various serogroups were responsible for the binding ofthis lectin. Other investigations have reported similar conclusions[39].

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In another investigation, ConA was reported to bind to isolated cell walls of various strains of Strep. mutans [41-42]. Streptococcus mutans grown in sucrose medium bound moreConA than those grown in glucose medium. After treatment with dextranase, the sucrose-grown cells bound two- to fourfold more of the lectin1421. The albumen gland of the edible snail, H. pomatia (HPA), contains an agglutinin that specifically bindsto group A human erythrocytes, witha specificity directedto terminal nonreducing N-acetyl-D-galactosamineresidues [43-46]. The lectins derivedfrom H. pomatia and the plants, Dolichos biforus (DBA) and Wisteriafloribunda (WFA), will specifically agglutinate group C streptococci owingto their high affinity for N-acetylgalactosamine, the major group-specific polysaccharide of group C streptococci. There are many other lectins that are effective in distinguishing group C streptococci from the other clinically significant 6-hemolytic streptococci H. pomatia (Table 1). In early investigations, crude extracts containing lectins were shown to identify group C streptococci specifically, without agglutination responsesfrom either groups A, B, C, F, or G streptococci. The lectins from D. biflorus and W. floribunda also agglutinate group C &hemolytic streptococci, without reactivity with other clinically important serogroups [47]. Prokop et al. [48] determined that group C streptococci were specifically agglutinated by the lectin from the H. pomatia. The activity of this lectin is directed toward terminal a- and 0-N-acetyl-D-galactosamineresidues. Group C streptococci were reported to be agglutinated by the lectin from the albumen gland of the snail Cepaea hortensis [49] and from the seeds of the tropical leguminous plant D. biforus [44,46]. One report has demonstrated, on the basis of more than 4O00 strains of P-hemolytic streptococci,that H. pomatia was highly specific in agglutination of group C streptococci [48]. More recently, an investigation that comprised 1045 strains of group C streptococci, includingStrep. equisimilis, Strep. zooepidemicus, and Strep. dysgalactiae,were agglutinated by the Helix lectin [48]. None of 12,264 strains of group A, 1346 strains of group G, 330 strains of group B, as well as other streptococcal groupswere agglutinated. A few strains ofStrep. anginosus (S. miller0 associated withgroup G-specific carbohydrate, agglutinated in the presence of the Helix lectin. Streptococcussanguis strains with groupH antigens were also agglutinated with this lectin [48]. The H. pomatia lectin can react, however, with other bacteria in addition to group C streptococci. The lectin has been reported to agglutinate all strains of Corynebacterium diphtheriae, some but not all Bacillus sp., Salmonella,Escherichiacoli 025, 058, 0117,0126, and most strains of Staph. auras [46,48].

Lectins in ClinicalBacteriology

I

I

I

I

I

I I

I I

I I

I I

I

I

I

I

I

I

I

I

I

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I

I

I I I I I l i i + +

I

I I + + + + i i + + +

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+ I

E

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Thefishes Salmo irideus,Luciopercalucioperca, and Rutilus rutilus contain a rhamnose-specific lectinthat agglutinates strainsof streptococcal group G, variant strains of groups A and C , some strainsof streptococcal groups D,E, L,0,S, and T, as well as some salmonellae ofthe groups B, C , D, and E [48]. Another lectin of R. rutilus is specific for D-glucosyl residues and agglutinates streptococci of group E, carrying &D-gluocosides and rhamnose. The lectin of Cyprinus carpi0will agglutinate another subgroup of group E streptococci and is specificfor D-glucose [48]. In contrast to the lectin from H. pomatia (HPA),these lectinsdo not agglutinate allof the strains mentionedand cannot be applied to routine diagnosis [48]. They can be used only for the determination of immunochemical end-grouping. The seed extracts of W.floribunda also specifically agglutinate group C streptococci, as shown by the investigations of Ottensooser and associates [47]. These authors emphasize the advantages of lectins in relation to the savings of time, such as pretreatment of bacteriafor immunization, injection of bacteriainto animals, and absorptions of antisera to eliminate nonspecific antibody. Our first introduction with lectins concernedthe development of a test that used three fluorogenic 4-methylumbelliferyl substrates and the lectin from D. biforus (DBA).The assay provided a rapid, simple, and specific means to identify groups A, B, C , F, and G streptococcal isolates from throat cultures [50]. The three methylumbelliferyl substrates used in that investigation provided a means to accurately identify groupsA, B, and F P-hemolyticstreptococciisolatesfrom throat cultures.Somestrainsof group C and G streptococci, however, could not be differentiated because of overlapping 4-methylumbelliferylsubstrate patterns. The useof soluble a rapid preparation of the lectin DBA as an agglutination reagent provided and specific agglutination response when mixed on a microscope slide with five or more colonies of group C organisms. Results were obtained within 30 sec of a hand rotation on a microscope slide. No agglutination was detected with group G or with groups A, B, or F streptococcal cells. The use of the combination of the methylumbelliferyl substrates and the lectin reagent provideda nonserological methodthat permitted accurate identification of isolated colonies of P-hemolytic streptococci obtained from throat cultures. The methylumbelliferyl-lectin protocol was later applied by other investigators for the identification of streptococci isolated from cows with mastitis [51]. As with the previous investigations [50],the use of the lectin D. biforus was required to identify and differentiate Strep. dysgalactiae (group C ) from the other groupable streptococci. The demonstration of species-specific enzyme activities of the streptococci with various Cmethylumbelliferyl-conjugated substrates in combination with the lectin agglutination


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permitted differentiation within1-2 hr at 37OC. This is incontrast with the standard identification techniques of these serogroups with biochemical tests that require an incubation periodof 4-24 hr. These investigations ledto the development of tests employing lectins as diagnostic tools for the identification of various serogroups of the 0hemolytic streptococci. Thus, it was later reported that crude extracts of arm, yielded DBA,whencoupled to polystyreneparticleswithaspace an effective lectin-latex microsphere reagent that specifically agglutinated group C streptococcal antigens prepared as nitrous acid, autoclaved, or enzyme extracts. The coated beads could also agglutinate suspensions of isolated colonies[52]. This latex-lectinpreparation could be usedto replace latex microspheres conjugatedto antibody to group C streptococci for use in test kits for the identification of /.%hemolytic streptococcifrom isolated colonies. Many lectins are extremely stable at ambient temperatures.The lectin of W. floribunda was absorbed onto polystyrene latex microspheres that were stained blue. Fifty microliters of these lectin conjugated microspheres were placed on cardboard slides and dried overnight at ambient temperature. The dried lectin reagent cards were mixed with 50 p1 of phosphate buffer (pH 7.2) at various times and tested with nitrous acid extracts of group C streptococci. Strong and highly specific agglutination responses were observed with the polystyrene lectin reagent driedon cards 7 months previously. No reactions were observed with group A streptococci or with 1). other clinically significant 0-hemolytic streptococci (Fig. The use of HPA was used as a membrane particle-capture assay to detect nitrous acid-extracted group C streptococci. The HPA lectin-latex microspherereagent was spotted onto aporousmembrane and dried. Group C streptococcal nitrous acid extractwas added onto a lectin-capture substrate and incubated for 5 min at room temperature. Peroxidase labeled HPA was then addedto sandwich the analyte, and color detection proceeded, as withan enzyme-linked immunosorbent assay (ELISA) format (Fig. 2). Holm and associates have shown that blocking the sialic acid of the type-specific polysaccharide of a type I11 strain of group B streptococcal cells by the sialic acid-specific lectinfrom the snail Cepaea hortensis, leads to the promotion of phagocytosis of a group B streptococcus of type Ia strain [53]. The effect of the lectin was dose-dependent and required the presence of complement. The sialic acid-specific lectin from C. hortensis and C. nemoralis agglutinates all groupB streptococcal strainsthat contain a type-specific polysaccharide[49,54]. However, group B streptococcithat lack a type-specific polysaccharide,as well as strainsof other streptococcal groups and many other bacteria, including species containing sialic acid, are not agglutinated [53].

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Figure 1 Reactivity of dried polystyrene latex microspheres conjugatedto Wisteria floribunda for a nitrous acid extract of group C streptococci. The microspheres have been dried on a cardboard card. The microspheres on the bottom left of the card have been mixed with a nitrous acid extract of group A streptococci and show no agglutination, whereas the microspheres on the bottom right have agglutinated in the presence of group C streptococcal extract.

A relatively wide spectrum of lectins were demonstrated to agglutinate various serotypes ofgroup B streptococci [S]. In some instances treatment of the bacterial cells with sialidase removedthe reactivity of various lectins with those cells.In other instances, agglutination with lectins occurred only after enzyme treatment ofthe bacterial cells. Group B streptococci can be specifically agglutinatedor labeled with fluoresceinconjugated lectins (Fig. 3) derived from certain plants of the Solanaceae family [56]. The pulp lectin from the tomato, Lycopersicon esculentum (LYE), as wellas the related plant, the potato, Solanum tuberosum (STA), canbe passively coupledto amide-modified polystyrene microspheres [56]. These reagents could be usedfor the specific identificationof group B streptococci grown in selective Todd-Hewittbroth, such as Lim’s broth, as well as nonselective broth [57]. These lectins do not agglutinate the other clinically significant serogroupable streptococci. The lectins could


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possibly be of valuefor their abilityto assist in the identification of group B streptococcal colonizationin women and infants. These two lectins have an affinity, in part, for N-acetylglucosamine, the major group-specific polysaccharide for group B streptococci. Either the lectin of the tomato (LYE) or potato (STA) conjugated to latex particleswill agglutinate group B streptococci. Particulate organisms, as well as nitrous acid, autoclaved,or enzyme extracts were agglutinated or precipitated, respectively, by STA. These streptococci, like thoseof group A and group C streptococci, contain relatively high amounts of N-acetyl-Dglucosamine,comparedwithmembersofgroups D, F, or G [58]. The unique presenceof 1,4-anhydroglucitol in the cells of group B streptococci [59] may, in part, be related to the affinity of the tomato and the potato lectins for group B streptococci. It has been shownthat tomato lectin binds oligosaccharides containing the repeating disaccharide (p-1,4-GlcNAc-P), or poly-N-acetyllactosamine sequence,requiring at leastthreerepeating disaccharide unitsfor interaction [60]. Although not precisely defined,the relevant antigenic determinantsof group B polysaccharide appearto be associated withthe rhamnose-glucitol

Figure 2 Microsphere-enzymatic procedure using H.pomatia lectin as a capture reagent for a nitrous acid extract of group C streptococci. The microparticle-capture membrane on the left did not indicate a positive reaction when group A nitrous acid extract was added to the membrane. A positive enzymatic response is seen on the membrane on the right when group C nitrous acid extract was added.

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Figure 3 Fluorescent response of group B streptococci labeled with fluoresceinconjugated L. esculenturn.

units or terminal rhamnose on side chains associated with N-acetylglucosamine [59]. It is possible that the tomato and potato lectin can interact with other substrates associated withgroup B streptococci. The lectin from the garden snail Cepaca hortensis has a specificity for sialic acid-containing polysaccharides and can react withgroup B streptococci [53]. Various typeableand nontypeable strainsof group B streptococci were examined to determine their respective reactivityto LYE coupled to polystyrene spheres (Table 2). All the strains grown in either Todd-Hewitt or Lim’s broth reacted with the lectin reagent. Nitrous acid, Streptomyces albus lysozyme enzyme, and autoclaved extracts of group B streptococcal cells yielded results similarto those with particulate group B streptococcal cells grown in these culture broths. None of the type l b strains grown on Columbia sheep blood agar, however, reacted with the LYE-conjugated microspheres. Observation of medium-dependent agglutination reactions have been previously reported with Legionella-lectin interactions [61]. The latter investigators [61] emphasizedthat when lectinsare employed as diagnostic reagents, growth conditions must be carefully standardized. Fluorescein-conjugated LYE labeled all the group B streptococcal cells grown from the blood agar medium.The serotype l b strains, however, did not fluoresce as brightly as did the other group B serotypes. No fluorescence or agglutination responsewere observed whenthe other serogroups of


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streptococci were examined. The lectin fromthe solanaceous plant Datura stramonium did not agglutinate group B streptococci nor the other serogroups tested. Datura stramonium, similar to LYE and STA, is a member of the Solanaceae familyof plants. This lectin, like that of the tomato and potato lectins, is specific for A, B, and 0 erythrocytes and binds to chitooligosacThe lack of group B streptococcal activity withD. stramocharides [a]. nium may be related to the inability of relatively small concentrations of N-acetyl-D-glucosamine to block the agglutination of human erythrocytes with this lectin, or to its lack of reactivity with dimers or trimers of Nacetyl-D-glucosamine [60]. It was previouslyreported that various commercially available serogrouping reagents cross-reacted with the Todd-Hewitt broth manufactured by Difco Laboratories, but not with that manufactured by Baltimore Biological Laboratories [62]. The tomato or potato lectins do not precipitate in inoculated Todd-Hewittbroth of either manufacturer. Three elderberry species contain two identical carbohydrate-binding sites per moleculeand exhibit a very high specificityfor the NeuSAw-2,6Gal/GalNAc sequence [63-651. Relative affinitiesfor various oligosaccharides were significantly different among them, suggesting differences the in detailed structure of the carbohydrate-binding sitesof these related lectins. Previous to these reports, no plant lectin had been reported to bind specifi-

Table 2 Agglutination of Group B Streptococci with Lycopersicon esculenturn or Solanum tuberosurn Lectin Coupledto Polystyrene Spheres Agglutination-response (no. agglutinatedlno.tested) tested Organisms Nontypeable la lb IC (WC) I1 111 Group A Group C Group F Group G

Lm’s

No. Columbia Todd-Hewitt sheep broth broth blood agar 2 4 4 4 4 4 28 20 20 20

0/2 3/4 0/4 4/4 4/4 4/4 0/28 0/20 0/20 0/20

2/2 4/4 4/4 4/4 4/4 4/4 0/28 0/20 0/20 0/20

2/2 4/4 4/4 4/4 4/4 4/4 0/28 0/20 0/20 0/20

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cally to sialic acid or to oligosaccharide units that included this carbohydrate residue [66]. Sialic acids represent about 30 derivatives of N-acetyl- or N-glycolylneuraminic acids [67]. Various sialic acid derivatives exhibit interesting species- and tissue-specific distribution. Sialic acids playan important role as receptors for viruses [68] as wellas inthe social behaviorof cells. Theyalso act as masking agents on antigens, receptors, and other recognition sites of the cell surface [ 161. There is a relatively small selection of lectins that specifically bind to sialic acids [67]. The lectin isolated from elderberry, Sambucusnigra (SNA),binds the sequenceNeuNAc a-2,6-~-Gal/~-GalGalNAc with high specificity [63]. This lectin has a relatively weak affinity for D-galactose or D-G~NAc,but does not bind to eitherNeuNAc or NeuNGuc. It has a high affinity for terminal sialic acid that is linked cr-2,6to D-galactose, compared with the a-2-3-linked’ isomer [63].On the basis of inhibitions with simple sugars, the elderberry bark lectin was demonstrated to be specific for D-galactose [63]. The lectin from the tulip (Tulipa; TUG) is derived from the bulb of the plant, a member of the family Liliaceae [69]. This lectin was reported to be the first lectin to be isolated from this plant family and is the first example of a phytohemagglutinin obtained from a monocotyledonous species other than the Graminaeae family associated with wheat, rye, barley, and rice [as]. Two lectins with different agglutinating affinities are associated with T. gesneriana bulbs [70]. One TUG lectin component will agglutinatetrypsinized human or rabbit erythrocytes [69], but will not agglutinate nontreated human red blood cells [69]. A second TUG lectin was previously shown to agglutinate the cells of the Saccharomyces genus at a concentraof Candida, E. tion of 15-30 pl/ml, but did not agglutinate various species coli, Staph. aureusor B. subtilis [71]. The lack of agglutination response of to group A streptococcal extracts with TUG-bound latex particles appears indicate that the lectin substrates may be either denaturedor not extractable. The group A streptococcal extracts, in contrast, agglutinated latex particles bound with antibodythat is directed to the specific group antigen or “C-substance” reportedto be N-acetylglucosamine [58]. In the literature, the tulip lectin has been reportednot to have an affinity for this glycoprotein [69,71]. According to one investigation, D-mannose, ~-mannose-6-phosphate, L-fucose, and L-fucosylamine were demonstrated to be potent inhibitors of this lectin in the binding to Saccharomyces cerevisiae cells [70]. In a hapten-inhibition investigation, agglutinationof human erythrocytes with TUG lectin was inhibited by N-acetylgalactosamine, lactose, fucose, and galactose [69]. ,

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It has been determined that the TUG lectin derived from the bulb, or the lectin from the bark of Sambucus nigra, the elderberry, will agglutinate both group A and C streptococci (see Table 1). Agglutination of group A streptococcal organismswas affected withtulip lectin at a concentration of 0.3 mg/ml on latex spheres. All of the 40 stains of group A streptococci grown on Columbia sheep blood agar agglutinated with this lectin reagent in 15-30 sec by manually rockingthe reactants on a glass microscope slide. Within this period, the reactions appeared at an agglutination reactivity strength of 2-3 + (Fig. 4). The tulip lectin reagent agglutinated12of the 40 (30%) test strains of, the group C streptococci. These relatively weaker agglutination responses, however, occurred after 30-60 sec of manual rocking ofthe reactants. The reactions ofthese group C strains produced only 1 agglutination responses within this observation period. All 40 strains of group A streptococci were also agglutinated in the presence of microspheres prepared with 0.01 mg of lectin per milliliter. These 2-3+ agglutination reactions were observed within 30-60 sec of manual rocking of the reactants on microscope slides. Furthermore, at this lower concentrationof tulip lectin, none

+

Figure 4 Agglutination of group A streptococci with Tulipa lectin conjugated on microspheres. The lectin-conjugated particles on the left were mixed with group C streptococci. The right side shows the lectin-microphase and group A streptococci.

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of the group C streptococcal strains agglutinated within60 sec of observation. Conjugation of the TUG lectin onto 0.2-pm polystrene microspheres also yields agglutination with group A streptococci and none with theother clinically significant serogroups.In contrast, the use of larger microspheres effected no differentiation between group A or C streptococci (Table3). None of the strains of group B, F, or G streptococci agglutinated in the presence of the TUG-latex reagent.The use of differentlots of Columbia sheep blood agar yielded the same agglutination patterns with these streptococci. Nitrous acid, enzyme, autoclave,or sonic-derived extracts of the various 8-hemolytic streptococcal serogroups did not produce responses when tested withthe TUG-latex reagent. These extracts, however, did produce a specific agglutination response when mixed with their respective commercially prepared serodiagnostic-latex reagent. Inhibition studies were performed to determine the effects of various sugars and glycoprotein inhibitors on the reactivity of TUG lectin, with either group A or group C streptococci. This inhibition protocol was performed by preparing twofold serial dilutionsof various sugars and glyco4. These preparationswere proteins in phosphatebuffer, as shown in Table respectively addedto 0.1 m1 of TUG-latex reagent. The latter contained 30 pg of lectin per milliliter. The lectin and inhibitors were mixed on a glass microscope slide with a wooden applicator stick for 10 sec. Four to six colonies of group A or group C streptococci were removed from a sheep blood agar plate with a wooden applicator stick and mixed into the lectinlatex inhibitor mixturefor 30 sec. Four to six colonies of groupA streptococci were also mixed into 0.1 ml of TUG-latex reagentthat did not contain any potential inhibitor solution to serve as a control. A determination of Table 3 Effect of Microsphere Size on Specificityand Reaction Time with Interactionof Tulip Lectin and Group A and Group C Streptococci ~~

Reaction time (sec) Serogroup A

Latex particle size (pm) 0.7 0.5 0.3 0.2

Serogroup C

30

60

90

30

60

90

3+* 2+ 2+

3+ 3+

3+ 3+ 3+ 3+

l+ l+

l+ l+

2+

l+

2+ 2+

-

-

-

2+ l+

-

'Strength of reaction: - ,no agglutination; 1+ ,many fine clumps;2 + , a few moderate-sized clumps; 3 ,many moderate-sized clumps.

+

Bacteriology Ledins in Clinical

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Table 4 Inhibitory Effect of Various Carbohydrate-Glycoproteins onthe Agglutination of Tulip Lectin to GroupsA and C Streptococci

Carbohydrate/glycoprotein

Minimal concentration requiredfor inhibition oftulip-mediated agglutination of group A streptococci (mM)'

Asialofetuin Fetuin N-Acetylmuramic acid N-Acetylneuraminic acid N-Acetyl-D-galactosamine Methyl-P-D-galactopyranoside

Lactose Mannose Fucose Group C streptococcal nitrous acid extract 'NI,

0.2 0.5 3.0 1.S

NI NI

NI NI NI

NI

no inhibition at concentrations less than 200 mM.

agglutination was made after manually rocking the slide for 1 min. The inhibition studies demonstrated the various sialoproteins are effective as inhibitors of agglutinationby TUG-microsphere reagent, with either group A or groupC streptococci (see Table 4).In contrast, various simple sugars or oligosaccharides do not inhibit this agglutination response. Groups A and C streptococci have been reported to contain slightly higher concentrations of the sialoproteinand N-acetylmuramic acidthan groups B, D, F, or G streptococci[58]. Furthermore, the lectin of Limaxflavus, which is very specific for sialicacid[72], will also agglutinate only groups A and C streptococci (see Table 1). Accordingly, there appearsto be no interaction of TUG lectin for either N-acetylglucosamineor N-acetylsialolactosamine (see Table 4). Thus, group A streptococcal binding sites associated withtulip lectin appear not to be entirely related to the polysaccharides established as the respective group-specific antigens [58]. Therefore, one may consider that these binding sites may be associated, in part, with these group-specific polysaccharides aswell as other substrates. All lectin molecules are multivalent and have two or more carbohydrate binding sites [8,17]. However, binding of lectins cannot always be fully correlated with their reported biochemical specificities [6,13,74]. An extensive determination of all potential inhibitors maynot always be possible. Lectin affinity may also be partly related to the method used to purify the lectin[7].

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A similar associationof binding of groups A and C streptococci was [75]. This lectin has an affinity reported with wheat germ agglutinin (WGA) for N-acetyh-glucosamine and its derivatives [76]. The wheat germ lectin was demonstrated to agglutinate all strains of groups A and C streptococci tested [75]. Some strains of groups B, E, H, L, 0, and S streptococci also agglutinate in the presence of this lectin. As with the tulip and Sambucus nigra lectins, the binding of groups A and C streptococci maynot depend on a terminal positionof GlcNAc [75]. Primary colonies of group A streptococci can be accurately identified with the use of tulip lectin asa lectin-bound latex reagent.The tulip lectinreagent thus can be applied as an alternative methodfor the rapid identification of group A streptococcal colonies. The lectin provides a relatively cost-effective meansto identify colonies ofgroup A streptococcal isolates. The use of the tulip lectin, similar to the previously reported D. biflorus lectin [52], affords the availability of a diagnostic reagentthat can be prepared with relatively less complexity than the production of an antibody for serogrouping procedures.Table 1 shows a summary of someof investigations on the interactions of various lectins with serogroups A, B, C, F, and G 8-hemolytic streptococci. Kohler and Nagai [48] have reported that some 8-hemolytic strains of Strep. anginosus (Strep. millerr] can react with HPA. They observed that not all strains reacting with group-specific anti-C antiserum were agglutinated with HPA. It was suggested that N-acetyl-D-galactosamineis not in the terminal position. The agglutination by the HPA by nongroup C Strep. milleri strains also indicated that other antigens may be involved. All eight strains of group C-associated Strep. anginosus strains examined reacted withH. pomatia bound to polystyrene spheres (Table5). This lectin reagent did not agglutinate group A, group F,or nongroupableStrep. anginoms strains. These reactions were similar when either nitrous acid extracts or nonextracted cells were used. The TUG lectin reacted with all eight strainsof group A Strep. anginosus and 25% of the group C streptoTable 5 Interaction of Various Lectins with Streptococcus anginosus

Serogroup of S. anginosus (no. testedho. reactive) Lectin

A

C

F

Nongroupable

Tulipa Sambucus nigra Helixpomatia

8/8 6/8 0/8

2/8 7/8 8/8

0/6 0/6 0/6

014 014 014


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coccal strains. No reactivity was observed with the group F or nongroupable strains. The SNA-latex reagent reacted with most the of group A and C strains of Strep. anginosus tested. This reactivity for non-Strep. anginosus group C streptococci on the SNA lectin was not previously observed [48], which suggests the presence of other SNA-binding substrates on the Strep. anginosus strains. The lectin from Misgurnus anguillicaudatushas a relatively strongaffinity for rhamnose [77]. This lectin, therefore, may be of value in the identification of streptococci [22]. D. Listeria

Recently, Sonnenfeldand Doyle [see 181 reported that the relatively common serotypes ofL. monocytogenes could be differentiated with the use of a panel of lectins.Four lectins, includingthe application of a lectinlike seed extract (Persea americana),were demonstrated to be useful in distinguishing the major serotypes of L. monocytogenes. The lectins included in the panelwere Griffonia simplicifolia, Vicia villosa, HPA, and ConA. The agglutination procedure required that the L. monocytogenes isolates be heated to expose the lecting-binding sites of the organisms. 111.

USE OF LECTINS FOR GRAM-NEGATIVE BACTERIA

A. Enterobacteriaceae

ConcanavalinA agglutinates a variety of gram-negative bacteria.The binding siteof this lectin appearsto be related to the carbohydrate moieties on the lipopolysaccharide molecule [74]. Polysaccharidesof various serotypes of Salmonella precipitate with ConA [78], and it can be applied in microscopic slide agglutination tests [79]. This lectin has been reported to react with Salmonella strains containing the0 : 1 factor, and it will also bind to E. coli [80]. This lectin, therefore, could be employedto classify lipopolysaccharides derived from Salmonella, although not all lipopolysaccharides presumed to contain cy-D-glucosyl side chains form a precipitate with ConA. No precipitate was observed when ConA was reacted withthe lipopolysaccharide fromSal. typhi [81]. The polysaccharide is reportedto contain side chains similar to Sal. typhimurium [78] with which faint precipitation was detected [78]. Concanavalin A was reported to precipitatelipopolysaccharide extracts obtained fromShigellaflexneri and Sal. abortivoequina, but not Sal. enteritidis [81]. Yersinia entercoliticastrains, possessing the antigenic factor 0 : 12 are agglutinated with ConA1791. In the genus Erwinia, the strainsof

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E. amylovora will agglutinate in the presence of ConA in contrast with E. caratovora and E. chrysanthemi [79]. Concanavalin A conjugated with fluorescein did not react with Pseudomonas aeruginosa, Haemophilus injluenzae,or Proteus mirabilis strains 1821. The applicationof lectins for the identification of bacteria commonly associatedwithocularinfectionshasbeendemonstrated.Somelectinbinding patterns were reported to be specific for the bacterial pathogens examined [82]. Litchi chinensislectin was demonstrated to be applicablefor the identiE. coli and of Proteus sp. isolates fication of many @-hemolytic strains of 1831. The H. pomatia lectin failed to precipitate the lipopolysaccharide derived from Sal. typhimurium rough mutants of type Ra and Rb, but was somewhat reactive withan SR mutant [84]. The H. pomatia lectin [48] and the lectin from the horseshoe crab Limuluspolyphemus [85] also have been reported to agglutinate E. coli and other members of the Enterobacteriaceae. Salmonella minnesota cells were agglutinated with wholeLimulus serum, but did not agglutinate inthe presence in L. polyphemus serum purified by affinity column procedure [86]. The sialic acid-binding lectinfrom the horseshoe crab Carcinoscorpius rotunda cauda, also known as carcinoscorpin, will agglutinate E. coli K12 and Sal. minnesotaR595 cells [86]. Vibrio choleraedoes not agglutinate in the presence ofthis lectin. B. Pseudomonas A lectin obtained fromthe potato was reported to distinguish strainsof P. solanacearum [87]. Strains of this bacterial speciesthat are pathogenic for the potato do not react with this lectin, whereas avirulent strains of this species are agglutinated. The investigators determined that an extracellular polysaccharide, associated with virulent strains, appears to inhibit the reactivity of the lectin with its ligand site on a lipopolysaccharide. Sixteen serotypes of P. aeruginosa were typed with a panel of lectins [88]. All serogroups were highly agglutinated by the lectin of Moringa olifera seeds, except serotype H7. Differentiation of the various serotypes was accomplished with agglutination (and precipitation) tests when a panel of lectins derivedfrom M. olifera, Artocarpus integrifolia,and Artocarpus Iakoocha and lipopolysaccharide extractsof various serotypes ofP. aeruginosa were used. Previous investigat.ionson 9 strains of P. cepacia with a relatively large panel of lectins indicated a heterogeneity of lectin-binding sites [89]. All


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strains did not react with the lima bean lectin. The latter research demonstrated that this lectin interacted only withP. cepacia, suggesting that this lectin may be species-specific. The investigators recommendedthat more pseudomonad isolates be examined to confirm this observation. C. Legionella

A panel of plant lectins gave differential reactivities with manysixofserogroups ofL. pneumophila tested [61]. The agglutinationpatterns appeared not to be related to the serogroup of these bacteria, as it was determined that five of the strains of serotype 1 had unique agglutination patterns. These investigations suggested that the cell surfaces of Legionellaceae do not contain carbohydrate groupsthat are accessible for binding by lectins. Some of these bacteria were agglutinated by lectinlike substances from Persea americana, Mangifera indica, Aloe arborescens, and Albizzia julibrissin. These plant extracts are associated with polyphenol derivatives, tannins, and may be capableof binding cell surfaces[W]. D. Neisseriaand RelatedBacteria

Among the gram-negative bacteria, the application of lectins as diagnostic reagents is especially associated with the members of the genus Neisseria. The use of wheat germ lectinto agglutinate strainsof N. gonorrhoeae has been reported [91-921. Nonencapsulated N. meningitidis and N. gonorrhoeae will also agglutinate inthe presence of this lectin, whereas encapsulated N. meningitidis will not [93]. This observation suggested that gonococcal agglutination with wheat germ lectin may be relatedto the lack of capsular material on the gonococci. Other investigations also have shown that wheat germ lectin is of use for the identification of N. gonorrhoeae [94]. From an panel of 14 lectins, all strains of the gonococcal isolates examined were agglutinated by wheat germ, soybean, and ricin lectins[95]. None of the differential patterns of gonococci agglutinationby lectins was related to GC serogroups. Theauthors concluded that GC serogroup antigenic determinants involveportions of sugar structure that arelarger than, or separate from, those recognized by lectin-combining sites. It was suggested that agglutination by these lectins is mediated by cell envelope lipopolysaccharides, and this interaction involved elements of common lipopolysaccharide core structures. A panel of 22 lectins was used to demonstrate the diagnostic effect of lectin on the identification of the family Neisseriaceae [96]. DeHormaeche et al. [97] determined that the lipopolysaccharide of N. gonorrhoeae, but not nonpathogenic neisseriae, contained an epitope consisting of N-acetylglucosamineand N-acetylgalactosamine, and was re-

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active with wheat germ agglutinin. Other investigators, using a battery of lectins, have suggested that the lipopolysaccharides from pyocin-sensitive and pyocin-resistant strains of N. gonorrhoeae possesses terminal GlcNAc or Gal(or GalNAc) residues[95,98]. The use of wheat germ agglutinin for the identification of N. gonorrhoeae was associated with nonspecific agglutination [94] and autoagglutination of gonococcal suspensions[96]. Doyle et al. [96] employed wheat germ and soybean agglutinins in conjunction with a a-glutamylaminopeptidase assay. All of the gonococcal strains and lectin-reactive meningococciwere distinguishable by the hydrolysis of a-glutamyl-8-naphythylamideby the former. This assay is applicable for distinguishing Moraxella catarrhalis, N. lactamica, and N. meningitidis. The autoagglutination phenomenon was overcome by DNase treatment the of bacterial cells. Other investigatorsused both wheat germ and soybean agglutinins in conjunction with various chromogenic substrates [99]. They identified N. gonorrhoeae and differentiated it fromother Neisseria sp. examined. Recently, an investigation of 140 strains of N. gonorrhoeae has shown that 4.9% of the strains are not agglutinated withWGA [l001 as previously reported [99]. Theuseoftenlectins,however,yieldeddiscriminating groups of serotypes 1A and 1B and prototroph and proline-dependent auxotypes [loo]. This method of typing was reproducible and may be considered for epidemiological applications. E. Campylobacter (He/icohcter)

In a recent investigation, it was determined that C. fetus strains lacking surface array proteins with type A lipopolysaccharide were agglutinated with Griffonia simplicifolia 11, H. pomatia, and wheat germ lectins. No agglutination was observed withC. fetus mutants that lacked surfacearray proteins with type B or type C lipopolysaccharide or strains containing a surface array protein layer[loll. The authors concluded that knowledge of the inhibition of lectin-binding to lipopolysaccharide by the surface array proteins will aid in the characterization of the lipopolysaccharide interaction. In another investigation, various serotypes of C. jejuni and C. coli displayed differential reactivities with galactose-binding lectins, the reactions of which appeared to be strain-specific[102]. Other investigators have used lectinsfor the differentiation of various Campylobacter sp. Strainsof C. jejuni and C. coli interact specifically with certain lectins and blood group antibodies [103]. Agglutination by lectins was also reported effectivefor the differentiation of C. jejuni and C. coli [104]. A simple and effective agglutination procedure using a panelof lectins wasdeveloped for differentiating strains of variousspeciesof

nical

Lectinsin

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Campylobacter 11041. All strains tested were agglutinated by the proteinreactive agglutinins of Mangifera indica (mango) and Perseaamericana (avocado). A large number of the Campylobacter strains also agglutinated in the presence ofConA and Triticum vulgaris. The reactions of the other lectins used in the investigation varied. Thedata implied that lectins could be used ina supplementary procedurefor fingerprinting individual isolates. IV. USE OF LECTINS FOR MYCOBACTERIA

Huorescein-conjugated ConA was effective in the visualization of Mycobacteriumfortuitum and M. chelonei in both pure culture and experimental keratitis samplesfrom corneal scrapings [105]. This lectin yields only minimal background stainingof corneal tissue 11061. Concanavalin A binds to nonreducing terminal D-arabinofuranosyl residues associated with the end chain of arabinogalactan of the cell wall ofM. bovis [1071. V. EPIDEMIOLOGICAL APPLICATIONS

Applications of lectinsfor epidemiological investigationsand strain characterization of Haemophilus ducreyi [108], Campylobacter jejuni [1031, and N. gonorrhoeae [109,110] have been reported. These investigations have been applied in certain instances for clinical studiesto differentiate between treatment failure and reinfection; to study transmission,and to investigate the presence of subtypes of various pathogens. In-depth coverage of this topic is found in Chapter 3. VI. CONCLUSIONS

The selection ofa lectin as a potential diagnostic reagentfor use in bacterithe selecology first requires that a reasonable protocol be followed. Thus, tion of a lectin or a panel of lectins may be based on the affinity of these lectins for a particular marker for a given bacterial species. The perfect exampleisclearlyseenwithgroup C streptococci and the reactivity of those lectinsfor N-acetylgalactosamine. This relation of lectin with group C streptococci yields a very effective means to discriminate one serogroup from other clinically significant serogroups of 0-hemolytic streptococci. This groupof streptococci contains relatively larger amounts of GalNAc in comparison withthat of other serogroups. The reactivity of the lectins that bind with this carbohydrate moiety is not specific, however, for only this one serogroup. These lectins can also bind to other gram-positive and gramnegativebacteria. Thus, theobservation of characteristicgram-positive morphology,catalasereactivity, and hemolyticresponseprovides the

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primary guidelines that establishes the use of these lectins for 8-hemolytic streptococci. Without these first-stage guidelines, a lectin,such as that of Helixpomatia, that differentiates group C streptococci from other &hemolytic streptococci can also interact, for example, withStaph. a u r m v11. On the other hand, the determination that LFA and TUG are relatively effective for their respective binding with group B and group A streptococci is not based on the reactivity of these lectins for the antigenic determinants generally associated withthe serogroups. Instead, reactivityof these lectins for the serogroups doesnot appear to be for the N-acetylglucosamine determinants, but possibly for sialic acid residues as well as other substrates. Furthermore, chemical modification ofthe lectin, the lectin concentration, as well as the test assay system are some ofthe factors that will influence the potential for a lectin to be usedas a diagnostic probe. Relative to microsphere size, it was recently reported that the use of relatively small-diameter polystyrene particles will decrease the sensitivity of a latex agglutination test[11 l]. For tulip lectin, however, small microspheres yielda highly specific response to group A streptococcal organisms. This, in part, may be due to possible differences inthe sialopolysaccharide concentration associated with groups A and C streptococci. Given the reported observations of lectin interactions with various bacteria, the most convenient means of testing lectins as a diagnostic tool is generally associated withthe application of isolated bacterial colonies. The direct detection of a particular bacterial agent in a clinical specimen with the useof a lectin assay may be difficult to attain. This is due to the potential for a relatively wide variety of carbohydratesubstrates to be available for lectin interaction ina given clinical specimen. Inoculation of clinical specimensonto tissue culturecells has permitteda specific and sensitive means to detect herpes simplex virus with use of fluorescein-conjugated H. pomatia lectin [1 121. The usefulness of lectinsin clinical microbiology is associated with an extremely broad spectrum of advantages that render them of value in routine diagnostic procedures. Manyof these diagnostic procedures associated with lectinsare relatively inexpensiveand can be rapidly completed. Lectins can also be used for light and electron microscopic investigations [l 131. Some of the significant advantagesare (1) their stability, (2) their activity in very small concentrations, (3) the commercial availability of many lectins, and (4) their ability to probe subtle surface structural differences between various isolates[22]. The cost-effectiveness of these unique naturally occurring cytochemical-histochemicaltools further emphasizes their value as diagnostic reagents for the clinical microbiologist. It was previously suggestedthat because of the binding specificity of

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lectins, they may be able to replace immune sera in detecting various carbohydrate residueson microbial cellsand, by extrapolation, particular microorganisms [19]. This chapter has emphasized that lectins are versatile reagents that can be applied in certain instances to (1) provide definitive identification, (2) characterize a strain of a particular organism, and (3) function as epidemiological markers. The effect of standard methodology and contemporary emphasis on molecular biology technology associated with monoclonal antibodies and nucleic acid probes has, in part, diverted attention from the potential of lectins in applied and clinical microbiology.Furthermore, the unique carbohydrate specificities of lectins in comparison with the specificity attained with the use of monoclonal antibodies or nucleic acid probes may also a be factor considered by various investigators. In contrast, an examination of the list of references on lectins inthe Cumulative Medical Index,as well as an increase in patented processes on diagnostic usesof lectins for the last 5 years, clearly indicatea significant increase inthe use of lectins in scientific research and in clinical microbiology. The emphasis of this chapter is on the application of lectins derived from plant seeds, pulp, bark, root, bulb, and invertebrate extractsfor the identification and characterizationof bacteria. The useof lectins obtained from certain bacteria may also beof value as diagnostic tools for clinical microbiology. The interaction of lectins from Pseudomonas aeruginosa, for example, may be of value for the identification of various E. coli strains [l 14,1151. The mannosephilic hemagglutinin of P. aemginosa is reported to agglutinate the cells of the enteropathogenic E. coli 0128 :B12 and 0 8 6 :B7. The binding of the lectin was shown by specific agglutinationof the E. coli strains by hemagglutination tests[l 141. The use of this bacterial lectin for the typing of these potential enteropathogens was consideredto have certain distinct advantages [1151. Although less specificthan antibodies, the bacterial lectin probe can be conjugated with a peroxidase signal [l 151. Furthermore, in the realm of clinical. microbiology, certain lectins [1 161, and they have been considered as may also have antibacterial activity carriers of chemotherapeutic agents[12]. There are now a wide variety of lectins available in unconjugated as well as conjugated forms from several commercial sources. Furthermore, panels of lectins under the trade name of Taxonolectins are commercially available for the identification and characterization of bacteria from E-Y Laboratories, San Mateo, California. The published reviews on the diagnostic applications of lectinsin microbiology aswell as the commercial availability ofa relatively wide source of lectins should provide ample opportunities to further assess the application of these unique chemical reagents as diagnostic probes.

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REFERENCES 1. Sumner JB, Howell SF. The identification of the hemagglutinations of the jack bean with concanavalin A. Bacterioll936; J 32:227-237. 2. Sharon N. Bacterial lectins, cell-cell recognition and infectious disease. FEBS Lett 1987; 217:145-157. 3. Sharon N, Lis H. Lectins as cell recognition molecules. Science 1989; 246: 227-234. 4. Ofek I, SharonN. Adhesins as lectins: specificity and role in infection. Curr Top Microbiol Immunol 1990; 151:91-113. 5. Mirelman D, ed. Microbial lectins and agglutinins: propertiesand biological activity. New York: John Wiley & Sons, 1986. 6. Damjanov I. Biology of disease. Lectin cytochemistry and histochemistry. Lab Invest 1987; 575-20. 7. Brown JC, Hunt RD. Lectins. Int Rev Cytol1972; 52:277-349. 8. Goldstein IJ, Hughes RC, Monsigny M, Osanna T, Sharon N. What should be calleda lectin? Nature 1980; 28966. 9. Goldstein IJ, Hayes CE.The lectins: carbohydrate-binding proteins of plants and animals. Carbohydr Chem Biochem1978; 35:601-615. 10. Liener IE, Sharon N, Goldstein IJ, eds. The lectins: properties, functions and applicationsin biology and medicine. Orlando: Academic Press,1986. 11. Lis H, Sharon N. Lectins: their chemistry and application to immunology. In: Sela M, ed. The antigens, v01 4. New York Academic Press, 1986:429529. 12. Lis H, Sharon N. Lectins as molecules and tools. Annu Rev Biochem 1986; 55:35-67. 13. Lis H, SharonN. Application of lectins. In: LienerIE, Sharon N, Goldstein IJ, eds. The lectins: properties, functions and applications in biology and medicine. Orlando: Academic Press, 1986293-370. Trans 1989; 17:ll-12. 14. Sharon N. Biomedical aspects of lectins. Biochem SOC 15. Franz H. 100 Jahre lectinforschung-eine bilanz. Naturwissenschaften 1990: 77:103-109. 16. Yeaton RW. Invertebrate lectins. 11. Diversity of specificity, biological synthesis and function in recognition.Dev Comp Immunoll981; 5:535-545. 17. Wu AM, Sugii S, Herp A. A guide for carbohydratespecificities of lectins. Adv Exp MedBioll988; 288:819-847. 18. Slifkin M, Doyle RJ. Lectins and their application to clinical microbiology. Clin MicrobiolRev 1990; 3:197-218. 19. Pistole TG. Interaction of bacteria and fungiwithlectinsandlectin-like substances. Annu Rev Microbiol 1981; 35235-1 12. 20. Hart DA. Lectins in biological system: applications to microbiology. Am J Clin Nutr 1980; 33:2416-2425. 21. Doyle RJ, Slifkin M. Applications of lectins in microbiology. ASM News 1989; 55~655-658. 22. Doyle RJ, Keller KF. Lectins in the clinical microbiology laboratory. Clin Microbiol News1 1986; 8:157-159.

Lectins

167

23. Doyle R, Keller K. Lectins in diagnostic microbiology.Eur J Clin Microbiol 1984; 3:4-9. 24. Doyle RJ, Birdsell DC. Interaction of concanavalin A with the cell wall of Bacillussubtilis. J Bacterioll972; 109:652-658. 25. Graham K, Keller K, Ezzell J, Doyle R. Enzyme-linked lectinosorbent assay (ELLA) for Bacillus anthracis.Eur J Clin Microbiol1984; 3:210-212. 26. Cole HB, Ezzell JW, Keller KF, Doyle RJ. Differentiation of Bacillus anthracis and other Bacillus species bythe use of lectins. J Clin Microbioll984; 19~48-53. 27. Doyle RJ, Keller KF, Em11 JW. Bacillus In: Lennette EH, Balows A, Hausler JR, Shadomy HD, eds. Manual of clinical microbiology, 4th ed. Washington, DC: American Societyfor Microbiology, 1985:211-215. 28. DeLucca AJ 2nd. Lectin grouping of Bacillus thuringiensis serows. Can J Microbiol 1984; 3O:llOO-1104. 29. Piller V, Piller F, Cartron JP. Comparison of the carbohydrate-bindingspecificities ofseven N-acetyl-D-galactosamine-recognizing lectins. Eur J Biochem 1990; 191:461-466. 30. Reeder NJ, Ekstedt RD. Study of the interaction of concanavalin A with staphylococcal teichoic acids. J Immunol 1971; 1%:334-340. 31. Hammerstrom S, Kabat EA. Studieson specificity and binding properties of the blood group A reactive hemagglutininfrom Helixpomatia. Biochemistry 1971; 10~1684-1692. 32. Owen P, Salton MRJ. Membrane asymmetry and expression of cell surface antigens ofMicrococcus lysodeikticusestablished by crossed immunoelectrophoresis. J Bacteriol 1977; 132:974-985. 33. Gilbride KJ, Pistole TG. Isolationand characterization ofa bacterial agglutinin in the serum of Limuluspolyphemus.Prog Clin Biol Res 1979; 29525-535. 34. Kloos WE, Jorgensen JH. Staphylococci. In: Balows A, Hausler JR, Shadomy NJ, eds. Manual of clinical microbiology, 4th ed. Washington, DC: American Society Microbiology,1985:143-153. 35. Wegrzynowicz PB, Hcezko J, Jeljaszewicz J, Neugebauer M, Pulverer G. Pseudocoagulase activity of staphylococci. J Clin Microbiol1979; 9:15-19. 36. Sperber WH, Tatime SR. Interpretation of the tube coagulase test for identification of Staphylococcus aureus. Appl Microbiol1975; 29502-505. 37. Davidson SK,Keller KF, Doyle RJ. Differentiation of coagulase-positive and coagulase-negative staphylococciby lectins and plant agglutinins. J Clin Microbiol 1982; 15547-553. 38. Ellwood DC, Hardie TM, Browning PM, Bowden GH. Carbohydrate composition of cell walls of Streptococcus mutansand Streptococcus sanguis. J Dent Res 1973; 52:955. 39. Kashket S, Guilmette KM. Aggregation of oral streptococci in the presence of concanavalin A. Arch Oral Bioll975; 20:375-379. 40. Hamada S, Gill K, Slade HD. Binding of lectins to Streptococcus mutans cells and type-specific polysaccharides,and effect on adherence. Infect Immun 1977; 18:708-716. 41. Staat RH, LangleySD,Doyle RJ. Streptococcus mutam adherence: pre-

168

42. 43.

44. 45. 46. 47. 48. 49. 50.

51. 52. 53. 54. 55.

56. 57.

Slifkin

sumptiveevidence for protein-mediated attachment followedbyglucandependent cellular accumulation.Infect Immun 1980; 27:675-681. Staat RH, Doyle RJ, LangleySD,Suddick RP. Modification of in vitro adherence ofStreptococcus rnutans by plant lectins. Adv Exp MedBioll978; 107~639-647. Kohler W, Prokop 0. Agglutinatin von streptokokken ver gruppe C durch ein agglutinin aus Helixpornatia. Z Immunitaetsforch Allerg Klin Immunol 1967; 133:30-53. Kohler W, Prokop 0. Agglutinationsversuche am streptokokken mit dem phytogslutinin aus Dolichos biflorus.Z ImmunitaetsforchAllerg Klin Immuno1 1967; 133:171-175. Kohler WD, Prokop 0, Kuhnemund 0. Routine identification of group C streptococci by means of an agglutinin (protectin) from the .albumen gland of the edible snail, Helixpornatia. J Med Microbioll973; 6:127-130. Prokop D,Kohler W. Agglutinations reactions en von mikroorganiumen mit Helix pornatia eiweiss drusnextract. Z Immunitaetsforsch Allerg Klin Immunoll967; 133:176-179. Ottensooser F, Nakamizo Y, Sat0 M, Miyamito Y, Takizawa K. Lectins detecting group C streptococci.Infect Immun 1974; 9:971-973. Kohler W, Nagai T. Reactions of the lectin anti-Ahp from the edible snail Helix pornatiawith N-acetyl-D-galactosamineof streptococci. Kitasato Arch Exp Med 1989; 62:107-113. Wagner M. Agglutination of bacteria by a sialic acid-specific lectin of the snail Cepaea hortensis.Acta Histochem 1982; 71:35-39. Slifkin M , Gil GM. Rapid biochemical tests for the identification of groups A, B, C, F, and G streptococci from throat cultures. J Clin Microbiol 1983; 18:29-32. Schanufuss P, Lammler C, Lobe1 B. A rapid differentiation of streptococci isolated from cows with mastitis. J ClinMicrobioll986; 24:1098-1099. Slifkin M, Gil GM. Identification of group C streptococci antigen extracts with lectin-bound polystyrene particles. J Clin Microbiol1984; 1993-84. Holm SE, BersholmA, Wagner M. A sialic-acid-specific lectinfrom Cepaea hortensis that promotes phagocytosis of a group-B, type Ia, streptococcal strain. J Med Microbiol 1985; 19:317-323. WagnerM. Interactions ofwheat-germagglutininswithstreptococci and streptococcal cell wall polymers. Immunobiology 1979; 15657-64. Dietz E, Bohmer G, Solter J, Uhlenbruck G. Adhesion of bacteria and tumors: inhibition by lectin blocking. In: B@-Hansen TC, Freed DLJ, eds. Lectins- biology, biochemistry, clinical biochemistry,v01 6. St Louis: Sigma Chemical CO, 1988:27-33. Slifkin M, Cumbie R. Identification of group B streptococcal antigen with lectin-bound polystrene particles. J Clin Microbiol 1987; 25:1172-1175. Lim DV, Morales WJ, Walsh AF, Kazanis D. Reduction of morbidity and mortality rates for neonatal group B streptococcal disease through early diagnosis and chemoprophlaxis. J Clin Microbiol1986; 23:489-492.

Lectins

Clinical Bacteriology

169

58. Pritchard DC, Coligan JE, Speed SE, Gray BM. Carbohydrate fingerprints of streptococcal cells.J Clin Microbiol 1981; 13:89-92. 59. Pritchard DG, Gray BM, Dillon HC. Characterization of the group-specific polysaccharide of group B streptococcus. Arch Biochem Biophys 1984; 235: 385-372. 60. Nachbar MS, Oppenheim DJ, Thomas JD. Lectins in the US diet. Isolation and characterization of a lectinfrom the tomato (Lycopersicon esculetum).J Biol Chem 1980; 225:2056-2066. 61. Doyle RJ, Nedjat-Haiem F, Miller RD, Keller KF. Interaction between plant agglutinins andLegionella species. J Clin Microbioll982; 15:973-975. 62. Slifkin M, Pouchet-Melvin GR. Evaluation of three commercially available test products for serogrouping beta-hemolytic streptococci. J Clin Microbiol 1980; 11 249-255. 63. Kaku H, Peumans WJ, Goldstein IJ. Isolation and characterization of a second lectin (SNA-11) present in elderberry (Sambucus nigraL ) bark. Arch Biochem Biophys 1990; 277:255-262. B, Peumans WJ. A lectin from 64. Broekaert WF, Nsimba-Lubaki M, Peters elder (Sambucus nigraL) bark. Biochem J 1984; 221:163-169. IJ, Broekaert W, Nsimba-LubakiM,Peeters B, 65. ShibuyaN,Goldstein Peumans WJ. Theelderberry (Sambucus nigraL) bark lectin recognizes NeuSAc(a2-6)GaVGalNAc sequence. J Biol Chem 1987; 262:1596-1601. 66. ShibuyaN,Goldstein IJ, Broekaert W, Nsimba-LubakiM,Peeters B, Peumans WJ. Fractionation of sialylated oligosaccharides, glycopeptides, and glycoproteinson immobilized elderberry(Sambucus nigraL) bark lectin. Arch Biochem Biophys 1987; 254:l-8. 67. Mandal C, Mandal C. Sialic acid binding lectins. Experientia 1990; 46:433441. 68. Weiss W, Brown JH. Cusack S, Paulson JC,Skehel JJ, Wiley DC. Structure of the influenza virus hemagglutinin complexed with its receptor sialic acid. Nature 1988; 333:426-431. 69. Cammue BPA, Peeters B, Peumans WJ. A new lectin from tulip (Tulipa) bulbs. Planta 1986; 169583-588, 70. Oda Y,Ichida S, Aonuma S, Shibahara T. Studieson chemical modification of Tulipa gesnerianalectin. ChemPharm Bull 1989; 37:2170-2173. 71. Oda, Y, Minami K. Isolation and characterization of a lectin from tulip bulbs, Tulips gesneriana. Eur J Biochem 1986; 159:239-245. W.Purification and macromolecular proper72. Miller RL, CollawanJF, Fish W ties of a sialic acid-specific lectin from the slug Limux~avus.Biol Chem 1982; 257~7574-7580. 73. Hsu SM, Ree HJ. Histochemical studies on lectin binding in reactive lymphoid tissues. J Histochem Cytochem1983; 31538-546. 74. Karayannopoulou G , Weiss J, Damjanov I. Detection of fungiin tissue sections by lectin histochemistry. ArchPathol Lab Med 1988; 112:746-748. 75 Wagner M. Interactionof wheat-germ agglutinin with streptococci and streptococcal cell wall polymers. Immunobiology1979; 15657-64. a

170

Slifkin

76. Allen A K , Neuberger A, Sharon N. ,me purification, composition and specificity of wheat-germ agglutinin. Biochem J 1973; 131:155-162. 77. Sakakibara F, Takayanagi G, Kawauchi H. An L-rhamnose-binding lectinin the eggs of Misgurnus anguillicaudatus. Yakugaku-Zasshi 1981; 101:918-925 (In Japanese). 78. Goldstein IJ, Staub A M.Interaction of concanavalin A with polysaccharides of salmonellae. Immunochemistry1970; 7:315-319. 79. LeMinor L, Tournier P, Chalon AM. Agglutinabilite pour la concanavaline a de divers bacilles a gram negatif. Etude chez lesSalmonella de la correlation avec la structure antigenique 0. Ann Microbiol(Paris) 1973; 124A:467-476. 80. Maruyuma HB, Arisawa M, Ono-Onitsuka M. Simplified assay for concanavalin A-dependent bacterial agglutination by using cell surface mutants. Infect Immun 1975; 11:1320-1324. 81. Doyle RJ, Woodside EE, Fishel CW. Protein-polyelectrolyte interactions. The concanavalin A precipitin reaction with polyelectrolytes and polysaccharide derivatives.Biochem J 1968; 106:35-40. 82. Avni I, Arffa RC, RobinJB, Rao NA. Lectins for the identification of ocular bacterial pathogens. MetabPediatr Syst Ophthalmol 1987; 10:45-47. 83. Uppal JB. Use of Litchi chinesis lectins (agglutinins)in diagnostic microbiology. J Pak Med Assoc 1985; 35:lOO-111. 84. Hammarstrom S, Lindberg AA, Robertsson ES. Precipitation of lipolysaccharides from rough mutants of Salmonellatyphimurium by an Ahemagglutinin from Helixpomatia. Eur J Biochem 1972; 25:274-282. 85. Pistole TG. Bacterial agglutinins from Limulus polyphemus- an overview. Prog Clin Biol Res 1979; 29547-553. 86. Dorai DT, SrimalS, Mahan S, Bachhanat BL,Bolgenesh TS. Recognitionof 2-keto-3-deoxyoctonate in bacterial cells and lipopolysaccharidesby the sialic acid binding lectinfrom the horseshoe crab Carcinoscorpius rotunda caudia. Biochem Biophys Res Commun 1982; 104:141-147. 87. Sequeira L, Graham TL. Agglutination of avirulent strains of Pseudomonas solanacearum by potato lectin. Physiol Plant Patholl977; 11:43-57. 88. Chatterjee BP, Guha AK, Pal P, Bhattacharya M. Lectin typingof Pseudomonas aeruginosa strains of different serotypes, Habs and Fisher types. Zentralblat Bakteriol 1989; 271:364-371. 89. Eaves DJ, Doyle RJ. Surface characteristics of Pseudomonas cepacia. Microbios 1988; 53:119-128. 90. Meade NA, Staat RH, Langley SD, DoyleRJ. Lectin-like activity from Persea americana.Carbohydr Res 1980; 78:349-363. 91. Senne JE. Lectin agglutination in Neisseriagonorrhoeae. ClinMicrobiol News1 1981; 3:lO. 92. Schaefer RL, Keller KF, Doyle RJ. Lectins in diagnostic microbiology: use of wheat germ agglutinin for thelaboratory identification of Neisseria gonorrhoeae. J Clin Microbioll979; 10569-672. 93. Frasch EC. Role of lipopolysaccharide in wheat germ agglutinin-mediated agglutination of Neisseria meningitis and Neisseria gonorrhoeae.J Clin Microbiol 1980; 12:498-501.


171

Neisseriugonor94. Curtis GDW, Slack MPE. Wheat-germ agglutination of rhoeue. A laboratory investigation. Br J Vener Dis 1981; 57:252-255. 95. Allen PZ,Connelly MC, Apicella MA. Interactions of lectins withNeisseriu gonorrhoeue. Can J Microbiol 1980; 26:468-474. inter96. Doyle RJ, Nedjat-Haiem F, Keller KF, Frasch CF. Diagnostic value of actionsbetweenmembersof the family Neisseriuceue and lectins.JClin Microbioll984; 19:383-387. 97. deHormaeche R D , Burdell C, Chong'H, Taylor DW, WildyP. Definition of a virulence-related antigen of Neisseriu gonorrhoeue with monoclonal antibodies and lectins. J Infect Dis1986; 153535-546. on lipopoly98. Connelly MD, AllenPZ. Chemical and immunochemical studies saccharides from pyocin 103-sensitive and resistant Neisseriu gonorrhoeue. Carbohydr Res 1983; 120:171-186. WK.Rapid confirmatory identification Neisseof 99. Yajko DM, Chu A, Hadley ria gonorrhoeue with lectins and chromogenic substrates. J Clin Microbiol 1984; 19~380-382. 100. Vazquez JA, Berron S. Lectinagglutinationtest

as on epidemiological marker for Neisseriu gonorrhoeue.Genitourin Med 1990; 66:302. 101. Fogg GC, Yang L, Wang E, Blaser MJ. Surface array proteins of Cumpylobucter fetus block lectin-mediated binding to type A lipopolysaccharide. Infect Immun 1990; 58:2738-2744. 102. Alaba A, Coker AD, Okotore RD. Interaction of lectins with the surface coats of Cumpylobucferspecies in Nigeria. In: BQS Hanson TC, Freed DLJ, eds. Lectins: biology, biochemistry, clinical biochemistry, v01 6. St Louis: Sigma ChemicalCO, 1988:565-570. 103. Wong KH, SkeltonSK, Feeley JC. Interaction of Cumpylobucferjejuni and Cumpylobucter coliwith lectins and blood group antibodies. J Clin Microbiol 1985; 22~134-135. 104. Corbel JJ, Gill KPW. Lectin agglutination of thermophilic Cumpylobucfer species. Vet Microbiol 1987; 15:163-173. Matoba AY, RobinJB. The useoffluorescein105. JacksonM,ChanR,

conjugated lectins for visualizing atypical mycobacteria. Arch Ophthalmol 1989; 197:1206-1209. 106. Robin JB, Schmiodt L, Haimov T, NielsenSA,

Salazar J. Fluoresceinconjugated lectin visualization of infectious microrganisms. In: Caranaga H, ed. Proceedings of the third world congress on cornea. New York: Raven Press, 198:485-489. 107. Goldstein IJ, Misaki A. Interaction of concanavalinA with an arabinogalactan from the cell wall of Mycobacterium bovis. J Bacterid 1970; 103:422425. 108. Korting HC, Abeck D, Johnson AP, Ballard RC, Taylor-Robinson, Braun-

Falco 0. Lectin typing ofHuemophilus ducreyi. Eur J Clin Microbial Infect Dis 1988; 7:678-680. 109. Schalla WD, Rice RJ, Biddle J W , Jean Louis Y, Larsen SA, Whittington WL. Lectin characterization of gonococcifrom an outbreak caused by penicillin-resistant Neisseriu gonorrhoeue.J Clin Microbiol 1979; 10:669-672.

Slifkin

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110. Schalla W D , Whittington WL, Rice RJ, Larsen SA. Epidemiologicalcharacterization of Neisseria gonorrhoeae by lectins. J Clin Microbiol 1985; 22: 481-483. 111. Bangs LB. New developments in particle-based tests and immunoassays. J Int Fed Clin Chem1990; 2:1-6. 112. Slifkin M,Cumbie R. Rapid detection of herpes simplex virus with fluorescein-labeled Helixpomatia lectin. J Clin Microbioll989; 27:1036-1039. 113. Taatjes DJ, Roth J, Peumans W, Goldstein IJ. Elderberry bark-lectin-gold techniques for the detection of NeuSAc (a-2-6) GalIGalNAc sequences: applications and limitations. HistochemJ 1988; 20:478-490. 114. Gilboa-Garber N, Nir-Mmahi I, Mizrahi L. Specific agglutination of Esche-

richia coliO,, B12 by the mannose-binding proteins of Pseudomonas aeruginosa. Microbios 1977; 189-109. 115. Garber N, Glick J, Gilboa-Garber N, HellerA. Interactions ofPseudomonas aeruginosa lectins withEscherichia coli strains bearing bloodgroup determinants. J Gen Microbiol 1981; 123:359-363. 116. Friis-Christiansen P, Thiel S, Svehag SE,Dessau R, Svendsen P, Andersen 0, Laursen SB, Jensenius JC. In vitro and vitro antibacterial activity of conglutinin, a mammalian plasma lectin. Scand J Immunol 1990; 31:453460.

5 lectin Specificities Relevant to the Medically Important Yeast Candida albicans HANS C. KORTINC University of Munich, Munich, Germany MARKUS W. OLLERT University of Hamburg, Hamburg, Germany

1. INTRODUCTION

The dimorphic yeast Candida albicansis an opportunisticpathogen of great medical importance in human diseases with underlying conditions that impair immunity [l]. Thus, infections with C. albicans are frequently seen in patients suffering from diseases such as acquired immunodeficiency syndrome (AIDS), diabetes, neutropenia, bums, and cancer [l-31. Candida albicans normally colonizes mucosal surfaces of the gastrointestinal tract and the oral cavity, and can leadto invasive and metastatic candidiasisthat originates from these locations under the named conditions [l]. Several properties allow C.albicans to colonize and invade host tissues. The most important of these properties seemsto be the capability to adhere to host cells, the production and secretion ofan acid protease,and the abilities for phenotypic switching and antigenic mimicry by C. albicans [2, 41. This yeast is characterized by its dimorphic growth, which is represented either by the blastospore (yeast) or the pseudohyphal (mycelium) growth phase (Fig. la) [l]. By consensus, the initiation of the pseudohyphal growth phase of C.albicans is considered to be a crucial step inthe establishment of invasive candidiasis[l]. The transition from yeast to pseudohyphal growth, a dynamicprocessreferred to as germination, isaccompaniedbydrastic changes in the cell surface architecture and composition, resulting in the expression of pseudohypha-specificneoantigenicepitopes [5-91. These changes also involve the differential surface expression of complex carbohydrates as integral parts of glycoproteins, proteoglycans, and glycolipids [l]. Because of a suggested relatedness of surface glycoproteins to mecha173

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Specificities Lectin

of Candida albicans

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nisms of candidal pathogenicity, there has been considerable interest in identifying the responsible molecules, defining their functional role, and dissecting them biochemically [2]. Most of the glycosylated structures on the candidal surface are embedded into the cell wall of the fungus, a polymeric structure mainly composed of mannoproteinsand chitin, which surrounds the plasma membrane (see Fig. lb) [l]. The glycoproteins of the candidal surface are recognized by soluble lectins and membrane-bound lectin-type receptors of host cells [lo]. On the other hand, complex carbohydrate structures on host cells are potential ligandsfor putative C . albicans cell surface lectins in host-parasite interactions, thus contributing to a proposed multireceptor concept of candidal attachment to host cells [10,l l]. Lectins are carbohydrate-binding proteins of defined specificity and nonimmuneorigin that agglutinate cells and precipitatecarbohydratebearing structures (see Chapter 1). Most of the lectinsnowknown are soluble factors and have been isolated from various sources [12]. If one extends the classic definition of lectins, a carbohydrate-specific monoclonal antibody that represents a homogeneous molecular population of defined specificity could also be applied to many of the techniques used in lectin studies, thus being a “lectin-like molecule.” Because soluble lectins usually do not enter cells, they can be usedto obtain information about the type, the structure, the abundance, and possibly the function of glycoconjugates on the cellular surface of medically important fungi, such as C. albicans. More recently, an accumulating number of membrane-bound lectin-type proteins (receptors) have been described that may playan important role in cell-cell and cell-parasite interactions, such as tissue evasionof inflammatory cells, homing of lymphocytes,and attachment of bacteria to mammalian cells [13-151. Similar mechanisms might also apply to the adhesion of C . albicans and, therefore, need careful evaluation.

Figure 1 (a) Dimorphism of Candida ulbicuns: elongated hyphal structures originate from round-shaped blastospores. Brightfield (A, C) and immunofluorescence (B, D)optics after staining of C. ulbicuns myceliawith the germtube-specific monoclonal antibody 1.183 and subsequent detection with fluorescein isothiocyanate-conjugatedgoatantimouseimmunoglobulin.Smallarrowsindicateparent blastoconidia and pseudohyphal segments originatingfrom the parent cell, neither of which were stained with the monoclonal antibody. (b) Transmission electron microscopy ofC. ulbicunspseudohyphae reacted with an immunogold-labeled monoclonal antibody againstthe C. ulbicuns C3d receptor. Intense stainingwas obtained in association with the outer fibrillar layer of C. ulbicuns, a heavily glycosylated heteropolymeric structure of the candidalcellwall that is mainly composed of mannoproteins. (From (a) Ref. 9; (b) Ref. 26.)

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The focusof this chapter is threefold. In two areas of interest,the use of soluble lectins as tools and probes for candidal research is discussed, whereas a third focus is directedat cellular lectins as important molecules in the pathogenicity ofCandida:

1. The application of lectin techniques, which, in part, are based on reversible interactions of lectins with their respective ligands, in the biochemical analysis ofC. albicans' surface structuresis reviewed. 2. The possibility of a lectin-typing scheme ofC.albicans is proposed. 3. A major focus is to highlight the importance of lectin-carbohydrate interactions, as mediatedby cell-bound molecules, in the pathogenicity of C. albicans. II. LECTIN TECHNIQUES IN THE BIOCHEMICAL ANALYSIS O F CANDIDA ALBICANS A.Lectin-Binding

Studies

Lectin-binding studies with whole fungi or isolated fractions thereof can provide first-line answersto the following questions: (1) Are carbohydrate moieties present on the cell surface? (2) What is the quantity ofthese carbohydrates? (3) Are they complexor hybrid? (4) What is the functionof these glycosylated molecules? Giventhe answers, the investigations can go one step farther with the analysis of the glycan structure by enzymatic digestion, using specific glycosidases that allow sequencingand determination of the nature of the glycosidic bonds (16). Other novel techniques, such as high-performance chromatography, anion-exchange, pulsed amperometric detection (HPAE-PAD)and nuclear magnetic resonance (NMR), to name a few, can be applied to further resolve the glycan structure [16]. A summary of the lectinsusedincandidalresearch, the techniquesin which they were applied, and of the experimental background is shown in Table 1. The binding of lectinsto whole cells ofC. albicans has been employed in ultrastructural studies of its fate inside human neutrophil phagolysosomes [17]. Neutrophils are considered a very important line of defense C. albicans [l].Therefore, determinationof the exact against infection with molecular mechanisms involved in neutrophil-mediated phagocytosis C. of albicans isof greatsignificance. By usinggold-labeledconcanavalin A (ConA)and wheat-germ lectin (WGA), it was possible to show a progresas evidenced by a diffuse staining sive loss of mannan-rich cell wall layers, pattern with ConA during the process of phagocytosis, whereas chitin was apparently unalteredand could be readily demonstrated by WGA [specific for N-acetylglucosamine (GlcNAc) within the 0-1,4-linked chitin back-

Table 1 Lectins Used in Different Techniquesto Study Glycoproteins of Candida

albicans Lectins used"

Ref.

ConA, WGA

COnA COnA COnA COnA COnA

COnA COnA

COnA

purposeb Experimental Gold-labeled; morphologicalstudy of Candida phagocytosis by PMNs: detection of mannan (ConA) and chitin (WGN FITC-labeled; distinction between extra- and intracellular C. albicans in phagocytosis studies Lectin blot: detection of high molecular mass germ tube-specific glycoproteins of C. albicans Lectin blot: detection of 43-kDa germ tube-specific Candida glycoprotein Lectin blot: detection of adhesionrelated proteins ofC. albicans Latex particle-, gold-, and peroxidasebased detection ofC. albicans glycoproteins relevant to plastic adherence Lectin blot: association of phosphorylation and glycosylation of proteins in the C. albicans cell wall Microtiter plate-immobilized: recognition of Candida D-mannan in a sandwich ELISA together with an antiserum Lectin affinity chromatography: separation of hormonal ligands ofC. albi-

17

18 6 19 20 21 22 23

27

cans ConA, LCA ConA, DBA, PHA, PNA, RCA-I,SOH,UEA-I, WGA ConA;WGA, LOTUS COnA ConA, DBA, LPA, LCA, PEA, UEA-I, WGA C o d , SBA, GS-I1

Lectin affinity chromatography: isolation of C. albicans C3d receptors; functional inhibition of C3d rosetting Inhibitors: inhibition ofC. albicans adhesion to human buccal cellsin vitro

25,26

Inhibitors: inhibition ofC. albicans adhesion to buccal epithelium Inhibitor: inhibition of C. albicans adhesion to fibrin-platelet matrices, corneocytes, and endothelium Inhibitors: inhibition ofC. albicans adhesion to human intestinal cells Lectin typing ofC. albicans

30

29

34-36 37 51

'See Appendix in Chapter 1 for lectin abbreviations. bPMN,polymorphonuclear leukocyte.

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bone1 in the cell wall remnants under these conditions [l71(See Appendix differentiate in Chapter 1). Similarly, fluorescein-labeled ConA was fu used between extracellularand intracellular C.albicans blastospores in phagocytosis assays[181. More frequently, lectins have been used to detect C.albicans glycoproteins following separation by gel electrophoresis [12]. Since most of the assumed C.albicans virulence factors, such as adhesionto host cells, protease secretion, phenotypic switching,and molecular mimicry, are somehow related to glycoprotein structures, there have been enormous efforts to obtain detailed studies of these molecules. The expression of germ tube-specific neoantigens on pseudohyphal cells of C.albicans is apparently important in establishing disease[l]. Such germ tube-specific high-relative molecular mass (M,) components of C. albicans were identified by the use of a polyclonal antiserum [6]. However, only quantitative differences in ConA binding in a lectin blot were detectable,therebysuggestingcommonoligosaccharidestructuresonblastospores and germ tubes, with antigenic variation inthe peptide backbone [6]. However, a more detailed lectin study and a structural analysis of the glycan structure would be required to substantiate these findings. Another germ tube-specific candidal glycoprotein of 43 kDa was expressed on the cell surface and was recognized by ConA binding [19]. Endoglycosidase H digestionof this- proteinproduceda39-kDaprotein not reactivewith ConA. The same 39-kDA protein appeared by treatment of the cells with tunicamycin, an inhibitor in the assembly of dolichol diphosphate-bound oligosaccharides leadingto the formation of peptide chains free of Winked carbohydrate units [121. The exact nature of the latter proteinand its function need further investigation. Strains of C.albicans, selected on their reduced agglutination with a polyclonal anticandidal antiserum, also exhibited reduced binding to buccal epithelialcells [20].Of the adhesion-related proteins identified on a matched pair of wild-type and mutant strains, all were glycosylated as shown by ConA stainingof candidal extracts[20]. It will be of great interestto isolate these adhesion-related proteins and to resolve their glycan structure in an attempt to define an exact role ofthe carbohydrate residues in the biology of Candida. Such studies will require further lectin-binding studies and ultimately the resolution of not only the glycan, but also of the peptide sequence. In an attempt to isolate the C.albicans' structures responsible for the attachment to plastic surfaces of prosthetic devices and catheters, which are major sources for the hematogeneous dissemination of the organism, Tronchin et al. [21] incubated C. albicans on plastic dishes, which led to the depositioa of adhesion-related extracellular glycoproteins on the plastic

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surface. The presence of these glycoproteins was established by light microscopy usingConAcoated latex beads as detection markersfor immobilized candidal mannoproteins [21]. To further resolve the molecular structures responsible for the phenomenon of plastic adherence ofC. albicans, Cod-sensitized gold particles were employed in transmission electron microscopy, which ledto the suggestion ofan important role for the external fibrillar layer of C . albicans germ tubes in this process [21]. Finally, the investigators succeeded in identifying a 68-kDa protein that mediates C. albicans adhesion to plastic, a protein that was intensely stainedby ConAperoxidase, thereby suggesting its mannoproteinaceous nature [21]. The presence of phosphorus asa minor componentof the cell wall of C.albicans prompted the assumption that it was present in phosphodiester on linkages among mannose residues [22]. The use of ConA in studies the distribution of phosphate, carbohydrate, and protein in the cell wall components, extracted from intact yeast or hyphal cells of C. albicans, showed that not all glycoproteins contained detectable phosphate [22]. Furthermore, a major phosphorylated protein did not contain any carbohydrate, as revealed by ConA blotting. Thesedata allowed the conclusion of differential modificationof proteins on the surface of C.albicans, one such modification beingthe phosphorylation of glycoproteins. A completely different methodological approach of lectin binding as an application to detect candidal structures is the use of ConA together with polyclonal antiserum in a sandwich enzyme-linked immunosorbent assay (ELISA) to detect D-mannopyranose units of C.albicans [23]. The rationale for using immobilized lectin in suchan assay system wasa previous observation that the recognition of D-mannan by immobilized C o d was equivalent to the quantitative precipitin reaction of the Same D-mannans in the fluid phase [23]. B. Lectin Affinity Chromatography

Lectin affinity chromatography is a widely used application for lectins in candidal research. From the reversible interaction of immobilized lectins mediated by at least two carbohydrate-binding sites with their oligosaccharide ligands, several potentiallyimportant glycoproteins have been isolated by this technique and further characterized [a]. Calderone et al. [25] used ConA-Sepharose to separate mannoproteins from nonmannosylated proteins in their successful attempt to identify the C3d receptor onC . albicans. The receptor for C3d, an activation product of the human complement system, on C.albicans represents one of the recently emerging molecular mimicry proteins onthe candidal surfacethat have been implicated as virulence factors of the organism [2]. The results obtained suggest. that the

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candidal C3d receptor is a mannoprotein, although the incubation of candidal pseudohyphae with soluble ConA over a wide concentration range did not inhibit receptor activity ainfunctional assay[25].These data imply that oligosaccharides recognized by ConAon the candidal surface are apparently not involved in binding the C3d component. Further studies with the isolated C3d receptor substantiated these findings by using Sepharose beads with various immobilized lectins (ConA, LCA, WGA, GS-l) [26]. The data indicated that the C3d receptor of C. albicans may contain mannose and glucose as oligosaccharide components, as only ConA- and LCAthe isolated receptor[26]. Sepharose ledto a significant agglutination with Lectin affinity chromatography has also been used to separate ligands that bind to C. albicans. A further example of a class of proteins that mimic the properties of mammalian proteins is the presence of binding sites for human luteinizing hormone (hLH) and the human chorionicgonadotropin (hCG) in microsomal and cytosol fractions prepared fromC . albicans [27].To rule out that trace amounts of hLH would exhibit differential binding to candidal proteins when compared with mammalian receptors, the hLH preparation was fractionated by ConA-Sepharose, which resolved three distinct peaks, presumably reflecting differences in their carbohydrate compositions. All three peaks, however, bound equally well to both candidal membranes and mammalian LH receptors [27].These data favor a concept of possible hormonal regulation of the yeastC. albicans by molecular mimicry, as was also suggested for the presence of steroid receptors in C. albicans [28]. 111. LECTIN-CARBOHYDRATEINTERACTIONS IN CANDIDA ALBICANS PATHOGENICITY

A. Lectinlike Molecules onthe Candida1 Surface

The interest in adhesion mechanisms of microorganisms to various surfaces has increased tremendously in recent years[10,15].This is also true for C . albicans, and evidence that adhesion to host cells may be the first step in candidiasis and may thereby mediate invasiveness of the organism is accumulating [for review, see101.Earlier studieson adhesion ofC.albicans to epithelial cells suggested a role of glycosides, containing L-fucose, GlcNAc, and D-mannose, as integral parts of epithelial receptors for different strains of C.albicans [lo].The roleof these sugar moieties in candidaladhesion was established in inhibition experiments using the respective sugars as LOTUS, ConA, and WGA (see Apand specific inhibitory lectins such pendix in Chapter 1 for definitions of lectin abbreviations) [29,30].More recently, two detailed studies on the role of complex carbohydrates and

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glycosphingolipids as host cell receptors extendedthe previous knowledge and provided additional thorough evidence for a lectin-based candidal adhesion as one link of a multireceptor adhesion mechanism [31,32]. In the first of these studies, human milk oligosaccharide probes were used to inhibit adhesion of C. albicans to human buccal epithelial cells 1311. The minimal structural requirement for activity was the Fuca-l,2Gal@determinant on tri- to heptasaccharides, a structural feature that corresponds to the H disaccharide sequence found on all blood group substances of the AB0 (H) system [3l]. Even very similar sequences with two vicinally linked fucosyl residueson a lactosamine backbone such the as blood group antigen Lewisb were inactive, whereasthe same difucosylationpattern on a lactose backboneexhibitedinhibitoryactivity[31].Theserecognition patterns clearly demonstratethe high discriminatory power and the fine-tuning involved in Candida-host adhesion, based on a lectin-type adhesion on C. albicans. The identification of the lectinlike structure on C. albicans responsible for the described specificity is still obscure. Clinical data, however, strongly support these findings because the saliva of AB0 secretors has an inhibitory effecton C.albicans adhesion to epithelial cells [33]. In a secondstudy, the glycosphingolipidlactosylceramide (Gal@-l, 4GlcO-1,lCer) acted as a possible adhesion receptor for the medically important yeasts C.albicans and Cryptococcusneoformans on human glioma brain cells [32]. The fungal binding was specific for lactosylceramide, for the removal of the terminal galactosyl residue resulted ina loss of binding activity[32].Similarly, a whole array of other glycosphingolipidswere inactive, indicatinga specificity for lactosylceramide recognitionby a putative C.albicans adhesion. The ubiquityof lactosylceramide in mammalian tissues could account for the multiorgan involvement in patients with disseminated fungal disease [32]. B. Mammalian Lectinsas Receptors for Candida albicans Glycoconjugates

It is evident from the previous paragraph that adhesion of C. albicans to

host cells is apparently mediatedby a multireceptor system. (Table2 gives a summary of carbohydrate moieties involved in candidal adhesion.) There is consensusthat one of the effector moleculesfor adhesion of C.albicans is a cell surface mannoprotein that interacts witha lectinlike receptoron the mammalian cells[lo]. Most of the investigators studyingthe role of mannoprotein as an adhesin of C.albicans used ConA as a specific inhibitor[34371. In this way, it was shown that ConA is able to inhibit adhesion of C. albicans to fibrin-platelet matrices [34] and to epithelial cells [35,37].0thers have suggesteda role for chitin as an adhesin of C.albicans to vaginal epithelial cells [38].

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Table 2 Carbohydrate Moieties Relevant to the Adhesive Propertiesof Candida albicans

Carbohydrate moieties

in C. albicans adhesion

Ref.

L-Fuc, GlcNAc, D - M ~ disaccharide) Fuw-l,2Gal/3 G@-l ,4Glc/3-1, lCer (lactosylceramide)

10 31 32

Manp/3-1,2Manpa-l,2Manpa-1,2Manpa-l,2Man 39,42 Manp/3-1,2Manp/3-1,2Manpa-l,2Manpor-l,2Manpa-l,2Man

(mannopentaose, mannohexaose) Cer, ceramide; Fuc, fucose;Gal, galactose, Glc, glucose; GlcNAc, N-acetylglucos-

amine; Man,mannose.

A recent study demonstratedthat mannan-mediated adhesionto buccal epithelial cells is achieved mainly the by C. albicans serotype A-specific determinants (antigen 6) [39]. Antigen 6 represents one antigenic formula used for the serological classification of medically important yeasts, such as C. albicans [39]. The presence of antigen 6 defines the serotype A C of. albicans, as opposedto the serotype B, which lacks this antigen [40]. Originally defined by polyclonal antibodies, antigen 6 can now be recognized by a lectinlike monoclonal antibody (MAb-6) [41]. Recently, the serotype A-specific determinantson C. albicans were identified as two oligomannosyl residues containing both &1,2 and a-1,2 linkages (Manp&1,2Manpa1,2Manpcu-l,2Manpar-l,2Maand n Mar@-l ,2Manpp-l,2Manpcu-I,2Manpcr1,2Manpa-l,2Man) [42]. Antigen6-deficient C. albicans serotypeA mutants, as well as strainsof C. albicans serotype B, exhibited significantly reduced adhesive properties compared with antigen 6-bearing species [39]. Although the effector molecules mediating serotype A specificity and adheas mannopentaose and sion to epithelial cells have now been identified mannohexaose, their respective lectinlike receptors on host cells can be only speculated on.In addition to antigen 6,another 6-1,2-linked oligomannosyl antigen [antigen 5; Manpfi1-(2manpfi-l),2Man;n = 0 51 seems to play a minor role inC.albicans adhesion to host cells [39,43].

-

IV. LECTINS AS MOLECULAR PROBES FOR TYPING CANDIDA ALBICANS

Because of the importance of C. albicans as an opportunistic infectious pathogen, it has long been desirableto establish highly specific procedures to type C. albicans species for epidemiological and for diagnostic purposes. Most of the currently available-typing procedures for Candidaalbicans

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have proved unsatisfactory in some respect [l].Serotyping basically distinC. afbicansby means of polyclonal antibodies guishes between two types of directed against candidal cell surface polysaccharides [1,40].Other typing systems do not exhibithigherdiscriminatorypower or, if they do, are simply not feasiblefor routine purposes[44,45].With the advent of hybridand serotypoma technology [46],potentially more accurate serodiagnostic ing reagents become available inthe form of carbohydrate-specific, lectinlike monoclonal antibodies [47].There has been considerable interest in obtaining such reagents with the aim of improving currently available serotyping systems[47]. Most recently, lectin typing has become very usefulin differentiating biotypesof other microorganisms,suchas Neisseriagonorrhoeae and Haemophilus ducreyi [48-501.As lectin typing in this context was both simple and efficacious, the method was also appliedto C.albicans isolates using 14 lectins [51].Sixteen different lectin types could be distinguished, the most frequent type representing 22% of the strains.A simplified-typing scheme based on three lectins (ConA, SBA, GS-11) seems to be almost as efficacious for epidemiological purposes[51].The reactionpatterns (lectin 3. types) found in 50 C. afbicansisolates are shown in Table V. CONCLUSIONS

Candida albicansis a majoropportunisticpathogen in humans.Because of its importance, the organism has received steadily growing interest among medical mycologists in recent years. The new research emphasis in this field has led to progress in understanding the various virulence factors of C . afbicans.One conclusive element ofthe numerous studies isthat glycoprotein structuresare very important in the virulence of C. afbicansand, therefore, need to beresolved further. Through the useoflectins,abetter understanding of C. afbicanscell surface glycoproteins has become available. However, the biological import of carbohydrate moieties on C.afbicans proteins is not clearly understood. It is most likely that some glycan structures on C. afbicans are involved in adhesion to mammalian tissue (e.g., serotype A determinant). On the other hand, there are many more glycoproteins on C. afbicans for which the carbohydrate residues are not functionally defined. The characterization of the biological role of these residues merits future research emphasis. Recently, the biological role of carbohydrate moieties on various mammalian proteins has been investigated. One study focused on the functional role of carbohydrates on the extracellular matrix protein laminin [52].The reported findings indicate that cell attachmentto glycosylated laminin wasthe same asto unglycosylated laminin. However, cell spreading and neurite outgrowth didnot occur

184

+

+

+

+ +

+ + + + + +

+

d

+ + VI

l-

00

+ +

+ + + + + + + + + + cu

+

+

+ + +

+

+ + + +

+ +

+

+ + + +

+ +

185

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on unglycosylated laminin, thereby showingthe potentially functional importance of carbohydrate residues on glycoproteins. Similar differential properties might also apply to carbohydrate residues on glycoproteins involved inthe virulence of C. albicans. REFERENCES 1. Odds FC. Candida and candidosis. 2nd ed. London: Bailliere Tindall, 1988. 2. Calderone RA. Host-parasiterelationshipsincandidosis. Mycoses1989; 32(s~ppl2):12-17. 3. Korting HC, Ollert M, Georgii A, Froeschl M. In vitro susceptibilities and biotypes of Candida albicans isolates from the oral cavities ofpatients infected with human immunodeficiency virus. J Clin Microbioll988; 26:2626-2631. 4. Ollert MW, Wadsworth E, Calderone RA. Reduced expression of the functionally active complement receptorfor iC3b but not for C3d on anavirulent mutant of Candida albicans. Infect Immun 1990; 58909-913. 5. Smail EH, Jones JM. Demonstration and solubilization of antigens expressed primarily on the surfaces ofCandida albicans germ tubes. Infect Immun1984; 45:74-81. 6. Sundstrom PM, Kenny GE. Enzymatic release of germ tube-specific antigens from cell walls of Candida albicans.Infect Immun 1985; 49:609-614. 7. Casanova M, Gil ML, Cardenoso L, Martinez JP, Sentandreu R. Identification of wall-specific antigens synthesizedduring germ tube formation by Candida albicans. Infect Immun 1989; 57:262-271. 8. Tronchin G, Bouchara JP, Robert R. Dynamic changesof the cell wallsurface of Candida albicans associated with germination and adherence. Eur J Cell Bioll989; 50:285-292. 9. Ollert MW, Calderone RA. A monoclonal antibody that defines a surface antigen on Candida albicans hyphae cross-reacts with yeast cell protoplasts. Infect Immun 1990; 58:625-631. 10. Ghannoum MA, Abu-Elteen K. Adherence of Candida albicans: influencing factors and mechanism(s1. In: Prasad R, ed. Candida albicans: cellular and molecular biology. Heidelberg: Springer-Verlag, 1991:144-163. . 11. Ollert M. Molekulare Mechanismen der Adhaerenz von Candida albicans an humane keratinotyten in vitro. Akt Dermatol 1993 (in press). 12.Beeley JG. Glycoprotein and proteoglycan techniques. In: Burdon RH, van Knippenberg PH, eds. Laboratory techniques in biochemistry and molecular biology, v01 16. Amsterdam: Elsevier, 1985. 13. Springer TA. Adhesion receptors of the immune system. Nature 1990;346: 425-433. 14. Butcher EC. Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity. Cell 1991; 67:1033-1036. 15. Hasty DL, Ofek I, Courtney HS, Doyle RJ. Multiple adhesins of streptococci. Infect Immun 1992; 60:2147-2152. I.

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16. GeisowM. Shifting gear in carbohydrate analysis. Biotechnology 1992; 10: 277-280. 17. Marquis G, Garzon S, Montplaisir S, Strykowski H, Benhamou N. Histochemical and immunochemical study of the fate of Candida albicans inside human neutrophil phagolysosomes. J LeukocyteBioll991; 50587-599. 18. Richardson MD, Kearns MJ, Smith H. Differentiation of extracellular from ingested Candida albicans blastospores in phagocytosis tests by staining with fluorescein-labelled concanavalin A. J Immunol Methods 1982; 52241-244. 19. Broom MF, Shepherd MG, Sullivan PA. Changes in cell envelope glycoproteins during germ-tube formation of Candida albicans.Microbios 1991; 67:721. 20. Fukayama M, Calderone RA. Adherence of cell surface mutants of Candida albicans to buccal epithelial cells and analyses of the cell surface proteins of the mutants. Infect Immun 1991; 59:1341-1345. 21. Tronchin G, Bouchara JP, Robert R, Senet J-M. Adherence ofCandida albicans germ tubes to plastic: ultrastructural and molecular studies of fibrillar adhesins. Infect Immun 1988; 56:1987-1993. 22. Casanova M, Chaffin WL. Phosphate-containing proteins and glycoproteins of the cell wallof Candida albicans.Infect Immun 1991; 59:808-813. 23. Tojo M, Shibata N, Osanai T, Mikami T, Suzuki M, Suzuki S. Sandwich enzyme-linked immunosorbent assay of D-mannansof Candida albicansNIH A-207 and NIH B-792 strains using concanavalin A and polyclonal rabbit anti-C. albicans antisera. Carbohydr Res 1991; 213:325-330. 24. Montreuil J, Bouquelet S, Bebray H, Fournet B, Spit G. Strecker G. Glycoproteins. In: Chaplin MF, Kennedy JF, eds. Carbohydrate analysis: a practical approach. Oxford: IRL Press,1986: 143-204. 25. Calderone RA, Linehan L, Wadsworth E, Sandberg AL. Identification of C3d receptors on Candida albicans.Infect Immun 1988; 56:252-258. 26. Linehan L, Wadsworth E, Calderone RA. Candida albicans C3d receptor, isolated by using a monoclonal antibody. Infect Immun 1988; 56:1981-1986. 27. Bramley TA, Menzies GS, Williams RJ, Kinsman OS, Adams DJ. Binding sites for LH in Candida albicans: comparison with the mammalian corpus luteum LH receptor. J Endocrinoll991; 130:177-190. 28. Loose DS, Feldman D. Characterization of a unique corticosterone-binding protein in Candida albicans.J Biol Chem 1982; 257:4925-4930. 29. Critchley IA, Douglas LJ. Role of glycosidesas epithelial cell receptors for C. albicans. J Gen Microbioll987; 133:637-643. 30. Sandin RL, Rogers AL, Patterson RJ, Beneke ES. Evidence for mannosemediated adherence ofCandida albicansto human buccal cells in vitro. Infect Immun 1982; 35:79-85. 31. Brassart D, Woltz A, Golliard M, Neeser J-R. In vitro inhibition of adhesion of Candida albicansclinical isolatesto human buccal epithelialcells by Fucarl2GalO-bearing complex carbohydrates.Infect Immun 1991; 59:1605-1613. 32. Jimenez-Lucho V, Ginsburg V, Krivan HC. Cryptococcus neoformans, Candida albicans,and other fungi bind specificallyto the glycosphingolipidlacto-

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34. 35. 36. 37. 38. 39. 40. 41. 42. 43 *

44. 45. 46. 47.

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sylceramide(GAl/31-4Glc/31-1Cer),apossibleadhesionreceptor for yeasts. Infect Immun 1990; 58:2085-2090. Blackwell CC, Thom SM, Weir DM, Kinane DF, Johnstone DF. Host-parasite interactions underlying nonsecretionof blood-group antigensand susceptibility to infections by C. albicans. In: Lark DL, ed. Protein-carbohydrate interactions in biological systems. London: AcademicPress, 1986:231-233. Maisch PA, Calderone RA. Roleof surface mannan in the adherence of Candida albicans to fibrin platelet clots formed in vitro. Infect Immun 1981; 32~92-97. Ray TL, Digre KB, Payne CD. Adherence of Candida species to human epidermalcorneocytes and buccalmucosalcells: correlation with cutaneous pathogenicity. J Invest Dermatol1984; 83:37-41. Rotrosen D, Edwards JE Jr, Gibson TR, Moore JC, Cohen AH, Green I. Adherence of Candida to cultured vascular endothelial cells: mechanisms of attachment and endothelial cell penetration. J Infect Dis1985; 152:1264-1274. Klotz SA Penn RL. Multiple mechanisms maycontribute to the adherence of Candida yeasts to living cells. Curr Microbioll987; 16:119-122. Segal E, Lehrer N, Ofek I. Adherence of Candida albicansto human vaginal epithelial cells: inhibition by amino sugars. ExpCell Biol 1982; 50:13-17. Miyakawa Y, Kuribayashi T, Kagaya K, Suzuki M, Nakase T, Fukazawa Y. Role of specific determinants in mannan of Candida albicans serotype A in adherence to human buccal epithelialcells. Infect Immun1992; 60:2493-2499. Hasenclever HR, Mitchell WO. Antigenic studies of Candida. I. Observation of two antigenicgroups in Candida albicans.J Bacteriol 1961; 82570-573. Kagaya K, Miyakawa Y, Fujihara H, Suzuki M, Soe G, Fukazawa Y. Immunologic significance of diverse specificity of monoclonal antibodies against mannans of Candida albicans.J Immunoll989; 143:3353-3358. Kobayashi H, Shibata N, Suzuki S. Evidence for oligomannosyl residuescontaining both 8-1,2 and ar-1,2 linkages as a serotype A-specific epitope(s) in mannans of Candida albicans.Infect Immun 1992; 60:2106-2109. Shibata N, Arai M, Haga E, Kikuchi T, Najima M, Satoh T, Kobayashi H, Suzuki S. Structural identification of an epitope of antigenic factor S in mannans of Candida albicans NIH B-792 (serotype B) and 5-1012 (serotype A)as p-l ,2-linked oligomannosyl residues.Infect Immun 1992; 60:4100-4110. Warnock DW,SpellerDCE,Day JK, Farell AJ. Resistogrammethod for differentiation of strains of Candida albicans.J Appl Bacteriol 1979; 46571578. Odds FC, Abbott AB. A simple systemfor the presumptive identification of Candida albicansand differentiation of strains within the species. Sabouraudia 1980; 18:301-317. Kohler G, Milstein C. Continuous cultures of fusedcells secreting antibody of predefined specificity. Nature 1975; 256:495. Tojo M, Shibata N, Kobayashi M, MikamiT, Suzuki M, Suzuki S. Preparation of monoclonal antibodies reactive with /3-1,2-linked oligomannosyl residues in the phosphomannan-protein complex of Candida albicansNIH B-792 strain. Clin Chem 1988: 34539-543.

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48. Schalla WO, Whittington W, Rice JC, Larsen SA. Epidemiological characterization of Neisseria gonorrhoeueby lectins. J Clin Microbiol1985; 22:379382. 49. Korting HC, Abeck D. Lektin Typisierung als leistungsftihiges epidemiologisches markersystem fur Neisseria gonorrhoeue-Infektionen. Zentralbl Bakterio1 Hyg 1988; A269:506-512. 50. Korting, HC, Abeck D, Johnson A p , Ballard RC, Taylor-Robinson D, BraunFalco 0. Lectin typing of Huemophilus ducreyi. Eur J Clin Microbiol Infect Dis 1988; 7:678-680. 51. Korting HC, Abeck D. The lectin type of Candida ulbicum-an epidemiological marker relevantto pathogenesis. Mycoses 1992; 35:89-94. 52. Dean JW 111, ChandrasekaranS, Tanzer ML. A biological roleof the carbohydrate moieties of laminin. J Biol Chem 1990; 265:12553-12562.


6 Lectin-Leishmania Interaction R. L. JACOBSON The Hebrew University-Hadassah Medical School, Jerusalem,

Israel

1. INTRODUCTION

Leishmania are digenetic (heteroxenous) parasiticprotozoa of humans and animals that are found alternatively as flagellated, motile promastigotes and paramastigotes inthe alimentary tract of phlebotomine sandflies, oras obligate intracellular aflagellate amastigotes in the phagolysosomes of host macrophages. The genusLeishmania is of the order Kinetoplastida, family Tryposomatidae. Thereare over 20 designated speciesand several unnamed species groupedinto two subgenera[l] (Table 1). The subgenusLeishmania has been further divided into three complexes, the L.(L.) donovani complex, for which development inthe sandfly vector is restrictedto the midgut and foregut of the alimentary canal (suprapylaria), and those Old World species outsidethis complex (e.g., L.(L.) major) and the New World complexes L.(L.) mexicana and L.(L.) hertigi. The other subgenus is L.(Viannia) braziliensis and is characterized by prolific and prolonged phases of development in the hindgut of the sandfly vector, with later migration of the flagellates to the midgut and foregut (peripylaria). This genus is restricted to the American tropics and subtropics. Other methods, such as isoenzyme profiles, serotyping, and kinetoplast DNA buoyant densities, have been used inattempts to characterizethe different species groups, and each method has its own criteria. Lectins, when used as taxonomic tools, follow more closelythe broad pathological conditions causedby the parasites than do some other methods. The leishmaniasesare a group of pathological conditions ranging from a simple cutaneous lesion caused, for example, by L. major, through to the 191

Jacobson

192

Table 1 Taxonomy of the genus Leishmania

Subgenus

Leishmania

Complex L. donovani

Species

L. archibaldi L. chagasi

L. tropica L. major L. aethiopica L. mexicana

Not pathogenic to humans

L. hertigi

Viannia

L. braziliensis

L. guyanensis

Unassigned

L. donovani L. infantum L. killicki L. tropica L. major L. aethiopica L. amazonensis L. garnhami L. mexicana L. pifonoi L. venezuelens& L. arabica L. gerbilli L. turanica L. aristidesi L. enriettii L. hertigi L. deanei L. braziliensis L. peruviana L. guyanensis L. panamensis L. Iaisoni

Source: Updated from Ref. 61.

disfiguring diffuse cutaneous leishmaniasis (L. aethiopica)and mucocutaneous leishmaniasis (L. braziliensis) and, finally, to the fatal visceral leishmaniasis (L. donovani). There is variation within species, as L. tropica is both cutaneous and occasionally visceralizing and L. donovani is found in a condition known as post-kala-mar dermal leishmaniasis. Both forms of the parasite invade macrophagesand, as primary contact is at the cell surface level, it is this host-parasite interfacethat has been extensively studied. Clear differences in the surfaces of the parasite have been observed among strains that are otherwise closely related. Soluble and membrane-bound proteins linkedto oligosaccharides and termed glycoconjugates, constitute someof the major componentsof surface material. Specific probes, such as lectins, have added extensively to knowledge of the cell surface carbohydratesand are being usedto study their function.

lectin-leishmania

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Released and cell membrane-bound carbohydrate determinants have both been cited as beingimportant in the biology of leishmanial parasites. Surface carbohydrates of leishmanial promastigotes are important for attachment to the vertebrate host macrophage[2-41 and the sandfly digestive tract [5]. It has also been proposed that they play a role in the protection and survival of the parasite at the onset and during the infection of the host macrophage[6,7] and against the digestive tract enzymes ofthe sandfly vector [8]. The antigenic determinantson the surface of the parasite are oligosaccharides, but the nature of their structural association with glycoproteins, glycolipids, or other moleculesisstillbeinginvestigated.Lectinprobes have been used to determine the presence of carbohydrate receptors on Leishmania. Dwyer[9]was the first to report the binding of lectins to saccharides on the surface of promastigotes. Agglutination techniques and fluorescein-labeled lectins have revealed that other carbohydrate residues are on the surface of the parasitesas well as in the excreted glycoconjugate. Differences have been foundthe in configuration and quantity of the carbohydrate moieties among species and strains [lo-121. The surface membrane carbohydrates of different strains of Leishauthors showed varimania have been examined with lectins [13-161. These ation in the carbohydrate determinants of the different species and have revealed inter- and intraspecific differences in promastigote surface carbohydrate moieties. Glucose or mannoselike residues are found on the surfaces of all Leishmania, but in greater abundancein the L. tropica strains [13]. Five L. tropica strains were tested for lectin-mediated agglutination with seven lectins and only ConA, RCA, and SBA, (see Chapter 1 for ab[171. Galactose residues as reflected by lectin agglubreviations) were positive tination, are common to all species, but to a smaller extent inL. donovani and L. mexicana strains. Eleven strains ofL. major were investigated,and it was reported that the surface components and strains of this species [181. display marked heterogeneity In retrospect, these earlier studies of surface and released carbohydrates used material harvestedat a given point duringthe parasite’s growth cycle, but did not investigate the infective potential of the parasites. It has since been shownthat stationary-phase (S-phase) promastigotes, which agglutinate with lectins differently from exponential phase parasites, are more infectiveto mice [191. The lectin RCA-I(RC&), which is specificfor galactose @Gal)and, but less so, for N-acetyl-D-galactosamine(GalNAc), agglutinated fewer cells from the S-phase of L. donovani. Promastigotes from this phase gave a greater parasite burden inthe liver of the infected mice [19]. The lectin of Arachis hypogaea (peanut; PNA),which is specific for Gal@-1,3GalNAc> @Gal,was used as a marker to show that S-phase

194

Jacobson

promastigotes that did not agglutinate with this lectin caused larger lesions in infected mice [20]. These promastigotesare the infective forms and the final stage of metacyclogenisis. More recently, a lipophosphoglycan (LPG) has been defined, consisting of a tripartite molecular structure [21]. This LPG consists ofa polymer of repeating phosphorylated saccharide units attached by a carbohydrate core to a novel lipid anchor. The prominent feature of LPG is the polymer of 16 phosphorylated disaccharidesof [PO4 + 6Gal(/31 + 4)Manall units. In L. major, the analogous portion is composed of a series of small oligosaccharides, consisting of several common hexoses and the pentose arabinose [22]. The structurally related molecule, phosphoglycan, is shed by promastigotes in vitro and is present in spent culture medium [23]. It has been suggested that LPG is a multifunctional molecule [21]. There is evidencethat in the natural habitat of the promastigote,the sandfly gut, the shed carbohydratesare found as a gellike matrix in both the midgut and the cardia in histological sections [24] and by immunocytochemical analysis [25]. The released glycoconjugate also enhances the survival of foreign promastigotes in the indigenous host, indicating a vector-specific function of the shed material[26].

II. LECTIN-MEDIATEDAGGLUTINATION A. Single Strains

The fact that lectins could agglutinate promastigotesand be used to determine the presence of carbohydrate receptors on Leishmania was fiist described in 1974 [9]. In this first report, the binding of lectinsto saccharides on the surface of promastigotes was described ina single species,L. donovani, with three lectins. Thisauthor was able to find evidence of glucose or mannose and N-acetylgalactosamine randomly distributed on the surface of the parasite using the lectins ConA and phytohemagglutinins M and P. There have been additional descriptions of single isolates and their reactions to various lectins, using a variety of techniques. These included agglutination with five lectins (ConA, RCA, WGA, SBA,and PHA-P) of different stages of growth of L. braziliensis [1l]; an agglutination and electronmicroscopic study of horseradishperoxidase (HRP0)-labeled ConA on the surface of L. braziliensis guyanensis [27]. Another study reported agglutination tests with 31 lectins of which 17 reacted with the promastigotes, and fluorescent microscopic examination of 6 fluorescein isothiocyanate (F1TC)-labeled lectinson the surface of L. tropica [15]. The original work on L. donovani clone l-S, Cl2, was expanded with agglutination tests with five lectins (ConA, PHA-P, SBA, WGA,and LOTUS) and use of ['HIConA

Lectin-Leishmania

195

and HRPO-ConA [lo]. The results of these studies showed that, whereas mannose and glucose and galactose were common to all promastigotes, GalNAc, GlcNAc, and fucose were notfound on the New World strains.It was also noted that three types of agglutination occurred, flagellar-flagellar, flagellar-body, and body-body, indicating that the oligosaccharides are widely distributed onthe surface of leishmanial cells [9,27]. It has also been reportedthat there are indicationsthat sugars similarto a-D-mannose and a-D-glucose,D-galactose,N-acetylgalactosamine,N-acetylglucosamine, and a-L-fucose are present on the surface of variousLeishmania species [28]. Mannose or glucose and galactose appear to be common membrane lectin receptors for most species of Leishmania; however, the literature is somewhat equivocal concerning the other sugars. The foregoing studies were basedon single isolates offour species.

B. Comparison of Strains and Species A comparison of five isolates L. oftropica from Iran and L. d. chagasi, L. m. mexicana, and L. m. amazonensis withsevenlectins (ConA, RCA, SBA, WGA, PHA, PWM, and LOTUS) indicated some species specificity [17]. All strains testedwerepositivewith ConA. All five strains of L. tropica were positive with RCAand SBA, but were negative withthe other lectins. Leishmania was positive with RCA and WGA, L. d. chagasi was positive for RCA; and L. m. amazonemis was negativeexcept for the mannose-glucose-specific lectin. It was then suggestedthat lectins couldbe used for taxonomic determinationof species. Lectin agglutination of promastigotes was linked to an existing serotype system [l31 and was also suggested as an additional taxonomic tool [13,14,16]. Table 2 is an attempt to tabulate all the lectin agglutination data currently available for which the strains are reasonably well documented, and the more popular lectinsare available to other researchers. In Table 2, the many variations of scoring results by individual lectinologists have been reduced to the simple formula of “+” for strong agglutination, ‘‘f ” for weak results,and “ - ” for nonagglutination. The main conclusions that can be drawn from this bodyof workis that the Middle-Eastern strains of cutaneous leishmaniasis, caused by either L. major or L. tropica, were nearly always strongly agglutinated with SBA (GalNAc)and UEA-l (Fuc) or UEA-II(GlcNAc),. In the New World, the one speciesthat has a clearly defined lectin profile is L. m. pifanoi [14]. The one strain of this species, LRC-LW, that was tested, was agglutinated by ConA,RCA, PNA, SOJ, SOH, UEA, but not with PHA or AAP [14]. No other New World species reflected such a diverse and rich pattern of oligosaccharides on their surfaces. The OldWorldspecies L. aethiopica would appear to be at the

196

Jacobson

Table 2 Lectin-Mediated Agglutinationof Leishmania Species

COnA D - M ~ RCA120 DGlc l-3GalNAc &&Gal

Ref.Designation Species Old World L. donovani

L. d. infantum L. major (USSR)

L. major (M.East)

L. major (Kenya) L. tropica

L. aethiopica New World L.b. braziliensis

L.b. panamensis

L.m. mexicana L.m. amazonensis L.m. pifanoi Key:

l-S 10 14 l-S.3S,L-51,LV-139 16 L. do 16 L. doT 13.34 LRC- L-52,L-133.L-210 16 L-l33 43 Sudanese 14 L V-I40 K263 16 54 LV9 14 LV252,LV-253,L-38 18 LRC-L38 18 Neal-P 14 Ko,Ha,Schwe,Ne,Ro,Ve LRC- L137,L287,L288,L31 13,18,34 18 L306,L464,L505,L23 L223 16 L251,L137 54 L448.L461 18 L1 19 18 LV-249,LRC-L39 14 LRC- L-32,L-289,L-39 13,34 16 LV556.L-32 L-36 54 LV- 1 16 14 LV-l,LV-15,LV-24,LV-26 13.34 LRC-L134,L147 M2903 M2903,LRC-L77 WR120

11 54 14 54

LRC-L94 LRC-L94,M379 L1 1 L1 1 LRC-L259 LTB0016 H21,M1696 LRC-L90

13 14 16 54 16 54 14 14

+ + + + + + + + + + + + a + a + + + + * + + + + + + */+ + + + + + + + * + + + +

nd

++ * + + + + + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + +

- ,no reaction; ,weak reaction; + ,strong reaction; nd, not done.

PNA fl-~-Gal

nd

+

nd nd

a/+ nd

+-

nd

+ + + + a/+ + nd + -/+ * + + nd

nd

-

nd

nd

*-

-

nd + nd

lectin-leishmania Interactions

197

Lectin SJH WGA SBA OD-G~NAC ULEX-I ULEX-I1 D-G~NAc BD-Gal L-Fucose (D-GlcNAc), L-Fucose NeuNAc DGlcNAC

+

nd nd nd

-

nd

-

nd nd

+

nd

-

+

nd

+

+ + + + + + nd +

nd

nd nd

-/* -

nd

-

-

+ + nd + + nd +

nd

nd nd

+

nd

*nd ++nd

+

nd nd

a/+

nd nd nd

nd

+

+

nd

+ +-

nd

+ *

-

+ + nd +

nd

-

-

nd

-

PHA D-GdNAC (D-GIcNAc~

+

nd

-

nd nd nd nd nd

f

nd nd nd

-

nd

-

nd

-

nd nd

nd

-

-

nd nd

+ + + + nd + + + + + nd -

nd nd nd nd

nd nd

-

nd nd

-

nd

nd nd nd

-

nd

-

nd nd

nd nd

nd

-

nd nd

f

nd

nd

nd

h

nd

nd

nd

nd -

f

nd nd

nd nd

-

nd f

nd nd

-

nd

nd -

+

-

-

-

+ +

nd

f

-

-

-

-

+

-

nd nd nd nd nd nd

-

nd nd nd nd

-

nd nd nd

-

nd nd

-

nd -

nd

nd

nd

-

nd nd nd nd

+

nd nd

-

-

+

-

-

-

nd

-

198

Jacobson

opposite end of the spectrum, as promastigotes of some strains of this species are strongly agglutinated only by RCA-I1 and very weakly so by ConA, PNA, SOJ, and SBA [13,14,16,28]. The question must arise whether enough lectins have been used to form a panel of lectins that could separate the main complexes, if not individual species. Thirty-one lectins were used to explore the surface membranes of L. tropica and Crithidia lucillae (an insect protozoan) [15],and 23 lectins were used bythe same group of researchers inan attempt to identify and classify different species [16]. Notwithstanding that some of the strains used have been reclassified as different species [i.e., L. major (simple cutaneous leishmaniasis) is now a species distinctfrom L. tropica and L. infantum (visceral leishmaniasis with an animal reservoir) is distinct from L. donovani (man the onlyreservoir)],these authors concluded that lectinagglutinations added to our general knowledge of the “calling card” of the leishmaniae, but could not be used definitivelyfor taxonomic classification[29]. In my own laboratory, six lectins (ConA, RCA, PNA, SBA, UEA-I, and UEA-11) have beenfound to be the most useful in membrane agglutination analysis. We have also used LAAto differentiate L. urabica, a rodent parasite found in Psammomys obesus, from human isolates of L. major from Turkestan, where the reservoir host is Rhombomys opinus. This lectin gave identical agglutination results for L. arabica from Saudi Arabia and for L. major strains in Israel, whether isolated from humansor the reserthe L. major strains voir host P. obesus. As there is such variation among and isolates, we decided to investigate 11 such strains withstandard a panel of lectins [18]. 1. Lectin Specificity of 1 1 Strains of Leishmania major

The results ofthe lectin-mediated agglutination tests are presented diagrammatically in Figure 1. Agglutination; shown on the vertical axis, is graded from 0.0 (no agglutination)to 4.0 (all parasites agglutinated). Little uniformity was seenin the lectin agglutination profilesof the strains, even those of the same serotype. Alectinspecific for 8-l-D-galactose, RCA-11, agglutinatedall the strains strongly. Concanavalin A, specific for D-mannose and D-glucose, caused different degrees of agglutination among the strains. Strain LRCL119 and the P strain were weakly agglutinated, whereas strains LRC-L23 and LRC-L38 reacted strongly. The PNA lectin, with an affinity for the galactose-N-acetylgalactosaminedimer, caused moderate agglutination in most strains, but only feebly agglutinated strain LRC-L119 and had no effect on strain LRC-L461. The SBA lectin,specific for N-acetylgalactosamine, also agglutinated most strains moderately, but did not agglutinate strain LRC-L38.LectinUEA-I,specific for L-fucose,agglutinated

BE


0

0

LE

LSC

ELL SCC

BE

d

E l 909 CSC 909

LE L

LE

ISC

ELL

d

BCC

E l

SE

PSC

so9 SOE LE L

LE L

200

Jacobson

strains LRC-L137 and LRC-L23, but reacted only weakly, or not at all, with the other strains, and UEA-11, specific for di-N-acetyl-Dchitobiose, failed to agglutinate strain LRC-L38 and the P strain, but agglutinated all other strains moderately. When promastigotes were incubated with fluorescein-labeled lectins, we were unable to detect any labeling, as monitored by microscopy, with those lectins that had also failedto agglutinate them. Inhibition of the lectins with the appropriate specific sugars revealed some unexpected findings.Of the 11 strains agglutinated by ConA, only2, strains LRC-L461 andLRC-L119,werenotinhibitedbya-l-D-mannopyranoside, but were with glucose. Lactose failed to inhibit UEA-I1 agglutinin infour out of eight strains checked, LRGL306, LRC-L464, LRC-L505, and LRC-L3 1. Two of these strains, LRC-L464 and LRC-L505, were inhibited from agglutinating by chitobiose, whereas the other two were not. All 11strains of L. major tested have 6-l-D-galactose moieties on their surface membranes, as evidencedby Ricinus 120 binding. These results were in agreement with previous studies[13,141. Concanavalin A has also Leishmania [13,14,16]. This usubeen reportedto agglutinate all species of ally indicatesthe presence of mannose and glucose residueson the surface to inhibit ConA membrane. The saccharide, a-l-D-mannopyranoside, failed agglutination of strains LRC-L119and L461, but was inhibited by glucose. These two Kenyan strains are radically different from other strains of L. major. Strain LRC-L119 does not release glycoconjugates detectable by our methods [4;30] and LRC-L461 is one of several strains of L. major that produces serotype B EF [31-331. The lectin UEA-I, which is specific only for L-fucose residues, reacted diversely with the 11 strains. Strains LRC-L38 and P, two lines derived from an original isolate,from a Rhombomys opinus caught inTurkestan, and both EF subserotype &, failed to agglutinate and, therefore, appear to lack L-fucose as terminal residues. Strains LRC-L448 and LRC-L461, both from Kenya and subserotype &Bz and Bz, respectively,alsolackedL-fucose.Veryweakagglutinationoccurred in the other Kenyan strain, LRC-L119, and the Israeli strain LRCL31, a strain representing the EF subserotype A,Bz. Conversely, strain LRC-L119, as reported [34], was not agglutinated by an UEA lectin that could be inhibited by lactose, but not fucose. This anomaly may have been due to the potency of the reagents used; as bothUEA-11, specific for GalNAc, and UEA-I did agglutinate cells from this strain (LRC-L119). The other Israeli strains showed various reactions to UEA-I, from weak in strain LRC-L306 subserotype AI, to very strong in strain LRC-L23, subserotype A.,. Strain LRC-L38 and other Turkestani strains of L. major also lack L-fucose residues [14]. This might explain some of their differences from strains found in the Middle East, as was suggested on clinical grounds [35].

lectin-leishmania

201

Strains LRC-L119and LRC-L461 also appearedto lack galactose(asshown by PNA agglutination reactions)or have considerably reduced amounts of it when it is coupled to part of the N-acetylgalactosamine dimer. However, both have sufficient 8-l-D-galactose and N-acetylgalactosamine to react with RCA-I1and SBA agglutinins. The promastigotes of these two strains, therefore, seem to have lectin profiles that differ considerably from the other L. major strains (see Fig. 1). Strain LRC-L38 and the P strain, both derived from the same isolate, were maintained in Jerusalem and London, respectively, for many years. Although these two lines have identical enzymeand serotype profiles (zymodeme LON 1) and EF subserotype (A,,), their reactions with lectinsare different. Promastigotes of LRC-L38 appeared to lack GalNAc on their surface membrane, whereas the promastigotes of the P strain bound well with SBA. Conversely, strain LRC-L38 bound strongly with ConA, showing an abundance of mannose and glucose residues, and P strain showed very weak agglutination with this lectin. There is alwaysthe possibility ofa human error, but it is unlikely here, becausethe enzyme profilesare identical and, more importantly, both had A., subserotypes. Therefore, this shows clearly that the strains have become different in their reactivity to some lectins, even though they are now grown underthe same conditions. The strainsLRC-L306 and LRC-L31 differ in both enzyme profileand serotype, yet their lectin profile is very similar. Although both are readily agglutinated by UEA-I1 lectin, neither reaction was inhibited by the addition of lactose or chitobiose, indicating that both have an unusual carbohydrate configuration on their surface membrane. If a contaminant was present in the UEA-I1 lectin it would be very difficult to ascertain which saccharide residue was responsible for the agglutination reaction. Some UEA-I1 commercial reagents may agglutinate cells with a saccharide chain but considering the weaker reactions these of ~-Fu~~tl,2GalP1,4GlcNAc, two strains gave with other lectins, thisconfigurationseems unlikely. This study showedthat L. major strains have a diversity in their lectin specificities, as theydo in their serotypes. There no is direct evidencethat a universal increase in one carbohydrate denotes the decrease in another. Nor does there appear to be a direct correlation betweenthe carbohydrate topography expressedon the surface membranesand released carbohydrate moieties as determined by lectins. The one lectin that seems to indicate a species specificity is SBA, which is specific for GalNAc, and the only exception was the strain LRC-L38, which wasagglutinated by neither UEA-I nor UEA-11. This diversity of lectin-mediated agglutination indicates that no single strain typifies this species with respect to its surface and released carbohydrates.

202

Jacobson

These diversities were so pronounced for these 11 strains that it was decided to investigate whether different-growing conditions could modulate the surface carbohydrates expressed by the promastigotes [36]. 2. Surface Carbohydrate Expression of Leishmania major in Two Media

The two media chosen were the biphasic (Novy, MacNeal, and Nicolle’s) (NNN) medium, rich in blood components and Schneider’s medium (SDM), fetal calf serum serves a semidefinedall liquid medium, in which inactivated as a protein source. A clonedlineof L. major, strain LRC-L137, was initiated and a duplicate series of culture tubes were inoculated with a standard dose of promastigotes from the S-phase of growth, washed free of medium components,.in phosphate-buffered saline (PBS),pH 7.2. Promastigotes were tested daily for their ability to react with the panel of lectins [36]. In theNNN medium between day3 and 4, there was an increase inthe number of promastigotes that could be agglutinated by the lectins RCA, PNA, SBA, and UEA-I (Fig. Z), followed by a decrease untilthe end of the growth cycle. In SDM, the promastigotes manifestedtrends similar to those inNNN for the lectin UEA-I, but with PNA, RCA, and SBA there was either an increase or agglutination remained the same during the stationary phase (see Fig. 2). In both media, there was some increase inthe number of cells agglutinated duringthe growth cycle when they were incubated with ConA andUEA-11. A s a population of cells,the promastigotes grownin NNN medium to stationary phase, showeda clear tendencyto lose their ability to bind lectins that were specific for galactose, N-acetylgalactosamine, and fucose. Although cells grown ina semidefined medium showed a similar trend for the fucose-specific lectin(UEA-l), they were bound moreto those lectins specific for either 6-l-galactoseor GalNAc (PNA, RCA,and SBA) during the stationary phase than they were during the exponential phase. This clearly indicates that the cell surface receptors can be affected by the nutrient contents of their surrounding medium,and that the promastigote population adapts either by selection, or changes metabolically withthe digestive tract of the sandfly [37] or with the extracellular milieu of the mammalian host. It has been suggestedthat the uptake of the natural carbohydrate diet of the sandfly in the wild may profoundly affect the abilityof the parasite to survive and be transmittedto the mammalian host [38].In another study, the presence of lectinlike receptors was reportedin Phlebotomus papatasi homogenates, the main vector of L. major [39]. This lectin activity was


203

X

.-

c

I

i o

Da Y Figure 2 Daily changes of lectin-mediated agglutination of promastigotes of L. major (LRC-L137) grown in two media:filledcircle, NNN; opencircle, SDM. Agglutination index; scale range:0, no agglutination; 4, all promastigotes agglutinated. (FromRef. 36.)

inhibited by only two oligosaccharides, trehalose and turanose, for which there is no specificplant lectin analogue. Trehalose the is main disaccharide found in the hemolymph of many insects as well as being widelydistributed in bacteria, yeasts, and fungi, whereas turanose can be found in the disaccharide fraction of honey [M].This would suggest that these inhibiting saccharides are either of insect originor are taken up as part of the sandfly carbohydrate diet [41]. The sequentially changing configurations of carbohydrate moieties that were found in L. major maywellbe in response to the presence of these lectinlike receptors in the heads and midguts of sandflies. Although agglutination of promastigotes by the lectins has increased our knowledge of the carbohydrate residues of the cells, the use of labeled lectins has permitteda more intensive investigation ofthe surface oligosaccharides.

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Jacobson

111. LABELED LECTINS A N D SURFACE TOPOGRAPHY

The use of labeled lectins, whether conjugated with fluorescent dyes, ferritin, horseradishperoxidase, or radioactivelabels,hasenabledvarious workers to map the surface membranes of leishmanial promastigotes. A. Fluorescent Labels

There have been two maintools used to detect the presence of fluorescent labels on the surface of promastigotes:the fluorescence microscopeand the fluorescence-activated cellsorter (FACS). The microscope has been useful in detectingthe region ofthe lectin receptorsand visualizing some receptors that gavenegativereactionsinagglutinationtests.TheFACS,withits argon laser, has been used to show kinetic labeling in different populations of cells, as wellas revealing insufficient lectin receptorsto cause agglutination [42]. l . FluorescenceMicroscopy

In an early study [15], a strain of L. tropica, (unfortunately unidentified), was compared with Crithidia IuciIIae, a nonpathogenic insect flagellate. Rather surprisingly, only fluorescein-RCA- and fluorescein-UEA labeled the parasites, whereas labeledPEA, LCA, and LAO were negative. This is rather an unusual finding, as these latter lectins are specific for mannose and glucose, which are common to almost everystrain of Leishmania ever tested. at both A Sudanese strain of L. donovani was comprehensively studied the amastigote (intracellular) and promastigote (extracellular) stages [43]. The six FITC-labeled lectins used were ConA, PNA, WGA, RCA, SBA, and LOTUS. In this elegant study, there was a clear distinction between the stages of the parasite accordingto their lectin binding. Whereas LOTUS and SBAwerenegative, indicating the absence of GalNAc and fucose, PNA was positive onlyfor promastigotes, and WGA was positive onlyfor amastigotes. Another important difference indicated inthis work was that, whereas FITC-ConA binding on promastigotes was inhibited by 0.8 mM D-mannose, only10 mM a-methylmannopyranose would inhibit the FITCConA binding to amastigotes. The authors also showed that the WGA binding on amastigotes was decreased by GlcNAc and not sialic acid, even after sialidase treatment. In a more recent study[M],three Indian strains have been investigated with eight FITC-lectins, including C o d , DBA, PHA-P, PNA, RCA-11, SBA,UEA-I, and WGA. The three strains investigated were a visceral L. donovani, a post-kala-mar dermal leishmaniasis L. donovani, and a cutaneous L. tropica. The FITC-labeled SBA, PNA, and WGA failed to

lectin-leishmania

205

label the visceral leishmanial strain, whereas the other two strains were labeled by allthe lectins. The authors point out that these resultsare not in agreement with their previously published results using lectin agglutination tests [45]. 2. Flow Cytometry The other use of FITC-lectins has been with studies of populations of parasitesusingflowcytometrywithfluorescence-activatedcellsorters (FACS). Both the kinetics of changing surface carbohydrates during the sequential growth of a parasite population and comparisons of different species have been actively pursued in laboratory. my We have made a comparison of the FITC-lectin receptors on six L. major strains, three isolated in Israel (LRC-L306, -L464, and -L23), two from Turkestan (LRC-L38 and P strain), and one from the Sinai Desert (LRC-L505). The FITC-lectins used were RCA, ConA, PNA, WGA, and SBA. All the parasites were grown in Schneider’s defined medium, with 10% fetal calf serum, until the stationary phase. Promastigotes were harvested, washedthree times in PBS,pH 7.2, and fixed in 1% formaldehyde in PBS, with 2% glucose and 0.1% sodium azide. The FITC-lectins have been standardized inour department, and are used as follows: FITC-PNA and FITC-WGA, 20 pg/ml; FITC-SBA, 10 pg/ml; FITC-RCA, 5 pg/ml; and FITC-ConA, 0.6 pg/ml.Theresultsshowedmarkedheterogeneity among the strains and little correlation with lectin-mediated agglutination results [36,42] (Table 3and see Fig. 1). Strains that were not agglutinated, such as LRC-38 by SBA, bound well with the FITC-labeled lectin.Furthermore, FITC-WGA labeled between and 20 40% of allthe cells tested in this series and, although there are almost noreports of this lectin agglutinating promastigotes, the presence of GlcNAc has been confirmed by other methods [22]. The use of flow cytometric techniques allowed to measure us the changes of the carbohydrates on the surface of the parasites during the various phases of growth [36,42]. Although very little variationfound was in FITCConA and FITC-RCA binding during the growth of strain LRC-L544 (a freshly isolated IsraeliL. major), indicating the constancy of mannose and galactose, other lectins showed definite changes. The FITC-SBA labeled twice as many cells at the early exponentialand late stationary phases (days 3, 9, and 12) as at the late exponential/early stationary phase (Fig. 3).This dramatic loss of GalNAc and surprising increase in GlcNAc (as seen with FITC-WGA) may indicatethe metacyclogenic stageof the parasite, as Seen in in vitro cultures. ASthe FITC-WGA and FITC-SBA gave such interesting resultsthe in foregoing study, I have been using these lectins with other strains and

206

Jacobson

Table 3 Percentage of Cells Labeled by FITC-Lectinsfor All Strains Tested Under the Same Growth Conditionsin Semidefined Media and Harvested at the Stationary Phase

Species and number LRC

FITC-Lectin RCA WGA PNA SBA COnA

L.major L306

L464 L23 L38 “P” L505 L. donovani L133 L. aethiopica L147 L. amazonensis L259 94.3 L. enriettii L327

70.9 39.0 69.7 45.6 60.0 27.6

79.7 84.2

0.11

99.0 60.5

1.02

91.2

0.64

91.3 75.4

0.38

96.4

90.5

86.6 56.7 75.3

1.2 0.1 0.0

19.4 46.2 40.7 41.6 52.8 36.4

0.9

56.5

39.7 39.8 21.7 39.6 30.0 38.0

85.3 95.0 90.9 81.2 82.4 84.7

87.0

49.9

40.0

67.5

Source: From Refs. 42 and 58.

100 90 80 70 60 50

WGA SBA PNA

40

30 20 10 0

Figure 3 Fluorescent lectin-labeling of L. major (LRC-LS44) promastigotes during the growth cycle. (From Ref.36.)

lectin-leishmania

207

species ofLeishmania grown in different media. The percentageL.ofdonovani (LRC-L133) promastigotes, grown in biphasic medium0, that are labeled with FITC-WGA increased with time until the late stationary g), when there was a rapid decline in the phase of growth (about day number of cells labeled. Whenthe same cells were cultured in a semidefined medium(SDM),only a steady increase of binding, during growth, was observed. The FITC-SBA did not label any L. donovani cells. When L. tropica (LRC-L36) promastigotes were similarly labeled, the dramatic reduction (that was also seen in L. major [36]) ofthe percentage of cellsthat lose the ability to bind to FITC-SBA during the stationary phase was also observed, but only inthe NNN medium. In the SDM medium, the promastigotes of this strain were poorly labeled with FITC-SBA and seemed to lose any GalNAc-binding sitesafter the fifth day in culture. These results indicate that the parasites adapt rapidly to the surrounding medium and changes occur inthe surface carbohydratetopography. These changesmay reflect the various sandfly diets that the parasitesare exposed to in nature. Table 3 summarizes the results from the FACS studies and shows the percentage of binding to each FITC-lectin that was used. All cells were grown in semidefined medium and harvested at the stationary phase of growth. Only those strains that were examined withthe panel of five lectins are tabulated. B. HorseradishPeroxidaseand Ferritin

Several authors have used horseradish peroxidase (HRPO) and diaminobenzidine (DAB)or ferritin and radioactive 3H-labeled lectinsto map the surface membranes of Leishmania [10,27,43]. These studies used electron microscopy to visualize the actual location of the lectin-binding sites on the surface of the parasites. When using ConA, an asymmetric coat of a-D-glucopyranosyl, a-D-mannopyranosyl,or 6-D-fructopyranosyl was distributed over the surface of L. braziliensis guyanensisto a mean thickness of 50 nm [27]. In a thorough investigation of the surface of L. donovani (clone l-S, Cl2-&HRPO-ConA,HRPO-SBA, and HRPO-WGA were and follow used (as wellas 3H-labeled lectins)to map surface carbohydrates the kinetics of binding [lo]. Dense ConA-HRPO-DAB reaction product was restricted to the pellicular and flagellar outer lamina. Cells treated with SBA-HRPO or DAB had no dense-staining products, but when used in combination(SBA-HRPO-DAB),largeamountsof the lectinreaction product werenoted on portions of the surface membrane covering the subpellicularmicrotubules.Similarresultswereobtainedwith WGAHRPO-DAB, with the additional observation that the lectin depositswere also found on the flagellar membrane, within the flagellar reservoir, and

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the membrane liningthis cavity. The amount of cell-bound [3H] ConA was determined from mean saturation-binding kinetics, which was calculated to be2.78-4.86 x pmol/lO*whichprobablyrepresents the minimal number of cm-mannose ligands sterically available for binding with the lectin and does not reflect the total number of moieties presenton the surface of the promastigote [lo]. In another study of L. donovani, WGA-HRPO and PNA-HRPO were used to study stage-specific conversion of amastigoteto promastigote and vice versa [43]. In this study, in which agglutination, FITC-lectins, and HRPO-lectins, all were appliedto the two stages, the authors showed that HRPO-PNA bound to the promastigote, but not to the amastigote. Conversely, HRPO-WGA bound homogeneously only to the amastigote and not to the mature promastigote. During stage conversion, from amastigote to promastigote, the HRPO-PNA was first found inside the flagellar pocket of the promastigote and, later, over the entire surfaceof the promastigote. This presumed loss of PNA-binding sites inthe infectious metacyclic form has been the subject of much debate in several papers [20,46,47], but the significance of WGA-binding sites has been mostly ignored [36]. C. Enzymelinked lectin Assay

The lectin receptors of formalin-fixed promastigotes were probed using enzyme-linked lectins. The parasites of L. d. donovani, L. d. chagasi and L. m. amazonensis were fixed in formalin and attached with poly-L-lysine to microtiter plates. The parasites were then incubated with enzyme-linked ConA, RCA, WGA, PNA, and SBA. Only the parasites of the L. d. chagasi strain reacted with SBA, but all three strains reacted with theother lectins. Trypsinization of the cells did not remove the lectin receptors [48] as had been previously shown[49]. IV. INFECTIVITY OF LEISHMANIA AND LECTINS

The fact that lectins agglutinated promastigotes differently, dependingon the phase of growth,was described over 10 years ago, and these differences were correlated withthe infectivity ofL. donovani promastigotes for golden hamsters [19]. A.

Leishmania donovaniand Ricinus communis lectin

The three lectins usedfor this study were ConA, RCA-I, and .RCA-11, and the strain of L. donovani was the Sudanese 3s strain. The results showed that 10-day cultured promastigote; were less agglutinated than 3-day cultured cells when incubated with low concentrations (25 pg/ml) of RCA-I

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and C o d . Of these10-daypromastigotes, about 15%couldestablish themselves in macrophages, whereas fewer than 2% of 3-day cultures would infect the mammaliancells. The authors concluded that, asRCA-IImediated agglutination did not change over time, a-D-galactose was constant, but as RCA-I binding did, GalNAc was the variant carbohydrate on the surface. B. Leishmania major and Peanut lectin

Another group, using L. major (Friedlin strain NIH, Clone lA), and the lectins ConA, RCA-11, SBA, WGA, UEA-I, and PNA at concentrations from 0.37 to 100 pg/ml, showed with this strain that exponential- and stationary-phase promastigotes varied only with PNA, and less so with RCA-I1[20,46]. Here,about 10% ofthe 3-day promastigotes were infective to mouse peritoneal cellsand to 50% of the 5-day-cultured cells. C. Leishmania donovani and Peanut Agglutinin

As PNA agglutinationwas thought to be a suitable marker for the infectivity (metacyclogenic) of cells, other groups triedto repeat the work [46] with different strains. AnL. donovani strain (MHOM/ET/67/HU3) was grown in a specialized medium (modified Gracie’s medium)from amastigotes derived from hamster spleen [47]. Three lectins (ConA, WGA, and PNA) were used to test the changes in agglutination between exponential- and stationary-phase promastigotes. TheWGA failed to agglutinate any cells, ConA binding was the same for both stages, but only about 20% of S-phase cells agglutinated with PNA (125 pg/ml) against almost 100% of the Ephase cells. The authors concluded that the change in surface galactose,as promastigotes matured toward metacyclogenesis,was probably a common phenomenon of the Leishmania. A method for purifying populations of PNA- promastigotes has been published in which sucrose gradients were used [50]. D. Leishmania enriettii and Other lectins

A different approach to the problem of variation on the surface of the promastigote duringmaturation was that of comparing an infective and a noninfective strain of the same species. Two stocks ofL. enriettii, with differing pathogenicity to guinea pigs, were studied according to their lectin-mediated agglutination with the following lectins: ConA, RCA-11, EUE, SOH, PNA, UEA-I, UEA-11,LOTUS, and LAA [51]. The FITC-UEA-I, FITC-PNA, and FITC-LOTUS complex, were also used to ascertain differences between the two stocks.

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The concentrationsof lectins in this study were high compared with other studies (see foregoing), as lo00 pg/ml was an average for most tests. The EVE and UEA-I were negativefor both stocks. At the same concentration ConA, PNA, LAA, and UEA-I1 were negative for the pathogenic stock and only LOTUS was negative for the nonpathogenic stock. .The FITCLOTUS complex labeled onlythe pathogenic stock, whereas FITC-UEA-I labeled neither stock. The main conclusion that can be derived from this study is that the two stocks differ quite radically in their surface carbohydrates. The apparent lack of ConA receptors (indicating loss of mannose residues)isquitedifferent from other studies, as the parasites of both stocks wereharvested at S-phase and, for most Leishmania strains, no difference has been reported. Although LOTUS lectin strongly agglutinated the pathogenic stock (fucose receptors), UEA-I failedto agglutinate either stock. Similarly, UEA-I1 and LAA [(GlcNAc)2and Fuc,Gal] agglutinated only the nonpathogenic stock, indicating receptors for the LOTUS lectin and N,N-diacetylchitobiose as membrane components of the New World nonpathogenicL. enriettii stock.

E.

Leishmania braziliensis panamensisand ConcanavalinA and Lentil Lectins

Another New World species of human originthat has been studiedfor differences in lectin binding, during in vitro growth,was L. braziliensispanamensis (strainsMHOM/PA/82/WR470 and MHOM/PA/83/WR539) [52]. The panel of FITC-lectins tested included PNA, WGA, SBA, UEA-I, DBA, ConA, and LCA.Only the two mannose-glucose-specific lectins (ConA and LCA) bound the promastigotes, and LCA bound to only the stationary phase parasites. Other lectins were used to study the glycopeptides of this parasite and this will be discussed later. F. Leishmania mexicana amazonensisand Other Ledins

One of the more comprehensive studies of the interaction between lectins purified and a New World strain of Leishmania entailed the use of 28 highly lectins, and a binding assay with '251-labeled lectins [49]. The strain used was L. mexicana amazonensis (Josefa strain), originally isolated from a human case of cutaneous leishmaniasis, and both amastigotes (with and without membranes) and promastigotes (infective and noninfective) were tested.NineGalNAc-binding [JCA, BSI( =GS-I), DBA,SBA, HPA, MPA, LBA, PHA, and WIF], four Gal-binding (AXP, PNA, RCA-I, and RCA-11), three GlcNAc-binding[AAP,BS-I1 (=GS-11), WGA], twoMan/ Glc-binding (ConA and LCA), and a sialic acid (LIP or 1imulin)-binding lectin, all were positive for the promastigotes. Except for LBA, RCA-11,

eractionsLectin-Leishmania

21 1

and LPA (which were not tested) they would also agglutinate with the amastigotes. The specific results indicatethat LBA, WIF, and WGA react only with infective forms, whereas PNAand AXP were more selectivefor infective forms (ten times more lectin required to agglutinate the noninfective forms). Hence, for this parasite, GalNAc, GlcNAc, and Gal residues are all increased inthe infective promastigote form. The discrimination of the binding capacity of amastigotes from promastigotes was most noticeable where BS-I (=GM), DBA, PHA, SBA, and WIF are highly specific for amastigotes and MPA for promastigotes. Lectins that did not react to either stage included LOTUS, UEA-I, SOJ, BPA, W A , STA, PWM, UEA-11, and GEC. The bindingand kinetics of 12SI-labeled lectins (WIF, WGA, and PNA) were also studied by the same group [49]. Infective and noninfective promastigotes were easily separated by this method. With these three lectins the number of binding sites per infective promastigote could also be calculated. The ratio of binding sites for WGA/WFA/PNA was 30 :7.9 : 3.1, indicating that for this strain of L. m. amazonensis, GlcNAc was more abundant than GalNAc and Gal. G. Leishmania major and Fluorescein lsothiocyanate Labeled-Lectins

The problem of what constitutes an infective (metacyclogenic) promastigote has also been studied by following the growth of the parasites in culture and sequentially labeling with FITC-lectins. The population of labeled parasites was then analyzed by flow cytometry (FACS) and samples of the populations injectedinto hamsters at each phase ofthe growth cycle [36]. The FITC-lectins used were PNA, WGA, and SBA. The parasite was a new human isolateof L. major (strain MHOM/IL/87/YD;LRC-L544). Promastigotes of the strain of L. major LRC-LW, grown in SDM, showed a diverse pattern of labeling when incubated with FITC-lectins during the growth cycle (see Fig. 3). Most ofthe cells (82-90%) were readily labeled with FITC-PNA throughout the entire growth period. When the cells wereincubated with FITC-WGA there was an increase inthe percentage of cells labeled, from 27% on day 3 to 61% on day 12. When the promastigotes were labeled with FITC-SBA, a reduction of binding was reported, from 75% on day 3 to 40% on day 6. This trend was reversed by day 9 with 80% of the cells being labeled (Fig. 4). When the parasites with the different carbohydrate configurations, were injected into golden hamsters, there was no appreciable differences in the lesions caused, regardless of the age of the culture (Table 4). The constancy of FITC-PNA binding in a virulent parasite population was surprising when compared

I

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ISBA

of fluorescence

Relativeintensity

Figure 4 Flow-cell cytometric histograms of fluorescent lectin labeling during the growth ofL. major LRC-L544 promastigotes. (From Ref.36.)

with the appearance of PNA- promastigotes in other strains of the same species [38]. The presenceof WGA receptors (GlcNAc),that increased over time and the decrease and reappearance of SBA receptors (GalNAc) during a long growth cycle, indicateda change in the surface carbohydrate configuration that overlapped themastigote surface carbohydrates describedfor virulent L. donovani [43]. Table 4 Infectivity to Hamsters ofL. major

(Strain LRC-L544) Promastigotes from Different Days of Culture ~~~______

Days after infection 21 42 63

Source: Ref. 18.

Days of growth 3

6

9

113 2/3 3/3 3/3

1/3 3/3 3/3

0/3

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V. LEISHMANIAL GLYCOCONJUGATES AND LECTINS

One of the most interesting developments in the study of leishmaniasis has been the exploration of the leishmanial glycoconjugates. The contact between the parasiteand the host macrophage or the sandfly gut microvilli is the cell surface. Within this surface membrane is anchored a unique lipophosphoglycan, which is released into the surrounding medium. This material, it has been suggested, is multifunctional [7] and has been used as the basis for serotyping (taxonomy)[131, cell biology,and vaccination studies. Lectins have been particularly useful in defining the oligosaccharides that are the distal portion of the molecule. A. Clycoconjugates and Surface Membranes

The presence of carbohydrate residues in the pellicular membrane (PM) was demonstrated by isolating the membrane from L. donovani (strain l-S, clone2-D),exponential-phasepromastigotes[53]. The membranes were solubilized in SDS,run on sodium dodecyl sulfate-polyacrylamidegel electrophoresis, (SDS-PAGE), and probed with FITC-lectins. The FITClectins used were ConA, DBA, RCA-I and 11, SBA, UEA-I, and WGA.All the lectins “stained”the PM bands, and it was suggested that PM carbohydrate ligands were sidechains on membrane glycopeptidesor glycoproteins. When lectin-ferritin conjugates were used, all the lectins bound only to the external lamina of the PM and, therefore, the carbohydrates’ spacial orientation was external to the membrane plane, and there was chemical asymmetry ofthe PM relativeto glycosylation [53]. Another methodof comparing speciesand strains for their membrane glycoconjugate components isthat of lectin blotting. This method has been used both on purified membranes [54] and extracted whole promastigotes [52,55]. Purified membranes of several species were probed with either 1z51-C0nAor by triple sandwich Western blots for LCA, SBA, PNA, and RCA-I1 [54]. The strains testedwere L. major; L. b. braziliensis, and L. b. panamensis; L. m. mexicana and L. m. amazonensis; L. tropica; L. donovani infantum and L. enriettii. The results showedthat the triple-sandwich technique for ConA gave nonspecific binding; therefore,the direct method was usedfor this lectin. Onlythe two species from the L. mexicana complex were essentially similar in the overall patterns. Concanavalin A and LCA bound to similar glycoproteins, with multiple components with relative molecular masses (Mr)ranging from 27,000 to 200,000. Only L. b. braziliensis and L. enriettii were exceptional with single componentsM,of52,000 and 200,000, respectively. HighM, doublet or triplet (160,000, 175,000,and 185,000) polypeptides are present in the other stocks. Leishmania tropica (LRC-L36) was the only strain that did not react withthe galactose family

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of lectins (RCA, PNA, or SBA). The PNA bound to only three strains: L. b. panamensis, L. enriettii, and L. major (LRC-L137). The other strain of L. major (LRC-L251), which had been in continuous culture for 13 months (and, therefore, nonpathogenic), was weakly positive for PNA, but negative when freshly isolated promastigotes were cultured from mouse lesions. Lectin-blotting with PNA selectively bindsto 28,000, 37,000, and 48,000M, membrane components of both the non-pathogenic and the weakly pathogenic strainsof L. major [54]. B. Glycoconjugates of Extracted Promastigotes

A different approach to the study of the constituent carbohydratesof the leishmanial glycoconjugates was used by extractinglate exponential-phase parasites by freeze-thawing, separating on SDS-PAGE, and running the components in Laemmli gels [55]. The lectins were radioactively labeled, using the Iodogena method,and used to stain the gels. Fourteen different strainsof Leishmania were probed with 14 lectins. The SBA, SNA, and Narcissus pseudonarcissus (daffodil; specificfor terminal a-mannose) did not bind to any component. The LYE, CAN, Sus scrofa (porcine lung lectin; PLL; p-Gal), and Colchicum autumnale (autumn crocus; Gal&4Glc > GalNAc > Gal) bound to the 1l-kDa band in all strains, as did most of the lectins. The PNA bound to only the 11-kDa band in L. b. panamensis and L. m. amazonensis. The GlcNAc-specific lectins DAS (the samefour bands for all species 11,19,50, and 86 kDa)and WGA (multiple binding to different bands accordingto species) showed a wide range of glycoconjugate specificities.A 62-kDa bandwas consistently labeled (8 of 14 species) withthe Man-Glc-specific lectin ConA,but not by PEA lectin. Other lectins used included STA, which bound to only the two strains tested from the L. donovani complex, and GS-I, which bound to the 24-kDa band of L. b. braziliensis, a 53-kDa band of L. m. mexicana, and a 63-kDa band of L. d. donovani. The conclusion drawn from this study, which sometimes agreed [l41and sometimes contrasted[13,36] with previous studies,was that it is difficult to extrapolate from lectin-mediated agglutination resultsthe identity of the carbohydratemoieties of individual glycoproteins [S]. In another study, extracted L. b. panamensis exponential-phase and stationary-phase parasites were blotted and probed with the peroxidaselabeledlectinsDBA,CS-I, CS-11, ConA, PNA,WGA,MPA,RCA-I, SBA, and UEA-I[52].Only the lectinsPNA,WGA, DBA, and MPA bound to the Western blots; PNA bound to two glycopeptides (M, 59 and 61 kDa) of stationary-phase promastigotes,but not to blots of exponential @)-phase cells. These unique glycopeptide bands ofthe S-phase were also

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intensely stained by the other lectins, butso were other bands from E-phase promastigotes. Especially noticeable were three high M, bands (152, 165, 170 kDa)that were stained withWGA and were unique to E-phase promastigotes. There were some differences between these results [52] and the other reports [54,55], but the methods and individual isolates were not identical, so it is difficult to make a direct comparison. C. ReleasedClycoconjugates

We have already seen how some leishmanial investigators feltthat the absence of receptors for PNA (i.e., PNA- cells), was indicative of infectivity [46], whereas others showedthat if macrophages had surface receptorsfor galactose, then this would bethe most likely candidate for an immunodominant carbohydrate [56]. In this latter study, only RCA-Iand RCA-I1 could precipitate the released glycoconjugate@F), whereas ConA, WGA, DBA, LOTUS, and SBA, all failed to precipitate EF from the L. major strain LRC-L137. The theory suggestedthat the leishmanial-released glycoconjugate was, therefore, a macrophage-conditioning agent, facilitatingthe rapid uptake ofcells, and alymphocyteinhibitor.RCA-I1was later used to purify, by affinity chromatography, the released glycoconjugate inan attempt to ascertain the saccharide and amino sugar content of the material from L. major and L. donovani [57]. The absence ofGlcNAc and the presence of xylose as reported [57] differ somewhat from the currently accepted chemical structure of the glycoconjugate of these two parasites [21,22]. In cooperation with Dr.L. F. Schnur, I have been studying the underlying fundamental differences in released glycoconjugates of Leishmania of the B serotype complex. This group of organisms includesL. donovani (LRC-L133), L. aethiopica (LRC-L147), L. amazonensis (LRC-L259), and the nonpathogenicL. enriettii (LRC-L327). Each of these four species have different subserotypes,so it was of interestto determine if their differences were detectable using FITC-lectins [58]. 0-~-Galactose(l3)~GalNAc is almost undetectable on the surface of stationary-phase promastigotes of serotype B strains whenlabeledwith FITC-PNA and FITC-SBA, so these carbohydrates couldnot account for the differences. a-D-Mannoseand a-D-glucose (FITC-ConA), (D-G~cNAc), (FITC-WGA), and 0-D-galactose (FITC-RCA-11), are all present,but only 50% of L. enriettii cells could be labeled with C o d , and only 50% of L. amazonensis were labeled with RCA. Thus, there was a difference in the mannose/galactose ratio for these two strains. The FITC-WGA labeling showed the greatest diversity among the strains. Over85% of L. amazonensis cells were labeled [with 50% inhibition by(GlcNAc),],whereasonly

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35% of L. enrieffiicells were labeled [only.lO% were partially inhibited by

(G1cNAc)Jwith the same FITC-WGA; 60% of L. donovani cells were labeled [with only 25% inhibited by (GlcNAc)J, whereas 75% L. aefhiopica promastigotes boundto this lectin and there was virtually no inhibition by the (GlcNAc)2 (Fig. 5). An examination of the histograms from the flow cytometry showed that the numbers of cells and the relative intensities of fluorescence were very different (Fig. 6). We believe these differences are the reason for the serotypical differencesfound among these strains[58]. VI. CYTOTOXICLECTINSANDLEISHMANIA

The cytotoxic effectof lectins on leishmanial parasites was first reported in taxonomicstudies,in which it was reported that cells agglutinated by RCA-I were not released by the addition of 0.5 M lactose nor was the pronounced toxic effect reversed [131. Parasites that have been exposed to mutagens and then grown selectively in culture media with different concentrations of RCA-I, resulted in the production of ricin agglutinin-resistant clones [59]. The mutagen used was N-methylnitroso-N'-nitroguanide, at a concentration of 3.5 pg/ml for 3.5 hr on L. donovani promastigotes. The parasites were subsequently exposed to 100 pg/ml of ricinon 2% agar-supplemented Dulbecco's modified Eagle medium (dDME) plates. Clones were selected on the basis of resistance to RCA-I agglutination, and one was subsequentlyfurther characterized (the R2D2clones). The clones retained their resistance to the ricin lectin, even when grown in normal condition, but became much more sensitive to agglutination with ConA. The R2D2 clonesand wild-type L. donovani promastigotes were exposed to ricin-gold labeling and only the wildtypecellswerelabeled.Thesecloneswerealsoincorporatedingreater numbers by macrophages than were the wild-type promastigotes, as shown by ['Hluracil labeling. The conclusions drawn from these studies indicate the R2D2 clones did not possess the glycoconjugate lipophosphoglycan, and that they would be useful in future investigations in dissecting the molecular structure of the parasite. VII. LECTINSANDTHE SAUROLEISHMANIA

In view of the new taxonomic status of the Leishmania, such as parasites of reptiles (theyare actually a different genus,Sauroleishmania), it was felt necessary to keep them separatedfrom the mammalian Leishmania [l]. Sauroleishmania tarenfolae senagalensis (G.lO) and S. adleri (LRCL123) promastigotes were exposedto 23 lectins of diverse specificities [16].

217


100 Q)

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80

c.

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v)

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a

40

0

0

ConA

Con m a n

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0 L.donovani L.aclhiopica L.amazonensis

L.enrieltii

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80

v)

6o

CI

0

a S

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-

RCA+gal

20 0

Q)

nRcA

40

Lrbmvani Laelhiopica Lamazonensis Lmriellii

80

W

v)

0

6o

tam WGA+GluNac

40

S

20 0 L.dorovani L.aelhiopica

L a m n e n s s i Lmrieltii

Figure 5 Fluorescent-lectin labeling of four B serotype Leishmania and the effect of inhibiting carbohydrates. Cross-hatched, labeledby FITC-lectin; black, labeled in presence of carbohydrate.

218

Jacobson L.donovani

L.aethiopica

L.amazonensis

L.enrietti

L

L

Relative intensity of fluorescence Figure 6 Flow-cell cytometric histograms of FITC-lectin labeling of four different species ofLeishmania. The four species areall B serotype.

There were no reactions to fucose- or N-acetylchitobiose-binding lectins and little reactivityto glucose or mannose-specific lectins, except for ConA. Four lectins, SRA (Gal > > Fuc), CLN (GalNAc), and RCA-I and RCA11, gave strong agglutination reactions with these two stocks. The authors concluded that the lectin profileof the Sauroleishmaniacould not be superimposed on any mammalianstrain 1161. A strain of S. agamae (LRC-L121) was tested for the ability of its promastigotes to agglutinate with UEA-II; a weak reaction was reported that was inhibited by 80 mM of lactose [34]. The carbohydrate moietiesof S. adleri glycoconjugates have been partially characterized by ConA affinity chromatography [60]. The results of this study demonstrated that the a-D-manno-pyranosyl and galactopyranosyl unitswere associated with the same polymer, and a galactomannan remained specifically attached to the ConA.

Lectin-leishmania Interactions

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VIII. CONCLUSION

Lectins have been used widely in the study of the leishmaniases both for their surface carbohydrate moieties and their glycoconjugates. The lectins have helped dissect the molecular structuresof both the promastigote and the amastigote. They have been used as taxonomic tools, infectivity and virulence markers, and for the purification of leishmanial products. The knowledge gained from the labeled lectins has greatly increasedthe understanding of carbohydrate configurationson the surface membranes of these ubiquitous parasites.The oligosaccharides on and within the surface membrane of the parasites are the key to their survival in the hostile environment of the macrophage phagolysosome or the alimentary tract of the sandfly vector. If the disease leishmaniasisand the epidemicsit causes are ever to be controlled, it may be through the basic understandingof the carbohydrate profiles of the glycoproteins and other glycoconjugatesthat have been characterized withthe aid of lectins. REFERENCES 1. Lainson R, Shaw JJ. Evolution, classification and geographical distribution. In: Peters W, Killick-Kendrick R, eds.The leishmaniasisin biology and medicine, vol. I London: Academic Press, 1989:l-120. 2. Bray RS. Leishmania mexicana mexicana: attachment and uptake of promastigotes to and by macrophages invitro. J. Protozooll983; 30:314-322. 3. Blackwell J. Receptors and recognition mechanisms of Leishmania species. Trans Roy SOCTrop Med Hyg 1985; 79606-612. 4. Handman E, Schnur LF, Spithill TW, Mitchell GF.Passive transfer of Leishmania lipopolysaccharide confers parasite survival in macrophages. J Immuno1 1986; 137:3608-3613. 5. Molyneux DH, Ryan L, Lainson R, Shaw JJ. The Leishmania-sandfly interface. Leishmania. In: Rioux J-A, ed. Taxonomie et phylogtnie. Application tcodpidtmiologiques. (Coll. Int. CNRWINNSERM 1984) Montpellier: I" EEE 1986:311-324. 6. El-On J, Bradley DJ, Freeman JC.Leishmania donovani:action of excreted factor on hydrolytic enzymes activity of macrophages of mice with genetically different resistance to infection. Exp Parasitol 1980; 49:167-174. 7. Turco SJ. The lipophosphoglycan of Leishmania. Parasitol Today 1988; 4: 255-257. 8. Schlein Y,Romano H.Leishmania major and L. donovani: effects on proteolytic enzymes of Phlebotomuspapatasi (Diptera, Psychodidae). ExpParasitol 1986; 62:376-380. 9. Dwyer DM. Lectin binding saccharideson a parasitic protozoa. Science 1974; 184~471-473.

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10. Dwyer DM. Leishmania donovani: surface carbohydrates of promastigotes. Exp Parasitoll977; 41:341-358. 11. Davidowicz K, Hernandez AG, Infante RB, Convit J. The surface membrane of Leishmania I. The effect of lectin on different stages of L. braziliensis. J Parasitol1975; 61:950-953. 12. Hernandez AG. Lectins as tools in parasite research. In: Chance ML,Walton BC, eds. Biochemical characterization of Leishmania. Geneva:UNDP/ WORLD BANK/WHO, 1982:181-196. 13. Jacobson RL, Slutzky GM, Greenblatt CL, Schnur LF. Surface reactions of Leishmania I. Lectin mediated agglutination. Ann Trop Med Parasitol 1982; 76~45-52. 14. Schottelius J. Lectin typing of Leishmania-strains from the New and Old World. In: Bag-Hansen TC, ed. Lectins: biology, biochemistry, clinical biochemistry, vol.2. New York: Walter deGruyter & CO,1982531-541. 15. Petavy A-F, GuegnotJ, Guillot J, Damez M. Coulet M. Fixation des lectines sur Leishmania tropicaet Crithidia lucillae.Protistologica 1978; 14: 103-108. 16. Gueugnot J, Guillot J, Damez M, Coulet M. Identification and taxonomy of human and animal leishmaniasis. Acta Trop 1984; 41:135-143. 17. Ebrahimzadeh A, Jones TC. A comparative study of different Leishmania tropica isolates from Iran: correlation between infectivity and cytochemical properties. Am J Trop Med Hyg 1983; 32:694-702. 18. Schnur LF, Jacobson RL. Surface reaction of Leishmania IV. Variation in the surface membrane carbohydrates of different strains of Leishmania major. Ann Trop Med Parasitol 1989; 83:455-463. 19. Doran TI, Herman R. Characterization of populations of promastigotes of Leishmania donovani. J Protozooll981; 28:345-350. 20. SacksD, Perkins PV.Development of infectivestage Leishmania within phlebotomine sandflies.Am J Trop Med Hyg 1985; 34:456-459. 21. Turco SJ. The leishmanial lipophosphoglycan: A multifunctional molecule. Exp Parasitol1990; 70241-245. 22. McConville MJ, Homans SW, Thomas-Oates JE, Dell A, Bacic A. Structure of the glycoinositol-phospholipids from Leishmania major. J Biol Chem 1990; 265:7385-7394. 23. Jaffe CL, Leonor Perez M, Schnur LF. Lipophosphoglycanand secreted acid phosphatases of Leishmania tropica share species-specific epitopes. Mol Biochem Parasitol1990; 41:233-240. 24. Walters LL, Modi GB, Tesh RB, Burrage T. Host parasite relationship of Leishmania mexicana mexicana and Lutzomyia abonnenci (Diptera: Psychodidae). Am J Trop Med Hyg 1987; 36:294-314. 25. Davies CRYCooper AM, Peacock C, Lane RP, Blackwell JM. Expression of LPG and GP63 by different developmental stagesof Leishmania major in the sandfly Phlebotomuspapatasi. Parasitology 1990; 101:337-343. 26. Schlein Y, Schnur LF, Jacobson RL. Released glycoconjugate of indigenous Leishmania major enhances survival of a foreign L. major in Phlebotomus papatasi. Trans R SOC Trop Med Hyg 1990,84:353-355.

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27. De Souza W, Brasil RP. An electron microscopicand cytochemical detection of concanavalin A receptors on the cell membrane ofLeishmania braziliensis guyanensis. Z Parasitenk 1976; 5O:l-9. 28. Dwyer DM, Gottlieb M. The surface membrane chemistry ofLeishmania. Its possible role in parasite sequestration and survival. J Cell Biol 1983; 23:3545. 29. Guegnot J, Coquillard P, Guillot J. Utilisation des lectines pour l’dtude des Trypanosomatidae. In: Rioux J-A, ed. Taxonomie et phylogdnie application Cco-CpidCmiologiques. (Coll. Int. CNRSANNSERM 1984 Montpelier: IMEEE 1986~77-84. 30. Schnur LF. The influenceof host type on the infectivity and attenuation of a leishmanial strain. In: Proceedings of the eleventh international congress on tropical medicine and malaria, Calgary. 1984:136. F) serotypes in Su31. Schnur LF, Zuckerman A. Leishmanial excreted factor @ dan, Kenya and Ethiopia.Ann Trop Med Parasitol 1977; 71:273-294. LD. Characterization of 32. Githure J, Schnur LF, Le Blancq SM, Hendricks Kenyan Leishmania spp. and identification ofMastomys natalensis, Taterillus emini and Aethomys kaiseri as new hosts of Leishmania major. Ann Trop Med Parasitol 1986; 80501-507. 33. Morsy TA, Schnur LF, Feinsod FM, Salem AM, Wahba MA, El Said SM. Natural infections of Leishmania major in domestic dogs from Alexandria, Egypt. Am J Trop Med Hyg 1987; 37:49-52. 34. Greenblatt CL, Meline D, Slutzky GM, Schnur LF, Levene C. Surface reactions of Leishmania 111. Ulex europaeus I1 lectin affinity for excreted factor (EF)serotype A strains. Ann TropMed Parasitoll984; 78%-107. 35. Adler S, Katzenellenbogen I. The problems the of association between particular strains of Leishmania tropica and the clinical manifestations producedby them. Ann Trop Med Parasitol 1952; 46:25-32. carbohydrateconfigurations dur36. Jacobson RL, Schnur LF. Changing surface ing the growth of Leishmania major. J Parasitol1990; 76:218-224. 37, Schlein Y. Sandfly dietand Leishmania. Parasitol Today 1987; 2: 175-177. 38. Schlein Y,Borut S, Greenblatt CL. Development of sandfly forms in Leishmania major in sucrose solution. Parasitol J 1987; 73:797-805. 39. Wallbanks KR, Ingram GA, Molyneux DH.The agglutination of erythrocytes and Leishmania parasites by sandfly gut extracts: evidence for lectin activity. Trop Med Parasitol 1986; 37:409-413. 40. Noller CR. Chemistry of organic compounds. Philadelphia: W B Saunders, 1965:419. Leishmania in phlebotomine sandflies In: Lum41. Killick-Kendrick R. Biology of sden WHR, Evand DA, eds. Biology of the kinetoplastida, vol. 2. New York: Academic Press, 1979:395-460. 42. Jacobson RL, Schnur LF, Greenblatt CL. Variation in Leishmania species expressed by antigenic glycoconjugates and excreted factor. In: Hart DT, ed. Leishmaniasis: the current status and new strategies of control. NATO-AS1 series A, vol.163, New York: Plenum Press,1987:401-408.

Jacobson

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RD.

43. Wilson ME, Pearson Stage-specific variations in lectin binding to Leishmania donovani. Infect Immun 1984; 46:128-134. 44. Ghosh DK, Ghosh AK, Ghosh KN,De A, Bhattacharya A. Kinetoplastic flagellates: surface-reactivecarbohydrates detected by fluorescein-conjugated lectins. J Parasitol 1990; 76:130-133. 45. Ghosh DK, Ghosh AK, De A, Bhattacharya A. Differentiation of pathogenic and non-pathogenic kinetoplastic flagellates by lectins. In: Bag-Hansen TC, ed.Lectins:biology,biochemistry,clinicalbiochemistry,vol. 4. St Louis: Sigma ChemicalCO, 1988559-564. 46. Sacks DL, Hieny S , Sher A. Identification of cell surface carbohydrate and antigenic changes between noninfective and infective developmental stages of Leishmania major promastigotes. J Immunol1985; 135564-569. 47. Howard KM, SayersG, Miles MA.Leishmania donovani metacyclic promastigotes: transformation in vitro, lectin agglutination, complement resistanceand infectivity. ExpParasitol 1987; 64:147-156. 48. Andrade PP, Schottelius J, Andrade CR. An enzyme-linked lectin assay for the study of lectin receptors of Leishmania. Braz J Med Biol Res 1988; 21: 517-521. 49. Saraiva EMB, Andrade A F B , Pereira MEA.Cell surface carbohydrate of Leishmania mexicana amazonensis:differences between infectiveand noninfective forms. Eur J Cell Biol 1986; 40:219-225. 50. Ready PD, Smith DF. Peanut agglutination and isolation of infective forms of Leishmania major. Trans R SOCTrop Med Hyg 1988; 82:418. 51. Schottelius J. Selective lectin reactions of two stocks of Leishmania enriettii with differing pathogenicity. Parasitol Res 1987; 73:l-8. 52. Grog1 M, Franke ED, McGreevy PB, Kuhn RE. Leishmania braziliensk protein, carbohydrate, and antigen differences between log phaseand stationary phasepromastigotesin vitro. Exp Parasitol 1987;63:352-359. . 53. DwyerDM. Structural, chemical and antigenic properties of surface membranes isolated from Leishmania donovani. In: Slutzky GM, ed. The biochemistry of parasites. Oxford: Pergamon Press, 1981:lO-28. 54. Jaffe CL, McMahon-Pratt D. The identification of membrane glycoconjugates in Leishmania species. J Parasitoll988; 74548-561. 55. Rossell RJ, Stevens AF, Miles MA, Allen AK. A comparison of the lectinbinding properties of glycoconjugates from a range of Leishmania species. Parasitol Res 1990; 76:294-300. 56. Slutzky GM, Greenblatt CL. Identification of galactose as the immunodominant sugar of leishmanial excretedfactor and subsequent labeling with galactose oxidase and sodium boro[’H]hydride. Infect Immun 1982; 37:lO-14. 57. Zehavi U, Abrahams JC, Granoth R, Greenblatt CL, Slutzky GM, El-On J. Leishmanial excretedfactors (EFS): purification by affinity chromatography. Z. Parasitenkd 1983; 69:695-701. 58. Jacobson RL, Schnur LF.Surface carbohydrates and shed antigenic glycoconjugate expression of serotype B Leishmania species. In: Sharon N, Lis H, Duskin D, Kahane I, eds. Proceedings of 10th international symposium of glycoconjugates. Jerusalem, 1989:182-183.

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59. King DL, Turco SJ. A ricin agglutinin-resistant cloneof Leishmania donovani deficient in lipophosphoglycan. Mol Biochem Parasitoll988; 28:285-294. 60. Palatnik CB, Previato JO, Gorin PAJ, Mendonca-Previato L. Partial characterization of the carbohydrate moieties inLeishmania adleri glycoconjugates. Mol Biochem Parasitoll985; 14:41-54. 61. Control of theleishmaniases.TechnicalReportSeries793,Geneva:World Health Organization, 1990.


7 Trypanosome-lectin Interactions JUSTUSSCHOTTELIUS Bernhard Nocht Institute for TropicalMedicine,

Hamburg, Germany MARTINS S. 0.AlSlEN University of Benin, Benin City, Nigeria

1.

INTRODUCTION

The discovery of lectins in mammalian tissues, following the observation that the survival of a serum glycoprotein is dependent on the structure of its carbohydratecomponent, has shown that lectins and their corresponding carbohydratestructures are information carriers. Thesecarbohydratestructures are involved in a biological recognition system in which the carbohydrate sequence representsthe information capable of triggering certain reactions. The natural reaction partners for these carbohydrates are animal and plant lectins, which occur either in soluble or bound forms. Lectins have been employed in the investigation of several protozoa. In this chapter, the use of lectins in the investigation of the developmental stages of Trypanosoma cruzi, the causative agent of Chagas’ disease, as well as T. rangeli, T. conorhini, T. dionisii, and T. vespertilionis, is described. Fromthe plethora of literature on this subject only a representative fraction could betaken into consideration. Lectins can be employed for the isolation of glycoconjugates from these parasitesand for interspecific and intraspecific differentiation; also,lectin-carbohydrateinteractionshave definite biological functions.For example, the investigation of the gut sections of Rhodniusprolimrs, a vector of T. cruzi, led to the observation of lectins that react with gut lumenforms of T. cruzi, but not with the blood forms. The developmental goal ofthe gut lumenforms is the production of infective, metacyclic flagellates in the hindgut. The inhibition of the gut lectin of Glossina morsitans with a sugar resulted in the inability of T. congolense to adhere to the gut epithelium of this vector. A s a result, the 225

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development of infective metacyclic flagellates was prevented. For Leishmania, lectins are also thought to play a role inthe transformation process of this flagellate inthe vector (see Chapter6). Lectin-carbohydrate interactions are integrated in the transformation processes of parasites in their vectors. The adhesion of pathogenic agents to the gut or salivary gland epithelia is only the initial step in a signal-connected process that triggers the transformation events. To understand the operative mechanismof this process, it would be of interestto know how external influences affect the intravectoral development of infective parasite stages that are eventually transmitted to the vertebrate host. Withthe aid of lectins, the changesthat occur in the carbohydrate structures on cell and tissue surfaces can be brought to light. The natural reaction partnersof these carbohydrate structures of the cell and tissue surface lectins that undergo changes during transformation processes, can be demonstrated with the aid of neoglycoproteins and neoglycoenzymes. Thus,tools are provided for unraveling the code systemon which lectin-carbohydrate interactionsare based. II. MEDICALLY IMPORTANT TRYPANOSOMES A. Trypanosoma (Schizotrypanum)cruzi Chagas, 1909

The American trypanosomiasisor Chagas’ disease is a human infection in Latin America causedby the protozoan Tvpanosoma cruzi, which is contaminatively transmitted by insects the of subfamily Triatominae [l]. According to statistics from the World Health Organization (WHO) [2],approximately 16-18million personsare infected with the parasite,and about 100 million persons live in endemic areasand are exposed to the risk of infection. Other than humans, the flagellate has been isolated from several mammals in South, Central, and North America, and 36 species of triatomid insects come into consideration as facultative vectorsof T. cruzi [3-61.It, therefore,means that the geographic distribution ofChagas’diseaseis smaller than the spread ofthe causative agent,the vectors, and the reservoir hosts. In Latin America,the parasite is involved in two .transmissioncycles: the so-called domestic cycleand the forest cycle [7].The two cycles may be connected by an intermediary one through which parasites from wild animals may be introduced into the domestic cycle.Tvpanosoma cruzi undergoes a developmental cycle.In humans and mammals, the pathogen exists in the blood only in the trypomastigote form and as amastigotes in the tissue. Whenthe blood formsare taken in bythe triatomid vectors during a blood meal, the parasite undergoes a series of developmental stages in the

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gut of the vector, inwhich the epimastigote and trypomastigote metacyclic forms are formed [8]. In culture medium[g], the epimastigotes are preponderantly formed. The clinical manifestations of Chagas’ disease in Latin America are related to their geographic origin [lo]. However, it is unclear if these differencesare in any way related to the differences in theT. cruzi species. B. Trypanosoma (Tejeraia) rangeli Tejera, 1920

The flagellate T.rangeli has similarly beenfound in many vertebratesand in humans with T. cruzi infections [4,1 l]. In contrast with T. cruzi, T. rangeli, inoculatively transmitted, most especially as the metacyclic infectious stages, develop in the salivary glands of the triatomid vectors. This species is neither pathogenic for humans nor for other vertebrates. It is important because, in Venezuelaand Colombia, the spread of T. cruzi and T. rangeli overlap in some areas and double infection with both species are known to occur [l l]: Whereas the trypomastigote blood forms of both flagellates are morphologically distinguishable, the culture forms can be distinguished only with much difficulty. Therefore, the question arises of how to differentiate the two culture forms, C. Trypanosoma (Megafrypanum) conorhiniShortt and Swaminath, 1928

In the epidemiology of Chagas’ disease, T. conorhini should be takeninto found in the New World. Some account [4,12]. This flagellate has also been authors [13,14] have demonstratedthat the natural vector of T. conorhini, Triatomarubrofasciata [l], canalsobeinfectedwith T. cruzi. Consequently, the question of similarity arises concerningthe differentiability of the cultural forms. Rats are reservoir hosts for T. conorhini, but mice and monkeys also show susceptibility to the parasite. These animals can also be infected withT. cruzi. Investigations [l21 have shownthat the vectors of T. cruzi can also be infected withT. conorhini. Whereas the blood forms of T. cruzi, T. rangeli, and T. conorhini are morphologically distinguishable, it is not always possibleto differentiate the cultural forms of these flagellate species. D. Trypanosoma (Schizotrypanum) vespertikonis Battaglia, 1904; Trypanosoma (Schizotrypanum) dionisiiBittencourt and Franca, 1905

The causative organism of Chagas’ disease can also be isolated from different bat species in South America [3,4,15]. Several trypanosomesthat are morphologically very similarto T. cruzi have been isolatedfrom a number

220

Schottelius and Aisien

of bats in South America. In the Old World,bat trypanosomes, namely T. vespertilionis and T. dionisii, are widely distributed, and their cultural forms are very similar to those of T. cruzi. It is not quite clear if bat infections with T. cruzi are of any epidemiological importance. Nevertheless, itcannot be ruled out that transmission of these parasitesfrom domestic animals and bats through triatomid vectorsto humans does occur when closely associated. E. Differentiation of Trypanosomes with Lectins

Several authors have investigated the possible contribution lectins could make in the differentiation of these flagellates; these include inter- and intraspecific differentiationof the foregoing Trypanosoma species; differentiation of T. dionisii and T. vespertilionis from T. cruzk lectin reactions of the developmental stages ofT. cruzi; intraspecific differentiationof the subpopulations of the trypanosomes of the T. cruzi complex relative to the geographic distribution; clinical manifestationof Chagas’ disease; and the occurrence ofthe parasites in a domesticor a forest cycle. 111. LECTIN REACTIONS OF THE DEVELOPMENTAL STAGES OF TRYPANOSOMA CRUZI

Lectins have been employed for comparative investigations ofthe developmental forms of T. cruzi [16,17]. The epimastigotes react with concanavalin A (ConA),but the trypomastigote blood formsfail to react with this lectin [16]. The epimastigote culture formsof the Tehuantrepec strain had a differential affinity for ConA, SBA, and WGA [l71 (lectin abbreviations are defined in the Appendix of Chapter 1). No reaction occurred with PHA before treatment of the cells with proteolytic enzymes. Epimastigotes and trypomastigote metacyclic forms ofthe T.cruzi strains Y,Cl, and F1 were this investigated with ConA[18]. All these developmental stages react with (-gp)was isolated lectin. By using a LCA column, a 90,000-Da glycoprotein from the epimastigotes of the Y2 strain [19]. This glycoprotein has also been detected inthe mastigote tissue forms, aswell as in the epimastigote and blood trypanosomes. Whereas a protein specificfor the epimastigotes could not be identified, one for the amastigote stage (gp 30,000 Da) and for the trypomastigote forms(gp 180,000 Da) were detected. Investigation of the amastigote forms of the Tehuantepec strain [20] with ConA, WGA,SBA, and PHA showed that the cells reacted only with ConA. At 37OC, a capping effectwas observed. With ConA a migration of the receptors similarto that which has been describedfor T. cruzi antibodies could be inducedon the epimastigotes [21]. This receptor mobilitywas

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also observed in the blood forms, demonstrating that the receptors are integral components of the surface coat. The lectin investigations of the amastigote, epimastigote, and trypomastigote forms of the strains Y, Cl, and MR showed that the developmental stages reacted distinctively with RCA, ConA, SBA, PNA,, and WGA [22]. No reaction was observed with LIP, UEA, and DBA. In the search for stage-specific lectin receptors, the epimastigote, amastigote, metacyclic trypomastigote, and trypomastigote bloodstream forms of strain Y were investigated with about 30 lectins [23]. Stage-specific lectins could not be found. Results from this study, however, showedthat the epimastigote form reacted with the minimal concentrationof WGA, GSA, SBA, and HPA; the amastigotes withPNA and PHA; the blood flagellates reacted with PHA; and the metacyclicforms with WIF. Otherauthors [24] also found such differences in lectin affinity with the amastigotes, epimastigotes, and metacyclicforms, as well as the bloodstream forms of strain Y with ConA, WGA,and SBA. These authors used a WGA column the blood forms. to isolate a glycoprotein called Tc 85, which occurs only in Tc 85 binds to ConA, but in contrast, not to LCA. The carbohydrate structures on the surfaces of T. cruzi developmental stages, aswell as their structural analogues formed during the transformation of the causative agents, are the receptors for the tissue-bound lectins in the different gut sections of the insects that function as vectors. Such lectins were found in the stomach, midgut, and hemolymph of Rhodnius prolixus [25]. The corresponding lectin receptors are absent on the surfaces of trypomastigote flagellates, but are present on the epimastigote forms. The reciprocal actions between the tissue-bound lectins of the insects and the carbohydrate structures on the parasites induce a chain reactionthat ends in the formation of infectious metacyclic forms. When Glossina morsitansis fed with glucosamine, lectin receptors are inhibited [26]. As a result, the formation of the metacyclic forms of T. congolense totally ceases. Investigationsinto the composition of the glycoconjugates of a clonefrom strain Y of T.cruzi showed that the epimastigote forms contain mannose, glucose, galactose, glucosamine, galactosamine, fucose, xylose, and ribose [27]. The same carbohydrate composition was found during a searchfor a glycoprotein specificfor the epimastigote stage [28]. N-Acetylneuraminic acidwas not detected, althoughthe investigation of diverse strains ofT. cruzi has shownthe occurrence ofthis carbohydrate in the parasite [29-331. In the aforementioned investigations,it was clearly demonstratedthat the reactions with WGA, LFA,and AAA were associated with sialic acid, as the enzymatic splitting of the acid from the cells resulted in the loss of the lectin reactions. Trypanosoma cruzi is incapableof sialic acid synthesis

230


[34]. Instead, the parasites take up sialized glycoconjugates from either their maintenance mediumor from the host [35-371. The selection mechanism for these glycoconjugatesand the process of integration into the parasite membrane is unclear. So also is its importance as an antigen and its transmission during transformation of the flagellates. It could be demonstrated that fetuin and other sialoglycoproteins stimulate cell infection [38]. With a ConA column, a 25-kDa glycoprotein was isolatedfrom epimastigotes [39]. This glycoprotein reacted with the antibodies from patients with Chagas’ disease of different clinical manifestations, a reaction considered to be of diagnostic importance. L-Fucose was described from epimastigotes of different strains of T. cruzi [a],but no reaction of radioactive iodine-labeled UEA-I with gel bands of T. cruzi extracts in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) could be observed. Similarly, other working groups could. not demonstrate reactions with either LOTUS or UEA-I [23,31,32]. The investigations with the iodine-marked lectins [ a ] for the T. cruzi showed labeling of electrophoretically separated cell extracts of differences inthe glycoconjugation betweenthe strains and the transformation stages. These authors found that “PNA strains” of the T. cruzi zymodeme Z1 reacted with two bands [41,42,43]. The T. cruzi strains of other zymodemes didnot show any reactions. These results are in agreement with lectin reactions ofT. cruzi strains [31-33,37,44]. Strains of the “PNA type” belong to zymodeme Z1and the ‘WGA type” to the zymodeme 22. The reactions of T. cruzi cell extracts with ConA [ a ] underlie the presence of a varietyof glycoconjugates, which could be used for the intraa column three stagespecific differentiationof T. cruzi strains. With ConA specific glycoproteins of 90, 85, and 55 kDa, respectively, were isolated from metacyclic forms ofT. cruzi [45]. A 72-kDa glycoprotein was isolated from its epimastigotes. Only this protein reacted with antiepimastigoteserum from rabbits, whereas the serum from Chagas’ disease patients reacted very strongly with the three glycoproteins,but only slightly with the 72-kDa antigen. A TC 85 glycoprotein was also isolated with a WGA column [46]. This glycoprotein loses its binding to the WGA column after treatment with sialidase. An investigation with exoglycosidases showed that fucose, sialic acid, and a Gala-l,3Gal unit are present in T. cruzi. Because this flagellate is incapable of sialoglycoconjugate synthesis, a sialized glycoconjugate must be present in the Tc 85 antigen, which the parasite must have incorporated into its cells. An antibody against Gala-l,3Gal epitope has A stage-specific 90-kDa glycobeen found in patients with Chagas’ disease. protein was found in the metacyclic forms of T. cruzi [47]. This gp90 is preceded by a gp75 precursor which can be inhibited with tunicamycin. Concanavalin A binds to gp90, but not to gp75.This gp90 appears to

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be important for the invasive ability of the metacyclic forms, because a monoclonal antibody 1G7, which reacts very well withgp90, partly inhibits penetration into cells. IV. INTERSPECIFIC DIFFERENTIATION

A. Trypanosoma cruzi and Trypanosoma rangeli

The comparative investigations with lectins of epimastigote culture forms of T. cruzi and T. rangeli showed that strains of T. rangeli react with only lectins of the mannose and glucose type (ConA, LCA, PEA). In contrast, T. cruzi strains react additionally with lectins of the galactose and Nacetylgalactosaminetype and withsialicacid-bindinglectins [29,31,37,44,48401. Only two strains of T. rangeli, Da 2114 and DA EP 230, show the lectin reactions typical for T. cruzi [31]. Because the epimastigote forms of T. rangeli react in agglutinationand fluorescence tests with only lectins of the mannose and glucose type and no labeling can be demonstrated on T. rangeli inelectron-opticalinvestigationswithgold-labeledlectins of RCA and SBA, these lectins are consequently of great importance in interspecific differentiation1511 (Table 1, Fig. 1). The investigation of the three T. cruzi strains, five strains of T. rangeli, and one strain of T. conorhini, with 27 radioiodinated lectins showedthat these trypanosomes apparently had receptors for all the lectins tested, although with differing affinities [52]. In this investigation, the possibility for interspecific differentiation was demonstrated. Among the developmental forms of T. cruzi and T. rangeli, only the flagellates ofT. cruzi, occurring inthe gut of reduviid bugs react withthe lectins fromRicinus communisand Glycine max (RCA-I1and SBA, respectively), whereasthe metacyclic salivary glandform of T. rangeli alsoreactswithRCA-I1 [50]. But inthesesalivaryglandformsof T. rangeli, binding with gold-labeled RCA-I1 could not be demonstrated[48]. Both Tvpanosoma species can also be distinguished on the basis of the fine structure of their kinetoplasts [53], as well as with the following methods: isoenzyme electrophoresis [54,55], restriction endonucleases [56], and monoclonal antibodies 1571. In comparison with the useof lectins, the aforementioned methods arevery cumbersome. It has been shownthat the trypomastigote metacyclic forms of T. rangeli possess an RCA-I1 receptor [50]. In contrast, the developmental forms of this parasite in blood, the gut, and hemolymph of reduviid bugs lackthis receptor. It cannot be ruled out that this receptor is vital for the adhesion of the flagellates to the epithelia of the salivary glands of the vectors, a prospect that, however, necessitates the presence of a corresponding lectin in the salivary glands.In T. congolense, the inhibition of the midgut lectinof Glossina morsitansby

o o + o o o o

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++a++++ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

oooo+oo

oooo+oo oooo+oo

+++++++ +++++++ +++++++ ++ooo++

++o++++ + + a + + + +

o+o+++o

+ooooo+ ++ooo++ ++ooo++

c-;c-;c-;c;c-;c-;c-;

Trypanosome-Ledin Interaction

c)

233

T. rangeli

l. cruzi

+

R. communis-'lectin G. max-lectin

L.-lectin

Figure 1 Differentiation possibilities of culture forms of T. cruzi and T. rangeli with lectins as well as tests for sialidase and sialic acid: ,positive reactions;0, no reaction.

+

234


D-glucosamine results in the inhibition of the metacyclic stage formation [26]. The interspecificdifferentiationof T. cruzi and T. rangeli is facilitated by the fact that onlytheculture forms of T. cruzi possesssialicacids that can be split by sialidase [23,29,30]. Sialidase abolishes reaction with antisialyl-specific lectinsof AAP [23,58,59], WGA [23,31,33,44] and LFA [31,49] (see Table 1). It is advisableto investigate the flagellates that are of importance in the epidemiology of Chagas’ diseaseby examination of the cell-freeculturefluids of the isolatedflagellates,withthe4-methylumbelliferylneuraminic acid test,for the presence of free sialidase[60,61]. Because onlyT. rangeli releases sialidaseinto the culture medium, it should be readily determined whether it is T. rangeli that has been isolated. Subsequent tests with lectins of LFA, RCA-11, or SBA will then show if T.cruzi is also present (see Fig. 1). In this way, it can be determined with ease if mixed infections of T. cruzi and T. rangeli are present. It has also been shown that culture formsof T. cruzi produce sialidase, which can be demonstrated by incubating the culture flagellatesincell-cellcontactwith erythrocytes [all. Enzyme damaged red blood cells agglutinate after incubation with PNA lectin; intact red blood cells do not show this reaction. B. Trypanosoma cruziand Trypanosoma conorhini

Results from several investigations have shown that the culture forms of T. cruzi and T. conorhini have numerous lectin reactions in common [31,62] (see Table 1). Investigations on the binding of 27 radioiodinated lectins[S21 have shown that, for all these lectins, binding sites have been foundon T. cruzi, T. conorhini, and T. rangeli. However, for differentiating these three trypanosome species, several lectins are unique: LYE, BAP, and SOJ for T. cruzi; ML-1-11-111 for T. rangeli; and PNA, UEA-I, and LOTUS for T. conorhini. The culture forms of T. cruzi react with lectins from HPA, in contrast with thoseof T.conorhini, which lackthis reaction [3 l] (see Table 1; Fig. 2). These differences can be used in agglutination and fluorescence tests for the differentiation of these two trypanosomes. Moreover, the epimastigote forms of T. conorhini show no agglutination reactions withthe antisialyl-specific lectins fromAAP and LFA, however, theydo absorb the two lectins and show positive fluorescence testsfor them. Treatment with pronase rendersthe parasites vulnerableto agglutination by the two lectins.. The absence of sialidaseon T. conorhini, as demonstrated by the Cmethylumbelliferylneuraminic acid test,and the failure to detect sialic acid, which can be liberatedby sialidase, providesa further possibility for the differentiation of this parasite from T. cruzi (see Fig. 2). Selective reaction of T. conorhini with the lectins from UEA-I, LOTUS, and PNA have been re-

Trypanosome-Lectin Interaction

235

T. conorhini

Sial idase Test

H..porhatialectin

T. cruzi PNA type

WGAPhilippinen type

T. conorhini

T. conorhini

Figure 2 Inter- and intraspecific differentiationof the epimastigote culture forms of T. cruzi and T.conorhini: positive reaction;0, no reaction.

+

236Aisien

and

Schottelius

ported [52]. In another report, the reaction of three strains of T. conorhini with PNA was described in which the anti-H-specific lectins reacted with only two strains[3 l] (see Table 1). C.

Trypanosoma conorhini and Trypanosoma rangeli

The interspecific differentiation of the epimastigote formsof these Tvpanosoma species has been facilitated by the fact that T. rangeli reacts with only lectins of the mannose and glucose type, whereas strains of T. conorhini react with other lectins (see Table1). In addition, that only T. rangeli releases sialidase into the culture medium, is another factor that can be used to differentiate these flagellates (Fig.3) [57]. D.

Trypanosoma vespertilionis and Trypanosoma dionisii

The epimastigote culture forms of both trypanosome species react with several lectins( C o d , LPA, LCA, PEA, HPA, SBA, RCA-11, M P ) [63]. Whereas T. dionisii strains react with WGA,T. vespertilionis strains agglu1; Fig. 4). The reactionof the two species tinate with PNA lectin (see Table AAP, LFA, and WGA ledto the demonwith the antisialyl-specific lectins [63,64]. As in T. cruzi, the presence stration of sialic acid in these flagellates [63]. In contrast of sialidasewas detected by the erythrocyte incubation test with the epimastigote culture forms, the amastigotes of T. dionisii strain P7 do not react with the antisialyl-specific lectins. Sialidase was also not detected in these amastigotes[63]. E.

Trypanosoma cruzi and Eat Trypanosomes

As shown bycomparing the results in Table1, the interspecific differentiation of European bat trypanosomes from T. cruzi using lectins,sialic acid, and sialidase has proved futile. Investigations of the autochthonous bat trypanosomes in Latin America with lectins for the differentiation of these species is stilloutstanding [3]. IV. INTRASPECIFIC DIFFERENTIATION A.

Trypanosoma cruzi (WCA Type, PNA Type)

In the course of the interspecific lectin differentiation of T. cruzi, it was observed that this parasite complex can be subdivided intraspecifically into WGA and PNA types [31,44] (see Fig.2). The reactionof the culture forms of T. cruzi with these two lectins has shown that the presence of sialic acid on the surface of theepimastigotestages [30] is ofgreatimportance.


I

T. ranaeli “

237

r

,“ T b o 1

T. conorhini

Sial idase Test

T

. conorh i n i

R. comnunis-lectin

G. max-lectin

l+,-, U T. rangeli

0

Figure 3 Interspecific differentiation possibilities of the culture forms of T. rangeli and T. conorhini: positive reaction;0, no reaction.

+

Whereas the PNA-type reacts with the PNA lectinand the antisialyl-specific lectins of LFA and AAP [49], binding to.the antisialyl-specific lectin of WGA, in-addition to the aforementioned lectins, is characteristic for the WGA-type. That the lectins of LFA, AAP, and WGA react only with the sialic acid of T. cruzi is evident because when the cells are treated with

=l-


238

T. vespertilionis

+

0

U T . dionisii

A. hypogaea-lectin

T . vulgaris lectin

Figure 4 Interspecific differentiation of the culture forms of T. vespertilionis and

T. dionkik

+ ,positive reaction;0, negative reaction.

sialidase, this treatment abolishes their ability to react with these lectins. However, reactivityis restored when these parasites are incubated in culture medium [33,37,63]. The Y-strain (WGA type), after treatment with sialidase becomes inactive, %ut subsequent incubation in culture medium restores their abilityto react with WGA [36].

Trypanosome-Lectin

239

Investigations show that the two lectin types of T.cruzi exhibit some differences in the incorporation of sialized glycoconjugates from culture medium or their hosts. The parasite must, of necessity, absorb these sialized conjugates from their host or from the culture medium because they lack the ability to synthesize sialic acid [34]. It is not clear, however, how the selection of these sialized glycoconjugates is realized [35]. The replacement of the lost sialic acid through incorporation of new sialoglycoconjugates proceeds slowly [65,66], a situation also applicable to T. cruzi 1251. The uptake of sialized glycoconjugates represents the uptake of foreign antigens. The fate of these antigens duringthe transformation of the parasites from the epimastigote to trypomastigote metacyclic forms,to blood forms, and then to tissue forms is unknown. Cell reaction with PNA, which is typical for the PNA type of T. cruzi, occursingeneralonly after the terminally bound sialic acid has been split away [67,68]. Because the WGA type of T. cruzi complex reacts with PNA only after treatment with sialidase [33], it can be assumed that the surfaces of the WGA type are better protected by sialic acidthan those of the PNA type. Thepartial removal of sialic acid from the erythrocytes’ cell surface facilitates their removal from the bloodstream [68]. It still needsto be investigated whetherthe trypomastigote blood forms of T. cruzi PNA type strains have a shorter stay in human and animal blood, or if they are less pathogenic in comparison with strains of the WGA-type. The subdivision of the epimastigote culture forms of T. cruzi strains of different originsinto two lectin types is in agreement with their subdivision into zymodemes Z1 and 22 [41,42,69-721. Analyses of detergent extracts made from eight strains of T. cruzi produced reaction with only PNA in the strains corresponding to zymodeme Z1 (PNA type) [40]. The intraspecific differentiation of the T. cruzi complex into two lectin types mirrors their reactions with monoclonal antibodies [73,74]. The twozymodemes have also been differentiated with antibodies [75]. It could be shown [76] that strains of T. cruzi, which belong to the two lectin types, are also in agreement with the two immunological groups A and B [77-791. The intraspecific differentiationof the T. cruzi complex with lectins apparently represents a differentiation between antigen types. However, it is not now possible to determine into how many antigenic typesthe New World strains of the T. cruzi complex can be allocated. The ConA precipitationof CHAPS detergent extract of radioiodinated epimastigote forms of four T. CmZi strains @-Brazil, G-Guatemala, Tehuantepec-Mexico,WA 301/130-Brazil) showed, in an SDS-PAGE gradient, that ConA receptorsare diverse, since they presented different band patterns. The RCA-Sepharose binding yielded similar results1631.


240

C. Trypanosomarangeli

The 12 investigated strains of T. rangeli, without exception, all react with C o d , PEA, and LCA.Otherlectinreactionshave not beenobserved [31,76].Intraspecific differentiation using lectins could not be established (see Table 1). Concanavalin A precipitation of CHAPS extracts from radioT. rangeli strain V (Venezuela) and iodinated epimastigote culture forms of Choachi 4 (Colombia) (see Table l), revealed no differences in band patterns detected inan SDS-PAGE gradient. However, differences were found in the isoenzymepatterns of T. rangeli strains [80]. D. Trypanosomaconorhini

Intraspecific differentiation of the three T.conorhini strains was possible only withthe anti-H-specific LOTUSand UEA-I, becausethe strains from the Philippines, but not those from Hawaii, react with them (see Table 1 and Fig. 2) [31].The extent of variability in the lectin reactions of this parasite is unknown, because only a few strains are available worldwide. By Cod-Sepharose binding of CHAPS extracts of radioiodinated epimastigotes of T. conorhini Hawaii, band patterns different from those of T. cruzi and T. rangeli strains were obtained from SDS-PAGE gradients.

E.

Trypanosoma vespertilionisand Trypanosoma dionisii

With the lectins presented in Table1, no intraspecific differences could be found between the strains of T. vespertilionis and T. dionisii. The lectin types of T. cruzi in relation to their geographic distribution, the clinical manifestation of Chagas’ disease, and their distribution in a domesticand sylvatic transmissioncycle are under investigation. 7.

United States

Here, Chagas’ disease presentsno medical problem, even though T. cruzilike flagellates [4]have been isolated in different states, from several wild animals, as well as in triatomid insects. The reason Chagas’ disease is of no significance in North America, despite the occurrence of the causative organism and its vector, is probably that the natural ecological balance between the wild animals and the triatomid-vectors has remained undisturbed. As a result, vectorsdo not become domesticated; consequently,no domestic cycle is established. The circumstances are totally the opposite for 16-18 million infected personsin Latin America [2] and approximately 100 million others at risk of infection?As shown by the lectin investigation of North American T. cruzi, two lectin typesare represented [31].A demarcation of the parasite into domestic and sylvatic cycles is nonexistent. Human

Trypanosomelectin Interaction

241

infection, therefore, is strictly zoonotic. The parasite has been isolated in three instances from humans,but this is very negligible in comparison with the prevailing situation in Latin America. 2. South America In the northern region of the Amazon in South America, only the PNA type of T. cruzi has been isolated [31,32]. Thesestrains were isolated from humans, domesticand wild animals, as well as triatomid insects. The occurrence of onlyone lectin type can be traced back to the absence ofa separate habitat for the reduviid bugs. A demarcation between the domestic and wildcyclesis not present. Even the randomly selected Venezuelan OPS strain of T. cruzi [31,32] showed no significant titer differences against PNA. This remarkable absenceof the WGA type is in agreement with the findings that in Venezuela only the zymodeme Z1 of T. cruzi is present [42]. Heart disease is a frequent cause of death, but megasyndromes are very rare in rural areas [81,82]. In Colombia, the situation is similar, in which the T. cruzi strains also belongto the PNA type[3 1,321.It is remarkable that in the northern regions of the Amazon, megaformations of the internal organs are unusual. The WGA type of T. cruzi is either rare or totally absent in these regions, with the PNA type preponderant. South of the Amazon region, megaformations of the internal organs have been found, particularly in central Brazilas well as in Minas Gerais, Goias, Bahia, and Sao Paulo areas [lo]. A comparison of the T. cruzi strains of patients from Iquatama (NW-Minas Gerais) [32]and Virgem da Lapa @E-Minas Gerais) [32] showed that the isolates from Iquatama belong preponderantlyto the PNA type, whereasthe strains from Virgem da Lapa were of the WGA type. The T. cruzi strains from Ribeirao Preto also belong to the PNA type, with the exception of two isolatesfrom bats and the Y-strain from Sa0 Paulo, which belongto the WGA type [32,33,44].It can be concludedfrom these results,that in the environs of RibeiraoPreto and Iquatama, the PNA type of T. cruzi is preponderant in the wild cycle and is transmittedto humans. In Ribeirao Preto, infection with T. cruzi is responsible for heart disin [83]. In this ease, a large number of persons, most of which result death area, the PNA type is also pathogenic for humans. In Minas Gerais, a transition from the PNA type to the WGA type occurs as one proceeds from the northwestern to the northeastern part. In Bahia and Sao Felipe, Salvador, the WGA type is restricted to humans (domestic cycle) and the PNA type to wild animals (forest cycle). This lectin characterization is in agreement withthe zymodeme typing[42] in which the domestic typeof T. cruzi belongs to the zymodeme 22 and the forest typeto zymodeme Z1. The three trypanosome strains isolated from naturally infected Tria-

242Aisien

and

Schottelius

toma infestam from a domestic cycle in Southern Brazilthe in state of Rio Grande do Sul,belong to the WGAtype[31,32,44].From12Chilean strains of T. cruzi, which belongto the domestic cycle,10 strains belong to the WGA type and only 2are of the PNA type [31,32]. That the latter two Chilean strains belongto the PNA type ofT. cruzi has also been confirmed by another working group [84]. The lectin characterization of the Chilean and Bolivian T. cruzi strains are also in good agreement with the isoenzyme characterization [72]. In Bolivia, T. cruzi from the domestic cycle belongsto both the PNA type, as well as the WGA type, a situation similarly true for T. cruzi strains from Peru. In contrast, the three T. cruzi strains from the domesticcyclein Ecuador all belongto the PNA type[31,32]. A correlation betweenthe two lectin types and the manifestation of Chagas’ disease is not so obvious in the Andes, probably reflecting the low number of investigated strains. Occasionally, in South America,there is a relation between the lectin type of T. cruzi complexes and the clinical manifestation of Chagas’ disease, aswell as a correlation betweenthe parasites and the forest or domestic cycles. The two lectin types correlate,to a large extent, with the zymodeme Z1 (PNA type) and 22 (WGA type). Forthe clinical manifestationof Chagas’ disease,the amastigote tissue formsor the bloodstream forms are responsible, but not the epimastigotes. Therefore, it can be presumedthat certain structuresof these developmental stagesT.of cruziare preserved dur[85,86]. ing the transformation process. Such structures have been described The intraspecific subdivision of the T. cruzi complex into two lectin types is a reflection of the fact that the culture flagellates react correspondingly differently with monoclonal antibodies [73,74]. The two zymodemes were also differentiated with monoclonal antibodies[75]. The T. cruzi strains Tulahuen and Esmeraldo, among others, were classified on this basis as belonging to zymodeme 22, which is in agreement with its allocation to the WGA type. Investigations into the nature of the antigens of different T. cruzi strains revealed that there are-threeimmunological groups (A,B, C), group C being the least represented [77-791. Strains Y , P-60, Tulahuen, and Maryland belong to type A, whereas strains FH4 and FH5 belong to type B [79]. On the basis of lectin investigations, it can be concluded that type A is equivalentto the WGA type, whereas typeB represents the PNA type [76]. With the intraspecific differentiation of the T. cruzi complex with lectins, there is a simultaneousdifferentiation between antigens types in these parasites. Only a few strains have been investigated from Costa Rica, Guatemala, and Mexico, most of them belongingto the PNA type. Because both types of lectins are present. in the United States, it can be concluded that the PNA type aswell as the WGA type of the T. cruzi complex are present in


243

North, Central, and South America, with one of the lectins being preponderant in particular regions. Most of the described experiments were carriedout with epimastigote cultural forms which do not come into question as pathogens. Therefore, it cannot be excluded that the investigation of trypomastigote bloodstream forms and amastigote tissue forms will lead to a better correlation with the geographic distribution of certain clinical manifestations of Chagas’ disease. REFERENCES 1. Lent H, Wygodzinsky P. Revision of the Triatominae (Hemiptera, reduviidae)

and their significance as vectors of Chagas’ disease. Bull Am Mus Nat Hist 1979; 193~125-520. 2. World Health Organization. Control of Chagas’ disease-report of a WHO expert committee. WHO Technical Report Series81 1.Geneva: World Health Organization, 1991. 3. Barretto MP. Epidemiologia. In: Brener Z, Andrade Z, eds. Trypanosoma cruzi e doenca de Chagas. Rio de Janeiro: Guanabara Koogan, 1979:89-151. A zoological monograph. Oxford: 4. Hoare CA. The trypanosomes of mammals. Blackwell Scientific,1972. 5. Sherlock IA. Vectores. In: Brener Z, Andrade Z, eds. Trypanosoma cruzi e doenca de Chagas. Rio de Janeiro: Guanabara Koogan, 1979:42-88. for research and train6. World Health Organization. WHO special programme ing intropical diseases. Chagas’ disease(1979-1981). 1983:139. la provincia de 7. CarvalloRU,deCelisMR.LaenfermedaddeChagasen Buenos Aires. Ministr Bien SOC La Plata 1972; 141. 8. Tafuri WL. Pathogenesisof Trypanosomacruzi infections.In:Lumsden WHR, Evans DA, eds. Biology of the Kinetoplastida,v01 2 1979547-618. 9. Maekelt A. El Cultivo “in vitro” de Trypanosoma cruzi. Caracas: Inst Med Trop FacMed, Univ Centralde Venezuela, 1981(monograph.) 10. Rezende de JM. Chagasic mega syndromes and regional differences. In: American trypanosomiasis research.PAHO Sci Pub11976; 318:195-205. 11. D’AllesandroA.Biologyof Trypanosoma(Herpetosoma)rangeli Tejera, 1920. In: Lumsden WHR, Evans DAYeds. Biology of Kinetoplastida. v01 1. London: Academic Press, 1976:32-403. 12. Dias E, Campos-Seabra CA. Sobre o Trypanosoma conorhini hemoparasito

do rat0 transmitido pelo Triatoma rubrofasciata. Mem Inst,Oswaldo Cruz 1943; 39:301-329. 13. Dias E, Neves 0. Determinacao do infeccao natural por 5 Schizotrypanum em

Triatoma rubrofasciata no estado de Pernainbuco.Mem Inst Oswaldo Cruz 1943; 39:331-335. 14. Lucena D. Infeccao natural do Triatoma rubrofasciata (de Geer) pelo. Trypanosoma.cruzi Chagas, 1909. Hospital (Rio de J) 1940; 18:91-93.

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15. Markinkelle CJ. Epidemiology of Chagas’ disease in Colombia. In: American trypanosomiasis research.PAHO Sci Pub1 1976; 318:340-346. 16. Alves MJ, Colli W. Agglutination of Trypanosoma cruziby concanavalin A. J Protozooll974; 21575-578. 17. Katzin AM, Del Pino EJ, Cunio RM, Raisman JS, Olmos J, Lajmanovich S, Gonzalez-Cappa SM. Receptores para lectinas enla superficie de epimastigotes de Trypanosoma cruzi. Medicine (Buenos Aires) 1979; 39:76-84. 18. Chiari E, de Souza W, Romanha AJ, Chiari CA, Brener Z. Concanavalin A receptors on the cell membrane of Trypanosoma cruzi. Acta Trop 1978; 35: 113-121. 19. Snary D, Hudson L. Trypunosoma cruzi cell surface proteins: identification of one majorglycoprotein. FEBS Lett 1979; 100:166-170. 20. Villalta F, Katzin AM, Leon W, Gonzales-Cappa ST. Concanavalin A binding receptors on Trypanosoma cruziamastigotes. J Parasitoll980; 66:1053-1055. 21. Szarfman A, Queiroz T. Mobility of concanavalin A receptors in Trypanosoma cruzi. J Parasitoll980; 66:1055-1057. 22. Araujo FG, Handman E, Remington JS. Binding of lectinsto the cell surface of Trypanosoma cruzi. J Protozooll980; 27397-400. 23. Pereira MEA, Loures MA, Villalta F, Andrade N B . Lectin receptors as markers for Trypanosoma cruzi. J Exp Med 1980; 152:1375-1392. 24. Katzin AM, Colli W. Lectin receptors in Trypanosoma cruzk an N-acetyl-Dglucosamine-containing surface glycoprotein specific for the trypomastigote stage. Biochim Biophys Acta 1983; 127:403411. 25. Pereira MEA, Andrade AFB, Ribeiro JMC. Lectins of distinct specificity in Rhodnius prolixus interact selectively with Ttypanosoma cruzi.Science 1981; 21 1:597-600. 26. Maudlin I, Welburn SC. The role of lectins and Trypanosoma genotype inthe maturation of midgut infections in Glossina morsitans. Trop Med Parasitol 1988; 39:56-58. 27. Ferguson MAJ, Snary D, AllenAK. Comparative compositions of cell surface glycoconjugates isolatedfrom Trypanosoma cruziepimastigotes. Biochim Biophys Acta 1985; 84299-44. 28. Snary D, Ferguson MAJ, Allen A, Sher A. Structure and function of a cell surface glycoproteinfrom Trypanosomacruzi. Mol BiolHost Parasite Interact 1984; 239-247. 29. Schottelius J. NANA specific lectins and the Aminoff test as a tool for the differentiation between T. cruzi and T. rangeli. Eur J Cell Biol Med 1983; 4(supp):18. 30. Schottelius J. Differentiation between Trypanosoma cruzi and Trypanosoma rangeli on the basis oftheir sialic acidcontent. Tropenmed Parasitoll984; 35: 160-162. 31. Schottelius J. Contribution to the characterization of South American Trypanosomatidue: I. The importance of lectins, neuraminic acid and neuraminidase for the differentiation of trypanosomes and Leishmania from the New World. Zoo1 Anz 1989; 223:67-81. 32. Schottelius J. Contribution to the characterization of South American Try-

Trypanosome-Lectin

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panosomafidae:11. The geographical distribution of the lectin types of the Trypanosomacruzi complex and their relationto the clinical manifestation of Chagas' disease. Zoo1 Anz 1989; 223:198-210. 33. Schottelius J, Uhlenbruck G. Comparative studies ofTrypanosomucruzi, and T. cruzi-like stocks from different South American countries using lectins. Z Parasitenkd 1983; 69:727-736. 34. Schauer R, Reuter G, Muhlpfordt M, Andrade A F B , Pereira MEA. The occurrence of N-acetyl- and N-glycolylneuraminic acid in Trypanosoma cruii. Hoppe Seylers Z Physiol Chem 1983; 364:1053-1057. 35. Confalonieri A N , Martin NF, Zingalis B, Colli W, Lederkremer M. Sialoglycolipids in Trypanosomacruzi. Biochem Int 1983; 2:215-222. 36. Previato JO,Andrade AFB, Pessolani MCW, Mendonca-Previato L. Incorporation of sialic acid into Trypanosoma cruzi macromolecules. A proposal for a new metabolic route. Mol BiochemParasitol1985; 16:85-96. 37. Schottelius J, Marinkelle CJ, Gomez-Leiva MA. Comparative investigations of Latin American trypanosomes with lectins and complement lysis test.Trop Med Parasitoll986; 3754-58. 38. Piras MM, HenriquesD, Piras R. The effect offetuin and other sialoglycoproteins on the in vitro penetration of Trypanosoma cruzi trypomastigotes into fibroblast cells. Mol BiochemParasitoll987; 22:135-143. 39. Scharfstein J, Luquetti A, Murta ACM, Senna M, de Rezende JM, Rassi A, Mendonca-Previato L. Chagas' disease: serodiagnosis with purified g p 25 antigen. Am J Trop Med Hyg 1985; 34:1153-1160. 40. Stevens AF, Miles MA, Allen AK. Trypanosomacruzk studieson the interactions of lectins with glycoconjugates of different zymodemes. Exp Parasitol 1988; 67:324-333. 41. Miles MA, Toye PJ, Oswald SC, GodfreyDG. The identification of isoenzyme patterns of two distinct strain-groups ofTrypanosoma cruzi circulating independently in a rural area of Brazil. Trans R SOCTrop Med Hyg 1977; 71:217225. 42. Miles MA, Povoa MM, Prata A, Cedillors RA, de Souza AA, Macedo V. Do radically dissimilar Trypanosomacruzi strains (zymodemes) cause Venezuelan and Brazilian forms of Chagas'disease. Lancet 1981;1:1338-1340. 43. Miles MA, S o w A, Povoa M, Shaw J, Lainson R, Toye PJ. Isoenzymatic heterogeneity of Trypanosoma cruzi in the first autochthonous patients with Chagas' diseasein Amazonian Brazil. Nature 1978; 272:819-821. 44. Schottelius J. The identification of lectins of two strain groups of Trypanosoma cpzi. Z Parasitenkd 1982; 68:147-154. 45. Harth G, Hidaris G, So M. Purification and characterization of stage specific glycoproteins from Trypanosomacruzi. Mol BiochemParasitol 1989; 33:143150. 46. Couto AS, Goncalves MF, ColliW,Lederkremer R" The N-liked wbohy&ate chain of the 85-kilodalton glycoproteinfrom Trypanosomacruzi trypomastigotes contains sialyl, fucosyl and galactosyl(ol-l,3)galactose units. Mol Biochem Parasitol1990; 39:lOl-108. 47. Yoshida N, BlancoSA, Araguth MF, Russo M, Gonzalez J. The stage-specific

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48. 49.

50.

51. 52. 53. 54. 55.

56.

57. 58. 59.

60. 61.


90 kilodalton surface antigen of metacyclic trypomastigotesof Trypanosoma cruzi. Mol BiochemParasitol1990; 39:39-46. Rudin W, Schwarzenbach M, Hecker H. Binding of lectins to culture and vector forms of Trypanosoma rangeli(Tejero, 1920 Protozoa, Kinetoplastida) and to structures of the vector gut. J Protozooll989; 36532-538. Schottelius J. Limaxflavus agglutinin: a new toxonolectin for the identification of Trypanosoma cruzi. the agent of Chagas’ disease. In: Bsg Hansen TC, van Driessche E, eds. Lectins: biology, biochemistry, clinical biochemistry, v01 5. Berlin: Walter deGruyter, 1986579-587. Marinkelle CJ, Vallejo GA, SchotteliusJ, Guhl F, de Sanchez N. The affinity of the lectins Ricinus communisand Glycine maximato carbohydrates on the cell surface of various forms of Trypanosoma cruziand Trypanosoma rangeli and the application of these lectins for the identification of T. cruzi in the feces ofRhodniusprolixus.Acta Trop 1986; 43:215-223. Schottelius J, Miihlpfordt H. Carbohydrates as markers for Trypanosoma cruzi and Trypanosoma rangeli. Hoppe Seylers Z Physiol Chem 1984; 365: 1061. Miranda-Santos IKF, Pereira MEA. Lectins discriminate between pathogenic and nonpathogenic South American trypanosomes. Am J Trop MedHyg 1984; 33:839-844. Muhlpfordt H. Vergleichende kinetoplastmorphologie verschiedener trypanosomenarten unter besonderer Beriicksichtigung von Trypanosoma cruzi. Tropenmed Parasitol1975; 26:239-246. Kreutzer RD, de Souza 0. Biochemical characterization of Trypanosoma ssp by isoenzyme electrophoresis.Am J Trop Med 1981; 30:308-317. Ebert F. Isoenzynme studies on Leishmania stocks from Peru by ultrathinlayer isoelectrofocusing.Trop Med Parasitoll987; 38:37-40. Frash ACC, Goijman SG, CazzulaJJ, Stoppani AOM. Constant and variable regions in DNA minicircle from Trypanosoma cruziand Trypanosoma rangeli: applications to species and stock differentiation. Mol Biochem Parasitol 1981; 4~163-197. Anthony RL, Cody TS, Constantine NT. Antigenic differentiation of Trypanosoma cruzi and Trypanosomarangeli bymeans of monoclonal-hybridoma antibodies. Am J Trop Med Hyg 1981; 30:1192-1197. Muhlpfordt H, Schottelius J. Agglutinationsverhalten von T. cruzi, T. cruzilike stiimmen, T. rangeli und T. conorhini mit dem lektin von Soja hispida und dem Aaptospapillata protektin. Tropenmed Parasitoll977; 28:l-7. Bretting H, Schottelius J. Immunfluoreszenzmikroskopischeunterscheidung zwischen T. cruzi, T. cruzi-like stiimmen, T. conorhini und T. rangeli mit dem protektin des schwammesAaptospapillata. Z Parasitenkd 1978; 57:213-219. Schottelius J. Thiobarbituric acid/menthylumbelliferyltest for thedifferentiation of Trypanosoma cruziand Trypanosoma rangeli.Zentralbl Bakteriol Hyg 1987; A265:522-523. Schottelius J. Neuraminidase fluorescencetest for theinterspecific differentiation of Trypanosoma cruzi Chagas, 1909 and Trypanosoma rangeli Tejero, 1920. Trop Med Parasitol 1987; 38:323-327.

Trypanosome-Lectin

247

62. Schottelius J, Muller V. Interspecific differentiation of Trypanosoma cruzi, Trypanosoma rangeli and Trypanosoma conorhiniby lectins in combination with complementlysis. Acta Trop 1984; 41:29-38. 63. Ziegenhagen S. Anwendung von lektinen und sialidase fur die inter- und intraspezifische differenzierung von arten der gattung schizotrypanum, megatrypanum, und herpetosoma (Trypanosomatidae, Kinetoplastidae).Universitat Hamburg, Fachbereich Zoologie, Diplomarbeit,1989. 64. Schottelius J, Koch 0, Uhlenbruck G. Differentiation of Trypanosoma cruzi Chagas, 1 9 0 9 and Trypanosoma vespertilionisBattaglia, 1904 by various lectins. Tropenmed Parasitol 1983; 34:89-92. 65. Warren L, Glick MC. Membranes of animal cells. 11. The metabolism and turnover of the surface membrane. J Cell Bioll968; 37:729-746. 66. Hughes RC, Sanford BH, Jeanloz RW. Regeneration of the surface glycoproteins of a transplantable mouse tumor cell after treatment with neuraminidase. Proc Natl Acad Sci USA1972; 69:942-945. 67. Prokop 0 , Uhlenbruck G. Lehrbuch der menschlichen blut- und serumgruppen. Leipzig: Georg Thieme, 1966. 68. Schauer R, Sialic acids: chemistry, metabolism and function. Cell Biol Monogr, v01 10. Wien: Springer Verlag,1982. 69. Miles MA, Lanham SM, de Souza AA, Povoa M. Further enzymatic characters of Trypanosoma cruziand their evaluation for strain identification. Trans R SOCTrop Med Hyg 1980; 74:221-237. 70. Barrett TV, Hoff RM, Mott KE, Miles MA, Godfrey DG, Teixeira R, Souza AA, Sherlock IA. Epidemiological aspects ofthree Trypanosoma cruzizymodemes in Bahia State, Brazil. Trans R SOCTrop Med Hyg 1980; 74:84-90. 71. Ebert F. The identification of two maingroups of Trypanosoma cruzistocks from Brazil by their isoenzyme patterns of isoelectrofocusing. Tropenmed Parasitol 1982; 33:140-146. 72. Ebert F, Schaub G. The characterization of Chilean and Bolivian Trypanosoma cruzi stocks from Triatoma infestansby isoelectrofocusing. ZParasitenkd 1983; 69:283-290. 73. Petry K,Baltz T, SchotteliusJ. Differentiationof Trypanosomacruzi,Trypanosoma marinkellei, T. dionisii, and T. vespertilionis by monoclonal antibodies. Acta Trop 1986; 435-13. 74. Petry K, Schottelius J, Baltz T. Purification of metacyclic trypomastigotesof Trypanosoma cruziand Trypanosoma dionisiifrom culture using an epimastigote-specific monoclonalantibody. Parasitol Res 1987; 733224-227. 75. Flint JE, Schechter M, Chapman MD, Miles MA. Zymodeme and speciesspecificitiesofmonoclonal antibodies raisedagainst Trypanosoma cruzi. Trans R SOC Trop Med Hyg 1984; 78:193-202. 76. Schottelius J. Trypanosoma cruzi, Trypanosoma rangeli und Trypanosoma conorhini: inter- und intraspezifische differenzierung ihrer kulturflagellaten mit hilfe von lektinen.Habilitationsarbeit, Universitat Hamburg, Fachbereich Zoologie, 1988. 77. Cappa SMG, Kagan IG. Agar geland immunoelectrophoretic analysisof several strains of Trypanosoma cruzi. Exp Parasitoll969; 2550-57.


248

78. Nussenzweig V, Deane LM, Kl6tzelJ. Differences in antigenicconstitution of strains of Trypanosoma cruzi. Exp Parasitoll963; 14221-232. 79. Nussenzweig V, Goble FC. Further studies on the antigenic constitutions of strains of Trypanosoma (Schizotrypanum)cruzi. Exp Parasitol 1966; 18:224230. 80. Ebert F. Isoenzymes of Trypanosoma rangelistocks and their relationto other trypanosomes transmitted by triatomine bugs. Trop Med Parasitol 1986; 37: 251-254. a epidemiologia de la enfermedad de Chagas en Venezuela. In: 81. Pifano F. L 82. 83. 84. 85. 86.

SociedadArgentina de Parasitologia-simposio intemacionalsobre enfermedad de Chagas, Buenos Aires, 1972217-223. Mizrahi CIH. Megaesofago. Arch Hosp Vargas 1962; 4299-319. Amorin DS, MancoJC, GalhoL, Net0 JM. Clinica: forma cronicac cardiaca. In: Brener Z, Andrade Z, eds. Trypanosoma cruzi e doenca de Chagas. Rio de Janeiro; Guanabara Koogan, 1979:265-311. Zimmermann D, Peters W, Schaub G. Differences in binding of lectin-gold conjugates by Trypanosoma cruzi and Blastocrithidia triatomae (Trypanosomatidae) in the intestine ofTriatoma infestans.Parasitol Res 1987; 745-10. Tachibana H, Nagakura K, Kaneda Y.Species-specific monoclonal antibodies from membrane antigens inall developmental stagesof Trypanosoma cruzi. Z Parasitenkd 1986; 72:433-441. Tachibana H, Montenegro LT, Kurihara K,Nagakura K, Kaneda Y,Komatsu N. Localisation of the Trypanosoma cruzi specific M, 25,000 antigen by immune electron microscopy using monoclonal antibodies.Z Parasitenkd 1986; 721701-707.

8 lectin Sorbents in Microbiology V. M. IAKHTIN Institute for Applied Science of Moscow University and Institute of Food Substances, Russian Academy of Medical Sciences, Moscow, Russia

1. INTRODUCTION

According to Kocourek and HofejSi, Zectins are proteins of nonimmunoglobulin origin that are capable of specific recognition or of a reversible binding to carbohydrates (glycosyl groups), without altering the covalent structure of the recognized glycosyl ligands[l ,Therefore, 2]. in accordance with these authors, lectins are carbohydrate-binding proteins, withat least one sitefor carbohydrate recognition (monovalent), such as some vegetable or microbial toxins, or receptor lectins (chemotaxis receptors and others). a domain or an Enzymes of carbohydrate metabolism possessing either epitope capableof specific carbohydraterecognition, independentof a catalytic center, should also be considered true lectins [3,4](see Chapter 1). Glycosyltransferases in the absence of acceptors will behave as true lectins in the recognition of donor carbohydrates [5-71.Lectinlike properties can be demonstratedby other microbial enzymes of carbohydrate metabolism, such as hexoseoxidase and someglycosidases [8-lo]. Otherexamples 1,well as those of lectinlike subof lectinlike proteins are known [l 1,12as stances of a nonproteinnature [12-151. Lectin preparations have now been obtainedfrom more than 500 dif220 species of higherand lower plants[12,16ferent species, including over 191. Unique biological properties,the polyfunctional nature of lectins, and their complexes with endo-or exogenic bioeffectors, indicatethe necessity to continue screening for new sources of these proteins [12,20],especially among marine organisms[21],microorganisms [22-241,and mammals. Lectins are important for a variety ofprocesses and uses [20,25]. Among traditional uses, are the simplificationand improvement of technol249

250

Lakhtin

ogies for isolation of lectins and isolectins from microorganisms, including the use of lectinsas bioeffectors and the use of lectin sorbents.Newer uses include (1) cloning of lectin genes in microorganisms (e.g., in bacteria and yeasts), in cells of higher plants, in invertebrates, or vertebrates; cloning with other effector genes; cloning of toxic fragments or subunitsof lectin molecules in combination with other effector genes; cloning of sorption domain genes (polysaccharides or glycosyl sorbents), together with genes of other proteins (e.g., of glycosidases or alkaline phosphatases); (2) hybridization of subunits of lectin molecules with effectors to obtain bi- or polyfunctional conjugates (chimeric molecules); (3) use of lectin-glycoconjugate interactions with involvement of liposomes (e.g., in micellar enzymology); (4) chemical modification of antigenic, glycolipid, adhesive, or some other specificity of fimbrial, receptor, toxic,or other lectinsfor regulation and control of effector properties, creation ofnewvaccines, (5); synthesis of peptides and glycopeptides characterized by the antigenic properties of microorganisms’ lectins to obtain artificial microbial vaccines or highly effective antimicrobial antibodies; (6) development of highly sensitive methods of microanalysis based on lectin-glycoconjugate interactions aimed at development of biosensor technology (e.g., biosensors of glucose on the basis of immobilized ConA), lectin variants of the enzyme-linked immunosorbent assay (ELISA), ELLA (see Chapter l), methods employing blotting techniques, affinity electrophoresis in gels, and lectin chromatography; hybridization (fusion) of cells (protoplasts) depending on lectin-glycoconjugate intercellular interactions to obtain new microbial products of biologically active substances. Lectins now find most of their applications in areas such as biotechnology,medicine, and biology [for somerecentreviews, see 12,20,25-271. Lectins are widely used when dealing with viruses, bacteria, protozoans, yeasts, and fungi [12,13,28]. Lectins also seem to be applicable in medical microbiology for visualization, typing,and determination of mutant strains or isolates, as well as for determination of taxonomic relations between microorganisms [14,15,28,29; see also other chaptersin this book]. Lectins are also used in analysis of the surfaces of microorganismsthat are important for agrobiology, such as rhizobial bacteria, phytopathogens, entomopathogens, pathogens for agricultural animals, food contamination, and brewer’s and distillery yeasts. The overwhelming majority of publications on applications of lectins in studies of microorganisms include data on the use of free (nonimmobilized) or labeled lectins in agglutination, in adhesion tests, and in those of complex formation on cellular surfaces of microbes, or in variantsof microassays of glycoconjugates. However, the data on the use of immobilized lectins (lectin sorbents) in investigation of microbial

lectin Sorbents

251

glycoconjugates, especially receptors and enzymes, are rather limited [12, 27,301.

The purpose of this review is to summarize findings connected with the use of immobilized lectins in handling microbial glycoconjuates. II. LECTIN SORBENTS IN RESEARCH O F MICROBIAL CLYCOCONJUCATES A. Advantages of Lectin Sorbents for the Study ofClycoconjugates

Lectin sorbents are preferable for study of glycoconjugates, rather than free or labeled lectins, because of the following features: 1. The possibility of purification

or immobilization of the investigated glycoconjugates ina complex biopolymer mixture:A glycoconjugate is immobilized through its carbohydrate moiety, which, as a rule, is re(aswith enzymes)or any other biologically mote from a catalytic center active site.As a result of such noncovalent-directedadsorption on lectin sorbent, one can obtain preparations of immobilized biologically active effectors (glycoproteins or glycosylated subunits and polypeptides: supramolecular receptor complexes; membranes, organelles, or cells) with their simultaneous purification from crude extracts, partially purified samples of glycoconjugates, cell or membrane-containing suspensions. 2. Lectins are convenient for the separation of glycoconjugates that are similarinphysicochemicalproperties(molecularmass,isoelectric point, amino acid composition,carbohydrate content or composition) owing to the peculiar oligosaccharide structures of ligands. Table 1 contains numerous examples of such separations of variousoftypes enzymes or isoenzymes,separationofantigenicglycoproteins and of polysaccharide-containing materials,and other uses. The possibilityof usingcombined(sequential)chromatography of glycoconjugates on lectin sorbents with preservationof the initial amountsof these glycoconjugates for multiple use is attractive. One can obtain a kit of tested glycoconjugatesthat have differentaffinity for an array of lectins, with the simultaneous resolution of glycoconjugate oligosaccharide types recognized bythe lectin sorbents chosen. 3. The repeated use of lectin sorbents during simple procedures of sorbent regeneration enhances their value. 4. Weak affinityinteractions ofglycoconjugateswithlectinsorbents

Lakhtin

252

Table 1 Interaction of Microbial Glycoconjugates with Lectin Sorbents

glycoconjugates Microbial

(GC)"

Cyaaobacteria Oscillatoriaprinceps Phosphorylase A2 from CF Phosphorylase A, from CF Mierophytic algae Phaeodactylum tricornutum Chlorophyllase from Mb

Lectin sorbent@) Ref.commentsb

conA+ (two forms) cod-

31 31

conA+ (two forms);

32

conABacteria Bacillus subtilis 3 16M Hemagglutinin (GP) fromCF B. subtilh168 a-Glc-containing teichoic acid from cell walls B. subtilis W23 Teichoic acid linkedto peptidoglycan fromcell walls B. sphaericus 1593 Larvicidal toxin (GP)from spore coat B. stearothermophilusATCC 12016 Glucoamylase from CF Bordetellapertussis Adenylate cyclasefactor from Mb Bordetella sp. Surface hemagglutininAeukocytestimulating factor Brevibacterium linensAC 480 a-Glc-containing teichoic acid from cell wall GlcNAc-containing teichoic acid from cell wall GalNAccontaining teichoic acid from cell wall Campylobacterfetus VC 119 Surface array protein (131 kDa) Capnocytophagaochracea 25 Mannan-containing polysaccharide from CF

conA+

33

conA+

34

WGA+

35

conA+

36

conA-

37

WGA+

38

Lectin+

39

cod+

40

WGA-

40

HPA-

40

conA-

41

conA+

42

lectin Sorbents

253

Table 1 (Continued)

Lectin sorbent(s)

mmentsb glycoconjugates (GC)' Microbial CellulomonasJimi Endo-b-l,4glucanases (56,58 kDa) from CF Endo-0-1 &glucanase (48 kDa) from CF Cellulomonassp. ATCC 21399 Endo-0-l,4glucanases (49,53,67, 78 kDa) from CF Endo-0- 1,4glucanase (1 18kDa) from CF Enterococcusfaecalis Kiel27738; E. hirae ATCC 9790 Escherichia coli12 Heat-labile enterotoxin/hemagglutinin from spheroplast periplasmic space Flavobacterium sp. Isoamylase fromCF Lactobacillus fermentum737 Adhesin (12-13 kDa, GP) from CF Leuconostoc mesenteroidesDSM 20343 (a-Glc-) 14containing lipoteichoic acids from cell walls Micrococcus lysodeikticus Lipomannan(s) from Mb F1-ATPase from Mb Mycobacterium leprae

0-N-Acetylglucosaminidase 0-Glucuronidase M. tuberculosis H37/Rv; H37/Raantigen (38 kDa, GP) fromCF Periodate-treated antigen Pneumocystis carinii(taxonomy not defined) Surface antigens(31-176 kDa) from infected ferret lung exudate Spiroplasma citri GP(84 kDa)from Mb

conA+

43

conA-

43

conA+

44

conA-

44

WGA'

46

(Glycogen-ConA)'

47

conA-

48

ConA+ (few forms)

45

Cod+ COnA -

49 49

conA+ conACOnA

50 50 51

+

COnAConA', WGA', PWM', SBA-, LOTUS

52

conA+

53

Lakhtin

254

Table 1 (Continued)

Lectin sorbent(s) Ref.commentsb glycoconjugates (GC)" Microbial Staphylococcus aureus830; Wood46 a-Ribitolacid teichoic from cell conA+ wall @-Ribitol teichoic acid from cell conAwall GlcNAc-containing 8-ribitol teiConA--WGA+ choic acidfrom cell wall S. faecium ATCC 9790 Autolysin latent form (130-kDa, ConA' GP) from cell wall S. mutans HS 6 (serotype"a") Glucosyltransferases (161, 174 ConA+ (nonspecific) kDa) from CF a-l,3-D-Glucan Synthase conA+ Streptococcus sp. (group b) Polysaccharides Ia, Ib, 11, I11 withWGA' specificity ofgroup B streptococci Streptococcus sp.(groupC)antigensDBA+ Protozoans Chlamydomonas reinhardtiiRC 3 Sexualagglutinin from gametic fla- C o d + gella Entamoeba histolytica a-Amylase (3 kDa) 1 from CF conA+ a-Amylase (15 kDa) from CF conAGiardia lambliaWB SurfaceGP:A, B, C, D from troWGA' phozoites or cysts Isotricha prostoma 8-Fructofuranosidase from cells ConA+ (three forms) Leishmania adleri(promastigotes) Intracellular galactomannans: Man/Gal = 12:l (Mo1:Mol) conA+ Man/Gal = 1:2 (M/M) conA+ Man/Gal (M/M) = 1:s conAL. braziliensis, L. rnexicana (promastigotes) Acidphosphataseisoenzymes fromConA+ Mb

54,55

56 58

58 59,60 61 62 63 63 64

65 66 66 66 67

lectin Sorbents

255

Table 1 (Continued)

Lectin sorbent@) glycoconjugates Microbial

Ref.commentsb (GC)"

L. donovani (promastigotes) Glycoconjugates (GC)from CF GC (10-130 kDa) GC (50-130 kDa) GC (43-130 kDa) GC (55-130 kDa) Acid phosphatase (110 130 kDa)

+

3 '-Nucleotidase/nuclease(43 kDa) Acid phosphatase (97 149 kDa) from strain Sudanese Phosphogalactan (23 kDa) Surface lipophosphoglycans(10-20 kDa) L. mexicana Acid phosphatase (70 72 kDa) Glycolipids L. mexicana rnexicana(amastigotes) Intracellular cysteine proteases: Enzymes ofgroups A and M Enzymes of groups B and C Enzymes of all groups L. tarentolae LV-414 (promastigotes) Gal-rich GP (22 kDa) from cell surface L. tropica (promastigotes) Acid phosphatase from CF Phosphoglycan from CF Leishmania sp. (L. mexicana amazonensis, L. mexicana mexicana,and others) Promastigote surface proteasehew tral Zn-containing metalloprotease (63 kDa, GP) Paramecium tetraurelia5 1S Ca+2-ATPase(68 + 53 kDa) from CF Tetrahymenapyr.$ormis HSM

+

+

8-N-Acetyl-D-galactosaminidaseA

conA+ LCA+ RCA-I+ PNA' ConA', LCA+, RCA-I+, PNA+

conA+ LCA', RCA-I-LCA'

68 68 68 68 68 68a 69,70

RCA-1' RCA-11' (two forms)

71

conA+ conA+

71a 71a

conA+ conALCA-, PNA-

72 72

conA+

73

LCA-B+ LCA-B+

74 74

ConA', LCA'

75,76

ConA+ (two forms)

77

COnA'

78

conA-

78

(M, 170 kDa) from CF

8-N-Acetyl-D-galactosaminidaseB (M, 93 kDa) from CF

Lakhtin

256

Table 1 (Continued) glycoconjugates Microbial

(GC)”

Trypanosoma brucei Variant surface GP (54 kDa) T. brucei bruceivariant AnTat 1.1 Surface GP (94,60 kDa) T. brucei bruceivariants Etat3-V1, ETat3-V2surface GP (57-58 kDa) T. congolense Lister 1/148; 423 Variant surface GP (57 kDa) T. cruzi Maracay (metacyclic cells) GP (64 kDa) from CF T. cruzi (trypomastigotes) Intracellular GP (85 kDa) T. cruzi (epymastigotes) Stage-specific surface GP (72 kDa) T. cruzi (metacyclic cells) Stage-specific surface GP (90,85, 55 kDa) Stage-specific surface GP (55 kDa) T. cruzi (epymastigotes) Lysosomal cysteineprotease (60 kDa) surface GC from cells growing in the presence offetal serum Surface GCfrom cells growing without fetal serum Yeasts Candida albicanstype A strain B 3 11 Cytoplasmic antigen(54 kDa, GP) Mannan C. albicans 49 18 C3d receptor(60 kDa) C. Iipobtica Liposan/proteoglucan (28 kDa) c . utillk exo-fl-D-~hcanasefrom cells exo-fl-D-Glucanase treated by aglucosidase C. wickerhamii NRRL Y-2563 P-Glucosidase (94kDa, GP) from CF

Lectin sorbent@) Ref. commentsb conA+

79

conA+ (two forms)

80

LCA+

81

LCA+, ConA+

81,82

WGA+

83

WGA+ (two forms)

84

WGA”C0nA’

85

WGA+

85

WGA+-ConA+

85

conA+ WGA’

86 87

ConA+, WGA-

87

conA-

88 88

conA+ c o d + , LCA+, WGA+, GS-I-

89

conA+

90

conA-

conA+

91 91

conA+

92

Lectin Sorbents

257

Table 1 (Continued)

Lectin sorbent(s)

mmentsb glycoconjugates (GC)' Microbial Ctyptococcusalbidus CCY 1 7 4 1 Endo-p-l ,rl-D-xylanase from cell wall p-Xylosidase from cell wall Kluyveromycesfragilis ATCC 12424 8-Fructofuranosidase (GP) from CF Lipomycesstarkeyi ATCC 20825 Dextranase (74 71 68 65 kDa, GP) from CF Paracoccidioides braziliensisB 339 Antigen A2 (43-72 kDa) from CF Rhodosporidium toruloidesI F 0

+

+

+

93 93 94

ConA', LCA+, WGA-

95

conA+

96

conA+

97

0880" 1057

Surface Ca+'-dependent signaling peptidase (63 kDa) from cells with mating type"a" Rhodotorula glutinis Acid phosphatase (93 kDa, GP) from CF Rhizopus sp. 0-Fructofuranosidase Saccharomyces diastaticus5 106-9A Glucoamylase (68 59 + 53 kDa, GP) S. cerevisiae y-Glutamyltranspeptidase (64 23 kDa, GP) fromcells Phospholipase B (GP) from CF Alkaline phosphatase (46 + 66 + 92 kDa, GP) from cell vacuoles Acid phosphatase (GP) fromcell wall Glucoamylase (79 75 72 69 kDa), from cells of wellsporulating diploidstrains SKI and AP 1 Endochitinase (14 kDa, GP) from cell wall Trimming glucosidaseI from cytoplasmic microsome Mb

+

+

+ + +

LCA'

98,99

conA+

100

conA+

101

LCA+ (two forms)

102

conA+ conA+

103

conA+

105

LCA'

106

conA+

107

cod+

108

104

Lakhtin

250

Table 1 (Continued)


(GC)"

8-Fructofuranosidase from cells Trehalase (acidform) from cells Trehalase (neutral form) from cells Exo-0-glucanase(45kDa) from spores Exo-P-l,3-~-glucanasefrom CF Form 59 kDa (GP) Form 43 kDa Carboxypeptidase Y (62kDa, GP) Ca+'-dependent cysteine protease from Mb of haploid a-cellsX2180-1B Three forms (GP) from CF Cytoplasmic form Cell wallmannoproteins (29kDa and others) from yeast strain X2180-1A Mannoproteins from CF of strains FY;NFY SchizosaccharomycespombeATCC 24843 Galactosyltransferase (61 kDa) from Golgi Mb of fission yeast 8-Fructofuranosidase from cytoplasm GP I and I1 (33-34kDa) from cell wall Schwanniomyces alluviusATCC 26074 a-Amylase (52kDa, GP) from CF Fungi AIIomyces arbuscula Ca+'-Activated neutral cystein protease (40kDa, GP) from mycelium Alternaria aIternata &Glucosidase (80 70 kDa) from CF Aspergillus candidusvar aureus Glucoamylase (73 kDA) from CF A. fumigatus NCPF 2140 Antigen GC from mycelial surface

+

Lectin sorbent(s) Ref. commentsb ConA', ConA- PA-11' conA+ conAconA-

109,100 110 109 109 111

conA+ conA conA+ conA+

112,113 112,113 114,115 116

ConA', LCA' ConA+, LCA+ conA+

117 117 118,119

conA+

120

ConA', GS-I+

121

conA+

122

conA+

123

cod+

124

conA+

125

conA+

126

conA+

127

conA+

128

lectin Sorbents

259

Table 1 (Continued)


(GC)'

A. fumigatus F92 Antigen (58 kDa, GP) responsible

for invasive aspergillosis,from mycelium A. niger Glucose oxidase Lactonase (70 kDa, GP) from CF GlucoamylaseG1 (52-110 kDa, GP) from CF fi-Glucosidase (GP)from CF 8-Fructofuranosidasefrom CF a-L-Arabinofuranosidase (61 kDa) from CF Some proteasesand amylases from CF A. oryzae S1-Nuclease (32 kDA) from CF RNAse T2 (36 kDa, GP) from CF a-Galactosidase from CF

8-N-Acetyl-D-glucosaminidase

Lectin sorbent(s) Ref. commentsb conA+

129

conA+ PA-11' conA+ conA+

100,130 110 131 132

conA+ conA+ ConA+ (three forms)

131,133 134 135

cod-

134

conA+ conA+ conA+ conA+

136,137 138,139 140 140

conA-

140

conA+

conA+

141 141

conA+

142

conA+

143

COnA'

144

from CF

Endo-8-N-acetyl-D-glucosaminidase from CF A. tamarii IP 1017-10 a-Galactosidase (56 kDa, GP) 8-Mannanase (53 kDa, GP) A . terreus Glucoamylasefrom CF of strain GTC 826 &Glucosidase from CF Botrytis cinerea /3-iV-Acetyl-D-glucosaminidase (70 kDa) from CF Cephalosporium acremonium237 a-Galactosidase/hemagglutinin (GP) from CF P-N-Acetyl-D-glucosainidase from CF Ceratocystis ulmi (Baisman); C. moreau Phytotoxic proteoglycan from CF

ConA', LCA' WGA', PHAconA+ conA+

145,146 145 147

Lakhtin

260

Table 1 (Continued)


(GC)”

Lectin sorbent($ Ref.commentsb

Chaetomium cellulolyticum Endo-j3-1,4-glucanase from CF cod+ cod+ &Glucosidase from CF Cellobiohydrolase from CF Cod+ .C. thermophile isolate 0-453 from oxidase Polyphenol CF cod+ Coccidioides immitis Antigen (120 kDa) 110 from the C o d ’ inner conidial wall Colletotrichum Iagenarium Cutinase “a” (60kDa) CFfrom conA+ Cordyceps ophioglossoides Galactomannan CF from cod+ complex with GP its isolated from Cod’ galactose-aminoglycan from CF Dichomitus squalens(Karst) Reid CBS 432.34 a-L-Arabinofuranosidase (60 kDa) Cod+ from CF Dictyostelium discoideum Alkaline phosphataseM ’Cod+ nucleotidase from Mb of vegetative cells Cyclic GMP-dependent, GMPcod+ specific phosphodiesterase from cells CyclicAMP-hydrolyzingphosphoCod’ diesterase from cells Cyclic nucleotide phosphodiesterConA’ ase (55 kDa) from CF Cyclic nucleotide phosphodiesterCod’ ase inhibitoryGP (47 kDa) from CF Trehalase (97 kDa, GP)from cellsConA’ ATCC 24697 a-Galactosidase from vegetative ConA’ cells Folate deaminase (40 kD) from CF LCA’ (two form) of cellsAX2 Adhesin(s) (95 45 kDa)withstage WGA’ specificity from plasma membrane of cells

+

+

148 148 148 149 150 151 152 152

153 154 155 155

156 157 158 159 160 161

261

Lectin Sorbents Table 1 (Continued)


Ref.commentsb (GC)"

Discoidin-I+, RCA-I+, Receptor inhibitor (80 kDa) of ABP+ cell-cell cohesion from cell Mb Stage-specific antigensand adhesin from Mb of wild-type cells NC4 (33, 31,28 kDa) Discoidin-I+ Developmentally regulated (adhesin 31 kDa and others) from Mb of wild type cells NC4 WGA' Stalk-specific antigen(34 kDa) from Mb of cells NC4 Fusarium moniliforme conA+ Phosphodiesterase/ phosphomonoesterase, four forms (100 kDa, GP) Macrophomina phaseo!inaJAR 125 conA+ Endo-fl-l,rl-glucanase(35 kDa) from CF Monilia sitophila; Monilia sp. conA+ fl-Glucosidase + endoglucanase + xylanase from CF Morchella esculenta y-Glutamyltranspeptidases I, 11, I11 conA+ from mycelium Mucorfragilis IF0 6449 conA+ fl-N-Acetyl-D-glucosaminidase (70 kDa) from CF M. hiemalis Endo-fl-N-acetyl-D-glucosamini- conA+ dase from CF M. muhei conA+ Lipase A (32 kDa, GP) from CF M. pusillus NCPF 2092 conA+ Antigenic GP from mycelial surface M. rouxii ATCC 24905 conA+ Mannoproteins (10-25 kDa) from surface of yeast-formcells conA+ Mannoproteins (10-30 kDa) from surface of mycelial form cells Neurospora crmsaStL 74A (wildtype strain) conA+ Laccase from CF

162

163 164 165

166 167 168 169 170 171 128 172 172

173

Lakhtin

262 Table 1 (Continued) ~


(GC)”

N. crassa IF0 6068 Endo-@-l,6-glucanase(47 kDa) conA+ from CF N. sitophila IAM 5502 Lectin (22 kDa) with complex speci-codficity from CF Nocardia asteroides R 399 Cell wall arabinogalactoglucan(s) conAPenicillium chrysogenum Glucose oxidasefrom CF cod+ Apoglucose oxidasefrom CF Cod+ P. funiculosum Endo-0-1,Cglucanase I (56 kDa) CO&+ from CF P, oxalicum @-Glucosidase(133 kDa) from CF of @-N-Acetyl-D-hexosaminidase(68 conA+ kDa) from CF of cells I F 0 5748 P. purpurogenum Endo-@-1,4glucanaseI1 (50 kDa, cod+ GP) from CF Petriellidium boydii NCPF 1001 Antigenic GP from mycelial surcod+ face Phanerochaete chrysosporium Ligninases I, I1 (39,40 kDa, GP) conA+ from CF Cod+ Ligninase I11 (43kDa) from CF conA+ Ligninases H1, H2, H6, H7, H8, H10 (38,38,43,42,42,46 kDa; respectively; GP) from CF of strain BKM Physarumpolycephalum Acid aspartyl protease (3 1 23 Cod+ kDa, GP) from plasmodial cytoplasm Phytophthora infestans Pectinesterase I(48 kDa, GP) from conA+ CF P. megasperma var. gbcinea Phytoalexin/elicitor (42 kDa,GP) conA+ from CF

+

~~

Lectin sorbent(s) Ref. commentsb 174 175 176 177 177 178

180 181 128 182 182 183

184

185 186

lectin Sorbents

263

Table 1 (Continued)


Ref.commentsb (GC)"

P. parasitica Cell wall /3-1,3(6)-glucans Nonidentifed GP Puccinia graminisvar. tritici Erics and Henn Elicitor (67 kDa, GP) from germ tube walls Rhynchosporium secalis(Oud.) Davis Phytotoxic GP from CF Schizophyllum commune Endo-8-1 ,CglucanaseI from CF Endo-P-l,rl-glucanase (41 39 kDa) from CF &Glucosidases (96 94 kDa) from CF Sclerotium rorfsiiCPC 142 Cellobiohydrolase (42kDa, GP) from CF Sporotrichum pulverulentumCM1 172727 (Phanerochaetepulverylentum) Cellobiose quinone dehydrogenases (58-60 kDa, GP) from CF: Form l a Forms lb, 2a, 2b Streptomycesji'avogriseus ATCC 33331 Cellobiohydrolase (46 kDa) from CF S. plicatus

+

+

Endo-N-acetyl-8-D-glucosaminidase H from CF Trichoderma koningiiIMI 73022 Endo-B-l,4glucanase from CF &Glucosidase (40 kDa, GP) from CF Trichodermareesei Endo-/3-1,4-glucanases from CF: Form 43 kDa (GP) Forms 56,58,59,60 kDa (GP) 8-Glucosidase (82 kDa, GP) from CF

C o dcod+

187 187

cod+

188

cod+

189

cod+ C o d + (three forms)

190 191

cod+

191

cod+

192

codCOXA+

193 193

C o d - . ConA+

194

(Yeast 8-fructofuranosidase glycopeptides cod)+

195

cod+

Cod+

196 196

COXA+ cod+ cod+

197 198 l99

+

Lakhtin

264

Table 1 (Continued)


(GC)"

Cellobiohydrolase 1(73 kDa, GP) from CF Cellobiohydrolase I1(56 kDa, GP) from CF T. viride Endo-8-1 ,4-glucanasefrom CF @-GlucosidaseA (98 kDa, GP) from CF

Trichophyton rubrum I F 0 9185 Protease (27 kDa) from CF T. rubrum Q 2873 Antigenic GP from mycelial surface

Lectin sorbent(s) Ref.commentsb conA+

200

conA+

201

conA+, c o d ConA+, UEA-I+, RCA-I- (two forms), PNA- (two forms), SBA- (two forms), WGA- (two forms)

202 203

conA+

204

conA+

128

Taxonomic groups of microorganisms (cyanobacteria, microphyticalgae, bacteria, protozoans, yeasts, and fungi) include species in alphabetical order. Glycoconjugates (GC)or glycoproteins (GP) were isolated from cultural fluids (CF), membranes (Mb),and cell walls. For the same source the order of GC and GP is the following: enzymes (oxidoreductases,transferases, esterases, glycosidases. other hydrolases, accordingto Enzyme Nomenclature.Orlando: Academic Press, 1984), antigens as GP, polysaccharide-containing materials. For example, see positions for Saccharomycer cerevisiae or for Dictyostelium discoideum. GC refers to the absence of identification (glycolipids, lipopolysaccharides,GP, complexes) GP reflects assays employing the Schiff reagent, phenol-sulfuric acid method(fot hexoses), amino acid analysis -(for amino sugars), gas liquid chromatography (for carbohydrate composition), endo- or exoglycosidase treatments (in addition to testing with lectins). When possible, the subunit masses are given in the brackets for isolated GP or CG. bAbbreviationsof lectinsare given in Chapter l of this book. Lectin' or lectin- means binding or absence of binding of GP or GC to the lectin. In the brackets results of lectinchromatogaphy are given: elution with saccharide can result in two or more forms; elution without saccharide probably reflects nonspecificinteraction). Lectin-combined chromatography is indicated by arrow. Other details are given in the text.

based on the delay of glycoconjugate elutionare revealed during lectin chromatography withoutcarbohydratein the eluant. 5 . A combination of small columns with lectin sorbents (especially analytical lectin sorbent columns for high performance liquid chromatography HPLC) can be used instead of labeled lectins for highly sensitive rapid analysesof glycoconjugates.

lectin Sorbents

265

111. APPLICATIONS OF LECTIN SORBENTS FOR THE STUDY OF MICROBIAL CLYCOCONJUCATES

Table 1 presents resultson the use of lectin sorbents in handling microbial metabolites: enzymes, antigens, toxins, (hem)agglutinins or lectins, (lipo)phosphoglycans, and other glycoconjugates(extracellular,cytoplasmic, bound to the surface of cells or organelles, as well as membrane-bound) from cyanobacteria (blue-green algae), other microphytic algae, bacteria, protozoans, yeasts, and fungi (thetotal of morethan 115 speciesand organism strains). A.

MicrophyticAlgalClycoconjugates

For the blue-green algae, Oscillatoria princeps, concanavalin A (ConA)Sepharose is used for phosphorylase isoenzymeseparation, phosphorylase A2 being boundto the lectin and eluted with 10 mM borate buffer, pH 6.3, in two forms[31]. The ConA-sorbent maybe useful alsofor purification of glycoproteins containing saccharide residues, from phicobilisome thylacoid membranes of blue-green algae, Anacystis nidulans R2 and Synechocystis sp. PPC 6714 (205). Another lectin, RCA-I-sorbent, could be useful for study of bacterial glycolipids. Similarly, the use of HPA-Sepharose for separation or isolation of blood group A glycolipids in95% tetrahydrofu(207). For the diathomic algae,Phaeodactyran in water has been reported lurn tricornuturn, ConA-Sepharose makes it possible to separate three fracactivity), a tions of chlorophyllase: nonbinding to lectin (5% of lectin-binding form (75%) that iselutedwith 0.2 methyl-a-D-mannopyand a noneluted form in the immobiranoside (Me-a-D-Manp,or a-"), lized state in a complex with lectin (15%) [32]. B. BacterialClycoconjugates 7.

hteraction of Putative Bacterial Glycoproteins with Lectin Sorbents

Larvicidal toxinfrom spores of Bacillus sphaerim contains 12% carbohydrate (w/w), enabling the toxin to bind specifically to ConA-Sepharose [36]. Hemagglutinin from culture fluids of B. subtilis 316M is also a glycoprotein that binds to ConA-glycosyl-Spheron (Chemapol, Czechoslovakia) and can be eluted by 0.1 methyl-a-D-glucopyranoside(a-MG) [33]. The purity of heat-labile Escherichia coli enterotoxin is increased 20-fold with use of ConA-Ultrogel for its preparation [46]. Glycosyltransferases from Streptococcus rnutans are separated on ConA-Sepharose by elution of the bound enzymes using high-ionic strength solvent, followed by elution in the presenceof 1 M a-MG [58].Numerousglycosyltransferaseshavea

266

Lakhtin

characteristic high affinity for immobilized ConA owing to the additional contribution of hydrophobic interactions [30]. For purification of a latent form of autolysin fromStreptococcusfaecium, tandem chromatographyon columns with hemoglobin-Sepharose and ConA-Sepharose is used, thereby permitting a 62-fold purification of the isolated zymogen and its 82% recovery in activity[56]. An affinity chromatography step on immobilized ConA is also used for removal of glycoconjugate admixtures by binding them to the lectin, as demonstrated by purification of a B. stearothermophilus nonglycosylated glucoamylase (70% of total activity) [37], or glycoproteins from Campylobacter fetus and Lactobacillus fermentum[41,48]. Different purified preparationsof bacterial endoglucanasesare distinguished accordingto their affinityfor immobilizedConA [43,44]. It is interesting that micrococcal ATPase, with a low carbohydrate content (0.4%), was not bound to ConA-sorbent [49], although another microbial glycoprotein ATPase(from the protozoan, Paramecium tetraurelia)was characterized by its affinity for the lectin [76]. In investigations connected with bacterial glycoprotein antigens, immobilized lectins are often used. They include ConA [51-531, WGA and PWM 1521, or DBA [61]. The procedure for purification of extracellular bacterial antigens is simplerthan that of bacterial surface antigens solubilized withurea, detergents, or other chaotropic agents, because lectin chromatography is performed inthe presence of detergents[27,52,53,208]. Nevertheless, in receptorglycoproteinsolubilizationwithhydrolases(e.g., phospholipases or some proteases),the presence of detergents during lectin chromatography is unnecessary[27]. 2. Potential Lectin Sorbents for the StudyofBacterial Glycoproteins and Other Glycoconjugates

Immobilized WGA can be considered a potential sorbent for purification of several larvicidal toxins of the genus Bacillus [209-21 l]. The toxins contain amino sugars, preponderantly the in form of N-acetyl-D-glucosamine[210, 21 l]. This amino sugaris present in both endoglucanase from Bacillus sp. KSM 635 and thiaminase I1 from B. aneurinolyticus [212,213]. Extracellular chitinasefrom B. circuhns MH K1, caldolysin from Thermus aquaticus T 351, and membrane-bound ampicillin acylasefrom Pseudomonas melanogenum IF0 12020 also contain carbohydrates [214-2161 that are probably reactive with lectin sorbents. Oligosaccharide structures of the majority of bacterial glycoproteins have not yet been studied; hence, it is impossibleto discuss the fine specificity of commercial lectins for bacterial glycans. However, it is known that glycoproteins from the surface array layer (Slayer) of B. stearothermophi-

Lectin Sorbents

267

ius NRS 2004/3a include asparagine-linked glycans, with a novel type of protein-carbohydrate linkage -asparagine-rhamnose trisaccharide -and other unusual glycans[217]. It cannot be excludedthat interaction of some bacillary-associated glycoproteins withConA can be explained bythe presence of asparagine-linked glycans, similar to the glycoproteins of animal and plant origin. For purification of surface glycoconjugates of various strains of the genus Bacillus or isolates of P. cepacia, other lectins mayalso be used that are not listed in Table 1: These include SBA, ABP, GS-I, and LBA U41. Bacterial glycoconjugate interactions with SBA and WGA can probably partially be attributed to the presence of galactose- or N-acetyl-D-glucosamine-containing polysaccharides[14,2181. It has been demonstratedthat, in addition to bacilli, several other strains of gram-positive bacteria belonging to other genera react with WGA [219]. The character of bacterial coagglutination, dependenton the presence of carbohydrates in the growth medium, pointsto the possibility of using lectins from one type of microorganism for the purification or immobilization of glycoconjugates of partner microbes [220]. Other examples of lectin binding to bacterial surfaces can be found in reviews[12-15,281. There are some other lectins that should be noted in connection with the investigation of bacterial surfaces. Among them are the L-rhamnose/ melibiose-, and L-arabinose-, and galactose-specific lectinfrom salmon ova of Oncorhynchus sp., whichis capable of agglutinating the bacterium, Vibrio anguillarum, a pathogen for fish [221]. The galactose-specific lectin CRG-I from the mussel Crenomytilus grayanus agglutinates gram-positive bacteria, whereas the adhesive lectin CRG-11, with a complex specificity, [222]. The from the same mussel source agglutinates gram-negative bacteria lectin from the legume white jacaranda, Swartzia pickelli fillip, agglutinates Yersiniapestis strains [223], and the novel rice lectin (a-"-specific) agglutinates an exopolysaccharide-containing agentofriceleafblight, Xanthomonascampestrisbryzae [224]. Other new lectinswithcomplex specificities, such asthe lectin from the Chinese horseshoecrab Tachypfeus tridentatus, and a lectin from human placenta, specifically interact with pneumococcal polysaccharidegroup C or P. ueruginosa polymers of mannuronic acid (alginate), respectively [225,226]. 3. hteraction of Bacterial Polysaccharide-Containing Polymers and TeichoicAcids with lectin Sorbents

Polysaccharide-containing bacterial polymers may be conveniently divided into extracellular and those composed of the cell wall (constituents of cell wall). An extracellularpolysaccharide from Capnocytophagaochracea, free of lipopolysaccharide, protein, and nucleic acid contaminants, was

268

Lakhtin

characterized by a strong affinity for ConA-Sepharose (elution only with 1-2 M a-"). This is in good correlation withthe high level of mannose content inthe polysaccharide (79% of total carbohydrates) [42]. Micrococcus luteus lipomannan(s), but not its ATPase F1, bound to ConA-Sepharose [49]. Nonimmobilized ConA also reacted withM. luteus lipomannan, but not with that of M. agilis [227]. Possibly, the aforementioned ConApolysaccharide-containing glycoconjugates include reactive bacterial branched a-mannans [for a review on polysaccharide specificity of ConA see 161. Concanavalin A-Sepharose and WGA-Sepharose have been applied to the purification of teichoic acids from cell walls of gram-positive bacteria, such as B. subtilis strains [34,35]. Interaction of teichoic acids with lectin sorbents requires the presence of either glucose residues accessible for ConA, asare found in B. subtilis, Brevibacterium linens, Enterococcus sp., and Staphylococcus aureus[34,40,45,55],or galactose residues accessible for RCA-I, 11, as in streptococcigroup N, sialidase-treated streptococcal group B, and Lactobacillus fermentum [57,59]. However, N-acetyl-Dglucosamine (G1cNAc)- or N-acetyl-D-galactosamine (Ga1NAc)-containing to immobilized WGA or variations of bacterial teichoic acids did not bind HPA [40], thereby pointing either to masking of these carbohydrate residues, or to high stereospecificity of lectinsfor locations of sugar combinations (i.e., clusters of GlcNAc residues) in glycans. Wheat-germ lectin reacts with teichoic acids from S. aureus strains H, 830, and Wood 46, but not with that from S. aureus 52 A2 [54,55,228]. Teichoic acids were eluted from WGA-Sepharose or WGA-Ultrogel with 0.1 M GlcNAc or 0.05 M HCl, respectively [54,55,59]. For elution ofteichoicacids from ConASepharose, the presence of 50 mM or even 2 mM a-MM (with latter in the presence with0.1% Triton X-100)may be enough[35,45]. Lectin sorbentsare effective inthe separation of teichoic acid molecule subpopulations.Combinedlectinchromatography on ConA-Sepharose (filtration without binding), following WGA-Sepharose binding has been used for isolation of a GlcNAc-containing ribitol teichoic acid fraction from S. aureus [55]. Separation of Enterococcus sp. lipoteichoic acid subfractions, according to their increasing numbers of glycosyl substituents in the chain, was reached during lectin chromatography on ConA-Sepharose [45]. Thus, lectin sorbents can be used for investigation of both a- and 0-anomeric forms of teichoic acids as well as glycosyl clusters within polymer chains of teichoic acids. Lectin sorbentsare capable of selective binding to capsular polysaccharides as well. For example, polysaccharide group XIV from Streptococcus (pneumococcus) sp., containingthe activity of human blood group antigen I(H)M,, exhibits selective reaction with RCA-I or SNA-I1 [229,230]. The

Lectin Sorbents

269

polysaccharide binding with the lectin requires no fewer than three carbohydrate residues, suggesting an oligosaccharide (or complex) specificity of RCA-I for the bacterialpolysaccharide[229].Capsularpolysaccharides from Escherichia coli K1, Neisseria meningitidisserogroup B, and group B streptococci, which contain sialic acids, should react with sialic acid-specific lectins, such as slug lectin, LFA [231,232]. Also one cannot exclude that periplasmic cyclic 0-1,2-glucans from gram-negative bacteria (Agrobacter[233]. This possibility iswell ium sp., Rhizobium sp.) can react with lectins supported by the findingsof specific bindingof different cyclodextrins with microbial lectinlikesorption domains of some enzymes[25]. Lectin sorbentsmay also be used in studying bacterial lipopolysaccharides as constituents of 0-antigens of enterobacteria, pseudomonads, and other gram-negative microbes. It ,was demonstratedthat, with a set of Rtype lipopolysaccharides ofmutant strains of the generaSalmonella and of E. coli K12, different selective reactions are observed for ConA, LCA, WGA, and RCA-I1 [234]. Other lectins (ATL, CRS, RCA-I,and Moringia olifera lectin or moringin) reacted with lipopolysaccharides from P.aeruginosa when tested in precipitation reactions [235]. Probably, animal blood proteins or membrane receptor lectins of hepatocytes or leukocytes will also beofsomeuse for reactingwithbacteriallipopolysaccharides [236,237]. The presence of sialic acids in lipopolysaccharides of Neisseria gonorrhoeue and members of the rhodobacteria [238,239] suggests possible uses of sialic acid-specific lectins for study of these glycoconjugates.

I

C. Protozoan Clycoconjugates

Examples of interaction between protozoan glycoconjugates and lectin sorbents are also presented in Table1. In addition to glycoproteins (enzymes, antigens, and others) that make up the major part of the tested glycoconjugates, lipid-containing glycans have also been studied (lipophosphoglycans and others) [71,74,75]. 7.

heraction of Protozoan Glycoproteins with Lectin Sorbents

Of six forms of a-amylase from Entamoeba histolyticu, form “e” (31 kDa) was purified with ConA-Sepharoseas the last step in chromatography followingSephadex,preparativeisoelectricfocusing, and DEAEkellulose chromatography [63]. During lectin chromatography, glycoconjugate admixtures with lower affinity for ConA were removed as a result of elution by a linear gradientof 0 to 1 M a-”. The amylase itselfwas characterized by strong affinity to lectin (elution only with1 M saccharide) [63]. For the P-fructofuranosidase of the holotrich diliate,Isotricha prostoma, from the rumen of infected sheep, the application of ConA-Sepharose as the last

270

Lakhtin

step of purification (after Sepharose, octyl-Sepharoseand DEAE-cellulose chromatography) madeit possible to isolate not only the major form of the enzyme (elution with100 mMa-"), but also to attain partial purification of two minor forms with higher affinity for ConA (elution with 120 and 200 mM saccharide, respectively) [as]. Carbohydrate composition of the purified enzyme indicated the presence of asparagine-linked glycans of a complex type [65]. For isolation of acid phosphatase from Leishmania tropica and L. donovani, LCA-Sepharose(afterT-&agarose)orRCA-I-agarose was used for elimination of nonbinding admixtures, such as phosphoglycans and proteins [70,74]. Specific interaction of promastigote acid phosphatase or 3 '-nucleotidasehuclease from the genus Leishmania with lectins might probably be explained by the presence of complex-type glycans linked to asparagine@)[67,68,240]. Asparagine-linked oligomannoside-type glycans may be involved in interaction of epimastigote proteases from Trypanosoma cruzi with ConA-sorbent [86]. Amastigote cysteine proteases from L. mexicana mexicana are heterogeneous in theircarbohydrate moietes, as based on various affinitiesto different lectin sorbents [72]. Acid phosphatase solubilized fromL. donovani membranes showed complete bindingto all the four lectin sorbents tested (see Table 1). Extracellular ATPaseand hexosaminidase fromParamecium tetraureliaand Tetrahymenapyriformis were separated into different formsby using immobilized ConA [76,78]. The high heterogeneity of protozoan surface glycoconjugates is due to the various glycans, a factthat is well supported using lectins [241,242; see Chapters 6and 7, this book].The same is also observed for leishmanial culture fluid glycoconjugates [68]. One ofthe causes of such heterogeneity in the latter example could be the ability of extracellular lipophosphoglycansto form stable complexes with hydrophobic proteins [71]. As potential lectin sorbents, inaddition to WGA, ConA, PSA, DSA, STA, and GS-I, new lectins, such as GNA, Colchicum autumnale lectin, and porcine lung lectin (PLL), areof interest for purification of leishmanial glycoconjugates [241]. The choice of lectin sorbent type isimportant for purification procedures. For example, trypanosomal surface glycoproteinsare characterized by a high affinity for immobilized ConA. The glycoproteins are not desorbed, even in the presence of detergent with 0.5-1.0 M ar-" [79,80,82]. However,withLCA-Sepharose,a Idfold purification of trypanosomal surface antigenwas achieved [81].Pneumocystis cariniiglycoproteins were not desorbed when WGA-Sepharose was used, although by chromatography on ConA-Sepharose the elution of glycoproteinswas attainable by 0.5 M CY-" at pH8[77].Lectintandemchromatography was useful for isolation of several protozoan glycoproteins [85]. Other combinations of lectin sorbentsare known [19,243-2451.

lectin Sorbents

271

In addition to the correct choice of lectin sorbent(s), optimal conditions of lectin chromatography of protozoan glycoconjugates are also important. For example, WGA-Sepharose trypanosomal glycoproteins can be desorbed with 0.1 M GlcNAc at neutral pH [83,84], whereas Giardia lamblia glycoproteins require acidpH for their desorption [M]. 2. heraction o f Other Protozoan CJycoconjugates with Lectin Sorbents

For purification of phenol-extracted protozoan glycoconjugates, the following immobilized lectins have been used: ConA [66,73,87], RCA-I1 [71], and WGA [87]. Interaction between polysaccharidecontainingprotozoan polymers and lectin sorbents depends on the glycan structure. For example, binding to ConA-Sepharose was due to the presence of mannose in galactomannans [66,73]. Affinity of glycoconjugates for ConA decreased and, finally, disappeared completely along with decreasing relative contentsof mannose in galactomannans [66]. Interaction with WGA-Sepharose was due to the presence of sialic acid in trypanosomal glycoconjugates [87]. Galactose-mannose disaccharide units of lipophosphoglycan from Leishmania sp. were bound to RCA-11-agarose and eluted in two forms: one desorbed with lactose, whereas another desorbed with Triton X-100 1711. The latter example demonstratesthe effectiveness of lectin chromatography for separation of protozoan glycoconjugates with hydrophobic domains. Additional data on lectin chromatographyof hydrophobic glycoconjugates can be found [207,208]. D. Yeast and Fungal Clycoconjugates

Lectin sorbents have frequently been used in studies of glycoconjugates from lower plants and fungi of such genera asCandida and Saccharomyces, Dictyostelium, Mucor, Penicillium, and Trichoderma (see Table 1). Most of the glycoconjugatesare represented by enzymes bound to microbial surfaces or secreted into culture fluids. 7.

OxidoreductasesandTransferases

Lectin sorbents can be used for the isolation and separation of multiple glycoconjugate forms, such as isoenzymes. White-rot fungal cellobiose quinone dehydrogenase is represented by the forms la, lb, 2a, and 2b, having relatively low contents of hexoses (0.3, 0.8, 0.9, and 1.6%, respectively) [193]. Interestingly, the form l a was not bound to ConA-Sepharose, whereas the forms lb and 2a were bound, and eluted with 50 mM CY-MG.The form 2b showedstrong affinityfor the lectin (elutionby only 0.5 M a-MG). All purified ligninase formsfrom the filamentous fungus containedcarbo-

272

Lakhtin

hydrates and showed binding to ConA-sorbents [182,183]. However, the 39- and 40-kDa protein forms were eluted by saccharide at acid pH, whereas the 43-kDa form was not dissociated from the lectin complex, thereby suggesting the possibility of a simple isoenzyme separation procedure [1821. During purification of mycelialy-glutamyltranspeptidase on ConA-Sepharose, a 102-kDa form was activated (recovery: 150070,purification by 17fold), whereasthe 155- and 219-kDa forms were purified only sixfold [168]. Numerous results have been obtained on interactions between oxidases and ConA-sorbents. For example, the fungal glucose oxidases revealed strong affinity to ConA-Sephadex [loo], ConA-silica (sorbent for HPLC), ConA-Sepharose [246], or ConA-yeast sorbent [247]. As a result, it was possible to remove glucose oxidase from crudepreparations of the admixtures with lower expressed affinity for the lectin [130,177], and thereby on active enzyme-ConA obtain a highly active, stable biocatalyst, based the complex [100,246-2491. Fungal polyphenol oxidase also has strong a affinity for ConA (low recovery of bound enzyme) [149]. It cannot be excluded ConA involve hydrothat interactions ofthe aforementioned oxidases with phobic or other noncarbohydrate-lectin contributions.For example, itwas suggested that the FAD cofactorof fungal glucose oxidase can increase the glycoprotein affinity for ConA [ 1771. The third group of examples demonstrates effectiveness of lectin sorbents for the isolation of oxidases and transferases, with a high recovery, by using a relatively low saccharide concentrationfor elution. Fungal lactase was simply purified using Celite, followed by ConA-Sepharose[173]. However, without Celite, the percentage of the ConA-binding portion of enzymatic activity was significantly decreased [173]. For purification of yeast y-glutamyltranspeptidase, the effective combinationof LCA-Sepharose with DEAE-Sephadex and Sephadex was used [102]. The LCA-sorbent permitted purification of this glycoprotein by 202-fold (elution with 10 mM CY-MM)[102]. The ConA-Sepharose was useful as the last step of yeast Golgi galactosyltransferase purification (the bound enzyme was eluted by0.1 M CY-" [121]). This transferase was also specifically bound to GS-I-agarose, thereby pointingto the presence of galactose residues in the glycoprotein [121]. The latter example demonstrates the effectiveness of lectin sorbents in studies of intracellular microbial glycoconjugates. 2. Esterases of Yeasts and Fungi

Yeast and fungal lipase purification schema can include combinations of ConA-sorbents with Sephacrylor DEAE-cellulose [103,1711. These lipases include forms that are distinguishable by their carbohydrate moieties and affinity for a combinationof lectins [103,171]. Several reports describe the interaction of microbial phosphatases with

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lectin sorbents. The presence of asparagine-linked glycans in acidor alkaline phosphatases enables yeast glycoproteins to specifically bindto immobilized LCA or ConA (elution with50-100 mM a-") [98,99,104].However, mannan associated with yeast phosphatase can influence bindingof the enzyme formsto ConA-sorbents [1051.Galactan associated with fungal ribonuclease gives rise to the multiple enzyme forms with different affinities for the lectin [137,139,182].In addition to possible polysaccharideassociated complexes with enzymes, it is possible other types of enzyme complexes, such as fungal alkaline phosphatase-5 '-nucleotidase or phosphodiesterase-phosphomonoesterase, can be copurified on different sorbents, including ConA-Sepharose [154,165].Certain esterases can also be separated directlyon lectin sorbents.For example, slime mold phosphodiesterase I bound to ConA-sorbent, whereas phosphodiesterase I1 did not bind to this lectin[155]. 3. Glycosidases of Yeasts and Fungi

Comprehensive information has been obtained about interactions of lectin sorbents with lower plant glycosidases (see Table 1). It wasshown that interactions of fungal or yeast glycosidases with ConA-sorbents were due to the presenceof different asparagine-linked glycans [101,124,132,141,146, 200,2521 or glucan-mannan associated with enzymes [91,94,112,113,122]. The very high level of carbohydrate heterogeneity in fungaland yeast glycosidases results in complex elution profiles in their lectin chromatography. For example, fungal endoglucanases, all being glycoproteins, have no affinity for ConA [120],low affinity for the lectin (elutionby 10-30 mM a-MM or a-MG) [148,181,190,191,197,198], or strong affinity (the absence of elution or partial elution by 0.5-1.0 M saccharide)[133,202]. Frequently, glycoprotein enzymes fromthe same sourcecan be simply separated: &glucosidasefrom lactonase [13l],endoxylanase from 0-xylosi[140,1451,6dase [93],a-galactosidase from /3-N-acetyl-D-glucosaminidase fructofuranosidase from amylase [121], a-L-arabinofuranosidase from rhamnosidase [135],and acid and neutral forms of trehalase [1091 (see Table 1). Strong affinityof glycosidases for ConA-sorbents has been used Interestingly, use for preparing the biocatalysts [100, 133,246-251,253,2541. of a kit of lectin sorbents allows a choice in type of sorbent(s) for glycosidase isolation[95,203],immobilization (foregoing),or investigation of glycan structure [95,140,145,203,252]. 4. Proteases and Other Enzymesof Yeast and Fungi

When proteases have been tested for affinity to ConA-sorbents, a strong affinity of the enzymes for the lectin was observed [97,116,125,252].The valylprotease from Candida tropicalis may be reactive with lectin sorbents

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[255]. Carboxypeptidase Y, immobilized on ConA-glycosyl-Spheron, can directly serve as a biocatalyst [252]. Slime mold folate deaminase was almost completely bound to ConA-Sepharose or LCA-Sepharose (in the latter, 70% enzyme activity was eluted with 50 mM a-") [160]. Baker's yeast asparaginasesI and I1 wereseparated usingboth of the lectin sorbents [l 171. Similar to other yeast enzymes, multiple forms of asparaginase I1 possess different contents of polysaccharide [l 171. Another baker's yeast enzyme,aspartyl-tRNAsynthetase,containscovalentlyboundglucose, which may interact with ConA [256]. Interestingly, the latter is the only enzyme described in the literature showing an interaction between ligase and lectin[ 19,271. 5. Surface and Other Glycoproteins of Yeast and Fungi

Glycoproteins are common cell surface antigens. Enzymes from cell walls as important cell antigens.A major antigen of or membranes can also serve C.albicans B31 1 was isolated using DEAE-cellulose, followed byfiltration through ConA-Sepharose [88]. Nonactive polysaccharide-containing material was eliminated byits binding to the lectin [88]. Solubilized by autolysis or by zymolase and Novozyme treatments, mannoproteins from cell walls of Schizosaccharomycespombe and Saccharomyces cerevisiaewere readily purified with Sephacryl and ConA-Sepharose [1 18,119,1231. Another antigen, A 2 , from the yeast form of the fungus Paracoccidioides braziliensis B339, was isolated using a combination of Bio-Gel P30 and immobilized ConA (strong affinityof the antigen to ConA,as elutionwas only achieved by 0.5 M a-MM or 0.7 M glucose) [96]. Immunoaffinity chromatography enabled the isolation of the diagnostic 43-kDa glycoprotein for mycoses from the antigen A2 complex [96]. Phytotoxin and nonidentified glycoproteins were isolated fromthe fungi Rhynchosporium secalisand Phytophthora parasitica by using immobilized ConA [187,189]. As a result of lectin chromatography, polysaccharides were separated from glycoproteins [1871. The ConA-reactive cell wall glycoproteins solubilized by zymolase treatment were also found in eight species of ascomycetous and imperfect yeasts, such asZygosaccharomyces rouxiiand Hansenula wingli [257]. The fungal membrane-bound glycoproteins usually require detergent solubilization for lectin chromatography. Such glycoproteins ofthe slime mold Dictyostelium discoideum are characterized by strong affinity for WGA-Sepharose (elution with 0.5 M GlcNAc in the presence of 0.5% sodium dodecyl sulfate) [157,161]. However, affinity of slime mold glycoproteins for discoidin-I-sorbent was lower (elution could be achieved by use of 0.3 M galactose in the presence of 0.05% Triton X-100) [162,163]. In addition to discoidin-I, other galactose-specificimmobilizedlectins (RCA-I and ABP) have been used for isolation of slime mold antigenic

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inhibitors of intercellular adhesion [1621. Lectin chromatography has also enabled the isolation of glycoprotein inducers of slime mold cell cohesion [161,1631 or purificationof slime mold stalk-specific 34-kDa antigen 11641. Mycelial surface antigens of the fungi Mucorpusillus, Petriellidiumboydii, and Trichophyton rubrum were solubilized withTriton X-100 and binding to ConA-Sepharose (elution with 0.2 M a-") 11281. All purified fungal glycoproteinantigensprobablycontainedasparagine-linkedglycans,as [1281. suggested by their carbohydrate composition 6. Other Polysaccharide-ContainingGlycoconjugates of Yeasts and Fungi

In addition to the foregoing polysaccharide-associated enzymes, there are several other polysaccharide-containing effectors that were tested for reactions with lectins. Liposan (83% carbohydrate and 17% amino acids) of theyeast C.lipolyticawas purified using ConA-Affi-Gel [W]. Strong affinity of liposan (which contains glucose, galactose, galactosamine, and galacturonic acid)for the lectin required0.1 M acetatebuffer (pH3.6) for glycoconjugate elution. Polysaccharide heterogeneity of ConA-Sepharose-purified mannoproteins (36-72% carbohydrates,including63% Manal ,2-linked residues and 4-26070 uronic acids) was observed for Mucor rouxii [172]. The glycoprotein elicitor, purified using ConA-Sepharose from germtube walls of the wheat stem rust fungus Puccinia graminis,contained associated galactan (47-77% carbohydrate), which can be partially removed (in the latter, the carbohydrate moiety included 47% galactose and 53% mannose) [188]. For elution of this elicitor, 0.2 M a-MM was sufficient. For Cordyceps ophioglossoidesmetabolites, ConA-Sepharose was useful for separation of galactomannan and a serine, threonine glycosylated glycoprotein 11521. Phytotoxic glycopeptide (83% hexoses, mainly rhamnose and mannose) of the Dutch elm disease pathogen Ceratocystis ulmi was purified with Bio-Rad AG-5OW-sorbent and ConA-Sepharose (elution with 0.1 M a-") 11471. Nocardia usteroides polysaccharide (which includes galactose, arabinose, glucose, and a polyol) and Grifola frondosa antitumor P-glucans didnot react with immobilized ConA [176,258]. When boundto ConA-Sepharose, the a-glucan admixture was characterizedby a low affinity to lectin (elution with 50 mM a-MM) 12581. 7. Potential Lectin Sorbents for the Studyof Yeast or Fungal Glycoconjugates

Other kinds of lectins may also be used. in yeast polysaccharide studies (2591. Mannan-specific or mannan-bindingplant lectins from bulbs of Ngrcissuspseudonarcissus and a .Hippeastrum hybr.selectivelyprecipitate branched a-l,6-mannans of Hansenula .capsulataand galactomannans of

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C. lipolytica, Torulopsis lactis condensiand T. gropengiesseri [259]. Additional data on polysaccharide specificities oftraditional or commercial lectins can be found in severalreviews [12,14,16,19] and in the Appendix of Chapter 1. For possible purification of fungal glycoconjugates, additional fucosespecific lectins may beof certain interest, such as UEA-I, LFAand other fungal lectins [260]. It should be notedthat LCA and PSA can also recognize fucose residues in different asparagine-linked glycans [243-2451. Recently, itwas shown that fungal surface glycoconjugates include a group of oliosaccharides that are similar to high plant glycoconjugates (oligomannoside glycans, glycans containing fucose, xylose, or mannose-6-P) [26Oa]. Specific interactions between fungal glycoconjugates and lectin sorbents which were described in the foregoing, would be anticipated, given the foregoing compositions.

W.

OTHER EXAMPLES OF THE USE O F LECTIN SORBENTSIN MICROBIAL BIOTECHNOLOGY

Lectin sorbentscan also be usedfor a “sandwich” variant of chromatography. For example,glycogen-Cod-Sepharose was used for purification of Flavobacterium sp. isoamylase [47]. Another example ofsubstrate affinity chromatography using a lectinsorbent as the substrate carrier can be illustrated for the fungal endo-P-N-acetylglucosaminidaseH from Streptomyces plicatus. This enzymewas purified on pronase glycopeptidesof yeast invertase-Cowl-Sepharose [195]. It is likely that microbial receptor antigens (glycoconjugates) immobilizedon lectin sorbent(s) could be used for immunization procedures, as well as for studies of biological receptor activity [89]. Similarly, purified human tumor-associated antigens have beenstudied [261]. For immobilization of microbial glycoconjugates, crude extracts of lectins can be employed [61,249]. The Cod-sorbents are also widely used as carriers of microbial cells, such asthe yeasts S. cerevisiae [262-2641 and Trichosporon cutaneum[263], on ConA-Sephaor the bacteriumS. aureus [265]. Such cell immobilization rose or ConA-silica can be applied for detection of microbes [262,265,266]; for investigation of metabolic reactions of microbes in the presence of antibiotics or other agents [263]; for quantitative detection of phenols or catechols [263]; or for isolation of cell populations and cell membranes. Occasionally, LCA-Sepharose has advantages over ConA-Sepharose for cell immobilization [267]. Lectin sorbentsare useful for detecting glycosidaseor glycosyltransferase action on substrates [25,243]. The use of lectin sorbents for HPLC is particularly attractivefor isolation and microassay of glycoproteins, glyco-

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peptides, or oligosaccharides [19,25,245,268-2701. It is important to develop different techniques for preparation of lectin sorbents for HPLC. For example, an affinity sorbent containingConA immobilized by a Co3+ complexwaseffective for the isolation of fungal &N-acetyl-D-glucosaminidase [271]. The other potential directionfor using immobilized lectins is the development of ELISA-like and other types of microassays of glycoconjugates or cells and membranes [19,25,272]. For example, microplates withConAor WGA-coatedwells can be used for quantitative immunodetection of streptococcal and yeast polysaccharides, or HIV-1 envelope glycoproteins [60,273,274]. Other lectin ELISA-like methods, such as ELLSA, or ELLBA (see Chapter 1) can be useful for detection of viral glycoproteins [275], bacterial cells[276,277], or protozoan cells [278]. Labeled lectins in immunosandwich techniques can be applied for fluorescence analysis of microbes on glass slides[279]. Blotting lectin techniquesare often used in investigation of viral, bacterial, or fungal glycoproteins [280-2831, or of bacterial cells [284]. All of these foregoing microassys permit one to obtain useful information on possible uses of lectin sorbents for isolation of microbial glycoconjugates. V. POSSIBILITIES FOR MICROBIAL LECTIN USE IN THE STUDY O F MICROBIAL GLYCOCONJUGATES

From Table 1 it can be seen that lectin sorbents are mainly represented by immobilized lectins from higher plants. The only exceptions are the immobilized bacterial lectin PA-11, which reacted with lower plant glycoproteins [23,110], or immobilized fungal discoidin-I, which reacted with [162,1631. endogenic fungal glycoproteins Apparently, lectins from various microorganisms can be used as successfully as other commercial lectins in studies of microbial glycoconjugates [12,23,24,285,286]. Well-studied lectins are the influenza virus hemagglutinin and bacterial protein toxins (lectin activity). Carbohydrate specificities of several other viruses [287,288], bacterial proteins [22,23,220,234,285, 288-2931, protozoan lectins [286,294], and lectins from yeasts and fungi [295,296] are also known. One may consider as examples of possible uses of microbial lectins in studies of microbial glycoconjugates the following: use of bacteriophages in analysis of bacterial R-type lipopolysaccharides [297,298]; interaction of lectins from streptococci and mycobacteria with bacterial polysaccharides [299-3011; study of receptor glycoconjugates from protozoans, yeasts, or bacteria with PA-Iand PA-I1 [l 101; interaction of extracellular lectin@) of S. cerevisiae with yeast cells [302,303]; use of fucose-specific lectins inthe

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study of fungal spores, flagellae, or rhizoids[260];interactionbetween extracellular lectins of the fungus, Sclerotium rolfsii, with bacteria [304]; Bacteroides fragilis adhesionimmobilization of microbial cells using a sorbent complex [305]; and carbohydrate-sensitive microbe-microbe coagglutination [220]. VI CONCLUSIONS

This review describes several possibilities for lectin sorbents in studies of microbial glycoconjugates. Optimal conditions of lectin chromatography and the diversities of lectins provide highly effective separation and immobilization of microbial glycoconjugates through their carbohydrate moieties. The potential for using lectin chromatography in glycoconjugate isolation techniques is not nearly exhausted. For example, for animal receptor glycoproteins the useof RCA-II- or UEA-I-sorbents enables the purification of glycoconjugate complexes 300- to 440-fold [306,307]. The use of combined chromatography WGA-Sepharose with ConA-Sepharose,tanor dem chromatography Affi-Gel S-145withWGA-Sepharoseenables the purification of glycoproteins up to 2000-fold [308,309]. Currently, lectin chromatography is also used for isolation of mammalian cell receptors of microorganisms (bacteria, or others) using immobilized microbial lectins, such asE. coli type 1 fimbriae [3 10,31l]. Isolation of intracellular receptor glycoconjugates by the use of lectinsorbents is also likely, because there are many microbial glycoconjugates with affinity for lectins [312]. For example, yeast Golgi galactosyltransferase or microsomal trimming glucosidase I can serve as receptors for endogenic or exogenic lectins, including C o d - and GS-I-sorbents [108,121]. Examplesof other intracellularreceptors and enzymes are also known [27,30]. Many external surface receptors of microorganism are carbohydratecontaining enzymes. A number of such microbial surface enzymes were given in this review: streptococcal autolysin [56]; protozoan acid phosphatase [67,71a], 3 '-nucleotidasehuclease [68a,240], site-specific neutral endopeptidase [7q,or glycoprotein immunologically relatedto acetylcholinesterase [313]; and yeast and fungal cell wallor membrane-bound enzymes (see Table 1). These surface enzymes can serve asimportant antigenic surface determinants[27]. Finally, lectin sorbents are also useful for purification of glycosylated recombinant viral and other microbial glycoproteins

m.

The results outlined this in review demonstrate applicationsand advantages of lectin-sorbents for solving problems of biotechnology, immunology, enzymology,and pharmacology of microbial glycoconjugates.

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ACKNOWLEDGMENT

I wish to thank Professors R. Doyle and K. Shakhanina for general support, and Dr. N. Kalinin for discussion. REFERENCES

1. Kocourek J, HofejSi V. A note on the recent discussion on definition of the term “lectin.” In: Bsg-Hansen TC, Spengler GA, eds. Lectins: biology, biochemistry, clinical biochemistry, v01 3. Berlin: Walter de Gruyter, 1983: 3-6. 2. Varbanets L. Lectins and aspects of their studies. Microbiol J (Kiev) 1990; N1,87-90 [in English]. 3. Lakhtin VM.Enzyxpes of carbohydrate metabolism, which contain lectin domains of sorption on polysaccharides. In: Proceedings ofthe first republic conference on investigation and application of lectins, v01 1. May 31-June 2, 1988,Tartu, Tallinn. Tartu: Tartu University, 1989:128-131 [in Russian]. 4. Lakhtin VM. Lectins and aspects of their study. Microbiol J (Kiev) 1989; 51(3):69-74 [in Russian]. 5. Parchment RE, Shaper JH. Glycosyltransferases as probes for nonreducing terminal monosaccharide residues on nitrocellulose immobilized glycoproteins: the /3-1,4-galactosyltransferasemodel.Electrophoresis 1987; 8:421427. 6. Hanover JA, Cohen CK, Willingham MC, Park MK. 0-Linked N-acetylglucosamine is attached to proteins of the nuclear pore. Evidence for cytoplasmic and nucleoplasmic glycoproteins. JBiol Chem 1987; 262:9887-994. 7. Russell RRB, DonaldAC, Douglas CW.Fructosyltransferaseactivity of glucan-binding protein from Streptococcus mutans.J Gen Microbiol 1983; 129: 3243-3250. 8. Zakharova IY, Kovalenko EA, Buglova TT. Lectin properties of galactosoxidase from Fusarium graminearum IMV-F-1060. Biochemistry (Mosc) 1986; 5131249-1255 [in Russian]. 9. Kovalenko EA, Pavlova IN. Biological properties of a-mannanase from Rhadococcus erythropolis. Microbiology (Mosc)1986; 55:81-85 [in Russian]. 10. Lakhtin VM, Zaprometova OM. a-Galactosidase of Cephalosporium acremonium 237 and its ability to act as a lectin. Biochemistry (Mosc) 1988;53: 1270-1277 [in Russian]. 11. Lakhtin VM. Lectins as metabolism regulators. Biotechnology (Mosc)1986; N6:66-79 [in Russian]. 12. Lakhtin VM. Lectins for investigation of proteins and carbohydrates. In: Klyosov AA, ed. Achievements in science and technique, seria biotechnology, v01 2.Moscow: Viniti, 1987:l-290 [in Russian]. 13. Pistole GT. Interaction of bacteria and fungi with lectinsand lectinlike substances. AMU Rev Microbiol 1981; 35:85-112. 14. Slifkin M, Doyle RJ. Lectins and their application to clinical microbiology. Clin MicrobiolRev 1990; 3:197-218.

280

Lakhtin

15. Kalinin NL, Lakhtin V M , Shakhanina KL. Perspective using of lectins for identification of the infection diseases agents.Proc Inter-Lec 11th. 1989; 30. 16. Goldstein IJ, Poretz RD. Isolation, physicochemical characterization, and carbohydrate-binding specificity of lectins. In: Liener IE, Sharon N, Goldstein IJ, eds. The lectins: properties, functions, applications in biology and medicine. Orlando: Academic Press, 1986:35-247. 17. Riidiger H. Preparation of plant lectins. Adv Lectin Res1988; 1:26-72. 18. Wu AM, Sugii B, Herp A. A guide for carbohydrate specificities of lectins. In: Wu AM, Adams LG, eds. Molecular immunology of complex carbohydrates. New York: Plenum Press, 1988:819-847. 19. Lakhtin VM. Lectins for investigation of glycoconjugates. R o c Inter-Lec 1 lth, 1989; 44. 20. Lakhtin VM. Biotechnological aspectsof lectins. In: Kocourek J, Freed D, eds. Lectins: biology, biochemistry, clinical biochemistry, v01 7. St. Louis: Sigma ChemicalCO, 1990:417-426. 21. Loyenko YN, Artyukov AA, Lyamkin GP, Glazkova VE, Rutskova TA. Lectins and agglutinins from marinealgae. Plant Resources(Leningrad) 1990; 26:263-274 [in Russian]. 22. Kovalenko EA. Extracellular bacterial lectins. Microbiol J (Kiev) 1990; N3: 92-99 [in Russian]. 23. Gilboa-Garber N, Garber N. Microbial lectins. In: Allen HJ, Kisalius EC, eds. Glycoconjugates: composition,structure and function. New York: Marcel Dekker, 1991:540-590. 24. Sharon N. Bacterial lectins,cell-cell recognition and infectious disease. FEBS Lett 1987; 217:145-157. 25. Lakhtin VM. Biotechnology of lectins. Biotechnology (Mosc) 1989; 9676691 [in Russian]. 26. Lis H, Sharon N. Applications of lectins. In: Liener IE, Sharon N, Goldstein IJ, eds. The lectins: properties, functions, applications in biology and medicine. Orlando: Academic Press,1986:293-369. 27. Lakhtin VM, Yamskov IA. Lectins for investigation of receptors. Uspekhi Khim (Mosc) 1991; 60:1777-1816 [in Russian]. 28. Antonjuk VA, Formaziuk VE, Levashev VS. Use of lectins in microbiology. J Microbiol EpidemiolImmunobiol (Mosc) 1987; N6:97-104 [in Russian]. 29. Doyle RJ, Slifkin M. Applications of lectins in microbiology. ASM News 1989; 55~655-658. 30. Lakhtin VM. Purification of enzymes with lectins. Biotechnology (Mosc) 1985; N5:ll-27 [in Russian]. 31. Fredrick JP.Affiity chromatographystudies of the de novo glucan synthesizing phosphorylase isozyme of blue-green algae.Plant Sci Lett 1975; 5:131-135. 32. Terpstra W. Identification of chlorophyllase as a glycoprotein. FEBS Lett 1981; 126~231-235. 33. Simonenko IA, Kovalenko EA,Lakhtin VM. Characterization of the Bacillus mesentericus extracellular lectin.Proc Inter-Lec 1 lth, 1989; 6 6 . 34. Doyle RJ, Birdsell DC, Young FE. Isolation of the teichoic acid of Bacillus subtilis 168 by affinity chromatography. Prep Biochem 1973; 3:13-18.

lectin Sorbents

281

35. Hancock IC. The biosynthesis of wall teichoic acid by toluenised cells of Bacillus subtilisW23. Eur J Biochem 1981; 119:85-90. 36. Narasu ML, Gopinathan KP. Purification of larvicidal protein from Bacillus sphuericus 1593. Biochem Biophys Res Commun1986; 141:756-761. Assignment of a p-nitrophenyl a - ~ 37. Suzuki T, Shinji M, Eto N. glucopyranosidase of Bacillus stearothermophilus ATCC 12016 to a novel exo-a-l,4glucosidase active for oligomaltosaccharides and a-glucans. Biochim BiophysActa 1984; 787:281-289. 38. Donovan MG, MasureHR, Storm DH. Isolation of a protein fraction from Bordetella pertussis that facilitates entry of the calmodulin-sensitive adenylate cyclase into animal cells. Biochemistry 1989; 2838124-8129. 39. Stompe H, Drescher M, Mebel S, Rustenbach S. Method for isolation of bacterial antigens. DDR Patent Application N 3095875. Nov 27, 1987; published May 24, 1989 [in German]. 40. Fiedler F, Bude A. Occurrence and chemistry of cellwall teichoic acidsin the genus Brevibacterium. J Gen Microbiol1989; 135:2837-2846. 41. Dubreuil JE, Logan SM, CubbageS, Eidhin DN, McCubbin WD,Kay CM, Beveridge TJ, Ferris, FG, Trust TJ. Structural and biochemical analysis ofa surface array protein of Campylobacterfetus. J Bacteriol 1988; 170:41654173. 42. Dyer JK, Bolton RW. Purification and chemical characterization of an exopolysaccharide isolated from Capnocytophaga ochracea. Can J Microbiol 1985; 31:1-5. 43. Gilkes NR, Langsford ML, Kilburn DG, Miller RC, Warren RAJ. Mode of action and substrate specificities of cellulases from cloned bacterial genes. J Biol Chem 1984; 259:10455-10459. 44. Poulsen OM, Petersen LW. Purification of two immunologically distinct endoglucanases without affinity for microcrystalline cellulosefrom Cellulomonas sp. ATCC21399. Biotechnol Bioengr1989; 34:65-71. 45. Leopold K, Fischer W. Separation of the poly(g1ycerophosphate) lipoteichoic 3cids of Enterococcusfaecalis Kiel 27738, Enterococcus hirae ATCC 9790 and Leuconostoc mesenteroidesDSM 20343 into molecular speciesby affinity chromatography on concanavalin A. Eur J Biochem 1991; 196:475-482. 46. Grasser-Regallet F, Scheftel JM, Monteil H. Isolation of heat-labile enterotoxin producedby a human strain of Escherichia coliby wheat germ agglutinin affinity chromatography. FEMS Microbiol Lett1986; 35:239-243. 47. Evans RM, Manner DJ, Stark JR. Partial purification and properties of bacterial isoamylase. Carbohydr Res 1979; 76:203-212. 48. Conway PL, Kjelleberg S. Protein-mediated adhesion of Lmtobaciilus fermentum strain 737 to mouse stomachsquamous epithelium. J Gen Microbiol 1989; 135:1175-1186. 49. Lim SH, Salton MRJ. F1-ATPase of Micrococcus&sodeikticus is not a glycoprotein. Biochim Biophys Acta 1981; 638:275-281. 50. Wheeler PR, Bharadwas VP, GregoryD. Affinity chromatographic purification of ~-~-acetamido-2-deoxyglucosidase, 8-D-glucuronidase and acid phosphatase in Mycobacterium leprae. J Gen Microbiol1982; 128: 1063-1071.

282

Lakhtin

51. Espitia C, Manacilla R. Identification, isolation and partial characterization of Mycobacteriumtuberculosis glycoprotein antigens. Clin Exp Immunol 1989; 77:378-383. 52. Gigliotti F, Ballou LR, Hughes WT, MosleyBD. Purification and initial characterization of a ferret Pneumocystis cariniisurface antigen. J InfectDis 1988; 158:848-854. 53. Simoneau P, Labarbe J.Detection of concanavalin A bindingprotein in the mollicute Spiroplasma citri and purification from the plasma membrane. Arch Microbiol 1989; 152:488-491. 54. Ndulue A, Flandrois JP, Marmet D. Abnormal affinity of Staphylococcus aureus N-acetylglucosamineribitol teichoic acidfor wheat germ agglutinin. J Chromatogr 1981; 209:323-328. Staphylococcus 55. NdulueAN, Flandrois JP. Immunochemicalstudiesof aureus Oeding-Haukenes antigen a,: a phosphorus-containing polysaccharide. J Gen Microbiol1983; 129:3603-3610. 56. Kawamura T, Shockman GD.Purification and some properties ofthe endogenous, autolytic N-acetylmuramoyl hydrolase of Streptococcus faecium, a bacterial glycoenzyme. J Biol Chem 1983; 258:9514-9521. 57. Wicken AJ, Knox KW. Characterization of group N Streptococcus lipoteichoic acid. Infect Immun1975; 11:973-981. 58. Tsumori H, Shimamura A, Mukasa H. Purification and properties of extracellular glucosyltransferasesfrom Streptococcus mutansserotype “a.” J Gen Microbioll983; 129:3251-3259. 59. Gray BM, Dillon HC, Pritchard DC. Interaction of group B streptococcal type-specific polysaccharides with wheat germ agglutinin and other lectins. J Immunol Methods 1984; 72:269-277. 60. Holm SE, Hakansson S. A simple and sensitive enzyme immunoassay for determination of soluble type-specific polysaccharidefrom group B streptococci. J Immunol Methods 1988; 106:89-94. 61. Slifkin M, Gil G. Identification of group C streptococcal antigen extracts with lectin-bound polystyrene particles. J Clin Microbioll984; 19:83-84. carbohydrate 62. Millikin BE, Weiss RL.Localization of concanavalin A binding in Chlamydomonasflagella.Cell Sci 1984; 68:211-226. 63. Nebinger P. Separation and characterization of four different amylases of Entamoeba histolytica. Biol Chem Hoppe-Seyler 1986; 367:161-167. 64. Ortega-Barria E, Ward HD, Evans JE, Pereira MEA.N-Acetyl-D-glucosamine is present in cysts and trophozoites of Giardia lambliaand serves as receptor for wheat germ agglutinin. Mol BiochemParasitol1990; 43: 151-166. 65. Dauvrin T, Thines-Sempoux D. Purification and characterization of a heterogenousglycosylatedinvertase from the rumen holotrich ciliate Zsotricha prostoma. Biochem J 1989; 264:721-727. 66. Palatnik CB, Previato JO, Gorin PAJ, Previato LM. Partial chemical characterization of the carbohydrate moieties in Leishmania adleri glycoconjugates. Mol Biochem Parasitol 1985; 14:41-54. 67. Avila JL. Hernhndez-Morales D, Polegre MA, Convit J. On the acid phos-

lectin Sorbents

283

phatase isoenyzmes existing in American Leishmania promastigotes. Comp Biochem Physioll989; B-94:335-342. 68. Bates PA, Gottlieb M, Dwyer DM. Leishmania donovani: identification of glycoproteins releasedby promastigotes during growth in vitro. Exp Parasitol 1988; 67~199-209. 68a. Campbell TA, Zlotnick GW, NeubertTA, Sacci JB, Gottlieb M. Purification and characterization of the 3 '-nucleotidase/nuclease from promastigotes of Lesihmania donovani. Mol BiochemParasitoll991; 47:109-118. 69. Lovelace JK, Dwyer DM, Gottlieb M. Purification and characterization of the extracellular acidphosphatase from Leishmania donovani. Mol Biochem Parasitoll986; 20243-251. 70. Lovelace JK, Gottlieb M. Evidence for phosphorylation of the extracellular acid phosphatase of Leishmania donovani. J Protozooll987; 34:78-79. 71. King DL, Chang YD, Turco SJ. Cell surface lipophosphoglycan of Leishmania donovani. Mol BiochemParasitoll987; 24:47-53. 71a. Menz B, Winter G, Ilg T, Lottspeich F, Overath P. Purification and characterization of a membrane-bound acidphosphatase of Leishmania mexicana. Mol Biochem Parasitol 1991; 47:lOl-108. 72. Robertson CD, Coombs GH. Characterization of three groups of cysteine proteinases in the amastigotes of Leishmania mexicana mexicana.Mol Biochem Parasitol 1990; 42:269-276. 73. Xavier MT,Previato JO, Gorin PAJ, Mendonca-Previato L. Chemical structures of a galactose-rich glycoprotein ofLeishmania tarentolae. Comp Biochem Physiol[B] 1987; 88:lOl-104. and secreted acid phos74. Jaffe CL, Perez LM, Schnur LF. Lipophosphoglycan phatase of Leishmania tropica share species-specific epitopes. Mol Biochem Parasitol 1990; 41:233-240. 75. Wright EP, Amin ERM. Leishmania infection: surfaces and immunity. Biochem CellBioll989; 67525-536. 76. Ip HS, Om A, Russell DG, Cross GAM.Leishmania mexicana mexicanagp 63 is a site-specific neutral endopeptidase. Mol Biochem Parasitol 1990; 40: 163-172. 77. Levin AE, Travis SM, De Vito LD,Park KA, Nelson DL. Purification and characterization of a calcium-dependentATPase from Paramecium tetraurelia. J Biol Chem 1989; 264:4544-4551. 78. Vick GW, Blum JJ. Secretion of hexosaminidase isozymes by Tetrahymena. J Protozool 1979; 26510-518. 79. Ferguson MAJ, Cross GAM. Myristylation of the membrane form of a Trypanosoma brucei variant surface glycoprotein. J Biol Chem1984; 259301 13015. 80. Boutignon F, Hublart M, GomesV, Mendonca-Previato L, Tetaert D, HuetDuvillier G, DegandP. Molecular heterogeneity of the isolated surface glycoprotein from variant AnTat l .l of Trypanosoma brucei brucei. Biol Cell 1988; W131-135. 81. Strickler JE, Mancini PE, Patton CL. Trypanosoma brucei brucei:isolation

284

82. 83. 84.

85. 86.

87. 88. 89.

90. 91. 92. 93. 94. 95. 96. 97.

Lakhtin of the major surface coat glycoprotein by lectin affinity chromatography. Exp Parasitoll978; 46:262-276. Reinwald E. Role of carbohydrates within variant surface glycoprotein of Trypanosoma congolense. Protection against proteolytic attack. Eur J Biochem 1985; 151:385-391. Castanys S, Gamarro F, Ruiz-PCrez LM, Osuna A. Purification of a glycoprotein excreted by Trypanosoma cruzi to increase the permeability of the host-cell membrane. Biochem Biophys Res Commun 1990; 166:736-742. Couto AS, Goncalves MF, Colli W, De Loderkremer RM. The N-linked carbohydrate chain of the 85-kilodalton glycoprotein from Trypanosoma cruzi trypomastigotes contains sialyl, fucosyl,and galactosyl a-l,3-galactose units. MolBiochem Parasitoll990; 39:lOl-108. Harth G, Haidaris CG, So M. Purification and characterization of stagespecific glycoproteinsfrom Trypanosoma cruzi. Mol BiochemParasitoll989; 33~143-150. Cazzulo JJ, Hellman U, Couso R, Parodi AJA. Amino acid and carbohydrate composition of a lysosomal cysteine proteinase from Trypanosoma cruzi. Absence of phosphorylated mannose residues. Mol BiochemParasitol 1990; 38:41-48. Previato JO, Andrade AFB, Pessolani MCV, Mendonca-Previato L. Incorporation of sialic acid into Trypanosoma cruzimacromolecules. A proposal for a new metabolic route. Mol Biochem Parasitoll985; 16:85-96. Greenfield RA, Jones JM. Purification and characterization of a major cytoplasma antigen ofCandida albicans.Infect Immun 1981; 34:469-477. Linehan L, Wadsworth E, Calderone RA. Candida albicans C3d receptor, isolated by using a monoclonal antibody. Infect Immun 1988; 56:1981-1986. Cirigliano MC, Carman GM. Purification and characterization of liposan, a bioemulsifer from Candida lipolytica. Appl Environ Microbiol 1985; 50:846850. Notario V, Villa TG, Villanueva JR. Purification of an exo-8-D-glucanase from cell-free extracts of Candida utilis. Biochem J 1976; 159555-562. Freer SN. Purification and characterization of the extracellular 8-glucosidase produced by Candida wickerhamii. Arch Biochem Biophys 1985; 243515522. Peciarovi A, Biely P. Affinity chromatographic purification of an endo-81,4-D-xylanase from Cryptococcusalbidus. Biochim BiophysActa 1982; 716: 391-399. Workman WE, DayDF. Purification and propertiesof the 8-fructofuranosidase from Khyveromyces fragilis. FEBS Lett 1983; 160:16-20. Koenig D, Day D. The purification and characterization of a dextranase from Lipomycesstarkeyi. Eur J Biochem 1989; 183:161-167. Puccia RS, Schenkman S, Gorin PAJ, Travassos LR. Exocellular components of Paracoccidioides brasiliensis: identification of a specific antigen. Infect Immun 1986; 53:199-206. Miyakawa T, Kaji M, Jeong YK, Tsuchiya E, Fukui S. Purification and characterization of a Ca+’-dependent membrane peptidase involved in the

Lectin Sorbents

98.

99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110.

111.

112.

113.

285

signaling of mating pheromone in Rhodosporidium toruloides.J Bacterioll987; 169:1626-1631. Trimble RB,Maley F, Watorek W. Subunit structure and carbohydrate composition ofthe extracellular acidphosphatase of Rhodotontla glutinis. J Biol Chem 1981; 256:10037-10043. Watorek W, Morawiecka B. Function of carbohydrate moiety in acid phosphatase of Rhodotorulaglutinis.Acta Biochim Pol 1984; 31:217-221. Hussain Q,Saleemuddin M. An inexpensive procedure for the immobilization of glycoenzymes on Sephadex G-50 using concanavalin A. Biotechnol Appl Biochem 1989; 11508-512. Yamashita I, Hatano T, Fukui S. Subunit structure of glucoamylase of Saccharomyces diastaticus.Agr Biol Chem1984; 48:1611-1616. Penninckx MJ, Jaspers CJ. Molecular and kinetic properties of purified y-glutamyltranspeptidase from yeasts (Saccharomycescerevisiae). Phytochemistry 1985; 24:1913-1918. Witt W, Mertsching A, KonigE. Secretion of phospholipase Bfrom Saccharomyces cerevisiae.Biochim BiophysActa 1984; 795: 117-224. Onishi H, Tkacz J, Lampen J. Glycoprotein nature of yeast alkaline phosphatase. Formation of active enzyme inthe presence of tunicamycin. J Biol Chem 1979; 254:11943-11952. BarbariC S, KomliC B, Ries B,Mildner P. Multiple forms of acidphosphatase from Saccharomyces cerevisiae.Adv ClinEnzymoll982;2: 143-149. Pugh TA, Shah JO, Magee PT, Clancy MJ. Characterization and localization of the sporulation glucoamylase of Saccharomyces cerevisiae. Biochim Biophys Acta 1989; 994:200-209. Correa JU, Elango N, Polacheck I, Cabib E. Endochitinase,a mannanassociated enzyme from Saccharomyces cerevisiae. J Biol Chem 1982; 257: 1392-1397. Bause E, Erkens R, Schweden J, Jaenicke L. Purification and characterization of trimming glucosidase I from Saccharomyces cerevisiae. FEBS Lett 1986:206-212. Londesborough J, Varimo K. Characterization of two trehalases in baker’s yeasts. Biochem J 1984; 219511-518. Gilboa-Garber N.Lectins ofPseudomonas aeruginosa:properties, biological effects and applications. In: Mirelman D, ed. Microbial lectins and agglutinins. New York: John Wiley & Sons, 1986:255-269. Del Rey F, Villa TG, Santos T, Garcia-Acha I, Nombela C. Purification and partial characterization of a new sporulation specific, exo-0-glucanase from Saccharomyces cerevisiae. Biochem Biophys Res Commun 1982; 105:13471353. Sanchez A, NebredaAR, Villanueva JR, Villa TG. Post-secretional modification of exo-&1,3-~-glucanasesfrom Saccharomyces cerevisiae. Biochem J 1983; 215:471-474. Sanchez A, Villanueva JR, Villa TG. Effect of tunicamycin on exo-p-1,3-~glucanase synthesisand secretion of cells and protoplasts of Saccharomyces cerevisiae. J Gen Microbiol1982: 128:3051-3060.

286

Lakhtin

114. Chu FK, Maley F. Stabilization of the structure and activity of yeast carboxypeptidase Y by its high-mannose oligosaccharide chains.Arch Biochem Biophys 1982; 214:134-139. 115. Turkova J, Fusek M, Maksimov JJ, Alakhov YB. Reversible and irreversible immobilization of carboxypeptidase Y using biospecific adsorbtion. J Chromatogr 1986; 376:315-321. 116. Mizuno K, Nakamura T, Takada K,Sakakibara S, Matsuo H. A membranebound, calcium-dependent protease of yeast a-cell cleaving on the carboxyl side of paired basic residues.Biochem Biophys Res Commun 1987; 144:807814. 117. Dunlop PC, Meyer GM, Ban D, Roon RL. Characterization of two forms of asparaginase inSaccharomyces cerevisiae.J Biol Chem 1978; 253:1297-1304. 118. Pastor FIJ, Valentin E, Herrero E, Sentandreu R. Structure of the Saccharomyces cerevisiae cell wall. Mannoproteins released by zymolyase and their contribution to wall architecture. Biochim BiophysActa 1984; 802:292-300. 119. Sanz P, Herrero E, Sentandreu R. Autolytic release ofmannoproteins from cell walls ofSaccharomyces cerevisiae.J Gen Microbiol1985;131:2925-2932. 120. Saulnier L, MercereauT, Vezinhet F. Mannoproteins from flocculating and non-flocculating Saccharomyces cerevisiaeyeasts. J Sci Food Agric1991; 54: 275-286. 121. Chappell TG, Warren G. A galactosyltransferase from the fission yeastSchizosaccharomycespombe.J Cell Bioll989; 109:2693-2702. 122. Moreno S, Ruiz T, Sanchez Y, Villanueva JR, Rodriguez L. Subcellular localization and glycoprotein nature of the invertase from the fission yeast Schizosaccharomycespombe. Arch Microbiol1985; 142:370-374. 123. De Mora JF, Valentin E, Herrero E, Sentandreu R. Glycoprotein molecules in the walls of Schizosaccharomycespombewild-type cellsand a morphologically altered mutant resistant to paplulacandin B. J Gen Microbiol1990; 136: 2251-2259. 124. Moranelli F, Yaguchi M, Calleja GB, Nasim A. Purification and characterization of the extracellular a-amylase activity of the yeast Schizosaccharomyces alluvius.Biochem CellBioll987; 69899-908. 125. Ojha M,Wallace CJA. NovelCa+'-activated neutral protease from an aquatic fungus, AIIomyces arbuscula.J Bacterioll988; 170:1254-1260. 126. Vazquez C, Martinez MJ, Guillen F, Reyes F. &Glucosidase of Alternaria alternata autolyzed cultures. In: Proceedings of the 14th congress on biochemistry. July 9-15, 1988, Prague, v01 4:148. 127. Kolhekar SR, Mahajan PB, Ambedkar SS, BorkarPS. Purification and characterization of glucoamylase from a higher yieldingmutant of Aspergillus candidus Link var. aureus. Appl Microbiol Biotechnol1985; 22:181-186. 128. Hearn WM, Mackenzie DWR. The preparation and partial purification of fractions from mycelial fungi with antigenic activity. Mol Immunol 1980; 17: 1097-1103. 129. Fratamico PM, Buckley HR. Identification and characterization of an immunodominant 58-kDa antigen ofAspergillus fumigatus recognized by sera of patients with invasive aspergillosis.Infect Immun 1991; 59309-315.

lectin Sorbents

287

130. Borchehert A, Larsson PO, Mosback K. Matrix for high performance liquid affinity chromatography. J. Chromatogr 1982; 244:49-56. 131. Bruchmann EE, Schach H, Graf H. Role and properties of lactonase in a cellulase system. Biotechnol Appl Biochem 1987; 9: 146-1 59. 132. Svensson B, LarsenK, Svendsen I. Amino acid sequence tryptic of fragments of glucoamylase G1 from Aspergillus niger. Carlsberg Res Commun 1983; 48~517-527. 133. Woodward J, Marquess HJ, Picker CS.Affinity chromatography of glucosidase and endo-8-glucanasefrom Aspergillus nigeron concanavalin A-Sepharose: implications for cellulase component purification and immobilization. Prep Biochem 1986; 16:337-352. 134. Serban M, Roseanu A. Separation of invertase by affinity chromatography on ConA-Sepharose. In: Proceedings of the 14th congress on biochemistry. July 9-15, 1988, Prague, v01 4:150. 135. Gunata Z, Brillouet JM, Viorin S, Baumes R, Cordonnier R. Purification and some properties of an a-L-arabinofuranosidase from Aspergillus niger. Action on grape monoterpenyl arabinofuranosylglucosides.J Agr Food Chem 1990; 38~772-776. 136. Shishido K, Habuka N. Purification of S1-nuclease to homogeneity and its chemical, physicaland catalytic properties. Biochim BiophysActa 1986; 884: 215-218. 137. Reddy LG, Shankar V. Influence of lectin concentration on the catalytic properties of S1-nuclease bound to concanavalin A-Sepharose. Appl Biochem Biotechnol 1989; 22:79-94. 138. Reddy G , Shankar V. Preparation and properties of RNAse T2 immobilized on concanavalin A-Sepharose. Appl Biochem Biotechnol 1989; 22:237246. 139. Kanaya S, Uchida T. An affinity adsorbent, 5’-adenylate-aminohexyI-Sepharose. 11. Purification and characterization of multi-forms of RNAse T2. J Biochem 1981; 90:473-481. 140. Hitomi J, Murakami Y, Saitoh F, Shigemitsu N, YamaguchiH. Purification and characterization of endo-0-N-acetylglucosaminidase of Aspergillus oryzae. J Biochem 1985; 98527-533. 141. Civas A, Eberhard R, Le DizetP, Petek F. Glycosidases inducedin Aspergillus tamarii. Secreted a-D-galactosidaseand 8-D-mannanase. Biochem J 1984; 219:857-863. 142. Ali S, Hossain Z, Mahmood S, Alam R. Purification of glucoamylase from Aspergillus terreus.World J Microbiol Biotechnol 1990; 6:16-18. 143. Workman WE, Day DF. Purification and properties of &glucosidase from Aspergillus terreus.Appl Environ Microbiol1982; 44: 1289-1295. 144. Grassin M, Dubourdieu MD. Acquisitions recentes sur lesenzymes extracellulaires de Botrytis cinerea. Rev Fr Ounol 1987; 27, (Supp1)N 108:34-36. 145. Zaprometova OM, Lakhtin VM, Ulezlo IV. A study of the interaction of a-galactosidase and 0-N-acetylglucosaminidasefrom Cephalosporium acremonium 237 with lectins. Biotechnology (Mosc) 1986; N3:57-61 [in Russian]. 146. Zaprometova OM, Ulezlo IV, Lakhtin VM. Structure and properties of a

Lakhtin

288

Cephalosporiumacremonium a-galactosidase.Glycoconjugate J 1990;7: 287-300. 147. Strobe1G, Van Alfen N,Hapner KD, McNeil M, AlbersheimP. Some phyto148. 149. 150. 151. 152.

153.

toxic glycopeptide from Ceratocystis ulmi, the Dutch elm disease pathogen. Biochim Biophys Acta1978; 538:60-75. Fahnrich P, Irrgank K. Affinity chromatography of extracellular cellulase from Chaetomium cellulolyticum.Biotechnol Lett 1984; 6:251-256. Ishigami T, Yamada Y. Purification and properties of polyphenol oxidase from Chaetomium thermophile, a thermophilic fungus. J Gen Appl Microbiol 1986; 32:293-301. Kruse D, Cole GT. Isolation of tube precipitin antibody-reactivefractions of Coccidioides immitis. Infect Immun 1990; 58:169-178. Bonnen AM, Hammerschmidt R. Cutinolytic enzymes from Colletotrichurn lagenarium. Physiol Mol Pathol 1989; 35:463-474. Kawaguchi N, Ohmori T, Takeshita Y,Kawanishi G, Katayama S, Yamada H. Occurrenceof Gal-&1,3-GalNAc-a-Ser/Thr in the linkage region of polylactosamine containing fungal glycoprotein from Cordycepsophioglossoides. Biochem Biophys Res Commun 1986; 140:350-356. Brillouet JM, Moulin JC, Agosin E. Production, purification, and properties of an a-L-arabinofuranosidase from Dichomitus squalens. Carbohydr Res

1985; 144~113-126. 154. Das DVM, Weeks G. Studies on the unmasking of membrane-bound alkaline 155. 156. 157. 158. 159.

phosphatase during the differentiation of Dictyostelium discoideum. Can J Biochem CellBioll984; 62:970-974. Bulgakov R, Van Haastert PJM. Isolation and partial characterization of a cyclic GMP-dependent GMP-specific phosphodiesterase fromDictyostelium discoideum.Biochim Biophys Acta 1983; 75656-66. Orlow SJ, Shapiro RI, Franke J, Kessin RM. The extracellular cyclic nucleotide phosphodiesterase of Dictyostelium discoideum. Purification and characterization. J Biol Chem 1981; 256:7620-7627. Franke J, KessinRH. The cyclicnucleotidephosphodiesteraseinhibitory protein of Dictyostelium discoideum. Purification and characterization. J Biol Chem 1981; 256:7628-7637. Killick KA.Trehalase from the cellular slime mold Dictyostelium discoideum: purification and characterization of the homogeneous enzyme from myxamoebae. Arch Biochem Biophys1983; 222561-573. Kilpatrick DC, Stirling JL. Properties and developmental regulation of an a-D-galactosidase fromDictyostelium discoideum.Biochem J 1976; 158:409-

417. 160. Bernstein RL, Tabler M, Westweber D,

Van Driel R. Extracellular folate deaminase of Dictyostelium discoideum. Biochim Biophys Acta 1981; 677:

295-302. 161. Saxe CL, Sussman M.Induction of stage-specific cell cohesion in Dictyostel-

iumdiscoideurn by a plasma-membrane-associatedmoietyreactivewith wheat germ agglutinin. Cell1982; 29:755-759.

lectin Sorbents

289

162. Ray J, Lerner RA. Abiologicallyactivereceptor for the carbohydratebinding protein(s)of Dictyostelium discoideum.Cell 1982; 28:91-98. 163. Breuer W, Siu CH. Identification of endogenous binding proteins for the lectin discoidin-I in Dictyostelium discoideum.Proc Natl Acad Sci USA 1981; 78~2115-2119. 164. Kubohara Y, Okamoto K. A stalk-specific wheat germ agglutinin binding protein, wst 34, in Dictyostelium discoideumcan be detected with antiserum raised against Dictyosteium mucoroides stalk. Biochem Cell Biol 1990; 68: 699-704. 165. Kanaya S, Yoshida H. Phosphodiesterase-phosphomonoesterases from Fusarium moniliforme. Separation and properties of four isoenzymes. J Biochem 1979; 85:791-797. 166. Roy U, Vora VC. Purification and properties of a carboxymethylcellulase from phytophathogenicfungus Macrophominaphaseolina. Indian J Biochem Biophys 1989; 26943-248. 167. Berry RK, Dekker RFH. Fractionation of the cellulolytic enzymes produced by a species of Monilia. Purification and properties of an extracellular &Dglucosidase. Carbohydr Res 1986; 157:l-12. 168. Moriguchi M, Yamada M, Suenaga S, Tanaka H, Wakasugi A, Hatanaka SI. Partial purification and properties of y-glutamyltranspeptidasefrom mycelia of Morchella esculents. Arch Microbiol 1986; 144:15-19. 169. Yamamoto K, Tsuji Y,Matsushita S, Kumagai H, Tochikura T. Purification and properties of 8-N-acetylhexosaminidasefrom Mucor fragilis grown in bovine blood.Appl Environ Microbioll986; 51:1019-1023. 170. Kadowaki S, Yamamoto K, Fujisaki M, Izumi K, Tochikura T, Yokoyama T. Purification and characterization of a novel fungal endo-8-Nacetylglucosaminidase actingon complex oligosaccharides of glycoproteins. Agr Biol Chem 1990; 54:97-106. 171. Huge-Jensen B, Galluzzo DR, Jensen RG. Studies on free and immobilized lipases from Mucor mihei.J Am Oil Chem SOC 1988; 65:905-910. 172. Yamada H, Hiraiwa M, Miyazaki T. Characterization of mannoproteins from yeast and mycelial forms of Mucor rouxii. Carbohydr Res 1983; 119: 129-140. 173. Grotewold E, Taccioli GE, Aisemberg GO, Judewicz ND.A single-steppurification of an extracellular fungal laccase. J Appl Microbiol Biotechnol1988; 4~357-363. 174. Hiura N, Nakajima T, Matsuda K. Purification and some properties of an endo-8-1,6-glucanase from Neurosporacrassa. AgrBiolChem1987;51: 3315-3321. 175. Ishikawa F, Oishi K. Production, purification, and characterization of Neurosporasitophila lectin. AgrBiol Chem 1989; 53:1769-1776. 176. Voiland A, Michel G . Characterization of 6-deoxy-~-altriolin the cell wall polysaccharide ofNocardia asteroidesR 399. Carbohydr Res 1985; 141:283288. 177. Krysteva MA,Papukchieva SP, Zlateva TP. Affinity chromatography purifi-

290

178. 179. 180. 181. 182. 183. 184.

185. 186.

187. 188. 189. 190. 191. 192. 193.

Lakhtin

cation of apoglucose oxidase from Penicillium chrysogenum.Rep Acad Sci Bulg 1990; 43(9):69-72. Sahasrabudhe NA, Laohke AH, Ranjekar PK. Characterization of the purified multi-functional cellulase component of Penicillium funiculosum. Biotechno1Lett 1987; 9:881-886. Copa-Patino JL, Rodriquez J, PCrez-Leblic MI. Purification and properties of a 8-glucosidase form Penicillium oxalicum autolysates. FEMS Microbiol Lett 1990; 67~191-196. Yamamoto K, Lee KM,, Kumagai H, Tochikura T. Purification and characterization of 8-N-acetylhexosaminidasefrom Penicillium oxalicum. Agr Biol Chem 1985; 49:611-619. Kamagata Y, Yachi M, Sasaki H, Takao S. Purification and properties of two endocellulases from Penicillium purpurogenum. Agr Biol Chem 1986; 50~2989-2995. Renganathan V, Miki K, Gold MH. Multiple molecularforms of diarylpropane oxygenase, an H202-requiring,lignin-degrading enzymefrom Phanerochaete chrysosporium.Arch Biochem Biophys 1985; 241:304-314. Tien M. Properties of ligninasefrom Phanerochaete chrysosporiumand their possible applications. CRC Crit Rev Microbiol 1987; 15:141-168. Murakami-Murofushi K, Takahashi T, Minowa Y, Iino S, Takeuchi T, Kitagaki-Ogawa H, Murofushi H, Takahashi K. Purification and characterization of a novel intracellular acid proteinase from the plasmodia of a true slime moldPhysariumpolycephalum. J Biol Chem 1990; 265:19898-19903. Forster H. Pectinesterases from Phytophthora infestans. Methods Enzymol 1988; 161:355-361. Parker JE, Schulte W, Hahlbrock K, Scheel D. An extracellular glycoprotein from Phytophthora megasperma f sp glycinea elicits phytoalexin synthesis in cultured parsley cells and protoplasts. Mol Plant-Microbe Interact 1991; 4: 19-27. Fabre I, Bruneteau M, Ricci P, Michel G . Isolement et etude structurale de glucanes de Phytophthoraparasitica. Eur J Biochem 1984; 142:99-103. Beissmann B, Reisner HJ. Isolation and purity determination of a glycoprotein elicitor from wheat stem rust by medium-pressure liquid chromatography. J Chromatogr 1990; 521:187-197. Mazars C, Hapner KD, Strobe1 CA.Isolation and partial characterization of a phytotoxic glycoprotein from culture filtrates of Rhynchosporium secalis (Oud). Experientia 1984; 40:1244-1247. Clarke AJ, Yaguchi M. The role of carboxylgroups in the function of endo8-1,rl-glucanase. Eur J Biochem 1985; 149:233-238. Willick GE, Seligy VL. Multiplicity in cellulases Schizophyllum of commune. Derivation partly from heterogeneity in transcription and glycosylation. Eur J Biochem 1985; 151:89-96. Patil RV, Sadana JC. The purification and properties of @-1,4-~-glucan cellobiohydrolase from Sclerotium rolfsiii: substrate specificity and mode of action. Can J Biochem CellBioll984; 62:920-926. Morpeth FF, Jones GD. Resolution, purification, and some properties ofthe

lectin Sorbents

194. 195.

196. 197. 198. 199. 200. 201. 202.

203. 204. 205. 206. 207. 208. 209.

291

multiple forms of cellobiose quinone dehydrogenase from the white-rot fungus Sporotrichumpulverulentum.Biochem J 1986; 236:221-226. Mackenzie CR, Bilous D, Johnson KG. Purification and characterization of an exoglucanase from Streptomycesflavogriseus. Can J Microbiol 1984; 30: 1171-1178. Greber UF, Kozulit B, Mosbach K. Purification of endo-0-N-acetyl-DglucosaminidaseH by substrate-affinity chromatography. Carbohydr Res 1989; 189:289-299. Wood TM, McCraeSI. Purification and some propertiesof the extracellular B-D-glucosidase of the cellulolytic fungus Trichoderma koningii. J Gen Microbiol 1982; 128:2973-2982. Niku-Paavola ML, Lappalsinen A, Enari TM, Nummi M. A new appraisal of the endoglucanase of the fungus Trichoderma reesei. Biochem J 1985; 231175-81. Tikhomirov DF, Nbtsubidze NN, Lakhtin W, Klyosov AA. Isolation of multiple forms of Trichoderma reeseiendoglucanase possessing high hydrophobicity. Biochemistry (Mosc)1987; 52: 1097-1 106[in Russian]. Chirico WJ, Brown D. Purification and characterization of a &glucosidase from Trichoderma reesei.Eur J Biochem 1987; 165:333-341. Salovuori I, Makarow M, RauvalaH, Knowles J, Kaiiriilinen L. Low molecular weight high-mannose type glycans in a secreted protein of the filamentous fungus Trichoderma reesei.Biotechnology 1987; 5: 152-156. Niku-Paavola ML, LappalainenA, Enari TM, Nummi M. Trichoderma reesi cellobiohydrolase11. Purification by immunoadsorption and hydrolytic properties. Biotechnol ApplBiochem 1986; 8:449-458. Kminkova M,KuEera J. Separation of cellobiase (EC 3.2.1.21) from the crude cellulase system (EC 3.2.1.4) of Trichodermaviride using affinity chromatography on Con Abound to agarose.J Chromatogr 1982;244:166168. Wilhelm M, Sahm H.Purification and characterization of two extracellular 0-D-glucosidasesfrom Trichodermaviride ITCC 1433. Acta Biotechnoll986; 6:115-121. Apodaca G, McKerrow JH. Purification and characterization of a 27,000-Mr extracellular proteinase from Trichophyton rubrum. Infect Immun 1989; 57: 3072-3080. Riethman HC, Mawhinney TP, Sherman LA. Characterization of phycobilisome glycoproteinsin the cyanobacterium Anacystis nidulans R2. J Bacteriol 1988; 170:2433-2440. Holo H, Broch-Due M,Ormerod JG. Glycolipids and the structure of chlorosomes in green bacteria. Arch Microbiol 1985; 143:94-99. Torres BV, Smith DF. Purification of Forssman and human blood group A glycolipids byaffinity chromatography on immobilized Helixpomatia lectin. Anal Biochem 1988; 170:209-219. Lotan R,Nicolson CL. Purification of cell membrane glycoproteinsby lectin affinity chromatography. Biochim BiophysActa 1979; 559:329-376. Muthukumar G, Nickerson KW. The glycoprotein toxin of Bacillus thurin-

Lakhtin

292

210. 21 1. 212. 213. 214. 215. 216. 217. 218. 219. 220. 221. 222. 223. 224. 225.

giensis subsp isruelensisindicates a lectinlike receptorin the larval mosquito gut. Appl Environ Microbiol 1987; 53:2650-2655. Pfannenstiel MA, Muthukumar G, Couche GA, NickersonKW. Amino sugars in the glycoprotein toxin from Bacillus thuringiensissubsp israelensis. J Bacterioll987; 169:796-801. Davidson EW. Variation in binding of Bacillus sphaericus toxin and wheat germ agglutinin to larval midgut cells of six species of mosquitoes. J Invest Pathol 1989; 53:251-259. Yoshimatsu T, Ozaki K, Shikata S, Ohta YI,Koike K, Kawai S, Ito S. Purification and characterization of alkaline endo-p-1,4-glucanases from alkalophilic Bacillus sp KSM-635. J Gen Microbiol1990; 136:1973-1979. Nakatsuka T, Suzuki K, Nakano Y, Kitaoka S. Physicochemical properties of intracellular thiaminaseI1 of Bacillus aneurinolyticus.Vitamins 1988; 62: 15-22. Yabuki M, UchiyamaA, Suzuki K, Ando A, Fujii T. Purification and properties of chitosanasefrom Bacillus circulansMH-K1 J Gen Appl Miqobiol 1988; 34:255-270. Cowan DA, DanielRM. Purification and some propertiesof an extracellular protease (caldolysin) from an extreme thermophile. Biochim Biophys Acta 1982; 705:293-305. Kim DJ, Byun SM. Purification and properties of ampicillin acylase from Pseudomonas melanogenum.Biochim BiophysActa 1990; 1W12-18. Messner P, Sleytr UB. Asparaginyl-rhamnose:anoveltype of proteincarbohydrate linkage in a eubacterial surface-layer glycoprotein. FEBS Lett 1988; 228~317-320. Ezzell JW, Abshire TG, Little SF, Lidgerding BC, Brown C. Identification of Bacillus anthracisby using monoclonalantibody to cell wall galactose-Nacetylglucosamine polysaccharide. J Clin Microbiol 1990; 28:223-33 1. Sizemore RK,Caldwell JJ, Kendrick AS. Alternate Gram staining technique using a fluorescent lectin. Appl Environ Microbiol1990; 56:2245-2247. Kolenbrander PE. Surfacerecognitionamong oral bacteria:multigeneric coaggregations and their mediators. CRC Crit Rev Microbiol 1989; 17:137159. Kamiya H, Muramoto K, Goto R, Sakai M, Ida H. Properties of a lectin in chum salmon ova. Nippon Suisan Gakkaishi 1990; 56: 1139-1 144. Loenko YuN, Glazkova VE, Mikhaylov VV, Artyukov AA, Rutskova TA, Ovodova RG. The selectivity of the interaction of lectins isolatedfrom Orenomytilusgrayanus with some microorganisms. Proc Inter-Lec 1lth, 1989; 45. Cavalcanti MSM, Almeda AMP, Coelho LCBB. Interaction of lectins with Yersiniapestis strains. ApplBiochem Biotechnol1990; 26: 125-131. Teraoka T, Sakakibara T, Den E, Hoskawa D, Watanabe M. A novel rice lectin specific to mannoside/glucoside residues in rice seedlings. Agr Biol Chem 1990; 54:3053-3056. Yang D, Wang Z-X, Zhang L-X.Isolation, purification and characterization of a lectin from the Chinese horseshoe crab Tachypleus tridendatus. Chin Biochem J 1990; 6505-510.

.

Lectin Sorbents

293

226. Ceri H, Hwang WS, Cheung H. Endogenous heparin-binding lectin activity in human placenta: purificationand developmental expression. Biochem Cell Biol 1990,68:790-795. 227. Lim SH, Salton MRJ. Comparisonof the chemical composition of lipomannan from Micrococcus agilis membrane with that of Micrococcus luteus strains. FEMS Microbiol Lett1985; 27:287-291. 228. Lotan R, Sharon N, Mirelman D. Interaction of wheat germ agglutinin with bacterial cellsand cell wall polymers. EurJ Biochem 1975; 55:257-262. 229. Wu A M , Sugii B, Gruezo FG, Kabat EA. Immunochemical studies on the N-acetyllactosamine /3-1,6-linked trisaccharide specificity of Ricinus communis agglutinin. Carbohydr Res 1988; 178943-257. 230. Kaku H, Peumans WJ, Goldstein IJ. Isolation and characterization of a second lectin (SNA-11) present in elderberry (Sambucus nigraL) bark. Arch Biochem Biophys 1990; 277:255-262. 231. Wessels MR, Rubens CE, Benedi VJ, Kasper DL. Definition of a bacterial virulence factor: sialylation of the group B streptococcal capsule.Proc Natl Acad Sci USA 1989; 86:8983-8987. 232. Mandal C, Mandal C. Sialic acid binding lectins. Experientia 1990; 46:433441. 233. Miller KJ, Kennedy EP, Reinhold W. Osmotic adaptation by gram-negative bacteria: possible role for periplasmic oligosaccharides. Science 1986; 231: 48-51. 234. Ahamed N M , Kuhn HM, Widemann C, Radziejewska-Lebrecht J, Mayer H. Specific interaction of R-type lipopolysaccharides with lectins versus their nonspecific reactivity with basic and hydrophobic proteins. In: Bag-Hansen TC, ed. Lectins: biology, biochemistry, clinical biochemistry, v01 2. Berlin: Walter de Gruyter,1982:341-350. 235. Chatterjee BP, Guha AK, Pal R, Bhattacharyya M. Lectin typing of Pseudomonas aeruginosa strains of different serogroups, Habs and Fisher types. Zentralbl BakteriolHyg [A] 1989; 271:364-371. 236. Parent JB. Membrane receptors on rat hepatocytes for the inner core region of bacterial lipopolysaccharides. J Biol Chem 1990; 265:3455-3461. 237. Wright SD, Ramos RA, Tobiai! PS, Ulevitch RJ, Mathison JC. CD14, a receptor for complexes of LPS and LPS binding protein. Nature 1990; 249: 1431-1433. 238. Apicella MA, Mandrell RE, Shero M, Wilson ME, Griffiss JM, Brooks GF, LammelC,Breen JF, Rice PA. Modification by sialicacidof Neisseria gonorrhoeae lipopolysaccharide epitope expression in humtin urethral exudates: an immunoelectron microscopic analysis. J Infect Dis 1990; 162506512. 239. Krauss JH, Reuter G, Schauer R, Weckesser J, Mayer H. Sialic acid-containinglipopolysaccharidesinpurplenonsulfurbacteria.ArchMicrobiol 1988; 150586589. regulated 2 4 0 . Sacci JB, Campbell TA, Gottlieb M. Leishmaniadonovani: changes in the level of expression of the surface 3'-nucleotidase/nucleae. Exp Parasitol 1990; 71:158-168.

Lakhtin

294

AK. A comparison of the lectinbinding properties of glycoconjugates from a range of Leishmania species. Parasitol Res 1990; 76:294-300. Grog1 M, Franke ED, McGreevy PB, Kuhn RE. Leishmaniabraziliensix protein, carbohydrate, and antigen differences between log phase and stationary phase promastigotesin vitro. Exp Parasitoll987; 6332-359. Merkle RK, Cummings RD. Lectin affinity chromatography of glycoproteins. Methods Enzymol1987; 138:232-259. Osawa T. Recent progress in the application of plant lectins to glycoprotein chemistry. Pure Appl Chem 1989; 61:1283-1292. Gallagher JT. Affinity chromatography of complex carbohydrates using lectins. In: Oliver RWA, ed. HPLC of macromolecules: a practical approach. Oxford: IRL Press, 1989:209-227. Iqbal J, Saleemuddin M. Activity and stability of glucose oxidaseand invertase immobilizedon concanavalin A-Sepharose: influenceof lectin concentrations. Biotechnol Bioeng 1983; 25:3191-3195. D’Souza SF, Nadkarni GB. Continuous conversion of sucrose to fructose and gluconic acid by immobilized yeast cell multienzynme complex. Biotechnol Bioengin 1980; 222179-2189. Hussain Q, Iqbal J, Saleemuddin M. Entrapment ofconcanavalin Aglycoenzyme complexes in calcium alginate gels. Biotechnol Bioengin 1985;

241. Rossell RJ, Stevens A F , Miles MA, Allen 242. 243.

244. 245. 246. 247. 248.

27~1102-1107. 249. Hussain Q, Saleemuddin M. Immobilization of glycoenzymes using crude concanavalin Aand glutaraldehyde. Enzyme Microbiol Technol 1986; 8:686690. R, Rinaldi A,Satta G . Highly efficient solubi250. Fadda MB, Dessi MR, Maurici

lization ofnatural lignocellulosic materials by a commercial cellulase immobilized on various solid supports. Appl Microbiol Biotechnol 1984;19:306311. 251. Woodward J, Zachry GS. Immobilization of cellulase through its carbohy-

drate side chains: a rationale for its recovery and reuse. Enzyme Microbiol Technol 1982; 4245-248. 252. Chu FK, Maley F, Tarentino AL. The use ofiodinated lectins for determination the degree of deglycosylation of high-mannose glycoproteins by endo-8N-acetylglucosaminidase H.Anal Biochem 1981; 116:152-160. 253. Iqbal J, Saleemuddin M. Sucrose hydrolysis using invertase immobilizedon concanavalin A-Sepharose. Enzyme MicrobiolTechnol 1985; 7:175-178. for renal clearance determina254. Day DF, Workman WE. A simple inulin assay tion using immobilized 8-fructofuranosidase. Ann NY Acad Sci 1986; 434: 504-507. 255. Abbasi A, Voelter W, Zaidi ZH. Isolation, purification, and properties of a

site-specific proteolytic enzymevalyl-proteinase from Candida tropicalis. Biol Chem Hoppe-Seyler1986; 367:441-445. 256. Colas B, Boulanger Y. Glycosylationofyeastaspartyl-tRNAsynthetase. Affinity labelling by glucose and glucose-6-phosphate. FEBS Lett 1983; 163: 175-180.

lectin Sorbents

295

257. Herrero E, Sanz P, Sentandreu R. Cell wall proteins liberated by zymolyase from several ascomycetous and imperfect yeasts. J Gen Microbiol 1987; 133: 2895-2903. 258. Mizuno T, Ohsawa K, Hagiwara N, Kuboyama R. Fractionation and characterization of antitumor polysaccharides from “Maitake,” Grifola frondosu. Agr Biol Chem 1986; 50:1679-1688. 259. Kaku H, Van Damme EJM, Peumans WJ, Goldstein IJ. Carbohydratebinding specificity ofthe daffodil (Narcihs pseudonarcissus) and amaryllis (Hippeastrum hybr) bulb lectins. Arch Biochem Biophys 1990; 279:298-304. 260. Guillot-Breton A, Damez M, Dusser M, Gaillard-MartinieB, Millet L. Use of lectins for a comparative study of cell wallcomposition ofdifferent anaerobic rumen fungalstrains. E M S Microbiol Lett 1990; 67:151-156. 260a. Amatayakul-Chantler S, Ferguson MAJ, Dwek RA, Rademacher TW, Parekh RB, Crandall IE, Newel1 PC. Cell surface oligosaccharides on Dictyostlium during development. J Cell Sci1991; 99:485-495. 261. Morgan AC, Woodhouse CS, Knost JA, Abrams PG, Clarke GC, Arthur LO, McIntyre R, OchsJJ, Foon KA, Oldham RK. Monoclonal antibodiesto human colorectal tumor-associated antigens: improved elicitation and subclass restriction. Hybridoma 1984; 3:233-245. 262. Mattiasson B, Johansson PA. A simple rapid methodfor quantifying microorganisms by their metabolic activitywhen bound to a specific adsorbent. J Immunol Methods 1982; 52:233-240. 263. Mattiasson B. Analytical applications of immobilized cells. In: Mattiasson B,ed.Immobilizedcellsandorganelles, v01 2. Boca Raton: CRC Press, 1983:95-123. 264. Warren GS, Fallon R. Reversible, lectin-mediated immobilization of plant protoplasts on agarose beads. Planta 1984; 161:201-206. 265. Mandenius CF, Welin S, Danielsson B, Lundstrom I, Mosbach K. The interaction of proteinsand cells withaffinity ligands covalently coupledto silicon surfaces as monitored by ellipsometry. Anal Biochem1984; 137:106-114. 266. Mandenius CF, Mosbach K. Detection of biospecific interactions using amplified ellipsometry. Anal Biochem1988; 170:68-72. 267. Mattiasson B. Reversible immobilization of enzymes with special reference to analytical applications. J ApplBiochem 1981; 3:183-194. 268. Ohlson S, Hansson L, Glad M, Mosbach K, Larsson PO. High performance liquid affinity chromatography: anew tool in biotechnology. Trends Biotechno1 1989; 7:179-186. 269. Welply JK. Sequencing methodsfor carbohydrates and their biological applications. Trends Biotechnol1989; 75-10. 270. Lee KB, Loganathan D, Merchant ZM, Linhardt RJ. Carbohydrateanalysis of glycoproteins. A review. Appl Biochem Biotechnol1990; 2353-80. 271. Berezin BB, Gevorkyan RG, Lakhtin VM, Yamskov IA. Affinity sorbent containing ConA immobilized via cobalt(3 +) complex. Proc Inter-Lec 1 lth, 1989; 7. 272. McCoy JP. Contemporary laboratory applications of lectins. Biotechniques 1986; 4:252-262.

296

Lakhtin

273. Tojo M, Shibata N, Osanai T, Mikami T, Suzuki M, Suzuki S. Sandwich enzyme-linked immunosorbent assay of D-mannansCandida of albicansNIH A-207 and NIH B-792 strains using concanavalin A and polyclonal rabbit anti-C. albicans antisera. Carbohydr Res 1991; 213:325-330. 274. Robinson JE, Holton D, Liu J, McMurdo H, Murciano A, Gohd R. A novel enzyme-linked immunosorbent assay (ELISA)for the detection of antibodies to HIV-l envelope glycoproteins basedon immobilization of viral glycoproteins in microtiter wells coated with concanavalin A. J Immunol Methods 1 9 9 0 ; 132:63-71. 275. Sjoblom I, Lundstram M, Sjiigren-JanssonE, Gloriso JC, Jeansson S, Olofsson B. Demonstration and mapping of highly carbohydratedependent epitopes in the herpes simplex virus type l-specified glycoprotein C. J Gen Virol 1987; 68545-554. 276. Graham K, Keller K, Ezzell J, Doyle R. Enzyme-linked lectinosorbent assay (ELLA) for detecting Bacillus anthracis.Eur J Clin Microbiol 1984; 3:210212. 277. Shakhanina K, Kalinin N, Ruanet V, Kuljakina M, Pavlova I, Lakhtin V. ELISHA-like methods of micrassay of anthrax agent basedon soybean agglutinin. Proc Inter-Lec llth, 1989;65. 278. Kalinin NL, Goncharov DB, Shakhanina KL, Safjanova VM. Comparative studies on the interaction between lectins and Leishmania in agglutination test and enzyme-linked lectin-biotin assay (ELLBA). Proc Inter-Lec 12th, 1990;62. 279. Lakhtin VM, Guseva NI, Fedurkina N V , Chibisova VA, Shakhanina KL. Differential affinity of pathogenic microbial species to a set of lectins, detected by the sandwich method withthe use of fluorescein isothiocyanate.J Microbiol Epidemiol Immunobiol (Mosc) 1985; N1:30-33. 280. Adam G, Heegard P, Bag-Hansen TC, Mundry KW. Lectins as probes for the assay of rhabdovirus infections in plants.J Virol Methods 1987; 17:263275. 281. Riethman HC, Mawhinney TP, Sherman LA. Phycobilisome-associated glycoproteins in the cyanobacterium Anacystis nidulans R2. FEBS Lett 1987; 215:209-214. 282. Gubler F, Hardham AR. Secretion of adhesive material during encystment of Phytophthora cinnamomizoospores, characterizedby immunogold labelling with monoclonal antibodies to components of peripheral vesicles. J Cell Sci 1988; 90~225-235. 283. Holden DW, Rohringer R. Proteins in intracellular washing fluid from noninoculated and rust-affected leavesof wheat and barley. Plant Physiol 1985; 78~715-723. 284. Liang R, Emerich DW. Analysis of lectin binding Bradyrhizobium by japonicum strains grown on nitrocellulose filters using peroxidase-labeled lectin. Anal Biochem 1987; 164:488-493. 285. Mirelman D, ed. Microbial lectinsand agglutinins: propertiesand biological activity. New York: John Wiley & Sons, 1986. 286. Schrinner E, Richmond MH, Seibert G, Schwarz U, eds. Surface structures

lectin Sorbents

287. 288. 289. 290. 291. 292. 293. 294. 295. 296. 297.

298. 299. 300. 301. 302.

303.

297

of microorganisms and their interactions with the mammalian host. Weinheim: Verlagsgesellschaft, 1988. Markwell MAK. Viruses as hemagglutininsand lectins. In: Mirelman D, ed. Microbiallectins and agglutinins:properties and biologicalactivity. New York: John Wiley & Sons, 1986:21-53. Weir DM. Carbohydrates as recognition molecules in infection and immunity. FEMS MicrobiolImmunol 1989; 47:331-340. Keusch GT, Donohue-Rolfe A, Jacewicz M. Sugar binding bacterial toxins. In: MirelmanD, ed. Microbial lectinsand agglutinins: properties and biological activity.New York: John Wiley & Sons, 1986:271-296. Uhlenbruck G. Bacterial lectins: mediators of adhesion. Zentralbl Bakteriol Hyg [A] 1987; 263:497-508. Ofek I. Lectinophagocytosis mediated bybacterial surface lectins. Zentralbl Bakteriol Mikrob Hyg[A] 1989; 270:449-455. Old DC. Bacterial cell envelopes in adhesion. In: Hancock I, Poxton I, eds. Bacterial cell surface techniques. Chichester: John Wiley & Sons, 1988:227240. Sharon N, Lis H. Lectins as cell recognition molecules. Science 1989;2 4 6 : 227-234. Pereira MEA. Lectins and agglutinins in protozoa. In:MirelmanD,ed. Microbiallectins and agglutinins:properties and biologicalactivity. New York: John Wiley & Sons, 1986:297-317. Ishikawa F, Oishi K. Chitin-binding lectins from fungi. In: Zikaris JP, ed. Chitin, chitosan, and related enzymes. Orlando: Academic Press, 1984:369382. Kocourek J, Freed D, eds. Lectins: biology, biochemistry, clinical biochemistry, v01 7. St. Louis: Sigma ChemicalCO, 1990. Norberg T, Ritzen H. Synthesis of methyl-ar-D-Glcp-ar-1,2-D-Galp-cw1,3-~-Glcp and an acyclic analogue thereof for probing the carbohydratebinding specificity of bacteriophage 4x174. Glycoconjugate J 1986;3:135142. Wollin R, Bruse GW, Jansson PE, Lindberg AA. Definition of the phage G 13 receptor as structural domains of trisaccharides inSalmonella and Escherichia colicore oligosaccharides. J Mol Recogn 1989; 2:37-43. Drake D, Taylor KG,BleiweisAS,Doyle RJ. Specificity of the glucanbinding lectin ofStreptococcus Cricetus.Infect Immun 1988; 56:1864-1872. Cowan MM, Parrish K, Kessler RE, Pyle CJ, Taylor KG, Ciardi JE, Doyle RJ. Glucan-binding factor in saliva. Infect Immun 1988; 56:2912-2917. Kundu M, Basu J, Chakrabarti P. Purification and characterization of an extracellular lectin from Mycobacterium smegmatis. FEBS Lett 1989; 256: 207-210. Al-Mahmood S, Giummelly P, Bonaly R, Delmotte F, Monsigny M. Kluyveromyces bulgaticus yeast lectins. Isolation of N-acetylglucosamineand galactose-specific lectins: their relation with flocculation. J Biol Chem 1988; 263: 3930-3934. Giummelly P, Separi F, Suptijah P, Bonaly R. Cell wall receptors of the

298

304. 305.

306.

307. 308. 309. 310. 311. 312. 313.

Lakhtin

lectins produced by Kluyveromyces bulgaricus. Yeast 1989; S(special issue): S309-3 1 1. Barak R, Chet I. Lectin of Sclerotium rolfsiii: its purification and possible .function in fungal-fungal interaction. J Appl Bacteriol1990;69:lOl-112. Guinet RMF, Rogemond V, Tessier F, Capdepuy M. Characterization and possible role of lectinlike adhesion in the Bacteroidesfragilis group. In: Proceedings of international seminar on biology of anaerobic bacteria. June 1718, 1986, Lille. Amsterdam. 198650-68. Szecowka J, Hallden G , Goldfine ID, Williams JA. Purification of the pancreatic cholecystokinin receptor. Regul Peptides 1989; 24:215-224. Duong LT,Hadac EM, Miller LJ, Vlasuk GP. Purification and characterization of the rat pancreatic cholecystokinin receptor. J Biol Chem 1989; 264: 17990-17996. Hill R, LevinJ. Partial purification of thrombopoietin using lectin chromatography. Exp Hematoll986; 14:752-759. Ushikubi F, Nakajima M, Hirata M, Okuma M, Fujiwara M, Narumiya S. Purification of the thromboxane A2/prostaglandin H2 receptor from human blood platelets. JBiol Chem 1989; 264:16496-16501, Giampapa CS, Abraham SN, Chiang TM, Beachey EH. Isolation and characterization of a receptor for type 1 fimbriae of Escherichia coli from guinea pig erythrocytes. J Biol Chem 1988; 2635362-5367. Geary SJ, Gabridge MG. Characterization of a human lungfibroblast receptor site for Mycopasmapneumoniae. Isr J Med Sci 1987; 23:462-468. Hart GW, Haltiwanger RS, Holt GD, Kelly WG. Glycosylation in the nucleus and cytoplasm. AnnuRev Biochem 1989; 58:841-874. Belzunces LP, Theveniau M, Masson P, Bounias M. Membrane acetylcholine esterase from Apis melliferahead solubilized by phosphatidylinositol-specific phospholipase Cinteracts with an anti-CRD antibody. Comp Biochem Physio1 B 1990; 95:609-612.

9 Microbial Lectins for the Investigation of Clycoconjugates K. L. SHAKHANINA and N. L. KALlNlN Gamaleya Institute of Epidemiology and Microbiology,Russian Academy of Medical Sciences, Moscow, Russia V. M. LAKHTIN Institute for Applied Science of Moscow University and Institute of Food Substances, RussianAcademy of Medical Sciences, Moscow, Russia 1. INTRODUCTION

This review contains data on interaction of lectins of viruses, rickettsias, bacteria, protozoans, microscopic fungi, and yeasts with solubleand receptor glycoconjugates(glycoproteins,glycolipids,lipopolysaccharides, and other biopolymers)containingpolysaccharides of vertebrates,invertebrates, plants, and microorganisms. The summary presented indicatesnot only carbohydrate (mono-and disaccharide) and oligosaccharide extended specificities of lectinsfrom different microorganisms,but also defines their specificitiestowardglycoconjugatepolysaccharidemolecules. The summary may be used as a basis for purification of receptors and other glycoconjugates and polysaccharideswith the helpofimmobilizedmicrobial lectins. In recent years more and more attention has been paid to carbohydrate-binding proteins, especially lectins. This is clearly evident as seen from numerous reviewsand books [l-l l]. Multiple conferences, symposia, seminars, and schools have been devoted to the study of glycoconjugates and lectins.Rapidgrowthin the numbersoffirmsproducingcomplex glycans,neoglycoconjugates,lectins,glycosidases,carbohydrate-binding toxins,monoclonalantibodies to carbohydrate-containingtargets,and publication ofnew scientific journals (e.g., Trends in Glycoscience and Glycotechnology) are additional evidence for the importance of studying glycoconjugates and their interactions with lectins.In fact, glycoconjugate recognition isthe basis of several key meFhanisms of biological recognition of living organisms. 299

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The least studied lectinsare still thoseof microbial originthat interact selectively with glycoconjugates of host organisms (initial infection processes in humans, farm animals, plants, and marine food organisms). Aspects of bacteria-mediated fixation of nitrogen in tubercles of plants, the obtaining of microbial insecticides, the phenomena of microparasitism, and selective coaggregationof microorganisms, all involve protein-glycoconjugate interactions [4,12-171. In many interactions between microbial lectins and cell surfaces, there is insufficientinformation available about the structures of receptor glycoconjugatesand the spectrum of possible glycoconjugate targets. Studyof the interaction of purified microbial lectins with a set of model glycoconjugates (glycoproteins, glycolipids, polysaccharides) with the known structures of glycans helps define the specificities of microbial lectins. Therefore,the main task of the present reviewtoisoutline the data on new preparations of microbial lectins that have not been considered in other reviews [12,18-221. II.

INTERACTION OF MICROBIAL LECTINS WITH CARBOHYDRATE-CONTAINING TARGETS

Agglutinins (mainly lectins) have been investigatedfrom about 150 microbial sources and isolated from only a few species. However, lectins from only 100 microorganism species have been characterizedfor monosaccharide specificities. There is little information about oligosaccharide specificities of microbial lectins or of their specificities for wholemoleculesof glycoconjugates and polysaccharides. The main sources of microbial lectins are from viruses (defined as a microorganism for purposes of this book), bacteria, protozoans, yeasts, and fungi, including mushrooms. The lectins that belong to each of these groups of microorganisms will be considered. 111. VIRALLECTINS

Table 1 shows some resultson the interaction of viral lectins with receptors and isolated glycoconjugates (natural or synthetic). Many viral proteins show hemagglutinating activities [23], although it is only recentlythat hemagglutinins of viral origin have begun to be considered as lectins [23]. Carbohydrate-containing targets for viral lectins, as a rule, include lipopolysaccharides, gangliosides,and glycoproteins (see Table 1). The mostthorough investigation was carriedout on the specificities of the hemagglutinins of influenza viruses for mammalian glycoproteins and gangliosides [34-391. All hemagglutinins of influenza virus types A, B, and C exhibit the properties of sialospecific lectins, interacting with terminal residues of N-acetylneuraminic or N-glycolylneuraminic acid in glycans.

Microbial

301

Table 1 Interaction of Viral Lectins with Glycoconjugatesand Polysaccharides

Source of lectins Bacterial viruses Bacteriophage G13 hemagglutinin Bacteriophage 4x174 hemagglutinin Coronaviridae Sheep encephalomyelitis virus and bovine corona virus hemagglutinin Hepadnaviridae Hepatitis B virusS protein Herpemiridae Herpes simplex virustype 1 and pseudorabies virus GP11-B, GPl11-C, GPV Lentiviridae Human immunodeficiency virus type 1 gp120 Reoviridae Reovirus type3 0 protein 1 Orbivirus of blue linguae of sheep hemagglutinin Rhabdoviridae Rabies virus hemagglutinin Vesicular stomatitis viral hemagglutinin Orthomyxoviridae Influenza virus type A Influenza virus type C hemagglutinin

Targets containing carbohydrates Ra, Rb,, Rb,-type LPS from mutant strains of Salmonella sp., Escherichia coli. Glc-or-l ,2-Gal-or-l,3-Glc-containing LPS of Salmonella

Ref.

24 25

9-0-Ac-NeuSAc-containing protein of surface structure of chicken erythrocytes and human sialidasetreated erythrocytes

26

NeuAc, NeuAc-Lac-containing surface structures of Vero cells

27

Sensitive to heparin surface structure of rabbit kidney cell lines RK-13

28

Antigen CD4and transmembrane gp41 ofT lymphocytes

14,29

Glycoprotein Am of humanerythrocytes Neu-Ac-a2,6, NeuG-a2,6-containing (GP); glycophorin, mucin of bovine submandibular gland

30

GangliosidesGTlb, GQlb, GDlb

32

GangliosidesGM, of goose erythrocytes

33

31

NeuAcor2,3(6) NeuG-a2,3(6)34,36,37 containing gangliosidesand GP Neu-Accontaining or,-macroglobulins NeuSAc-or-containing neopolysac38 charide 9-0-Ac-NeuAc-containing protein of 39 surface structure of chickenerythrocytes

302

Shakhanina et al.

However, the hemagglutinin of influenza virus typeA predominantly recognizes CO-acetylated groups of N-acetylneuraminic acid. Influenza virus type C hemagglutinin shows a high affinity for 9-O-acetylated derivatives of sialic acid [37-391.Moreover, influenza virus lectinsare capable of recognizing microdomainsand clusters fromthe sialic acid residues within the elongated glycans, including sets of sialic residues. Some viral lectins are specific for polysialogangliosides(e.g.,rabiesvirushemagglutinin), and other viral lectinsfor mannosialogangliosides (suchas the vesicular stomatitis virus hemagglutinin)[32,33]. Specificities of viral lectinsfor lipopolysaccharide and neopolysaccha[24,25].Because of the rides with known structures have also been studied density of glycan clusters, their lengths and glycoconjugate structures, and composition variations, is it possibleto detect glycans with maximal affinity for lectins, as has been demonstratedfor hemagglutinins of bacteriophage G13 and influenza viruses [24].Chapter 2 of this book provides a detailed examination of the interaction between lectins, viruses, and viral-infected cells. 111. BACTERIAL LECTINS

Significantly more papers have been publishedon specificities of bacterial lectins [15-22,24,41-44].This may be because the researchers began to consider carbohydrate-binding toxins of bacterial origin as lectins [41,53,54]. Among the sources of bacterial lectins, several investigators report nearly 100 strains from more than 70 species of organisms. However, most of the lectins have not been purified, or the purified lectins have not been exhaustively investigatedfrom the viewpoint of their specificities for glycoconjugates [B].Table 2 gives data on the known specificities of bacterial lectins. As can be seen from Table 2, bacterial lectins represent preparations with different biological activities and cellular distribution. Adhesins may be found on microbial surfaces containing fimbrial and nonfimbrial appendages. Some are extracellular toxins,and some are enzymes (see Chapter 1 for a discussion ofthe definition of a lectin). Allthe microbial lectins, as a rule, show hemagglutinating properties. Toxic hemagglutinins usually include RNA-N-glycosidase or ADP-ribosyltransferase activities [41],although frequently proteolytic activity is also detectable in microbial hemagapart from glutinins [18,1391.Some glycosidasesand glucose oxidases have, [M,117,1791. their catalytic centers, lectinlike domains From the examples of coliform bacteria, pseudomonads, and cholera vibrios one can observe that each of the microorganisms synthesize a set of lectins with various specificities for glycoconjugate targets (see Table 2).

3

a

Table 2 Interaction of Lectins of Bacterial Origin with Glycoconjugates and Polysaccharides

Lectin sources

Carbohydratecontaining targets

Ref.

Actinomyces naeslundii WW45 fmbriae

Gal-@1 ,3-GaINAc/GalNAc-/31,3-Gal-containing glyco-

42

A . viscosus T14V fimbriae

Gal-@l,3-GaINAdGalNA~-@l ;3-Gal-containingpoly-

Aeromonas caviae, A . hydrophila, A . veroni, A. sobria hemagglutinins pilins/adhesins Agrobacterium tumefacienslectins

saccharides Sensitive to mannose of animal erythrocytes and lines of mammalian cells CY-L-FUC-BSA; fucan; chondroitin-sulfate; pectin of

lipids

B L

6

a2

42 45

46

&NS

Azospirillum bradiense hemagglutinin BaciIIus mesentericus316M extracellular hemagglutinin Bacteroidesfragiiis adhesin (70 kDa) B. (Prevotella)Ioescheii adhesin Bordetella bronchisepticaadhesin B. pertussis pertussis toxin (B subunit oligomer) (11-22 kDa) adhesin (filamentous hemagglutinin) Bradyrhizobiumjaponicum Campylobacterjejuni extracellular enterotoxin

a-L-Fuc-containing substances of erythrocytes NeuG1; NeuAc-containing substances of rabbit erythrocytes a-GlcN; a-GaW-Sepharose; polymers of the bacterium Enterococcus hirae; epithelial intestinal cell line of human Pro- and eukaryotic cells NeuAc-Lac-Gp-containing sialogangliosides of mycelia; mucin of bovine submandibular gland Neu5Ac-a2,6contaiNng GP; some asialo-GP; thermoprocessed fetuin; proteins of goose erythrocytes Substances of complement-3/integrin aM &; CDllb CD 18 of macrophages Lac-Sepharose; @Gal;Lac-containing polymers of soybean cell line Gangliosides SRL

+

47 48

49,50 51

52 53-57 58,59 60,61

62,62a (continued)

W

0 W

304

a

305

306

d

0

..

..

308

Shakhanina et al.

The ability of a microorganismto produce surface lectins signifies the role of lectins in the ecology of microbes, especially for biological recognition of host cells [4,17,18,22]. One can also see from Table 2 that bacterial lectins are capable of recognizing not only several types of monoand disaccharides (specificities of lectinsfor simple sugars)or oligosaccharides (fine specificities of lectins),but also macromolecular glycoconjugatesand polysaccharides. From the example of monosaccharides coupled with suitable carriers it can be observedthat the lectin-inhibiting effectiveness of the neoglycoconjugates shows lo3- or 10'-fold increase when compared with free carbohydrates [ m , 1861. The effectiveness of such conjugates depends not only on the monosaccharide type,but also on its density and distribution over the carrier surface. Lectins of Actinomyces are capable of recognizingthe same structures of disaccharide fragments within different targets, such as glycolipids and polysaccharides [166-1 681. Pertussis toxinand elder lectins react with such glycoproteins as fibrinogen, transferrin, and phosvitin, containing Neu-S-Ac-a2,6-residues of sialic acid(but not Neu-5-Ac-a2,3-residues). Fetuin, containing equal numbers of both types of these sialic acid residues, exhibits a higher affinityfor the toxin than for the elder lectins. Human fibrinogen was characterized by maximal a f f i ~ t for y the toxin. Fibrinogen glycoprotein containsfour identical biantennary asparagine-bound glycans, with terminal sialic acid residues. More weakly reacting with the toxin were human transferrin and chicken phosvitin, which possess two biantennary or one triantennary sialylated glycans, respectively. In its ability to react with sialoglycoproteins, pertussis toxin could be distinguished from a sialospecific plant lectin from wheat embryos [53,54]. Bacterial lectins also frequently show a high selectivity for glycolipids (globosides and gangliosides). With these receptors, lectins of microorganisms areoften distinguishable from those of plant or animal origin [10,17]. Whereas cholera toxin has a maximal affinity for monosialoganglioside GM,, the tetanus, botulinum, and gas gangrene toxins show high affinities for gangliosidescontaining three to four sialicacidresidues.Here, the increased selectivity of the microbial lectin for glycolipids may be enhanced because of variations in the structure of the lipid component. Thermolabile enterotoxins of various serotypes ofEscherichia coli differ in theirlevels of affinity for a wide range of gangliosides [92]. Oral streptococcal lectins [l21 may be considered an example of the high-level selectivity of microbial lectins toward polysaccharides. Ofa set of glucans, with varying contentsof a-1,6; a-1,4; a-3; and a-1,2 linkages, the lectin reacted only with those glucans containing more than 80% of the a-1,6 linkages; the m&mal affinity for lectins was a glucan containing

Microbial Ledins

309

95% of cu-1,6 linkages. Only linear glucans of the foregoing type with a relative molecular mass(M,) of over 5 x l@ kDa were ableto induce rapid aggregation of the streptococci. From Table 2, one can see that a single microbial lectin can show not only selective reaction with a definite type target (glycoprotein, glycolipid, polysaccharide)but can also recognizeglycoconjugates of various other types. Cholera toxin, for instance, may exhibit a high specificity toward some glycolipidsand polysaccharides [1661681. The ability of a microbial lectin to react selectively with glycoconjugates is determined by the domain and epitope structures of the lectin molecule. Epitope organization of the cholera toxin carbohydrate-binding subunit [1871, and the size and structure of carbohydrate-binding fragments or subunits of diphtheria, pertussis, and other microbial protein toxins also vary. FilamentousBordetella hemagglutinin contains binding sitesfor heparin or glycoconjugates containing terminal residues of galactose that are not adjacent to each other [59]. Interaction of Clostridium toxin A with antibodies does not interfere with hemagglutinating activity of this toxinlectin, which is characterized by a h,igh selectivity toward complex glycans [68,69]. Therefore, variations in selectivityof microbial lectins toward glycoconjugates may occur because of the existence of a variety of sites with different specificitiesfor glycoconjugates and because ofstructural changes within these sites. Lectins from actinomycetes, streptomycetes, and Sclerotium rorfsiiinteract differently with various gram-positive and gram-negative bacteria[166,182,183]. IV. PROTOZOAL LECTINS

Currently, protozoal lectins have been studied in limited detail relative to interactions with other microorganisms. Lectins from protozoa are also characterized by the aforementioned general regularities: productionof a system of lectins with different carbohydrate specificities bya microorganism of one species or strain and the ability of the same lectin to interact 3). In addition with various types of carbohydrate-containing targets (Table to the glycoproteins and neoglycoproteins listed in Table 3 that interact with protozoal lectins, thereare reports on the interaction betweenGiardia lamblia lectin and Salmonella lipopolysaccharides [1431. V. YEAST LECTINS

As with the protozoal lectins, little information is available concerning carbohydrate complexes with yeast lectins [154,1551. The most thorough study has been given to the preparations of lectins from the pathogenic

Shakhanina et al.

310

yeast species Candida albicans,the cause of candidiasisin humans, as well as lectins of yeasts widely usedin the food industry, produced by strains of Saccharomyces cerevisiae (Table 4). It is worth mentioning that the spectrum of biologicalactivitiesofyeastlectinsalsoincludeskilleractivity toward other yeast species. Thus, extracellular lectinfrom Pichia anomala shows anticandidal activity owing to selective interaction with@-l,6-glucan of C. albicans [160]. Another significant process of biological recognition between yeast gametes of different sexes is controlled by participation of yeast a-mannan-sensitive lectins[1621.

Table 3 Interaction of Lectins of Protozoan Origin with Glycoconjugates and Poly-

saccharides

Carbohydratecontaining Lectin Entamoeba histolyticalectin

Surface adhesin

Asialoorosomucoid; Gal, GalNaccontaining polymers ofCH0 hamster cell line N-, 0-glycan of cell surface CH0 of hamster cell line, mucin of rat intestine

144

145-147

Giardia lamblia

Portland 1 strain surface lectin (57-78kDa) Tagerin/lectin activated by trypsin (28-30kDa) Leishmania braziliensis(NR) surface lectin L. donovani transporter of

148,149 Glucose/mannose-containing polymers of intestinal epidermal cells of mammals Mannose-&phosphatecontaining materials of rabbit erythrocytes GlcNAc-BSA, N-acetylglucosamine 150 glucose, mannose, galactosecontaining materials of macrophage 577408 91 Glucose-containing ligands

glucose Paramecium tetraurelialectins

Gal-BSA, Man-BSA, dextran-BSA

151

of apex of trichocysts Glycophorins, GlcNAc-Sepharose (140,70,35 kDa) Shizont soluble antigen Sialoreceptors of human erythro(175 kDa) of camp strain cytes Pneumocystis cariniiadhesins 8-Gal-BSA, Man-BSA a-Fuc-BSA or amino sugar-BSA

Plasmodium falciparum

152 153 110

Microbial Lectins

311

Table 4 Interactionof Lectins of Yeast Origin with Glycoconjugates and Polysac-

charides Lectin sources Ref.

Carbohydrate-containing targets

Candida albicans extracellular L-Fucose-containing substances of 156 lectinofGDH 2346 strainbuccalandvaginalepithelialcells of mammals Extracellular lectin of N-Acetylglucosamine-containingma157 terials of epithelialcells of mamGDH 2023 strain C. albicans and Cryptomals. Gal-Dl, 4-Glc-Dl containing coccus neoformans glycolipids adhesins Kluyveromyces bulgaricus ex- 0-GlcNAc,aGal-containingmaterials158,159 tracellular lectins sheep rabbit and of erythrocytes, yeasts Pichia anomala extracellular/31,6-glucan-containingsubstances of 160 anti candidai toxin cell wall of Candida albicans Saccharomycescerevisiae ex-Galactose-andlactose-sensitivepoly-161 tracellularhemagglutinin of mers of animalerythrocytes CD115 strain Mannose-containing polymers of 162 Sex agglutinin (22 kDa) yeasts

VI. FUNGAL LECTINS Lectins of fungi have been less well investigated than those of higher plants [lo]. Many of the fungal lectins studied belong to the group of chitinbinding proteins [163,164]. Data on the interaction of fungal lectins with other carbohydrate-containing targets are presented in Table 5. Many fungal lectins are characterized bya high affinityfor sialomucins ofthe bovine submaxillary gland [164,169,170,178], whereas lectins from Athelia rorfsii and Rhizoctonia solanimycelia showa high affinity for sialomucins ofthe pig intestinal mucous membrane [172]. Some fungal lectins also interact well with human erythrocyte glycoproteins, bovine fetuin [ l a ,1721, and glycosaminoglycans of the chondroitin sulfate type[170]. Of considerable interest are data onspecific interaction of fungal lectins with polysaccharides. Lectins from Rhizoctonia solani react well with galactose and N-acetylgalactosamine-containingpolysaccharides of bacterial and plant origin [166,1811. Lectins from Phanerochaete chrysosporium and the bacterium Streptomyces murinus [179,183] show affinity for B-

Table 5 Interactions of Lectins of Fungal Origin with Glycoconjugates and Polysaccharides

2 N

Lectinsources

Chrysosporiumkeratinophilum (Frey) conidial hemagglutinin Arthrobotrys ellipsospore extracellular hemagglutinin Arthrobotrys oligospora surface adhesins Athelia rolfsii micellar hemagglutinin (17 kDa) Beauveria bassiana micellar hemagglutinins Cephalosporiumacremonium extracellular agalactosidasehemagglutinin Clitocybegeotropa, Laccaria emethystina, and Photoliota squarrosa lectins Conidiobolus obsurus lectins/adhesins of spore surface Ganodermafucidummicellar hemagglutinin (17 kDa) Epidermophytonfloccosum, Microsporum cank, M, cookei, and M. fulvum extracellular hemagglutinins Neurospora sitophila extracellular hemagglutinin (22 kDa) Phanerochaete chrysosporiumextracellular cellobiose oxidase with domain for sorption on glucan (flavin domain of enzyme) Pythium aphanidennatum lectin Rhizoctonia crocorum micellar hemagglutinin (11 kDa) R. solani micellar hemagglutinin (13 kDa) Sclerotium ro@ii extracellular lectins (55 + 60kDa)

Carbohydrate-containing targets

Ref.

N-Acetylneuraminic acid and Caz+-sensitivesubstances of rabbit erythrocytes, much of submandibular bovine gland Mucin of submandibular bovine gland and chondroitin sulfate containing substances of chicken erythrocytes 2-Deoxy-~-glucosesubstances of cell surface of Trichostrongylus colubriformb Mucin of pig intestine; asialofetuin-sensitivesubstances of trypsin-treated erythrocytes of pigs Monosaccharide-insensitivesubstances of animal erythrocytes Branched B1,rlglucan potato starch

169

L-Fucosecontaining substances of spores, flagella, sporangia and rhizoids of fungal strains from intestine of sheep GlC-BSA, GlCNAc-BSA Sheep erythrocytes N-Acetylneuraminicacid-containing substances of rabbit erythrocytes; mucin of bovine submandibular gland; GP of human erythrocytes Mu.& of bovine submandibular gland; GP of human erythrocytes 81,CGlucan (cellulose) Fucosecontaining substances of Lepidum sativum Mucin of pig stomach; fetuin-sensitive substances of trypsintreated rabbit erythrocytes Gum arabic; N-acetylgalactosamine-sensitivesubstances of rabbit erythrocytes Surface polymers of E. coli

170 171 172 173 174 175 176 177 178

163 178

171,179 172 181 182

Microbial Lectins

313

1,4-glucans or 8-1,3(6)-glucans. The lectinof Cephalosporiumacremonium binds specifically with a-1,4-glucan [174]. Among fungal lectins, simi2) are some enzymes, such lar to some lectins from bacteria (see Table as cellobiose oxidase and a-galactosidase, with a specificity for glucans [174,179]. Both of these enzymes of carbohydrate metabolism are characterized by the presence of carbohydrate-binding sites inaddition to a catalytic center[1791. As can be seen from Table 5 , lectins of fungi may be applied to the investigation of glycoconjugates, polysaccharides,and receptors of microorganisms. With the help of fungal fucose-specific lectins, one can detect considerable structural subtleties between eight strains of fiveother fungal species belongingto the genera: Neocallimastix, Piromonas, and Sphaeromonas [175]. VII.

CONCLUSIONS

The foregoing summary was devotedto interaction of lectins and agglutinins from more than 100 microbial sources, with glycoconjugates, polysaccharides, and cellularreceptors.Mostmicroorganismsseemcapableof synthesizing a group (or a system) of lectins characterized by various carbohydrate specificities, various cellular distributions, and a variety of biological activities. The set of lectins can vary mutants in and recombinant microorganisms, and they also dependon the life cycles of microorganisms.The ability of a microbial lectin to recognize carbohydrates generally increases within the range: free mono- and disaccharides (simple carbohydrates isolatedoligosaccharidesmono-,di-, and oligosaccharideswithinglycoconjugatesandpolysaccharides).Microbiallectinsareablenotonlytorecognize various typesof natural carbohydrate-containing targets (glycoproteins,gangliosides,globosides,lipopolysaccharides,glycosaminoglycans, and polysaccharides), but they can also recognize differences in carbohydrate-containing targets within each type.Factors that can potentially affect theaffinity of a microbial lectinfor complex glycansas constituents of carbohydrate-containing targets may the be following: (1) types of terminal and internal carbohydrate residues; (2) presence of terminal residues of carbohydrates of the same type in clusters, accessible to lectin (nearest neighbors withinone glycan in a glycoconjugate, such as a polysialoganglioside or sialylated antenna(e) in polyantennary asparagine-bound glycans of glycoconjugates. Shark glycans bound with serine or threonine in glycoproteins represent another example; (3) the degree of polysaccharide branching. Further refinement of lectin specificity to glycans within carbohydratecontaining targets may be residues bearing hydrophobic groups (methyl, acetyl, and other alkyl substituents), or charged groups (sulfate, phosphate,

314

Shakhanina et al.

and others). Relative to standardization of the specificity of microbial lectins for glycoconjugates and polysaccharides, of crucial importanceare the studies devotedto interaction between the lectins and neoglycoconjugates. The data on the specificity of microbial lectins may be taken into consideration when purifying or immobilizing the glycoconjugates. Thus, sorbents that are prepared with the fimbrial lectinsof bacteria are successfully used for obtaining the complementary receptorsfrom tissues of mammals [76,84].Exotoxin from Pseudomonas aeruginosa-Sepharose is employed for isolationofreceptorglycoproteins ofmammaliancelllines [187]. Lymphocyte receptors can be purified with immobilized pertussis toxin [188]. Sorbents based on bacterial adhesins are frequently used for immobilization of microbial cells[49,112].Microbial lectins are also promising for studies on carbohydrate-containing biopolymers. Thus, immobilized discoidin 1 (lectin from Dictyosteiium discoideum) reacts selectively with endogenous receptor glycoproteins [189].Bacterial and fungal lectins [102,1761. form complexes with endogenous polysaccharides A s a whole, the results indicate that microbial lectins can be used in structural analyses of carbohydrate-containing biopolymers, irrespective of their origin from animal, plant, or microbe. Microbial lectins (including bacterial toxins and others) are produced by many commercial firms; the list of such lectin preparations is being constantly expanded. REFERENCES 1. Lakhtin VM. Biotechnological aspects of lectins. In: Kocourek J, Freed D, eds. Lectins: biology, biochemistryand clinical biochemistry,v01 7.St Louis: Sigma ChemicalCO, 1990:417-426. KL. A review of identification of 2. Kalinin NL, Lakhtin VM, Shakhanina infectious agents with lectins. Proc Inter-Lec llth, 1989; 30. 3. Doyle RJ, Rosenberg M, eds. Microbial cell surface hydrophobicity. Washington, DC: American Society for Microbiology, 1990:425. 4. Sharon N, Lis H. Lectins as cell recognition molecules. Science 1989; 246: 227-234. 5. Mandal C, Mandal C. Sialic acid binding lectins. Experientia1990; 46:433441. 6. Slifkin M,Doyle RJ. Lectins and their application to clinical microbiology. Clin Microbiol Rev 1990; 3:197-218. 7. Sharon N, LisH. Lectins. London: Chapman t Hall 1989. 8. Freed D,Bag-Hansen TC, eds. Lectins: biology, biochemistry and clinical biochemistry, v01 6.St Louis: Sigma ChemicalCO, 1989. 9. Kocourek J, Freed D, eds. Lectins: biology, biochemistry and clinical biochemistry, v01 7. St Louis: Sigma ChemicalCO, 1990. 10. VanDriessche E, Franz H, Beeckmans S, Pfuller U, Kallikorm A, Bag-

Microbial Ledins

11. 12.

13. 14. 15.

315

HansenTC.Lectins:Biology-Biochemistry,ClinicalBiochemistry,Vol 8. Textop Publishers, Copenhagen,1993. Kalinin N, Doyle RJ. Distribution of glucan-binding lectin on the surface of Streptococcus. Proceedings of 13th international lectin meeting. Berlin: Abstract book 1991:36. Sharon N. Bacterial lectins,cell-cell recognition and infectious disease.FEBS Lett 1987; 217:l-13. Sharon N. Biomedical aspects of glycoconjugate recognition. Biomedical aspects of lectins. Biochem SOC Trans 1989; 17:ll-12. Weir DM. Carbohydrates as recognition molecules in infection and immunity. FEMS MicrobiolLett 1989; 47:331-340. Old DC. Bacterial cell envelopes in adhesion. In: Hancock I, Poxton I, eds. Bacterial cell surface techniques. Chichester: John Wiley & Sons, 1988:227-

m.

16. Ofek I. Lectinophagocytosis mediatedby bacterial surface lectins. Zentralbl Bakteriol Hyg [All 1989; 270:449-455. 17. Karlsson KA. Animal glycosphingolipidsas membrane attachment sites for bacteria. Annu Rev Biochem 1989; 58:309-350. 18. Mirelman D, e d . Microbial lectinsand agglutinins: properties and biological activity. New York: John Wiley & Sons, 1986. 19. Sharon N. The lectins. In: Liener IE, Sharon N, Goldstein IJ, eds. Lectins: properties, functions and applications in biology and medicine. Orlando: Academic Press, 1986. 20. Uhlenbruck G. Bacterial lectins: mediators of adhesion. Zentralbl Bakteriol Hyg [A] 1987; 263:497-508. 21. Gilboa-Garber N, Garber N. Microbial lectins. In: Allen HJ, Kisailus EC, eds. Handbook of glycoproteins. New York: Marcel Dekker,1989. 22. Nimich W. Adhesins ofEscherichia coli. Acta Biotechnol 1990; 10:151-161. 23. Markwell MAK. Viruses as hemagglutinins and lectins. In: Mirelman D, ed. Microbial lectins and agglutinins. New York: John Wiley & Sons, 1986:2153. 24. Wollin R, Bruse GW, Jansson P, Lindberg S. Definition of the phage G13 receptor as structural domains of trisaccharides in Salmonella and Escherichia coli core oligosaccharides. J Mol Recogn 1989; 2:37-43. 25. Norberg T, RitzenH.Synthesisofmethyl a-D-glyCOpyranOSyl-(1-2)-a-Dgalactopyranosylacyclicanaloguethereof for probing the carbohydratebinding specificity of bacteriophage 4x174. Glycoconjugate J 1986; 3:135142. 26. Schultze B, Gross HJ, Brossmer R, Klenk HD, Herrber G . Hemagglutinating encephalomyelitis virus attaches to N-acetyl-9-0-acetylneuraminic acidcontaining receptors on erythrocytes: comparison with bovine coronavirus and influenza C virus. VirusRes 1990; 16:185-194. 27. Komai K, Kaplan M, Peeples ME. The vero cell receptor for the hepatitis B virus smallS protein is a sialoglycoprotein. Virology 1988; 163:629-634. 28. SawitzkyD, Hampl H, Habermehl KO. Comparison of heparin-sensitive attachment of pseudorabies virus (PRV)and herpes simplex virus type1 and

Shakhanina et al.

316

identification of heparin-binding PRV glycoproteins. J Gen Virol 1990; 71: 1221-1225. 29. Berman PW, Gregory TJ, Riddle L, Nakamura G, Change M. Protection of

chimpanzees from infection by HIV-l after vaccination with recombinant glycoprotein gp120 but not gp160. Nature 1990; 345:622-625. 30. Paul RW, Lee PW. Glycophorin isthe reovirus receptor on human erythrocytes. Virology 1987; 159:94-101. 31. Eaton BT, Crameri GS. The site of blue tongue virus attachment to glycophorins from a member of animal erythrocytes. J Gen Virol 1989; 70:33473353. 32. Superti F, Hauttecoeur B, Morelec MJ, Goldoni P, BizziniB, Tsiang H. Involvement of gangliosidesin rabies virus infection. J Gen Virol 1986; 67: 47-56. 33. Mastromarino P, Conti C, Goldoni P, Hauttecoeur B, Orsi N. Characteriza-

tion of membrane components ofthe erythrocytes involvedin vesicular stomatitis virus attachment and fusion at acidic pH. J Gen Viroll987; 68:23592369. 34. Suzuki Y,Nagao Y,Kat0 H, Matsumota M, NeromeK, Nakajimo K, Nobu-

sawa E. Human influenza A virus hemagglutinin distinguishes sialyloligosaccharides in membrane-associated gangliosides as its receptor which mediates the adsorption and fusion processes of virus infection. Specificity for oligosaccharides and sialic acidsand the sequence to which sialic acid isattached. J Biol Chem 1986; 261:17057-17061. 35. Sauter NK, Bednarski MD, Wurzburg BA, Hanson J, Whitesides G, Skehel J, WileyD. Hemagglutinins from two influenza A virus variants bind to sialic acid derivatives with millimolar dissociation constants: a 500-MHz proton nuclear magnetic resonance study. Biochemistry 1989; 28:8388-8396. 36. Higa HH, Rogers GN, Paulson JC. Influenza A virus hemagglutininsdifferentiate between receptordeterminants bearing N-acetyl-N-glycollyl-and N,Odiacetylneuraminic acids. Virology1985; 144:279-282. 37. Pritchett TJ, Paulson JC. Basis for the potent inhibition of influenza virus infection by equine and guinea pig a2-macroglobulin. JBiol Chem 1989; 264: 9850-9858. 38. Rogers GN, Herrler G, Paulson JC, Klenk HD. Influenza C virus uses 9-0acetyl-N-acetylneuramic acidas a high affinity receptor determinant for attachment to cells. J Biol Chem 1986; 261:5947-5951. 39. Herrler G, Reuter G, Rott R, Klenk HD, Schauer R. N-Acetyl-9-0-

acetylneuramic acidthe receptor determinant for influenza C virus is differa entiation marker on chicken erythrocytes. Biol Chem Hoppe-Seyler 1987; 368:451-454.

40.

Ysen-CaldenteyM,Adams-Burton CR, Hart LT, Laine RA. Cell-surface Anaplasma marginaleproteins and interaction with bovine erythrocytes. Glycoconjugate J 1988; 2351. 41. Keusch GT, Donohue-Rolfe A, Jacewicz M. Sugar binding bacterial toxins. In:Mirelman D, ed. Microbiallectins and agglutinins. New York: John Wiley 8c Sons, 1986:271-296.

Microbial Lectins

317

42. Kolenbrander PE. Surfacerecognitionamong oral bacteria:multigenetic coaggregation and their mediators. CRC Crit Rev Microbiol 1989; 17:137159. 43. Nimmich W, ZinglerG, Falkenhagen U, Naumann G. Hemagglutinationand fimbrial antigens of urinary Escherichia coli. Zentralbl Bakteriol Hyg 1990; 272:411-418. 44. Knowles J, Lehtovaara P, Teeri T. Seculase family and their genes. Trends Biotechnol 1987; 5:255-261. 45. Clark RB, Knoop FC, Padgitt PJ, Hu PH, Wong JD, Janda J. Attachment to mesophilic Aeromonas to cultured mammalian cells.Curr Microbioll989; 19:97-102. 46. Depierreux C, Michel MF, Monsigny M, DelmotteF. Evidence of Agrobacterium tumefaciens surface lectins. Proceedings of the 19th FEBS meeting, July 2-7,1989, Rome. 1989:TH85. 47. Nikitina VE, Italianskaya JV. Fucose-specific lectin of Azospirillum brasilense. Abstracts of proceedings ofthe 10th international lectin meeting, July 3-8, 1988, Prague. 198959. 48. Simonenko IA, Kovalenko EA,Lakhtin VM.Characterization ofthe Bacillus mesentericus extracellular lectin.Proc Inter-Lec 1 lth, 1989; 96. 49. Guinet RMF, Rogemond V, Tessier F, Capdepuy A. Characterization and possible role of lectin-like adhesin in the Bacteroidesfragilis group. Proceedings of international seminar on biology of anaerobic bacteria, June 17-18, 1986, Lille. 1986:60-68. 50. Pruzzo C, Guvnan CA, Dainelli B. Incidence of hemagglutination activity among pathogenic and non-pathogenic Bacteroides fragilis strains and role of capsule and pili in HA and adherence. FEMS Microbiol Lett 1989; 59: 113-118. 51. London I, Allen I. Purification and characterization ofa Bacteroids loescheii adhesin that interacts with procaryoticand eucaryotic cells. J Bacteriol 1990; 172~2527-2534. 52. Ishikawa H, Isayama Y. Evidence for sialylglycoconjugates as receptors for Bordetella bronchiseptica on swine nasal mucosa. Infect Immun 1987; 55: 1607-1609. 53. Heereze LD, Armstrong GD. Comparison the of lectin like activity of pertussis toxin with two plant lectins that have differential specificities for 42-6) and a(23)-linked sialicacid.BiochemBiophysRes Commun 1990;172: 1224-1229. 54. Tyrrell GJ, Reppler MS, Bonnah RA, Clark C, Chong P, Armstrong C. Lectin like properties of pertussis toxin. Infect Immun 1989; 57:1854-1857. 55. Bernadette MJ, Quentin-Millet J, Arminjon F. Patent N 2597605.16.04.1986, France. 56. Chong P,Klein M. Singlestep purification of pertussis toxin and its subunits by heat-treated fetuin-Sepharose affinity chromatography. Can J Biochem Cell Biol 1989; 67:387-391. 57. Clark CG, Armstrong GD. Lymphocyte receptor for pertussis toxin. Infect Immun 1990; 58:3840-3846.

Shakhanina et al.

318

58. Relman D, Tuomanen E, Falkow S, Golenbock D, SaukkonenK, Wright S. Recognition of a bacterial adhesin by an integrin: macrophage CR3 (ay & CD1lb/CD18) binds filamentous hemagglutininof Bordetella pertussis. Cell 1990; 61~1375-1382. 59. Menozzi FD, Ganties C. Locht C. Interaction of the Bordetella pertussis filamentous hemagglutinin withheparin. FEMS MicrobiolLett 1991; 785964.

60. HOSC, Schindler M, Wang JL. Carbohydrate binding activitiesofBradyrhi61. 62. 63. 64.

65.

66. 67. 68. 69. 70. 71. 72.

73.

zobium japonicum. 2. Isolation and characterization of a galactose-specific lectin. J Cell Biol 1990; 111:1639-1643. Ho SC, WangJL, Schindler M. Carbohydrate binding activitiesof Bradyrhizobium japonicum. 1. Saccharide-specificinhibition of homotypic and heterotypic adhesion. J Cell Biol 1990; 111:1631-1638. Daikoku T, Kawaguchi M, Takama K, Suzuki S. Partial purification and characterizationof the enterotoxin produced by Campylobacterjejuni. Infect Immun 1990; 582414-2419. Kamata Y,Kozaki S, Sakaguchi G.Effects of pH on thebinding of Clostridium botulinum type E derivative toxin to gangliosides and phospholipids. FEMS MicrobiolLett 1988; 55:71-76. Sugii S, Kozaki S. Hemagglutining and binding properties of botulinum Gtoxin. Biochim BiophysActa 1990; 1034:176-179. Krivan HC, Clark GF, Smith DF, Wilkins TD. Cell surface binding site for Clostridium diffice enterotoxin: evidence for a glycoconjugate containing the sequence Galal,3 GalPl,4GlcNAc. Infect Immun 1986; 53573-581. Krivan HC, Wilkins TD. Clostridium dvficile enterotoxins carbohydrate sequence structure and functions. Infect Immun 1987; 55:1873-1877. Kamiya S, Reed PJ, Borriello SP. Analysis of purity of Clostridium dvflcile toxin A derivedby affinity chromathography on immobilized bovine thyroglobulin. FEMS MicrobiolLett 1988; 56:331-336. Rothman SW, Gentry MK, Brown JE, Foret DA, Stone MJ, Strickler MP. Immunochemical and structural homology of toxins Aand B of Clostridium difficile. Toxicon 1988; 26583-597. Tucker KD, Wilkins TD. Toxin A ofClostridium difficilebind to the human carbohydrate antigens. I, X and Y. Infect Immun 1991; 59:73-78. Baldassarri L, Pantosti A, Caprioli A, Mastrantonio P, Donelli G. Hemagglutination and surface structures in strains of Clostridium spiroforme. FEMS MicrobiolLett 1989; 60:1-4. Johnstone SR, Morrice LM, Van Heynigen S. The heavy chain of tetanus toxin can mediatethe entry of cytotoxic gelonininto intact cells. FEBS Lett 1990; 265:lOl-103. McConnell MM,Chart H, Rowe B. Antigenic homology within human enterotoxigenic Escherichiacoli fimbrial colonization factor antigens: CFA/I, coli-surface associatedantigens (CS)l, CS2, CS4 and CS17. FEMS Microbiol Lett 1989; 61:105-108. Yamamoto T. Bacterial adhesins and pathogenicity. Jpn J Bacteriol 1987; 42~627-651.

Microbial Lectins

319

74. Hanson MS, Brinton CC. Identification and characterization of E. coli type I pilus tip adhesion protein.Nature 1988; 332:265-268. 75. Abraham SN, Sun D, Dale JB, Beachey E. Conservation of the D-mannoseadhesion protein among typeIfimbriated members ofthe family Enterobacteriaceae. Nature 1988; 336:682-684. 76. Beachey EH, Giampapa CS, Abraham SN. Specificity of bacterial lectins. Am Rev Respir Dis 1988; 1382345448. 77. Wold AE, Thorsen M, Hull S, Svanberg-Eden C. Attachment of Escherichia coli via mannose or Galal+4 Galp-containing receptors to human colonic epithelial cells. Infect Immun 1988; 56:2531-2537. 78. Sherman P, Soni R, Del Vedoro S, Veeser J. Effect of oligomannoside type glycopeptides indiarrheal disease ofrabbit induced by Escherichia colistrain RDEC-l. FEMS Microbiol Lett1989; 61:121-126. 79. Stromberg N, Marklund BI, Lund B, Ilver D, Hamers A, Gaastro W, Karlsson KA, Normark S. Host-specificity of uropathogenic Escherichia coli depends on differences in binding specificityto Galal,4 Gal-containing isoreceptors. EMBOJ 1990; 9:2001-2010. 80. Senior D, Baker N, Cedergren B, Falk P, Larson G, Lindstedt R, Eden C. Globo-A-a new receptor specificity for attaching Escherichia coli. FEBS Lett 1988; 237:123-127. 81. Schmoll T, Hacker J, Goebel W. Nucleotide sequence of the sf& gene coding for the S-fimbrial protein subunit of E. coli. FEMS Microbiol Lett 1987; 41~229-235. 82. Sjoberg PO, Lindahl M, Porath J, Wadstrom T. Purification and characterization of CS2 a sialic acid-specific haemagglutinin of enterotoxigenic Escherichia coli. Biochem J 1988; 255:105-111. 83. Parkkinen J, Korhonen TK,Pere A, Hacker J, Soinila S. Binding sites inthe rat brain for Escherichia coli. S fimbriae associated withneonatal meningitis. J Clin Invest 1988; 81:860-865. 84. Conway PL, Welin A, Cohen PS. Presence ofK88-specific receptors in porcine ileal mucus is age dependent. Infect Immun 1990; 58:3178-3182. 85. Metcalfe JW, Krogfelt KA, Krivan HC, Cohen PS, L a w PC. Characterization and identification of a porcine small intestine mucus receptor for the K88ab fimbrial adhesin. Infect Immun1991; 59:91-96. 86. Brooks DE. Glycoprotein A acts as a receptor for E. coli K99binding. Biophys J 1984; 49198. 87. Lindahl M, Carltedt I. Binding of K99 fimbriae of enterotoxigenic Escherichia colito pig small intestinal mucin glycopeptides.J Gen Microbiol1990; 136:1609-1614. 88. Kyogashima M, Ginsburg V, Krivan HC. Escherichia coli K99 binds to Nglycolyl-GM, found in piglet small intestine. Arch Biochem Biophys 1989; 270~391-397. 89. Lindahl M, Wadstrom T. Binding to erythrocyte membrane glycoproteins and carbohydrate specificity of F41 fimbriae of enterotoxigenic Escherichia coli. FEMS MicrobiolLett 1986; 34297-300. 90. Griinberg J, Perry R, Hoschiitsky H, JannB, Goldhar J. Non-fimbrial blood

320

91. 92.

93. 94. 95.

96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106.

Shakhanina et al.

group N-specific adhesin (NFA-3)from Escherichia coli020-KX104-H causing systemic infection. FEMS Microbiol Lett 1988; 56241-246. Baly DL, Horuk R.The biologyand biochemistry ofthe glucose transporter. Biochim BiophysActa 1988; 947571-590. Fukuta S, Magnani JL, Twiddy EM, Holmes R, Ginsburg V. Comparison of the carbohydrate-binding specificities of cholera toxin and Escherichia coli heat-labile enterotoxins LTh-I, LT-2 and LT-2b. Infect Immun 1988;56: 1748-1753. Sugii S, Tsuji T. Binding specificities of heat-labile enterotoxinsisolated from porcine and human enterotoxigenic Escherichia coli for different gangliosides. Can J Microbiol 1989; 35:670-673. Sugii S, Tsuji T, Honda T, Miwatani T. Hemagglutinating activity of the &subunits ofthe heat-labile enterotoxin isolated from human enterotoxigenic Escherichia coli. FEMS Microbiol Lett1988; 49:463-465. Sugii S, Tsuji T. Hemagglutinating activity of the &subunits of the heatlabile enterotoxin isolated from chickenenterotoxigenic Escherichia coli. FEMS MicrobiolLett 1989; 57:105-108. DeGrandis S, Law H, Brunton J, Gyles C, Lingwood C. Globotetraosylceramide is recognized by the pig edema disease toxin. J Biol Chem 1989; 264: 12520-12523. Serra MT, Castreana MC, Tejerina G. Hemagglutinating activityin phytopathogenic bacteria surface compounds. J Basic Microbioll987; 27: 147-153. Korhonen TK, Haahtela K, Pirkola A, Parkkinen J. N-Acetyllactosaminespecific cell-binding activity in a plant pathogen, Erwina rhapontici. FEBS Lett 1988; 236:163-166. Yamazaki Y, Ebisu S, Okada H. Partial purification of a bacterial lectin like substance from Eikenella corrodens.Infect Immun 1988; 56:191-196. Faghri MA, Helman SW, Wallace JH. Proceedings ofthe 86th annual meeting of American Societyfor Microbiology, March23-28, 1986, Washington. 1986:35. Cottin J, Loiseau 0, Robert R, Mahaza C, Carbonnelle B, Senet J. Surface Listeria monocytogenescarbohydrate-binding components revealed by agglutination with neoglycoproteins. FEMS Microbiol Lett 1990; 68:301-306. Kundu M, Basu J, Ghakrubarti P. Purification and characterization of an extracellular lectin from Mycobacterium smegmatis. FEBS Lett 1989; 256: 207-210. Watanabe T, Shibata K, Yukitake H. Hemagglutinating activity of Mycoplasma salivarum and its attachment to sheep red blood cells. Microbiol Immunol1990; 34:439-446. Young TF, Erickson BZ, RossRF, Wannemuehler Y. Hemagglutination and hemagglutinin inhibition of turkey red blood cells with Mycoplasma hypopneumoniae. Am J Vet Res 1989; 50:1052-1055. RobertsDD,OlsonLD,BarileMF,Ginsburg V, KrivanH.Sialicaciddependent adhesionof Mycoplasma pneumoniaeto purified glycoproteins. J Biol Chem 1989; 264:9289-9293. Krivan HC, Olson LD, Barile MF, Ginburg V, Roberts. Adhesion ofMyco-

Microbial Lectins

107.

108.

109.

110. 111.

112. 113.

114.

115. 116.

117.

118. 119.

120.

121.

122.

32 1

plasma pneurnoniae to sulfated glycolipids and inhibition by dextran sulfate. J Biol Chem 1989; 264:9283-9288. Paruchuri DK, Seifert HS, Ajioka RS, Karlson KA, So M. Identification and characterization of a Neisseria gonorrhoeae gene encoding a glycolipidbinding adhesin. Proc Natl Acad Sci USA 1990; 87:333-337. Deal CD, So MY, Seifert HS. Neisseria gonorrhoeaelectin useful as a vaccine and diagnostic marker and means for producing this lectin. Enzyme Microbiol Techno1 1989; 11:779. Nyberg G, Stromberg N, Jonsson A, Karlsson K, Narmark S. Erythrocyte gangliosides act as receptors for Neisseria subfava. Identification of the Sio-1 adhesin. Infect Immun 1990; 58:2555-2563. Vierbuchen M, Ortmann M, Uhlenbruck G. Endogenous carbohydrate binding proteins in Pneurnocystis carinii. Infect Immun 1990; 58: 143-146. Murakami Y, Amano A, Takagaki M. Lectin-like substances, adhesins and pathogenicity. FEMS Microb Lett 1990; 72:275-280. Stinson M, Safulke K, Levine M. Hemagglutinins and carbohydrate specificity. Infect Immun 1991; 59:102-108. Gilboa-Garber N. Lectins of Pseudomonas aeruginosa:properties, biological effects and applications. In: Mirelman D, ed. Microbial lectins and agglutinins. New York: John Wiley & Sons, 1986:255-269. Garber N, Guempel U, Gilboa-Garber N, Doyle R. Specificity of the fucosebinding lectin of Pseudornonas aeruginosa. FEMS Microb Lett 1987; 48:33 1334. Beuth J , KO H, Uhlenbruck G, Pulverer G. Lectin-mediated bacterial adhesion to human tissue. Eur J Clin Microbiol 1987; 6591-593. Allured V, Brandhuber B, McKay D. Structure and mechanism of exotoxin A of Pseudornonas aeruginosa. In: Bonavida B, Collier R, eds. Membrane mediated cytotoxicity. New York: Alan R Liss, 1987:3-7. Gilbert HJ, Hall J , Hazlewood GP, Ferreira L. The N-terminal region of an endoglucanase from Pseudomonas fruorescens subspecies cellulosa constitutes a cellulose binding domain that is distinct from the catalytic centre. Mol Microbiol 1990; 4:759-767. Daly JG, Stevenson RM. Characterization of the Renibacterium salrnoninarum haemagglutinin. J Gen Microbiol 1990; 136:949-953. Kristiansen K, Baloda SB, Larsen JL, Wodstrom A. Toxins putative cell adhesins and fibronectin binding properties of Salmonella dublin. Acta Patho1 Microbiol Immunol Scand [B] 1987; 9557-63. Lindberg AA, Brown JE, Stromberg N, Westling-Ryd H, Schultz J , Karlsson K. Identification of the carbohydrate receptor shiga toxin produced by Shigella dysenteriae type S . J Biol Chem 1987; 262: 1779-1785. Jacewicz M, Feldman H , Donohue-Rolfe A, Balasubramanian K, Kensch G. Pathogenesis of shigella diarrhea. XIV. Analysis of shiga toxin receptors on cloned HeLa cells. J Infect Dis 1989; 159:881-889. Velazco MI, Waldvogel FA. Monosaccharide inhibition of Staphylococcus aureus adherence to human solid phase fibronectin. J Infect Dis 1987; 155: 1069- 1072.

322

Shakhanina et al.

123. Beuth J , KO H , Schumacher-Perdreau F, Peters G, Heczko P, Pulverer G. Hemagglutination by Staphylococcus saprophyticus and other coagulasenegative staphylococci. Microbiol Pathol 1988; 4:379-383. 124. Beuth J , KO H , Ohshima Y, Yassin A, Ulenbruck G, Pulverer G. The role of lectins and lipoteichoic acid in adherence of Staphylococcus saprophyticus. Zentralbl Bakteriol Hyg [A] 1988; 268:357-361. 125. Murray PA, Levine MJ, Reddy MS, Tabak L, Bergey E. Preparation of a sialic acid-binding protein from Streptococcus mitis KS 32 AR. Infect Immun 1986; 53:359-365. 126. Murray PA, Levine MJ, Tabak LA, Reddy MS. Specificity of salivarybacterial interactions: I1 Evidence for a lectin on Streptococcus sanguis with specificity for a NeuAca2+3Gal@l+ 3GalNAc sequence. Biochem Biophys Res Comm 1982; 106:390-396. 127. Cowan MM, Taylor KG, Doyle RJ. Role of sialic acid in the kinetics of Streptococcus sanguis adhesin to artificial pellicle. Infect Immun 1987; 55: 1552- 1557. 128. Drake D, Taylor KG, Bleiweis AS, Doyle RJ. Specificity of the glucanbinding lectin of Streptococcus cricetus. Infect Immun 1988; 56:1864-1 872. 129. Cowan MM, Parrish K, Kessler RE, Pyle C, Taylor K, Ciardi J , Doyle RJ. Glucan-binding factor in saliva. Infect Immun 1988; 56:2912-2917. 130. Landale EC, McCable MM. Glucan-binding lectins. Proceedings of the 86th annual meeting of American Society for Microbiology, March 23-28, 1986, Washington. 1986:39. 131. Sanchez J, Svennerholm AM, Holmgren J. Genetic fusion of non-toxic heatstable enterotoxin-related decapeptide antigen to cholera toxin B subunit. FEBS Lett 1988; 241:llO-114. 132. Liang X, Lamm ME, Nedrud JG. Cholera toxin as a mucosal adjuvant. Glutaraldehyde treatment dissociates adjuvanticity from toxicity. J Immunol 1989; 143~484-490. 133. Pacuszka T, Bradley RM, Fishman PH. Neoglycolipid analogues of GM1 as functional receptors of cholera toxin. Biochemistry 1991 ;30:2563-2570. 134. Schengrund CL, Ringler NJ. Binding of Vibrio cholerae toxin and the heatlabile enterotoxin of Escherichia coli to GM 1 and nonlipid oligosaccharide polyvalent ligands. J Biol Chem 1989; 264: 13233-13237. 135. Parkinson ME, Smith CG, Garland PB, van Heyningen S. Identification of cholera toxin-binding sites in the nucleus of intestinal epithelial cells. FEBS Lett 1989; 242:309-3 13. 136. Monferran CG, Roth GA, Cumar FA. Inhibition of cholera toxin binding to membrane receptors by pig gastric mucin-derived glycopeptides; differential effect depending on the ABO blood group antigenic determinants. Infect Immun 1990; 58:3966-3972. 137. Iwanaga M, Nakasone N, Ehara M. Pili of Vibrio cholerae 01 biotype El Tor: a comparative study of adhesive and nonadhesive strains. Microbiol Immunol 1989; 33:l-9. 138. Banerjee KK, Ghosh AN, Dutta-Roy K, Pal SC, Ghose A. Purification and characterization of a novel hemagglutinin from Vibrio cholerae. Infect Immun 1990; 58:3698-3705.

Microbial Lectins

323

139. Hase CC, Finkelstein RA. Comparison of the Vibrio cholerae hemagglutinid protease and Pseudomonas aeruginosa elastase. Infect Immun 1990; 58:40114015. 140. Yu C, Lee AM, Roseman S. The sugar-specific adhesiorddeadhesion apparatus of the marine bacterium Vibrio furnissii is a sensorium that continuously monitors nutrient levels in the environment. Biochem Biophys Res Commun 1987; 149~86-92. 141. Bassler B, Gibbons P, Roseman S. Chemotaxis to chitin oligosaccharides by Vibrio furnissii, a chitinivorous marine bacterium. Biochem Biophys Res Commun 1989; 161 :1 172- 1 176. 142. Saada AB, Terespolski Y, Adoni A, Kahane I. Adherence of Ureaplasma urealyticum to human erythrocytes. Infect Immun 1991; 59:467-469. 143. Pereira MEA. Lectins and agglutinins in protozoa. In: Mirelman D, ed. Microbial lectins and agglutinins. New York: John Wiley & Sons, 1986:297-317. 144. Petri WA, Smith RD, Schlesinger PH, Murphy C, Ravdin J. Isolation of the galactose-binding lectin that mediates the in vitro adherence of Entamoeba histolytica. J Clin Invest 1987; 80: 1238-1244. 145. Ravdin JI, Stanley P, Murphy CF, Petri W. Characterisation of cell surface carbohydrate receptors for Entamoeba histolytica. Infect Immun 1989; 57: 2 179-21 86. 146. Ravdin J J , Jackson TF, Petri WA, Murphy C, Ungar ByGathiran V, Skilogiannis J, Somjce A. Association of serum antibodies to adherence lectin with invasive amebiasis and asymptomatic infection with pathogenic Entamoeba histolytica. Infect Immun. 1990; 162:768-772. 147. Chadee K, Johnson ML, Orozco E, Petri W, Pavdin J. Binding and internalization of rat colonic mucins by the galactose/N-acetyl-D-galactosamine adherence lectin of Entamoeba histolytica. J Infect Dis 1988; 158:398-406. 148. Farthing M JG, Pereira MEA, Keusch GT. Description and characterization of a surface lectin from Giardia lamblia. Infect Immun 1986; 51:661-667. 149. Ward HD, Lev BI, Kane AV, Keuch G, Pereira M. Identification and characterization of tagerin: a mannose-6-phosphate binding, trypsin-activated lectin from Giardia lamblia. Biochemistry 1987; 263669-8675. 150. Hernandez AG, Rodriguez N, Stojanovic D, Candelle D. The localisation of a lectin-like component on the Leishmani cell surface. Mol Biol Rep 1986; 1 1 :149- 1 53. 151. Haacke-Bell B, Plattner H. Secretory lectins contained in trichocyst tips of Paramecium. Eur J Cell Biol 1987; 44:l-9. 152. Jungery M, Boyle D, Pate1 T, Pasvol G, Weatherall D. Lectin-like polypeptides of P. falciparum bind to red cell sialoglycoproteins. Nature 1983; 301: 704-705. 153. Orlandi PA, Sim B, Kim L, Chulay J , Haynes J. Characterization of the 175-kilodalton erythrocyte binding antigen of Plasmodium fakiparum. Mol Biochem Parasitol 1990; 40:285-294. 154. Nakajima T, Giummelly P. Yeast lectins. Trends Glycosci Glycotechnol 1989; 1:E31-E34. 155. Calderone RA, Braun PC. Adherence and receptor relationships of Candida albicans. Microbiol Rev 1991; 55: 1-20.

324

Shakhanina et al.

156. Critchley IA, Douglas LJ. Role of glycosides as epithelial cell receptors for Candida albicans. J Gen Microbiol 1987; 133:637-643. 157. Jimenez-Lucho V, Ginsburg V, Krivan HC. Crytococcus neoformans, Candida albicans, and other fungi bind specifically to the glycosphingolipid lactosylceramide (Galol-4 Glcol- lCer), a possible adhesin receptor for yeast. Infect Immun 1990; 58:2085-2090. 158. Al-Mahmood S, Giummelly P, Bonaly R, Delmotte F, Monsigny M. Kluyveromyces bulgaricus yeast lectins. Isolation of N-acetylglucosamine and galactose-specific lectins: their relation with flocculation. J Biol Chem 1988; 263: 3930-3934. 159. Giummelly P, Separi F, Suptijan P, Bonaly R. Cell wall receptors of the lectins produced by Kluyveromyces bulgaricus. Yeast 1989; 5:S309-S3 1 1 . 160. Sawant AD, Ahearn DG. Involvement of a cell wall receptor in the mode of action of an anticandida toxin of Pichia anomala. Antimicrob Agents Chemother 1990; 34: 133 1 - 1335. 161. Basu J, Kundu My Mukherjee K, Chakrabarti P. Release of a lectin from a fatty acid auxotroph of Saccharomyces cerevisiae grown in presence of oleic acid. Biochem Biophys Res Commun 1986; 136596-602. 162. Watzele M, Klis F, Tanner W. Purification and characterization of the inducible “a” agglutinin of Saccharomyces cerevisiae. EMBO J 1988; 7: 1483-1488. 163. Ishikawa F, Oishi K. Chitin-binding lectins from fungi. In: Zikaris J, ed. Chitin, chitosan and related enzymes. Orlando: Academic Press, 1984:369382. 164. Ishikawa F, Oishi K. Production, purification and characterization of Neurospora sitophilia lectin. Agr Biol Chem 1989; 53: 1769-1776. 165. Toda K. Molecules of cell adhesion of mycomyces. Seikagaku (Jpn) 1986; 58~1139-1149. 166. McIntire FC, Crosby LK, Barlow JJ, Matta K. Structural preferences of 0-galactoside reactive lectins on Actinomyces viscosus T 14V and Actinomyces naeslundii WV445. Infect Immun 1983; 41:848-850. 167. Brennan MJ, Joralmon RA, Cisar JO, Sandberg A. Binding of Actinomyces naeslundii to glycosphingolipids. Infect Immun 1987; 55:487-489. 168. McIntire FC, Crosby LK, Vatter AE, Cisar J, McNeil M, Bush C, Tjoa S, Tennessey P. A polysaccharide from Str. sanguis 34 that inhibits coaggregation of Str. sanguis 34 with Actinomyces viscosus TMV. J Bacteriol 1988; 170~2229-2235. 169. Chabasse D, Robert R. Detection of lectin from Chrysosporium keratinophilum (Frey) carmichael and Anixiopsis stercororia (Hansen) hansen by inhibition of haemagglutination. Ann Inst Pasteur Serol Microbiol [B] 1986; 137: 187-193. 170. Yamanaka K, Wakabayasi K, Saito T. Capture of pine-wilt nematodes by Arthrobotrys ellipsospora Y 4007. Mucin-specific hemagglutination and its role in the capture. Agr Biol Chem 1988; 52:675-683. 171. Wharton DA, Murray DS. CarbohydrateAectin interaction between the nematophagous fungus, Arthrobotrys oligospora, and the infective juveniles of Trichostrongyluscolubrijiormis (Nematoda). Parasitology 1990; 101:101-106.

Microbial Lectins

325

172. Kellens JT, Allen AK, Peumans WJ. Isolation and characterization of lectins from Rhizoctonia crocorum and Athelia rolfsii. J Gen Microbiol 1989; 135: 3 127-3 132. 173. Peczynska-Czoch W, Mordaraski M. Hemagglutinin from insect pathogen Beauveria bassiana. Proceedings of the 10th international lectin meeting, Prague. 1988~61. 174. Zaprometova OM, Ulezlo IV, Lakhtin VM. Structure and properties of a Cephalosporium acremonium a-galactosidase. Glycoconjugate J 1990; 7: 287-300. 175. Guillot J , Breton A, Damez M, Dusser M, Gaillord-Martinse B, Millet L. Use of lectins for a comparative study of cell wall composition of different anaerobic rumen fungal strains. FEMS Microb Lett 19%; 67: 151-156. 176. Latge J-P, Monsigny M, Prevost M-C. Visualization of exocellular lectins in the entomopathogenic fungus Conidiobolus obscurus. J Histochem Cytochem 1988; 36:1419-1424. 177. Kino K, Yamashita A, Yamaoka K, Watanabe J , Tanaka S, KO K, Shimizu K, Tsunoo H. Isolation and characterization of a new immunomodulatory protein, Ling Zhi-8(LZ-8), from Ganoderma Zucidium. J Biol Chem 1989; 264:472-478. 178. Bouchara JP, Robert R, Chabasse D, Senet J. Evidence for the lectin nature of some dermatophyte haemagglutinins. Ann Inst Pasteur Ser Microbiol 1987; 138:729-736. 179. Gilboa-Garber N, Garber N. Microbial lectins. In: Allen H , Kisailus E, eds. Glycoconjugates: composition, structure and function. New York: Marcel Dekker, 1991540-590. 180. Longman D, Callow J. Specific saccharide residues are involved in the recognition of plant rest surfaces of zoospores of Pythium aphanilermatum. Physiol Mol Plant Pathol 1987; 30: 139-150. 181. Vranken AM, Van Damme EJ, Allen AK, Peumans W. Purification and properties of a N-acetylgalactoseamine specific lectin from the plant pathogenic fungus Rizoctonia solani. FEBS Lett 1987; 216:67-72. 182. Barak B, Chet I. Lectin of Sclerotium rolfsii: its purification and possible function in fungal-fungal interaction. J Appl Bacteriol 1990; 69:101-1 12. 183. Suzuki K, Kosai M, Yokomizo K, Uyeda M, Shibata M. SAF a new cell aggregation factor produced by Streptomyces murinus strain no A-2805. Agr Biol Chem 1987; 51:3017-3025. 184. Jungery M, Pasvol G, Newbold C, Weatherall D. A lectin-like receptor is involved in invasion of erythrocytes by Plasmodium falciparum. Proc Natl Acad Sci USA 1983; 80:1018-1022. 185. Takata I, Chida K, Gordon M, Myrvic Q. L-Fucose, D-mannose, L-galactose and their BSA conjugates stimulate macrophage migration. J Leukocyte Biol 1987; 41 :248-256. 186. Tamplin ML, Ahmed MK, Jalali R, Colwell R. Variation in epitopes of the p subunit of El Tor and classical biotype Vibrio cholerae 0 1 cholera toxin. J Gen Microbiol 1989; 135:1195-1200. 187. Thompson MR, Forrista J , Kauffmann P , Madden T, Kazak K, Marris R,

Shakhanina et al.

326

Saelinger C. Isolation and characterization of Pseudomonas aeruginosa exotoxin A binding protein from mouse LM cells. J Biol Chem 1991; 266:23902396. 188.

Beuth G, KO HL, Roszkowski W, Roszkowski K, Ohshima Y. Lectins: mediators of adhesion for bacteria in infectious diseases and for tumor cells in metastasis. Zentralbl Bakteriol Hyg 1991; 274:350-358. 189. Ray J, Lerner R. A biologically active receptor for the carbohydrate-binding protein(s) of Dictyostelium discoideum. Cell 1982; 28:91-98.

10 Lectin-Blood Group Interactions C. LEVENE Ministry of Health, Jerusalem, Israel NECHAMA GILBOA-GARBER and NACHMAN C. GARBER Bar-Ilan University, Ramat-Gan, Israel

1.

INTRODUCTION

The description of a hemagglutinating factor in an extract of Ricinus communis seeds by Stillmark in 1888 [ 11 stimulated extensive searches for phytohemagglutinins that might be used for the detection of human blood groups. More than half a century passed until this goal was achieved [2-41 and, as a result, in 1954, Boyd and Shapleigh [5] named these factors Zectins, to indicate their selectivity. Later on, the lectin spectrum was expanded to include additional plant [6-121, animal [8,9,12], and microbial [9,14] hemagglutinating proteins. Lectins are now known to be ubiquitous, generally multivalent , and relatively stable proteins, exhibiting noncatalytic selective and reversible carbohydrate-binding sites. The latter enable them to agglutinate target cells and precipitate glycosylated macromolecules, such as antibodies, and to play important roles in various biological systems, in and between cells, as well as between cells and the extracellular matrix [ 151. They have proved to be very useful not only for blood grouping, but also for the advancement of various domains of science and medicine [ 13,141. Their importance is due to their selectivity and physiological functions, resembling those of antibodies, hormones, and the positioning (not catalytic) domains of enzymes [ 14,151. Because of the internationally accepted lectin definition (see Chapter l), that limits their specificities to carbohydrates [la], lectins can now be used only for the detection and study of glycosylated blood group antigens. If the lectin definition is expanded to include all the “proteins that exhibit noncatalytic selective and reversible binding to target cellular and extracel327

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lular molecules,” then perhaps nonglycosylated blood group antigenic epitopes may also be detected by lectins. Many lectins are not useful as classic blood group reagents because they react with saccharides that are present on all human erythrocytes, or with structures that are not involved in blood group determination [ 171, Other lectins, behaving like blood group-specific antibodies, do interact with the immunodominant sugars, including Dgalactose (Gal), L-fucose (Fuc), N-acetyl-D-galactosamine (GalNAc), Nacetyl-D-glucosamine (GlcNAc), and N-acetylneuraminic acid (NANA or NeuNAc) [ 111. The interactions of both the lectins and antibodies are reversible and specifically inhibitable by soluble blood group substances and competing lectins or antibodies. However, lectins are generally less specific than antibodies [ 1 1,181, as they exhibit satellite interactions with additional glycosylated components of the cell surface. Consequently, they are useful as blood group reagents only following standardization with the appropriate red blood cells (RBCs) and careful titration for proper dilution [1 1,191. The same extracts may contain several lectins (isolectins) with similar or different carbohydrate and blood group specificities [8- 141: Evonymus europaeus seeds contain separable anti-B and anti-H, Salvia horminum seeds contain separable anti-Tn and anti-Cad [1 11, Griffonia simplicifolia seeds contain at least four different lectins, each with a different blood group specificity, such as for B, Tk, Leb, and Y [11,20-251, and even from the bacterium Pseudomonas aeruginosa different lectins have been defined in the same extract [14]. Lectin specificity for glycosylated antigens depends not only on the presence of the compatible immunodominant saccharide in a terminal position, but also on its anomeric configuration, the nature of the subterminal or penultimate structure, and the branching of the macromolecule [11,13, 14,18,26,27]. Additional important factors are the site of the saccharide attachment to this structure, the number and location of the antigenic sites, the membranal components that bear them (glycoproteins or glycolipids) and their length, as well as the amount of steric hindrance caused by vicinal structures [ 1 11. Thus, two lectins that exhibit a similar carbohydrate specificity may differ in reactivity with the ABO and other antigens. An example is the GalNAc-specific lectins of Dolichos biflorus and Salvia sclarea. The first reacts with A, and Tn cells, whereas the second reacts with only Tn cells [l 11. Knowledge of the carbohydrate specificity of a hemagglutinating lectin is an advantage and a very important tool for determination of the structure of the target glycosylated antigen. The use of lectins allowed the demonstration that the immunodominant sugars of the A, B, and H antigens are N-acetyl-D-galactosamine,D-galactose, and L-fucose, respectively (Table 1) [28-361. These discoveries were followed by an advanced research into the

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Table 1 A Simplified Representation of the Structural Relations Among Someof the A, B, H, i, I, P, P,, and Pk Human Glycolipid Blood Group Antigens

Structure

Antigen Globo series P‘ P

Forssman Globo H

Galal,4Galfll,4Glcfll,lCer GalNAcfll,3Galal,4Galfll,4Glcfll,lCer GalNAcal,3GalNAc/31,3Galal,4Galfl1,4Glcfll,lCer Fucal,2Galfll,3GalNAcfll,3Gala1,4Galfll,4Glcfl1,1Cer

Lacto series Paragloboside Om) type 1 type 2 p, Om) type 2

B

Galfll,3GlcNAcfl1,3Galfll,4Glcfl1,1Cer Galfll,4GlcNAcfl1,3Galfll,4Glcfl1,1Cer Galal,4Galfll,4GlcNAcfll,3Galfll,4Glcfl1,1Cer Galfl1,4GlcNAcfll,3Gal@l,4Glcfll,lCer Fucal,2 Galal,3Galfll,4GlcNAcfll,3Galfll,4Glcfll,1Cer Fucorl,2

A

i

GalNAcal,3Galfl1,4GlcNAcfl1,3Gal/31,4Glc/31,lCer Fucal,2 Galfll,4GlcNAcfl1,3Galfll,4GlcNAcfll,3Galfl1,4Glcfl1,1Cer Galfll,4GlcNAcfl1,6

I

Gal@l,4GlcNAcfl1,3Gal~l,4GlcNAcfll,3Gal/31,4Glcfll,lCer

Source: Data adaptedfrom Refs. 41,48.

detailed structuresof the blood group antigens and their subgroupsand to an understanding of their enzymatic and genetic bases[34-531. Lectins can also be used for the demonstration of the structure of additional new blood group antigens of individualdonors and for studies of associations between related blood groups [48-501; between blood groups and certain diseases, especially cancer [39,54-581; as well as between blood groupsand adhesins [59-661 or antigens [67,68] of human microbial pathogens. Moreover, lectins may also be usedto characterizethe functions of blood group antigens. One exampleof the latter is the report that the anti-H lectin of Ulex europaeus (at subagglutinating concentrations) affects human RBC Ca2+uptake [69]. This finding indicatesthat the ARH complex, which is a part of the cell membrane major protein band 3 (anion-transport glycoprotein)and is also present in band4.5 (the glucose-transporter glycoprotein), as well as in glycosphingolipids, may be involvedthe in recognition sitesfor the stimulation of Ca” influx [69]. Interaction of lectins with it may affect both the cation influx [69] and the osmotic fragilityof the RBCs [70].

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II. LECTINPREPARATIONS

Crude extracts obtained from plants, animals, and microorganisms are suitable for blood group antigen detection, but it is better to use them following purification. The purification may be achieved by heating the crude extract (depending onthe thermal stabilityof the lectin) at 65OC,precipitation of the [l 1,13,64]. lectin by ammonium sulfate,and then affinity chromatography 111. THE LECTIN HEMAGGLUTINATINGACTIVITY AND ITS DEPENDENCE ON ERYTHROCYTE TREATMENT BY ENZYMES

The lectin hemagglutinating activity depends on the presence ofthe specific saccharide residues on the erythrocyte surface in an unmasked and unblocked form. It also depends on the lectin insensitivity to the repulsing effect of the sialic acid residues present on the erythrocyte surface glycoprountreated RBCs, whereas others teins [11,711.Some lectins may agglutinate will agglutinate only enzyme-treated cells (Table2). Lectins reacting with both untreated and enzyme-treated (e.g., the ABH antigens may agglutinate sialidase or protease) RBCs. These lectins generally exhibit higher activity with the latter cells, since the reduction of the negative charges (of sialic A second group of lectins includes those acid) facilitates hemagglutination. that .agglutinate only untreatedRBCs. Among these lectins are sialophilic ones reacting with sialoglycoproteins, such as the glycophorins, that bear the M and N antigens and disappear following sialidase or protease treatment of the cells. A third group of lectins include those that agglutinate Table 2 Agglutination of Untreated, Sialidaseand Papain-Treated Human Erythrocytes by Lectins and Antibodies*

Hemagglutination Cell treatment

Untreated

Sialidase

Papain

+

+

-

LectidAb Cod

Anti-A Ab Anti-B Ab Anti-M and N Abs PNA SBA, PA-I lactuca Ulva Lygos sphaerocarpa Lygos monosperma

1

+ -

4-

-

-/*

+ -

+ +

-

'Ab, antibody; lectin abbreviations supplied in Appendix to Chapter 1.

Interactions Lectin-Blood Group

33 1

erythrocytes onlyafter removal of sialic acid or certain other carbohydrates (by sialidase or other glycosidases, respectively). These lectins may also agglutinate defectiveRBCs owing to either exposure to bacterial enzymes or genetic mutation affecting glycosyl or sialyltransferases (e.g., Tn and HEMPAS) (see section XI.I.1 for discussion of HEMPAS)[11,54,55]. The RBC treatment with sialidaseand glycosidases unmask subterminal carbohydrates, revealingT and related cryptantigens[l 1,5 1,521, which then may bind with specific lectins not reacting with untreated cells (e.g., peanut lectin) [1 1,5 l]. These lectinsdo not agglutinate RBCs following proteolytic treatments that remove not only sialic acids, but also additional glycOsylated branches mounted on the cell surface glycoproteins (e.g., glycophorins). Lectins of the fourth group react with both sialidase- or papaintreated cells much more strongly than with untreated cells. The enzyme treatments enable their contact with glycolipid antigens of the cell surface (e.g., part of ABH, I, and P systems) [36-48], without disturbanceby sialic acid residues or their negative charge [l l]. Examples of lectins of this fourth group are those of the soybean [l l], P. aeruginosa [63,64,72], and Ulva [73]. Only rare lectins selectively agglutinate papain-treated cells. An example isthe lectin ofLygos monosperma[1 l]. As in antibody-induced hemagglutination, there is no direct relation between the agglutination intensity and the antigen abundance or density on the cell surface. Thelatter may be better estimated by determination of the lectin adsorption onto the cells using hemagglutination inhibition tests or adsorption tests with unlabeledor variously labeled lectins, respectively. IV. THE LECTIN CARBOHYDRATE SPECIFICITY AND ITS RELATIONTO BLOOD GROUPSELECTIVITY

Inhibition of the hemagglutinating activity of lectins by sugarsor saccharides is used for determination of their carbohydrate specificities. Most lectins are classified according to their major carbohydrate specificity into several main groups [13,26,27], and in each group, there are subtle differences of specificity at the following levelsof variance: A. The Most Preferred Carbohydrate Derivative

Generally, lectinsthat exhibit a preferential specificity for immunodominant saccharides may be useful for antigen typing(see Appendix in Chapter 1). B. SecondaryCarbohydrateBinding

Lectins that exhibit a similar preferentialcarbohydrate affinity may vary in their satellite interactions with additional carbohydrates and their deriva-

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tives. For example,the lectin of Streptomyces sp. (which exhibits significant antiB specificity) [74,75] and Pseudomonasaeruginosa (PA-I, which shows a slight B preference) [63-661, both bind D-galactose, but differ in their relative affinitiesfor L-rhamnose and D-galactose derivatives. C.

Differential Preference Betweenthe Primary and the Secondary Carbohydrates

Lectins that bind the same major carbohydrate, but vary in their differential preference for it, may differ in their bloodgroup specificity. For example, thosethat bind galactose and GalNAc with low differential preference (e.g., Sophorajaponica or Pseudomonas PA-I lectins)are not as promising reagents for the differentiation between Aand B antigens, as that of Streptomyces sp., which exhibits a much higher affinity for galactose than for GalNAc. There are other lectins that exhibit dual specificities, including those of Crotalaria striata (anti-A,B) [49] and Moluccella laevis(anti-A,N) ~761.

D. Intensity of theAffinity for the Preferred Carbohydrate

Lectins that bind the same carbohydrate vary in their affinity for it, as measured by equilibrium dialysis [77] or hemagglutination inhibition tests. High carbohydrate affinity (low association constant and high sensitivity to inhibition by low carbohydrate concentrations) [78], as also do steric differences in structure, may be associated with lower blood group specificity (Fig. 1). E. Specificity for the Anomeric Configuration of the Carbohydrate

Lectins may differ in preferences for the sugar anomeric configurations, from profound cr or @ preference, to relative insensitivityto the differences between them. This property determines lectin bloodgroup selectivity, bethe same terminal cause, as described in Tablel , diverse antigens may bear dominant carbohydrate in a different configuration (e.g., cr-galactose of groups B, Pk,and PI antigens, in contrast with @-galactoseof I and T, or the a-GalNAc of A and Tn, as opposed to the 8-GalNAc of P and Cad). Salvia sclarea lectin, which is specific for cr-GalNAc, reacts with Tn, but not Cad RBCs [79], whereas the lectin from Leonurus cardiaca, which is specific for P-GalNAc, reacts withCad, but not with Tn erythrocytes [80]. Lectins that are insensitive to the anomeric configuration of the carbohydrate react with more antigens and, hence, may be less specific for blood group determination.

I

I

I I

I I

I I

I I

I

I

I

I

I

3 9

\ \ \ \ \ \

I

I

I

I

I

I

l I

Levene et al.

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F. lectin Repulsion by Molecules Present in the Vicinity of the Target Carbohydrate

In addition to the masking and hindering effects of sialic acids and their charges, the binding of lectins to their specific carbohydrates may vary in the presence of adjacent carbohydrates or other molecules. For example, the L-fucose-binding lectin of Ulex europaeus, and P. aeruginosa (see Fig. as opposed to O(h), Bombay 1) strongly agglutinate L-fucose bearing O(H), erythrocytes [73]. However, theydiffer in hemagglutinating activity toward A, B, and AB erythrocytes: hemagglutinationby Ulex-I is strongly reduced by the presence of the galactose and the GalNAc adjacent to the fucose in the B and A antigens, whereas PA-I1 reacts with fucose of the H gene product even in AB erythrocytes. As seen in Figure1, PA-I1 hasan advantage in detectingthe hidden H gene product or shorter H chains, but is not useful for differentiation between 0, B, A, or AB erythrocytes [73]. The special pattern of PA-I1 may be due to its outstandingly high affinity for fucose [78], interaction withshorter H chains, as well as its smaller sizeor different steric structure. G. lectin Interactions with longer, Branched, and Adjacent Molecules

Generally, lectins exhibit stronger bindingto branched glycoconjugates or to a complex of vicinal branches of oligosaccharides than to unbranched chains[18].Thispropertyisalsorepresentedintheirinteractionswith blood .group antigens. One example is the group of lectins that exhibit stronger interaction withHI [81] than with Hi antigenic structure. Another one is the evidencefor the noninvolvement of medium-sized glycosphingolipids inthe Dolichos bifroruslectin hemagglutination [82]. All the foregoing aspects of lectin specificities and their usefulnessfor blood group detection are closely related. Lectins exhibiting specificities for carbohydrates that are not immunodominant, for example, those extracted from Canavaliaensiformis,Lens culinaris,and Pisum sativum(pea), are generally not or are lowly specific for blood group antigens. On the other hand, lectins that bind galactose or GalNAc may preferentially interact with one or several of the following antigens: A, B, P, PI, Pk, I,i, T, Tn, and Cad, as well as with the ground substance of Bombay RBCs. Higher selectivity is a property of those lectins that differentiate between the carbohydrates and their derivatives (or related carbohydrates) and exhibit high sensitivity for the anomeric configurationof the specific carbohydrate and the adjacent components. Those lectins specificfor a-galactose would react better withB, PI, and Pkantigens, whereasthose choosing the &configuration of this sugar might be better reagentsfor I and T antigens


335

and agglutinate 001)Bombay erythrocytes. BothDolichos biforus @BA) and Falcata japonica lectins agglutinateAI (a-GalNAc) erythrocytes more strongly than A2 [83], but they differ in agglutination of Cad (p-GalNAc) erythrocytes. Although DBA strongly agglutinates these cells,the Falcata japonica lectin induces weaker agglutination of them [83]. V. MULTlSPEClFlClTYEXHIBITED IN SIMULTANEOUS INTERACTIONS OF LECTINS WITHSEVERAL BLOOD GROUP ANTIGENS

Because there are several blood group antigensthat are determined by the same terminal immunodominant carbohydrate (someexamplesarepresented in Table 1 and there are additional ones amongthe cryptantigens), lectins specificfor this carbohydrate interact with several of these antigens. Thus, salmon roe lectin [84] interacts with B, P,, and P' (a-galactose), as well as weakly with A and P antigens (a-GalNAc).Crotalariastriata exhibits a dual, anti-A and B specificity [l 1,491. In these examples, the agglutinating activity toward all the erythrocyte types is completely absorbed by RBCs of any one of the reactive blood groups. Dolichos biforus, Falcata japonica, and Helixpomatia react with AI, Tn (both aGalNAc), and Cad (PGalNAc) antigens [l 1,831. Similarly, the PA-I lectin of P. aeruginosa detects T, I, Pk, B, P, antigens and O(h)erythrocytes (both a- and pgalactose). It also reacts withA and P (which are a- and p-GalNAc, respectively) [65,66]. Such lectinsare not useful as trivial unequivocally monospecific blood group reagents, but may be of great help in identification of certain blood groups under special circumstances, for example, Tn and Cad antigens in erythrocytes other than AI (by Dolichos biforus) and Th in erythrocytes other than A, (by Vicia cretica) [l l], N in erythrocytes other than A (by Moluccella laevis) [l 1,761, T in untreated erythrocytes (by soybean and PA-I lectins), and Pk antigen in 0 erythrocytes (bythe fimbriae of pyelonephritic Escherichia coli strains [59] or the PA-I lectin of P.aemginosa [65,66]). VI. LECTINSEXHIBITINGBLOODGROUPSPECIFICITY FOR A COMBINATION OF TWO ANTIGENS

Some lectins induce the highest agglutination of erythrocytes bearing a combination of two closely constructed antigens. Examples are the anti-B/ A I lectin of Sophora japonicaand other lectins [85,86] and the anti-HI activity of several anti-H lectins [81,86-901, including that of Erythrina corallodendron (ECorL) [87]. The latter lectin, which does not recognize fucose (but binds LacNAc), probably reacts with domains producedthe by

+

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combination of the LacNAc of the I antigen, and the fucose of the H antigen present in its vicinity. Additional examplesare the anti-B1 and HI lectins ofthe ova of the Black Sea breamSpondyZiosoma canthams [M], as well as several anti-AIand anti-PI activities [90]. VII. LECTIN APPLICATION FOR DETECTION A N D STUDY O F ABO(H) A N D LEWIS BLOOD CELL ANTIGENS A N D SOLUBLE SUBSTANCES

The AB0 blood group antigens were the fiist to be identified serologically [91] and, then, 50 years later, their structures were studied.by means of antibodies, lectins, glycosidases, and glycosyltransferases [28-43]. Theyare inherited in a codominant fashion following classic Mendelian genetic laws. Their locuswas assignedto the long arm of chromosome 9, closely linked to the locus codingfor the RBC enzyme adenylate kinase [92]. The worldwide frequency of the AB0 blood groupsis well-established [92].The O(H)is of the highest frequency: 44% (Caucasians) to 56% (Mexicans); next isthe A antigen: 44% (Caucasians) to 27% (all others). TheB phenotype frequency is 9% (Caucasians)to 25% (Orientals),and the AB is about 5%. The AB0 antigens are the most important in blood transfusions. These antigens are also present on many other cells and tissues of the same individual (granulocytes do not produce them) [93,94]. Their compatibility is very important for blood transfusions [91,95] and for transplantation of highly vascularized allografts, especially renal allografts. A n AB0 incompatibility may result in a swift rejection of kidney allografts. The special importance of this system intransfusion and transplantation is due to 1. The presence of considerable amounts of alloantibodies (anti-A in B subjects, antiB in A subjects, and both in 0 individuals) 2. The ability of these antibodies to react with the donor’s cells and bind complement, resulting in intravascular hemolysis and damage to the kidneys 3. The large number of antigen sites of A (A,:810,000-1.170 million; Az: 240,000-290,000; B:610,000-830,000) [57]. The AB0 blood group antigensare also foundon cells of other organisms: plants, animals, and microorganisms [67,68,96], as well as in body fluids of most persons, (the “secretors”) [19,97,98]. In secretory cells ofthe body, expression ofthe H antigenis controlled by the independently inherited autosomal/dominant gene Se (present in about 80% of the popula(Sese) are “nonsecretors.” The human tion). People who lack this gene blood group H determinant, which serves as a common precursor for the action of specific glycosyltransferases that construct the A and B blood


337

groupantigens,is a terminal ~-fucosyl(al,2)galactosyl the synthesisof which is catalyzed by GDP-a1,2-fucosyltransferases[99]. These enzymes are coded by Hh and Sese structural genes. The enzyme coded by the H gene may be readily detected in hematopoietic tissues and plasma in all individuals except the rare Bombay and para-Bombay phenotypes [99]. In secretory fluids and tissue cells, expression ofthe H antigen is determined by the Se gene,whichdetermines the formation of a distinct a1,2fucosyltransferase. Nonsecretorsare homozygous for a null allele at the Se locus, whereas Bombayand para-Bombay individuals are homozygous for a null allele at the H locus. A cloned human DNA restriction fragment in transfected murinecells determines the H-enzyme[loo], which differs from the Se-enzyme in several properties, especially in higher affinities for the substrates. Because in nonsecretors (sese) the H-specific transferase is not found in tissue fluids, A and B substances are not formed in their secretions, even though the A and B enzymes may be present in serum and secretions. In red cellsand other hemopoietic tissues, the presence A ofand B enzymes lead to formation of A and B antigens, irrespective of secretor status. A, B, and H antigens found in saliva and other secretions are glycoproteins, but in plasma they are mainly glycosphingolipids.The soluble A and B antigens may inhibit hemagglutination and hemolysis. This ability was already recognized in 1910[loll. It has been suggestedthat in vivo the natural presence of soluble antigens may decreasethe destructive potential of the corresponding antibody in transfusionsand in hemolytic disease of the newborn[102]. In someblood group systems,suchas Lewis (Le), plasma containing soluble antigens can be administered to neutralize antibody activityand permit transfusion of an antigen-positive blood[1021. The secretor status may be determined by a hemagglutination inhibition test. In this test, the saliva of the examined individual is used with antiserum (anti-Aor anti-B), which agglutinates thisperson’s erythrocytes, or with anti-H lectin of UZex europaeus [l 1,19,103]. In RBCs the ABO(H) determinants on glycoproteinsor glycolipids have been shown to be structurally related oligosaccharide chains. The precursor chainsare also common to Lewis, P, and Ii systems, but bear a different terminal carbohydrate that maybea-fucose (H), a-galactose(B, P’, and P1),a-GalNAc (A), [la]. 6-GalNAc (P), or &galactose (Ii) (see Table 1) The glycolipid erythrocyte antigens may be divided into those acquired from the plasma in “secretors” (Lewis antigens, ABH type 1, and IHLeb type 1) and to those being integral components of the RBCs: P, P1,P‘, ABH (type 2), and Ti. As seen in Table 1, only the H structure (Fuca-l2GalB1,R)is a precursor to the B (Galal,3Fuca1,2]Galpl,R)and A (GalNAcal,3Fucal,2]Gal(3l,R)structures [28-341 as well as to the Lewis a and b structures. These determinants are present on various carbohydrate

Levene et al.

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core chains. Polymorphism is generated by branching and elongation (including repetitions),on the one hand,and by variations in the combination of the determinant and carrier chains,on the other [37-41]. There are four types of core structures in A subjects recognized by monoclonal antibodies [37-41,1051: Type 1: GalPl,3GlcNAcPl,R 2: GalPl,4GlcNAcPl ,R = LacNAcPl ,R 3: Galpl,3GalNAcal,R 4: GalP1,3GalNAcpl,R Type 1 chain is present in glandular tissues and secretions. The Se transferase acts on type 1 substrates found in secretions. This chain type issecreted into the plasma by nonhematopoietic cells and is present in erythrocytes by adsorption as the Lewis antigens.Thetype 2 chainis the substrate for the H-transferase. In RBCs, only type2 chain paraglobosides are formed, and theylead to formation of subtypeantigenswithunbranched (H, and H*) and branched (H, and H4)species [40]. In the Hzto H4 types there are longer polymers of i and I antigens. Polyglycosylceramides (containing terminal LacNAc) function as precursor structures for the H-fucosyltransferase and later on for the A and B GalNAc and galactose transferases (respectively), resulting in several H (and accordingly A and B) subtype structures[40,106]: Subtype H, Fucal,2LacNAcP1,3Lac,Cer H, (i) Fuccrl,2LacNAc~1,3LacNAc~l,3Lac,Cer H, (I) Fuca1,2LacNAcP1,6

Fucal,2LacNAc~l,3LacNAc~l,3Lac,Cer H4(I) a more complex branched precursor There is a difference inthe distribution of the H, toH4subtypes in red cells: in RBCs of fetus and newborns almost exclusively, the unbranched H, and Hz structures are found with the i phenotype. Mature red cells possess a high concentration of the complex branched structures H3 and H4, with the I phenotype. The expressionof type 1 chain H in secretions is related to the H and Se genes, whereasthat of type 2 chain H in red cells is dependent onlyon the H gene [107]. The quantity of H antigen in various tissues is much higher in blood group 0 than in A or B individuals. Human blood group A RBCs contain A-active glycolipids of all four types [37,40]. The type 3 chain glycolipid structures are characterizedbyhaving an internal A trisaccharide and, therefore, are found only in A individuals. This repetitive A (GalNAcal,

.


339

3~uccu1,2]Galfll,3-GalNAccul,3~uccul,2]GaU?l-R) chain is especially abundant in Al erythrocytes.Type 3 chain H (Fuca1,2Galfl1,3GalNAcal, 3[Fuc~~l,2]Galfll,4GlcNAcfll,3Gal~l,4Glcfll,lCer) is present exclusivelyin A erythrocytes asa precursor to the repetitive A structure. It is preponder[a,1081. The Al enzyme is quantitatively and qualitaant in Az erythrocytes tively different from A, enzyme in transfer activity of a-GalNAc to type 2 chain H, type 3 chain H, and globo H. The A,-specific antigens in erythrocytes and tissues are repetitive A type 3 and 4 chains. It was pointed out [106,109] that the precursor to type 3 chain H, which is galactosyl-A antigen, hasa terminal disaccharide Galfl1,3GalNAcal,R, identical with that of the Thomsen-Friedenreich T and is known antigen [106,110,11l], which isGalfl1,3GalNAcal,OSer/Thr, to prevail in cancerous tissues [55,56]. Clausen et al. [l061 produced two monoclonal antibodies and showed that one of them cross-reacted with both T antigen on desialylated glycophorinA (similar to anti-T and peanut lectin) and the galactosyl-A on glycolipids. The second antibody did not cross-react withT antigen, as it recognized the entire galactosyl-A structure. The globo-H(the third typeofH)antigen Fum1,2Galfll,3GalNAcfll, 3Galal,4Lacfll,1,Cer has been discovered aas major component of human teratocarcinoma. In AB heterozygotes there is a competition betweenthe A and B transferases for the H antigen precursor structures, resulting in less AI and A, antigen production inAIB and AzB than in AIO and AZO,and reduced B level, respectively. It is suggested [92]that UDP-galactose hasan inhibitory effect on the A GalNAc-transferase, especiallyof the A,-transferase. As a result, mostA,B persons develop anti-A, in their serum [92]. Since sera of people possessing A blood group normally contain anti-B antibodies and those of B type normally contain anti-A, there is plentyof sera that may be used for the determination of B and A blood groups, respectively [19]. There is a shortage of anti-H sera for detection of O(H) and O(h) blood and recognition of secretors and nonsecretors of ABH substances. Anti-H is found in the serum of persons who have the rare O(h) Bombay, but it is regularly present together with both anti-A and anti-B. Therefore, anti-H lectins are in routine use in blood banks. They are useful for recognition of Bombay 001) blood and identification of A subtypes (AI, Az, and so on) together with anti-A lectins, and for recognition of H secretors and nonsecretors. All lectin A and B reagents have the drawback that cord blood and samples from AB donors often give poor reactions. A general comparison between lectins and antibodies as blood group reagents shows that, . a l though the latter are more specific, lectins have the following advantages:

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1. Availability when there is a limitation in specific sera because of rarity of people producing the antibodies [e.g., O(h) individuals as a source of anti-H, or p individuals as a source anti-Pk] of 2.Higherstabilityunderunfavorableconditions(hightemperature,extreme pH, contamination) during shipment, storage, and use 3. Availability in large, standardized stocks enabling long-term use of the same preparations,as opposed to heterogeneous human sera 4. Special specificities that are more useful than antisera for certain purposes [e.g., determinationof secretor status (with Ulex-I) in individuals who differ in theirABO(H) blood groups (with no need of preliminary blood group determination), and discrimination between AI and A, individuals (with Ulex-Iand Dolichos biflorus lectins] 5. Higher security in handling them compared with antibody-containing blood plasmapreparations (risk of viral infections) 6. Possibility to inhibit or reverse hemagglutinationand easily removethe agglutinins from the cells by simplesugars, and saccharides) A. Anti-H Lectins

The human blood group H antigen is determined by a terminal a-L-fucose bound (1 2) to galactose (asdescribed in Table 1) of the glycosphingolipid or glycoproteingroundsubstances. Its synthesisiscatalyzedbya special GDP-fucosea-(1,2)-fucosyltransferase, coded by the H or se genes. The discovery of anti-H in seed extracts was a veryimportant finding for bloodgroupinvestigatorsbecause anti-H sera are rare and are usually impure (contaminated with anti-A or anti-B). Renkonen, in 1948 [3], was the first to find anti-H in seed extracts of Cytisus sessilifolius, Laburnum alpinurn, and Lotus tetragonolobus. Four years later, Cazal and Lalaurie [86] found the anti-H activity in Ulex europaeus (UEA-I). In parallel with the discovery of UEA-I, which is a plant lectin, an animal anti-H lectin (in serum of the eel Anguilla anguilla)was found very early,and its neutralization by simple sugars was described [28]. The UEA-I lectin, which is L-fucose-specific,the is most highly useful and reliable reagent for detection of the H blood group on cells and for discrimination between persons of the O(H) type and other blood groups and between secretors and nonsecretors [l 1,19,97]. This proved to be a fortunate circumstance, because this lectin has a strong affinity for the H-type 2 determinant (see Table l), but does not recognize the H-type 1 determinant [l 121. Radioimmunoassays with a wide range of chemically modified structures related to the H-type 2 human blood group determinant by the UEA-I revealed that the binding of Fuccw1,2Galpl,4GlcNAc~,OMe lectin involves a wedge-shaped amphiphilic surface that extends on one side

Interactions Lectin-Blood Croup

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of the molecule from the methoxy aglycone to OH-3 of the P-galactose unit. A cluster that involves OH-3,OH-4, and OH-2 of the a-L-fucose unit along with OH-3 of the 0-galactose unit provides the polar interactions with the lectin. However, only OH-3 and O H 4 of the a-fucose are indispensable to complex formation and are thought to provide the key polar interactions [43]. UEA-I is the most frequently usedanti-H lectin in blood banks (suffering from shortage of anti-H antibodies) for O(H) and A2 detection inRBCs and for determination of secretor status [19]. The other anti-H lectins arenot identical in theircarbohydrate and RBC specificities. They exhibit differences in sugar specificity and affinity, as well as in sensitivity to inhibition by the soluble blood group substances.The heterogeneity of the plant anti-H group lectins has been discussed in several important papers that emphasize their individual advantagesand disadvantages [l 131221. First, it was shown [l 181 that the anti-H hemagglutinins in extracts of plant seeds could be subdividedinto two groups: eel serum type (including Lotus tetragonolobus [28,123] and UEA-Iinaddition to the eelserum hemagglutinins), fucose-specific lectins and Cytisus (which includesCytisus sessilifolius and Laburnum alpinurn)type [99,103] hemagglutinins, specific for both salicin and (D-G~cNAc),. This division was based on inhibition assayswithsimplesugars and saccharides,such as fucose and di-Nacetylchitobiose. However, even among the same groups of hemagglutinins, there are subtle differencesin specificities [l 14,1151. The L. tetragonolobus lectin (LOTUS)was also shown, by Pereira and Kabat [ W ] , to be strongly precipitated by human ovarian cyst H substance, by human saliva A2 substance, and by ovarian cyst Le" substance. With use of ovarian AI and A, blood group substances in insoluble forms, it was shown that this hemagglutinin also can distinguish human A2 blood group substance from Al. It does not precipitate with the latter, whereas its precipitation curve with A2 substance showstoitbe 65% as active as blood group H substance. The B substance also didnot precipitate withthe purified lectin. This lectin is inhibited only by oligosaccharides with a type 2 chain. Addition of a second fucose to the GlcNAc increases the inhibitory power. The striking specificity ofthe lectin of L. tetragonolobus for type 2 chains (which contain fucose residues on C-2 of the GalPl,4GlcNAc, with or without a second fucose on the GlcNAc) and their failure to react with type 1 chains, (substituted by Fuc on the Gal, with or without a second Fuc on the GlcNAc) makes this lectin, according to those investigators,an extremely valuable reagent in studies of the structures of blood group oligosaccharides [108]. In contrast with the eel-type lectins, those of the Cytisus type are inhibited best by di-N-acetylchitobiose [31,113], the structure of which is not found in the carbohydrate chain of any known blood group determinant structure. Extractsof U.europaeus seeds were shownto contain both

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types of anti-H hemagglutinins [116,117] (i.e., eel serum typeand the Cytisus type). Treatment of human O(H) erythrocytes with H-decomposing enzyme (a-L-fucosidase)from Bacillus fulminans destroyedthe agglutinability of the cells not only by eel serum-type hemagglutinins but also by Cytisus-type hemagglutinins[1 171.This clearly indicatesthat the L-fucosyl residue is important, even for the H-specificity of the Cytisus-type hemagglutinins. Lactoseand its derivatives, which werenot inhibitory against the eel serum-type hemagglutinins, gave weak, but definite, inhibition against the Cytisus-typehemagglutininsof C. sessilifolius, Laburnum alpinum, Cerastium tomentosum, andUlex-I1 [1171. Inhibition of the anti-H hemagglutinins by blood group substances (at concentrations lower than 10 mg/ ml) showed that, although blood group H substance is equally inhibitory against both eel serum-type and Cytisus-type hemagglutinins, blood group A and B substances are generally more inhibitory against Cytisus-type hemagglutinins (minimum amounts completely inhibiting four hemagglutinating doses: A, 0.02; B, 0.01; H, 0.01 mg/ml) than against eel serum-type hemagglutinins (0.63, 0.16, 0.01 mg/ml, respectively). This phenomenon was attributed to the fact that eel serum-type hemagglutinins are more strongly affected by steric hindrance, caused by the a-GalNAc or a-Gal joined (1 -+ 3) to the terminal galactose unit in the carbohydrate chain of the blood group determinant, than the Cytisus-type hemagglutinins. Blood group Le" substance showed weak,but definite, inhibitory activity against C. sessilifolius hemagglutininand Ulex-11, but it was not inhibitory against eel serum, Ulex-I, and Laburnum alpinum hemagglutinins. Accordingly, the latter two are considered to be more suitable reagentsfor identification of secretors. An additional factor of heterogeneity was described by Chessin and McGinnis in 1968 [125], who forwarded evidencethat Cytisus and Laburnum lectins detect H antigen on O(H) erythrocytes in association with the I antigen. An association that had been observed earlier with anti-HI cold antibodies [126]. As described before, these reports were followed by additional observations of combined HI specificities of some of the anti-H lectins [81]. We have also recently described HI preference in the LacNAc-binding lectin ofErythrina corallodendron (ECorL) [87,127]. This lectin exhibits preferencefor adult O(H) erythrocytes, but is relatively insensitive to the soluble blood group substances.In studies with synthetic macromolecules [128], thislectinexhibitedpreferential affinity for synthetic IantigencontainingLacNAcin a forked structure. However, in hemagglutination tests [87], it did not prefer I antigen alone, because it exhibited onlya weak reaction withadult O(h), Bombay type, erythrocytes (possessing I antigen without the fucosyl of the H antigen). Such cells are agglutinated by anti-I sera more strongly than are O(H)I cells [11,871. This controversy indicatesthat ECorL preferentially binds either to a combined


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structure produced when the I and H antigens are closely situated [81,87], or to a mixed product of the I and H antigens. The mixed product could be the H3 and H4 structures formed on the branched LacNAc (I antigen)bearing ground substance glycolipids. It is possiblethat EcorL and the other so-called anti-HI, ,anti-AI, anti-B1 reagents interact better with the longer and branched chains (produced byI transferase) than with the shorter ones (of i-transferase). The stronger interaction of anti-IreagentswithO(h) Bombay than with O(H) erythrocytes may be related to the same phenomenon. Anti-H activity was described in Erythrina subrosa by Bhatia and Boyd in 1962 [1291and in E. lysistemon by Moore in 1981 [130]. However, these two lectins were reported to exhibit similar activities toward 0 1 and Oii cells. Another group of fucose-specific anti-H lectins includes G$the fonia simplicifolia IV lectin (GS-IV), described by Shibata et al. [20] and Kalades et al.[25], and the Ulva lactucalectin (ULL), described by GilboaGarber et al. [73,131]. These two lectins may be used as anti-H reagents with human erythrocytes (the latter only with papain-treated cells). With soluble blood group substances (or saliva), both lectins exhibit highest sensitivity to Leb and Y substances.Salivasofnonsecretorindividualsare clearly detectedby these lectins because they do not inhibit them (Table3). On the other hand, salivas of Le(a-b +) individuals (Se+ Lewis-positive) inhibit them most strongly. But, theydo not differentiate between salivas of se Le(a +b -) (Lewis-positive, nonsecretors)and SeLe(a-b -) (secretors, Lewis-negative), being partially inhibited by both. Namely, they are not useful for determination of secretors who are Lewis-negative and nonsecretors who are Lewis-positive [73,131]. Last to be mentioned is the fucose-binding lectin of the bacterium Pseudomonas aeruginosa which differentiates between H-transferase-deficient (Oh) and positive (OH,A,B, and AB) RBCs. This lectin is not useful Table 3 Inhibition of the Hemagglutinating Activity of UEA, GS-IV,and ULL by Saliva of Different H and Lewis Phenotypes Lectin inhibition'

GSIV and

Phenotype Nonsecretor Le(a -b -) Secretor Le (a -b -) Nonsecretor Le (a+ b -) Secretor Le (a- b +)

UEA

-

++++ ++++

'+ ,Inhibition, graduatedfrom + to + + + + . -, No inhibition.

ULL

-

++ ++ ++++

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for detection of secretors becauseit is neutralized by most salivas, probably owing to its high sensitivity to the presence of other fucose or mannose residues [65,78].Additional anti-H lectins are currently described. An example is the fucose-specific lectin fromthe ova of the sea bass Dicentrarchus labrax [1321. B. Use of Lectins for Detection of H-Deficient Bombay-Type Phenotypes

The first H-deficient phenotype was reported in Bombay in 1952 [l 1,191. The classic Bombay phenotypes are totally devoid of H antigen, both on red cells and in secretions (because of a double dose of the recessive h gene). These erythrocytes react withanti-I sera even more stronglythan do O(H)I cells [l lA ]. similar phenomenon was also observed with the Ricinus communis lectin [1 l] and the PA-I lectin ofP. aeruginosa [66].Increase of I antigen during H-deficiency has been described in several publications [11,38,99].These observations may be ascribed to one or several of the following reasons:

1. Additional I antigen productionwhen there is no competition with the fucosyltransferaseon the ground structure [1 l]. 2. Reduced steric disturbanceto the anti-I reagent by the fucosyl residue in the I antigen vicinity. 3. Reduction information of H3 and H,, subtypes on the I antigen, by its fucosylation. 4. Cointeraction of the anti-I with the #?-galactosyl residue of the un(see maskedground structure Gal#?l,4GlcNAc#?1,3#?lactosylceramide Table 1). The latter is very similarto the Ii antigen system terminiand may function asan additional branch.

C. Anti-Lewis Lectins The first antigens of Lewis system were discovered duringthe years 1946Le" in 1946 [l331 and Leb in 1948 [134]. A year later [l351 a 1949 [57]: related Le" antigen was discovered. TheLe" and Lebare coded by one gene Le [57].The Lewis antigens detectable on the human red blood cells afe not produced in them[135-1391,but are glycosphingolipids [1401 from the plasma lipoproteins that adsorb onto RBCs [135-1391.The Lewis system was later shown to include several additional tissue antigens that are excreted into secretions (e.g., saliva) as glycoproteins and to plasma as glycolipids. The latter are carried by lipoproteins and, subsequently, adsorbed onto the RBCs [92]. ErythrocyteLe(a-b -) canbeconverted to Le(a+ b -) or to Le(a- b + ) by exposure to plasma, but not to saliva


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(which contains higher concentrations of Lewis substances). The different Lewis antigens are produced by the action of one enzyme on different substrates. The gene Le, which codes the enzyme, is thought to be located on chromosome 19 linked to the C3complement locus. Its frequency in whitesis 90%.It codes for a specific cr-l,4-~-fucosyltransferasethat is related to the HSe-specific transferase intransfer of L-fucose from GDP-Lfucose. It differs from the latter enzyme inthe linkage form:a1,4instead of al,2 of H, and in the receptor sugar: P-GlcNAc, instead of the @-galactose, following it in the paragloboside or glycoprotein chains. The Lewis a and b antigens in saliva, milk, submaxillary glands, gastric mucosa, kidney, and cyst fluids are type 1 [140,141],whereas the Lewis X and Y antigens are type 2. Their structures are presented in Table 4. As may be seen in this table, Le"shares withthe ABO(H) systemthe common unfucosylated type l paragloboside precursorsubstrate to which the Lewis enzyme adds fucose. Whenthe Lewis enzymeadds fucoseto the fucosylated (by the Se enzyme) H, A, or B substances, the structures that possess two L-fucose residues (of HSe and Le) already exhibit Leb antigenicity of the types: HLeb, ALeb, or BLeb, respectively[92]. Inthesecretions of nonsecretors(sese)onlyLe"is formed, but in secretors (Se) the Lewis fucosyltransferase competes withthe HSe fucosyltransferase (and the subsequent A and B glycosyltransferases) for the type 1 chain precursor. As a consequence, the respective amounts of Le" and type 1 H substances formed in secretions are determined by the ratio of these two fucosyltransferases. Individuals possessing both Se and Le genes have lower amounts of A- and B-bearing type 1 plasma glycolipids than those with Se without Le. Their Le product (mainly Leb) is high because H, A, and B type 1 chains may be utilized as substrates for the Lewis fucosyltransferase to form Leb. However, following fucosyltransferase by Table 4 Structures of h, H,Lewis

Chain type

1 1 1 1 2 2 2 2

a, b, X and Y Glycoconjugates

Antigens

Structure"

h

Gal/31,3GlcNAc/31,3R Fucal,2Gal/31,3GlcNAc/31,3R Gal~1,3(Fucal,4)GlcNAc/3l,3R Fuccrl,2Gal/31,3 (Fuccrl,4)GlcNAc/31,3R Gal/31,4GlcNAc/31,3R Fucoll,2Gal/31,4GlcNAc/31,3R Gal/3l,4(Fucal,3)GlcNAc/31,3R Fuca1,2Gal/3l,4(Fucar1,3)GlcNAc~1,3R

H Le" Leb h

H Le" LeY

'R, Lactosylceramide or other precursor.

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the Le enzyme, the precursor can no longer be used as substrate for H and A or B transferases owing to chain-termination signals by the Lewis transferase [141].The sameLe effects on the concentration of H, A, and B type 1 chain substancesare also found with glycoproteins.The synthesis of is affected by the type 2 chain H, A, or B soluble antigens in secretions not the Le gene product. Le"is considered as present in every individual who inherits the Le gene 1921. It has been reported that 90% of all Caucasians' cord bloods, which initially behave as Le(a- b -) phenotypes exhibit Le" antigen [142]. These newborns secrete Le" substance in their salivas, Lewis but glycosphingolipids become detectable in their plasmas and in their red bloodcells as a Le(a+ b -x +) phenotype after approximately 10 days of life. In LeSeH individuals, the Le(a -b -x +) phenotype at birth changes to Le(a +b +x +) after 10 days and finally to their true Le(a -b +x +) phenotype by 2 years of age. Individuals whoare lele stay Le(a-b -x -) phenotype for all their lives 1921. Theantigenicdeterminantof Le" is a trisaccharide structure 3-fucosyl-N-acetyllactosamine,which is formed by the 1-3 fucosylation of a type 2 blood group backbone chain (Galpl-4GlcNAc), a stage-specific a embryonicantigen [143,144].It is also found ingranulocytesandin number of normal tissues and malignant tumors from tissues that do not 1144-1471. normally express this antigen The Lewis erythrocyte antigens are detectable by means of antisera that differentiatebetween them. L-Fucose-binding lectins may be insensitive to the differences between them because they also bind strongly to the H antigen molecules, which are much more numerous on the red blood cell surface [57].However, these lectins may detect the soluble Le" and Leb Griffonia simplicifolia antigens in secretions. Two such lectins, those of [20,148-1501and Ulvalactuca [73,131],wereshown to exhibitmuch stronger specific inhibition by salivas of SeLe(a-b+) containing Leb substance than by those of either nonsecretors Le(a+ b -) containing Le" or secretors lele, containing neither of them. Such lectins may differentiate between the difucosylated Leb and Y structures and the monofucosylated Le" and Le" antigens [20,148-1501(see Table 4)because they bindand their hemagglutinating activities are inhibited by the difucosylated compounds much more strongly than by the monofucosylated ones. Body fluids that 3 lack the A, B, H, Le", do not inhibit the three lectins represented in Table and Leb specificity[15l]. D. The Simultaneous Use of Anti-H and Anti-Lewis Lectins for the Detection of Le" and Lebin Salivas Because Ulex europaeus lectin detects type 2 H soluble blood group substance, but not Lea or Leb substances, whereas GS-IV [20,148-1501and

ULL [73,131]detect both H and the Lewis fucosyl residuesand react most

Interactions lectin-Blood Group

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strongly with Leb and Y, a complementary use of both lectin types may confirm the results, as described in Table 3. E. Anti-ALectins

The A gene codesfor an a-N-acetylgalactosaminyltransferasethat transfers GalNAc from UDP-GalNAc to H-antigen (see Table 1). There are two types of transferases: one is coded by the A, gene, and the other by the A, gene, leading to formation of A, (7940%) and A2 (19-20%) phenotypes, respectively [92]. The A, and A2 enzymes differ in several properties [92]. A, actson the H,-H, precursor, whereas Azacts mainlyon the unbranched H, and Hz [108]. A s a result, A2 redcells have mostly Aon H structures 1 and 2, with more unconverted H3and H4antigen sites available [40]. Hence, A, RBCs are agglutinated strongly by anti-A, lectin of Dolichos bifoms and poorly by anti-H lectin of Ulex europaeus, whereas Az RBCs are very weakly agglutinated by D. bifoms, but are strongly agglutinated by U. europaeus lectin (Fig. 2). The RBCs of newborn infants behave as the A2 phenotype, whereas those of adults express higher branching and exhibit are complex [36], the A, phenotype. The multicomponents of these antigens

" " "_ " "

"_""""""""" _".

""""__"""_ """"""_""

A2

BLOOD GROUP D. bifloms L. alpinurn 63 C. sessilifolius E 4 L. tetragonolobus Q U. europaeus

Figure 2 Comparison of the hemagglutinating activity (titration scores) of Dolichos biforus toward 0, A,, and A, adult human erythrocytes with those of wellknown anti-H/HI lectins 181,871. The sources of lectins are indicated under the figure.

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and l-8% of A2 individuals produce an alloantibody against A,. There are additional subtypes of A: A3, A,, and A m d , which exhibit mixed field agglutination (only selected cells are agglutinated among many unagglutinated ones) with anti-A or anti-A,B. Additional subtypes (e.g., A,,,, Ay, 4,) adsorb only the anti-A, without agglutination[92]. A special kind ofa weaker A has been reported incis AB phenotypes, found in individuals withthree A B 0 genes instead of two [92].This situation is due to an unequal crossover or a structural mutation leading to a simultaneous inheritanceof the A and B genes on one chromosome instead of the usualAB phenotype, inwhich each chromosome carries only one of the A and B alleles [92]. In certain diseases,there is an expression of acquired A-like antigens, which also may be detected by the anti-A lectins. An example isthe report on such a phenomenon in human cancer in which Forssman glycolipid was expressed inthe gastric and colonic mucosa[1521. Because the immunodominant sugar of A blood group is a-GalNAc (see Table l), most lectins that detect it are specific for this carbohydrate. As described before, some of these lectins may also react with additional substances containing GalNAc, such as the Forssman antigen [l531 (see Table l), the Cad and Tn antigens [11,83],and even with antigens deterand 1 B [l 1,49,120] antimined by terminal a-Gal, including the N [l 1,76 gens. The anti-A specificity of lectinswas one of the first to be recognized, by Boyd in extracts from Phaseolus limensis and P. lunatus [2].Since that time many other anti-A lectins have been described in plantsand in other organisms [l 1,49,153-1621,including the plant lectins ofDolichos biflorus [4,161],Moluccella laevis [12,76],Crotalaria striata (anti-A, antiB) [49, 1601, Griffonia simplicifolia GS-I (4)[12,24],Phlomis fruticosa (anti-A, antiB) [1201, Vicia cracca[121,Hyptis suaveolens [ 1611, Viciaperegrina,V. villosa, .V. americana, Amphicarpaeabracteata, A . edgeworthy, Crotalaria aegyptiaca, C. vitellina,Lathyrus sylvestrus [103];and the animal lectins of the snails Helixpomatia [12,156-1581,H. hortensis [12],H. aspersa [12], H. leucorum, Caucasotachea atrolabiata, Otala lactea, Eobania vermiculata, Cepaea nemoralis, Euhadra periomphala, E. callizona [1031,and of the butterclam Saxidomus giganteus[103,161]. The anti-A lectins prepared from the albumin gland and ova of the snails Helixpomatia, H. hortensis, and H. aspersa [156-1581are also used extensively, especially in automated techniques in blood banks in Europe and the United Kingdom. The most widely used lectin in blood grouping laboratories is from D. biflorus [4,11,35,37,163,164]. This lectin was dein seeds of the horse gram [4,35].Undiluted scribed by Bird in 1951 [4] extracts react as anti-A, but when diluted, they react as a potent anti-A, [l1,351. Anti-A,-specific agglutininwas also described inthe butter clam S. giganteus [159].Human anti-A, antibody is difficult to prepare and often


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is a poor reagent; therefore, the foregoing described anti-A, lectins are highly valuable. The H. pomutiu lectin (HPA) consists of six subunits, each containing one carbohydrate-binding site [156-1581, whereas the D. biforus lectin @BA) consists of four subunits. Although both lectins have combining sites specific for terminal nonreducing a-linked GalNAc [156-158,1631, theirspecificities are considered to beoverlapping, but not identical [18,153,165]. The HPA lectin exhibits a broader carbohydrate range than does DBA [1651. Use of labeled lectins also allowsthe determination of the number of lectin molecules bound on one cell surface. Sung et al. [l651 showed that maximal numbers ofHPA and DBA molecules bound to human genotype A 0 RBCs were 3.8 x lo5 and 2.7 X lo5 per RBC (the number of HPA receptor sites on AIB cellswas 3 X 105-5 x lo5 sites per RBC), respectively. The binding of one type of lectin may influence the binding of another type: HPA was found to inhibit the binding of DBA, but not vice versa. Sialidase treatment of RBCs resulted in increased binding of both HPA and DBA, but through different mechanisms. An equal number (7.6 x Id) of new HPA sites were generated on genotypesA 0 and 00 RBCs by this treatment. These new sites accounted for the enhancement (A0 cells) and appearance (00 cells) of hemagglutinability of HPA. Similar treatment did not generate newDBA sites, but increased DBA affinity for the existing receptors. As a result, genotype A 0 cells increased their hemagglutinability by DBA, whereas00 cells remained unagglutinable. Sung et al. [l651 reported that the binding of the lectin of Limulus polyphemus (LPA), with specificity for N-acetylneuraminic acid, had no effect on the binding of HPA to the RBC (indicating no steric hindrance for this pair of lectins). The binding of DBA was enhanced by this lectin, possibly owingto a reduction of the density ofthe negative electrical charge of the N-acetylneuraminic acid inthe vicinity of the DBA receptors (facilitating the binding of the negatively charged DBA moleculesas a result of a decreased electrostatic repulsion).In addition to A,, DBA may also react weakly withAIB cells in those cases of “strong B” when the B gene product expression is very high and competes with the expressionthe ofA. Recently, it was shown that medium-sizedglycosphingolipids are not involvedin DBA hemagglutination [82]. F. The Simultaneous Use of Anti-H and Anti-A Lectins for the Detection of A Blood Subgroups

As described in the foregoing, the distinction of A, or AIB RBCs from A,

or A,B, or weaker subgroups,is an important advantage of DBA. Although the use of this lectin alone (stronger agglutination ofAI than of A2 cells) is

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sufficient, complementary tests with one of the anti-H lectins (see Fig. 2, stronger reaction with Az or AzB cells)done in parallel confirmthe results. Anti A + H specificity was described in several algae (e.g., Lyngbya majuscula and Ulva arasakii)[1621.

G. Anti-B Lectins The B genecodes for an cm-galactosyltransferase which transfers Dgalactose from UDP-galactose to the H-antigen. B antigens of H, and Hz structures have been isolated[92]. Subgroups of B are very rare [141]. The subgroups B, (reacts in mixed field pattern with anti-B reagent), B, (weak agglutination), and B, (unagglutinated by anti-B or anti-A,B, but adsorbs anti-B) are examples [92]. Weaker B may also found be in the unusual AB phenotypes that exhibit a cis-AB genetic unit, formed as previously described [92]. Sera of these individuals may contain a weak anti-Bthat reacts with all ordinary B-cells, but not cis-AB. Therefore, it was suggested that their B antigen is only a segment ofthe normal B antigen. The appearance of an acquired B-like antigen on erythrocytes [58,1661671 wasdescribed in A individuals suffering from certain alimentarytract microbial infections. It may be caused by enzymes from some strains of Escherichia coli, Clostridium tertium, or Proteus vulgaris. These enzymes deacetylate the terminal and immunodominant a-GalNAc of the blood group Aantigen,resultingin the appearance of terminal cy-D-galactosamine.This product issimilar to a-galactose, the immunodominant sugar of blood group B antigen. The cells that bear this new antigen are agglutinated by antiB reagents and by the patient’s own plasma which contains antiB antibodies (the patient is A). The result is polyagglutination because the transformed cells are also agglutinated by plasma of all the potential blooddonors (of A or even 0 blood groups). This appearance of new B antigen is restricted to RBCs carrying an A antigen. According to Judd [167], only 5% of A, individuals may develop acquiredB antigen. Enzymatic reacetylation of the acquired B-like antigen on the affected A cells resulted in disappearance of the acquired B antigenand reappearance of A reactivity withDolichos biflorus lectin [1681. Another type of acquired B antigenis associated with passiveadsorption of bacterial B-like antigen during intestinal infections. This type, which is referred to as “passengers” variety of acquired-B [169], may occur in both group A or 0 subjects from infections with P. vulgaris or E. coli Ow [170]. Both forms of the acquired-B antigens described are transient and disappear following elimination of the microbial infection. There is no good anti-B lectin in widespread use in the blood banks because most of the seed extracts reported either have apoor activity and


351

cross-react withgroup A cells, or are unstable. Anti-B has been reported in Caragna frutex [103], Aristolachia galeata [161], Griffonia simplicifolia [22-24,1611; in the fungi, Fomes fomentariusand Marasmius oreades[161]; in the algaePtilota plumosa [162,1711, Bryopsis hypnoides, and Laurencia undulata [162]; eggs of the fishes [84,161,172]: Clupeas harengus (herring) [161], Plecoglossus altivelis, ayu fish [173], and the Salmonidae [84,161, 172,1741, including the salmon Salmo solar (the salmon anti-B was also [174]). S. evaluated as an anti-Breagentinautomatedbloodgrouping irideus, and the trout S. trutta [160], in female gonads of the perch Perca fluviatilis [161]; in the snails, Iphygenaplicatula, Leciniaria biplicata, and Balea perversa [103]; in the hemolymph of the crab Charybdis japonica [175], and in the culture medium ofthe bacterium Streptomyces sp. [74,75]. In addition to these antiB lectins, there are the lectins that react with the anti-B both A and B (but not 0)RBCs. An interesting point concerning specificity of the Plecoglossus altivelis (ayu fish) egg lectin and that of the bacterium Streptomyces sp. is that L-rhamnose (and its oxy-derivative L-mannose) inhibitedboth much more strongly than D-galactose, which is the B immunodominant sugar. The lectin of Sophora japonica[176], which nicely discriminates between B and 0 cells, also reacts with A cells (although not so strongly as with B) and exhibits anti-I activity[85]. Attempts to make an effective anti-B from extracts of Griffonia simplicifolia have been reported by Judd [12,21] and by Murphy and Goldstein [23]. They A cprevent the reactions used GS-Iand added a small quantity of D - G ~ ~ N to with A RBCs. This treatment hasnot produced a good antiB reagent when fresh extractswere used, becausethis lectin reacts withboth A and B RBCs; however, aged preparations could be used as anti-B. Additional anti-A and anti-Blectinswerealsodescribed,including: Calpurinaaurea,Caragna frutex, Coronilla varia, Crotalaria brevifolia, C. striata, C. usraemonesis, Phlomis fruticosa, P. samia, and P. viscosa. Anti-B1 and anti-HI activity was also described inthe ova of the bream Spondyliosoma cantharus[88]. VIII. LECTINAPPLICATION FOR THEDETECTION AND STUDYOF li ANTIGENS

The I antigen was first describedbyWiener et al. in 1956[177]. They used nonspecific cold agglutininsand showed that the erythrocytes of five individuals were not agglutinatedby them (theI designated the individuality are encounof the five among22,000 blood donors). Cold anti-I agglutinins tered in serafrom healthy subjectsand in sera from patients suffering from acquired hemolytic anemia. Increased titers and thermal rangeof autoanti-1 has long been associated with Mycoplasma pneumoniae infections [178, 1791. Anti-i antibodieswere also describedby Marsh and Jenkins[180,18l],

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who have also madethe important observation of I deficiency in cord and newborn RBCs, which are rich in i [181].They have further shown that during the first 18 months of life, the i antigen is gradually lost as I is gained reciprocally. Soon after it was found that there is a great serological diversity in I antigens and anti-I sera. The association between the Ii antigens and the AB0 system was pointed out by Tippett [182],who in 1960 had already noted that the presence of AB0 antigens affected the interactions of the RBCs with anti-I and described an antibody that reacted preferentially with AI cells. The serum containing that antibody failed to react with adult Ai or 0 1 cells and was not inhibited by A secretor saliva. She rightly suggested that I synthesis might be a leadingstep in the formation of AB0 antigens [182].In 1964, a serum with the same specificity was reported by Gold [l831 and, later on, anti-IB, -IH, -iH, -ILeb, and even anti-IP, have been described [180],leading to the proposal of mosaic structure of Ii blood groups and to the understanding that these antigens are intercalated with the ABH, Lewis, and P, antigens. In 1990, the International Society for Blood Transfusion (ISBT) Working Party on Terminology for Red Cell Surface Antigens designated Ii as a blood group collection and has given it The biochemical relation the number 207: I is 207-001and i 207-002 [184]. between these two antigens is based on Marsh’s observation [l811 that all RBCs possessboth I and i in converse ratio: normal adult cells are generally rich in I, but very poor in i, whereas cord cells and rare adult cells of i phenotype possessabundant i receptors. It is clear that i is the precursor of I, and that Ii antigens are internal precursor structures in the biosynthetic pathways leading to the ABH determinants [38,141,185]and are integral components of the RBC membrane. Theyare expressed on both glycoproteins and glycolipids [186,1871.Anstee [ l861 estimated the total number of potential Ii sites (accordingto ABH) exceeded 2 million per red cell (75% carried on the glycoproteins band 3 and 4.5, in 2 : 1 ratio, and 25% on glycolipids [1801). The Ii activity resides in the core structures of type 2 chains Hz, HS, and H4,and is directly relatedto the presence of apoly-N-acetyllactosamine molecule in linear (i) and branched (I) chains.The i activity is defined by a nonbranching core structure, containing at least two repeating LacNAc units (found in HZ,but not in HI, which contains only one LacNAcunit). Thebranchedcorestructures,which are probablyformed by a 1 6 LacNAc-branching glycosylaminyltransferase [1091,are reactive with anti-I sera. These structures may be further glycosylated to form the H antigen by fucosylation (and afterward also A and B), or they may remain either exposed or be sialylated. Fucosylation of the core structure suppresses Ii activities, which are stronger in the unsubstituted structures(e.g., in Bom-


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bay-type H-negative cells [141]). AI transferases can convert linear (i-like) and branched (I-like)H structures to Al, whereas A2 transferases efficiently convert only linear chains (i-like) to A [36,104,141]. Yokoyama [l881 reported that bloodgroupsubstances,includinggangliosides that inhibit anti-A and anti-B, also inhibited anti-I. The I antigen is considered a public antigen, absent onlyin 1 : 10,OOO in the white adult population [186]. Very rare I -i-phenotypes have also been described [189]. They have in their sera alloanti-I of low titers (not suitable as a reagent). Anti-i antibodies may occur in hemopathies. Alteration of the I receptor in vitro was described by Schmidt etaal. in system containing mycoplasmal[96] or clostridial filtrates. These microbial effects were greater on group A than on group 0 cells [96]. Changes inI in leukemia have also been reportedand are related to a simultaneous lossof A and I [141,190]. The depressed I reactivities on this, other hemopathies, and additional malignant diseasesare generally associated with increasedi reactivity. In the same diseases, enhancedI without changes in i have also been reported. Sialidase or protease treatment unmasks I and i determinants on 01 and Oi RBCs and removes steric hindrance, enablinga better accessibility to the anti-I reagents [191]. Although there is an association between M. pneumoniae infections and anti-I cold agglutinins,the interaction of this microorganism with human erythrocytes is mediated by sialic acid linked (012-3)to the long backbone carbohydrate chains of buried Ii antigens on both glycoproteins (bands 3 and 4.5) and glycolipids (gangliosides) [62]. Inhibition studies showed that M. pneumoniae, in its adhesive specificity, may not distinguish between the branched carbohydrate backbones ofthe I type and the linear structuresof the i type [62]. There is, generally, a shortage of anti-I antibodies. Anti-I antibodies, which are mostly the cold autoantibodiesthat are observed in chronic cold hemagglutinindisease [177], are heterologousandrecognize the whole and branched structure or only the pl,6 branch. Some sera exhibitant", anti-IH specificities [141]. Such sera recognize branched structures in which it is thought that one of the branches has beenfurther modified bytransferases to P,or H, respectively (as described previously). There is alsoa shortage of pure anti-I lectins. Thereare certain lectins that also react as anti-HI [81,115] or anti-B, anti-A, or anti-I [85,89,90] and even PI. Bird [1151, as well as Voakand Lodge [81], described anti-HI or HI/H activity in several seeds previously consideredto be only anti-H [81]. They showedthat the lectins of Lotus tetragonolobus, Cytisus sessilifolius, and Laburnum alpinum gave lower titration scores with OHii cells than with OH1 cells. Tippett [l821 described anti-AI and Chien et al. [85] described anti-B(A)I activity ofthe Sophora japonica lectin. Sudakevitz et al. [87] have recently shown that the lectin of Erythrina corallodendron

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alsoexhibits an HI preference.Thislectin,despiteitsspecificity for LacNAc, which is most closely related to the I and i terminal carbohydrate structures, behaves asan anti-H reagent and agglutinatesOh1 cellsnot only more weakly than O(H)I (as opposed to classic anti-I) but also weaker than O(H)i. The galactophilic lectin PA-I ofPseudomonas aeruginosa has recentlybeenshown to agglutinateI-positiveerythrocytesconsiderably more stronglythan i cells, and O(h) Bombay erythrocytes slightly stronger than Om) [66]. A lectindisplayinganti-ispecificity wasdescribedby Moore in seeds of Pterocarpus angolensis [191]. This lectin was found to resemble human anti-i serum in stronger agglutination of cord erythrocytes of A, B, AB, and 0 groups compared withadult erythrocytes of the same types. Its carbohydrate specificity is Man> Glc = GlcNAc. IX. LECTIN APPLICATION FOR THE DETECTION A N D STUDY OF P,, P, P‘, and LUKE ANTIGENS

The P blood group system was discovered by Landsteiner and Levine in 1927 [l931 in the course of a systematic attempt to identify new alloantigens by immunization ofrabbits with human erythrocytes. The new antigen was named P [194], but later it was changed to PI (now designated bythe code number 003001 in the numerical nomenclature recently adopted by the P was found ISBT [184,195], when a secondantigen, nowknownas [186,196]. Later on, a third, rare antigen, P’, was also described[197-1991. Additional two genotypes related to this group of antigensare p and Luke [195,200]. P,Pk,and Luke have a globoside structure and, therefore, they have been assignedto the globoside collectionof antigens number209, and given the ISBT numbers 209001, 209002, and 209003, respectively [184, 1951. The P system antigensare integral componentsof the RBC membrane [45,46,104,194]. They were also found on skin fibroblasts and B lymphocytes, granulocytes, blood platelets, and other cells [200]. On these cells the P’ antigen is not rare [194]. Characteristically, the P system antigens are determined by well-defined oligosaccharide sequences linked to glycosphingolipids. The sequence and steric attachment of the carbohydrate molecules are brought about by gene-specified glycosyltransferases. The PI antigen (its gene is assigned to chromosome 22) is widespread in distribution [195,200] (Table 5), not only in human beings but also in other organisms (e.g., in pigeons and in the fluid of hydatid cysts isolated from patients with Echinococcus granulosus[200-2021). The P antigen (its gene is in chromosome 6) is of somewhat lower distribution (see Table5), and the Pkphenotype israre [203]. The structure of these antigens is known from the studies of Morgan and Watkins [202], who defined the trisaccharide structure inhibiting antiP, by using P, substance from hydatid cyst


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Table 5 The Phenotypes of the P System, the P Antigen Combinations that Determine Them, and Their Frequencies

Phenotype P1 P2 P,Pk

P system antigens detected"

PI, p, (pk?) P, (pk?)

hpk)

p,, p' Pk

P

None

Frequency (Olo) 75 25 Very rare

Very rare Very rare

'(P'?), suggested asan intermediate to P, but generally not

found or detected at very low level by special techniques [194].

fluid for hydrolysis and carbohydrate analysis [201], and Voaketal. [84,199], who suggested that a galactose is also involved in the Pkspecificity, by use of the galactophilic lectins of salmon and trout. Naiki and Marcus [45,46] completed the structural studies by using glycosphingolipids of known structure to inhibit the anti-P,, anti-Pk, and P. As described in Table 1, the common basic precursor of these antigensis lactosylceramide (CDH = ceramide dihexoside). Fromthis basic precursor,there is a divergence to two series of antigens catalyzed by theappropriate glycosyltransferases: 1. The globoside series by the addition of galactose (in an a l , 4 linkage) to form the ceramide trihexoside (CTH) exhibiting Pkantigenicity and, then, addition of GalNAc (in01,3 linkage) to form the P antigen. 2. The paragloboside series-in which P,, the definitive antigen of the P system,and p are assigned. P, has a terminal a-Gal linked 1,4 to subterminal PGal, much like Pk.It differs from the latter in possessing intermediate LacNAc. It is formed by addition of galactose to the B1,4 linkage) and, then, another galactose (ina l , 4 terminal GlcNAc (in linkage). Inthe absence ofthe latter enzyme, the paragloboside may be sialylated by addition of NeuNActo the terminal galactose inan a(2,3) position and formation of sialosylparagloboside. Thelatter reaction is not considered to be related to the P system and the product is viewed as a p determinant [46,195]. Persons homozygous for the p gene have red cells that test negatively for PI, Pk,and P determinants, but show relative abundanceof the sialylated (a2,3) paragloboside. A p individual was first described by Levine et al. in 1951 [l961 as Tj(a-): T for tumor and j to represent the patient's name. Tj(a-) was renamed p

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when Sanger showed that this phenotype was related to the P system ~91. The Luke antigen, now designated 209003, was characterizedby antiLuke, described by Tippett et al. in 1965 [204] and was shown to be related to the P and AB0 systems. All p and P‘ red cells are negative with antiLuke antibody. Most P,-positive samplesare Luke positive, but some P,negative, P,-positive bloods are Luke-negative/Luke(w). The association with the AB0 is that the Luke- and Luke(w) phenotypes were more commonly found among Al than among A2, B, or 0 persons. Namely, the production of the AB0 system on the same RBC membrane glycosphingoliof the Luke antigen.As pids as theP system antigens affects the expression shown in Table 6 [41,205], the Luke antigen is probably identical with SSEA-4, which is derived from P antigen by both 0-galactosylation (1,3 linkage to the GalNAc), leadingto SSEA-3 structure, and then a sialylation (a2,3 linkage). Hence, thereare relationships betweenthe P system andthe stage-specific embryonic antigens SSEA-3 and SSEA-4 (see Table 6). The P and Luke antigensare well developed in cord blood, whereasP1 antigen appears in younger fetuses, then decreases in older fetuses and newborns (resemblingAI, which isnot fully developedat birth) [19]. The P systemisalsorelated to the Ii, Lutheran, and Aubergerantigens[19]. Aswasdiscussed for the ABH system (designated 110 in the numerical nomenclature adoptedby the ISBT [184]),the enzymes ofthe I system may also modify some ofthe P antigen-bearing oligosaccharides [l041to create larger branched structures. The determination of P‘ and p is clinically important because all the subjects of these types have “natural” antibodies anti-P, P,, and Pk (the latter only in p) that cause problems in blood transfusions, may cause abortions in the first trimester, and the surviving newborns may suffer from hemolytic disease [19,206]. Several lectins interact with the P system antigens [12]. Since, as described in Table l , two of these antigens contain terminal Galal-4Gal-R Table 6 The Relations Between the P‘, P, SSEA-3, and SSEA-4 Antigens

Antigen

Structure

P‘ CTH

Galal,4Gal/31,4Glcl,lCer

Globoside P

GalNAc/31,3Galal,4Galfll,4Glcl,lCer Gal/31,3GalNAc/31,3Gal a1,4Galfl1,4Glcl,lCer NeuNAca2,3GAl/31,3GalNAcfll,3Gal al,4Galgl,3Glc1,lCer

SSEA-3 SSEA4 Source: Refs. 41,205.


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(P'

residues and P,), and the third one (P) contains GalNAcb13Gal, galactophilic lectinsthat bind both galactose and GalNAc may react with all of B and A antigens, but may exhibit preferthem. Such lectins also react with ential affinity for one of them. This was the case with the antiB, P, P,, and P' activity ofthe salmonid roe extracts.The lectins ofthe salmonid roe [84,161,172] exhibit weakest reaction with A,pp cells. The P fimbriae of uropathogenic E. coli strains beara lectin or lectinlike adhesinthat exhibits a preferential affinity for P' antigen [59-611, and the PA-I lectin of P. aeruginosa exhibited the weakest agglutination with Opp and i cells [65,66], behaving as an antiB, P(P')I. With both the salmonid and the Pseudomona's lectins, B RBCswere strongly agglutinated, irrespective of their P phenotype [12,66], their anti-B and antiP, activitieswereshown to be inseparable, and their agglutination of B/P,/P'-bearing erythrocytes was abolished by the addition of galactose. The pseudomonal PA-I lectin combined specificityto B,P(P')I is also analogousto that of antibodies exhibiting anti-IP activities [207,208]. X. LECTINAPPLICATIONFORTHEDETECTIONAND STUDY OF MN, S W ) BLOOD GROUP ANTIGENS

The MN system was discoveredas the second bloodgroup system, 27 years after the AB0 system, by Landsteiner and Levine [193,209]. They distinguished, by rabbit antibodies produced against human erythrocytes, three distinct phenotypes:M + N - ,M +N + , and M -N + . In 1974, Walsh and Montgomery [210] described the S antibodyandantigen, and in 1951, Levineet al. [211] describedanti-s. By meansof the anti4 and anti-s antibodies, three phenotypes were identified: S +S - ,S + S + ,and S -S . The M and N antigens (which sharea common tetrasaccharide component [212]) were assigned to two codominant allelesand so also were the S and S antigens. Subsequently family studies demonstrated that the MN and Ss loci were closely linkedon the long arm of chromosome four [213,214], and that crossing over between them is a rare event [19,214]. However, there are many genetic variants of the major MNSs antigens. In 1953, Wiener etal. [215] described anti-U activitythat identified two phenotypes: U + and U - . Red blood cells ofthe U - phenotype have also been shown to be S -S - (such a phenotype has beenfound only rarely in black individuals), but not vice versa, since U+ may also be found in S - S - RBCs. The Ss and Uu loci were also reported to be apparently very close, since crossing over between themwas not reported [214]. The frequency of the four common haplotypes recognizedMS,MS, NS,and Ns is presented in Table 7. The MNSs genes are involved in the coding for highly homologous

+

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Table 7 Frequencies of the MNSs Phenotypes in the White Population

Phenotype M+N+S+s+ M+N+S-s+ M-N+S-s+ M+N-S+s+ M+N-S-s+ M+N-S+sM-N+S+s+ M+N+S+sM-N+S+sAll types of S-s-

Frequency (Vo) 24 22 15 14 8 6 6 4 1

Very rare

Source: Adapted from Ref. 214.

sialoglycoproteins (SGPs) 1214,216-2211, each gene involved in the coding for a distinct RBC membranal SGP, known as glycophorin A and glycophorin B (syn: a-MNSGP and MsSGP), respectively [216-2211. It was shown that the M and N genes code for the production of different polypeptide chains [216,219]. There are also MN hybrids, produced when crossing over occurs within the gene, rather than between genes, and newgenes (produced by an unequal crossing over and gene fusion) [214,221], as well as several low-frequency allelic genes [214,220,222] and an Mk operator gene have beenfound [223-2251 that affect both MNand Ss loci presenton the same chromosome, resulting in no production of MNSGPand SsSGP 12141. The MNSs antigens are already well developed in RBCs at birth and also appear on human lymphocytes [226] and erythropoietic cells, but not [227]. on human circulating platelets The MNSGPsare composed ofa protein backbone with 16 short oligosaccharide chains attached within the first 50 amino acids of the N H 2 terminus [228-2361. Fifteen of these chainsare in the form of a tetrasaccharide [212,232], as described byThomas and Winzler [232] (Fig. 3 and Table 8) or related compounds. These oligosaccharides are attached by alkalilabile linkagesto serine orthreonine residues present alongthe amino acid 11-15 sugar residues chain. The additional oligosaccharide chain consists of and is linked to asparagine at position 25 by an alkali-stable linkage. All the oligosaccharide chains bear NeuNAc residues, which are important constituents of the MN antigens. Sialidase treatment ofthe cells abolishesthe MN antigenicity and reveals the T cryptantigen [52,53,111,228-2341. As seen in Table 8, the resulting T antigen can be further converted to Tn by removal of galactose. The determinants of the M and N antigens recognized

359


F

antisen

Leu (NHz terminal) Ser

I I Tetraose-Thr I Tetraose-Thr I Tetraose-Ser

Glu

M antiaen

I I Thr-Tetraose I Thr-Tetraose I Ser-Tetraose

Figure 3 Comparison of the amino acids sequences ofM and N SGPs at positions 1-5 from the NH,-terminus: Leu, leucine;Thr, threonine; Ser, serine, Glu, glutamic acid; Gly, glycine; Tetraose, tetrasaccharide of Thomas and Winzler. (Adapted from Ref. 216.)

by the human andrabbit antisera are formed by interactions of some of the carbohydrates with a componentof the polypeptide chain. An interaction between the free terminal amino group with NeuNAc residue (at positions 2-4) is suggested. The MNSGPs in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the RBC membranal proteinsare stained by periodic acid-Schiff (PAS)stain and are noted asthe major PAS-l, and PAS-2 SGPs. Tomita and Marchesi [216] established the amino acid sequenceof the MN SGPmonomer(relativemolecularmassapproximately31,000) that bears60%(ofits total mass)carbohydrates(the SGPa makes up approximately 2-4%of the total protein mass of the RBC membrane). PAS-l is a dimer of PAS-2 which is glycophorin or A MNSGP. Its polypeptide backbone consists of 131amino acids in three domains: first, the external hydrophilic (1-72 from the NH,-terminus), then the hydrophobic segment (residues 73-95) that spans the lipid bilayer, and the third domain extending into the cytoplasm of the RBC. The Mand N antigensare defined Table 8 Structure of the Alkali-Labile Tetrasaccharideof Thomas and Winzler and Its Relation to T and Tn Cryptantigens

Antigen

Structure

Thomas & W i d e r : NeuNAc a2,6 GalNAc aSerine / Threonine NeuNAc a2,3 GalS1,3 T Tn Adapted from Refs. 52.53.212.219-221.

GalSl,3GalNAc aSerine / Threonine GalNAc aSerine / Threonine

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Levene et al.

by the first eight amino acidsand the carbohydrate side chainsattached to the amino acidsat positions 2, 3, and 4. These antigensdiffer in two amino acids at positions 1 and 5 from theNH,-terminus [216,219,221,228,235,236]. These differencesare coded bythe M and Ngenes. The M antigen SGP has serine at position 1 and glycine at 5, whereas the NSGP has leucine and glutamic acid, respectively[216,219,228] (see Fig. 3). Both M and N SGPs are sensitive to proteolytic enzymes. Trypsin removes the 1-39 fraction, whereas ficin removes the1-56 fraction. The S and S antigens are also defined by amino acid differences (inS there is methionine,whereasin S there is threonine at residue 29) and glycosylation at the region 25-29 of glycophorinB. Approximately 70,00080,OOO copiesofGP6 are presentinRBCmembranes,comparedwith 500,000-900,000 for GPa [217]. In S-s-URBCs, there is a 15% decrease inthe total membrane NeuNAc. Since glycophorin B has leucineand glutamic acid at positions 1 and 5 (from the NH2-terminus), respectively, duplicating the NH2-terminal sequence of 1-25 amino acids of NSGP, it also exhibits N, but no M, activity. This W’ is present, regardless of the MN type of the blood [214]. However, the asparagine residue at position 26 is not glycosylated in GP& The U antigen is located on the SsSGP at a site closerto the RBC membranethan the S or S and is presumably defined only by an amino acid sequence, independentof the carbohydrate chains. Compared with the MN antigens, the Ss antigens are considerably shorter and are not abolished by sialidase treatment. They and the N-like ‘N’ antigen also exhibit sensitivity to proteolytic cleavage by chymotrypsin and pronase [237], but not by trypsin (which cleavesthe M and N antigens of the MNSGP). The U antigen, which is the product of the U gene, is not affected by any of these enzymes; therefore, it was suggested that it is probably located closerto the RBC membrane surfaceon the SsSGP. The complete amino acid sequences of glycophorin B have also been established [238] and the cDNA clones ofboth glycophorins A and B were that sequenced [239-242]. They showeda very strong homology, indicating the two proteinsare coded by separate,but closely linked genes. Theexactfunctionof the MN and SsSGPsisnotyetclear [214], because, in contrast to glycophorins C and D (exhibiting the high incidence Gerbich antigenicity), their absence in En(a-) individuals (lacking glycophorin A [237,243,244], S -S -U - (lacking glycophorin B), and MkMk (who lack both SGPs [225]) individuals is not associated with any disease or RBC abnormality. However, in these RBCs, there is an increased glycosylation of other glycoproteins (especially band 3) as a compensation [243,244]. Dahr et al. suggested [ M ] that band 3 and glycophorin A form a complex during biosynthesisand that the presenceof glycophorin A in it hampers band3 glycosylation.


361

There are several microorganisms that use the SGPs as receptors for their adhesins that enable their binding to the target host cells. Some of them bindto sialic acid residues (e.g., influenza virus [245], encephalomyocarditis virus [246], and a certain strain of E. coli [247]). The malarial parasite Plasmodium falciparum [248] interacts with glycophorin A or B through theirO-glycans.Thisparasitefails to invade Tn erythrocytes, which lack both sialic acidand galactose [249,250]. Lectins that react with the SGPs also vary in their binding to different components [222,235]. Many plant lectins agglutinateMN cells, but only a few are commonly used. Among them, are those of Vicia graminea [235] and Maclura aurantiaca,which bind to the tetrasaccharides nearthe NH2terminal end of the M or N SGP, and those of Phaseolus vulgaris (as well as ricin and ConA) that bind to the longer N-linked oligosaccharide. The alkali-labile tetrasaccharides of GP6 chains also bind the lectins of Vicia and Maclura [222]. A.Anti-NLectins

The lectin of Vicia graminea agglutinates N + RBCs quite strongly. This was described by Ottensooser and Silberschmidt in 1953 [251]. Since then this lectin has become a very useful reagent for N detectionin most bloodgrouping laboratories [222,252-2551. In addition to Nantigenon the NSGP, it detects the 'N' on the SsSGP; therefore, it may differentiate S S - from S - S in M N - RBCs [221,222]. It is not considered a pure anti-N, because it agglutinates all typesof sialidase-treated cells and reacts with N-antigen precursor and T antigens, both on RBCs [255] and in cyst fluids of malignant ovarian clear cell carcinoma [256,257]. OtherVicia sp. exhibiting a similar anti-N activity includeV. leganaya, V. picta [222], and V. unijuga [258-2601. These are not routinely widely used. Additional seeds reportedto exhibit anti-N activity include several species of Bauhinia: B. candicans, B. variegata, B. bonatiana, and B. purpurea [12,222].Fletcher[261] reported that anti-Nspecificityin B. variegata could be obtainedafter addition of 0.1 M glucoseto the preparation. The lectins ofMoluccella laevis [222,262] and one component inG$fonia simplicifoliaextract [263] exhibit anti-A+N specificity. They may be B and 0 RBCs. used for N-antigen detection only in

+

+

+

B. Anti"Lectins

Anti" lectins have been found in three varieties Iberis of seeds: I. amara, I. umbellata, and I. semperivens [222,264]; in four varieties of Japanese radish seeds, and in turnip [222,264-2661. A blood group "specific hem-

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Levene et al.

agglutinin was also described in a pyelonephritogenic E. coli [267]. These lectins are not yet usedfor routine blood grouping. C. Anti-MN Lectins

The Maclura aurantiacalectin, madefrom Osage orange, appearsto detect the tetrasaccharide side chains attached to MNSGP or SsSGP [222,243,2571; however, it is unable to distinguish betweenan MSGP and NSGP. It is useful in screeningfor RBCs deficient in MNSGP and for those having variant forms of this molecule [222]. Variants that are characterized by soja ( m a ) and Sophora reduced NeuNAc levels may be detectedGlycine by japonica lectins [268,269]. The Phaseolus vulgaris lectin (PHA) bindsto the alkali-stable chains, found as a single copy (attached to the asparagine residue)and to band 3 glycoproteins 1222,2431. The Ricinuscommunis lectin exhibits a similar behavior [222]. The PHA does not bind to the SsSGP because it lacks the alkali-stableoligosaccharide[222].Anti-MNlectinshavealsobeendescribed in bacteria, and the latters' specificity is mainly for the sialic acid moiety [247]. D. Use of Lectins for the Detection of MN- and %Deficient En(a-) and M' Phenotypes

In certain rare phenotypes, either one or both of the two major SGPs is absent. The En(a-) cells, which lack the entire MNSGP (glycophorin A), were first described in 1969 by Darnborough et al. [270]. Such individuals produce a mixture of antibodies against different domainsthe of MNSGP, including anti-En"and anti-Wrb [222]. These antibodies agglutinate all human RBCs exceptthose of En(a-). By treatment of the RBCs with trypsin and papain 12711, it was possible to define the antigenic portions of the MNSGP that bind these different antibodies [l 1,272,2731. The antibodies do not agglutinate protease-treatedcells [271]. There are also rare phenotypes lacking the SsSGP. In S -S -U RBCs, found almost exclusively in black populations [219], there is a decreasein total membraneNeuNAc.TheypossessnormalMNSGP, but exhibit no detectable normal glycophorin B. The M' phenotype, described in 1964 by Metaxas 12231, is associated with no production of MN and SsSGPs [222-2241. The RBCs of homozygous MkMk[225] exhibit 30% NeuNAc levels and demonstrate no PAS-1, PAS-2, PAS-3, or component C bands[219]. In En(a-), even in the heterozygous state, and in Mk,the RBC band 3, which normally contains 5-20070of the total membrane carbohydrates, becomes more heavily glycosylated [243]. The increase isin attached galac-


363

tose and GlcNAc residues[249]. This compensatingalteration may function instead of the SGPs in enabling normal appearance and life span of the En(a-) and M’ erythrocytes, by minimizing their direct contacts with their environment [219]. The lectins of Vicia gramineaand Maclura aurantiaca agglutinate the En(a-) cells more weaklythan those withthe MNSGP [222,243]. They are also usefulfor the detection of M‘ RBCs [223,224]. The RBCs of heterozyV. graminea and M. aurantigous M kpersons show reduced reactivity with aca lectins, and those of the homozygotesfail to react withthe Vicialectin, Neither VicianorMaclura but give markedly reduced reactions Maclura. with agglutinate the cells from MkMk individuals following their treatment with trypsin [219]. The soybean lectin [274], which (asshown in Table 2) differentiates between untreated erythrocytes (rich in sialic acid) and those treated by sialidase or papain (poor in sialic acid) was useful for detection of En(a-) and U - cells, that are poor in sialic acid[275]. It even enables detection of red cells heterozygousfor En and M k [1l]. XI.

LECTINAPPLICATIONFORTHEDETECTION AND EXPLORATION OF CRYPTANTICENS AND POLYACCLUTINATION

PolyagglutinableRBCs are those that are agglutinated by a large percentage of ABO-compatible sera from normal healthy individuals, but not by their own sera or those from cord blood. This phenomenonwas first described in 1925[276-2781, and is called the Thomsen-Hubner-Friedenreich phenomenon or simply T-polyagglutinationafter Thomsen. The in vitro change was considered to be due to the action of bacterial glycosidases, which alter the RBC membrane by removing carbohydrate residues, thereby exposing cryptantigens. The first in vivo polyagglutination was described by Levine and Katzin in 1938 [279] in a child suffering from a pneumococcal infection. In vivo polyagglutination is a rare condition and can be caused when offending microorganisms are present in the bloodstream [280], but the same effect occurs if theorganisms are at an extravascular site, and theirsecreted enzymes gain access to the bloodstream. Passive coating ofRBCswith bacterial productsthat have T and Tn antigen structures[67,281] can rarely be confused with true polyagglutination, as these coatedRBCs can be agglutinated by specific antibacterial antibodies or naturally occurring polyagglutinins present inthe patient’s serum[282]. The first use of a lectin to recognize polyagglutinable RBCswas in 1964, when Bird discovered that T-polyagglutinable red cells were aggluti-

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nated by Arachis hypogaea[283]. This finding initiated the investigationof polyagglutinable red cells with many different lectin preparations. Of those tested, Glycine soja, Salvia sclarea,S. horminum, CS-11(Griffoniasimplicifolia), Dolichos biflorus, Vicia cretica, and Leonurus cardiaca have been of great value in their recognition and classification [284]. The use of lectins allows early recognition of cryptantigen exposure, but only if the RBCs become truly polyagglutinable in vivo can they combine withthe natural polyagglutinins present inthe patient's serumto cause [285-2871. mild to severe clinical disease There are two ways of classifying polyagglutination. One is based on the reactions of the RBCs with specific lectins (Table g), and the other is based on the following etiological factors:

I. Acquired Microbial A. 1. Action of enzymes causing exposure of cryptantigen sites on the RBC membrane. Types: T, Tk,acquired-B, Th, probably Tx,and VA. B. Nonmicrobial caused by somatic mutation (Tn). 11. Inherited HEMPAS, NOR,and Cad. A. T-Polyagglutination

The receptors for T-polyagglutination are cryptantigens that are masked on normal red cells. They are present on the alkali-labile tetrasaccharide Table 9 Classification of RBC Polyagglutination and Cryptantigens Using Specific Lectins

RBC polyagglutination type ~

~

~~

Lectins

T

Tk

Arachis hypogeae Glycinesoja

+

+

GS-ZZ"

vicia creticab Salvia sclarea Salvia horminum Leonurus cardiaca Dolichos b$'orusb

+ +

-

Th

Tx

Tn

-

+ +

Cad

+ -

'OS-11, Griffoniasimplic$olia. with RBCs that are not A, or A,B.

-

3-

+ +


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side chains of the RBC found on glycophorins A and B [216,229]. The T-polyagglutination of RBCs is caused by the action of sialidase, which specifically removes NeuNAc, exposing P-D-galactose. Clinically, T-cryptantigen exposureand T-polyagglutination have usuallybeenfoundinindividualswhohavehadvariousbacterialorviral infections that produce sialidase. It is typically transient and observed in patients with sepsis, wound infections, urinary tract and lung infections, as well as ulcerative or obstructive lesionsof the gastrointestinal tract. Some recorded cases have hemolytic anemia [285-2891, and a few rare cases have been published with disseminated intravascular coagulation (DIC), renal failure [287], and even death [290]. Hemolytic uremic syndrome(HUS) has been described after pneumococcal infections where T-polyagglutination has been detected, and some of these patients diedof the disease [291]. In those patients for whom transfusion is indicated, only washed, packed red cells should be infused, whereas fresh plasma and other products should be avoided, as they contain natural polyagglutinins [287-2881. The T-antigen receptor, when present in the correct steric configuration [292] is recognized by Arachis hypogaea [283], which recognizes 8linked D-galactose. Other lectins, including Glycinesoja [12], also recognize these polyagglutinable RBCs. B.

Tk Polyagglutination

In 1972, Tk polyagglutination was described by Bird and Wingham [293]. The following organisms have been found to cause thisform of polyagglutination: Bacteroides fragilis, Serratiamarcescens,Aspergillusniger, and Candida albicans[ 1671. All ofthese secrete endo-and exo-$galactosidases [294,295] that can exposethe Tk cryptantigen. No clinical hemolysis has been described in patients with Tk polyagglutination [167,2931. Tk RBCs have been detected in patients with a variety of infections, rangingfrom ulcerating or obstructive lesions the of gastrointestinal tract to septicemia. The Tk-transformed RBCs are agglutinated by naturally occurring anti-Tk antibodies. These RBCs are also agglutinated byGriffoniasimplicifolia (GS-11) [21] and Arachis hypogaea lectins, but not by Glycine soja. Judd et al. [21] have suggested that GlcNAc is probably the immunodominant sugar in Tk specificity. This view is supported by their observation that the GS-I1 lectin is inhibitedby this saccharide, but notby GalNAc or D-galactose. C. Th Polyagglutination

Th polyagglutinationwas first identified in vivo by Bird al. et in 1978 [296] in a patient with peritonitis. These investigators suggested that the exposed

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Levene et al.

Th cryptantigen could have been the result of microbial enzyme action. Since then, several additional cases have been described withTh cryptantigen exposure [284,297-300; Levene C, unpublished results]. Bird reported ten cases, one of which was associated with a weak Tk, and another with Tn. Some cases have had a persistent Th cryptantigen exposure. Bird recorded Th on the red cells of a healthydonor for 12 months, and Okubu et al. [301] reported Th on the cells of a patient, with idiopathic thrombocytopenic purpura, that lasted for 3 years. Levene [unpublished results] has seen three patients in the course of routine blood-grouping examinations. In one patient,the Th lasted for 1 month, in another 2 years,and in athird for at least 3 years. There were no clinical symptoms in any of the latter three cases. Th polyagglutination was recentlyfound in a patient who had peritonitis from a rupture of his colon [302]. This Th polyagglutination probably caused severe intravenous hemolysis, which contributed to his death. Th polyagglutination in vitro was first described by Levene [299] when an enzyme producedby Corynebacterium aquaticum caused normal RBCs to be Th-transformed.The results of a study on the characterizationof this enzyme showed it to be a sialidase [303]. These findings suggestedthat Th transformation ofredcellsexpresses an earlystage of, oraweakened expression.of,T transformation of RBCs. The author's explanation of the reactions of Th cells with the lectins Arachis hypogaea and GQcine soja was that sialidase treatment of RBCs first demasks galactosyl residues. The demasking of these galactosyl residues would explain why A. hypogaea, known to be specific for terminal galactoside residues, reacts with these G. soja, whichpreferentiallyrecognizes cellsbeforetheirreactionwith disaccharide sequences (Gal-GalNAc)that appear slightly later in the process of hydrolysis. Although the results pointedto the changes being dueto a sialidase, and maybe a quantitative action, it could not be excluded that this was not a "variant type" of sialidase. Bird [personal communication] has suggested that Th could be due to the removal of c~-2,6NeuNAc from alkali-labile tetrasaccharide side chains of the RBC glycoproteins. Th cells are agglutinated by A . hypogaea and V. cretica [304], but not by G. soja nor by GS-11. D. Tx Polyagglutination

In 1982, a new form of cryptantigen exposure, Tx,was recognized by Bird et al. [305] on the RBCsof children who had pneumococcal infections. Tx-converting enzymes have been found invitro in supernatants of pneumococcal cultures byboth Bird et al. [305]and also by Levene [unpublished results].


367

In addition to the cases of Tx cryptantigen exposure by Bird in 1982 [305], five cases have been observed in Israel. Three of these were found in one family [306]. The propositis had fever and acute hemolysis, whereas the other two siblings had no clinical symptoms. The Tx transformation on their RBCs wastemporary and lasted 4 to 5 months. The finding of another two patientswith Tx cryptantigenon their RBCs was coincidental, with no clinical significance [Levene C, unpublished results]. Tx RBC wereagglutinated by A. hypogaeae, but not by G.soja, GS-11, Salvia sclarea, or V. cretica. The use of V. cretica to distinguish Tx from Th is important, but the AB0 groups of the RBCs must be known, as this lectin also reacts with group A, RBCs [307]. E. Acquired-BPolyagglutination

Acquired-B polyagglutination is caused by the action of microbial enzymes from strains of E. coli, C. tertium, and P. vulgaris on RBCs of group A. These enzymes cause deacetylation of a-D-GalNAc. Acquired-B has been observed in patients with bowel disorders, septicemia, and gangrene, and has also been observed in healthy individuals. In none of the cases has there beenany association with anemiaor problems relatingto transfusion. Such transformed RBCs will not be typed correctly with the routine AB0 reagents. This subject has been extensively reviewed by Garratty et al. [l661 and Gerbal et al.[308]. F. VA Polyagglutination

A rare condition, VA polyagglutination (found in Vienna),was originally seen in a patient who had chronic hemolytic anemia, and his RBCs were weakly agglutinated by most normal human sera[309]. The distinguishing feature of this form of polyagglutination from T, Tn, and Tk RBCs was that there was no reaction with the lectins of D. biforus or A. hypogaea. The VARBCs had a reduced H antigen and, in this first case, showed a stippled appearance with H. pomatia by immunofluorescence. G. Passive AcquiredPolyagglutination

Acquired antigens may also result from passive adsorption of bacterial antigens onto RBCs. The lipopolysaccharides from many genera of bacteria including Escherichia, Klebsiella, Proteus, and Salmonella have A, B, H, T, and Tn-like antigenic structures[67,281]. When these bacterial antigens are adsorbed onto RBCs, they can cause the coated RBCs to be agglutinated by lectins causing incorrect results, because they recognize them as though they are truly polyagglutinable.

Levene et al.

368

H. TnPolyagglutination

Tn is an acquired nonmicrobial form of polyagglutination caused by a somatic mutation of a faulty hemopoietic stem cell clone that was first described by Moreau et al. in 1957 [310]. The Tn cryptantigen was shown by Dahr et al. [249] to be a part of the alkali-labile tetrasaccharide of Thomas and Winzler [232]. Springer et al. [231] producedthe Tn structure by treating T-activatedRBCs with 8-D-galactosidase. Cartron et al. [31l] demonstrated that Tn RBCs have a deficiency of 3-8-galactosyltransferase (T-transferase); therefore, the transfer of galactose to the C-3 position of GalNAc is blocked, and thus the GalNAc is exposed as the immunodominant structure (see Table 8). Tn RBCs have A-like properties, whichare probably dueto this terminal GalNAc[311]. Clinically, most of the cases of Tn have been associated with a mild anemia, leukopenia,and thrombocytopenia. Two populationsof RBCs are present in the patient’s blood, one of normalRBCs and the other from the faulty stem cell clone [312,313]. Once this form of polyagglutination appears, it is usually permanent.It has been described in patients with myelofibrosis [314] and in patients who subsequently developed leukemia[315]. A few reports of transient Tn exposure in infants have appeared[316-3 181. These infants had a transient deficiency of T-transferase. Tn polyagglutination has also been detected on the RBCs of some healthy blood donors [314,319] and, according to Ness [320], they should be carefully followed. Tn RBCs react withthe lectins ofSalvia sclaria,S. horminum, and G.soja, but not with A. hypogaea (see Table 9). 1.

Inherited HEMPAS, NOR, and Cad polyagglutination

7. HEMPAS

The acronym HEMPAS stands for hereditary erythroblastic multinuclearity with a positive acidified serum and test,is also known as type I1 congenital dyserythropoietic anemia (CDA11). It is a rare condition, and the red cells from these patients are agglutinated by most ABO-compatible seraat room temperature, and by one of three sera when they are acidified. The reactive antibody, anti-HEMPAS, is an IgM antibody that fixes complement [321]. The sialic acid level of HEMPAS cells is saidto be lower than normal, the i-antigen is present, and the H-antigen is depressed [322]. The lectin of H. pomatia has been reportedto react weakly with these cells,but there are no specific anti-HEMPAS lectins. 2. NOR Hams et fiist described the NOR (found in Norfolk, Virginia)form of inherited polyagglutination in 1979 [323]on the RBCs of a patient andfour other family members.The polyagglutinable red cells were agglutinated by

al.


369

75% of normal adult sera, but not with the serum from cord blood. These cells were not agglutinated by any of the lectins routinely used to identify polyagglutinable red cells. 3. Cad

Polyagglutination causedby the Cad receptorwas first described by Cazal et al. in 1968 [324]. It was considered to be an inherited Mendelian dominant, low-incidence antigen. The Cad antigen is variably expressedon the RBCs of unrelated persons, and graded from 1 to 3 with the use of the lectin D. biforus. It is knownthat Cad and Sd" have a similar immunodominant sugar [325], and lectins complexing with GalNAc combine with them. XII.

USE OF LECTINSTO SEPARATE MIXED RED CELL POPULATIONS

Rare bloods contain two or more red cell populations. These may be chimeras or mosaics [326]. The first can be found inthe blood of newborns who had a twin witha different blood group. Mosaic populations may betodue somatic mutation(e.g., Tn), transfusion, or bone marrowtransplantation. They are demonstrated by mixed-field agglutination when typed with routine blood group reagents. Diagnosis of these conditions and separation of the two RBC populations often poses a difficult problem [ll]. It is often necessary to separate the two populations to examine the blood groups or various additional genetic markers. Examples of mixtures are A,/O and Tnhormal RBCs found in twin chimera and mosaic, respectively. For the RBC separations in these specific cases, lectins are superior to antibodies, since the disaggregation of the sedimented agglutinatedRBCs is much easier using the specific sugar. Dolichos biforus was useful for separation of A, from 0 cells [327], whereas SBA [328] was efficient in separating Tn from normal cells. Solutions of a-D-GalNAc were usedfor the disaggregation in both instances. XIII.

USE OF A LECTIN FOR THE RECOGNITION OF IMMUNOGLOBULIN IN BLOOD PLASMA

The lectin from the seeds of the jackfruit, Artocarpus heterophyllus (integrifolia), was first described in 1955 by Bird [329]. This lectinwas named jacalin by Roque-Barreira and Campos-Net0 in 1985 [330]. It precipitates IgA (IgA, > IgAa and IgD, but not IgG and IgM [330-3321. The significance of this finding in relation to transfusion is that it will facilitate the recognition of patients and donors who are IgA-deficient.These IgAdeficient patients can develop anti-IgA from either transfusion or preg-

370

nancy, and if they are transfused with blood products they can develop severe anaphylaxis [333].

Levene et al.

that contain IgA

XIV. USE O F LECTINS IN BLOOD AUTOMATEDGROUPING MACHINES

The lectins ofHelrkpomatia [1541, Dolichos bifoms [4], salmon roe[1741, and Glycine soja [272], all have been used in automated blood-grouping [121]. With each lectin and every batch,the optimal dilution must be chosen after testing with control cells for the specific blood group antigen to be detected. A. Helix pomatia lectin

The H. pomatia lectin, which strongly agglutinates group A RBCs, also cross-reacts withgroup B, and reacts strongly with T-activatedRBCs of all AB0 groups. The lectin dilutionfor machine use must be chosen so that it will react stronger with A, and A2 cells and weaker with A, and A, cells. The cross-reaction withB cells is easily dilutedout, but care must betaken in interpreting the results with AB0 groups other than A or AB, as Ttransformed RBCs will also be agglutinated. B. Dolichosbiflorus lectin

In many centers, this lectin is used for the distinction of AI and A2 cells. When it is routinely used in blood-grouping machines, it has also allowed the detection of Tn and Cad1 RBCs indonors who are not group A. C. Salmon Roe lectin

The useof salmon roe lectin asan antiB reagent was examined by Downie et al. in 1977 [174]. D. Glycine soja lectin

Machine use ofthis lectin is not usual, but, if introduced, it can allow the recognition of some polyagglutinable RBCs and RBCs with an abnormal membrane.Hodson [334] screenedbloodsamples from 24,736 random blood donors in the United Kingdom with an autoanalyzer and found 22 positively reacting RBCsthat included heterozygotesfor En, MiV, and Mk. W . USE OF LECTINS IN THE PREPARATION O F BONE MARROWFOR TRANSPLANTATION

The findingthat the soybean lectin, amongother lectins, specifically bound and agglutinated subpopulations of murine lymphocytes suggested that it could be used to separate peripheral T-cells [335]. In 1976, Reisner et al.


371

[336] separated murine Tand B lymphocytes usingSBA and, in 1978 [337], (GVHD) they were able to remove the cells causing graft-versus-host disease from murine marrow. Later, they showed that SBA agglutinated about of the pluripotential stem 80% of human bone marrow cells, leaving most cells(colony-formingcells)in the SBA- fraction [338]. Thisfraction, which wasalmost depleted of T-cell alloreactivity, couldfurther be depleted of T-cells by rosettingwith sheep red blood cells [339]. Reisner et al., in 1981 [340], also reportedthe first in vivo use of these procedures with humans. They fractionatedthe marrow of a paternaldonor prepared for his child who had acute leukemia. The marrow was successfully transplanted without GVHD. In 1984, Reisneret al. [341] summarized the work in this field and concluded that by using transplants of lectinGVHD waspreseparatedE-rosette-depletedbonemarrowspecimens, vented. To kill neoplastic cells in marrowsto be usedfor autologous transplantation, highly specific monoclonal antibodies have been coupled to A chains of ricin. Krolick et al., in 1980 [342], employed this technique with monoclonal antibodies against murine B lymphocytes, and other workers used monoclonal antibodies against other leukemic antigens, which were also coupled to ricin [343-3451. These studies suggested that such techniques could be used to remove tumor cells from autologous marrowtransplants. In 1987,Morecki et al. [346]showed that SBA couldbeused for effective purgingof a breast cancercell line. They showedthat a depletion (of three to four orders of magnitude) ofthe tumor cells could be achieved without impairing stem cell activity. This was an important observation in relation to autologous bone marrow transplants given to patients with advanced breast cancer. This method has been used in Jerusalem with autologous marrowtransplants infused to patients with advanced breast cancer, with some success [Slavin, personal communication]. Springer et al. [15,347,348] have shown that carcinoma cells, especially those of the breast, have exposed T and Tn cryptantigens, and these cells are agglutinated by SBA. X V I . USE OF LECTINS FOR THE EXPLORATION

AND DIAGNOSISO F DISEASES ASSOCIATED WITH BLOOD GROUPS

Lectins have been used to detect clinical conditions in which there have been changes in the RBC membrane [284,349]. Most are related to the exposure of cryptantigens, either because of the action of enzymes secreted by bacteria or viruses, or because of a nonmicrobial somatic mutation such as Tn. The Tn cryptantigen has been found onthe RBCs of some cases of

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Levene et al.

leukemia [3 15,3201, and T and Tn cryptantigens are known to be present on tissue from carcinomaof the breast and other carcinomas [347,350]. The examinationof RBCs with lectins for the detectionof cryptantigen exposure or polyagglutination should be made in a patient with severe infection with unexplained anemia, with or without hemolysis [285-2891, who may also have leukopeniaor thrombocytopenia [349]. Hemolytic uremic syndrome (HUS) should be suspected in cases in whom there is renal dysfunction in addition to the foregoing [284], as microbial enzymes can expose T-receptorson renal glomerular cells [291]. Tn has been found on the RBCs of a patient with myelofibrosis [314] and some patients with leukemia [315,320], and, because Tn has beenfound on the RBCs of some normal blood donors [315,319], Ness has suggested that these individuals should be carefully followed, in case they develop a leukemic state [320]. The T and Tn cryptantigens are exposedon malignant cells of cancer of the breast [350] and other carcinomas [55,347,348].Consequently, it was consideredimportant to examine the exposure of these and other cryptantigens on the RBCs, and several prospective studies on blood samples from donors, hospitalized patients [298], patients at high risk for infections and malignant states [299,351],and from some specific groups with known malignant diseases, such as breast cancer [352], lymphomas [353,354], and carcinoma ofthe colon [Levene N, unpublished observations]. The results of these studies have shown an increased cryptantigen exposure on the RBCsof these high-risk groups. Whereas tests with lectins cannot be considered diagnosticof the underlying disease, theydo provide important information relating to future transfusion. Any patient with cryptantigen exposure on his or her RBCs should be carefully monitored because, if true polyagglutination develops,there is a risk of mild or severe intravenoushemolysis.If transfusion therapy is required,thenpacked RBCs should be used, and fresh plasma or plasma products avoided. This is because fresh plasma contains naturally occurring anti-T and other antibodies to cryptantigens which,if transfused to these patients, could exacerbate any hemolysisof polyagglutinable RBCs [349]. REFERENCES 1. Stillmark H. ifber ricin, ein giftiges ferment aus densamen von Ricinus comm. L. und einigen anderen Euphorbiaceen. Inaugural Dissertation, Dorpat University, 1888. 2. Boyd WC, RegueraRM. Hemagglutinatingsubstances for humancells in varioas plants. J Immunoll949; 62:333-339. 3. Renkonen KO. Studies on hemagglutinins present in seeds of some represen-


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tatives of the family of Leguminosae. Ann Med Exp Biol Fenn 1948; 265672. for human red blood corpuscles in 4. Bird GWG. Specific agglutinating activity extracts of Dolichos biflorus. Curr Sci 1951; 20:298-299. 5. Boyd WC, Shapleigh E. Specific precipitating activity of plant agglutinins (lectins). Science 1954; 119:419. 6. Kriipe M. Inkomplette hiimagglutinine in pflanzenextrakten. Immunobiology 1954; 111~22-31. 7. Makela 0 . Studies in hemagglutinins of Leguminosae seeds. Ann Med Exp Biol Fenn 1957;35 (suppl11):l-133. 8. Prokop 0,Uhlenbruck G, Raven JL, trans. Human blood and serum groups. New York: John Wiley & Sons, 1969. 9. Gold ER, Balding P. Receptor-specific proteins. Plant and animal lectins. New York: American Elsevier,1975. 1977; 22-8. 10. Bird GWG. Lectins in blood banking: a brief review. Biotest Bull 11. Bird GWG. Lectins in haematology and blood banking. In: Greenwalt TJ, ed. Methods in haematology, v01 17. Blood transfusion. Edinburgh: Churchill Livingstone,1988:125-148. 12. Judd WJ. The role of lectins in blood group serology. CRC Crit Rev Clin Lab Sci 1980; 12:171-214. 13. Liener IE, Sharon N, Goldstein IJ, eds. The lectins: properties, functions and applications in biologyand medicine. New York: Academic Press,1986. 14. Gilboa-Garber N, Garber N. Microbial lectins. In: Allen HJ, Kisailus EC, eds. Glycoconjugates: composition,structure, and function. New York: Marcel Dekker, 1992:540-590. 15. Gilboa-Garber N, Garber N. Microbial lectincofunction with lytic activities as a model for a general basic lectin role. E M S Microbiol Rev 1989; 63: 211-222. 16. Goldstein IJ, Hughes RC, Monsigny M, Osawa T, Sharon N. What should be called a lectin?Nature 1980; 28556. 17. Goldstein IJ, Hollerman CE, Smith EE. Protein-carbohydrate interaction. 11. Inhibition studies on the interaction of concanavalin A with polysaccharides. Biochemistry 1965; 4:876-883. 18. Kabat EA. Dimensions and specificities of recognition sites on lectins and antibodies. J Supramol Struct 1978; 8:79-88. 19. Race RR, Sanger R. Blood groups in man, 6th ed. Oxford: Blackwell Scientific Publications, 1975:9-13. 20. Shibata S, Goldstein IJ, Baker DA. Isolation and characterization of a Lewis b - active lectin from Griffoniasimplicifolia seeds. J Biol Chem 1982; 257: 9324-9329. 21. Judd WJ, Beck ML, Hicklin BL,Shankar Iyer PN, Goldstein IJ. BSI1 lectin: a second hemagglutinin isolated from Bandeiraea simplicifolia seeds with affinity for type 111polyagglutinable redcells. Vox Sang 1977; 33:246-251. 22. Hayes CE,Goldstein IJ. An alpha-D-galactosyl-binding lectinfrom Bandeiraea simplicifolia seeds, Isolation by affinity chromatography and characterization. J Biol Chem 1974; 249:1904-1914.

374

Levene et al.

23. Murphy LA, Goldstein IJ. Five alpha-D-galactopyranosyl-bindingisolectins from Bandeiraea simplicifolia seeds. J Biol Chem 1977; 252:4739-4742. 24. Wood C, Kabat EA, Murphy LA, Goldstein IJ. Immunochemical studiesof the combining sites of two isolectins,A4 and B4, isolated from Bandeiraea simplicifolia. Arch Biochem Biophys1979; 198:1-1 1. 25. Kaladas PM, Kabat EA, Shibata S, Goldstein IJ. Immunochemical studies on the binding specificity of the blood group L e b specific lectin Griffonia simplicifolia IV. Arch Biochem Biophys 1983; 223:309-318. 26. WU A M , Sugii S, Herp A. A guide for carbohydrate specificities of lectins. Adv Exp MedBioll988; 228:819-847. 27. Wu A M , Sugii S. Coding and classification of D-galactose, N-acetyl-D-galaCtosamine and B-~-Galp-[1-3(4)]-/3-~-GlcpNAc, specificities of applied lectins. Carbohydr Res 1991; 213:127-143. 28. Watkins WM, Morgan WTJ. Neutralization of the anti-H agglutinin in eel serum by simple sugars.Nature 1952; 169:825-826. 29. Morgan WTJ, Watkins WM. The inhibition of the haemagglutinins inplant seeds by human blood group substances and simple sugars.Br J Exp Pathol 1953; 34~94-103. 30. Kabat EA. The blood group substances. New York: Academic Press, 1956: 1-330. 31. Watkins WM, Morgan WTJ.Further observations on the inhibition of blood group specific serological reactions by simple sugars of known structure. Vox Sang 1962; 7~129-150. 32. Lloyd KO, Kabat EA, Layug EJ, Gruezo F. Immunochemical studies on blood groups. XXXIV. Structures of some oligosaccharides produced by alkaline degradation of blood group A, B and H substances. Biochemistry 1966; 5:1489-1501. A, ed. Glyco33. Watkins WM. Blood-group specific substances. In: Gottschalk proteins:their composition, structure and function, 2nded.Amsterdam; Elsevier Publishing,1972:830-891. 34. Ginsburg V. Enzymatic basis for blood groups in man. Adv Enzymol 1972; 36~131-149. 35. Bird GWG. Relationship of the blood sub-groups A,, A2 and AIB, A2B to haemagglutinins present inthe seeds of Dolichos biforus. Nature 1952; 170: 674. 36. Fujii H, Yoshida A. Multiple components ofblood group A and B antigens in human erythrocyte membranes and their difference between AI and A2 status. Proc Natl Acad Sci USA 1980; 77:2951-2954. 37. Hakomori S, Stellner K, Watanabe K. Four antigenic variants of blood group A glycolipid: examples of highly complex, branched chain glycolipid of animal cell membrane. Biochem Biophys Res Commun 1972; 49:1061-1068. 38. Hakomori S. Blood group ABH and Ii antigens of human erythrocytes: chemistry, polymorphism, and their developmental change. SeminHematol 1981; 18~39-62. 39. Hakomori S. Philip Levine award lecture: blood group glycolipid antigens and their modifications as human cancer antigens. Am J Clin Path01 1984; 82~635-648.


375

40. Clausen H, Levery SB, NudelmanE, Baldwin M, Hakomori S. Further char41. 42.

'

43.

44. 45.

acterization of type 2 and type 3 chain blood group A glycosphingolipids from human erythrocyte membranes. Biochemistry1986; 25:7075-7085. Hakomori S. Glycosphingolipids.Sci Am 1986; 254:44-53. Hindsgaul 0 ,Norberg T, Pendu JL, Lemieux RU. Synthesisof type 2 human blood-group antigenic determinants.The H, X,and Y haptens and variations of the H type 2 determinant as probes for thecombining site of the lectin I of Ulex europaeus. Carbohydr Res 1982; 109:109-142. Hindsgaul 0 , Khare DP, Bach M, Lemieux RU. Molecular recognition 111. The binding of the H-type 2 human blood group determinant by the lectin I of Ulexeuropueus.Can J Chem 1985; 63:2653-2658. Gillard BK, Blanchard D, BouhoursJ-F, Cartron, J-P, van Kuik JA, Kamerling JP, Vliegenthart JFG, Marcus DM. Structure of a ganglioside withCad blood group antigen activity. Biochemistry1988; 27:4601-4606. Naiki M, Marcus DM. Human erythrocyte P and Pkblood group antigens: identification as glycosphingolipids. Biochem Biophys Res Commun 1974; 60:1105-1111.

46. 47. 48. 49. 50. 51.

52.

Naiki M, Marcus DM. An immunochemical study of the human blood groups P,, P, and P' glycosphingolipid antigens. Biochemistry1975; 14:4837-4841. Marcus DM, Kundu SK. Immunochemistry of the P blood group system. In: Sandler SG, Nusbacher J, Schanfield MS, eds. Immunobiology of the erythrocyte. New York: Alan R Liss, 198055-65. Marcus DM. A review of the immunogenic and immuno-modulatoryproperties of glycosphingolipids. Mol Immunol1984; 21:1083-1091. Bird GWG. The haemagglutinins of Crotuluria striata. Further evidence of similarity of the A and B agglutinogens. Vox Sang 1956; 1:167-171. Bird GWG. The red cell. Br Med J 1972; 1:293-297. Uhlenbruck G, Pardoe GI, Bird GWG. On the specificity of lectins with a broad agglutination spectrum. 11. Studies on the nature of the T antigen and the specific receptors for the lectin Aruchis hypogaea (ground nut). Immunobiology 1969; 138:423. Springer GF, Tegtmeyer H, Huprikar SV. Anti-N reagents in eludidation of the genetical basis of human blood-group MN specificities. Vox Sang 1972;

22~325-343. 53. Springer GF, YangHJ. Isolation and partial characterization of blood group

M- and N-specific glycopeptidesand oligosaccharides from human erythrocytes. Immunochemistry1977; 14:497-502. 54. Bird GWG, WinghamJ. The action of seed and other reagents on HEMPAS erythrocytes. Acta Haematoll976; 59174-180. 55. Springer GF. T and Tn, general carcinoma autoantigens. Science 1984; 224: 1198-1206. 56. Uhlenbruck G. Lectins, tumor markers, and metastasis: a review from my view. In:Lectins:biology,biochemistry,clinicalbiochemistry, v01 7. St. Louis: Sigma Chemical Co.1990:13-16. 57. Hardman JT. Clinical relevance of ABHand Lewis blood group systems. In:

Wallace ME, Gibbs FL, eds. Blood group systems: ABH and Lewis. Arlington, Va: American Association of Blood Banks, 1986:83-134.

Levene et al.

376

58. Anstall HB. ABH antigensin disease. In: WallaceME, Gibbs F‘L,eds. Blood

group systems: ABH and Lewis. Arlington, Va: American Association of Blood Banks, 19861135-155. 59. Kiillenius G, Mollby R, Svenson SB, Winberg J, Lundblad A, Svensson S, Cedergren B. The P’ antigen as receptor for the haemagglutinin of pyelonephritic Escherichia coli. FEMS MicrobiolLett 1980; 7:297-302. 60. Vliisanen V, Elo J, Tallgren LC, Siitonen A, Makela PH, Svanborg-Eden C, Kallenius G, Svenson SB, Hultberg H, Korhonen T. Mannose-resistant haemagglutination and P antigen recognition are characteristic ofEscherichia coli causing primary pyelonephritis. Lancet 1981; 2:1366-1369. 61. Korhonen TK, Viiisiinen V, SaxCn H, Hultberg H, Svenson SB. P-antigenrecognizingfimbriae from human uropathogenic Escherichia coli strains. Infect Immun 1982; 37:286-291. 62. Loomes LM, Uemura K-I, Feizi T. Interaction of Mycoplasma pneumoniae with erythrocyte glycolipids of and I i antigen types.Infect Immun 1985; 47: 15-20. 63. Gilboa-Garber N. Inhibition of broad spectrum hemagglutinin from Pseudomonas aeruginosa by D-galactose and its derivatives. FEBS Lett 1972; 20: 242-244. 64. Gilboa-Garber N. Pseudomonas aeruginosalectins. Methods Enzymol 1982; 83:378-385. 65. Gilboa-Garber N, Sudakevitz D, Sheffi M, Levene C, Sela R. Blood group 66.

67. 68. 69.

70. 71.

specificity ofthe Pseudomonas aeruginosalectins PA-Iand PA-11. Isr J Med Sci 1992; 28:65. Gilboa-Garber N, Sudakevitz D, Levene C, Sela R. Preferential interactions of PA-I lectin of Pseudomonas aeruginosawith terminal galactosyl residues of blood group antigens B, Pk, Bombay PI, I(i) and T. Proceedings 15th international congress biochemistry, Jerusalem.1991:328. Springer GF, Williamson P, Brandes WC. Blood group activity of gramnegative bacteria. J ExpMed 1961; 113:1077-1093. Springer GF, Williamson P, Readler BL. Blood group active gram-negative bacteria and higher plants. AnnNY Acad Sci 1962; 97:104-110. Engelmann B, Schumacher U, Duhm J. Role of ABH blood group antigens in the stimulation of a DIDS-sensitive Ca” influx pathwayhuman in erythrocytes by Ulex europaeus agglutinin I and a monoclonal anti-A, antibody. Biochim BiophysActa 1991; 1091:261-269. Gilboa-Garber N,Blonder E. Augmentedosmotic hemolysis ofhuman erythrocytes exposedto the galactophilic lectin ofPseudomonas aeruginosa. Isr J Med Sci 1979; 15537-539. Chien S, Jan K-M. Red cell aggregation by macromolecules: roles of surface absorption and electrostatic repulsion. J Supramol Struct 1973; 1:385-

-

409. 72. Gilboa-Garber N. Purification and properties of hemagglutinin from Pseu-

domonas aeruginosa and its reaction with human blood cells. Biochim Biophys Acta 1972; 273:165-173. 73. Gilboa-Garber N,Citronbaum R, LeveneC, Sela R. H blood group detection


377

by the L-fucose binding lectin of the green marine alga Ulvu lactuca. Dev Comp Immunol 1988; 12:695-705. 74. Fujita Y, Oishi K, Aida K. Sugar specificity of anti-B hemagglutinin produced by Streptomyces sp. Biochem Biophys Res Commun 1973; 53:495-501. 75. Fujita Y, Oishi K, Suzuki K, Imahori K. Purification and properties of an anti-B hemagglutinin produced by Streptomyces sp. Biochemistry 1975; 14: 4465-4470. 76. Bird GWG, Wingham J. Agglutinins for antigens of two different human blood group systems in the seeds of Moluccella laevis. Vox Sang 1970; 18: 235-239. 77. KochibeN, Furukawa K. Purification and properties of a novel fucosespecific hemagglutinin AIeuriu of aurantiu.Biochemistry 1980; 19:2841-2846. 78. Garber N, Guempel U, Gilboa-Garber N, DoyleRJ. Specificityof the fucosebinding lectin of Pseudomonas aeruginosa. FEMS Microbiol Lett 1987; 48: 331-334. 79. Bird GWG, WinghamJ. Seed agglutinin for rapid identification of Tn polyagglutination. Lancet 1973; 1:677. 80. Bird GWG, Wingham J. Anti-Cad lectin from seeds of Leonurur curdiucu. Clin Lab Hematol 1979; 157-59. 81. Voak D, Lodge TW. The demonstration of anti HI/HI-H activity in seed anti-H reagents. Vox Sang 1971; 20:36-45. C, Larson G. Deantigenation of 82. Falk P, Hoskins LC, Lindstedt R, Svanborg

human erythrocytes by bacterial glycosidases-evidence for the noninvolvement of medium-sized glycosphingolipids inthe Dolichos biflorus lectin hemagglutination. Arch Biochem Biophys 1991; 290:312-319. 83. Nakajima T,Kogure T, Furukawa K. Specificityof hemagglutinin ofFulcata japonica which reacts with blood group active N-acetyl-D-galactosamineresidues. Exp Clin Immunogenet1986; 3:187-194. 84. Voak D, Todd GM, Pardoe GI. A study of the serological behaviour and nature of the anti-B/P/Pk activity of Salmonidae roe protectins. Vox Sang 1974; 26:176-188. 85. Chien S-M, Lemanski

T, Poretz RD. The anti-I activity of the Sophora japonica lectin. Immunochemistry1974; 11:501-506. 86. Cazal P, Lalaurie M. Recherches sur quelques phytoagglutinines spkcifiques des groupes sanguinsABO. Acta Haematol 1952; 8:73-80. 87. Sudakevitz D,Gilboa-Garber N, Levene C, Sela R, Bhattacharyya L.Erythrina lectins detect the H/HI blood groups. Zintralbl Bakteriol Hyg 1991; 275:343-350. 88.

Rogers DJ. Lectin-type agglutinins, with anti-B1and anti-HI activity, from the ova of the black sea-bream, Spondyliosomu cantharus. Med Lab Sci

1978; 353239-245. 89. Rosenfield RE, Schroeder R, Ballard R, vander Hart M, Moes M, van Log-

hem JJ. Erythrocyte antigenic determinants characteristicof H, I in the presence ofH(IH), or H in the absence ofi [H(-i)]. Vox Sang 1964; 9:415-419. 90. Issitt PD. Applied blood group serology, 3rd ed. Miami: Montgomery Scientific Publications, 1985.

378

Levene et al.

91. Landsteiner K. fiber agglutination serscheinungen normallen menschlichen blutes. WienKlin Wochenschr 1901; 14:1132-1134. 92. Pittiglio DH. Genetics and biochemistry of A, B, H and Lewis antigens. In: Wallace ME, Gibbs FL, eds. Bloodgroup systems: ABH and Lewis. Arlington, Va: American Associationof Blood Banks. 1986:l-56. 93. Kelton JG, BebenekG. Granulocytes do not have surface AB0 antigens. Transfusion 1985; 25567-569. 94. Dunstan RA, Simpson MB, Borowitz M. Absence of ABH antigens on neutrophils. Br J Haematol 1985; 60:651-657. 95. Mollison PL. ABO, Lewis, Ii and P groups. In: Blood transfusion in clinical medicine, 6th ed. Oxford: Blackwell Scientific Publications, 1979:239-291. 96. Schmidt PJ, McGinniss MH. Cell surfaces, blood groups and microorganisms. Nature 1967; 214:1363. 97. Boyd WC, Shapleigh E. Separation of individuals of any blood group into secretors and nonsecretors by use of plant agglutinin (lectin). Blood1954; 9: 1195-1198. 98. Miyazaki S, Nakajima T, Furukawa K. Monoclonal anti A and anti A, B antibodies from a mouse immunized with A secretor saliva. Exp Clin Immunogenet 1991; 8:16-23. 99. Watkins WM. Biochemistry and genetics of the ABO, Lewis and P blood group systems. Adv Human Genet 1980; 1O:l-136. 100. Rajan VP,Larsen RD, Ajmera S, Ernst LK,LoweJB.Aclonedhuman DNA restriction fragment determines expression of a GDP-L-fucose: &Dgalactoside 2-a-L-fucosyltransferase in transfected cells. J Biol Chem 1989; 264:11158-11165. 101. Moss WL. Studies on isoagglutinins and isohemolysins. Bull Johns Hopkins HOSP1910; 21:63-70. 102. Denborough MA, Downing HJ, DoigAG.Serumblood group substances and AB0 haemolytic disease.Br J Haematol1969; 16:103-109. 103. Nance ST.Serology of the ABH and Lewis blood group systems. In: Wallace ME, Gibbs FL, eds. Blood group systems: ABH and Lewis. Arlington, Va: American Association of Blood Banks, 198657-81. 104. Graham HA. An overviewof the biochemistry of the Lewis,ABH and P systems. In: Bell CA, ed. A seminar on antigens on blood cells and body fluids. 33rd annual meeting of the American Association of Blood Banks. Washington DC, 1980249-257. 105. Abe K, Levery SB, Hakomori S. The antibody specific to type 1 chain blood group A determinant. J Immunoll984; 132:1951-1954. 106. Clausen H, Stroud M, Parker J, SpringerGF, Hakomori S. Monoclonal antibodies directed to the blood group A-associated structure, galactosyl-A: specificity and relation to the Thomsen-Friedenreich antigen. Mol Immunol 1988; 25:199-204. 107. Kumazaki T, Yoshida A. Biochemical evidence that secretor gene, Se, is a structural gene encoding a specific fucosyltransferase. Proc Natl Acad Sci USA 1984; 81:4193-4197.


379

108. Clausen H, Holmes E, Hakomori S. Novel bloodgroup H glycolipid antigens exclusively expressed in blood group A and AB erythrocytes (type 3 chain H): 11. Differential conversion of different H substrates by A, and A2 enzymes and type 3 chain H expression in relation to secretor status. J Biol Chem 1986; 261:1388-1392. 109. Clausen H, Hakomori S. ABH and related histo-blood group antigens; immunochemical differencesin carrier isotypesand their distribution. Vox Sang 1989; 56~1-20. 110. Vaith P, Uhlenbruck G. The Thomsen agglutination phenomenon:a discovery revisited50 years later. Immunobiology 1978; 154:l-15. 111. Springer GF, Desai PR. Humanblood group MN and precursor specificities: structural and biological aspects. Carbohydr Res1975; 40:183-192. 112. Sugii S, Kabat EA, Baer HH. Further immunochemical studies on the combining sites of Lotus tetragonolobus and W l a europeus I and I1 lectins. Carbohydr Res 1982;999-101. 113. Osawa T.Inhibition tests of plant agglutininsfrom Laburnum alpinum and Cytisus sessilifolius with various sugars and sugar derivatives. Indication of the type of linkage in certain disaccharides. Biochim Biophys Acta 1966; 115: 507-510. 114. Flory LL. Comparison of lectin anti-H reagents. Vox Sang 1%7; 13:357-361. 115. Bird GWG, Wingham J. Anti-H from Cerastium tomentosumseeds. A comparison with other seed anti-H agglutinins. Vox Sang 1970; 19132-139. 116. Matsumoto I, Osawa T. Purification and characterization of an anti-H(0) phytohemagglutinin of Ulex europaeus. Biochim Biophys Acta 1969; 194: 180-1 89. 117. Matsumoto I, Osawa T. Purification and characterization of a Cytisus type anti-H(0) phytohemagglutinin from Ulex europaeus seeds. Arch Biochem 1970; 140:484-491. 118. Matsumoto I, Osawa T. On the specificity of various heterologous anti-H haernagglutinins. Vox Sang 1971; 21548-557. 119. Bird GWG. Heterogeneity of anti-H lectin. Rev Fr Transfus Immunohematol 1976; 19:175-183. 120. BirdGWG.Lectins.In:Greenwalt TJ, SteaneEA,eds.CRC handbook series in clinical laboratory science, section D, blood banking, v01 1. Cleveland, Ohio: CRC Press, 1977:459-473. 121. Bird GWG. Lectins in immunohematology. Transfusion Med Rev 1989;3: 55-62. 122. Bird GWG, Wingham J. Anti-H from Cerastium tomentosumseeds. A comparison withother seed anti-H agglutinins. Vox Sang 1970; 19:132-139. 123. Yariv J, Kalb AJ, Blumberg S. Lotus tetragonolobus L-fucose-binding proteins. MethodsEnzymoll972; 28:356-360. 1%. Pereira ME, Kabat EA. Blood group specificity of the lectin from Lotus tetragonolobus. Ann NY Acad Sci 1974; 234:301-305. 125. Chessin LN, McGinniss MH. Further evidence for the serologic association of the Om) and I blood groups. Vox Sang 1968; 14:194-201.

380

Levene et al.

126. Voak D. Anti-HI, a new cold antibody of the H,O,I complex. A preliminary report. Scand J Haematoll964; 1:238-239. 127. Gilboa-Garber N, Mizrahi L. A new mitogenic D-gdactosephilic lectin iso128.

129. 130. 131. 132. 133. 134.

lated from seeds ofErythrina corallodendron.Comparison with soybeanand Pseudomonas aeruginosalectins. Can J Biochem 1981; 59:315-320. Bhattacharyya L, Haraldsson M, Sharon N, Lis H, Brewer CF. Binding and precipitating activities ofErythrina lectins with complex typecarbohydrates and synthetic cluster glycosides. A comparativestudy of the lectins from E. corallodendron, E. cristagalli, E. flabelliformis and E. indica. Glycoconjugate J 1989; 6:141-150. Bhatia HM, Boyd WC. Inhibition reactions of fourteen “nonspecific” seed extracts. Transfusion 1962; 2:106-109. Moore HH. Anti-H lectins from the seeds of three Erythrina trees indigenous to Zimbabwe. S Afr J Med Lab Techno1 1981; 27:33-34. Gilboa-Garber N. The lectin of Ulva lactuca detects the L-fucose residues of H and Lewis blood types in saliva. Proceedings Gentner symposium on the “biology of complex carbohydrates.” Rehovot, Israel,198755. Topliss JA, Rogers DJ. An anti-fucose agglutinin in the ova ofDicentrarchus labrax. Med Lab Sci 1985; 42:199-200. Mourant AE. A “new” human blood group antigen of frequent occurrence. Nature 1946; 158:237-238. Andersen PH. The blood group system L. A new blood group L2. A case of epistasy within the blood groups. Acta Pathol Microbiol Scand 1948; 25:

728-731. 135. Andersen PH, Jordal K. An incomplete agglutinin related to L- (Lewis) system. Acta Pathol Microbiol Scand1949; 26:636-638. 136. Sneath JS, Sneath PHA. Transformation of the Lewis groups of human red cells. Nature 1955; 176:172. 137. Makela 0,Makela P. Leb antigen. Studies on its occurrence in red cells, plasma, and saliva. Ann Med Exp Fenn 1956; 34:157-162. 138. Sneath JS, Sneath PHA. Adsorption of blood-group substances from serum on to red cells. Br Med Bull 1959; 15:154-157. 139. Makela 0,Makela PH, Kortekangas A.In vitro transformation of the Lewis blood groups of erythrocytes. AnnMed Exp Fenn1967:159-164. 140. Marcus DM. Discussion: the nature of the Le“ and Leb antigens in human plasma. Ann NY Acad Sci 1970; 169: 161-163. 141. Salmon C, Cartron J-P, Rouger P. The human blood groups. New York: Masson Publishing, 1984. 142. Schenkel-Brunner H, Hanfland P. Immunochemistry of the Lewis blood

group system. 111. Studies on the molecular basis of the Le” property. Vox Sang 1981; 40:358-366. 143. Gooi HC, Feizi T, Kapadia A, KnowlesBB, Sloter D, Evans MJ. Stagespecific embryonic antigen involves (1-3) fucosylated type 2 blood group chains. Nature 1981; 292:156-158. 144. Hakomori S, Kannagi R. Glycosphingolipidsas tumor-associated and differentiation markers. JNCI 1983; 71:231-251.

lectin-Blood Group Interactions

381

145. Lloyd KO. Blood group antigens as markers for normal differentiation and malignant changesin human tissues.Am J Clin Pathol 1987; 87:129-139. 146. Fox N,Damjanov I, Knowles BB, Solter D. Immunohistochemical localization of the mouse stage-specific embryonic antigen 1 in human tissues and tumors. Cancer Res 1983; 43:669-678. 147. Garin-Chesa P, Rettig WJ. Immunohistochemical analysis of LNT, NeuAc 2-3 LNT, and Le“ carbohydrate antigens in human tumors and normal tissues. Am J Path01 1989; 134:1315-1327. 148. Spohr U,Hindsgaul 0, Lemieux RU. Molecular recognition11. The binding of the Lewis b and Y human blood group determinants by the lectin IV of Griffoniasimplicifolia. Can J Chem 1985; 63:2644-2652. 149. Spohr U, LemieuxRU. Synthetic, conformational, and immunochemical studies of modified Lewis b and Y human blood-group determinants to serve as probes for the combining site of the lectin IV of Griffonia simplicifolia. Carbohydr Res 1988; 174:211-237. 150. Vandonselaar M, Delbaere LTJ, Spohr U, Lemiew RU. Crystallization of the lectin IV of Griffonia simplicifolia and its complexes with the Lewis b and Y human blood group determinants. J BiolChem 1987;262:1084810849. 151. Vicari G, Kabat EA. Immunochemical studies on blood groups. XLII. Isolation and characterization from ovarian cyst fluid of a blood group substance lacking A, B, H, Lea and Leb specificity.J Immunoll969; 102:821-825. 152. Hakomori S, Wang M, Young WW. Isoantigenic expression of Forssman glycolipid inhuman gastric and colonic mucosa:its possible identity with “Alike antigen” human in cancer. Proc Natl Acad sci USA 1977; 72:3023-3027. 153. Baker DA, Sugii S, Kabat EA, Ratcliffe RM, Hermentin P, Lemieux RU. Immunochemical studies on the combining sites of Forssman hapten reactive hemagglutinins from Dolichos biflorus and Helixpomatia, and Wistariafloribunda. Biochemistry 1983; 222741-2750. 154. Prokop 0 , Uhlenbruck G, Kohler W. A new source of antibody-like substances havinganti-blood group specificity. A discussion on the specificity of Helix agglutinins. Vox Sang 1968; 14:321-323. 155. Galbraith W, Goldstein IJ. Phytohemagglutinin of the lima bean (Phaseolus lunatus).Isolation, characterization, and interaction with type A blood group substance. Biochemistry 1972; 11:3976-3984. 156. Hammarstrom S, Kabat EA. Purification and characterization of a bloodgroup A reactive hemagglutininfrom the snail Helixpomatia and a study of its combining site. Biochemistry1969; 8:2696-2705. 157. Hammarstrom S, Kabat EA. Studieson specificity and binding properties of the blood group A reactive hemagglutininfrom Helixpomatia. Biochemistry 1971; 10~1684-1692. 158. Hammarstrom S, Westoo A, Bjork I. Subunit structure of Helixpomatia A hemagglutinin. ScandJ Immunoll972; 1:295-309. 159. Johnson HM. Human blood group A, specific agglutinin ofthe butter clam Saxidomus giganteus.Science 1964; 146548-549. 160. Nguyen OK, Guillaume J-L, Hoebeke J. A blood group A specific lectin

Levene et al.

382

from the seeds of Crotuluriu striata. Biochim Biophys Acta 1990; 1033:210213. 161. JuddWJ.

Lectins and polyagglutination.In: Petz LD,Swisher SN, eds. Clinical practice of blood transfusion, 2nd ed. New York: Churchill Livingstone, 1989:137-151. 162. Rogers DJ, Fish BC. Marine algal lectins. LectinRev 1991; 1:129-142. and characterization of a lectin (plant 163. Etzler ME, Kabat EA. Purification hemagglutinin) with blood group A specificity from Dolichos biforus. Biochemistry 1970; 9:869-877. 164. Etzler ME. Horse gram (Dolichos biforus) lectin. Methods Enzymol 1972;

28~340-344. 165. Sung LA, Kabat E A , Chien S. Interactions of lectins with membrane receptors on erythrocyte surfaces. J Cell Biol 1985; 101:646-651. 166. Garratty G , Willbanks E, Petz LD. Acquired-B antigen associated with Proteus vulgurisinfection. Vox Sang 1971; 21:45-46. 167. Judd WJ. Microbial-associated forms of polyagglutination (T,Tk and ac168. 169. 170. 171. 172. 173.

quired-B). In: Beck ML, Judd WJ. eds. Polyagglutination. WashingtonDC: American Association of Blood Banks, 1980:23-53. Gerbal A, Ropars C, Gerbal R, Cartron JP, Maslet C, Salmon C. Acquired B antigen disappearance by in vitro acetylation associated with A, activity restoration. Vox Sang 1976; 3164-66. BirdGWG.Erythrocytepolyagglutination. In: Greenwalt TJ, SteaneEA, eds. CRC handbook. Series in clinical laboratory science, section D; blood banks. Cleveland: CRC Press, 1977:443-454. Beck ML. Blood group antigens acquired de novo. In:Garratty G, ed. Blood group antigens and disease. Arlington, Va: American Association of Blood Banks, 1983:45-47. Rogers DJ, Blunden G, Evans PR. Ptilotuplumosu, a new source of a bloodgroup B specific lectin. Med Lab Sci 1977; 34: 193-200. Anstee DJ, Holt PDJ, Pardoe GI. Agglutinins from fish ova defining blood groups Band P. Vox Sang 1973; 25:347-360. Sakakibara F, Kawauchi H, Takayanagi G . Blood group B-specific lectin of Plecoglossus ultivelis (ayu fish) eggs. Biochim Biophys Acta 1985; 841:103-

111. 174. Downie DM, Madin DF, Voak D. An evaluation of salmon anti-B reagentin manual and automatedblood grouping.Med Lab Sci 1977; 34:319-324. 175. Umetsu K, Yamashita K, Suzuki T. Purification and carbohydrate-binding

specificities of a blood type B binding lectin from hemolymph of a crab (Churybdisjuponicu).J Biochem 1991; 109:718-721. 176. Poretz, RD, Riss H, Timberlake JW, Chien S-M. Purification and properties of the hemagglutinin from Sophora japonica seeds. Biochemistry 1974; 13: 250-256. 177. Wiener AS, Unger LJ, Cohen L, Feldman J. Type-specific coldautoantibod-

ies as a cause of acquired hemolytic anemia and hemolytic transfusion reactions: biologic test with bovine red cells. Ann Intern Med 1956; 44:221-240.


383

178. Horstmann DM, Tatlock H. Cold agglutinins: a diagnostic aid in certain types of primary atypical pneumonia. Am J Med Assoc 1943; 122:369-370. 179. Peterson OL, Ham TH, Finland M. Cold agglutinins (autohaemagglutinins) in primary atypical pneumonia.Science 1943; 97:167. 180. Beck ML. The I blood group collection. In: Moulds JM, Woods LL, eds. Bloodgroups: P,I,Sda and Pr. Arlington, Va: AmericanAssociationof Blood Banks, 1991~23-52. 181. Marsh WL, JenkinsWJ. Anti-i: a new cold antibody. Nature 1960; 188:753. 182. Tippett P, Noades J, Sanger R, Race RR, Laima S, Holman CA, Buttimer RJ. Further studies of the I antigen and antibody. VOXSang 1960; 5:107121. 183. Gold ER. Observations on the specificity of anti-0 and anti-A, sera. Vox Sang 1964; 9~153-159. 184. Lewis M, Anstee DJ, Bird GWG, Brodheim E, Cartron J-P, Contreras M, Crookston M, Dahr W, Daniels GL, Engelfriet CP, Giles CM, Issitt PD, Jorgensen J, Kornstad L, Lubenko A, Marsh WL, McCrearyJ, Moore BPL, Morel P, Moulds JJ, Nevanlinna H. Nordhagen R, Okubo Y, Rosenfield RE, Rouger P, Rubenstein P, Salmon C, Seidl P, Sistonen P, Tippett P, Walker G, Young S. Blood group terminology 1990. Vox Sang 1990; 58:152169. 185. Feizi T. The blood group Ii system: a carbohydrate antigen system defined by naturally monoclonal or oligoclonal autoantibodies of man. Immunol Commun 1981; 103127-156. 186. Anstee DJ. Blood group-active surface molecules of the human red blood cell. Vox Sang 1990; 58:l-20. 187. Childs RA, Feizi T, Fukuda M, Hakomori S-I. Bloodgroup I activity associated with band 3, the major intrinsic membrane protein of human erythrocytes. Biochem J 1978; 173:333-336. 188. Yokoyama M. Close relationship between A and I blood groups. Nature 1965; 206:411-412. 189. Joshi SR, Bahtia HM. A new red cell phenotype I - , i - : red cells lacking both I and i antigens.Vox Sang 1979; 36:34-38. 190. Thomas AB. The i antigen complex: a new specificity unique to dividing human cells. Eur J Immunoll974; 4:819-824. 191. Moore HH. A lectin displaying anti-i like specificity from the seeds ofPterocarpus angolensis.S Afr J Med Lab Techno1 1983; 29:43-44. 192. Doinel C, Ropars C, Salmon C. Effects of proteolytic enzymes and neuraminidase on the I and i erythrocyte antigen sites. Quantitative and thermodynamic studies. Immunology1978; 34:653-662. 193. Landsteiner K, Levine P. Further observations on individual differences of human blood. Proc SOC Exp Biol Med 1927; 24:941-942. 194. Marcus DM, Kundu SK, Suzuki A. The P group system: recent progress in immunochemistry and genetics. Semin Hematol 1981; 18:63-71. 195. Anstall HB, Blaylock RC. The P blood group system: biochemistry, genetics, and clinical significance. In: MouldsJM, Woods LL, eds. Blood groups: p,

Levene et al.

384

I, Sd" and Pr. Arlington, Va: American Association of Blood Banks, 1991: 1-22. 196. Levine P, Bobbitt OB, Walker RK, Kuhmichel A. Isoimmunizationof a new blood factor in tumor cells. Proc SOC Exp Biol Med 1951; 77:403-405. 197. Matson GA, Swanson J, Noades J, Sanger R, Race RR. A "new" antigen

and antibody belonging to the P blood group system. Am J Hum Genet 1959; 11~26-34. 198. Kortekangas A E , Kaarsalo E, Melartin L, Tippett P, Gavin J, Noades J, 199. 200. 201. 202. 203. 204. 205.

Sanger R, Race RR. The red cell antigen P' and its relationship to the P system. The evidence ofthree more Pk families. Vox Sang1965; 10:385-404. Voak D,Anstee DJ, Pardoe GI. The a-galactose specificity of anti-P'. Vox Sang 1973; 25:263-270. Issitt PD. The P blood group system. In: Issitt PD. ed. Applied blood group serology, 3rd ed. Miami: Montgomery Scientific Publication, 1985:203-218. Cameron GL, Staveley JM. Blood group P substance in hydatid cyst fluids. Nature 1957; 179:147-148. Morgan WTJ, Watkins WM. Blood group P, substance. I. Chemical properties. Proceedings 9th congress International Society BloodTransfusion. Mexico, 1962. 1964:225-229. Naiki M, Kat0 M. Immunological identification of blood group P' antigen on normal human erythrocytes and isolation of anti-Pk with different affinity. Vox Sang 1979; 37:30-38. Tippett P, Sanger R, Race RR, SwansonJ, Busch S. An agglutinin associated with the P and the AB0 blood systems. Vox Sang1965; 10:269-280. Tippett P. Contributions of monoclonal antibodies to understanding one new and some old bloodgroup systems. In: Garratty G, ed. Red cell antigens and antibodies. Arlington, Va: American Association of Blood Banks, 1986:8398.

206. Iseki S, Masaki S, Levine P. A remarkable familywith the rare human isoantibody anti Tj' in four siblings: anti Tja and habitual abortion. Nature 1954; 173~1192-1193. 207. Allen FH Jr, Marsh W L , Jensen L, Fink J. Anti-IP: an antibody defining 208.

209. 210. 211. 212. 213.

another product of interaction between the genes ofthe I and P blood group systems. Vox Sang1974; 27:422-426. Booth PB. Anti ITPI: anantibody showing a further association betweenthe I and P blood group systems. Vox Sang 1970; 19:85-90. Landsteiner K, Levine P. A new agglutinable factor differentiating individual human bloods. Proc SOC Exp Biol Med 1927; 24600-602. Walsh RJ, Montgomery C. A new human isoagglutinin subdividingthe MN blood groups. Nature 1947; 160504. Levine P, Kuhmichel AB, Wigod M, Koch E. A new blood factor, S, allelic to S. Proc SOCExp Bio Med 1951; 78:218-220. Adamany AM, Kathan RH. Isolation of a tetrasaccharide common to MM, NN and MN antigens. Biochem Biophys Res Commun1969; 37:171-178. German J, Metaxas MN. Metaxas-Buehler M. Louie E, Chaganti RSK. Fur-


214. 215. 216. 217. 218. 219.

220. 221. 222. 223. 224. 225.

226. 227. 228.

385

ther evaluation of a child withthe Mkphenotype and atranslocation affecting the longarms of chromosomes2 and 4. Cytogenet Cell Genet1979; 25:160. Holliman SM. The MN blood group system: distribution, serology and genetics. In: Unger PJ, Laird-Fryer B, eds. Blood group systems: MN and Gerbich. Arlington, Va: American Association of Blood Banks, 1989:l-29. WienerAS,Unger LJ, Gordon EB. Fatal hemolytic transfusion reaction caused by sensitization to a new blood factor U. J Am Med Assoc 1953;153: 1444-1446. Tomita M, Marchesi VT. Amino-acid sequence and oligosaccharide attachment sitesof human erythrocyte glycophorin.Proc Natl Acad Sci USA1975; 72:2964-2968. Furthmayr H.Glycophorins A, B and C: a family of sialoglycoproteins. Isolation and preliminary characterization of trypsin derived peptides.J Supramol Struct 1978; 9:79-95. Tomita M, Furthmayr H, Marchesi VT.Primary structure of human erythrocyte glycophorinA: isolation and characterization of peptides and complete amino acid sequence. Biochemistry1978; 17:4756-4770. Rolih SD. Erythrocyte antigens of the MN system and related structures. In: Bell CA, ed. A seminar on antigens on blood cells and body fluids. 33rd annual meeting of the American Association of Blood Banks. Washington, DC. 1980:209-247. Issitt PD. The MN blood group system. Cincinnati, Ohio: Montgomery Scientific Publications, 1981. Rolih S. Biochemistry of MN antigens. In: Blood group systems: MN and Gerbich. Arlington,Va: American Association of Blood Banks, 1989. Issitt PD. Applied blood group serology, 3rd ed. Miami: Montgomery Scientific Publications, 1985. Metaxas MN, Metaxas-Buhler M.M': an apparent silent allele at the MN locus. Nature 1964; 202:1123. Metaxas MN, Metaxas-Buhler M, Romanski Y. The inheritance ofthe blood group gene Mk and some considerations on its possible nature. Vox Sang 1971; 20~509-518. Tokunaga E, Sasakawa S, Tamaka K, Kawamata H, Giles CM, Ikin EW, Poole J, Anstee DJ, Mawby WJ, Tanner MJA. Two apparently healthy Japanese individuals of type MkMk have erythrocytes which lack both the blood group MN and Ss active sialoglycoproteins. J Immunogenet 1979; 6: 383-390. Stoltz JF, Streiff F, Genetet B. Demonstration of M antigen on human lymphocytes by liquid phase electrophoresis.Vox Sang 1974; 26:467-469. Simpson MB, Dunstan RA, Rosse W, Munro AC, Fraser R, Nichols ME. Status of the MNSs antigens on human platelets. Transfusion 1987; 27:1518. Wasniowaska K, Drzeniek Z, Lisowska E. The amino acids of M and N blood group glycopeptides are different.BiochemBiophysResCommun 1977; 76:385-390.

386

Levene et al.

229. Anstee DJ. Blood group MNSs-active sialoglycoproteins of the human eryth230. 231. 232. 233. 234. 235. 236.

rocyte membrane. In: Sander SG, Nusbacher J, Schanfield MS, eds. Immunobiology ofthe erythrocyte. New York: Alan R Liss, 1980:67-98. Springer GF, Huprikar SV. On the biochemical and genetic basis of the human blood-groupMN specificities. Haematologia 1972; 6:81-92. Springer GF, Desai PR. Common precursors of human blood group MN specificities. Biochem Biophys Res Commun1974; 61:470-475. Thomas DB, WinzlerRJ. Structural studies on human erythrocyte sialoglycoproteins: alkali-labile oligosaccharides.J Biol Chem 1969; 2445943-5946. Springer GF, Ansell NJ. Inactivation of human erythrocyte agglutinogensM and N by influenza virusesand receptor destroying enzyme.Proc Natl Acad Sci USA 1958; 44:182-189. Kim Z, Uhlenbruck G. Untersuchungen iiber T-antigen and T-agglutinin. Z ImmunitatsforschExp Klin Immunoll966; 130:88-99. Lisowska E. Reaction of erythrocyte mucoproteins withanti-N phytagglutinins from Vicia gruminea seeds. Nature 1963; 198:865-866. Lisowska E, Duk M. Effect of modification of amino groups of human erythrocytes on M, N and Nvg bloodgroup specificities. Vox Sang 1975; 28:

392-397. 237. Dahr W, Uhlenbruck G, Knott H. Immunochemical aspects of the MNSsblood group system. J Immunogenet 1975; 2:87-100. 238. Blanchard D, Dahr W, Hummel M, Latron F, Beyreuther K, Cartron J-P.

Glycophorins B and C from human erythrocyte membranes:purification and sequence analysis.J Biol Chem 1987; 2625808-5811. 239. Siebert PD, Fukuda M. Isolation and characterisation of human glycophorin A cDNA clones bya synthetic oligonucleotideapproach: nucleotide sequence and mRNA structure. Proc Natl Acad Sci USA 1986; 83:1665-1669. 2 4 0 . Siebert PD, Fukuda M. Molecular cloning ofa human glycophorinB cDNA nucleotide sequence and genomic relationship to glycophorin A. Proc Natl Acad Sci USA 1987; 84:6735-6739. 241. Rahuel C, London J, d'Auriol L, Mattei MG, Tournamille C, Skrzynia C, Lebouc Y, Galibert F, Cartron J-P. Characterisation of cDNA clones for human glycophorin A: use for gene localisation and for analysis of normal and glycophorin A-deficient (Finnish type) genomic DNA. Eur J Biochem 1988; 1721147-153. 242. Tate CG, Tanner MJA. Isolation of cDNA clones for human erythrocyte membrane sialoglycoproteinsa! and 6. Biochem J 1988; 254:743-750. 243. Tanner MJA, Anstee DJ. The membrane change inEn(a-) human erythrocytes. Absence of the major erythrocyte sialoglycoprotein. Biochem J 1976; 153:271-277. 244.

Dahr W, Uhlenbruck G, Leikola J, Wagstaff W, Landfriet K. Studies on the membrane glycoprotein defect of En(a-) erythrocytes. I. Biochemical aspects. J Immunogenet 1976; 3:329-346. 245. Winzler RJ. A glycoprotein in human erythrocyte membranes. In: Red cell membrane. Philadelphia: JB Lippincott, 1969: 157-171.


387

2 4 6 . Alloway GP, Burness AT. Site ofattachment of encephalomyocarditis virus on human erythrocytes. J Viroll986; 59:768-770. 247. Jokinen M, Ehnholm C. Viiisiinen-Rhen V, Korhonen T, Pipkorn R, Kalkinen N, Gahmberg CG. Identification of the major human sialoglycoprotein from red cells, glycophorin A, as the receptor for Escherichia coli IH 11165 and characterization of the receptor site.Eur J Biochem 1985; 147:47-52. 248. Pasvol G. Receptorson red cells for Plasmodium fulcipurum and their interaction with merozoites.Philos Trans R SOC Lond1984; B307:189-200. 249. Dahr W, Uhlenbruck G, Bird GWG. Cryptic A-like receptor sites inhuman erythrocyte glycoproteins: proposed nature of Tn-antigen. Vox Sang 1974; 27:29-42. 250. Dahr W, Uhlenbruck G, Gunson HH, Van Der Hart M. Molecular basis of Tn-polyagglutinatinability. Vox Sang 1975; 29:36-50. 251. Ottensooser F, Silberschmidt K. Haemagglutinin anti-N in plant seeds. Nature 1953; 172:914. 252. Duk M, Lisowska E, Kordowicz M, Wasnioska K. Studieson the specificity of the binding site of Viciugrumineu anti-N lectin. Eur J Biochem 1982; 123: 105-1 12. 253. Lisowska E. Reaction of eryihrocyte mucoproteins with anti-N phytohemagglutinins from Viciugrumineu seeds. Nature 1963; 198:865-866. 254. Romanowska E. Reaction of M and N blood-group substances natural and degraded with specific reagentsof human and plant origin. Vox Sang 1964; 8:578-588. 255. Prigent MJ, Blanchard D,Cartron J-P.Membrane receptorsfor Vicia grumi256.

257. 258. 259. 260.

nea anti-N lectin and its binding to native and neuraminidase-treated erythrocytes. Arch Biochem Biophys 1983; 222:231-244. Yanagi K, Ohyama K, Yamakawa T, Ohkuma S. Isolation and characterization of blood group N antigen precursor glycoproteins with Thomsen-Friedenreich Q activity, N antigen precursor glycoproteins and T-active glycoproteins from cyst fluids of malignant ovarian clear cell carcinoma. Int J Biochem 1990; 22:1015-1028. Yanagi K,Ohyama K, Yamakawa T, Watanabe H, Hirakawa S, Ohkuma S. Biochemical characterization of glycoprotein componentsin human ovarian cyst fluidsby lectins. Int J Biochem 1990; 22:659-663. Moon GJ, Wiener AS. A new source of anti-N lectin: leaves of the Korean Viciu unijugu. Vox Sang 1974; 26:167-170. Ohkuma S, Ohyama K, Akitoshi Y, Okada Y, Kawanishi M, Matsuda K. Immunochemical property of anti-N hemagglutinin of leaves of Viciu unijuga. J Clin ExpMed 1978; 107:631-633. Yanagi K, Ohyama K, Yamakawa T, Hashimoto K, Ohkuma S. Purification and characterizationof anti-N lectin from Viciu unijuguleaves. Int J Biochem

1990; 22~43-52. 261. Fletcher G. The anti-N phytoagglutinin of Buuhiniu vuriegutu. Aust J Sci 1959; 22:167. 262. Bird GWG, Wingham J. Agglutinins for antigens of two different human

388

Levene et al. blood group systems in the seeds of Moluccella laevis. Vox Sang 1970; 18:

235-239. 263. Judd WJ, Murphy LA, Goldstein IJ, Campbell L, Nichols ME. An anti-B 264. 265. 266. 267. 268. 269. 270. 271. 272. 273. 274.

reagent prepared from a-D-galactopyranosyl-bindingisolectins from Bandeiraea simplicifoliaseeds. Transfusion 1978; 18:274-280. Allen NK, Brilliantine L. A survey of hemagglutininsin various seeds. J Immunoll969; 102:1295-1299. Nakajima H,Nakayama T. Anti-M lectin of Iberis amana L. Rep Jpn Natl Res Inst Police Sci 1968;21:111. Okada T, Nakajima H. Anti-M lectin of Japanese radish. Rep Jpn Natl Res Inst Res Inst Police Sci 1970; 23:207. Vaisanen, V, Korhonen T, Jokinen M, Gahmberg CG, Ehnholm C. Blood group M specific haemagglutination in pyelonephritogenicEscherichia coli. Lancet 1982; 1:1192. Bird GWG, Wingham J. N-Acetyl-neuraminic (sialic) acidand human blood group antigen structure. Vox Sang 1970; 18:240-243. Furthmayr H.Structural comparison of glycophorins and immunochemical analysis of genetic variants. Nature 1978; 271519424. Darnborough J, Dunsford I, Wallace JA. The En" antigen and antibody. A genetical modification of human red cells affecting their blood grouping reactions. Vox Sang 1969; 17941-255. Uhlenbruck G . Action of proteolytic enzymes on the humanerythrocyte surface. Nature 1961; 190:181. Bird GWG, Wingham J. The action of seed and other reagents on En(a-) erythrocytes. Vox Sang 1973; 2448-57. Issitt PD, Pavone BG,RolihSD.New conceptsof the structure ofEn" abstracted. Transfusion 1979; 19:662 BirdGWG. Specificityof Glycine soja agglutinins.Experientia 1963;19:

625. 275. Furuhjelm U,Myllyla G , Nevanlinna HR, Nordling S, Pirkola A, Gavin J,

Gooch A, SangerR, Tippett P. The red cell phenotype En(a-) and anti Ena: serological and physicochemical aspects. Vox Sang1969; 17:256-278. 276. Hiibener G . Untersuchungen uber isoagglutination mit besonderer beriicksichtigung scheinbarer abweichungenvom gruppenschema. Immunobiology 1925; 45:223-226. 277. Thomsen 0 . Ein vermehrungsfiihiges agens als veriinderer des isoagglutina278. 279. 280. 281.

torischen verhaltens derroten blutkorperchen eine bisher unbekannte quelle der fehlbestimmung. Immunobiology1927; 52:85-90. Friedenreich V. The Thomsen hemagglutination phenomenon. Copenhagen: Levin and Munskgaard, 1930. Levine P, Katzin EM. Temporary agglutinability of redblood cells. Proc SOC Exp Biol Med 1938; 39:167-169. Bird GWG. Clinical aspects of red blood cell polyagglutinability of microbial origin. In: SalmonC, ed. Blood groups and other red cell surface markers in health and disease. New York: Masson, 198255-64. Springer GF, Desai PR, Murthy MS, Yang HJ, Scanlon EF. Precursors of


282. 283. 284. 285. 286. 287. 288. 289. 290. 291. 292. 293. 294. 295. 2%. 297. 298. 299. 300.

389

blood group MN antigens as carcinoma associated antigens. Transfusion 1979; 19~233-249. Chorpenning F W , Dodd MC. Polyagglutinable erythrocytes associated with bacteriogenic transfusion reactions. Vox Sang 1965; 10:460-471. Bird GWG. Anti-T in peanuts. Vox Sang 1964; 9:748-749. Bird GWG. Lectins and red cell polyagglutinability: history, comments and recent developments. In:Beck ML, Judd WJ, eds. Polyagglutination. Washington DC: American Association of Blood Banks, 1980:71-90. Rickard KA, Robinson RJ, Worlledge SM. Acute acquired haemolyticanaemia associated with polyagglutination. Arch Dis Child1969;44:102-105. Moores P, Pudfin D, Pate1 PL. Severe hemolytic anemiain an adult associated with anti-T. Transfusion 1975; 15:329-333. Levene C, SelaR, Blat J, Friedlaender M, Manny N. Intravascular hemolysis and renal failure in apatient with T polyagglutination.Transfusion 1986; 26: 243-245. Van Loghem JJ Jr, Van der Hart M, Land ME. Polyagglutinability of red cells as a causeof a severe hemolytictransfusion reaction. Vox Sang 1955;5: 125-128. Gray JM, Beck ML, Oberman HA. Clostridial-induced type I polyagglutinability with associatedintravascular hemolysis. Vox Sang1972; 22:379-383. Judd WJ, Oberman HA, Flynn S. Fatal intravascular hemolysis associated with T-polyagglutination.Transfusion 1982; 22:345-346. Klein PJ, Bulla M, Newman R A , Muller P, Uhlenbruck G, Schaefer HE, Kruger G, Fisher R. Thomsen-Friedenreich antigen in haemolytic-uraemic syndrome. Lancet 1974; 2:1024-1025. Lotan R, Skutelsky E, Danon D, Sharon N. The purification, composition and specificity of the anti-T lectin from peanut (Arachis hypogueu). J Biol C h m 1975; 250:8518-8523. Bird GWG, Wingham J. Tk: a new form of red cell polyagglutination. Br J Haematol 1972; 23:759-763. Doinel C, Andreu G, Cartron J-P, Salmon C, Fukuda MN. Tk polyagglutination produced in vitro by an endo-0-galactosidase from Escherichiafreundii. Vox Sang 1980; 38:94-98. Judd WJ. The role ofexo-0-galactosidases in Tk-activation. Transfusion 1980; 20:622. Bird GWG, Wingham J, Beck ML, Pierce SR, Oates GD, Pollock A. Th a new form of erythrocyte polyagglutination. Lancet1978; 1:1215-1216. Veneziano G, Rasore-Quartino A, Sansone G. Th erythrocyte polyagglutination. Lancet 1978; 2:483. Rawlinson V, Stratton F. Incidence of T activation in a hospital population. Vox Sang 1984; 46:306-317. Levene NA. Polyagglutination in Israel; MD thesis Hebrew University, Hadassah Medical School [in Hebrew].1984:l-68. Herman JH, Whiteheart W, Shirey RS, Johnson RJ, Kickler TS, Ness PN. Redcell Th activation: biochemical studies. Br J Haematol 1987; 65:205209.

390

Levene et al.

301. Okubu Y, Seno T, Yamaguchi H. A persistent type of erythrocyte polyagglutinability Th. Transfusion 1984; 24:277-278. 302. Levene NA, Levene C, Gecker K, Sigler E, Merhav H, Berrebi A.Th polyagglutination with fatal outcome in apatient with massiveintravascular hemolysis and perforated tumor of colon. Am J Hematol1990; 39215-216. Levene C, Manny N, Yew Wah L, Bird GWG, 303. Sondag-Thull D, Levene NA, Schechter Y, Francois-Gerard C, Huet M, Blanchard D. Characterization of a neuraminidase from Corynebacterium aquaticum responsible for Th polyagglutination. Vox Sang 1989; 57:193-198. 304. Bird GWG, WinghamJ. Vicia creticaa powerful lectinfor T and Th but not for Tk or other polyagglutinable erythrocytes. J Clin Pathol 1981; 3469-70. 305. Bird GWG, WinghamJ, Seger R, Kenny AB.Tx a “new” red cell cryptantigen exposed by pneumococcal enzymes. Blood Transfus Immunohematol 1982; 25:215-216. 306. Wolach B, Nahum S, Bird GWG, Moulds JJ, Bar-Shany S, Ben-Porath D, Levene NA, Sela R, Levene C. Tx polyagglutination in three members ofone family. Acta Haematol 1987; 78:45-47. 307. Yang E, Moore BPL, Liew YW, Bird GWG. Further observations on the Vicia creticalectin. Transfusion 1986; 26:306. 308. Gerbal A, MasletC,SalmonC.Immunologicaspectsof the acquired-B antigen. Vox Sang 1975; 28:398-405. 309. Graninger W, RameisH, Fischer K, Poschmann A, Bird GWG, WinghamJ, Neumann E.T A , ” a new type of erythrocyte polyagglutination characterized by depressed H receptorsand associated with hemolytic anemia.I. Serological and hematological observations.Vox Sang 1977; 32:195-200. 310. Moreau R, Dausset J, Bernard J, Moullec J. Anemie hemolytique acquise avec polyagglutinabilite hematies par un nouveau facteur present dans le serum humain normal (anti-Tn).Bull SOCMed Hop Paris 1957; 73569-587. 311. Cartron JP, Andreu G, Cartron J, Bird GWG, Salmon C.Demonstration of T-transferase deficiency in Tn-polyagglutinable blood samples. Eur J Biochem 1978; 92:lll-119. 312. Myllyla G, Furhjelm U, Nordling S, Pirkola A, Tippett P, Gavin J. Sanger R. Persistent mixed field polyagglutinability: electrokinetic and serological aspects. Vox Sang 1971; 20:7-23. 313. Bird GWG. Significant advances in lectins and polyagglutinable red cells. Presented at theXVII congressof the International Society of Blood Transfusion. Paris, Libraire Amette. 1978:87-98. 314. Bird GWG, Wingham J, Richardson SGN. Myelofibrosis, autoimmune hemolytic anemia and Tn polyagglutinability. Haematologia 1985;189-102. 315. Bird GWG, Wingham J, Pippard MJ, Hoult JG, Melikian V. Erythrocyte membrane modification in malignant disease of myeloid and lymphoreticular tissues I. Tn-polyagglutination in acute myelocytic leukemia. Br J Haematol 1976; 33~289-294. 316. Wilson MH, Cott ME, Sotus PC. Probable Tn-activation inutero. [abstract]. Transfusion 1980; 20:622. 317. Rose RR, Skradski KJ, Polesky HF, Beck ML, Judd WJ, Rogers E. Tran-


391

sient neonatal Tn-activation: another example [abstract]. Transfusion 1983;

23:422. 318. Schultz M, Fortes P, Brewer L, Miller A, Beck M. “In utero” exposure ofTn and Th cryptantigens [abstract]. Transfusion 1983; 23:422. a nonmicrobial form of polyagglutination.In: Beck ML, 319. BeckML.Tn: 320. 321.

322. 323. 324.

Judd WJ, eds. Polyagglutination. Washington DC: American Association of Blood Banks, 1980:55-70. Ness PM, Garratty G, Morel PE, Perkins HA. Tn-polyagglutination preceding acute leukemia. Blood 1979; 54:30-34. Crookston JH, Crookston MC,BurnieKL,Francombe WH, Dacie JV, Davis JA, Lewis SM. Hereditary erythroblastic multinuclearity with a positive acidified serum test:a type of congenital dyserythropoeitic anemia.Br J Haematol 1969; 17: 11-26. Rochant H, Gerbal A. Polyagglutinabilitie due a L’antigen Hempas. Rev Fr Transfus Immunohematoll976; 19:239-245. Harris PA, Kacmarski GR, Moulds JJ, Bird GWG,Shaha NG. An inherited RBC characteristic NOR, resulting in erythrocyte polyagglutination. Transfusion 1979; 19:664. Cazal P, Monis M, Caubel J, Brives J. Polyagglutinabilite hereditaire dominante: antigene prive (Cad) correspondent a un anticorps public et a une lectine deDolichos biflorus. Rev Fr Transfus Immunohematol 1968; 11:209-

221. 325. Blanchard D,Cartron JP, Fournet B, Montreuil J, van Halbeek H, Vliegen-

thart JFG. Primary structure of the oligosaccharide determinant of blood group Cad specificity. J Biol Chem 1983; 358:7691-7695. 326. Bird GWG. Human blood group mosaics and chimaeras. Biotest Bull 1985;

2:215-224. 327. Booth PB, Plaut G, James JD, Ikin EW, Moores P, Sanger R, Race RR. Blood chimerismin a pair of twins. Br Med J 1957; 1:1456-1458. 328. Bird GWG, Wingham J. The M, N, and Nvg receptorsof Tn-erythrocytes. Vox Sang 1974; 26:171-175. 329. Bird GWG. Haemagglutinins in Indian plants. Army Med Corps J (India) 1955; 9~17-25. 330. Roque-Barreira MC, Campos-Net0A. Jacalin: an IgA-binding lectin. J Immunol 1985; 134:1740-1743. 331. Kondoh H, Kobayashi K, Hagiwara K, Kajii T. Jacalin, a jackfruit lectin

precipitates IgA, not IgA, subclass on gel diffusion reaction. J Immunol Methods 1986; 88:171-173. 332. Autcouturier P, Mihaesco E, Mihaesco C, Preud’hoomme JL. Characterization of jacalin, the human IgA and IgD binding lectinfrom jack fruit. Mol

Immunol187;24:503-5 11. 333. Vyas GN, Holmdahl L, Perkins HA, Fudenberg HH. Serologic specificity of human anti-IgA and its significance intransfusion. Blood 1969; 34573-581. 334. Hodson C. The incidence and characterisation of variant red cell sialoglycoproteins in a UK population. Thesis, Lancaster, 1985. Quoted in:Bird GWG. Lectins in immunohematology.Transfus Med Rev 1989; 33-62.

Levene et al.

392 335.

Schnebli HP, Dukor P. Plant agglutinins usedto distinguish between different classesof mouse lymphocytes.Eur J Immunol 1972; 2507-609. 336. Reisner Y,Ravid A, Sharon N. Use of soybean agglutininfor the separation of mouse B and T lymphocytes. Biochem Biophy Res Commun 1976;72: 1585-1591. 337. Reisner Y, Itzicovitch L, Meshorer A, Sharon N. Hemopoieticstem cell

transplantation using mouse bone-marrow and spleen cells fractionated by lectins. Proc Natl Acad Sci USA 1978; 75:2933-2936. 338. Reisner Y,O’Reilly RJ, Kapoor N,Good RA. Allogeneic bone marrow transplantation using stem cells fractionated by lectins: VI. In vitro anlaysis of human and monkey bone marrow cellsfractionated by sheep red blood cells and soybean agglutinin. Lancet1980; 2:1320-1324. 339. Reisner Y,Hodes MB, O’Reilly RJ, Good RA. Enrichment for CRU-C from murine and humanbonemarrowusingsoybeanagglutinin.Blood 1982;59:

,

360-363.

340. Reisner

341.

342. 343. 344. 345.

346. 347.

348.

Y, Kapoor N, Kirkpatrick D, Pollack MS, Dupont B, Good RA, O’Reilly RJ. Transplantation for acute leukemia with HLA-A and B nonidentical parental marrow cells factionated with soybean agglutininand sheep red blood cells. Lancet 1981; 2:327-331. Reisner Y. Kapoor RA, Good RA, O’Reilly RJ. Allogeneic bone marrow transplantation in mouse, monkeyand man using lectin-separatedgrafts. In: Slavin S, ed. Tolerance in bone marrowand organ transplantation. Amsterdam: Elsevier Scientific,1984:293-308. Krolick K A Y Villemez C, Isakson P, Uhr JW, Vitetta ES. Selective killing of normal or neoplastic B cells by antibodies coupled to the A chain of ricin. Proc Natl Acad Sci USA 1980; 775419-5423. Raso V, Ritz J, Basala M, Schlossman SF. Monoclonal antibody-ricin chain conjugation selectively cytotoxicfof cells bearing the common acute lymphoblastic leukemia antigen. Cancer Res 1982; 42:457-464. Seon BK. Specific killing of human T-leukemia cells by immunotoxins prepared with ricin A chain and monoclonal anti-human T-cell leukemia antibodies. Cancer Res 1984; 44:259-264. Sienna S, Villa S, Bonadonna GU, Bregni M, Gianni AM. Ex vivo depletion of human. bone marrow T lymphocytes by soybean lectin fractionation followed bytreatment with anti-pan-T-cell(CDS) ricinA- chain immunotoxin. Transplantation Proc 1987; 19:2735-2737. Morecki S, Pavlotzky F, Margel S, Slavin S. Purging breast cell cancer cells in preparation for autologous bone marrow transplantation. Bone Marrow Transplantation 1987; 1:357-363. Springer GF, Desai PR, Robinson MK, Tegtmeyer H, Scanlon EF. The fundamental and diagnostic role ofT and Tn antigens inbreast carcinoma at the earliest histologic stage and throughout. In: Dao T, Brodie A, Ip C, eds. Tumor markers and their significance in the management of breast cancer.. Prog Clin Biol Res 1986; 204:47-70. Springer GF, Desai PR, Wise W, Carlstedt SC, TegtmeyerH, Stein R, Scanlon EF. Pancarcinoma T and Tn epitopes: autoimmunogens and diagnostic

,

p

Lectin-Blood

349. 350. 351. 352. 353. 354.

393

markers that reveal incipient carcinomas and help establish prognosis. In: Herberman RB, Mercer DW, eds. Immunodiagnosisof cancer, 2nd ed. New York: Marcel Dekker,1990587-612. Levene C, Levene NA, Buskila D, Manny N. Red cell polyagglutination. Transfus Med Rev 1988; 2:176-185. Anglin JH Jr, Lerner MP, Nordquist RE. Blood group-like activity released by human mammary carcinomacells in culture.Nature 1977; 269:254-255. Buskila D, LeveneC, Bird GWG, Levene NA.Polyagglutinationin hospitalized patients: a prospective study. Vox Sang 1987; 52:99-102. Buskila D, LeveneC, Bird GWG, Levene NA. Exposure of cryptantigens on erythrocytes inpatients with breast cancer. Cancer1988; 61:2455-2459. Tibebu M. Polyagglutination in lymphomas, bone marrow transplants and beta thalassemia major. Thesis for Haematology specialization: Hadassah University Medical School, Jerusalem,1987. Levene NA. Polyagglutination in patients with lymphoma [Hebrew]. Thesis for basic sciences, Internal Medicine, Ben Gurion University, Beer Sheva, 1988.


Index

Actinomyces, interaction with concanavalin A, 143 Agglutinin, comparison with lectin,1 Bacillus cereus,interaction with lectins, 40 Bacillus spp, and soybean agglutinin, 144,154

Bacillus subtilis, interaction with lectins, 29-37 Bacterial polysaccharides, complexes with lectins, 267,268 BELLA, biotin-lectin assay, 15 Blood banking, automated and lectins, 369 Blood group(s): antigens, 329 combinations of two antigens, 335 diseases diagnosedby use oflectins, 370

effects of sialidaseon lectin reactivity, 330 lectin reactivity after proteolysis, 330 Lewis, 335 multispecificity of lectin interactions, 334 role ofAB0 in tranfusions, 336

Bone marrow cells, separation by SBA, 370 Boyd and Shapleigh, coined word lectin, 1 Cad: definition, 368 interaction withDBA, 368 Campylobacter spp (Helicobacter spp) Campylobacterfetus and Campylobacterjejuni, 162 interaction of lectins with Campylobacter coli, 162 Candida albicans: adhesion to epithelial cells,180, 181

affinity chromatography of oligosaccharides, 179 binding to Con A and WGA, 59, 177,178

binding to hormones, 180 C3d receptor on, 179 dimorphism, 175 interaction with mammalian lectins, 181

interaction with tulip.lectin,154 lectin typing, 177, 182-185 395

396 [Candidaalbicans] surface glycoproteins, 173, 178 ultrastructure as revealed by lectins, 174 Carbohydrates: anomeric configurations and interaction with lectins,332 associated with bloodgroups, 331 lectin bindingon blood groups, 331 primary and secondary on blood groups, 332 CELLA, colloidal gold-lectin assay,l5 Cell walls: of bacteria, 24 bacteriophage receptors, 24 of fungi, 39 Concanavalin A: affinity purification of teichoic acids, 4,268 bacteriophage binding sites,4,25, 26 on cell surface of Bacillis subtilis, 35-37 complex with glycoproteins of protozoa, 269 detection of microbes,276 ferritin labeled, 39 fluorescein labeled,32-33,75 interaction with Bacillus subtilis, 144 interaction with Envinia amylovora, 159 interaction with Envinia carotovora, 159 interaction with Envinia chrysanthema, 159 interaction with Leishmania, 198, 200,210 interaction with Listeria monocytogenes, 159 interaction with Micrococcus lysodeikticus, 145 interaction with Mycobacterium and Actinomyces, 4, 143 interaction with Mycobacterium bovis, 163

Index

[Concanavalin A] interaction with Mycobacterium chelonei, 163 interaction with Mycobacteriumfortuitum, 163 interaction with Mycobacterium paratuberculosis, 143 interaction with Salmonella abortivoequina, 159 interaction with Salmonella enteritidis, 159 interaction with Shigellaflexneri, 159 interaction with Staphylococcus aureus, 145 interaction with Streptococcus mutans, 145, 146, 150 interaction with trypanosomes, 228 interaction with Yersinia enterocolitica, 159 and linear teichoic acids,8, 18 probe for insertion of cell wall,30, 34 probe for 'fate of wall, 30,34 probe for fungal walls, 39 reactions in gels,18 sorbent for fungal glycoconjugates, 271 Corynebacterium diphtheriae,interaction with Helixpomatia, 146 Discoidin: multifunctional lectin, 2 purification by use of WGA, 274 ELISA, 98,277 ELLA, enzyme-linked lectinosorbent assay, 4,15, 1 4 4 ELLBA, 277 Enterobacteriaceae, 146, 154, 159, 160,165 Epidemiologicalapplications of lectins: Campylobacterjejuni, 163 Haemophilis ducreyi, 163 Neisseria gonorrhoeae,163 I

Index

397

Erwinia: Erwinia amylovora, 160 Erwinia carotovora, 160 Erwinia chrysanthemi, 160 Escherichia coli, 146, 154, 160-161, 165

interaction of detergent sensitive mutants with ConA, 37-39 FACS, use of lectin probes for Leishmania, 204-207 FELLA, fluorescence and lectins, 15 Forssman antigen, saccharides,82, 86

Fungi, interaction with lectins, 39 Glossina morsitans, interaction with lectins, 229 Glycans: affinity chromatography, 87 biosynthesis, 73,76 interaction with HPA, 77 multiantennary complexes, 73-75, 80

N-linked, 72,79,84,96 0-linked, 72,81,84 oligosaccharide composition,72 unusual, 79 Glycoconjugates: microbial, 299 of microorganisms and viruses, 299

Glycoproteins: amylases fromEntamoeba complex, with C o d , 269 from protozoa, 269 . Glycosidases of fungi, complexes with lectins, 273 Glycosyltransferases: cellular, 78 viral specified, 76 Gram stain, lectin determination, 42

Haemophilus ducreyi, 163 Haemophilus influenzae,160

Haemophilus spp agglutination by lectins,128-129 cell envelope, 126 epidemiology and lectins, 127 interaction with lectins,12A-130 Hemagglutination, inducedby lectins, 330,333

Hemagglutinin, from Streptomyces,24 Helixpomatia: Bacillus anthracis,144 Campylobacterfetus, 162 Corynebacteriumdiphtheriae, 146 Listeria monocytogenes, 159 Staphylococcus aureus,145 Streptococcus anginosus,158 Streptococcus,group A, 147 Streptococcus,group B, 147 Streptococcus,group C, 146. 147, 149,163

Streptococcus, group F, 147 Streptococcus,group G, 147 HEMPAS: definition, 367 role in lectin reactivity,368 Herpes simplex virus,164 HIV, antigen detection with lectin, 23, 86

Horseradish peroxidase-lectin, as probe forLeishmania, 204 Hydrophobins, 12 Immunoglobulins, detectionby use of lectins, 368 Insect protozoan, interaction with lectins, 198 Lectin@),151, 153-155,158-163, 165 abbreviations of, Appendix (Chapter 1) advantages in diagnostic microbiology, 164 agglutination of gonococci, 116 agglutination of microorganisms, 143,145,146-149

aggregation ofLeishmania, 198, 208

398

Index

[Lectin(s)l with anti-A activities,346 with anti-B activities,349 with anti-H activities, 339 with anti-Lewis activities,344 with anti-M activities,361 as antimicrobials, 7 with anti-MN properties,361 with anti-N activities,360 applications in microbiology,19-21 as blood group reagents, 4,327-380 bacterial, 302-309 binding to combinations of blood groups, 335 Bombay phenotype, 343 bound to magnetic spheres,23 C-lectins and S-lectins, 7 cytochemistry, 87 definition, 1-3 derivatives, 13 detection ofMN and Ss blood group antigens, 356 detection of I-antigen,351 enzyme-linked sorbent assay, 144 epidemiologic applications, 111 factors governing reactivityof, 19 fluorescent, 144, 149, 152, 159, 164 in general virology,85 glycoconjugates, 213-216 gonococcal serovars, 118-121 hydrophobic effect, 12 identification of HSV,96 as immunefactors, 6 influence on microbial physiology, 40 as insect toxins,7

interaction withGlossina, 229 interaction with Leishmaniaspp.. 200

interaction with Sauroleishmania, 218

interaction with Trypanosoma, 228 isolation and purification, 12 latex microspheres, 149, 151, 156, 157,158

as mediators of adhesion, 22 membrane-particle capture, 149

[Lectin(s)] methylumbelliferyl-conjugated substrate, 148 microbial glycoconjugates, 19 of fungi, 310 of microorganisms,300 of protozoa, 309 of yeasts, 309 patented processes, 165 peroxidase-labeled lectins, 149 peroxidase conjugate, 87 precipitation, 151, 159, 160 probes for cryptantigens and polyagglutination, 362 purification of viral components, 88

reactive carbohydrates,9, 10 . receptors on microbes, 16, 17 screening, 3 sources and functions, 3, Appendix (Chapter 1) specificities, 8, Appendix (Chapter 1) and T-polyagglutination, 362 and Th polyagglutination, 365 and Tk polyagglutination,363 and Tx polyagglutination,365 use in study of P- and LKE antigens, 353 valency, 5 and VA polyagglutination, 365 viral, interaction with glycoconjugates, 300 Lectinophoresis, 15 Lectin sorbents: features and properties,251 for hydrophobic glycoconjugates, 27 1

interaction with microbial glycoconjugates, 252-264 uses instudy of glycoconjugates, 25 1

Legionella, 152, 161 Leishmaniaspp. : agglutination with RCA,193 antigenic determinants, 193 diseases due to, 191 effects of cytotoxic lectins.215

'

Index

399

[Leishmaniaspp.] general properties, 191 lectin reactivity compared with serotyping, 197 lectin-mediated agglutination, 195, 196

lectins and surface topography,204 species and strain dependency of interaction with lectin surface carbohydrates, 202 Lipooligosaccharide(s): gonococcal, 112 interaction with lectins, 113, 115 Lipophosphoglycan, ofhishmania, 194

Lipopolysaccharides, 16 of Campylobacter,162 complexes with lectins,269 mutations, 18 of Neisseria, 161 as 0-antigens, 269 of Pseudomonas, 160 of Salmonella, 159-160 of Shigella, 159 Lipoteichoic acids electron microscopic detection,36, 37

interaction with ConA, 19 purification by use of lectin chromatography, 268 streptococcal group N antigen, 28 Micrococcus lysodeikticus, 145 Microorganisms: lectin-reactive sites, 16 lectin receptors, 17 Moraxella catarrhalis,162 Mycobacterium bovis, 163 Mycobacterium chelonei,163 Mycobacteriumfortuitum, 163 Mycobacterium paratuberculosis, 143

Neisseria: Neisseria gonorrhoeae,161, 162 Neisseria lactamica,162 Neisseria meningitidis,161, 162

Neisseriae: epidemiology and lectins, 116 interactions with lectins,112-124 serovars, 119-122, 125 NOR, definitions, 368 Oral streptococcal lectins,308 Persea americana,inhibits streptococcal adhesion,22 Pneumococcal polysaccharides, interaction with lectins,22 Proteases of fungi, interaction with lectins, 273 Proteins: carbohydrate-dependent epitopes, 93

glycosylation of viral,72 immunological properties and lectins, 93 larvacidal toxin,42 Mycoplasma, 41 purification by use of lectins, 8893

Streptococcusfaecalis, 42 of viruses,68 Proteus mirabilis, 160 Pseudomonas: Pseudomonas aeruginosa, 160,165 Pseudomonas capacia,160 Red cell, separationby use of lectins, 368

RELLA, radiolabelsand lectin assays, 15

RGD, sequence of adhesin,2 Ribitol teichoic acids of staphylococci, 15

Saccharomyces cerevisiae,154 Salmonella, 146, 159, 160 Sauroleishrnania, interaction with lectins, 216 SELLA, saltenhanced lectinosorbent assay, 15 Shigellaflexneri, 159

400

Index

Staphylococcus: coagulase negativeStaphylococcus, 144,145

Staphylococcus aureus,145, 146, 154, 164

Staphylococcus epidermidis, 145 Stillmark, discoverer of lectins,1 Streptococcus Cricetus,glucan-binding lectin, 8 Streptococcus spp, interaction with lectins, 145-159, 163, 164 Streptococcus anginosus(S. milleri), 146, 148, 149 Streptococcus dysgalactiae, 146, 148 Streptococcus equisimilis,146 Streptococcus group A, 146-148, 150,151, 153-157, 164

Streptococcus group B, 146-153, 156-158, 164

Streptococcus group C, 146-149, 151,155-159, 163-164

Streptococcus group D,157 Streptococcus group E,148, 158 Streptococcus group F, 146-148, 151-153,156-157

Streptococcus group G, 146-148, 151-153,156-158

Streptococcus group H,158 Streptococcus group L, 158 Streptococcus group 0 , 158 Streptococcus mutans,154 Streptococcus sanguis,146 Streptococcus zooepidemicus, 145 Sumner and Howell first study of ConA-bacteria interaction, 143 Surface array, lectin probe inCampylobacter. 42 Tannins, 161 Teichoic acids, 144 bacteriophage receptors,24 conformations, 27 interaction with lectins,24,26,267 purification by use of lectins,27,28 ribitol-containing, 268 solubilities of ConAcomplexes,29,30

Transfection and oncogenes, 77 Treponema spp: characteristics, 130 epidemiologic applications of lectins, 132 interaction with lectins, 131-132 surface structures, 130 Triticum vulgaris, see Wheat germ agglutinin (WGA) Tvpanosoma spp: developmental cyclesand lectin reactivity, 222 general characteristics,225 intraspecies differentiation, 228242

lectin reactivity, 228 lectins and interspecific differentiation, 230-238 medically important, 226 Vibrio cholerae,160 Viral glycoconjugates, interaction with lectins, 76 Viral spikes, glycoproteinnature, 69 Viral taxonomy, use of lectins, 70-71 Virus adsorption complex (VAC), 68 Viral infection, inhibitionby lectins, 95

WELLA (WELLBA): glycoprotein ofSpiroplasma, 61 Western blot lectin assay,13, 15, 38,86

Wheat germ agglutinin(WGA) or (Triticum vulgaris), 145, 157, 162

adhesin inhibitor of Chlamydia,22 agglutination of neisseriae,122123

Campylobacterfetus, 162 chitin as receptor, 39 colloidal gold,34, 39 Gram stain, 42 interaction with discoidin,274 interaction withLOS, 113 interaction with viral proteins,85

401

Index

[Wheat germ agglutinin(WGA)] interactions with neisseriae,20 Neisseria gonorrhoeae, 161 Neisseria meningitidis, 161 probe for fungal walls, 39 probe for staphylococcal teichoic acid, 34,38

[Wheat germ agglutinin(WGA)] as sorbent for protozoan glycoconjugates, 270 as sorbent for teichoic acids, 268 Staphylococcus aureus, 145

Yersinia enterocolitica,159

Lectin-Microorganism Interactions

Interactions I

Fundamental Interactions

Affective Interactions

Fundamental Interactions

Designing Interactions

Empowering Interactions

Virus-Insect-Plant Interactions

Particle-Lung Interactions

Spacecraft-Environment Interactions

Aerosol-cloud-climate interactions

Spacecraft-Environment Interactions

Laser-Plasma Interactions

Fluid-Structure Interactions

Interactions in ultracold gases

Protein-Solvent Interactions

Atom-photon interactions

Stockley's Drug Interactions

Membrane Protein-Cytoskeleton Interactions

Drug-Nucleic Acid Interactions

Classroom Interactions in Literacy

Sound-Flow Interactions

Modulational interactions in plasmas

Drug-Membrane Interactions

Photon-hadron interactions

Protein-Solvent Interactions

Proboscidean and Paleoindian interactions

Stockley's Herbal Medicines Interactions

Metal Complex - DNA Interactions

Lectin-Microorganism Interactions

Interactions I

Fundamental Interactions

Affective Interactions

Fundamental Interactions

Designing Interactions

Empowering Interactions

Virus-Insect-Plant Interactions

Particle-Lung Interactions

Spacecraft-Environment Interactions

Aerosol-cloud-climate interactions

Spacecraft-Environment Interactions

Laser-Plasma Interactions

Fluid-Structure Interactions

Interactions in ultracold gases

Protein-Solvent Interactions

Atom-photon interactions

Stockley's Drug Interactions

Membrane Protein-Cytoskeleton Interactions

Drug-Nucleic Acid Interactions

Classroom Interactions in Literacy

Sound-Flow Interactions

Modulational interactions in plasmas

Drug-Membrane Interactions

Photon-hadron interactions

Protein-Solvent Interactions

Proboscidean and Paleoindian interactions

Stockley's Herbal Medicines Interactions

Metal Complex - DNA Interactions

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