Lectins: Vol. 3 Proceedings of the Fifth Lectin Meeting Bern, May 31–June 5, 1982 [Reprint 2020 ed.] 9783112315941, 9783112304792

Citation preview

Lectins Biology, Biochemistry, Clinical Biochemistry Volume 3

Lectins

Biology, Biochemistry, Clinical Biochemistry Volume 3 Proceedings of the Fifth Lectin Meeting Bern, May 31 - June 5,1982 Editors T. C. Bog-Hansen G. A. Spengler

W DE G Walter de Gruyter • Berlin • New York 1983

Editors Thorkild Christian Bog-Hansen, cand. scient., lie. techn. The Protein Laboratory University of Copenhagen Sigurdsgade 34 DK-2200 Copenhagen N Georg Alexander Spengler, PD Dr. med. Institute for Clinical Protein Research University of Bern Tiefenauspital CH-3004 Bern

Library of Congress Cataloging in Publication Data Library of Congress Cataloging in Publication Data Lectin Meeting (5th: 1982: Bern, Switzerland) Proceedings of the Fifth Lectin Meeting, Bern, May 31 - June 5,1982. (Lectins-biology, biochemistry, clinical biochemistry; v. 3) Bibliography: p. Includes indexes. 1. Lectins-Congresses. I. Bog-Hansen, T. C. (Thorkild Christian), 1939 . II. Spengler, G. A. (Georg Alexander), 1928. III. Title. IV. Title: Proceedings of the 5th Lectin Meeting. V. Series. QP552.L42L4 1982 599'.029 83-5134 ISBN 3-11-009504-1

CIP-Kurztitelaufnahme der Deutschen Bibliothek Lectins, biology, biochemistry, clinical biochemistry: proceedings of the . . . Lectin Meeting. - Berlin; New York: de Gruyter. NE: Lectin Meeting Vol. 3 = 5 . Bern, May 31 - June 5,1982. - 1983. ISBN 3-11-009504-1

Copyright © 1983 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm or any other means nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Gerike, Berlin. - Binding: Dieter Mikolai, Berlin. - Printed in Germany.

Preface

At the end of the Fourth Lectin Meeting in Copenhagen in June 1981 we decided that future meetings should be organized at varying locations and on a collaborative basis.

Therefore, from

May 31 through June 5, 1982, the Fifth International Lectin Meeting 'INTERLEC 5' was held in Switzerland in cooperation of the Universities of Copenhagen and of Bern. Over 100 participants attended the following sessions:

Detec-

tion of Cancer Markers; Biological Function and Biological Effects of Lectins; and Lectins as Specific Tools.

Eighty five

communications (lectures and poster presentations) were given, the majority of which appears in this volume.

Although the

scope of topics in the present book is wide again - as it should be from a meeting covering the whole range of recent progress in the rapidly developing field of lectins - it is noteworthy that a great many contributions were centered on interactions of lectins with normal or malignant cells, opening new and exciting aspects for the application of these natural tools, e. g. in transplantation immunology and tumor diagnosis. In addition to the scientific sessions, the Third Meeting of the International Working Party on Standardization of Lectins for Diagnosis was held, and a test program for con A from different sources was set up. After four successive meetings in Copenhagen, it was indeed a challenge to organize 'INTERLEC 5' in Bern. It was for the large number and the scientific quality of the presentations, as well as for the high spirits that prevailed throughout the entire meeting that this event was particularly rewarding and unforgettable for the organizers. We wish to express our sincere appreciation to all participants of 'INTERLEC 5' and to thank Dr. B.Nordbring-Hertz, Lund, for her helpful assistance in planning the meeting.

VI

'INTERLEC 5' was generously supported by the following institutions and companies (in alphabetical order):

The Authorities

of the State and of the City of Bern; Behringwerke AG, Marburg, Lahn; Boehringer Mannheim GmbH, Hamburg; Koechst-Pharma AG, Zurich;

Medac GmbH, Hamburg; Miles Italiana S.p.A., Cavenago

Brianza, Milano; Riact, Copenhagen.

Their help has been greatly

appreciated. 'INTERLEC 5' will be held in 1984, and we look forward to meet you again at our next lectin meeting. Copenhagen and Bern, January, 1983 T.C.B0g-Hansen and G.A.Spengler

VII

Obituary

During preparation of this volume, we recieved notice that one of the devoted participants of the INTERLEC meetings had unexpectedly deceased.

Prof.Dr.med. Gunther Hermann, Docteur de

l'Université de Paris, mention Sciences, Chief of the Immunological Division at the Surgical University Hospital in Cologne, was born September 20, 1924, in Stuttgart and died August 20, 1982, in Avignon. We mourn for Gunther Hermann and we shall always remember him as a distinguished scientist and a devoted friend of the lectinologic community. Copenhagen and Bern, January, 1983 T.C.B0g-Hansen and G.A.Spengler

T A B L E

OF

C O N T E N T S

INTRODUCTION: DEFINITION OF LECTIN A note on the recent discussion on definition of the term "lectin" J. Kocourek, V. Horejsi

3

PART I. BIOLOGICAL EFFECTS OF LECTINS Mechanisms of induction of transplantation tolerance in mice by lectin treatment I. Hilgert, V. Horejsi, H. Kristofova, P. Angelisova

9

The effect of lentil lectin treatment on skin allograft survival in mice and rats W. Harel, D. Nelken

17

Possible involvement of lectins in bacteria-induced histamine release in intrinsic asthma P. Stahl Skov, C. Jensen, S. Norn, T. C. B0g-Hansen, H. L®wenstein, C. Koch, H. Permin, N. H«iiby

27

T cell stimulating lectins are mitogen inducers but not mitogens B. M. Stadler, J. Liechti, G. D. Bonnard

37

Membrane alterations and induction of responsiveness to interleucin 2 in lymphocytes by lima bean lectins W. G. Bessler, H. Kraut, D. Busing, W. Muller-Hermes, H. Peters

45

T lymphhocyte-mediated cytolysis. and non-mitogenic lectins mediate interactions? G. Berke

55

IV. How do mitogenic lymphocyte-target

Lectin coated beads stimulate single cells from mouse embryos to spread S. J. Kimber, M. Azim, H. Surani

63

X Lectin-mediated endocytosis of Helix pomatia lectin by Zajdela hepatoma cells K. W. Lempfrid, H.-P. Weil, G. Hermann

73

PART II. LECTINS AS SPECIFIC TOOLS Lectins diagnostic G. Coggi,in P. Dell'Orto,pathology E. Bonoldi, P. Doi, G. Viale

87

Reactions with lectins on benign and malignant lesions of the human breast. Relation with the presence of differentiation antigens of the mammary gland P. Hageman, L. Bobrow, M. A. van der Valk, W. Misdorp, J. Hilkins

105

Ultrastructural study on the presence of receptors for several lectins in human breast cancer J. Calafat, H. Janssen

119

Ultrastructural localization of peanut agglutinin receptors on cells of human mammary tumors J. Calafat, P. C. Hageman, H. Janssen

131

Endocrine therapeutical studies on carcinogen-induced rat mammary carcinomas characterized by hormone-dependent lectin-binding sites M. Vierbuchen, P. J. Klein, S. Rossel, J. Fischer, G. Uhlenbruck

145

The characterization of milk proteins by lectins and antibodies in hormone dependent breast cancer P. J. Klein, M. Vierbuchen, J. Fischer, G. Farrar, G. Uhlenbruck

157

Histochemical and biochemical characterization of glycoprotein components in normal gastric mucosa, intestinal metaplasia and gastric cancers with lectins J. Fischer, P. J. Klein, M. Bierbuchen, R. Fischer, G. Uhlenbruck

167

Affinities of Ricin 120 and misteltoe lectin 1 for membrane components of liver and nervous tissues B. K. Groeger, L. G. Williams, J. Pigott, P. Ziska, D. S. 0'Dell, D. J. Williams, H. Franz, P. L. Debbage

179

Stability and steric hindrance of lectin-labelled gold markers in transmission and scanning electron microscopy M. Horisberger, M. Tacchini-Volanthen

189

Lectin binding sites on semithin sections of epoxy-embedded tissues G. Viale, P. Dell'Orto, R. Colombi, V. De Gennaro, A. Comi, G. Coggi

199

XI

Comparison of lectin binding sites in the kidneys of different animal species H. Holthofer, A. Miettinen, I. Virtanen

205

Receptors for Helix pomatia lectin on normal human lymphocytes and lymphocytes from patients with chronic lymphocytic leukaemia G. Hermann, J. Lembke, H.-P. Weil, D. Gerecke

213

Lectin binding patterns of normal and leukaemic and myeloid cells M. Harding, D. Crowther, J. T. Gallagher

225

lymphoid

The binding and stimulation of human leukocytes by the five pokeweed lectins M. J. Waxdal, B. F. Haynes, B. J. Fowlkes, C. T. Thomas, B. J. Mathieson, A. Fauci

235

The use of Pa-4 for the identification and removal of monocytes from human peripheral blood mononuclear cells C. Murre, M. J. Waxdal

243

Analysis of subpopulations of human peripheral blood leukocytees and splenic cells isolated by peanut agglutinin B. J. Mathieson, B. F. Haynes, M. J. Waxdal, B. J. Fowlkes, C. A. Thomas, S. 0. Sharrow

251

Detection and characterization of murine thymocyte delineated by lectins and flow microfluorometry B. J. Fowlkes, B. J. Mathieson, M. J. Waxdal

263

subsets

The late appearance of a PNA "dull" subset in the mouse thymus development D. Drakeford, W. M. Leiserson, S. 0. Sharrow, B. J. Mathieson

275

Characterization of peanut agglutinin receptors as possible expression sites of sialic acid during the maturation of mouse thymocytes J. Favero, J.-C. Bonnafous, J. Dornand, R. L. O'Brien, J.-C. Mani

283

Effect of 32 purified animal and plant lectins on human T lymphocytes F. Licastro, M. Chiricolo, L. Barbieri, F. Stirpe, A. Falasca, C. A. Rossi, C. Franceschi

293

Lectins as probes of blood platelet activity K. J. Clemetson, J. L. McGregor, E. F. Luscher

303

Sialic acid as a cell surface binding site for wheat germ agglutinin J. T. Gallagher, M. Harding, R. E. Dale

311

XII

Interaction of human young and old erythrocytes with WGA. A fluorescence study D. Bladier, M.-A. Deugnier, M. Caron

319

Labeling of rat spermatogenic cells with fluorescein isothiocyanate conjugated lectins R. Malmi, K.-O. Soderstrom, K. Karjalainen

327

Affinity of ten insolubilized lectins towards various glycopeptides with the N-glycosylamine linkage and related oligosaccharides H. Debray, A. Pierce-Cretel, G. Spik, J. Montreuil

323

Herpesvirus glycoproteins from wild-type and ricinresistant BHK cells. Characterization of glycopeptides by conA-Sepharose F. Dall'Olio, F. Serafini-Cessi, M. Scannavini, G. Campadelli-Fiume

351

Con-A-reactivity of serum and leucocyte alpha-1 anti-trypin. Electrophoretic heterogeneous alpha-1 antitrypsins studied by crossed affinity Immunoelectrophoresis. M. M. Andersen, S. Noack

361

Seminal acid phosphatase. chromatography M. Iwasa, K. Sagisaka

371

Purification by lectin affinity

Isolation and characterization of Poa pratensis acid phosphatases interacting with conA I. Lorenc-Kubis

379

WGA affinity Immunoelectrophoresis of glycophorin from human erythrocyte membranes. Capacity measurements of WGA gel and binding constant determination N. H. H. Heegaard, U. Christensen, 0. J. Bjerrum

387

Dissociation constants for concanavalin A at various pH values studied by affinity electrophoresis K. Takeo, M. Fujimoto, A. Kuwahara

397

Rapid methods for the demonstration of sugar residues in tissue extracts, fluids and lectins in plant extracts J. Kohn, J. Raymond, A. Voller, P. Turp

405

Hemadsorption lectin test (HALT): A simple sensitive and fast solid phase method for studying lectins J. A. Kint

415

PART III. ANIMAL LECTINS Endogeneous lectins on human peripheral leukocytes C. Kieda, M. Monsigny, M. J. Waxdal

monomuclear 427

XIII I n v o l v e m e n t of a b e t a - g a l a c t o s e - s p e c i f i c m a c r o p h a g e in binding and phagocytosis of e r y t h r o c y t e s E. Muller, C. Schroder, M. W. Franco, A. K. Shukla, R. Schauer

lectin 435

Isolation of a m e m b r a n e lectin from the m a r m o s e t cell 70N2 J. M. Decker

line 443

Studies on Lectins LV. Subcellular location of an endogenous lectin in fish oocyte J. Nosek, A. Krajhanzl, J. Kocourek

453

Lectins from tunicates and cyclostomes. characterization G. R. Vasta, J. J. M a r c h a l o n i s

461

A biochemical

Is the selectivity of proteid yolk precusor uptake insects based on a lectin-type binding reaction? D. Stynen, A. De Loof PART IV. BACTERIAL

in 469

LECTINS

Bacterial surface lectins T. W a d s t r o m , T. J. Trust, D. E. Brooks

479

The biological functions of Pseudomonas a e r u g i n o s a N. Gilboa-Garber

lectins

N o n m a n n o s e - s p e c i f i c lectins on e n t e r o t o x i g e n i c E. coli rocognizing different g l y c o c o n j u g a t e s T. W a d s t r o m , A. Faris, M. Lindahl, B. Lonnerdahl, B. Ersson PART V. PLANT

495

503

LECTIN

D i s t r i b u t i o n and function of the lectin in the genus glycine S. G. Pueppke

120,000 dalton

soybean 513

M a n n o s e - s p e c i f i c lectins and the r e c o g n i t i o n of pea roots by Rhizobium leguminosarum J. W. Kijne, I. A. M. van der Schaal, C. L. Diaz, F. van Iren

521

Pea lectin binding by Rhizobium I. van der Schaal, J. Kijne, C. Diaz, F. van Iren

531

Possible role of lectins in binding rhizobia to host B. Solheim

roots

P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of an endogenous root lectin from Pisum sativum L. M. Hosselet, E. van Driessche, M. van Poucke, L. Kanarek

538

.. 549

XIV

Lectin release from imbibed soybean seed and its possible function H. Causse and P. Rouge

559

Structural relationships and properties of legume lectins L. M. Shannon

573

Speculations about a physiological role of some plant lectins W. Peumans, H. Stinissen, A. Carlier

583

In vivo synthesis and processing of cereal H. Stinissen, W. Peumans, A. Carlier

593

lectins

The alpha and beta subunits of pea lectin are the result of a post-translational cleavage of a precursor chain M. Lauwereys, E. van Driessche, A. Strosberg, R. Dejaegere, L. Kanarek

604

Light and electron microscopic localization of lectins in plant cells and tissues J.-F. Manen, A. Pusztai

611

A s p e c t r o p h o t o m e t r y study of the carbohydrate site of peanut lectin J. Ohanessian, M. Caron

623

binding

A critical study on the purification of potato lectin (Solanum tuberosum L.) E. van Driessche, S. Beeckmans, R. Dejaegere, L. Kanarek .. 629 A galactomannan precipitating lectin in Sphenostylis stenocarpa seeds T. Animashaun

639

Further investigations on misteltoe lectin I (ML I): Effect of A-chain on cell-free protein synthesis H. Franz, A. Kindt, P. Ziska, H. Bielka, R. Renndorf, L. Venker

645

Purification and partial characterization of a Streptococcus mutans cell-agglutinating substance from Caucus carota B. Matiasson, M. Ramstorp, P. Carlsson, D. Bratthall

653

APPENDIX Red cell ghosts: A tool for purification of lectins A. Faure, J. Delrieu, M. Caron

661

The molecular basis of bacterial adherence to epithelial cells Y. Eshdat, N. Sharon

667

XV Changes in lectin receptors during lymphocyte differentiation: Application to bone marrow transplantation Y. Reisner

677

Report on the third session of the International Working Party on Standardization of Lectins for Diagnosis T.C.Btfg-Hansen

699

AUTHOR INDEX

701

SUBJECT INDEX

705

INTRODUCTION DEFINITION OF LECTIN

A NOTE ON THE REGENT DISCUSSION ON DEFINITION OS1 THE TERM "LECTIN" Jan Kocourek, Vaclav Horejsi Department of Biochemistry, Charles University, Albertov 2030, 128 40 Praha 2, Czechoslovakia

Recent years brought a rapid increase of our knowledge of lectins. In spite of their widespread occurrence in Nature, their extensive study as model ligand-binding proteins and their frequent use as specific tools in biological research, at present there exists some ambiguity as to the accurate delineation of the term "lectin". The original Boyd's definition (1) is rather broad and at present it seems necessary to re-define the term more specifically. Efforts in this direction were initiated by Goldstein et al. (2) who stressed sugar-binding and cell agglutinating properties as a basis for the definition. Their definition supplemented by several comments was also published by the Nomenclature Committee of IUB in its Newsletter 1981 (3). The comments specify in a more detail the scope of the definition; sugar-binding proteins with a single binding site are not considered as lectins, whereas sugar-specific enzymes, transport proteins and toxins may be classified as lectins, provided they have multiple sugar-binding sites. The authors of the present paper have suggested a different definition (4), in which emphasis is put on the sugar-binding properties and the lack of enzymatic activity toward carbohydrate structures, whereas the number of sugar-binding sites or agglutination/precipitation activities are not considered. The Nomenclature Committee of IUB has given preference to the Goldstein's et al. definition (5) essentially because they considered the criterion of cell agglutination

© Walter de Gruyter & Co. 1983, Berlin • New York Lectins, Vol. Ill

4

and/or glycoconjugate precipitation as operational and practical to apply; our definition was found too broad and the criterion of absence of enzymatic activity difficult to apply. However, we still believe that the definition should be based solely on the carbohydrate-binding and not on agglutination or precipitation properties and, also, that sugar-specific enzymes and transport proteins (as well as carbohydrate-specific immunoglobulins) should not be considered as lectins, mainly because: (1) Cell agglutination is a complex phenomenon depending on the properties of the protein, the cells and of the medium used. Thus the selection of agglutinable cells and optimum conditions may be difficult. Moreover, cell agglutination does not represent - at least in a vast majority of cases the true physiological activity of lectins. This renders the agglutination-criterion both impractical for general use and rather artifactitious. (2) Exclusion of monovalent carbohydrate-binding proteins is not logical as they may be structurally and functionally closely related to other multivalent proteins or they may form multivalent aggregates. (3) Carbohydrate-specific enzymes and transport proteins (regardless of the number of their binding sites) constitute distinct groups of proteins with well defined specialized functions; their classification as lectins might lead to confusion. (4) The definition should reflect the assumed (and in many cases well documented) carbohydrate-recognition functions of lectins in vivo. Thus, we suggest a modified version of our original definition (4) which is based on the above considerations: Lectins are proteins of non-immunoglobulin nature capable of specific recognition and reversible binding to carbohydrate moieties of complex carbohydrates without altering covalent structure of any of the recognized glycosyl ligands.

5

Comments s 1. A lectin molecule contains one or more sugar-binding sites; lectins may (but do not have to) agglutinate cells or precipitate glycoconjugates as a consequence of binding to their glycosyl moieties. 2. Lectins may possess various biological activities - they can be toxins, hormones etc.; they can be enzymes activity of which is not directed towards sugar molecules. 3. Lectins will usually bind sugars (i.e. low-molecular-weight carbohydrates) which specifically inhibit their interaction with soluble or cell-bound glycosylated macromolecules. 4. Lectins may occur in any type of organisms; they may be soluble or membrane-bound, may be glycoproteins or other posttranslationally modified protein derivatives. 5. Carbohydrate-specific immunoglobulins and any carbohydrate-specific enzymes are not to be classified as lectins. Also carbohydrate-binding proteins complexing only with free sugars but not with complex carbohydrates (some transport proteins, chemotaxis receptors, repressors of operons of carbohydrate metabolism etc.) are not lectins.

References 1.

Boyd, W.C. in The Proteins, Vol. 2, Part B (Neurath, H. and Bailey, K., Eds.), pp. 756-844, Academic Press, New York 1954.

2.

Goldstein, I. J., Hughes, R. C., Monsigny. LI., Osawa, T., Sharon, N. : Nature (London) 28£, 66 (1980).

3.

Newsletter 1981 of the Nomenclature Committee of IUB (NC-IUB) and IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN): Eur. J, Biochem. 114. 1-4 (1981; Arch.Biochem. Biophys. 206, 458-462 (l9Sl5; Hoppe-Seyler's Z. Physiol. C h e m . 1 % 2 . I-IV (1981); J. Biol. Chem. 256. 12-14 (1981).

4.

Kocourek, J., Hofejsi, 7. : Nature (London) 290, 188 (1981).

Dixon, H. B. P, : Nature (London) 2%2, 192 (1981).

PART I BIOLOGICAL EFFECTS OF LECTINS

MECHANISMS OF INDUCTION OF TRANSPLANTATION TOLERANCE IN MICE BY LECTIN TREATMENT Ivan Hilgert, Väclav Horejsi, Hana Kristofovä, Pavia Angelisovä Institute of Molecular Genetics, Czechoslovak Academy of Sciences, Videnskä 1083, Praha 4, Czechoslovakia

Administration of some lectins, e.g. Con A or PHA in vivo, results in prolongation of skin or organ graft survival time in such lectin treated recipients (1, 2). Lentil lectin (LCA) was found to be especially efficient in induction of transplantation tolerance in mice (3). In our initial experiments we were able to induce permanent survival of the B10.D2 mouse strain skin grafts in B10.D2(M504) recipients (abbreviated designation M504) after a twenty-dose treatment with LCA. This remarkable effect qualifies LCA as one of the most efficient immunosuppressive (or tolerogenic) substances known so far; a recent study indicates that the efficiency of LCA in rats can be still markedly increased by combination with antithymocyte serum (Harel and Nelken, these proceedings). Therefore, it was of interest to study in more detail the mechanisms underlying these in vivo effects. We tried to answer especially the following questions: (1) What is the immunosuppressive efficiency of LCA in various genetically well defined mouse strain combinations with histocompatibility disparity of different strength? What is the most efficient schedule of lectin treatment? (2) What is the effect of LCA on various functional characteristics of the recipient's lymphocytes? (3) What is the role of active suppressor cell mechanisms in this type of immunological tolerance? (4) What is the fate of LCA in the organism? Has LCA special affinity toward some tissues?

© Walter d e Gruyter & Co. 1983, Berlin • N e w York Lectins, Vol. Ill

10 Materials and methods LCA and Con A were prepared by affinity chromatography on Sephadex G-100 (4, 5).

Two to three months old female mice of

various inbred strains were obtained from the animal house of our Institute.

Skin grafting was performed by the standard Q method (6). In adoptive transfer experiments 10° spleen cells from the tolerant animals were taken 10 days after the last LCA dose and injected into sublethally irradiated recipients which were subsequently given a skin graft. LCA doses administered i.p. or i.v. were dissolved in 0.5 ml saline, the s.c. doses in 0.1 ml saline. Cytotoxicity of LCA toward spleen cells was estimated in vitro by the 51Cr-release test (7). Stimulation of spleen cell suspensions with LCA was measured by incorporation of ^H-thymidine. Mixed lymphocyte cultures (MLC) consisted usually of 3 components: responder spleen cells, stimulator (either 20 Gy irradiated allogeneic cells or a lectin) and modulator cells. Total culture volume was 0.2 ml; cultures were harvested 48 hours after a 16 h pulse with 36 kBq of H thymidine and mean cpm of cell incorporated H were measured. Stimulation index was calculated as a ratio of cpm from experimental culture to control unstimulated (or syngeneic cell "stimulated") culture. As a rule, MLC consisted of 10® stimulator cells, 5 x 10"* - 10® responder cells and 5 5 x 1 0 modulator cells. Modulator cells were prepared by cultivation of 1.5 x 10 /ml spleen cells for 40 hours in the presence of lectins (0.5 - 25 yg/ml) or without lectins (controls). After culture the cells were washed with 0.3 M methyl-a-D-glucopyranoside and pretreated with mitomycin C (50 yg/ml, 30 min, 37 C) .

Mitomycin treatment was omitted in the case of modula-

tor cells which were subsequently used in the cultures tested for cell mediated cytotoxicity (CMC) development.

Cultures

for generation of cytotoxic lymphocytes were 51 performed essentially according to Ferguson et al. (8), Cr-labelled EL-4 tumor cells were used as target cells.

The target vs. effector

11

c e l l r a t i o was 1:20 - 1 : 2 . LCA and bovine serum albumin 131 were l a b e l l e d by I using the chloramine T method ( 9 ) . r a t e of elimination of these r a d i o l a b e l l e d proteins a f t e r j e c t i o n i n t o mice was estimated by the whole-body measurement at v a r i o u s i n t e r v a l s .

(BSA) The in-

radioactivity

D i s t r i b u t i o n o f the

labelled

p r o t e i n s w i t h i n the body was f o l l o w e d a f t e r k i l l i n g the a n i mals

(24 hours a f t e r the i n j e c t i o n o f the l a b e l l e d

protein),

d i s s e c t i o n and s e p a r a t e measurement o f r a d i o a c t i v i t y i n d i f f e r ent organs and t i s s u e s . TABLE 1 Mouse s t r a i n

combinations used (donor -

recipient)

2R - B10.A

15

A.TL - A.TH

2

B10.AKM - B10.BR

16

A.TH - A.TL

3

B10.BR - B10.AKM

17

B10.AQR - B10.AKM

4

R103 - M504

18

B10.AKM - B10.AQR

1

5

B10.D2 - M504

19

B10.AQR - 4R

6

R103 - B10.D2

20

B10 - B10.A

7

B10.D2 - R103

21

B10.HTT - B10.D2

8

B10.A - B10.BR

22

B10.D2 - B10.A

9

B10 - 5R

23

B10.HTT - B10.A

10

B10.A - 4R

24

3R - B10.A

11

4R - B10.A

25

5R - B10.A

12

4R - B10.AQR

26

4R - B10

13

B10.AQR - B10.DBA/1

27

B10.AQR - B10.A

14

B10.DBA/1 - B10.AQR

28

B10.A - B10.AQR

Results E f f i c i e n c y o f LCA i n d i f f e r e n t s t r a i n combinations. ent mouse s t r a i n combinations w i t h v a r i o u s g e n e t i c at the H-2 l o c i were t e s t e d

(Table

1).

28 d i f f e r disparities

12

The efficiency of LCA was clearly dependent on the nature of antigenic disparity between the donor and recepient. In some strain combinations (No. 1-12, Table 1) LCA treatment resulted in permanent survival of the grafts (>100 days) in 80-100% recipients if LCA was administered daily (1 mg, i.p., the doses on the days 1, 3, 5, 7, 9 and 11 were given i.v., in animals bearing the grafts for more than 40 days the lectin was administered alternatively i.p. and s.c. until the rejection of the graft). In all these cases the difference between the donor and recipient is at the H-2DL locus only (No. 1-7) or H-2DL+ some of the H-2J, E, C, and S loci (No. 8-11); only in strain combination No. 12 the donor and recipient differ at the H-2K, J, C, S, DL loci. In other strain combinations involving differences at multiple H-2 loci including H-2K and/or H-2I (No. 13-26) or H-2K only (No. 27, 28) LCA treatment resulted always in marked prolongation of graft survival times as compared to controls (2-4 fold increase of the mean survival time depending on the particular strain combination but permanent survival of skin grafts was achieved only exceptionally. Thus, LCA treatment can be used to overcome the H-2DL antigenic barrier but is less efficient in the case of H-2K or H-2A antigenic disparities.

Cellular mechanisms involved in maintenance of LCA-induced transplantation tolerance.

Involvement of active suppressor

cell-mediated mechanisms was tested by adoptive transfer experiments.

B10.D2 grafts survived permanently in 70% of M504

recipients which were sublethally irradiated and reconstituted Q

with 10 spleen cells from M504 recipients bearing B10.D2 grafts as a result of LCA treatment. Only approximately 10% of R103 strain grafts survived permanently in such reconstituted recipients indicating specificity of the state of tolerance but also some degree of nonspecific inhibitory mechanisms involved. Transplantation of third-party test allografts (R103) onto recipients bearing a B10-D2 graft for >100 days

13

resulted in a rapid rejection of these grafts without affecting the survival of the original B10.D2 grafts. Effect of LCA on spleen cells in vitro. Mitogenic stimulation of murine spleen cells with LCA was typically dose-dependent (maximum stimulation index approximately 51 at 5 yg/ml in a 5% serum containing medium). The apparent decrease of mitogenic activity at higher concentrations was at least in part due to the cytotoxicity of LCA (approximately 65% of cells were killed after a 24 hours culture in the presence of 0.5 mg/ml LCA; virtually all cells were killed by Con A under similar conditions). Mitogenic response to LCA and Con A of the spleen cells obtained from the M504 mice which were given single 1 mg i.p. dose of LCA (48 hours before testing) was decreased by 93% as compared to untreated controls. There was essentially no mitogenic response in the case of spleen cells from the animals which received a 10-dose LCA treatment (each i.p. dose 1 mg, administered daily, treatment ceased 48 hours before testing). Spleen cells from the B10.A mice which received single 1 mg i.p. dose of LCA manifested a higher proliferative response (1.7 fold as compared to untreated controls) after stimulation with allogeneic B10 cells in MLC. The effects of modulator cells (i.e. spleen cells pre-cultured in the presence of the lectin) on several in vitro characteristics of murine spleen cells were tested. There was a marked difference between the effects of B10.A modulator cells generated in vitro either by LCA or Con A. Whereas addition of Con A-generated modulator cells suppressed the mitogenic response of syngeneic responder cells to LCA stimulation (by 55%), LCA-generated modulator cells enhanced this response (7.5 fold). Similarly, Con A-induced B10.A modulator cells suppressed, whereas LCA-generated modulator cells enhanced the blastogenic response of syngeneic responder cells in MLC toward allogeneic B10 stimulator cells (Hilgert et al., to be published). Both Con A- and LCA-induced modulator cells

14

suppressed the development of CMC in MLC (by 59% and 65%, respectively, as compared to the cultures containing "modulator" cells pre-cultivated in the absence of any lectin). Similar effect (40-45% suppression of CMC) was observed also when modulator cells generated in vivo were used, i.e. the cells obtained from the spleens of animals pretreated with a single 1 mg LCA dose 2 days before testing or after a 10-dose LCA treatment (ceased 6 days before testing). Distribution of LCA in vivo and the rate of its elimination. 131 The rate of elimination of I-labelled LCA was similar to 1 31 that of I-labelled BSA (approximately 80% of a 1 mg dose administered i.v. was eliminated after 48 hours; LCA administered i.p. or s.c. was eliminated approximately 2-3 times more slowly). Both LCA and BSA were distributed relatively homogeneously throughout the body but LCA possessed increased affinity for spleen, lymph nodes and lung (relative concentration per mg of the tissue was 3.1, 1.5,and 2.9 times higher in these organs, respectively, than the mean concentration in the body), whereas the level of BSA was elevated in blood only. Discussion Our results provide at least to some extent the answers to the questions raised in Introduction.

First, it seems clear

that the immunosuppressive efficiency of LCA depends on apparent strength of histocompatibility barrier between the donor and recipient.

The H-2DL antigenic disparity can be relative-

ly easily overcome by LCA treatment, whereas H-2K and H-2A antigens represent stronger histocompatibility barrier;

the

effects of non-H-2 histocompatibility antigens were not followed in this study.

Interestingly, long-term tolerance could be

induced only in the B10.D2 - M504 (mutant) strain combination by a limited number of LCA doses.

Two in vitro observed

15

effects may be directly related to the in vivo efficiency of LCA: first, cytotoxicity of LCA at higher concentrations might cause partial depletion of alloreactive cells. Second, the suppressive effect of LCA-generated modulator cells on CMC development might indicate that also in vivo development of alloreactive cytotoxic T cells may be inhibited. This conclusion is also in agreement with our experiments on adoptive transfer of LCA-induced tolerance. This suppressor cell mechanism may be sufficiently efficient for maintenance of B10.D2 graft survival on the M504 recipient after a 20-dose LCA treatment, but the necessity for continuous supply of LCA in other strain combinations tested (with apparently stronger histocompatibility barrier) seems to indicate, that suppressor cells induced in such recipients are not able to "manage" the rejection reaction and that LCA might also block directly some membrane bound or soluble complex carbohydrate molecule(s) taking part in the rejection reaction. The lectin might bind to the histocompatibility antigens of the graft and thus block their recognition. We cannot rule out this effect of LCA in some strain•combinations (it might be in agreement with our results on the rate of LCA elimination, which also indicate the necessity for continuous application of LCA to maintain its high (blocking?) level in organism) but the experiments on retransplantation of long-term tolerated B10.D2 grafts onto untreated M504 recipients, transplantation of grafts presoaked in LCA solution or transplantation of grafts from LCA pretreated donors did not reveal apparently any modification of normal immunogenicity of such "LCA-pretreated" grafts (I. Hilgert, unpublished results). Increased affinity of LCA for spleen and lymph nodes (and lung) may support rather the notion that the recipient's immune system is the prime target of LCA action.

In summary, induction of suppressor cells inhibiting graft rejection reaction and direct cytotoxicity seem to be involved in immunosuppressive activity of LCA observed in vivo.

Signi-

16

ficance of other possible effects such as direct blocking of some cell surface or soluble molecules by LCA and some kind of antigenic modulation of the graft remains to be evaluated as well as the roles of antigen-specific and non-specific suppressor mechanisms.

References 1.

Markley, K., Thornton, S., Smallman, E., Markley, P.: Transplantations 8, 258-264 (1969).

2.

Rosenau, W., Haber, J., Goldberg, M.: 624-626 (1972).

3.

Hilgert, I., Horejsl, V. , Angelisovâ, P., Kristofovâ, H.: Nature (London) 284, 273-275 (1980).

4.

Tichä, M., Entlicher, G., Kostlr, J., Kocourek, J.: Biochim. Biophys. Acta 221, 282-289 (1970).

5.

Agrawal, B.B.L., Goldstein, I.J.: 147, 262-271 (1967).

6.

Billingham, R.E., Medawar, P.B.: 392 (1951).

7.

Wigzell, H.:

8.

Ferguson, R., Anderson, S.M., Schmidtke, J.R., Simmons, R.L.: J. Immunol. 117, 2150-2157 (1976).

9.

Hunter, W.M., Greenwood, F.C.: 496 (1962).

Transplantation 13,

Biochim. Biophys. Acta J. Exp. Biol. 28, 385-

Transplantation 3, 423-431 (1965).

Nature (London) 194, 495-

THE EFFECT OF LENTIL LECTIN TREATMENT ON SKIN ALLOGRAFT SURVIVAL IN MICE AND RATS

W. Harel and D. Nelken Laboratory of Immunohematology and Department of Immunology, Hadassah Hebrew University Medical Center, Jerusalem, Israel

I t i s well known that concanavalin A (Con A) activated lymphoid cells may suppress in vitro primary and secondary plaque-forming cell responses of mouse spleen cells to heterologous red blood cells (1) and are capable of suppressing cell mediated immune responses in vitro (2).

I t was shown

that Con-A inhibited the generation of cytotoxic lymphocytes and that this inhibition was c r i t i c a l l y dependent upon the concentration and time of addition of Con A or Con A activated cells (2).

In vivo experiments

showed that intraperitoneal injections of Con A markedly prolonged skin allograft survival in mice (3) and heart allograft survival in rats (4). In a recent article Con A was shown to inhibit immunologically specific cytolytic a c t i v i t y , without any toxic effect on target or effector cells and without interfering with 51 C r release once the l y t i c lesion has been induced.

I t was suggested that the inhibition of cytolysis was caused by

immobilization in the lymphocyte membrane of various protein moieties by the lectin which directly interfered with events at the effector cell surface (5). Others have found that the inhibitory effect of Con A on cytotoxic effector cells was dependent upon treatment of the target cells with the lectin.

When the targets were preincubated with the lectin, or when the

lectin was present in the medium, effector cells were able to k i l l the targets, with a dependence upon the presence of specific histocompat i b i l i t y antigens on the target cells (6,7). observed with phytohaemagglutinin (PHA).

© Walter de Gruyter & Co. 1983, Berlin • New York Lectins, Vol. Ill

Similar effects were also

When injected intraperitoneally

18

under appropriate conditions, i t effectively inhibited the rejection of skin allografts, even across the H-2 barrier in mice (8,9).

In a recent

study, lentil lectin was shown to have a similar effect in preventing the rejection of skin allografts in mice (10).

I t was shown that the immuno-

suppressive efficiency of l e n t i l lectin depends on the strength of the histocompatibility barrier between donor and recipient in mice.

Lentil

lectin was able to overcome the H-2DL antigenic disparity much better than the H-2K and H-2A disparity (11). The present report deals with skin allografting experiments in mice and rats treated with various combinations of lentil lectin and anti thymocyte serum.

I t was found that such a treatment may induce a state of allograft

acceptance in closely related mice and rats.

In adoptive transfer

experiments, cells from the treated animals were able to prolong skin graft survival in transplanted secondary hosts.

Materials and Methods Lentil lectin was prepared as described elsewhere (12).

In short, 250g

of l e n t i l s were homogenized in a Sorvall OMNI-mixer and centrifuged.

The

supernatant was fractionated by addition of s o l i d ammonium sulfate to obtain the material which precipitated between one and two thirds saturation.

The precipitate was dialized against Tris buffer (0.05M,

pH 8.1), and passed through a column of DEAE cellulose (Whatman DE-52). The lectin appeared in the f i r s t protein peak (0D 280 nm) passing unbound through the column. erythrocytes (13).

Activity was detected by hemagglutination of rabbit The product was compared with a commercial lentil

lectin, obtained from Pharmacia Fine Chemicals, Uppsala, Sweden, and showed a similar potency in hemagglutination tests and an identical pattern in acrylamide gel electrophoresis. Anti rat thymocyte serum (ATS) was prepared in rabbits by the injection of rat thymocytes emulsified in complete Freund's adjuvant at four week intervals.

Two weeks after the third immunization the rabbits were bled,

the serum separated, pooled and frozen at -20°C in 10ml aliquots until use.

The cytotoxic titer against rat thymocytes with guinea-pig

19

complement was adjusted to 1:64 in the lymphomicrocytotoxicity test employing the eosin staining method (14). were not used. experiments.

Antisera with weaker titers

A single serum pool was used in each series of Inbred male Lewis (RT-1 1 ), Fisher (RT-1^) rats and inbred

d

BALB/c (H-2 ) and C57B1 (H-2t>) mice were used throughout this study.

Results In a recent report i t was shown that lentil lectin was able to induce skin allograft tolerance in mice (10).

In our laboratory, treatment with

8-10 daily doses of lentil lectin alone following transplantation, resulted in prolonged skin allograft survival but a state of tolerance was never achieved (15,16).

Using two mice strains with strong H-2 d i f f e r -

ences, BALB/c and C57B1, the longest mean skin graft survival time in lentil lectin treated recipients was 22.3+3.81 days (Table 1 group 3) compared to 10.6±1.78 (Table 1 group 8) in untreated control mice.

In

experiments with rats differing in minor histocompatibility antigens (Lewis to Fisher) treatment with lentil lectin increased the mean survival time to up to 25.1+5.33 days (Table 2 group 2) compared to 10.3±1.46 (Table 2 group 6) in the untreated controls.

In another series of

experiments the syngergistic effect of rabbit anti rat thymocyte serum and lentil lectin was examined.

ATS was injected at the time of the operation

followed 48 hours later by the injection of lentil lectin and 48 hours thereafter by another injection of ATS and so on. was given, 4 of which were ATS and 4 lentil lectin.

A total of 8 injections This combined

treatment consistently resulted in prolonged skin graft survival.

In mice

the skin allografts survived for about 24 days (Table 1 groups 6,7) and in rats for an observation period over 75 days (Table 2 groups 4,5).

In

passive transfer experiments we found that spleen cells from ATS and lentil lectin treated Fisher rats with an established Lewis skin graft (Table 2 group 5) were able to prolong graft survival time when transferred to untreated syngeneic rats transplanted with Lewis skin grafts.

The transplants were performed 24 hours after transfers of 5xl0 7

spleen cells (Fig. 1).

The mean survival time of these transplants was

20 Table 1 Skin a l l o g r a f t s u r v i v a l in mice (C57B1 to BALB/c) treated with anti thymocyte serum and l e n t i l l e c t i n

Group no.

ÛT

.r û

No. of mice

Mean s u r v i v a l time ± S.D.

5mg/kg, 10 i . p . i n j e c t i o n s every 24 h s t a r t i n g at transplantation time

25

14.2 ± 2.76

15mg/kg as group 1

18

16.8 ± 2.94

25mg/kg as group 1

15

22.3 ± 3.81

50mg/kg as group 1

13

15.1 ± 4.14

15

18.3 ± 3.77

0.25ml/mouse 8-10 1.p. i n j e c t i o n s every 24-48 hours

b

0.25ml x 5 i . p . i n j e c t i o n s ; on days 0, 2, 4, 6, 8 after transplantation

25mg/kg x 5 i . p . i n j e c t i o n s on days 1, 3, 5, 7, 9 after transplantation

20

24.7 ± 5.12

7

0.25ml x 5 i . p . i n j e c t i o n s as group 6

25mg/kg 5 i . v . i n j e c t i o n s as group 6

14

23.6 ± 5.74

8

Control :

0.5ml of 0 9% NaCl 10 i . p . i n j e c t i o n s every 24 h

30

10.6 ± 1.78

c

Skin g r a f t s (1 cm in diameter) prepared from the back of the donor mice were transplanted onto the backs of the tested r e c i p i e n t s . Grafts with at l e a s t 50% necrosis were considered to be rejected. p < 0.001 between groups 1,2,3,4,5,6,7 and group 8. No s t a t i s t i c a l l y s i g n i f i c a n t difference was found between group 6 and 7. S.D. = Standard Deviation ATS = Anti Thymocyte Serum LCA = Lens C u l i n a r i s A g g l u t i n i n ( l e n t i l l e c t i n ) i . p . = i n t r a peritoneal i . v . = intra venous

21

Table 2 Skin allograft survival in rats (Lewis to Fisher) treated with anti thymocyte serum and lentil lectin Group no.

ATS

LCA

No. of rats

Mean survival time ± S.D.

5mg/kg 8 i . p . injections on consecutive days

30

22.3 ± 4.72

5mg/kg 8 i . v . injections on consecutive days

16

25.1 ± 5.33

lml/rat 8 i . p . injections on consecutive days

18.6 ± 4.94

lml/rat 4 i.p. injections on days 0, 2, 6, 10 after transplantation

5mg/kg 4 i . p . injections on days 1, 4, 8, 12 after transplantation

lml/rat 4 i . p . injections as group 4

5mg/kg 4 i . v . injections as group 4

Control:

lml of 0.9% NaCl, 8 i . p . injections on consecutive days

10

9

22

23

32.1 ± 4.17 > 75 all grafts > 75

10.3 ± 1.46

Skin grafts (1 cm in diameter) prepared from the chest of the donor rats were transplanted to the chest of the recipients. Grafts with at least 50% necrosis were considered to be rejected. p < 0.0001 between groups 1,2,3,4,5 and group 6. S.D. = Standard Deviation ATS = Anti Thymocyte Serum LCA = Lens Culinaris Agglutinin (lentil lectin) i.p. = intra peritoneal i . v . = intra venous

22 22.2±2.50, compared with 15.0±0.82 in the control group in which transferred c e l l s were from untreated and untransplanted Fisher rats. In several other Fisher rats which showed a perfect take of the Lewis skin allograft (Table 2 group 5), a second Lewis skin graft was performed 8-10 weeks after the f i r s t graft.

The second graft was rejected in a l l

cases and caused the simultaneous rejection of the f i r s t allograft 10-15 days after the operation.

When rats, which showed a perfect take of the

f i r s t Lewis skin allograft were transplanted with a second skin graft from an unrelated Sabra rat, the second graft was always rejected within 7-10 days.

In these rats, however, the f i r s t skin graft was not

rejected (Fig. 2).

Fisher

2

TWO MONTHS AFTER ATS & LECTIN TREATMENT

spleen

(ESTABLISHED GRAFT)

1

24 HOURS BEFORE TRANSPLANTATION

Lewis Fig. 1:

4

Fisher

Passive transfer of 5x10^ c e l l s from treated rats with established grafts to newly transplanted rats, causes prolongation of the skin graft survival time.

23

Fisher

TAKE

l " 2

Fig. 2:

n d

GRAFT'-

ATS

GRAFT:

NO

REJECTION

& LECTIN

TREATMENT

TREATMENT

Transplantation of second set skin grafts to treated rats with an established f i r s t graft. When the second graft i s from an un unrelated donor (Sabra), i t i s rejected within 7-10 days, while the f i r s t graft survives. When the second graft i s the same as the f i r s t , both grafts are rejected within 10-15 days.

24

Discussion Our results indicate that the combination of anti thymocyte serum and lentil lectin causes a state of acceptance of skin allografts from Fisher to Lewis rats.

Cells from the treated rats are able to prolong skin graft

survival when transferred to syngeneic recipients transplanted with the same donor type grafts.

No real tolerance i s achieved, however, since a

second allograft from the strain i s rejected and seems to cause the rejection of the f i r s t otherwise surviving allograft. In a combination of two mouse strains differing in all the H-2 loci (C57B1/6 x BALB/c) we were unable to achieve permanent acceptance of the skin allografts by our lentil lectin treatment.

These results are in

agreement with the recent work of Hilgert, Horejsi et al. who have shown that only in certain genetic combinations lentil lectin treatment could overcome the histocompatibility barrier (11).

The same authors suggest

that a suppressor cell mechanism i s at least p a r t i a l l y responsible for the allograft acceptance in the lentil lectin treated mice.

We are at present

examining this point in rats and mice. In our present report we were able to reduce the lentil lectin dose and at the same time s i g n i f i c a n t l y improve the results of the skin allografts by using a combination of lentil lectin and anti thymocyte serum.

This

synergistic effect of lentil lectin and anti thymocyte serum points to the eventual clinical f e a s i b i l i t y of this treatment in an attempt to improve allograft transplantation.

References 1.

Rich, R.R., Pierce, C.W.:

2.

Peary, D.L., Pierce, C.W.:

3.

Markowitz, H., Person, D.A., Gitnick, G.L., R i t t s , R.E.: Science 163, 476 (1969).

4.

Weil, R. I l l , Nozawa, M., Chernack, W., Weber, C., Reemtsma, K., Mcintosh, R.: Transplantation 17, 600-604 (1974).

5.

Tartof, D.:

6.

J. Exp. Med. 137, 649-659 (1973). J. Exp. Med. 140, 356-369 (1974).

Cellular Immunology 50, 48-57 (1980).

Berke, G., Hu, V., McVey, E., Clark, W.R.: 776-781 (1981).

J. Immunol. 127,

7.

Berke, G., Hu, V., McVey, E., Clark, W.R.: 782-787 (1981).

8.

Stefani, S . S . , Moore, C.D.:

9.

Rosenau, W., Habler, J . , Goldberg, M.: 624-626 (1972).

J. Immunol. 127

J. Immunol. 104, 780-784 (1970). Transplantation 12,

10.

Hilgert, I . , Horejsi, V., Angelisova, P., Kristofova, H.: Nature 284, 273-275 (1980).

11.

Hilgert, I . , Horejsi, V., Kristofova, H., Angelisova, P.: Lectins, Biology, Biochemistry, Clinical Biochemistry, Vol. 3, 1982. T.C. Bog-Hansen and G.A. Spengler (Eds.), Walter de Gruyter, Berlin - New York.

12.

Howard, I . K . , Sage, H.J., Stein, M.D.: 1590-1595 (1971).

13.

Howard, I . K . , Sage, H.J.:

14.

Mittal, K.K., Mickey, M.R., Singal, D.P., Terasaki, P . I . : Transplantation 6, 913-927 (1968).

15.

Harel, W., Nelken, D.:

Transplantation 32, 69-71 (1981).

16.

Nelken, D., Harel, W.:

Transplantation 33^, 560-561 (1982).

J. Biol. Chem. 246,

Biochemistry 8, 2436-2441 (1969).

Supported by grants from the Lil and Ben Stein Transplantation Foundati from Alan Liker and the Shaklee Fund.

POSSIBLE RELEASE

INVOLVEMENT IN

INTRINSIC

OF L E C T I N S

IN B A C T E R I A - I N D U C E D

HISTAMINE

ASTHMA

B0g-Hansen,

P. Stahl S k o v , C. J e n s e n , S. N o r n , T . C . s t e i n , C h r . K o c h , H. P e r m i n , N. H a i b y

H.

Lawen-

D e p a r t m e n t of P h a r m a c o l o g y a n d P r o t e i n L a b o r a t o r y , U n i v e r s i t y of C o p e n h a g e n , D e p a r t m e n t of P a e d i a t r i c s T G , R i g s h o s p i t a l e t and Statens S e r u m i n s t i t u t , Copenhagen, Denmark

A pathogenic sic asthma a close toms

role

temporal

and acute

vious

study

several mine

of p a t i e n t s

acute

urinary

attacks

therefore

tion ria

are

by

metabolites

to the

strom

seem

n i s m of the

we

Eshdat

cell

surface and

studied

release

has

could

The

investigated - an

lectins the

might

mechanism (4,

6). reac-

of a

direct

Since

many

bacte-

(see

also

Wad-

(7)

in

non-immunological

1ectin-mediated.

© Walter d e Gruyter & Co. 1983, Berlin • N e w York Lectins, Vol. Ill

of

during

IgE-mediated

consisting

surface.

from hista-

finding

Sharon, Gi1boa-Garber

whether be

been

to e x i s t

of

release

symptoms.

mechanism

basophil

to c o n t a i n

and Trust,

proceedings),

the

histamine

asthmatic

release

mechanisms

the

pre-

that

of h i s t a m i n e

involvement

is s u p p o r t e d

(5). B a c t e r i a - i n d u c e d

with

known

The

symp-

found

of h i s t a m i n e

and a non-immunological

interaction

release

(3-4).

we

of

show

In our

disease

histamine

different

caused

(1-3).

asthma

intrin-

asthma

exacerbations

infections

intrinsic

in v i t r o

in the

intrinsic

excretion

contribute

of b a c t e r i a l Two

with

is s u s p e c t e d with

between

tract

of b a c t e r i a

in the

release

children

relationship

leukocytes

increased

since

respiratory

strains

basophil

of m i c r o o r g a n i s m s

disease,

these mecha-

28 Materi als

Lectins. Difco lens

Con

A,

Pharmacia

Laboratories, culinaris

wheat

germ

communis

AB,

Detroit,

agglutinin

agglutinin

agglutinin

Sweden, USA,

(LCA),

(WGA),

(RCA)

poke IBF,

peanut

from

phytohemagglutinin weed

lectin

E.Y.

mitogen

Clichy,

(PWM)

France,

(PNA)

and

Laboratories,

(PHA), and

and ricinus

San

Mateo,

USA. Carbohydrates. and

D-( + ) - g l u c o s e , D - ( + ) - g a l a c t o s e ,

histamine

release

of

histamine

release

were

read

histamine content

amounted

to

30 Inhibition were

studies

performed

for e x a m i n i n g

by a d d i t i o n

of

a lectin-mediated

specific

D-glucoside, ¿c-methyl-D-mannoside,

reaction

carbohydrates:

¿ + n

i Q i ». 2

E H

+


, 77-82 (1976) .

9.

Hammarström, S., Hellström, U., Perlman, P., Dillner, M.L.: J. Exp. Med. 138, 196-203 (1973).

10. Axellsson, B., Kimura, A., Hammarström, S., Wigzell, H., Nillsson, K., Mellstedt, H.: Eur. J. Immunol. _8, 757-764 (1978) . 11. Prokop, 0.', Graffi, A., Hoffmann, F., Schnitzler, St., Dt. Gesundh.-Wesen 23,

1926-1929

(1968)

12. Harding, M., Crowther, D., Gallagher, J., this volume.

LECTIN BINDING PATTERNS OF NORMAL AND LEUKAEMIC LYMPHOID AND MYELOID CELLS

M. H a r d i n g ,

D. Crowther and J . T .

Gallagher

C a n c e r R e s e a r c h Campaign Department of Medical Oncology, Manchester U n i v e r s i t y and C h r i s t i e Hospital & Holt Radium I n s t i t u t e , Wilmslow Road, Manchester M20 9BX, E n g l a n d

Leukaemia

a r i s e s p r e d o m i n a n t l y in two c e l l l i n e a g e s ,

and in

e a c h the d i s e a s e may follow an a c u t e or c h r o n i c c l i n i c a l c o u r s e . the lymphoid m a l i g n a n c i e s , in the m a j o r i t y of c a s e s ,

c h r o n i c lymphocytic l e u k a e m i a

express

leukaemia

(CLL)

s u r f a c e immunoglobulin

c o n s i d e r e d to be B lymphocyte d i s o r d e r s

(1).

Acute

and

( c h a r a c t e r i s e d by sheep red c e l l r o s e t t i n g ) poor long term s u r v i v a l

in both c h i l d r e n

phenotype,

the uncommon T

and B c e l l t y p e s have a

(9)

and a d u l t s

(16).

ALL c e l l s which l a c k s u r f a c e m a r k e r s of T or B lymphocytes defined by the common ALL a n t i g e n ,

a glycoprotein

small numbers of normal bone marrow c e l l s of common or n u l l significantly cells,

(i.e.

(15).

(9,16).

malignant,

is limited to the l a - l i k e a n t i g e n

immature lymphocytes)

and in c o n s e q u e n c e ,

f e a t u r e s of p r o g n o s t i c

value.

To f u r t h e r c h a r a c t e r i s e

alterations

s a c c h a r i d e composition of leukaemic c e l l s ,

(13)

are

P a t i e n t s with ALL types h a v e a

By comparison with

s u r f a c e phenotyping of the myeloid l i n e a g e ,

lymphoid

both normal

in the plasma

Results h a v e been compared with those of t h e i r mature

quantitatlectins.

counterparts,

namely p e r i p h e r a l blood lymphocytes for ALL and CLL and

© Walter de Gruyter & Co. 1983, Berlin • New York Lectins, Vol. Ill

surface

membrane

we h a v e studied

(AML).

and

( e x p r e s s e d a l s o by

t h e r e a r e few c e l l

i v e l y the c e l l s u r f a c e b i n d i n g of s e v e r a l f l u o r e s c e i n c o n j u g a t e d

c y t e s for a c u t e myelogenous l e u k a e m i a

Common

found a l s o on

devoid of a l l s u r f a c e m a r k e r s )

better prognosis

cells,

are

lymphoblastic

(ALL) is c l a s s i f i e d on the b a s i s of c e l l s u r f a c e

which h a s important i m p l i c a t i o n s for p r o g n o s i s :

Of

granulo-

226 Materials and Methods Cells from 11 patients with AML, seven with ALL (one of T cell phenotype) and four with CLL were studied at the time of d i a g n o s i s , before therapy was s t a r t e d .

A further patient with T cell CLL was

included in view of the r a r i t y of this malignant phenotype;

although

he had been treated for one y e a r , his d i s e a s e was uncontrolled. Cells from f i v e normal donors were used for comparison. Normal lymphocytes and leukaemic c e l l s were isolated from p e r i p h e r a l blood by density g r a d i e n t centrifugation on Ficoll 1.077: Flow L a b o r a t o r i e s ) . Boyum (8). al (5)-

(density

Monocytes were removed a s described by

Granulocytes were s e p a r a t e d by the method of Barrett et

An aliquot of each sample was treated with Clostridium

p e r f r i n g e n s neuraminidase (Sigma Chemicals) 0.05 units/10 7 cells/ml for 30 mins at 37°C in PBS/BSA (phosphate buffered s a l i n e , pH 7 . 2 , 0.1% w/v bovine serum albumin). by Trypan blue exclusion.

Cell v i a b i l i t y was >95% a s measured

Leukaemic samples r a r e l y included morph-

ologically normal lymphocytes.

Those that did were only a n a l y s e d if

there was c l e a r s e p a r a t i o n of the cell populations on the b a s i s of light s c a t t e r , such that the leukaemic c e l l s could be a n a l y s e d ately.

separ-

All cell samples were a d j u s t e d to 2 x 10^ cells/ml in PBS/BSA

and stored on ice. Lectins were obtained commercially a s a f f i n i t y p u r i f i e d , cein conjugated p r e p a r a t i o n s ,

fluores-

v a l u e s in parentheses are the molar

ratios of fluorescein ¡protein ( F : P ) .

Wheat germ a g g l u t i n i n , WGA

( 1 . 3 : 1 ) and Ricinus communis a g g l u t i n i n ,

RCA, ( 6 . 4 : 1 ) from Miles

Chemicals L t d . , Helix pomatia a g g l u t i n i n , HPA (0.56:1) from Polysciences, Glycine max (soybean a g g l u t i n i n ) ,

SBA, ( 3 . 9 : 1 ) from Vector

Laboratories and Wisteria floribunda a g g l u t i n i n , WFA, ( 4 . 5 : 1 ) from E-Y L a b o r a t o r i e s . 105 c e l l s (50 ul of 2 x 10 6 cells/ml) were added to lectin dilutions in 100 ul PBS/BSA.

After 30 mins incubation at 4°C the

cell s u r f a c e fluorescence was measured in a Biophysics Cytofluorograf 4800A (Ortho Instruments) interfaced to a Hewlett P a c k a r d 9845S computer for d a t a accumulation (7).

Mean cell fluorescence was

227 corrected for c e l l autofluorescence,

the F : P r a t i o of each lectin

the c e l l s u r f a c e a r e a c a l c u l a t e d from the measured a v e r a g e diameter.

and

cell

S p e c i f i c i t y of lectin binding was confirmed by >90%

reduction in fluorescence of c e l l s incubated with lectin in the presence of an appropriate s a c c h a r i d e

inhibitor.

Results are expressed as the r e l a t i v e number of lectin molecules bound per unit surface a r e a as c e l l diameters v a r i e d from 7u for PBL to 17u for some AML c e l l s . given in the legends to F i g s .

Lectin concentrations used are

1 and 2.

>70% s a t u r a t i o n of lectin binding

They were selected to give

sites.

Results The l e c t i n s studied comprised two groups, which bound to untreated c e l l s of a l l types,

WGA-F and RCA-F,

although a f t e r

neura-

minidase treatment RCA-F binding i n c r e a s e d ( r e s u l t s not shown) WGA-F binding was reduced ( F i g . l ) .

In c o n t r a s t ,

WFA-F bqund s i g n i f i c a n t l y only to d e s i a l y l a t e d

HPA-F, SBA-F and

cells.

RCA is thought to have an extended c a r b o h y d r a t e binding (A) and shows the highest a f f i n i t y for a s p a r a g i n e linked with terminal g a l a c t o s e r e s i d u e s .

and

site

sequences

The i n t e r a c t i o n of RCA-F with

untreated c e l l s is shown in F i g . l .

There was a c l e a r

distinction

between the high density RCA-F binding g a l a c t o s e sites on normal lymphocytes and the lower density on lymphoid leukaemic c e l l s . the myeloid s e r i e s normal granulocytes showed s i g n i f i c a n t l y

In

more

residues recognised by RCA-F per unit s u r f a c e a r e a than did AML cells.

The a v e r a g e binding site density was s l i g h t l y g r e a t e r on

lymphocytes than g r a n u l o c y t e s ,

but appeared similar between AML and

ALL c e l l s . WGA i n t e r a c t s with both s i a l i c acid residues s a c c h a r i d e s of N-acetyl glucosamine

(GlcNAc).

(6,17)

Fig.l

and oligo-

shows that WGA-F

binding to a l l c e l l types was reduced by neuraminidase

treatment.

The percentage reduction was similar for each sample of lymphoid cells.

Untreated lymphocytes had a higher density of WGA-F binding

s a c c h a r i d e s than either

ALL or CLL c e l l s .

more marked a f t e r d e s i a l y l a t i o n .

This distinction

In c o n t r a s t ,

became

untreated normal and

leukaemic c e l l s of the myeloid l i n e a g e showed similar densities of

228 WGA-F b i n d i n g

sites.

was a s i g n i f i c a n t on AML c e l l s : slightly

However,

reduction

a f t e r neuraminidase

in WGA-F b i n d i n g

b y comparison WGA-F b i n d i n g

reduced b y the enzyme treatment

RCA-F 2 m9

WGA-F 2 Mg

treatment,

per unit s u r f a c e to g r a n u l o c y t e s

(Fig.l).

there area

was

The d e n s i t y

only

of

WGA-F 2 Mg Neuraminidase

0.5 t

•

I =

0 PBL ALL CLL

PBL ALL CLL

PBL ALL CLL

1.0-

T> 0 O Ru ti 1 us ru t i lu Vici a cr acca ;

Dr. J. Kocourek

Wistaria

Dr. T.

floribunda;

Kurokawa

Vicia v i l l o s a ;

Dr. H. W i g z e l l

Abrus precatorius (agglutinin), Abrus precatorius (Abrin C), Adenia digitata (modeccin 4B), Adenia digitata (modeccin 6B), Hura crepitans (latex), Hura crepitans (seeds), Momordica c h a r a n t i a , Ricinus communis (R60), Ricinus communis (R120), Vicia sativa;

Our

Bandeiraea

Commercial

gare

s i m p l i c i f o l i a , Triticum

vul-

laboratories

tions

prepara-

295 Human lymphocytes. 3 0-4 0 ml of heparinized venous blood were collected from a total of 10 blood donors. The peripheral blood lymphocytes were separated by density gradient centrifugation through Ficoll-Hypaque (2) . Enriched T lymphocytes were obtained by incubating lymphocytes with 2-aminoethylisothiouronium bromide hydrobromide (AET)^treated sheep red blood cells (SRBC), according to Pellegrino et al. (1975) (3) with minor modifications as described elsewhere (4). Briefly 2.5 x 10 /ml lymphocytes were incubated with AET-SRBC (0,5%) for 15 min at 37°C in RPMI 1640 plus 10% of foetal calf serum. After incubation in an ice bath for 10 min, the rosetted cells (T lymphocytes) were separated from non-rosetted cells (B lymphocytes) by a Ficoll-Hypaque gradient. Preparation of T lymphocytes were 90% pure as judged by re-rosetting. 5 Effect of lectins. 0.1 ml of cell suspension ( 1 x 1 0 T lymphocytes) in complete medium (RPMI 1640, containing 2mM glutamine, 100 U/ml penicillin, 100 ^ug/ml streptomicin and 10% inactivated pooled human AB serum) was distributed in triplicate in microplate wells (Falcon Plastics, Los Angeles) and 0.1 ml of complete medium containing 2 ^ug/ml of PHA (PHA-P, Difco De troit) or 0.01,0.1,1,10 and 100 ^ug/ml of the 32 lectins tested was added. T lymphocyte cultures were incubated in a humidified3 atmosphere of 3 5% C0 9 and 95% air for 72 hours. Methyl- H-thymidine ( H-TdR, the Radiochemical Center, Amersham, spec. act. 5 Ci/mmol) was added for the last 6 hours (0.5 ^uCi/well). The inhibitory experiments with Dolichos biflorus, Perca fluviatilis and Momordica charantia lectins were performed by ad ding each lectin to 1 x 10^ T lymphocytes stimulated with PHA or ConA (10 yug/ml, ConA 3x crist.,Miles, Lausanne) as described above. Inhibitory lectins were added at the initiation of the culture period in appropriate amounts to reach final concentrations of 0.0 1 ,0.1 ,1 ,1 0 and 100 ^ug/ml. At the end of the incubation period the T lymphocytes were harvested and washed

296

on glass fiber filters with the aid of a multiple cell culture o harvester (Skatron, Norway) and H-TdR incorporation (expressed as cpm) was measured by liquid scintillation counting as previ^ ously described (5). Data evaluation. The results obtained in cell cultures were expressed as Stimulation Index (S.I.). S.I. was calculated as the ratio between cpm of stimulated and unstimulated cultures: g j

=

cpm of cultures stimulated with lectin cpm of cultures without lectin

Results To test the mitogenic and/or the toxic activity of the lectins included in the present study more than ten experiments were performed. In each experiment a negative (T lymphocytes without mitogen) and a positive control (T lymphocytes plus PHA) were always included. In the different experiments the "^H-TdR incor poration ranged from 200 to 4 ,000 cpm in negative controls (mean 1,120 cpm) and from 44,880 to 232,015 cmp (mean 99,840 cpm) in positive PHA-stimulated control cultures. 3 TABLE 2. Lectins which do not affect the H-TdR incorporation of human purified T lymphocytes. Lectin concentrations (/ug/ml) 0. 01

0 .1 (a) 1 _ 1 .13 11

Arachis hypogaea Coregonus lavaretus M.

0. 68

Helix pomatia

0. 47

Pisum sativum

0. 66

Sarothamnus scoparius Triticum vulgare Ulex europaeus

0. 81 1.01 0. 74

Vicia villosa

0. 53

1 0..73 0.. 97

10 0 .58

1 00 0. 84

0 . 68

0. 22

0 .46

0. 33

0. 51 0. 57

0..57 0..43 0..79

0 .52

0. 49

0. 68 0. 50 0. 20

1 .. 01 0..29 0,.34

0 .82 1 . 00 0 .34

1 .27 0. 88 0. 65

0 .30

0. 38

1 .1 2 0. 85

(a) Results are expressed as stimulation index(see Mat.and Met)

297

Table 2 shows the results obtained with a group of lectins which do not affect the ^H-TdR incorporation of the T lymphocytes. Although using a wide range of lectin concentrations (0.01-100

,ug/ml) the S.I. values are close to 1. 3 Twelve lectins were found which caused a H-TdR incorporation significantly higher than that observed in control unstimulated cultures. The results of these experiments are shown in Table 3. TABLE 3. Lectins which have mitogenic effect on human purified T lymphocytes.

Lectin concentrations ( yug/ml) 0..01 Crotalaria júncea Glycine max Hura crepitans (latex) Hura crepitans (seeds)

0.. 1

1

0..55 (a) 54.. 91 189..87 0..85 0.. 78 0..69 2,.84 45..10 132..59 2,.74

51 .00 .

Phaseolus lunatus Vicia sativa

0.,56 0..69

103..73 0..76 1 .. 25 0.. 68 1 .67 .

Datura stramonium Erythrina indica

0..77 0..78

1 .. 02 4 .. 98

1 .. 92 22,.59

Ononis spinosa

1 .26 .

1 .66 .

6..24

Phaseolus coccineus Robinia pseudoacacia

. 1 .58 2.. 97 1 .59 .

Rutilus rutilus

10

1 00

32,.77 0..81

7 .. 63 4.,15 15,. 67

.57 1 57. 98.. 24 16,.86 1 1.47 . 2..41 9..69 52,.36

. 1 .70 2.. 97 64..15 0..11 0..10 0.. 55

74.. 08 278 .37 132..1 25 0..80 . 48 .74 . 1 05. , 78 17.. 1 3 0.. 34 4 .. 28 1 4.,04 2..75 0.. 04

(a) Results are expressed as stimulation index (see Materials and Methods); A = lectins which are mitogenic even at the highest concentrations tested; B = lectins which bring about a decreased H-TdR incorporation at higher concentrations, after a peak of optimal activity.

298 The mitogenic power of many lectins (Crotalaria juncea, Hura crepitans from latex, Hura crepitans from seeds, Vicia sativa, Ononis spinosa, Phaseolus coccineus and Robinia pseudoacacia) is quite strong. The mitogenic activity of other lectins such as Glycine max and Datura stramonium is weak, while other lectins such as Erythrina indica, Phaseolus lunatus and Rutilus rutilus have, in these experimental condition, an intermediate mitogenic power. These mitogenic lectins have been further divided into two sub groups. In the first lectins are mitogenic even at the highest concentrations tested, while in the second lectins bring about 3 a decreased incorporation of H-TdR at higher concentrations, after a peak of optimal activity. 3

Many lectins caused a decreased H-TdR incorporation below the level observed in control unstimulated cultures. They are listed in Table 4. TABLE 4. Lectins with toxic effecton human purified T lymphocytes. Lectin concentrations ( ^ug/ml) 0. 1 (a)_ 2. 1 3 — 0. 02 0. 02 0. 02 0. 02 0. 02 0. 02

_1 0. 62

10 0.50

1 00

0. 01 0. 02 0. 04

0.02 0.02 0. 01

0.03 0. 01 0.01

0. 38 0. 93

0. 36 0. 39

0. 36 0. 24

0.18 0.22

0.13

0. 55

0. 49

0. 28 0. 02 0. 02 0. 1 1 0. 06 0. 50 0. 39

0.08 0.01

0.07 0.02 0. 06

0. 01

Abrus precatorius (agglutinin) Abrus precatorius (Abrin C) Adenia digitata (modeccin 4B) Adenia digitata(modeccin 6B) Dolichos biflorus Momordica charantia Perca fluviatilis Ricinus communis (ricin 60) Ricinus communis (ricin 120) Vicia cracca Wistaria floribunda

0. 02 0. 04 0. 54 —

• -

1. 04

0. 65

0.03 0.09 0.49

0.27

0.10 0.13

(a) Results are expressed as stimulation index (see Materials and methods).

299 Some of these lectins such as ricin, abrin and modeccin are well known inhibitors of protein synthesis (6—8)• The toxic activity of three of these lectins has been confirmed by their addition to T lymphocytes previously stimulated by PHA and ConA (Fig. 1). In this case the inhibition of 3H-TdR incorporation is dose-dependent and Momordica charantia lectin appears to be the strongest inhibitor. The results obtained with Bandeiraea simplicifolia (data not shown) were not clear enough to allow an assignment to one of three aforementioned group of lectins.

Discussion The main results of the present study may be summarized as fol lows.

ration of PHA and ConA stimulated lymphocytes. Results are given as per cent of control values.

300

First, as far as we know, the mitogenic activity for human T lymphocytes of Datura stramonium, Erythrina indica, Hura crepitans (from latex), Ononis spinosa and Rutilus rutilus lectins is described for the first time. The mitogenic activity of other lectins has been previously described (9,10). It is interesting to note that two of the most potent mitogenic lectins derive from the same plant, i.e. Hura crepitans. Preliminary data suggest that the two lectins which can be purified from the seeds and from the latex respectively are different although very similar molecules. (Barbieri et al., unpublished results). Particularly interesting seems also to be the lectin from Rutilus rutilus which is of animal origin and has also toxic properties in a cell-free system (as will be later discussed). Further studies are needed to ascertain whe ther the mitogenic activity of some of these lectins is restricted to the T lymphocytes. Many mechanisms have been proposed to account for the bell-sha ped responses observed with mitogenic lectins (11). Taking into account the dose-response curves the mitogenic lectins have been divided in two subgroups, a first one in which the 3 H-TdR incorporation is always higher than that observed in unstimulated control cultures, even at the highest concentration used, and a second subgroup in which high lectin concen3 trations cause a H-TdR incorporation lower than that of control cultures. This subdivision is proposed here as a working hypothesis to ascertain whether it can help in finding differences among the lectins of the two subgroups in terms of cell binding and toxicity, preferential stimulation of lymphocyte subpopulations with suppressor activity etc. Second, we observed that the lectins with the highest inhibito ry power on in vitro protein synthesis, such as abrin, ricin and modeccin, are also highly toxic for intact T lymphocytes. However apparent contradictions between the toxic activity in a cell-free system and on intact lymphocytes were also obser_ ved. In fact the lectins from Crotalaria juncea, Hura crepitans (latex), Hura crepitans (seeds) and Rutilus rutilus are

301

all mitogenic (and in the case of the first three lectins among the strongest mitogens) for T lymphocytes and still they exert a strong inhibitory effect on protein synthesis in a cell-free system (1, and unpublished results). This strongly suggests that these lectins bind to cell receptors but either any or very few molecules are able to enter within the cells or that, even if they are able of entering, they are rapidly inactivated. The mechanisms and the structural requirements which determine and control the penetration of a lectin into a cell and the subsequent fate within the cell (enzimatic clea vage, breakdown of disulphide bridges etc.) seem to be critical as far as the overall biological effect (stimulation, killing etc.) on the cell is concerned. Third, a group of lectins which did not affect the "^H-TdR incorporation in intact lymphocytes was identified. It is interesting to note that, apart from Coregonus lavaretus maraena lectin, .which has a weak inhibitory activity, all other lectins of this group are also inactive as protein synthesis inhibitors in a cell-free system (1). However, there are also lectins, such as those from Dolichos biflorus and Perca fluviatilis, which exert a toxic effect on the spontaneous blastogenesis of T cells and totally inhibit the "^H-TdR incorporation of PHA or ConA stimulated T lymphocytes at concentrations (100 ^ug/ml) that do not affect at all protein synthesis (1). Thus, the mechanism by which these two lectins exert a toxic effect on intact lymphocytes is different from that of the majority of the other toxic lectins, i.e. ribosome inactivation and consequently inhibition of protein synthesis (6). In conclusion our study suggest that different and apparently opposite properties such as the capacity to stimulate cell mitosis and to inhibit protein synthesis may belong to the same molecule and stress the importance of the experimental system used to assess the lectin activity. Moreover we hope that our approach i.e. the study of the effects exerted by lectins on a homogeneous population of intact purified human T cells, to-

302

gether with the study of the effects exerted by the same lectins on protein synthesis in a cell-free system, may help in the understanding of the structural requirements and the type of cell interactions that control binding, penetration, activation or inactivation, and consequently the final biological effect exerted by each lectin.

This work was supported by grants n. 81.01371.96 and number 81.01469.96 from Consiglio Nazionale delle Ricerche to prof. C. Franceschi and F. Stirpe, respectively, within the "Progetto Finalizzato sul controllo della Crescita Neoplastica", and by grant n. 81.00340.04 to Prof. C. A. Rossi. References 1. 2.

Barbieri, L., Lorenzoni, E., Stirpe, F.: Biochem. J. 182, 633-635 (1979). Böyum, A.: Scand. J. Clin. Lab. Invest. 21, Suppl. 97, 77-89 (1968).

3.

Pellegrino, M.A., Ferrone, S., Dierich, M.I., Reisfeld, R.A.: Clin. Immunol. Immunopathol. 324-333 (1975).

4.

Licastro, F., Franceschi, C., Chiricolo, M., Battelli, M.G., Tabacchi, P., Cenci, M., Barboni, F., Pallenzona, D.: Carcinogenesis 3, 45-48 (1982).

5.

Franceschi, C., Licastro, F., Paolucci, P., Masi, M., Cavicchi, S. and Zannotti, M.: J. Ment. Defic. Res. 22, 179-1 91 (1 978).

6.

Olsnes, S., Pihl, A.: In Cuatrecasas, P. (Ed.), the Specificity and Action of Animal, Bacterial and Plant Toxins 129-173, London: Chapman and Hall (1977).

7.

Olsnes, S., Haylett, T., Refsnes, K.: J. Biol. Chem. 253, 5069-5073 (1978).

8.

Gasperi-Campani, A., Barbieri, L., Lorenzoni, E., Montanaro, L., Sperti, S., Bonetti, E., Stirpe, F.: Biochem. J. 174, 491-496 (1978).

9.

Falasca, A., Franceschi, C., Rossi, C.A., Stirpe, F.: Biochim. Biophys. Acta 5J7J7, 71-81 (1 979).

10.

Falasca, A., Franceschi, C., Rossi, C.A., Stirpe, F.: Biochim. Biophys. Acta 632, 95-105 (1980).

11.

Hume, D.A., Weidemann, M.J.: Mitogenic Lymphocyte Transformation, Elsevier, North Holland Biomedical Press, Amsterdam. New York. Oxford 1980.

LECTINS AS PROBES OF BLOOD PLATELET ACTIVITY Kenneth J. Clemetson*, John L. McGregor*, and Ernst F. Lüscher* •Theodor Kocher Institute, University of Berne, P.O. Box 99, CH-3000 Berne 9, Switzerland. **INSERM Unité 63, 22 ave du Doyen Lépine, F-69500 Bron and Faculté de Médicine, Alexis Carrel, rue Guillaume Paradin, F-6900 Lyon, France.

Blood

platelets

form

an

ideal

test-system

for

studying

the

effect of lectins in inducing cellular activity and in inhibiting its induction by physiological stimulators. they

show

where

three

the

major

resting

activation

discoidal

stages:

platelet

is

In vitro,

a) Shape

change,

transformed

to

a

spherical form, which rapidly develops long pseudopods; b) the platelets storage

become

"sticky" and aggregate; c) the contents of

organelles,

released.

many of which are also stimulators,

are

This latter stage may produce an enhanced aggrega-

tion, the so-called second wave.

In addition, in vivo, plate-

lets can adhere to exposed subendothelium, a process in part dependent on a plasma glycoprotein, the von Willebrand factor. These

processes

various on

the

lectins effects

can and of

be

either

several

stimulated

studies

have

lectins on platelet

or been

activity

inhibited made,

by

either

(1-3) or to

find out which lectins bind to which platelet membrane glycoproteins

(4-7). However, little work has been done on corre-

lating the binding of a lectin to a particular with the biological effect produced.

© Walter d e Gruyter & Co. 1983, Berlin • N e w York Lectins, Vol. Ill

glycoprotein

304 Considerable

details

are

known

major platelet glycoproteins These

come

from

glycoproteins 1

mann s with

studies

thrombasthenia, The

role

of

some

of

syndrome, and

acid

GPIb,

Ilia)

against

sialic

the

in the functions detailed above.

GPIIb

directed

major

the

on platelets with genetically

(Bernard-Soulier

antibodies

15).

of

(8-11)

specific

containing

absent

and

Glanz-

and

studies

glycoproteins glycoprotein

(12— (GPIb)

is known to be the von Willebrand factor receptor and to contain

a

thrombin

binding

site,

while

GPIIb

and

Ilia

complexes

and

probably

constitute

receptors,

possibly

via

form

2+ Ca

-

dependent

telet

aggregation

ding.

the

pla-

fibrinogen

bin-

Glycoprotein V is the only thrombin-cleavable

protein

and

is

indispensable

for

thrombin-induced

glyco-

activation

(16) .

Methods and Materials Platelets for of

the the

(17). by

were Central

Swiss

from

Laboratory

Red

Cross

citrate-treated

of

and

the

Blood

washed

sonication

and

(6).

glycoproteins

differential In

by

order

to

endogenous

blood

Transfusion

Ca

Service described

from washed platelets

centrifugation avoid 2+

collected

as • previously

Platelet membranes were prepared

described

peptin

isolated

as

degradation

-activated

previously of

membrane

proteases,

leu-

(100 ng/ml, Sigma) was added before sonication.

Lectin-affinity membranes

chromatography

solubilized

bound to Sepharose (4,6) or purchased

in

1%

was

carried

sodium

out

with

deoxycholate

on

4B either prepared as previously from

Pharmacia

platelet lectins

described

(Uppsala, Sweden) or Medac

(Hamburg, F.R.G.). Two-dimensional by

the method

gel

electrophoresis

of Wang and Richards

or by the method of O'Farrell

(19).

was (18)

carried

out

(unreduced,

either reduced)

Lectin binding components

305

were identified by treating the fixed, washed gels 125 I-labelled lectins as previously described (20). Aggregation platelet

studies.

The

effects

agglutination/activation

activation

induced

by

of

or

lectins

in

physiological

with

in

inducing

inhibiting

platelet

agents

were

studied

by

adding different concentrations of lectin to 200 pi of plateg

lets (5x10 /ml).

When inhibition of other agents was tested,

the agent was added lectins

5 sec after the lectin.

The effects of

on platelets which had been desialated by

treatment

with neuraminidase (Behringwerke) (6) were also checked. Results and Discussion The effects of various lectins on platelets have been studied by

several

these

groups

lectins

little attempt

(1 - 3 ) but

bind

have

not

and

been

glycoprotein

to

which

so well characterized

and

has been made to correlate binding of lectins

with the effects produced. attempted

the

the

major

In Table I such a correlation is

glycoproteins

bound

and

effects

on

platelets are shown for those lectins which have been studied in

the most detail.

drawn

from

In part, the effects on platelets

previously

work

(1-3)

complemented

by

our

are own

studies,reported here,while the results for the glycoproteins bound come principally chromatography platelet though on

the

into

various it

conclusions two

separated

gel electrophoresis

platelets

broad

studies with lectin-affinity 125 (4,6) and binding of I-labelled lectins to

glycoproteins

acrylamide

from our

classes.

lectins

is

produce

nevertheless

and

to

see

These

are

by

two-dimensional

poly-

(results reported here).

that

a wide range of possible the

firstly,

to

Al-

effects

draw

certain

lectins fall

roughly

those

lectins

which

induce aggregation but only at relatively high concentrations and which are not strong inducers of the platelet release re-

306 TABLE I Lectin

Major GP bound

Direct effect

Lens culinaris

lb, lib, Ilia (4) Aggregation

Inhibition of

(2)

Second wave^

Concanavalin A Ilia, 111b

Shape change, release (2)

Aggregation"^

Wheat germ agglutinin

Strong aggregation and release (3)

vWf induced aggregation

la, lb, Illb (4)

Ricinus communis lectin I*

la, lb, Illb (V)

Aggregation and release (1,2)

Soy bean agglutinin*

None,glycolipid?

No shape change but Aggregation (1)

Peanut agglu-- Ib (5,6) tinin*

Strong aggregation and release (1)

vWf induced aggregation

Maclura aurantiaca

lb, high mol.wt. component (5)

Strong aggregation and release

vWf induced aggregation

Phaseolus vulgaris

Ib, Ila, Illb

Aggregation + release (1)

Aggregation^

*

With platelets treated with neuraminidase

'

Induced by ADP, epinephrine or arachidonic acid

action and secondly, lectins which induce strong aggregation and release first agents

at low concentrations.

group

tends

but

not by

to

inhibit

In inhibition studies the

aggregation

ristocetin/von

by a wide

Willebrand

factor

range while

second group inhibits ristocetin/von Willebrand factor aggregation other

strongly

agents.

but has

little

The main members

effect

of the

induced

on aggregation

first group

of the

are

by

Lens

culinaris lectin, concanavalin A and Phaseolus vulgaris lectin while wheat germ agglutinin agglutinin platelets)

and are

Ricinus the

(with normal platelets) and peanut

communis

principal

lectin

lectins

of

(with the

desialated

second

group.

307 Fig. 1 shows the results obtained with Lens culinaris

lectin

as representative of the first group while Fig. 2 shows those obtained

with

peanut

lectin as representative

of the

second.

It is of course not yet possible to exclude that lectins bind also to glycolipids and thus affect platelet function.

Soybean

agglutinin may be a lectin which falls in this category. addition,

because

glycoprotein they of

the

the lectins bind to more than one

is

some

overlapping

in

the

effects

induced first

release

group

culinaris

of

storage

inhibit

granules

aggregation.

though

This

can

the

lectins including wheat germ agglutinin and

lectin

be

lectins

despite

bind

to

glycoprotein

V

(the

cleavable glycoprotein) albeit relatively weakly. obtained

which

None of the lectins tested had any effect on

fact that several Lens

of

there

produce.

thrombin

most

In

interpreted

as

follows;

those

thrombin-

The results lectins

which

bind strongly to GPIb such as wheat germ agglutinin or, after neuraminidase both

aggregation

gation

if

culinaris though

they

which

Lectins

weak

treatment, and can

lectin

be

used

to

or

agglutinin, strongly

and at

GPIIb are

inhibit

a high and/or

concanavalin

amounts

A

required,

vWf-induced

enough

induce

adrenalin proteins

terms

or

as

Lens

generally

induced by agents such as ADP,

thrombin.

bound

results some of

compared brand

such

aggregation,

are

and

The

the

obtained

relationship

effect using

produced

monospecific

between

alonly

differences

in

the

sites

to

binding

antibodies.

factor-induced

effect

which

collagen,

the

is in good

glyco-

agreement

or monoclonal

bodies directed against specific glycoproteins are

aggre-

concentration.

GPIIIa but

induce

inducers of release. They inhibit aggregation or second-

wave of aggregation

with

release

bind

larger

peanut

can

(12-15). be

antiThere

explained

on

the

glycoprotein

Thus,

the

inhibition of von Wille-

platelet

for

in

lectins

aggregation by lectins binding

GPIb supports earlier suggestions

(21) that

it is the outer,

308

100 n Fig.1

T 1

0

Fig. 2

A

2

1

1 3

A

T 1

1 4

T

r 2

4

Fig. 1: Effect of Lens culinar^s lectin on ADP/fibrinogeninduced platelet aggregation (xlO platelets/ml, 4)iM fibrinogen). A: Aggregation curve with ADP added at arrow 2. B: Aggregation curve with Lens culinaris lectin (final concentration (200jig/ml added at arrow 1, followed by 5)iM ADP at arrow 2. Fig. 2: Effect of peanut lectin on ristocetin-induced aggregation of neuraminidase treated platelets resuspended in plasma. A: Aggregation curve with ristocetin (final concentration, 1.5 mg/ml) added at arrow 2. B: Aggregation curve with peanut agglutinin (20|ig/ml, final concentration) added at arrow 1 followed by ristocetin as in A at arrow 2. carbohydrate-rich

part

of

Willebrand

factor

lectin

concanavalin

by

and

a

gives

variety further

mediated ducing The

the

A

to

platelets blocking

inhibiting idea

sites

that

the

involved of

Lens

platelet

von

between

this

aggregation.

fibrinogen

von

aggregation factor)

agglutination

GPIIb

may

in

culinaris

Willebrand and

on the platelet

molecules

causing

is

effects

(excluding the

fibrinogen

by

in

formation

binding

which

The

agents

weight

dimeric either

binding.

complex

fibrinogen

between act

by

of

GPIb

surface

then

form

The

binding

Ilia,

(22).

bridges

lectins

sites

is

pro-

may

directly

309 or

by

preventing

complex

It

is

not

why

second

clear

wave

served with

is

two

different

the

aggregation

the

platelets, can

clear

with

affected

antibodies

Lens

but

produced

are by

between

culinar is

similar

directed

mechanisms be

formation

In

have

whereby

Ilia.

only

the

been

ob-

GPIIb/IIIa.

fibrinogen

inhibited.

and

lectin

results

against

involved

GPIIb

Possibly

only

one

i.e.

and ADP released

from

conclusion,

it

is

now

that many of the effects of lectins on platelets can be

explained

in

terms

of

the

glycoproteins

to

which

they

bind

but that not all of the problems have yet been solved.

Acknowledgements This

work

was

supported

by

grants

from

the

Swiss

National

Science Foundation and INSERM CRL 817016. References 1.

Patscheke, H., Brossmer, R., and Wörner, P. Biophys. Res. Commun. , 7_5, 200-206, (1977).

2.

Patscheke, H. and Wörner, P. 402, (1977).

3.

Greenberg, J.H. and Jamieson, G.A. Acta, 345, 231-242, (1974).

4.

Clemetson, K.J., Pfueller, S.L., Lüscher, E.F. and Jenkins, C.S.P. Biochim. Biophys. Acta, 464, 493-508, (1977) .

5.

Judson, P.A. and Anstee. D. In: Proc. of the 27th Meeting, Protides of the Biological Fluids, H. Peeters (Ed.) Pergamon, Oxford, New York 1980 pp. 871-874.

6.

Clemetson, K.J., Nairn, H.Y. and Lüscher, E.F. Natl. Acad. Sei. USA, 78, 2712-2716, (1981).

7.

Hagen, I. and Gogstad, G. In: Lectins, Biology, Bio chemistry, Clinical Biochemistry, T.C. B?ig-Hansen (Ed) Vol 1, de Gruyter, Berlin, New York 1981 pp 347-357.

8.

Jenkins, C.S.P., Phillips, D.R., Clemetson, K.J., Meyer D., Larrieu, M.-J. and Lüscher, E.F. J. Clin. Invest. 57, 112-125, (1976).

9.

Hagen, I., Nurden, A., Bjerrum, O.J., Solum, N.O. and

Thromb. Res.

Biochem. 11, 391-

Biochem. Biophys.

Proc.

310

Caen, J.P.

J. Clin. Invest.

£5, 722-731, (1980).

10.

Nurden, A.T. and Caen, J.P. 250, (1979).

Semin. Haematol.

16^, 234-

11.

Phillips, D.R. and Poh Agin, P. 535-545, (1977).

12.

Ali-Briggs, E.F., Clemetson, K.J. and Jenkins, C.S.P. Br. J. Haematol. 48, 305-318, (1981).

13.

Jenkins, C.S.P., Ali-Briggs, E.F. and Clemetson, K.J. Br. J. Haematol. 49, 439-447, (1981).

14.

Ruan, C., Tobelem, G., McMichael, A.H., Drouet, L., Legrand, Y., Degos, L., Kieffer, N., Lee, H. and Caen, J.P. Br. J. Haematol. 49, 511-519, (1981).

15.

Kornecki, E., Lee, H., Tuszynski, G.P. and Niewiarowski, S. Fed. Proc. 41, 528 (1982) abstr.

16.

Berndt, M.C. and Phillips, D.R. 59-65, (1981).

17.

Massini, P. and Liischer, E.F. 372, 109-121, (1974).

18.

Wang, K. and Richards, F.M. 8018, (1974).

19.

O 1 Farrell, P.H.

20.

McGregor, J.L., Clemetson, K.J., James, E., Capitanio, A., Greenland, T., Liischer, E.F. and Dechavanne, M. Eur. J. Biochem. 116, 379-388, (1981).

21.

Jamieson, G.A., Okumura, T. and Hasitz, M. Haemostas. 42, 1673-1678, (1979).

22.

Nachman, R.L. and Leung, L.L.K. 263-269, (1982).

J. Clin. Invest.

J. Biol. Chem.

60,

256,

Biochim. Biophys. Acta J. Biol. Chem.

J. Biol. Chem.

252, 8005-

2j>0, 4007-4021, (1975).

Thromb.

J. Clin. Invest. 69,

SIALIC ACID AS A CELL SURFACE BINDING SITE FOR WHEAT GERM AGGLUTININ John T. G a l l a g h e r * ,

Margaret Harding* and Robert E.

Dale#

* C a n c e r Research Campaign Department of Medical Oncology, #Paterson

Laboratories,

Christie Hospital & Holt Radium I n s t i t u t e , and Manchester Wilmslow Road, Manchester M20 9BX, E n g l a n d .

University,

The combining site of wheat germ a g g l u t i n i n (WGA)consists of three i d e n t i c a l s u b - s i t e s each complementary to N-acetylglucosamine

-GlcNAc-:

the free s u g a r is a poor i n h i b i t o r of lectin binding to g l y c o c o n j u g a t e s but the d i - and t r i s a c c h a r i d e forms (chitobiose and chitotriose) potent i n h i b i t o r s

suggesting that high a f f i n i t y binding is

are

associated

with l i g a n d s that i n t e r a c t simultaneously with two or more of the available subsites ( 1 , 2 ) .

In glycoproteins terminal GlcNAc residues

in a s p a r a g i n e - l i n k e d g l y c a n s are not r e a c t i v e with WGA although lectin will

precipitate

with

the isolated t r i s a c c h a r i d e

GlcNAc which forms the l i n k a g e sequence to a s p a r a g i n e

the

man-GlcNAc(3,7).

In

native glycoproteins the proximity of this sequence to the core protein may impair recognition by WGA.

The interaction of WGA with cell

s u r f a c e s is i n h i b i t e d by treatment of c e l l s with

neuraminidase

suggesting that s i a l i c acid may be an important s u g a r in the binding process.

(5,6,9)

When used as a s a c c h a r i d e inhibitor of WGA,

acid has very low a c t i v i t y .

(3)

sialic

However, multiple i n t e r a c t i o n s with

s i a l y l residues may be favoured at c e l l s u r f a c e s because of the location of t h i s s u g a r at the terminal non-reducing end of c a r b o h y d rate c h a i n s .

In an attempt to elucidate the roles of s i a l i c acid and

GlcNAc we have c a r r i e d out k i n e t i c and inhibition studies on the influence of neuraminidase on WGA binding to the surfaces of K562 cells. Materials and Methods Cells used were an erythroleukaemic cell l i n e ,

K562, derived from

a patient with chronic myeloid leukaemia in b l a s t c r i s i s . binding of F-WGA (Miles Chemical C o . ,

© Walter de Gruyter & Co. 1983, Berlin • New York Lectins, Vol. Ill

molar r a t i o ,

(8)

fluorescein

The :

312 protein

1.3:1)

was determined

as described

p r e v i o u s l y (4).

v a r y i n g c o n c e n t r a t i o n s of F-WGA were a d d e d to K562 c e l l s 150 u l ) a n d i n c u b a t e d

a t 4°C f o r 30 m i n u t e s .

l e c t i n w a s t h e n m e a s u r e d b y flow c y t o m e t r y . (GlcNAc a n d c h i t o b i o s e ) cells.

Cell Sugar

Briefly, (1C)5 c e l l s in

surface-associated inhibitors

were mixed with t h e l e c t i n b e f o r e a d d i t i o n of

In k i n e t i c a n a l y s e s

l e c t i n b i n d i n g to c e l l s w a s e f f e c t i v e l y

s t o p p e d b y d i l u t i n g t h e i n c u b a t i o n t e n f o l d w i t h cold p h o s p h a t e saline

(PBS)

(pH 7 . 2 ) .

buffered

C o m p e t i t i v e i n t e r a c t i o n b e t w e e n F-WGA a n d

n a t i v e form of t h e l e c t i n w a s c a r r i e d out b y f i r s t i n c u b a t i n g

the

the cells

for v a r y i n g times with the f l u o r e s c e i n - d e r i v a t i v e and then a d d i n g two to t e n f o l d e x c e s s of t h e u n c o n j u g a t e d f o r m . be f o u n d in t h e f i g u r e l e g e n d s .

Neuraminidase

Further details

a may

( T y p e IV - from

Sigma C h e m i c a l Co. ) t r e a t m e n t w a s c a r r i e d out b y i n c u b a t i n g

10? c e l l s

w i t h 0 . 0 5 u n i t s of enzyme in a t o t a l volume of 1 ml of PBS.

Maximum

d e c r e a s e in F-WGA b i n d i n g c a p a c i t y

was achieved under these con-

ditions . Results S p e c i f i c i t y of i n t e r a c t i o n of WGA w i t h t h e K562 c e l l s u r f a c e w a s established

by t h e complete i n h i b i t i o n of l e c t i n b i n d i n g

in t h e

of c h i t o b i o s e a n d t h e s i g n i f i c a n t b u t l e s s complete i n h i b i t i o n GlcNAc.

presence by

(Fig.1)

D i s t i n c t i v e b i n d i n g c u r v e s were o b t a i n e d when c o m p a r i n g and neuraminidase-treated a plateau

between

(Fig.2).

in t h e f o r m e r b i n d i n g

1 u g a n d 5 u g of l e c t i n ,

treated cells binding range

cells:

w h e r e a s in

increased quite s h a r p l y over this

control

approached the concentration

The d e g r e e of i n h i b i t i o n of b i n d i n g to t r e a t e d

was dependent upon the lectin c o n c e n t r a t i o n ,

64% i n h i b i t i o n

cells

at 0.1

ug

d e c l i n i n g to 39% a t 5 u g . Kinetic a n a l y s i s considerably cells

(Fig.3).

i n d i c a t e d t h a t t h e r a t e of WGA b i n d i n g

more r a p i d to u n t r e a t e d t h a n to

neuraminidase-treated

In t h e t r e a t e d c e l l s f a s t a n d slow p h a s e s of

b i n d i n g process were observed with each p h a s e r e p r e s e n t i n g of t h e b i n d i n g c u r v e .

In t h e c o n t r o l c e l l s ,

w a s c o m p l e t e a t 1 min ( f a s t p h a s e ) tatively less significant

(Fig.3).

was

however,

70% of

a n d t h e slow p h a s e w a s Further complexity was

the a b o u t 50% binding quanti-

evidenced

313 when b i n d i n g (Fig.4).

was e x a m i n e d o v e r

In the c o n t r o l

achieved

within

enzyme-treated that

different

iments ation

(Figs. as t h i s

selective

5 sees w h e r e a s cells

led

lectin

analysis

of

were

significant

saturation

sites o c c u p i e d

tration

lectin

concentration

agglutination.

so some p o t e n t i a l

fast phase

of b i n d i n g lectin

was not a remain

in

exper-

control

popul-

preventing

concentration.

cells

we c o u l d employ

sites w o u l d

sites

was

emphasise

used in the k i n e t i c

at low

The l a t t e r

We would

f o r the u n t r e a t e d

5 ug was used f o r the n e u r a m i n i d a s e - t r e a t e d the h i g h e s t

site occupancy

25% o c c u p a n c y .

1 ug w a s chosen virtual

short time increments of 5 sees

60% b i n d i n g

the c o r r e s p o n d i n g

to o n l y

concentrations

3-5). gave

very

population,

because

without

this

was

causing

'saturating'

concen-

unoccupied.

100

•s 50

\

50

100

2.5

5

GlcNAc Chitobiose

C o n c e n t r a t i o n of Inhibitor(mM)

Fig.l.

Saccharide

inhibition

of

F-WGA b i n d i n g

to K562 c e l l s

GlcNAc ( ) and D i , N - a c e t y l c h i t o b i o s e ( ) were mixed with the l e c t i n ( l u g / 1 5 0 u l ) b e f o r e a d d i t i o n of K562 c e l l s . For each s a c c h a r i d e c o n c e n t r a t i o n i n c u b a t i o n s with c e l l s were f o r 30 mins at A°C. C e l l f l u o r e s c e n c e was measured b y f l o w c y t o m e t r y .

314

Fig.2.

I n f l u e n c e of n e u r a m i n i d a s e on F-WGA b i n d i n g

Neuraminidase-treated( ) and untreated cells ( ) were i n c u b a t e d with the s t a t e d r a n g e of F-WGA c o n c e n t r a t i o n s for 30 mins at 4 ° C . 10^ c e l l s were mixed with the a p p r o p r i a t e l e c t i n c o n c e n t r a t i o n in a t o t a l volume of 150 ul of PBS.

2

-3

2-5

ng F-WGA When the f l u o r e s c e i n - c o n j u g a t e d

WGA (1 u g ) w a s i n c u b a t e d

with

K562 c e l l s for 30 mins a n d then a t e n f o l d e x c e s s of n a t i v e WGA a d d e d , no d e c r e a s e in cell f l u o r e s c e n c e ,

a n d hence no e o u i l i o r i u m

bound a n d f r e e forms of the lectin w a s o b s e r v e d

( r e s u l t not

between shown).

However, in view of the complex b i n d i n g k i n e t i c s of WGA we d e c i d e d to e x p l o r e the p o s s i b i l i t y dependent,

i.e.

that i r r e v e r s i b i l i t y

of b i n d i n g may be time

t h a t a c e r t a i n r e s i d e n c e time of the lectin at the

cell s u r f a c e is n e c e s s a r y for i r r e v e r s i b l e b i n d i n g .

Cell

suspensions

were t h e r e f o r e i n c u b a t e d with F-WGA for v a r i o u s times a n d the

bind-

ing p r o c e s s i n t e r r u p t e d by a d d i t i o n of an e x c e s s of n a t i v e WGA. n a t i v e l e c t i n s h o u l d both compete with F-WGA for unoccupied s i t e s a n d e s t a b l i s h an e q u i l i b r i u m with bound F-WGA if l e c t i n

The

binding binding

315

Fig.3.

Rate of b i n d i n g

of

F-WGA

F-WGA w a s a d d e d to u n t r e a t e d { ) and n e u r a m i n i d a s e treated ( ) c e l l s at a l e c t i n c o n c e n t r a t i o n of 1 ug/150 ul and 5 ug/150 ul r e s p e c t i v e l y . At the times i n d i c a t e d F-WGA b i n d i n g w a s stopped b y a d d i t i o n of 10 volumes of PBS. 100 a>

0

1

2

3

4

5

Incubation time (mins.)

Fig.4.

Rate of

Conditions

b i n d i n g of

F-WGA

as in F i g . 3 e x c e p t

much s h o r t e r

tirr:e increments

v/ere

used.

316 is r e v e r s i b l e . Fig.L,

The r e s u l t s showed that in c o n t r a s t to the d a t a

the b i n d i n g c u r v e s for u n t r e a t e d a n d

c e l l s were almost i d e n t i c a l

(Fig.5).

At the 5 s e c .

time p o i n t ,

c o n c e n t r a t i o n of bound F-WGA on the u n t r e a t e d c e l l s (Fig.4).

In c o n t r a s t ,

effectively

the b i n d i n g c u r v e s

n e u r a m i n i d a s e - t r e a t e d c e l l s were almost i d e n t i c a l ,

the

( F i g . 5 ) w a s only

a b o u t 25% of the v a l u e o b s e r v e d when F-WGA b i n d i n g was s t o p p e d by d i l u t i o n

in

neuraminidase-treated

for

i r r e s p e c t i v e of

whether the l e c t i n b i n d i n g p r o c e s s w a s i n t e r r u p t e d by d i l u t i o n or e x c e s s n a t i v e lectin

(Fig.4)

(Fig.5).

Discussion N e u r a m i n i d a s e treatment c l e a r l y d e c r e a s e s cells

(Fig.2)

indicating

an important role for s i a l i c a c i d in the cell

s u r f a c e r e a c t i v i t y of t h i s l e c t i n . f i n d i n g s of other workers of p a r t i c u l a r

F-WGA b i n d i n g to K562

These r e s u l t s a r e in a c c o r d

(see Introduction).

S i a l y l r e s i d u e s may be

s i g n i f i c a n c e at low lectin c o n c e n t r a t i o n s

s i n c e the

neuraminidase-inhibition

e f f e c t w a s more pronounced at 0 . 1 ug

5 ug of lectin

However,

(Fig.2).

d a t a i s complex.

1

2

than

i n t e r p r e t a t i o n of F-WGA b i n d i n g

The lectin r e c o g n i s e s both s i a l i c

a c i d a n d GlcNAc

r e s i d u e s a n d it i s c o n c e i v a b l e that removal of s i a l y l g r o u p s

0

with

3

may

4

Incubation time (mins.)

Fig.5.

Competitive i n h i b i t i o n of F-WGA b i n d i n g

F-WGA w a s a d d e d to u n t r e a t e d ( ) and neuraminidasetreated ( ) c e l l s u s i n g lectin c o n c e n t r a t i o n s of 1 u g / 1 5 0 ul a n d 5 u g / 1 5 0 ul r e s p e c t i v e l y . At the s p e c i f i e d times, ( i d e n t i c a l to those in F i g . 4 ) 10 ug of n a t i v e lectin w a s a d d e d to the i n c u b a t i o n m i x t u r e a n d c e l l f l u o r e s c e n c e determined i m m e d i a t e l y .

317 e x p o s e GlcNAc in

sites

F-WGA b i n d i n g

balance

between

cryptic and

sites.

it c o u l d

loss of Sialic

lectin

by

sialyl acid

concentrations.

may be i n f l u e n c e d

suggest

that

acid.

involves

proximal

to the membrane

of e x c e s s treated

native

and

binding

lectin

primary

quantitatively represents and

F-WGA i n t e r a c t i o n

lectin

yielded cells

of

3 & A) •

the b i n d i n g

with

sialic

is r e v e r s i b l e

acid

so that

fluorescence declines and

f r e e forms.

' lectin-binding

i r r e v e r s i b i l i t y and 30 min. contrast,

similar

(Fig.5)

were determined (Figs.

stage

acts m a i n l y

with

to

of

neuramlectin

the

binding

to GlcNAc.

We

most n o t a b l e the l e c t i n

in

and

more important

WGA r e c o g n i t i o n

of

sialic

in

the

sites

consisting

principally

with c e l l

surfaces by

after

dilution

process

more

of

with

that

of

not

native

lectin

influence cell

interrupting

curves

are

the b i n d i n g

results

point

cells

native

when

addition in

F-WGA

lectin

the

is

these added,

between

contact acquired

a pre-incubation

fluorescence cells

almost

inter-

of

a progressive,

after

is,

values. in the

identical

process

of In

main,

irrespective

(cf.Figs.4

to c o n s i d e r a b l e

of

the

is e s t a b l i s h e d of

to

the

occupancy

prolongation undergo

to n e u r a m i n i d a s e - t r e a t e d

our

than

an e x c e s s of

binding

with

rather

Initially,

addition

populations

indicate

to u n t r e a t e d

complexes

lectin

contrasted

two c e l l

results

residues.

However,

addition

In c o n c l u s i o n ,

r a t e s of

and this

in these

These

when

so that b i n d i n g

the method of

net

as a new e q u i l i b r i u m

site'

F-WGA d i d

binding

irreversible, of

Additionally,

between

at

interaction

resistant

of WGA b i n d i n g ,

differences observed

rates

native

of

untreated

conspicuous

bound

matrix.

surface

the

readily

f a c t o r s and the r a t e of

an i n t e r a c t i o n

cells,

r a t e of and

of

chains

residues. Interruption

sites

and

of

interact

impede WGA b i n d i n g

the f a s t component

cells,

at the p e r i p h e r y may

a

expression

s u g a r on s a c c h a r i d e

GlcNAc

steric

may

The s l o w e r component,

GlcNAc

time,

by

Changes

may thus comprise

sites

to be m a i n l y

residues

neuraminidase-treated

cell

These

the p e r i c e l l u l a r

WGA to s i a l y l

to the l e c t i n .

accompanying

Accessibility

inidase,

diffusion through

and

localised

glycocalyx.

sites, presumed

untreated

sites

is a t e r m i n a l

internal

of

available

neuraminidase

be p r e f e r e n t i a l l y

surface-associated low

not n o r m a l l y

induced

and

5).

heterogeneity

318 in F-WGA b i n d i n g s i t e s at the K562 c e l l s u r f a c e . b i n d i n g p r o c e s s by k i n e t i c a n a l y s i s , neuraminidase-induced

Dissection of the

competitive inhibition

and

inhibition indicates that s i a l y l residues

the p e r i p h e r y of the g l y c o c a l y x

a r e the most

at

rapidly-recognised,

q u a n t i t a t i v e l y p r e d o m i n a n t s i t e s a n d t h a t following d e s i a l a t i o n g r o u p of r e l a t i v e l y i n a c c e s s i b l e ,

which a p p e a r l e s s r e a d i l y s a t u r a b l e than the s i a l y l c a r e f u l choice of the e x p e r i m e n t a l c o n d i t i o n s low lectin c o n c e n t r a t i o n )

a

s l o w - r e a c t i n g GlcNAc s i t e s a r e sites.

exposed

By

(short incubation

time,

it seems p o s s i b l e t h a t WGA may be u s e d

a s e l e c t i v e probe of cell s u r f a c e s i a l i c

as

acid.

References 1. A d a i r ,

W.L. & K o r n f e l d , S .

J.Biol.Chem.

249,

2. Allen, A . K . , Biochem.J.

3. B h a v a n a n d a n , J.Biol.Chem.

V.P. & Katlic,

E.B.,

J.Biol.Chem. Biochemistry, 7. G o l d s t e i n , Biochim.

Li,

(1973)

(1979)

J.T.,

Swindell,

R. & Crowther,

222-226 (1977)

1107-1116 (1973) 1312-1323

12,

Hammastrom, S. & S u n d b l a d ,

Biophys.

Blood, 45,

A.W.

E. & K o r n f e l d , S.

P.

I.J.,

8. Lozzio, C . B . 9. S t a n l e y ,

IV,

252,

6. C u a t r a c a s a s ,

N.

4000-40C8

G., Gallagher,

Flow Cytometry Briles,

A. & S h a r o n ,

155-162

254,

4. B l a c k l e d g e , 5.

4696-4704

Neuberger,

K51,

(1974)

Acta 405,

& Lozzio,

53-61

B.B.

(1975)

321-334

P. & C a r v e r ,

Proc.Natl.Acad.Sci.

J.P.

U.S.,

(1977)

74,

5056-5059

G.

(1975)

D.(1980)

INTERACTION OF HUMAN YOUNG AND OLD ERYTHROCYTES WITH WGA. A FLUORESCENCE STUDY.

Dominique Bladier0,Marie-Ange Deugnier®°and Michel Caron° "Laboratoire de Biochimié et Immunochimie (Pr P.Cornillot) 9

"Laboratoire de Microscopie Quantitative (Pr J.-C.Bisconte) U.E.R. Biomedicale,74,rue Marcel Cachin-93012 BOBIGNY,France

Introduction During its lifespan in the circulation,the macromolecular components of the red blodd cell (RBC) are submitted to progressive modifications leading to the alteration of its physicochemical and functionnal properties and ultimately resulting in its removal by the macrophages.Erythrocyte aging,among other events,gives rise to the homogeneous loss of membrane fragments (1) and to modifications of some membrane dynamic properties (2).Changes at the membrane level have an even higher importance since it is the membrane which interacts with the extracellular environment.The role attributed to the surface glycoconjugates in such interactions led us to examine these structures and their properties in relation with the age of the cells. The aim of the study,reported here,was thus to investigate the interaction of human RBC with rhodamine labelled WGA,looking for possible differences between young and old cells.

Material and methods Cell labelling.Young and old human 0 erythrocytes,obtained from healthy donors of the blood bank (Centre Departemental de transfusion Sanguine,Bobigny,France)were separated accor-

© Walter de Gruyter & Co. 1983, Berlin • New York Lectins, Vol. Ill

320

ding to Murphy (3).Rinsed RBC were incubated with the rhodamine wheat germ agglutinin conjugate (WGA-T,IBF) for 30 min at 20°C.A subagglutinating concentration,i.e. 9;6ug of WGA-T per 1C>8 cells,was used for all experiments.Cells were washed twice with 0.9% NaCl solution before being resuspended in lmL of phosphate buffered saline solution (PBS).Fluorescein diacetar te (FDA) was used as a freely diffusing intracellular probe, at 6 yg per 10®RBC (4).The labelling was performed for 10 min at 37°C and followed by 3 whashings with PBS.The labelled samples were kept at 4°C until the analysis. Fluorescence measurements.Suspensions of erythrocytes were mounted on a glass slide covered with a coverslip and examif ned under a microscope equipped with a laser illumination.The basic instrument has been previously described (5) and detail of the computer control is given in (6).Briefly,the laser beam (Spectra Physics,Aemission=514.5 nm) is focused through the microscope objective (xl25) to a 3.5 ym diameter spot, which allows cell by cell measurements.Light pulses of preselected duration (500 msec) and intensity (30viW) are delivered using an accousto-optical modulator (S0R0) monitored by a PDP 11-34 (DEC) computer.The fluorescence emission is detected by a photomultiplier (Leitz,MPV2) also connected to the computer.The fluorescence intensity values,stored for further processing,are proportionnal to the number of fluorescent sites per illuminated cell location. Data analysis.Two types of analysis have been made: i)determination of the mean (-sd) fluorescence intensity of a given cell population.In this case,one measurement per cell was performed and about 100 cells were examined.A histogram can be plotted with these 100 values. ii)determination of the reproducibility of the measurement on a single cell.In this case,5 successive measurements were performed per cell and about 20 cells per sample were assayed

321

It should be pointed out,that before each of the 5 measurements the chosen cell was repositionned at the center of the microscopic field,where the laser beam is focused,thus resulting in the eventuality that the 5 locations,successively examined,are not exactly superimposable.The results are expressed as follows: for each cell,the differences between the 5 measured values of fluorescence intensity were determined 2 by 2.Ten numbers are thus obtained.The set of numbers,calculated for the 20 assayed cells,are then distributed,into classes of 1 arbitrary unit wide,on a histogram.

Results 1)Mean fluorescence intensity.of the RBC populations.lt was measured:i)on young and old WGA-T-labelled RBC.The results obtained from 5 samples are listed in table l.It appears that the mean values found for the old RBC populations are significantly higher than that found for the young ones.A typical his togram of fluorescence distribution is shown in figure 1 B. ii)on old FDA-labelled RBC,figure 1 A.Very similar fluorescence distributions were obtained for both WGA-T and FDA labelled populations,figure 1 A and B. 2)Reproducibility of the measurements on a single cell.It was tested on both FDA and WGA-T labelled RBC.Typical histograms are presented in figure 1 A' and B' respectively.These histograms are very different,suggesting that the reproducibility of the measurements on a single cell is better on FDA labelled RBC than on WGA-T labelled RBC.

Discussion The results obtained in this study of WGA interaction with

322 TABLE 1 Mean value of fluorescence intensity old and young RBC. Sample N°

m

Old Cells sd

(arbitrary units) of WGA-T labelled

n

m

Young Cells sd

n

1

51

13.5

lOO

41

9

102

2

59

10

104

52

10

99

8.5

100

3

61

12

100

50

4

65

14

lOO

58

10

100

5

61

14

101

47

10

101

n=number of measurements per sample. The mean value of autofluorescence was less than 11 a.u.In all cases, the difference between fluorescence intensity of young and old RBC are significant (p 0.001)

young and old human RBC reveal a double heterogeneity of WGA binding:on a single cell,and within a same cell population. i)Heterogeneity on a single cell:it was established by comparing the measurements obtained on FDA(a freely diffusing intracellular probe)and WGA-T labelled RBC.It can be seen that the reproducibility of the measurements on the FDA labelled RBC is rather good,fig.1 A'.This means that whatever each of the 5 cell locations successively illuminated,the corresponding measurements are very close together.This is true for each of the 20 analysed cells.The distribution of the FDA probe can thus be considered to be homogeneous within the cell.On the contrary,the wide histogram exhibited by WGA-T labelled RBC,fig.1 B' reveals that the measurements,obtained for esch of the 5 cell locations successively illuminated,are different.The distribution of the lectin,at such subagglutinating concentration,can thus be considered to be heterogeneous at the surface of the cell,due to the presence of clusters.Whether these clusters are induced by the lectin interaction,or preexist,is currently under study.It cannot be excluded that differences in some

323

w J J w u Dk o « H m

20

20 !

10"

10 "

a D

nJ

2

20

60

Bin '

100

0

F L U O R E S C E N C E

60

w a

Ik

Id 65 I'OO I N T E N S I T Y (a.U.)

60 .

A'

40.

40 .

20-

20

H p4 o OS

w «

s D

z

X

-10

0

10

- t h

0

-Ur-

-20

0

' l"l. f I 20

VARIATION OF FLUORESCENCE INTENSITY (a.u.)

Fig. 1

Fluorescence of FDA and WGA-T labelled RBC:

Above: Histograms of fluorescence distribution, one measurement per cell (100 cells per sample), classes of 4 a.u. wide. A = FDA labelled RBC; B = WGA-T labelled RBC. Below: Reproducibility of the measurements: the difference of fluorescence intensity between the 5 successive measurements per cell (20 cells per sample are distributed into classes of 1 a.u. wide). A* = FDA labelled RBC; B' = WGA-T labelled RBC.

324

dynamic properties of the cell membrane with aging(2)could also participate in the mechanism of cluster formation. ii)heterogeneity within a same cell population:old RBC populations are characterized by both higher mean values of fluorescence intensity and wider fluore scence distribution.As WGA bin ding to cells is known to be biphasic,due to the presence of high and low affinity binding sites(7)this difference may Be due either to a higher density of WGA binding sites on old RBC or to modifications in the balance of the number of high and low affinity binding sites,or to variations of the affinities. In the interaction between the cell surface and W-a-Mjn-(|-»«)/

9

i - G « l - ( l - » 4 ) 0-GIcNAe-(l->2)-o-.Man-I.I-3) x 0-Gk-N A c - ( 1 -4)-0-MaJl-< 1 -4)-ClcNAe i-Ga1-(l-4)-0-C!cNAc-(!-»2)-o-Mar.-(l-6)/

10 0-G.il-< 1 —II-1• ClcNAf-( 1 - 2 ) - a - » l a n - ( 0-Cal-(l-4)-,J-GlcNAc--n-(l-4)-,l-GlcNAc- 3 ) > )|J-Man-(1~4)- G!cNAc

Fig. 1.- Interaction of insolubili zed lectins N-glycosylamine linkage.

UEA,

LTA

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FR"

FNR

FNR

FNR

FNR

FE

FR"

FR*

FNR

FNR

FNR

FNR

FE

FE

FR

FE

FNR

FNR

FNR

FNR

FNR

FNR

FR

FR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FR

FE

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FR"

FNR

FNR

FNR

FNR

FE

FE

FE

FR

FE

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

LCA

VFA

PSA

FNR

FNR

FNR

FR"

FR +

FNR

FNR

FNR

FNR

FNR

FR"

NO

ND

ND

ND

FNR

ND

ND

ND

ND

FE lOmM

FE

FE

FE

Con A FE iomM "MG FE iomM «MG

RCA, RCA || G S A „ WGA

*(MG FE iomM VMG FE 10 mM FNR MG FE 10m M «MG FNR

+

FE 10mM of MG FE lOmM «iMG

with glycans

from g l y c o p r o t e i n s w i t h

the

340

GLYCAN STRUCTURE 12

tt-NeuAc-(2-+6)-0-Ojl-(l-*4)-í3-GlcNAc-(W2)-a-Man-(l-*3K \-Man-(l-4)-ClcN-Ac O-Min-(l~6K

13

c-N:uAc-C-J)-((-C3l-(l-4)-;-GlcNAc-(l-:)-e-Man-(l-3)-(!-Mjn-(l->-o-Man-(l~3) i-Man-(l-4)-CkNAt

15

(-GJI-(I-4)

16

Ca,

-„'J

-JI-GlcNAc--(l~2)-a-Man-(1-3>. J-Clc\'Ac-(l-:)-o-Xbn-(l-0)/

M:.n-(l"-l)-P-C;icNAc-U-'4)- > i ClcSAc-(l".l)-Asn

_-*H>-ClrNAe--«-S1ai!-

17

J-Gal-d-MlCj

. -i-C.lcNAc-(1-2)-«-M»n-{l-3). }0-Man-(!--GlcNAc-(l->41-J-GlcNAc-(l-4)-AJll -Í-C.ICNAC-(I-:)-O-MJH-(I-6/

18

| 1.6

•-Fuc

-|>dcNAc-(l-2)-n-M:m-(l-3)^ ^ . . G I I N A V - 0 "4)-j)-M3n-(l-4)-()-CkNAc-(l-4)_/)-GIcNAc- ()-4)-A»n "~^--0-GlcNAc-(l~2)-a-M»n-2)-a-Maa-< 1-3). V-M>n--Man--0-

(1—2)-a-Man-(l—6)'

Cal-(l-4)-0-G!cNAc-(l-M).^

o-NcuAc-C-6)-()-Gal-(l—ll-fl-ClcNAc-ll-D-a-M.ni-0-3). p-Mun-(l—4)—0—GlcNAc-(l—4)-0-GlcNAc-(l—3)-Asn a - N t u A c - ( 2 — 3 ) - P - G a t - ( l — 4 ) - 0 - GlcN A c - ( 1 — ? ) - « - M a n - ( l —

31

t -Gal-( 1 - 4 ) - 0- GlcNAc-(l Man—(I—4)-iJ-GlcNAc-(l—4)-|3-GlcNAc-( I—4)-A»n i-Gal(l-4)-S-GlcNAc-(l-:)--Man-(I-6K

.

P - G a l ^ l ^ - i - C l c N A c - d - t ) ^

32

i

GlcNAc-(l-4K^

K-ClcNAo (1-3)

aMjil-(l-J)^

(1- C : i c N A c - ( l - 4 t

a -M.m ( 1 - 4 ) - 0 - G l c W U - ( l - 4 > - 0

CltNAf-d-tt-Avl

(1 G t c N A c - t l - . ' > - n - M j n - ( l — < ! > /

F i g . 3 . - I n t e r a c t i o n of i n s o l u b i l i z e d N-glycosylamine linkage.

lectins

Con A

LCA

VFA

PSA

UEA,

LTA

FR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FE

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FI^R

FNR

FNR

FNR

FNR

FR

FE

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FR

FE

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FR

FE

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

ND

FR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

ND

FE

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FR

FE

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FR

FNR

FNR

FNR

RCA, RCA || GSA|| WGA

-

with glycans from glycoproteins with the

346

GLYCAN STRUCTURE 33 J-GkNAc-(I-2)-«-Man-(l-3). \3). 0-Man-(l"4)-GlcNAc P 'GlcMAc-(l-2)-ii~Man-(!-«/ 39 i-GlcNAc- ( l - > : ) - a - M a n - ( l - 3 ) ^ i - G L N A c - i 1-41-11-Man-( l - 4 ) - f f - C l c N A c - ( l - 4 ) - U - G ! c N A c - ( ! - 4 1 - A s n o-Man-(l-O) -a-Man-(l-G)/ a-MznAWr

F i g . 4 . - I n t e r a c t i o n of i n s o l u b i l i zed l e c t i n s N-glycosylamine l i n k a g e .

RCA, RCA,, G S A „ W G A

UEA,

LTA

FNR

FNR

FNR

FR"

FNR

FNR

FNR

FE

FNR

FNR

FNR

FNR

FNR

FNR

FE

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FE

FNR

FNR

Con A

LCA

VFA

PSA

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FNR

FR

FNR

FNR

FNR

FNR

FR

FNR

FNR

FNR

-

FR +

FE 0.3M «< MG

FNR

FE lOmM VMG

with glycans

from g l y c o p r o t e i n s w i t h

the

348

glycopeptides such as those found in hen ovalbumin

(Structure

39). Structure 16 which possesses also a g-N-acetylglucosamine residue linked to C-4 of the g-linked mannose residue but belongs to the N-acetyllactosaminic type family, presents a weaker affinity for WGA, and structure 18 which just differs from structure 16 by the presence of an a-L-fucose residue in C-6 position on the N-acetylglucosamine residue involved in the N-glycosylamine bond is no more recognized by insolubilized WGA. The lectin does not present any affinity for oligomannosidic-type glycopeptides

(Structure 37). As found also by

Yamamoto it at. (13), the N-N'-diacetylchitobiose moiety has to be intact, for, oligosaccharide 21 which possesses the 8 - Nacetylglucosamine residue in C-4 position on the B-mannose residue, but where the g-GlcNAc-(l -*• )-Asn sequence is lacking is not retained on WGA-Sepharose. It can also be noted that sialylated glycopeptides oligosaccharides

(Structures 1, 29, 30) or sialylated

(Structure 2) are not recognized by insolubi-

lized WGA. These interactions involve a charge effect and depend on a mechanism called by Monsigny it at. (14) "avidity effect"

: only glycopeptides with a high density of terminal

non-reducing N-acetylglucosamine or N-acetylneuraminic acid residues interact with WGA.

GA^^on^a

iLmptLc-iloila.

agglutinin.-

This

insolubilized

lectin represents a very useful tool for the fractionation of glycopeptides or oligosaccharides with unmasked N-acetylglucosamine residues in a terminal non-reducing position and the affinity of the lectin increases with the number of such residues : glycopeptide 19 is more retarded on the column than glycopeptide 33. But, at least two of such N-acetylglucosamine residues are necessary for the interaction to take place, for glycopeptides 15, 16, 17, 18 or 39 with only one unmasked Nacetylglucosamine residue are not retained on the column. In the case of structures with two terminal non-reducing N-acetylglucosamine, GSAjj presents a greater affinity for the glycopeptide than for the related oligosaccharide

(Glycopeptide 19

349

compared to o l i g o s a c c h a r i d e 20 or 2 1 ) . This can a l s o be e x p l a i ned by the f a c t t h a t the g l y c a n - a m i n o a c i d l i n k a g e l e a d s t o s t r u c t u r e s more r i g i d than those of the o l i g o s a c c h a r i d e s . Le.ct£m

w^th

a ipzailtcity

{¡oh. L-^uco,se.-

Lotui

te.£fiagonolobu.i

a g g l u t i n i n or U£ex euAopzui a g g l u t i n i n I have no a f f i n i t y f o r g l y c o p e p t i d e s or o l i g o s a c c h a r i d e s of the N - a c e t y l l a c t o s a m i n i c t y p e , even i f t h e s e s t r u c t u r e s p o s s e s s one or two f u c o s e r e s i dues c t - ( l •*• 3) or a - ( l 6 ) - l i n k e d to N - a c e t y l g l u c o s a m i n e r e s i dues as i n g l y c o p e p t i d e 5 o r 6. As p r e v i o u s l y d e s c r i b e d by P e r e i r a t£ a.1. (15) , the s p e c i f i c i t y of t h e s e two l e c t i n s seems to be d i r e c t l y towards the a - ( l -*• 2 ) - l i n k e d fucose r e s i d u e s found i n blood-group H a c t i v e substances.

Conclusions This work completes and e x t e n d s our p r e v i o u s r e s u l t s o b t a i n e d by i n h i b i t i o n of the a g g l u t i n a t i o n of human-red blood c e l l s induced by l e c t i n s ( 7 ) . These r e s u l t s c o n f i r m t h a t l e c t i n s , c o n s i d e r e d " i d e n t i c a l " i n terms of monosaccharide s p e c i f i c i t y , p o s s e s s the a b i l i t y to r e c o g n i z e f i n e d i f f e r e n c e s i n more comp l e x s t r u c t u r e s and t h i s can be used to f r a c t i o n a t e m i x t u r e s of N - g l y c o p e p t i d e s of v a r i o u s o r i g i n s .

Acknowledgements This work was supported i n p a r t by the Centre N a t i o n a l de l a Recherche S c i e n t i f i q u e ( L a b o r a t o i r e Associé n° 217) and by the D é l é g a t i o n Générale à l a Recherche S c i e n t i f i q u e e t Technique (Grant 7 9 - 7 - 0 6 6 9 ) . We thank Drs G. S t r e c k e r , B. Fournet and A. Kobata f o r t h e i r generous g i f t s of o l i g o s a c c h a r i d e s and glycopeptides.

350 References

1.

Lotan, R., Nicolson, 329-376 ( 1979) .

2.

F i n n e , J . , K r u s i u s , T . , J ä r n e f e l t , J . : 27th I n t e r n a t i o n a l C o n g r e s s o f P u r e and A p p l i e d C h e m i s t r y ( e d . A . V a r m a v u o r i ) Pergamon P r e s s , O x f o r d - N e w Y o r k , p p . 1 4 7 - 1 5 9 , 1980.

3.

D a l l ' O l i o , F . , S e r a f i n i - C e s s i , F., Scannavini, C a m p a d e l l i - F i u m e , G. : T h e s e p r o c e e d i n g s .

4.

O g a t a , S . , Muramatsu, 687-696 ( 1 9 7 5 ) .

5.

Krusius, T . , Finne, 117-120 ( 1 9 7 6 ) .

6.

Debray, H . , M o n t r e u i l , J. : L e c t i n s - B i o l o g y , B i o c h e m i s t r y , C l i n i c a l B i o c h e m i s t r y , V o l . 1, p p . 2 2 1 - 2 3 0 , W a l t e r de G r u y t e r , B e r l i n - N e w York ( 1 9 8 1 ) .

7.

Debray, H., Decout, D., S t r e c k e r , G., Spik, J . : E u r . J . B i o c h e m . 117, 41-55 ( 1 9 8 1 ) .

8.

Montreuil, J. (1980) .

9.

March, S . C . , P a r i k h , 60_, 149-152 ( 1974) .

10.

Strecker, ( 1979) .

11.

Koide, N., (1974) .

12.

K o r n f e l d , K . , Reitman, M . L . , 256 , 6633-6640 ( 1981) .

13.

Yamamoto, K . , T s u j i , T . , M a t s u m o t o , B i o c h e m i s t r y 2Q_, 5894-5899 ( 1 9 8 1 ) .

14.

Monsigny, Delmotte,

15.

P e r e i r a , M.E.A., K i s a i l u s , E . C . , Gruezo, F . , Kabat, A r c h . B i o c h e m . B i o p h y s . 185 , 108-115 (1978 ) .

G.,

: Adv.

G.L.

T.,

J.,

: Biochim.

Kobata,

Rauvala,

Carbohyd. I.,

Montreuil,

Muramatsu,

T.

Biophys.

A. H.

: J.

M., 78,

: FEBS-Letters

71,

G.,

Montreuil,

Chem. B i o c h e m . _37» P.

: Biochimie

: J.

Biol.

: Anal. 6J_,

Chem.

Kornfeld,

M., Roche, A . C . , Sene, C . , F . : E u r . J . B i o c h e m . 104,

559,

Biochem.

Cuatrecasas, J.

Acta

I.,

R.

157-223 Biochem.

1199-1246

249,

4897-4904

: J.

Biol.

Osawa,

T.

Chem.

:

Maget-Dana, R . , 147-153 ( 1 9 8 0 ) . E.A.

:

HERPESVIRUS GLYCOPROTEINS FROM WILD-TYPE AND

RICIN-RESISTANT

BHK CELLS. CHARACTERIZATION OF GLYCOPEPTIDES BY CON A-SEPHAROSE

F. Dall'Olio, F. Serafini-Cessi Istituto di Patologia Generale, Università di Bologna, Italy

M. Scannavini and G. Campade11i-Fiurne Istituto di Microbiologia e Virologia, Bologna, Italy

Herpes simplex virus-1

(HSV-1) specifies the synthesis of seve-

ral antigenically distinct classes of glycoproteins

(1, 2),

which mainly contain N-linked glycans. In the infected cells both the mature forms gB, gC, gD and gE, and their partially glycosylated precursors (gA-pgB, pgC, pgD and pgE) accumulate. Sialylated complex-type glycans are present in the mature forms whereas mannose-rich glycans predominate in the precursors

(3)

In a preceding paper (4) we reported that the synthesis of HSV-1 glycoproteins is altered in a mutant cell line (Ric 14) defective in the N-acetylglucosaminyltransferase

(5), which is

required for the processing of mannose-rich to complex-type glycans. Mature forms of HSV-1 glycoproteins were absent in Ric 14 cells whereas underglycosylated proteins accumulated. In the present study the detailed structure of glycans synthesized in HSV-l-infected Ric 14 and parent BHK cells were examined using a combined technique of ion-exchange and Con A-Sepharose binding.

© Walter de Gruyter & Co. 1983, Berlin • New York Lectins, Voi. Ili

chromatography

352 Materials and Methods

Radiolabeling of infected cells. R 2 Ric 14 (5) and BHK cells in 25 cm Falcon flasks were

infected

with HSV-l(MP) at an imput multiplicity of 10 PFU/cell. The cells were labeled between 5 and 18 h after infection with minimum essential medium containing one-half the usual concentration of glucose, 1% foetal calf serum and 4 jiC/ml of 14

C-glucosamine.

Polyacrylamide gel electrophoresis of HSV glycoproteins. Cells in Tris/HCl, pH 7, were solubilised with

2% SDS and 5%

[} -mercaptoethanol , and heat denatured for 2 min.

Samples were

run in a slab gel containing 8.5% acrylamide and analyzed autoradiography of the dried slab

by

(3).

Digestion of glycoproteins with endo-|3-N-acetylglucosaminidase H. Portions of sonicated cells were dialyzed against 0.1 M citrate buffer, pH 5.5. 300 Ail aliquots of the suspensions

(protein

concentration 1.5 mg/ml) were incubated at 37° C with 0.025 U of endo-j3-N-acetylglucosaminidase H (Seikagaku Kogio Co., Tokio). At the indicated times 50 Ail aliquots were withdrawn and proteins precipitated with 50 Ail of 12.5% TCA. The release of acid-soluble labeled glycans was measured on 50 Ail of the supernatant. Separation and analysis of glycopeptides. Infected labeled cells were harvested in 0.1 M phosphate buffer, pH 7.9, and digested with pronase (3). Pronase-digests were

chromatographed on 1 x 7 5 cm column of Bio-Gel P-10. The

fractions corresponding to elution position of acidic and neutral glycopeptide

markers (from 4800 to 1500 daltons) were

pooled. A portion Sephacel

column

(2 m l ) of this pool was loaded on a DEAE-

(1x15

c m ) equilibrated w i t h 0.05 M

acid. A f t e r a w a s h i n g w i t h H^O

acetic

(10 ml), a salt g r a d i e n t

(50 ml

0.05 M acetic a c i d — • 50 ml 0.05 M acetic acid and 0.5 M N a C l ) w a s applied to the column. Fractions of 1 ml w e r e and the r a d i o a c t i v i t y

of 0.1 ml w a s m e a s u r e d . The central

tions of each peak w e r e pooled. One p o r t i o n for n e u r a m i n i d a s e another portion -Sepharose.

treatment as previously

(1 m l ) w a s

described

(2 m l ) for affinity c h r o m a t o g r a p h y

The column

( 0 . 5 x 1 0 cm) was .washed w i t h

pH 7.4, c o n t a i n i n g 0.1 M NaCl and 0.02% MnCl^;

collected

it w a s subsequently

and then w i t h 200 mM

eluted with

a-methyl-mannoside

NaN 5 mM

frac-

used

(3) and on Con ATris/HCl,

, CaCl2, MgCl2

and

a-methyl-mannoside

in the same

buffer.

Results Glycoprotein Fig.

analysis.

1 shows the p a t t e r n of e l e c t r o p h o r e t i c a l l y

coproteins synthesized

in H S V - 1 ( M P ) - i n f e c t e d

Fig. 1. S D S - P A G E of g l y c o p r o teins from H S V - 1 ( M P ) - i n f e c t e d R Ric l ^ a n d BHK cells labeled with C-glucosamine

Ric

separated R

14 and

gly-

BHK

Fig. 2. A c i d - s o l u b l e glycans released by endo-|9-N-acetylg l u c o s a m i n i d a s e H from infected-Ric 14 (O), and BHK (•)

354 cells. Tn the mutant cells only the partially glycosylated precursors (pgD, pgB and gA) accumulated, whereas the fully glycosylated forms (gB and gD) were absent. Since previous observations (3) indicate that precursors of HSV-glycoproteins, namely gA and pgD contain glycans of the mannosyl-rich type, they should be sensitive to endo-^-N-acetylglucosaminidase H. This enzyme cleaves between the two N-acetylglucosamine

residues

in high-mannose glycans (four to nine residues) (6). Fig. 2 shows the results of a typical digestion experiment of 14 R C-glucosamine-labeled HSV glycoproteins from BHK and Ric 14 cells. The amount of acid-soluble glycans released by the enzyme from glycoproteins of mutant cells was significantly

high-

er than that from wild-type cells. This result confirms that mannosyl-rich glycans predominate in precursors glycoproteins of mutant cells and suggests that glycan processing is arrested before the action of N-acetylglucosaminyltransferase Glycopeptide

characterization.

Both neutral

(N) and acidic

(A) pronase-glycopeptides

by preparative Bio-Gel filtration

T.

obtained

(free from undigested mate-

rial and low molecular weight molecules) were analyzed on DEAESephacel

(Fig. 3). Five peaks N, ATa, Alb, ATT and AITI were

separated. N was not absorbed by the column and emerged with the neutral mannosyl-rich glycopeptide marker. The most acidic glycopeptide AIII was eluted together with a triantennary marker (fetuin glycopeptide) and ATI with a diantennary

glycopeptide

(prepared from human transferrin). In the elution position of slightly acidic glycopeptides two distinct peaks were obtained, ATa and Alb. Tn the BHK sample all peaks were present and AITI was the most represented. In the Ric 14 sample N represented the major peak; among the acidic glycopeptides only Ala and

355

t ract i o n n u m b e r

Fig. DEAE-Sephacel chromatography of pronase-glycopeptides from C-glucosamine-labeled HSV-infected BHK (•) and Ric 14 (o) cells. The arrows represent, from left to right, the elution positions of ovalbumin-, human transferrin- and fetuinglycopeptide. The horizontal bars indicate the fractions of each peak which were pooled and chromatographed on Con A-Sepharose. All! were present but the latter was small in amount. After neuraminidase treatment the acidic glycopeptides were all split in two peaks when rechromatographed on DEAE-Sephacel: one emerged as desialylated

glycopeptide

(unretained) and the other

one emerged as free sialic acid. This shows that the negative charge responsible for absorption to the DEAE column was due to sialic acid. The amount of radioactivity cleaved by neuraminidase as sialic acid from ATTI, ATI, Alb and Ala was 40%, 25%, 13% and 10%, respectively. These results reinforce the indication that the structures of AIIT, ATT and A1

(a and b) are, re-

spectively, those of triantennary, biantennary and monoantennary glycans. Since the processing of mannosyl-rich

to complex-

type glycans changes the affinity of glycopeptides to ConA

(7),

356 Table. Comparison of glycopeptides from HSV-1(MP)-infected BHK and Ric 14 cells separated with DEAE-Sephacel chromatography.

Glycopeptide

%

14

C-glucosamine incorporated

BHK

NANAase suscept ibi1i ty

Ric 14

Elut i on from Con ASepharose by Me-Man (mM)

16

51

-

200

Ala

5

20

+

200

Alb

18

All

20

AITT

40

N

Proposed structure

O JD-e-e

« - • - • - a

unbound

•N-Acetylglucosamine, A s ialic ac id.

OMannose,

• Galactose,

the five glycopeptides were further characterized by Con Aaffinity chromatography using two different concentrations of a-methyl-D-mannoside

(Me-Man) as specific eluent. N, Ala, Alb

and AIT were all retained by Con A-Sepharose; N, and Ala were eluted by 200 mM Me-Man, whereas ATb and ATI were eluted at the lower concentration

(5 mM Me-Man); AITI from both kinds of

cells was not retained by the column. The table summarizes the experimental data and shows the relative distribution of the glycopeptides

in each cell type. On the basis of the results

a putative structure of the glycopeptides is proposed.

357 Discussion

The combined technique of ion-exchange separation and Con A-affinity chromatography was a good approach for the structural analysis of pronase-glycopeptides from HSV-infected cells. Neu raminidase treatment and DEAE-Sephacel chromatography

provided

informations on the number of sialic acid residues which accounts for the antennary structure of complex-type glycans, while the binding to Con A-Sepharose correlated with processing of the oligomannosyl core. It was shown that in N-linked glycans the presence of the oligomannosyl moiety at the nonreducing terminal position results in a strong binding to Con A and that any addition of sugars to the mannosyl core causes a progressive decrease in the association constants of the binding between glycopeptide and lectin The results reported here indicated that

(7). (i) AITI was not bound

by Con A-gel as expected for triantennary glycans (8, 9);

(ii)

diantennary All was retained by Con A-Sepharose and was eluted with a low concentration of a-methyl-mannoside;

(iii) Alb was

eluted by Con A-Sepharose like a diantennary glycans; after neuraminidase treatment the amount of label emerging by DEAESephacel in the elution position of sialic acid was half of that released with the same treatment from All. All together these results suggest that Alb has a diantennary structure in which only one branch carries the terminal sialic acid;

(iv)

Ala was strongly retained by Con A column like neutral mannosyl rich glycans (N), suggesting that it still retains the mannosyl cluster which is usually trimmed during the assembly of complex-type oligosaccharides. On the other hand its behaviour on DEAE-Sephacel indicated the presence of terminal sialic

358 acid,

in that b e f o r e n e u r a m i n i d a s e d i g e s t i o n

acidic s t r u c t u r e

it b e h a v e d

(bound to the DEAE c o l u m n ) but a f t e r

the single radiolabeled peak w a s split m a j o r one w a s neutral

digestion

in two f r a c t i o n s ,

(unbound to the D E A E ) w h e r e a s

the

one coeluted with u n l a b e l e d sialic acid. W h e n Ala was to high resolution gel filtration p o s i t i o n of the g l y c o p e p t i d e s m a n n o s y l - r i c h glycans results).

it emerged

remarkably

in the

the other

subjected

elution

larger than

(five m a n n o s e r e s i d u e s )

For these reasons we suggest

oligo-

(unpublished

that Ala has a h y b r i d -

- t y p e structure similar to that present glycoproteins.

as

in Rous sarcoma

Hunt and W r i g h t ( l O ) showed

in this virus

virus the

presence of a h y b r i d - t y p e glycan w i t h an o l i g o m a n n o s y l

core

(Man GlcNAc - A S N ) c h a r a t e r i s t i c 5 2

plus

"branch" sugars acidic

of neutral

structures,

(NeuNAc-Gal-GlcNAc) c h a r a t e r i s t i c

of

complex

structures. R

The reduction of c o m p l e x - t y p e g l y c a n s sistent with

the g l y c o s y l t r a n s f e r a s e

in Ric 14 cells deficiencies

of

these

mutant cells and shows the relevance of p o l y b r a n c h e d to the b i n d i n g of ricin by the cell This

is con-

glycans

surface.

investigation was supported by g r a n t s from C o n s i g l i o

zionale delle Ricerche and M i n i s t e r o della Pubblica

Na-

Istruzione.

References

1. Spear, P. G.: J. Virol.

17_, 991-1008

(1976);

2. Baucke, R. B., Spear, P. G.: J. Virol.

32,

779-789

( 1979);

3. S e r a f i n i - C e s s i , F., C a m p a d e l l i - F i u m e , G.: Arch. Virol. 331-343 (1981); 4. C a m p a d e l 1 i - F iume, G., Poletti, L., D a l l ' O l i o , F., - C e s s i , F.: J. V i r o l . 43, (1982) (in press);

70,

Serafini

359 5. V i s c h e r ,

P.,

Hughes,

R.

C.:

Eur.

J.

Biochem.

117,

275-284

(1981) ; 6.

Tarentino, (1974) ;

A.

L.,

Maley,

F.:

J.

Biol.

Chem.

249,

811-816

7.

Baezinger, ( 1979 ) ;

J.

U.,

Fiete,

D.:

J.

Biol.

Chem.

254,

2400-2407

8.

Krusius, T., Finne, 120 (1976) ;

9.

Debray,

10.

Hunt,

L.

H., A.,

J.,

Montreuil, Wright,

S.

Rauvala,

J.: E.:

H.:

Biochimie, J.

Virol.

FEBS

Lett.

7_1_,

117-

60_, 6 9 7 - 7 0 4

( 1978);

39,

(1981).

646-650

C O N - A - R E A C T I V ITY ELECTROPHORETIC CROSSED

Mette

OF

LEUCOCYTE

aj-ANT ITRYPSIN.

a^-ANTITRYPSINS

STUDIED

BY

IMMUNOELECTROPHORESIS.

Andersen,

The Finsen Denmark.

AND

HETEROGENEOUS

AFFINITY

M.

SERUM

Susanne

Laboratory,

The

Noack Finsen

Institute,

Copenhagen,

Introduction

In a p r e v i o u s contain tor). cell

study

(1)

a^-antitrypsin

We

also

line,

6 0 ctjAT's

showed

HL-60 have

we

(2)

similar

sis

of

by

sis

(B0g-Hansen

about dy

the

more

closely

to

compare

was

undertaken.

in o r d e r

to

Materials

from

We

compare

Concanavalin

was

the

A

does

affinity give

involved

molecular

variants defined

also

more

studied

this

with

a^AT

and

HL-

in c r o s s e d

Im-

(2).

precise (2).

present

serum

leukemic

Analy-

Immunoelectrophore-

ctjAT's from

normal

inhibi-

Leucocyte

heterogeneous

not

leucocytes

promyelocyte

morphologies

very

with

(conA)

Sigma,

Difco

(6))

human

a^-protease

information

In o r d e r in t h e s e

the

to

stu-

a^AT's

present

study

ajAT-deficient

sera

a,AT.

Methods

Chemicals, from

human

traditional

al.

them

and

being

subpopu 1 ations

various

and

Fine

et

the

that

called

a j - a n t i t r y p s in.

precipitate

both

oijAT's

(also

that

synthesizes

munoelectrophoresis these

demonstrated

(aj A T )

Lab.,

(Batch

no

Sweden;

Porcine

U.S.A.,

and

Detroit,

GL

23289)

pancreatic

Trypsin

U.S.A.

© Walter d e Gruyter & Co. 1983, Berlin • N e w York Lectins, Vol. Ill

1:250,

Five

was

from

elastase "Difco"

normal

human

Pharmacia (type

III)

certified sera

were

362 obtained viduals

from with

a normal

volunteers genotype

serum

trophoresis

adjusted

(both

1 mg/ml)

thereafter.

DAKOPATTS, Cells: HL-60 of

Human

by

1):

gel

without

to

The

der

minimize

and

were

on

the

width

of

placed

Prealbumin,

at

used the

as

out

in

and

the

conA

gel.

by

normal

containing

Both

in

centration out.

All

dation 5

elec-

Sepharose.

trypsin °C,

or

and

This

elastase

run

immediate-

were

1640

as

described

with

10%

respectively

al.

This (6)

was

or

as

(1)

and

from

a first

in

of out

for

of

the

the

internal

edge

the

dimension

second

the

was

antigen

con-

and

in

The

gel

added

cast.

into

a

well

gel

was

electrophoresis

sec-

plate

was

containing

or-

gel

secondary

electrophoresis

dimension

gel

5 mm

antigen.

of

elec-

Immunoelectrophowas

conA-containing

standard,

between

used

applied

cathodic

space

affinity

mostly

cut

as

a

dimension

sections

plates

(2).

procedure

were

sections

pg/ml

either

a two-step

electrophorized

(i.e.

and

50

described

performed

as

were

FCS

respective

gel

as

(R

and

was

conA-reactivity

included

prepare

slice

performed

into

anti-

gel.

the

Technical

Biue

antigen(s)

First

coefficient

% was

gels.

to

indi-

agarose

The

traditional in

used

by

separated

the

border

body

the

et

width

remaining

from

anti-prea1 bumin

obtained

RPMI

antigen(s)

gel

the

cut

followed

were in

secondary

resis.

on

with

sera

Denmark.

conA).

of

were

i h 37

and

electrophoresis

fractions

transferred

tions

ratios

streptomycin

the

sera

purified

mixed

anti-a^AT

leucocytes

Firstly

trophoresis

to

was

volume

B0g-Hansen

agarose

taining

1:1

and

was

immunoe1ectrophoresis.

described (Fig.

in

Rabbit

normal

ajAT

chromatography

1 mg/ml,

propagated

penicillin

free

by

8 otj A T - d e f i c i e n t

The

to

Copenhagen,

cells,

Affinity

ZZ. Serum

followed

ctjAT,

ly

pool.

and

the

two-step

standardized fractions

= Lo/Lr-1)

specific

details

at

were

(7).

as

140

the ?

ug/cm

defined

by

conA

in

in

all

(6).

con-

throughtheir

retar-

a-methy1-D-mannoside

displacer

otherwise

technique

second

(MM)

dimension

363

—

1. dimension

-j-

—

F i g . 1: Two-step crossed a f f i n i t y Immunoelectrophoresis (schematic drawing), a the controlplate, i l l u s t r a t i n g the p o s i t i o n of the heterogeneous molecul a r variants of a protein after electrophoretic separation (A, B, C and D) and the p r e c i p i t a t e pattern of these molecular variants after second dimension e l e c t r o p h o r e s i s . b> the f i r s t step of t h i s technique with electrophor e t i c separation of multiple protein samples in p a r a l l e l (controls are run next to t e s t samples). £ the second step, in which a molecular variant (A) has been transferred to a secondary plate where f i r s t and second dimension electrophoresis has been performed. Results i-

g^AT f r o m normal

mal

serum was a p p l i e d

ly

or d i l u t e d

trophoresis, tions, these

fig.

(R(Sj) 3 pi, tion

30 pi

2 a.

= 0.62

low a m o u n t s ,

(1:100)

either

however directly

same i n

R ( S 2 ) = 1 .48 + / - 0 . 0 4 ,

or d i l u t e d

30 pi

fraction

(R + / R(S3)

higher

(1:10),

frac-

S-)

normal

of

sera

= 2.39

amounts,

a fourth

was p r e c i p i t a t e d

e.g.

frac-

in the

first

if

specific

displacer

fraction

was not

defined

d e p e n d e d on t h e

length

the

it

relative

size

eluted

Immunoelec-

of t h i s

precipitate,

was n o t

in

direct-

The r e t a r d a t i o n

since

it

affinity

nor-

conA-reactive

individual

applied

If

u1,either

coefficients all

serum was

The f o u r t h since

in three

serum.

was not p r e s e n t . affinity

gel

eg 0 , 3

in t r a d i t i o n a l

The r e t a r d a t i o n were t h e

+/- 0.02,

If

appeared.

dimension

in

t h e otjAT was d i v i d e d

fractions

+/- 0.07).

serum and a ^ A T - d e f i c i e n t

which d i f f e r e d

and t h e

retardation

in of

of the g e n e r a l individual this

conA-

sera.

fraction

wellBoth

depended

364 on t h e t e c h n i c a l

details

The c o n A - r e a c t i v i t y lar

to t h a t

less

than

nient

in

of

relative

normal

values

these

producing

coefficients

sizes

experiments.

serum otjAT. S i n c e

3 ul-amounts

retardation

the

of oijAT f r o m a j A T - d e f i c i e n t

normal

10 % of

of

were t h e

the

sera

sera

was

simi-

concentration

was

were r u n most

4 fractions

(fig.

2 b).

of t h e

3 genuine

fractions

same as

i n normal

serum.

conveThe

and

their

F i g . 2: Crossed a f f i n i t y immunoelectrophoresis of 0,3 gl normal seKum (a) and 3 ul a,AT-deficient serum (b). Both plates contained 0,36 gl/cm a n t i a,AT plus in (a) 0,06 Ml/cm , and in (b) 0,14 pl/cm anti-prealbumin. A total of three f r a c t i o n s (S, to S O was seen in normal serum and four f r a c t i o n s (Sj to S 4 ) in a^AT-deficient serum (see t e x t ) . V!hen normal divided the

serum a^AT was r u n

into

three

serial

relative

sizes

of

varied front

within

gelslice

precipitate

leg)

leg)

where

intermediate tive

almost

gelslice

distribution

gelslices

(not

of

shown

o f 5 mm g e l

conA-fractions

it

relatively

gelslice

contained

ajAT

a^AT,

to

the The rela-

to the that

3). the

and

to

but t h e

demonstrated

S^)

(fig.

more a j A T ( S j )

intermediate

that

and

and S3 d o m i n a t e d .

more t o t a l This

being

seen

S2

(a^AT c o r r e s p o n d i n g

and S^ was

in f i g u r e ) .

was

(Sj,

a j AT c o r r e s p o n d i n g

no S^ was p r e s e n t

Sj,

technique

the e l e c t r o p h o r i z e d

(containing

contained

t h a n t h e most c a t h o d i c hind

sections

the three

systematically

The most a n o d i c

in the t w o - s t e p

S,

other has

365

the h i g h e s t the t h r e e Similar

and S3 the l o w e s t e l e c t r o p h o r e t i c

mobility

among

fractions.

results

were o b t a i n e d w i t h ctjAT from

cijAT-deficient

sera.

b

a

s W&

^

„

PA

2

3

mm

||B|

S

¿'sfiH'

|

fpffsif

2

$

PA

S 3 S i

3

3

. •

Fig. 3: Crossed a f f i n i t y Immunoelectrophoresis (the two-step technique) of serum a,AT. (a) and (b) i l l u s t r a t e the distribution of conA-reactive fractions (5,, Sp and S 3 ) in the anodic and cathodic fractions, respectively. In the f i r s t step 3 pi normal serum was applied. The second step was performed with 3 gelsections of 5 mm each. Antibody: 0,14 ul/cm anti-a,AT and 0,06 gl/cm anti-prealbumin. 2.

g^AT-elastase

and o ^ A T - t r y p s i n

acted w i t h e l a s t a s e

reaction

p r o d u c t s . a^AT

produced two p r e c i p i t a t e

Immunoelectrophoresis,

peaks

one w i t h s i m i l a r m o b i l i t y

bility

and one w i t h

Serial

sections

The oijAT h a v i n g m o b i l i t y

were as

follows:

to serum cx^AT had the

as serum oijAT.

to the c t j A T - e l a s t a s e or the two a j A T -

of s l o w e r m o b i l i t y

fined conA-reactivity.

had d i f f e r e n t

Only one ( o r two b a d l y The r e t a r d a t i o n

and l e s s

separated)

c o u l d be seen w i t h t h e s e otjAT's and t h e i r

were o f t e n d o u b l e c o n t o u r e d . 2,20.

(^-mo-

l e n g t h of the a j A T -

The r e s u l t s

corresponding

same t h r e e c o n A - r e a c t i v e f r a c t i o n s The oijAT c o r r e s p o n d i n g

from 1,19 to

produced

as serum a j A T , one w i t h

p r o t e a s e p a t t e r n s were p e r f o r m e d .

tion^)

a^M

^-mobility.

of 5 mm t h r o u g h the e n t i r e

trypsin fractions

crossed

as serum

and one w i t h (J-moti 1 i t y . a^AT r e a c t e d w i t h t r y p s i n t h r e e p e a k s , one w i t h m o b i l i t y

in

re-

de-

frac-

precipitates

coefficient

varied

366

a-, AT

/

PA B PA

B

1-6

Le LS

L

D

6 LS L

4

L4

—- L'-j

'0

E

1-2-1

l3 L7 (J" íy H

367

_3-

HL-60

grown In

cells.

in f e t a l

all

calf

samples

thesized

by t h e s e

wide

nity

with

strong

H L - 6 0 a ^ A T was

if

specific

affinity

precipitate

In t h e most

coefficient

a fraction

were p r e s e n t

with could

anodic

syn-

a^AT

one o r after

was p r e s e n t ,

a lower

conA-reactivity. due t o

conA. conA-affidespite dimen-

having

as w e l l

Whether

was

two

in the f i r s t

to

be d e t e r m i n e d

this

a fraction

similar not

Sj

lab.

be

was o m i t t e d

was f o r m e d

gelslices

to

towards

smaller

displacer

was

in our

it

of

reactivity

of

line

one y e a r proving

containing

area

dation of

sections

electrophoresis gel.

cell

The c o n A - r e a c t i v i t y

5 mm g e l

precipitates

no v i s i b l e sion

more t h a n

a^AT was p r e s e n t ,

cells.

all

The p r e c i p i t a t e

human p r o m y e l o c y t e

serum f o r

taken

heterogeneous, very

This

as

S2

the wide

retartraces

and

S3

precipita-

tes . 4.

Leucocytes.

could within

be d i s t i n g u i s h e d a single

fractions

reacting

(fig.

fractions

found

(R(Lj)

0,05),

to

with

= 0 +/-

fig.

conA-reactive

4).

Up t o

5 fractions

section

(fig.

4C).

= Sp

anodic

4 F-H).

three +/-

(L4

retardation

7 different

(fig.

cathodic

similarly

undefined pitate

in the

The m o s t

of

5 mm g e l

were f o u n d

were p r e s e n t A-C).

A total

part

part

that

L5

of

of

= S2

of

0,00,

Lg

very

= S3)

pattern

R(L2)

the

and

(fig.

with

0,01,

4 a^AT

rather

contoured

leucocyte

+/-

they

contained

preci-

a^AT

low c o n A - r e a c t i v i t y = 0,21

present

serum-a^AT

1eucocyte-ajAT

and a d o u b l e of

were

3

the a j A T - p r o t e a s e s ,

In the middle or

the

t h e a^AT

coefficient zero

and

All

fractions

pattern

were

R(L 3 )

=

0,45

4B-F.

F i g . 4: Crossed a f f i n i t y Immunoelectrophoresis (the two-step technique) of leucocyte otiAT. The upper p i c t u r e i l l u s t r a t e s the control plate (the 5 mm gel s e c t i o n s t r a n s f e r r e d from the corresponding experimental plate are marked A-H). Plates A-H i l l u s t r a t e the various conA-reactive f r a c t i o n s within each gel s l i c e . Antigen: 3x10 leucocytes. Antibody: control plate 0,43 Ml/cm and A-H 0,14 pl/cm a n t i - a , A T . A l l plates 0,06 pi anti-prealbumin. The prealbumin p r e c i p i t a t e (PA) i l l u s t r a t e d on plate A but has been cut o f f from plates B-H.

368 Discussion

Serum lar

ajAT

was

variants

recently reover

been

fraction

S2

content

of

decreased The high

of

of

the

both

Bayard

et

that

uniformity fraction

acid

Sj

this

had

glycans

S3

could of

fraction most

since the

(S^)

probably

it

varied

general

serum

appearing

when

represent

in

a^AT

individual

within

each

with

chains.

due

to

since

higher sialic

mobility

and

conA-affinity seen

was

according

precipitate.

with

the

applied

in

unspecificica11y

sera

probably

because

the

major

glycoproteins

affinity

precipitate

(e.g.

o^-macrog1obu1in)

to

A fourth

two-step

fracti-

technique,

producing are

en-

the

the

conA-

excluded

by

procedure. presence

rously

of

tion

such

conclusions

otj A T - d e f i c i e n t (fig.

al.(10)

2a,

found

centration explained

sera

the

they

non-reactive

moreover

conA-reactivity the

to

the

as

the

found to

The

unchanged

the

their

same of

was

due

if

In

basis

ever,

same

in

whether

latter

ours,

a

conA.

pattern

small This of

elastase

of

free

a,AT

are of

the of

run

in

could

a^AT

after

trypsin

compared despite

to

their

fraction (1

(0,93

% of

concentra-

conA can

et

conbe

pi). the

Howtotal

reproduce.

reaction was

erwith

Hinnerfeldt

applied

not

to

serum

higher

aj AT

fourth

fraction

lead

normal

Since

serum

we

or

could

of

serum

fractions.

amount

conA-reactivity

surplus

entrapment

comparison

study

4 conA-reactive

was on

unspecific

eg.

b).

otjAT)

had

mo-

electrophoretic be

serum

never

be

workers

biantennary

a^AT,

was

The

very

triantennary,

electrophoretic

a^AT

this

molecu-

have

carbohydrate higher

This

fraction

increased

These

having

fraction

fractions. on

of Sj

distinct results

(5).

residue

fraction

provide

al.

for

and

three

Similar

found

biantennary

other

into

methods.

con A - r e a c t i v i t y .

amounts

size on

by

They

showed

sialic

fourth

trapped,

divided

N-acetylglucosamine

than

would

be

evidence

having

results

mobility

to

present

obtained

variant.

intercalated

acid

the

provided

molecular

Our

found

by

used.

serum

with

protease

Some

otjAT

a^AT.

theoretical

This

higher

could molar

369 ratios vity

of e n z y m e s .

compared

acted

with

to

the

this

Baumstark

mine

and

with

elastase.

peptide

of

dues

cleares

aj AT c o u l d sidues,

and

probably

from a^AT.

way

have

to t h e s e

This

could

thetic

products

strong to

reactivity

lack

of

Leucocyte Three was

a^AT

fractions

similar

ning

three

could

be

break

down

Thirdly (2)

sialic

of m o s t

products

from

similar with with

and o t h e r s steps

(11,

that and

4000 from

aj AT

(37

resi-

of e i t h e r

effect

enzyme

with

on c a r b o h y d r a t e

do

or c o n t i n u o u s

on t h e

re-

that to

serum a^AT or

with

very

to

devoid

HL-60

conA

syn-

synthesis could

and o n e

protease.

low

of

The be

due

fraction The

components

of c a r b o h y d r a t e a^AT,

studied. remai-

conA-reactivity

leucocyte

synthesize

of

in s e r u m a ^ A T .

of t h e o t h e r a j A T ' s

reacted

zero

steps

conA-

glycans.

12), t h i s aj AT c o u l d

compared

heterogeneous

multiple

present

specific

of a ^ A T

leucocytes

a

a jAT

of t h i s aj AT w i t h

to t h a t of a ^ A T

if

than

residues

differed

fractions

different

cleave

(3) f o u n d

exhibited

present

types

were

at

may from

be d u e to e i t h e r

acid

in c o m p l e x

synthetic

indirect

reacts

et a l .

interaction

cells

being

glycan

about

interaction

peptides.

reactivity. a^AT

MW

The

with

peptide

a 4000-5000

of

(7)).

inter-

N-acetylglucosa-

enzymes

Banda

conA-reactienzymes

In a g r e e m e n t

after

both

by H L - 6 0

with more

of 8

(8))

otjAT s y n t h e s i z e d

a^AT

loss

most

Gann

both

of a j A T .

elastase

a peptide

in t h i s

linked

net

changed

that

per m o l e o^AT

removed

et a l .

had

and

size

elastase

(Carell

residues

(4) f o u n d

(Feste

a^AT

indicating

residues

similar

macrophage trypsin

et a l .

trypsin

of a j A T

remaining

carbohydrate

10 h e x o s e

Although sites

The

serum a^AT

as be

be

residues.

suggested stored

or

at

by

us

different

o^AT.

C o n e 1us ion. aj AT f r o m a ^ A T - d e f i c i e n t reactive

fractions

as

normal

sera

contained

s e r u m ot,AT

(S,,

the S~

same and

three S,).

conA-

370 2.

Seven

individual

stinguished

in h u m a n

vity.

of t h e

a^AT One

Three

in s e r u m variant

3_. with

or v e r y The a j A T conA.

no d e f i n i t e

similar

of a j A T

basis

of t h e i r

similar

by t h e i r

molecular

could

to t h e

variants

di-

conA-reacti-

variants

retardation with

be

of

coefficients.

trypsin

or

elasta-

in l e u c o c y t e s

had

conA-reactivity.

synthesized

One

on

were

to otjAT r e a c t e d

remaining low

variants

leucocytes

variants

as d e t e r m i n e d

was

se. T h e t h r e e zero

molecular

variant

by

was

retardation

HL-60 similar

reacted

cells to

coefficients

heterogeneously

Sj , b u t f o r could

be

the

remaining

given.

Acknowledgements This cil

study

was

supported

(grant

no.

12-1054).

by t h e

Danish

Medical

Research

Coun-

References 1.

Andersen,

M.M.

Scand.

2.

Andersen,

M.M.

(submitted

3.

Banda, M.J., Clark, 1563-1570 (1980).

4.

B a u m s t a r k , J.S., Babin, D.R., Lee, C.T., Allen, B i o c h i m . B i o p h y s . A c t a 62J3, 2 9 3 - 3 o 2 ( 1 9 8 0 ) .

R.C.

5.

B a y a r d , B., K e r c k a e r t , J . - P . , L a i n e , J. B i o c h e m . J_24, 371 - 3 7 6 ( 1 982 ).

A.

6.

B0g-Hansen, T.C., nol . 4, suppl . 2,

7.

B 0 g - H a n s e n , T . C . , P r a h l , P., L 0 w e n s t e i n , M e t h o d s 22!, 2 9 3 - 3 0 7 ( 1 978 ) .

8.

C a r r e 11 , R . W . , B o s w e l l , D . R . , G r e n n a n , S . O . , O w e n , B i o c h e m . B i o p h y s . R e s . C o m m . _93, 4 0 2 - 3 9 9 ( 1 9 8 0 ) .

9.

F e s t e , A., (1981).

10.

Hi n n e r f e l d t , F., A l b r e c s e n , J . M . , B 0 g - H a n s e n , T . C . Elect r o p h o r e s i s '82, e d . S t a t a c h o s , W. de G r u y t e r , B e r l i n , (in p r e s s ) .

11.

I k u t a , T., O k u b o , Biochem. Biophys.

12.

Wilson, G.B., Walker, Proc. Soc. Exp. Biol.

Gann,

J.

E.J.,

I m m u n o l . _1_5, 3 9 9 - 4 0 7 to

Scand.

Werb,

Z.

J. c l i n . J.

A.,

Bjerrum, 0., Ramlau, 141 - 1 4 7 ( 1 975 ) .

J.C.

J.

Biol.

Chem.

Exp.

( 1 982 ). lab.

Med.

Hayem,

J.

Invest.). 152,

Scand. H.

256,

J.

Eur. J.

Immu-

Immunol. M.C.

6374-6380

H., K u d o , J . , I s h i b a s h i , H., I n o u e , R e s . C o m m . _1_04, 1 5 0 9 - 1 5 1 6 ( 1 982 ). J.H., Watkins, J.H., Wolbroch, M e d . 164, 1 0 5 - 1 1 4 ( 1 9 8 0 ) .

T. D.

SEMINAL ACID PHOSPHATASE. Purification by lectin affinity chromatography.

Mineo Iwasa and Kaoru Sagisaka Department of Legal Medicine Gifu University School of Medicine, Gifu, 500 Japan

Acid phosphatase (AcP,E.C.3.1.3.2.)from human prostate gland has been known to be a phosphaoric monoester hydrolase (5) which is used for identification of seminal stains in forensic practice (3). AcP is widely distributed in animals,plants and microorganisms (7,10). As for human body, AcPs from prostate and seminal plasma have the same specificity of substrate but a few differences in the pattern of polyacrylamide electrophoresis were pointed out (6). On the reactivity with lectins, human urinary AcP was demonstrated to have different activity to Con A from serum AcP (4). On the other hand, some plant AcPs identified as glycoproteins were inhibited or activated by Con A ( 8,9). From these results lectins have been used to isolate glycoproteins in affinity chromatography. In this paper, we report the reaction of AcP from seminal plasma with 5 different lectins and purification of AcP by affinity chromatography with CanavaJLLa. gladcata

DC lectin.

Materials and Methods Preparation of lectins. Ground seeds of PhainoluA coc.dine.Lii, L. , I/¿gna Ccutiang

Endl. varis Sinensis , Cytliai

icopcvU.uA Link. , WiAta/uA

bsia.chbo£>iyA Sieb et Zucc.and CanavaLLa. gladiaXa

© Walter de Gruyter & Co. 1983, Berlin • N e w York Lectins, Vol. Ill

DC

(Can G) were ex-

372 tracted with 10 volumes of saline at room temperature for 60 min. The suspensions were centrifuged and the supernatants were defatted with an equal volume of ethyl ether. Purification of Can G was performed by affinity chromatography (1). The defatted extract was precipitated with 60 % saturated (NH.JzSO., and precipitate was applied onto a Sephadex G-200 column (Fig.l). Elution was carried out stepwise with 1.0 M NaCl and 1.0 M NaCl containing 0.1 M a-methvl-D-mannoside as specific displacer. AcP activity of Can G was eluted as a single peak with maximum activity in tube number 55. Inhibition tests revealed that only the specifically displaced fraction had lectin activity and reacted with AcP of seminal plasma. The affinity purified lectin was coupled to agarose by the cyanogen bromide method (2). -0.7

Fig.l. Affinity chromatography of (NBU)2SO„ fraction of Can G lectin on Sephadex G-200. The dotted line is the activity of Can G AcP. The arrow indicates addition of a-methyl-D-mannoside. The fraction indicated with the black bar contained Can G lectin with agglutinin activity (the second peak).

¿0.6

c 3 0.5 o

I OA

e §0.3 «VI g 0.2 fl

A

o 0.1 in

n
Sieb et Zucc., 15 %). Of the five lectin extracts tested, only Can G

374 lectin reduced the activity of AcP. When the lectins of less than 5 mg/ml were added, AcP activity of seminal plasma changed scarcely. However, adding of the lectins of concentration of 10 mg/ml or more resulted in increasing or reducing of the AcP activity. Fig.2. Inhibition of AcP activity with various lectins. O O ,Phcu,zoZiH cocclne.uA L.; O-Q ,1/igna. Ccutiang Endl. varis Sinensis; •—W,Cytl&u6 ¿copa/UuS Link.; a—ft, Ultita/uA bnachbottyi Sieb et Zucc.;#—•,CcLna.vaLia. gladCata DC. 0 I 0

, 5

. 10

, , 15

20

Lectin ( m g / m l )

Fig.3. Crossed immunoelectrophoretic patterns of seminal plasma (a) and the supernatant of seminal plasma precipitated with Can G lectin (b). The gel plates were stained for AcP.

a \ b Crossed Immunoelectrophoresis was performed of the supernatants and the height of the AcP peak was remarkably reduced with Can G lectin(Fig.3). When gel plates were stained with Coomassie brilliant blue, about 15 precipitation peaks were observed and the peaks other than AcP were not affected by the lectins. Fractionation of seminal plasma. Three protein peaks were observed by gel filtration of seminal plasma on Sephadex G-200. The AcP assay revealed that some activity was eluted with the first peak and most of the activity with second peak. By addition of pure lectin to the active fractions, we found that the activity of both fractions was some what reduced (Fig.4). The rate of reduction was 3.6 % at 1.5 mg/ml of lectin added and

375

13.7 % at 2.0 mg/ml or more. 85

Fig.4. Inhibition of the two fractions possessing AcP activity separated by Sephadex G-200 gel filtration with purified Can G lectin. O—O »Fraction 1; Fraction 2.

0

0i5

1.0

1.5

20

25

Lectin (mg/ml) Isolation of AcP by affinity chromatography. By elution with a linear 0-0.1 M a-m.ethyl-D-mannoside gradient, the AcP fraction was recovered with the eluent at low concentration (Fig.5). The specific activity of AcP was elevated (54 fold) by this method. Rabbits immunized with AcP-coupled gels produced potent antibody to AcP. A week activity to one of the seminal proteins was absorbed with serum.

0.1

1.0

3 (A

O c c m E

ot I

0.05 « £ E 20 ^

05

S n
1 -p

•H

O

•H

MH •H

0 a) eu

W

-p to x: c tn •H •H -p (I) ü S a) rH M (0 Q) rH > 0 •H U •P A) (d H tí 0 1 S

-p c ai -p

tí

0

0

Ifl -p

-H C

0 XI 0 10 1

a) -p id SH TI >1 si 0 XI M Id u

a 0 •rl -P

W

X CU

tí

\

TI

•r| id

01

3-

id

.tí

•rl

a ai —. W rH I o CM Id W 2

•H

E

TN

id

-P

tí

>I •P

•H

0 A

O

CO •

•

10 R^ CN -p œ a: di o- a e •H -p -p TI td id : 387-398 (1979). 33. Sun, S.M., Mutschler, M.A., Bliss, F.A., Hall, T.C.: Plant Physiol. 61: 918-923 (1978). 34. Talbot, C.F., Etzler, M.E.: Biochemistry 17: 1474-1479 (1978). 35. Tully, R.E., Beevers, H.: Plant Physiol. 58: 710-716 (1976). 36. Van Dreissche, E., Smets, G., Dejaegere, R., Kanarek, L.: Planta 15J3: 287-296 (1981) . 37. Youle, R.J., Huang, A.H.C.: Plant Physiol. 5J3: 703-709 (1976).

A SPECTROPHOTOMETRY STUDY OF THE CARBOHYDRATE BINDING SITE OF PEANUT LECTIN

Jacqueline Ohanessian, Michel Caron Laboratories of Biomolecular Structural Chemistry and Immunochemistry UER Biomédicale, 74, rue Marcel Cachin, 93012 Bobigny Cedex, France.

Introduction There i s a need for rapid and s e n s i t i v e assays of the interactions of l e c t i n s with carbohydrates (1,2). The difference spectrophotometric method i s one of the useful tools for the study of s p e c i f i c binding of sugars to a l e c t i n . As pointed out e a r l i e r (3-6) changes in the spectrum of the l e c t i n from Arachis hypogaea (peanut) may be used to calculate a s s o c i a t i o n constants. Difference spectra i n t e n s i t i e s dependent on the amount of sugars bond are obtained with s p e c i f i c sugars, but not with non s p e c i f i c sugars. The binding of lactose to the l e c t i n i s accompanied by pronounced changes dependent on the temperature, the a f f i n i t y decreasing with increasing temperature (5). The interactions between the l e c t i n and d i f f e r e n t mono- and o l i g o saccharides are related to the structure of the sugars (6). The present study i s undertaken to ascertain whether these sugars interacts with the binding s i t e of the l e c t i n .

Materials and Methods Peanut l e c t i n i s p u r i f i e d by a f f i n i t y chromatography on desialylated red cell ghosts as previously described (7) and dialyzed extensively to remove the eluting sugar. Sugars are commercial products of analytical grade except f o r methyl-7-deoxy-L and D-glycero-e-D-galacto-heptopyranosides synthetized as described by Lemieux and a l . (8). Measurements are performed with a concentration of l e c t i n in the range 0 . 5 - 1 mg/ml, concentrations of sugars of 1-10.10

M, in NaCl 0.15 M at 10°C.

© Walter de Gruyter & Co. 1983, Berlin • New York Lectins, Vol. Ill

624

625

626 Absorbance and difference spectra are recorded between 260 and 300 nm using a temperature-controlled Cary 219 spectrophotometer. The intensities of difference spectra are determined between the typical positive and negative peaks (3,5). By plotting the change in difference spectrum aA versus the sugar concentration, a typical saturation curve is obtained. Linearisation according to Matsumoto (3) results in a straight line. The slope and the intersections obtained with this representation, calculated by the least-squares method, permit the determination of the binding constant (6).

Results and Discussion By plotting [sugar]/AA as a function of [sugar] we investigated the association constants for sixteen mono- and oligosaccharides at a constant temperature of 10°C. The resulting data are summarized in Table 1. With D-galactopyranose (conformation

4

C,) K = 1200 M 1 -i

constant measured for L-galactopyranose is 170 M

-1

while the

. The other saccha-

rides studied possess structural configuration differences and are substituted on C(6), C(4), C(2) and C(l) (see Table 1). The following conclusions were drawn with respect to the importance of the configuration of these atoms for

the interaction with the carbohydrate

binding site of peanut lectin. Atom C(6) An extracyclic chain is necessary for the carbohydrate lectin interaction and the orientation of the hydroxyl group on C(6) is essential. A substitution of the hydroxymethyl group decreases the strength of the association. Atom C(4) A free axial hydroxyl on C(4) is needed for binding. Atom C(2) A C(2) equatorial hydroxyl group is not essential for the interaction but the axial position of this group decreases the binding. Atom C(l) 0(1) methylated : Methylation favorises association. Among methyl-D-

627 galactosides differing only in the configuration about the anomeric C(l), there seems to be a slight preference for the a anomer. 0(1) involved in glycosidic bond : for the oligosaccharides studied the second residue appears to be involved in the association. The linear chain of the gluconic acid prevents the binding. Furthermore, the 3(1-4) linkage seems to give better binding than the a(l-6) linkage. These results suggest that the configuration on C(l) is important. Comparing their effectiveness to interact with the carbohydrate binding site of peanut lectin, the only sugars to have a more marked affinity than D-galactose appeared to be galactosides substituted on C(l).

Conclusion Our results show the following details : the sequence C(4)-0(4), C(5), C(6)-0(6) of the galactopyranosyl ring is involved in the interactions in the carbohydrate binding site of peanut lectin as postulated by Lotan et al.(9). Furthermore, for di- and oligosaccharides, it appears that the configuration on C(l), the nature of the osidic bond and of the second residue may influence the strength of the association.

References 1. Takeo, K., Fujimoto, M. and Kuwahara, A. (1982). These proceedings. 2. Wu, A.M. (1982) These proceedings. 3. Matsumoto, I., Jimbo, A., Kitagaki, H., Golovtechenko-Matsumoto, M. and Seno, N. (1980) J. Biochem. 88, 1093-1095. 4. Neurohr, K.J., Young, N.M. and Mantsche, H.M. (1980) J. Biol. Chem. 255, 9205-9209. 5. Caron, M., Ohanessian, J. and Becquart, J. (1981) 1st European symposium on Carbohydrates and Glycoconjugates (Vienna) Abstract G.S. 3. 6. Caron, M., Ohanessian, J., Becquart, J. and Gillier-Pandraud, H. Biochim. Biophys. Acta (In press). 7. Caron, M., Ohanessian, J., Faure, A. and Felon, M. (1981) C.R. Acad. Sci. Paris 292, 183-186.

628 8. Lemieux, R.U., Boullanger, P.H., Bundle, D.R., Baker, D.A., Nagpurkar, A. and Venot, A. (1978) Nouv. J. Chim. 2, 321-329. 9. Lotan, R., Skutelsky, E., Danon, D. and Sharon, N. (1975) J. Biol. Chem. 250, 8518-8523.

A CRITICAL STUDY ON THE PURIFICATION OF POTATO LECTIN (SOLANUM TUBEROSUM L.) Edilbert Van Driessche'®'^ , Sonia Beeckmans1®" , Robert Dejaegere^ and Louis Kanarek'®' t Laboratorium voor Chemie der Proteinen, and ^Laboratorium voor Plantenfysiologie; Vrije Universiteit Brüssel, Instituut Moleculaire Biologie, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium

Introduction The purification of the lectin from potato tubers has been described by different groups (1-5). Most of these methods are based on affinity chromatography, taking advantage of the sugar specificity of the lectin (NAG^; x 2-5). During the isolation of proteins from phenol-rich tissues such as potatoes, special attention has to be paid to the inhibition of phenol oxidases. Indeed, during the extraction, endogeneous phenols are quickly enzymatically oxidized to quinones which can irreversibly modify proteins by covalent binding to reactive groups such as sulfhydryl-, amino- and imino groups. These modifications result in most instances in reduced solubility and stability of the proteins or in complete loss of activity due to precipitation and/or inhibition of the proteins (6-8). The problems mentioned above can best be avoided either by eliminating the phenols or by inhibiting the phenolase activity with reagents such as sodium metabisulphite (Na 2 S 2 0 5 ), diethyldithiocarbamate (DIECA) or thiourea (6, 7) . Until now, exclusively Na2S20,- was used as antioxidant (1, 2, 4, 5). Previously we noticed (2) that when Na2S 2 0^ (2 mM) had been added during the extraction of the tubers, the purified lectin displayed some unexpected spectral properties,

© Walter de Gruyter & Co. 1983, Berlin • New York Lectins, Vol. Ill

630 i.e. the UV-absorption spectrum had a maximum at 271 nm, and more curiously the ratio of the absorbances at 280 to 250 nm was 1.1 - 1.3. Later we found that all the hemagglutinating activity was lost when ^ 2 8 2 0 ^ (2 mM) was added during the dialysis of the ammonium sulphate precipitate of the extract. In this paper, we describe a new preparation method for potato lectin and we compare the effect of the use of different phenolase inhibitors on the activity and the spectral properties of the lectin, model peptides, enzymes and amino acids.

Materials and Methods 1. Purification of potato lectin : potatoes were washed, peeled, cut in fine pieces and homogenized in 0.1 M sodiumacetate buffer containing 9 g/1 NaCl and 0.2 g/1 NaN^. The pH of the buffer was adjusted to 3.8 or 5.4 and either 2 mM Na2S20j-, or 1 mM DIECA, or 5 mM thiourea were added. Extractions were allowed to proceed at 4°C for 6 hours, the slurry was passed through cheesecloth and centrifuged for 30 minutes at 7000 r.p.m. Solid ammonium sulphate (243 g/1) was added to the clear supernatant in order to get 40% saturation at 4°C. The ammonium sulphate pellet was collected by centrifugation and dialyzed against the respective extraction buffers at room temperature. Precipitated proteins were removed by centrifugation and the clear supernatant was slowly applied to a chitin column which was equilibrated with the same buffer. The column was then washed with buffer until the absorbance of the effluent had dropped to zero. The column was then washed with 1 M C a C ^ in buffer and finally with saline. The lectin was eluted with 0.1 M acetic acid. 2. SDS-electrophoresis was performed on 10% polyacrylamide slab gels according to Laemmli (9), or on linear 12.5%-25% polyacrylamide gradient gels. Molecular weights were determined from the migration relative to standard proteins with known molecular weight.

631

3. Hemagglutinating activity was tested using rabbit red blood cells. 4. Chitin was prepared as follows : commercial (Sigma Chem. Comp.) chitin was exhaustively washed with 0.1 N HC1, water, 0.1 N NaOH, water, and was deproteinized with pepsin and trypsin.

Results 1. The effect of Na 2 S 2 0 5 > DIECA and thiourea on N-acetyltryptophanairide, N-acetyl-tyrosinamide, model peptides and proteins : The deleterious effect of ^2820,. on the UV-absorption spectrum of N-acetyl-tryptophanamide is shown in Fig. 1. Similar

Fig. 1.

The effect of 20 mM metabisulphite on the UV-absorption spectrum of N-acetyl-tryptophanamide in 0.1 M sodium-acetate buffer, pH 5.4. Incubation times : At = 0 h (initial spectrum) (—); 1 h 30 (-•-); 3 h ( ); 4 h 30 ( ); 10 h ( ); 25 h ( ) and 46 h (final spectrum) ( — ) .

632 changes were observed in a broad pH-range with tryptophane containing model proteins such as melittin (1 tryptophane, no tyrosine) (10), lysozyme, pig heart fumarase and purified potato lectin. No spectral changes were observed with N-acetyl-tyrosinamide or bovine pancreas ribonuclease A (6 tyrosines, no tryptophane) (11). Concomitant with the induction of changes in the UV-absorption spectrum, loss of activity of lysozyme, fumarase and potato lectin was observed. On the other hand, neither DIECA, nor thiourea had any effect on the spectral properties of the above mentioned probes. The action of the three phenolase inhibitors was also tested on a model S-S-bridge (5,51-dithio-bis(2-nitrobenzoate)) and on a cysteine containing peptide (glutathion). The extent of modification of cysteinyl groups (-SH) was estimated (according to Ellman (12)) from the measurement of the residual -SH groups of glutathion after incubation with phenolase inhibitor (Scheme 1 and Table 1). The extent of modification of the Scheme 1. "00C

COO"

COO" + Glu-Cys-Gly — • SH

1

5,5 -dithio-bis (2-nitrobenzoate)

COO"

Cys-S-S-^-N02 + Gly

glutathion

2-nitro-5-thio benzoate anion

model S-S-bridge with phenolase inhibitor (R) was estimated directly from the generation (Scheme 2) of 2-nitro-5-thiobenzoate anion (see Table 2). Scheme 2. "00C

COO"

02N-^^-S-S-^yN02

COO" +

R

»

COO"

R-S-^^-N02 +

5,5'-dithio-bis (2-nitrobenzoate)

"S^^-f^

2-nitro-5-thio benzoate anion

From these results it is clear that only thiourea does not interfere with S-S bridges or sulfhydryl groups.

These

results are further confirmed by the fact that thiourea is

633 the only phenolase inhibitor tested which does not inactivate lysozyme, an S-S containing enzyme (13), or fumarase, an enzyme with essential SH-groups (14). Table 1.

The effect of phenolase inhibitors on cysteine. [2-nitro-5-thiobenzoate anion] (mM) pH

no addition

thiourea

DIECA

Na

2S2°5

3.8

6.56

6.51

3.68

6.25

5.4

6.56

6.49

3.53

6.38

7.0

6.47

6.28

4.41

6.35

8.0

6.16

6.02

5.44

6.09

Glutathion (6.5 mM) in buffer (0.1 M sodium-acetate pH 3.8 and 5.4, or 0.1 M Tris-acetate pH 7.0 and 8.0) was incubated with 5 mM of respectively thiourea, DIECA or metabisulphite during 3 hours. The concentration of the remaining free -SH groups was determined spectrophotometrically at 412 nm according to Ellman (12) by adding 30 fil of the glutathion solutions to 3 ml of 5 mM DTNB in 0.1 M potassium phosphate buffer pH 7.0; a solution containing 5 mM DTNB in 0.1 M potassium phosphate buffer pH 7.0 and 50 ¡iM of the corresponding phenolase inhibitor was used as bianco. The concentration of 2-nitro-5thiobenzoate anion was estimated from its specific absorbance : e = 13600 M ^ m " 1 (12) . 412 nm Table 2.

The effect of 50 pM phenolase inhibitors on 5 mM DTNB (5,5'-dithio-bis(2-nitrobenzoate)) in 0.1 M potassium phosphate buffer pH 7.0. [2-nitro-5-thiobenzoate anion]

(fM) Thiourea

0

Diethyldithiocarbamate

30

Sodium metabisulphite

90

The concentration of 2-nitro-5-thiobenzoate anion was estimated from its specific absorbance : e =* 13600 M 1 cm 1 (12).

4 1 2

n m

634 2. Comparison of the effect of ^26205» DIECA and thiourea on the activity of potato lectin in extracts : In order to compare the effect of adding ^2820^, DIECA or thiourea during the extraction on the activity of potato lectin, tubers were homogenized in saline and extracted for 30 minutes. The coloured extract was passed through cheesecloth and centrifuged at 15000 r.p.m. for 20 minutes. The supernatant was devided into different fractions which were adjusted to pH 3, 4, 6 (0.1 M sodium-acetate, final concentration), 8 and 9 (0.1 M Tris-acetate, final concentration) and incubated with 5 mM Na n £„O c , 5 mM DIECA or 5 mM thiourea. ¿ZD After respectively 2 1/2 and 24 hours of incubation, the phenolase inhibitors were removed by dialysis against the corresponding buffer and finally against saline containing 0.02% NaN 3 . From the results in Table 3 it is clear that ^2820,. should be avoided as anti-oxidant for the preparation of potato lectin and should be replaced by DIECA or by thiourea.

Table 3.

The effect of different phenolase inhibitors on the agglutinating activity of potato lectin in extracts. Titer Thiourea

Diethyldithiocarbamate

Metabisulphite

pH

2.5 h and 24 h incubation

2.5 h and 24 h incubation

2.5 h 24 h incubation

3 .0

128

128

16

1

128

128

8 - 16

0

128

128

0

0

.0

128

128

0

0

9 .0

128

128

0

0

6 .0 00

4,.0

635 3.

Purification of potato lectin :

The lectin was prepared as described in Materials and Methods. The progress in the purification is shown in Fig. 2.

1 2

3

4

KD •m 92 -60

KD 92 —

ft»

% » »

» « *

I

48 5

.

-40 — 35

60 —

-.23 3

48.5 — 4035 — 23 3 -

1 2 3 4 5 6 7 8 9 10 11 1213 A

Fig. 2.

B

The progress in the purification of potato lectin as demonstrated by SDS-PAGE on

(A) 10% Polyacryl-

amide and (B) a linear 12.5%-25% gradient slab gel. Lane_in_gel A B 1 2 3 4 5 6 7 8 9 10 11 12 13

2 3 4 1

Sample Preparation with DIECA (pH 5.4) elution from chitin (0.1 M acetic acid) fraction I from Fig. 3A (pure lectin) fraction II from Fig. 3A Preparation with thiourea (pH 5.4) elution 1 from chitin (0.1 M acetic acid) elution 2 from chitin (0.1 M acetic acid) fraction I from Fig. 3B (pure lectin) fraction II from Fig. 3B 1 M CaCl2 wash Preparation with thiourea (pH 3.8) elution 1 from chitin (0.1 M acetic acid) elution 2 from chitin (0.1 M acetic acid) fraction I from Fig. 3C (pure lectin) fraction II from Fig. 3C Preparation with metabisulphite (pH 3.8) purified lectin

636 The lectin is strongly bound to the chitin matrix and is eluted as two fractions. A first lectin containing peak is eluted immediately with 0.1 M acetic acid (elution 1), while a second peak can only be released after an overnight incubation period in 0.1 M acetic acid (elution 2). When the eluted fractions are further chromatographed on Sephadex G-150 (Fig. 3), highly pure potato lectin is obtained as can be seen in Fig. 2A, lanes 2, 6, 11. The UV-absorption spectrum of potato lectin which has been prepared with DIECA, thiourea or Na2S20,- during the extraction is shown in Fig. 4. Lectin which has been isolated with DIECA or thiourea displays a normal protein absorption spectrum with a maximum at 280 nm and a 280/250 nm absorption ratio of 2.

Fig. 3.

Gel filtration of affinity purified potato lectin on Sephadex G-150 in 0.1 M sodium acetate containing 9 g/1 NaCl and 0.2 g/1 azide, pH 5.4 (A: 1 mM DIECA and B: 5 mM thiourea used in the extract at pH 5.4) or pH 3.8 (C: 5 mM thiourea used in the extract at pH 3.8).

637

Fig. 4.

The UV-absorption spectrum of potato lectin in phosphate buffered saline. Ultra-pure lectin prepared with DIECA or thiourea (—); lectin prepared with iretabisulphite ( ); pure lectin prepared with DIECA and completely inactivated with metabisulphite ( ) .

Discussion In this paper we clearly demonstrate that Na2S20j_ should be avoided as anti-oxidant for the preparation of proteins from phenol-rich tissues and should be replaced by thiourea which was shown to have no effect on either tryptophane, tyrosine, S-S bridges or cysteine. Although DIECA can react with both cystine and cysteine, it can be successfully used for the preparation of potato lectin which is free of SH-groups, but rich in S-S bridges (1). Since DIECA does not affect the activity of purified potato lectin, it may be assumed that the cystines are well buried in the interior of the molecule. Although potato lectin can be isolated to purity according to available literature procedures (using Na2S20j_), the specific activity of such preparations is only about 25% when compared to lectin prepared as described above. The reduced specific activity could be ascribed to the modification of an essential

638

tryptophane residue whose presence has been demonstrated by Ashford et al. (15) in the carbohydrate binding site. Although two active peaks are consecutively eluted from the chitin column with 0.1 M acetic acid, electrophoretic analysis demonstrated that these two fractions are indistinguishable, suggesting that the difference in elution has to be attributed to heterogeneity of the chitin matrix rather than to the presence of isolectins which differ in their affinity for carbohydrate .

References 1. 2. 3.

Allen, £.K. and Neuberger, Biochem. J. 135, 307-314 (1973) Van Driessche, E. and Kanarek, L. : Arch. Intern. Physiol. Bioch. 82, 414-415 (1975) Delmotte, F., Kieda, C. and Monsigny, M.: FEBS Lett. ^3, 324-330 (1975)

4.

Desai, N.N. and Allen, A.K.: Anal. Bioch. 93, 88-90 (1979)

5.

Owens, R.J. and Northcote, D.H.: Phytochem. 1_9, 1861-1862 (1980) Anderson, J.W.: Phytochem. 7, 1973-1988 (1968) Mayer, A.M. and Harel, E.: Phytochem. 1_8, 193-215 (1979)

6. 7. 8.

Patil, S.S. and Zucker, M.: J. Biol. Chem. 240, 3938-3943 (1965) 9. Laemmli, U.K.: Nature (Lond.) 227, 680-685 (1970) 10. Habermann, E. and Jentsch, J.: Hoppe-Seyler1s Z. Physiol. Chem. 348, 37-50 (1967) 11. Richards, F.M. and Vfyckoff, H.W. : The Enzymes 4 (3rd. ed.) (P.D. Boyer, ed.) pp. 647-806 (1971) 12. Ellir.an, G.L.: Arch. Biochem. Biophys. 82, 70-77 (1959) 13. Imoto, T., Johnson, L.N., North, A.C.T., Philips, D.C. and Rupley, J.A.: The Enzymes 2 (3rd. ed.) (P.D. Boyer, ed.) pp. 665-868 (1972) 14. Hill, R.L. and Teipel, J.V7. : The Enzymes 5 (3rd. ed.) (P.D. Boyer, ed.) pp. 539-571 (1971) 15. Ashford, D., Menon, R., Allen, A.K. and Neuberger, A.: Biochem. J. 199, 399-406 (1981)

A G A L A C T O M A N N A N P R E C I P I T A T I N G L E C T I N IN STENOCARPA

Theresa

SPHENOSTYLIS

SEEDS

Animashaun

B i o c h e m i s t r y D e p a r t m e n t , U n i v e r s i t y of Ife, Ile-Ife, Nigeria

Introduction H a e m a g g l u t i n a t i o n a c t i v i t y was r e p o r t e d in e x t r a c t s of S p h e n o stylis s t e n o c a r p a seeds by S c h e r t z e_t a_l ( 1 ) . In the paper the a g g l u t i n a t i o n of red blood cells by saline from S p h e n o s t y l i s s t e n o c a r p a lionaceae)

present extracts

(Hochst. ex A. Rich) Harms

seeds was e x a m i n e d f u r t h e r . The

(Papi-

haemagglutination

a c t i v i t y of this e x t r a c t was shown to be i n h i b i t e d by D - g a l a c tose and its d e r i v a t i v e s . Some D - g a l a c t o s e b i n d i n g lectins, from R i c i n u s c o m m u n i s

(2) and B a n d e i r a e a B i m p l i c i f o l i a

e.

(3),

p r e c i p i t a t e v a r i o u s g a l a c t o m a n n a n s . 3. s t s n n r a r p a e x t r a c t was found to p r e c i p i t a t e a g a l a c t o m a n n a n from A f z e l i a a f r i c a n a (Caesalpiniaceae)

s e e d s . The p o t e n t i a l use of this

Sm

polysaccha-

ride in p u r i f y i n g the lectin from S. s t e n o c a r p a seeds is d i s cussed .

M a t e r i a l s and M e t h o d s P r e p a r a t i o n of S. s t e n o c a r p a e x t r a c t . S. s t e n o c a r p a seeds w e r e p i c k e d l o c a l l y and i d e n t i f i e d by M r . I.B. F a r e m i . The

seeds

w e r e g r o u n d , d e f a t t e d w i t h p e t r o l e u m ether and s t i r r e d for h o u r s at 4°C w i t h 5 v o l u m e s

(wt/vol) of 0 . 8 5 % s a l i n e . The

n a t a n t , o b t a i n e d a f t e r s p i n n i n g the s u s p e n s i o n at 20 m i n , is r e f e r r e d to as the P r e p a r a t i o n of

10,000g

18 supe for

extract.

a f r i c a n a g a l a c t o m a n n a n . A. a f r i c a n a

© Walter de Gruyter & Co. 1983, Berlin • New York Lectins, Vol. Ill

seeds

640 were kindly donated by the Seed Section, Forestry Research Institute of Nigeria, Ibadan. After removal of the aril and husk, A. africana seeds were ground and defatted. Two grams of the meal were stirred in 100 ml water with heating. The polysaccharide was precipitated

from the hot-water extract by 40% ethanol

and resuspended in water. The carbohydrate concentration was determined by the anthrone procedure

(4).

Haemagglutination studies. Haemagglutination studies were performed in V-shaped micro-titre plates with 1.5% formalinised red blood cells

(5). The titre value was determined with doub-

ling dilutions of S. stenocarpa extract. The concentration of sugar which reduced the titre value by one was said to have inhibited haemagglutination by 50%

(6).

Agar-gel double diffusion studies. Agar-gel plates contained 1.5% agar

(Oxoid Ltd.) and 0.01% sodium azide

(Difco Laborator-

ies) in PBS (0.1 M sodium chloride, 0.025 M phosphate, pH 7.4). The plates were incubated in a humid atmosphere at room temperature for 48 hours. Examination of affinity chromatography media. Sepharose 6B was purchased from Pharmacia while immobilised-lactose was from Pierce Chemical Co. Acid-treated Sepharose was prepared ing to Ersson e_t a_l(7). 0.5 ml of the affinity

accord-

chromatography

media being tested was incubated with 0.5 ml of S. stenocarpa extract for 1 hour. The titre value of the supernatant was determined and compared with a control containing the same dilution of extract.

Table I:- Titre Values of S .stenocarpa Extract Blood Type

Titre Value

A1

A2

B

0

Bat

25

25

25

26

0

641 Table II:- Concentration of Sugar Producing 50% Inhibition of Agglutination of 0 Red Blood Cells by S. stenocarpa

extract

mM D-galactose

3.0

N-acetyl-D-galactosamine

0.2

Lactose

0.8

Raffinose

3.0

Results Saline extracts of S. stenocarpa seeds agglutinated 0 red blood cells with a titre value of 2^ (Table I). The extracts agglutinated A^ , Ag and B cells to the same or to a lesser extent, but did not affect the sedimentation pattern of chicken and fruit-bat

(Eidolon helvum) erythrocytes. There was no clear-

cut difference between the agglutination of A^and A^ red blood cells by the extract. The agglutination of 0 erythrocytes by a

2

2

dilution of S. stenocarpa extract was totally abolished by

50 mM N-acetyl-D-galactosamine, D-galactose, lactose or raffinose. 50 % inhibition of 0 red blood cells was achieved in the presence of 0.2 mM N-acetyl-D-galactosamine, 0.8 mM lactose, 3 mM raffinose or 3 mM D-galactose

(Table II).

The polysaccharide isolated from A. africana seeds has been shown to be a galactomannan

(8). S. stenocarpa extract formed

precipitin bands in agar gel double diffusion plates with 13 to 0.4 mg/ml A. africana galactomannan

(Figure I). The galacto-

mannan did not precipitate dilutions of the S. stenocarpa extract. Various reagents were examined for their potential to purify the lectin from S. stenocarpa extract by affinity

chromatogra-

phy. The lectin did not bind to Seharose or acid-treated

Seph-

arose. The amount of lectin bound to immobilised-lactose was very low (approximately 20y^g protein per ml of gel).

642

@

®© o Figure I:- Diffusion of S. stenocarpa extract and A. africana galactomannan in agar gel. Centre well - S. stenocarpa

extract,

outside wells

(1 - 6) - doubling dilutions of A. africana

galactomannan

(13 - 0.4 rag/ml).

Discussion It has been confirmed that S. stenocarpa extracts human erythrocytes

agglutinate

(Table I). This haemagglutination is rela-

tively non-specific and unlike the findings of Schertz et a_l (1) the extract did not preferentially agglutinate h^ rather A^ cells. Haemagglutination was inhibited not only by N-acetylD-galactosamine and D-galactose but also by the fl- and the vLD-galactoside containing disaccharide lactose and

trisacchar-

ide raffinose. Some galactose-binding lectins have been purified by affinity chromatography on Sepharose e.g. ricin abrin

(10) and lectin from Pseudomonas aeruginosa

(9)>

(11). Tomita

et al (12) found that only three of the four galactose-binding

643 lectins tested bound to Sepharose. Acid-treated Sepharose has been used to purify lectin from Erythrina corallodenron

(13).

However,¡the lectin from S. stenocarpa did not bind to Sepharose or acid-treated Sepharose and the yield from

immo&ilised-

lactose was poor. Lectins from R. communis

(2) and B. simplicifolia

(3) precipi-

tate galactomannans from several sources such as guar gum, locust bean and Cassia alata. Moreover, cross-linked guaran has been used to purify both these lectins and that from Echinocystis labata

(14). Guaran immobilised in polyacrylamide gel was

used to prepare a high-capacity adsorbent for B. simplicifolia lectin

(15). Since S. stenocarpa extracts interact with A. af-

ricana galactomannan

(Figure I), the possibility of isolating

S. stenocarpa lectin employing cross-linked or immobilised A. africana galactomannan will be explored.

Acknowledgements I am grateful to Messrs A. Abimbola, 0.0.0. Binutuand O.M. Udeagu for technical assistance and to Mr. B.M. Salami for the photography

References 1.

Schertz, K.F., Boyd, W.C., Jurgelsky, W., Cabanillas, E.: Econom. Botany J_4, 232-239

(1960)

2.

Van Wauwe, J.P., Loontiens, F.G., De Byuyne, C.K.: Biochim. Biophys. Acta 3J_3, 99-105 (1973)

3.

Hayes, C.E., Goldstein, I.J.: J. Biol. Chem. 24_9,. 19041914 (1974)

4.

Viles, F.J.Jr., Silverman, L.: Anal. Chem. 21, 950-953 (1949)

5.

Butler, W.T.: J. Immunol. 90, 663-671

6.

Schimizu, S., Ito,M., Niwa, M.: Biochim. Biophys. Acta 500, 71-79 (1971)

(1963)

644 7.

E r r s o n , B., A s p b e r g , K., P o r a t h , J.: B i o c h i m . Acta 3_1_0, 446-452 (1973)

Biophys.

8.

Ikhile, P., A n i m a s h a u n , T.: S u b m i t t e d to X l t h ional C a r b o h y d r a t e S y m p o s i u m (1982)

Internat-

9.

O l s n e s , S., Pihl, A.: B i o c h e m i s t r y _1_2, 3121-3126

10.

O l s n e s , S., Pihl, A.: Eur. J. B i o c h e m . 35,

11.

G i l b o a - G a r b e r , N., M i z r a h i , L., G a r b e r , N.: FEBS 28, 93-95 (1972)

12.

T o m i t a , M., K u r o k a w a , T., O n a z a k i , K., Ichiki, N., Osawa, T., Ukita, T.: E x p e r i e n t i a 28, 24-26 (1972)

13.

G i l b o a - G a r b e r , N., M i z r a h i , L.: Can. J. B i o c h e m . 59., 315-319

(1973)

179-185

(1973?

Letts.

(1981)

14.

L o n n g r e n , J., G o l d s t e i n , I.J.: FEBS L e t t s . 68, ( 1976)

31-34

15.

H o r i s b e r g e r , M.: C a r b o h y d r a t e

(1977)

Res. ¿ 3 , 231-237

.FURTHER INVESTIGATIONS ON MISTLETOE LECTIN I (ML I): OF A-CHAIN ON CELL-FREE PROTEIN SYNTHESIS

1

2

EFFECT

H. Franz, Annemarie Kindt and P. Ziska H. Bielka, R. Renndorf and Liselotte Venker

Staatliches Institut fur Immunpraparate und Nahrmedien, 1120 Berlin-Weißensee, DDR 2 Zentralinstitut fur Molekularbiologie, Abteilung Zellphysiologie, Akademie der Wissenschaften der DDR, 1115 Berlin-Buch, DDR On the 4 th lectin meeting we presented a method for simultaneous reduction of mistletoe lectin I (ML I) and affinity chromatography of its A- and B-chains (1). In analogy to ricin and abrin we expected toxophoric properties of the A-chain of ML I. It is the aim of this paper to describe the inhibition of protein synthesis in cell-free system by the A-chain of ML I. Material and methods 1. Preparation of the A- and B-chains of ML I ML I was prepared as described earlier (2) the procedure for the isolation of both chains was slightly modified in comparison to our first communication. The lectin was dissolved in phosphate buffered saline (PBS), pH 7.2, (1 mg ML I/ml) and applied to a column of partially hydrolized Sepharose 4B. Non-bound proteins were washed out with PBS. About 10 mg of ML I were bound to 50 ml of partially hydrolized Sepharose 4B. Then ß-mercaptoethanol (5%) was applied to the column. After appearence of the ß-mercaptoethanol in, the effluent the flow was stopped and the reduction of ML I in the column was allowed to proceed overnight at room temperature. Thereafter the

© Walter de Gruyter & Co. 1983, Berlin • New York Lectins, Vol. Ill

646 column was washed with PBS and the effluent was applied to a second column of partially hydrolized Sepharose 4B in order to eliminate residual amounts of the B-chain. The A-chain passed the columns together with 3-mercaptoethanol. The B-chain remained bound and was eluted with 0.1 M D-galactose. The fractions were dialysed against PBS or 0.05 M potassium acetate to remove 3-mercaptoethanol and D-galactose. The chains were identified by SDS-PAGE according to the method of WEBER and OSBORN (3). If the B-chain fractions contain small amounts of A-chain the whole procedure has to be repeated. The protein content of the preparations was measured by the method of LOWRY et al. with bovine serum albumin as standard (4). Cell-free protein synthesis systems. Two different translation systems have been used in this study: a. A polysomal system from rat liver as described in (5) (but 100 |il samples only) containing 10 jig of polysomes (prepared with some modifications according to (6)), 20 (il of rat liver cytosolic fraction (corresponding to 20 |j.g protein; 105 000 x g supernatant in 50 mM Tris/HCl, pH 7.6, 1 mM EDTA, 6 mM 3-mercaptoethanol by Sephadex G-10 filtration) and, besides the other ingredients as described in (8), 1 |iC (^H) leucin (68 Ci/mmol). The samples were incubated at 37°C for 30 min without (controls) or in the presence of varying amounts of the A-chain samples (see Table 1) and then furthermore treated and counted for radioactivity as described in (5). b.

A rabbit reticulocyte lysate system.

The lysate was prepared with some modifications following the procedure of CRYSAL et al. (7) (the buffer for lysis of the reticulocytes contained additionally 5 mM Hepes/KOH, pH 7.0). Immediately before use the lysate was filtered through Sephadex G-25, equilibrated and eluted with a buffer containing 25 mM KC1, 10 mM NaCl, 3 mM Mg(0Ac)2, 1 mM ATP, 1 mM GTP, 0.25 mM

647 dithioerythritol, 1 mM glycose and 20 mM Hepes/KOH, pH 7.2, following JAGUS and SAFER (8). To 1.0 ml of the filtered lysate were added 25 )il of a hemin stock solution and 20 |il of a creatine phosphokinase stock solution as described by SAFER et al. (9) to give final concentrations of 25 jiM and 0.2 mg/ml, respectively. 2.5 of 2 M KC1 plus 10 mM Mg (0Ac)2, 1 of 400 mM creatine phosphate, 1 |il of 0.5 M putrescine acetate, pH 7.0, 1 |il of 0.2 mM spermine plus 2 mM spermidine, 2 p.1 of a mixture containing all amino acids (1 mM each) , and 3 )il of U ( 1 4 C) leucine (0.6 |i.C; 213 mCi/mmol) were added to 40 p.1 of the gel filtered lysate. A-chain preparations were added as specified in Table 2. The volume was completed to 57.5 |il by addition of aqua bidest. Incubation was at 37°C for 30 min; thereafter, two 20 samples were removed and pipetted into filter paper discs. The discs were processed for determination of hot TCA-insoluble radioactive material by liquid-scintillation counting.

Results and discussion 1.

Reduction of ML 1 under mild conditions:

Attempts to split ML I using &-mercaptoethanol only in absence of denaturating reagents were not successfull. Therefore, we tried to separate A- and B-chains by reducing Sepharose-bound ML I. This procedure is based in the hypothesis that only the B-chain is responsible for lectin activity. The result was the liberation of a single protein, which passed the column and another one, which was eluted by D-galactose solution. This leads to the following conclusions: ML I consists of two different chains. The A-chain does not bind D-galactose. The B-cha in is fixed to the carrier and therefore represents the virtual lectin component in ML I. Binding to a D-galactose bearing carrier effects alterations in the conformation of ML I which permit to reduce the S-S-bound. Denaturating reagents like 6 M urea can be avoided. This method

648 O) (1) M m i rH H a) u

H 0 4-1 M 0 -P c 0\O 0 o

H H H

1

m

(N!

LD

1

ai

1 id en U) id

o CN ro •H

m

o

\

o LD LO (Ti (M

rH

o o n ^D LD

o in CN

o

O IO rH

tn C

Oì

rH

O in 00 m

O f IT) O ro

o

o o m

m 0

ft

a) rH -P U) •H e

•H

—

M a)

>

-p •H u H Q) 4H -P 4-1 Iti W (-1

m

o

id

Cn C

H 0 4-1 S-L 0 4-1 C 0\° 0 a

H c 0 •H -P (0 SH •4J U -H ai io o o tu co UI • o o CO U C ai e u o co O "0 en >•Gì U k4 Q.4J 4-1 a < "O w -H C T3 X) o c ^ t !c O C rH O Ü C0 -H -C U •— C0O) 2 Q.4J W —^ kl • Q)ai m ai^ rH O ai C Ä X — >M TI "H ai *tH N a* >.jî 4-1 -O E rH C O • 3 ai CO J3 \ C o kj ^c u c cp ai o E e 1000 rad) t o t a l body i r r a d i a t i o n may well be needed to ensure engraftment of HLA-disparate c e l l s in m u l t i t r a n s f u s e d immunocompetent leukemic h o s t s . I t i s hoped that continued p r o g r e s s of t h i s l i n e of work should permit us in the near f u t u r e to apply marrow t r a n s p l a n t s to a l l i n d i v i d u a l s and not j u s t to the 40% of i n d i v i d u a l s p o s s e s s i n g matched s i b l i n g d o n o r s . Use of such t r a n s p l a n t s w i l l a l s o enable us to e x p l o r e the a l l o g e n e i c advantage f o r promoting leukemia r e s i s t a n c e in d i s e a s e d human h o s t s . Furthermore, a b r o g a t i o n or a m e l i o r a t i o n of GVH d i s e a s e a s a c o m p l i c a t i o n of marrow t r a n s p l a n t a t i o n , should reduce the a c u t e m o r b i d i t y and m o r t a l i t y a s s o c i a t e d with t h i s procedure to between 5 and 10% and, t h e r e f o r e , may u l t i m a t e l y permit us to c o n s i d e r t r a n s p l a n t s f o r a number of other blood d i s e a s e s and c o n g e n i t a l enzymopathies which a r e not a c u t e l y l e t h a l but r a t h e r l e t h a l over the c o u r s e of y e a r s (25) .

696 References 1.

Camitta B.W., Blood 4 5 , 355

Rappeport (1975).

2.

Thomas E . D . , B u c k n e r C . D . , C l i f t R . A . , F e f e r A . , J o h n s o n F . L . , Neuman P . E . , S a l e G . E . , S a n d e r s J . E . , S i n g e r J . W . , Shulman H . , S t o r b R . , Weiden P . L . : New E n g . J . Med. 3 0 1 , 597 ( 1 9 7 9 ) .

3.

Gale R . P . , G., Fahey (1976) .

4.

Neiman P . E . , Thomas E . D . , R e e v e s W . C . , Ray C . G . , S a l e G . , L e r n e r K . G . , B u c k n e r C . D . , C l i f t R . A . , S t o r b R . , Weiden P . L . , Fefer A.: T r a n s p l a n t . P r o c . 8 , 663 ( 1 9 7 6 ) .

5.

Thomas E . D . , Storb R., Clift R.A., Fefer F . L . , Neiman P . A . , L e r n e r K . G . , G l u c k s b e r g C.D.: New E n g . J . Med. 2 9 2 , 8 3 2 ( 1 9 7 5 ) .

6.

Lowenberg B . , D i c k e K . A . , v a n Bekkum T r a n s p l a n t . P r o c . 8 , 527 ( 1 9 7 6 ) .

7.

Y u n i s E . J . , Good R . A . , S m i t h J . , A c a d . S e i . USA 7 1 , 2 5 4 4 ( 1 9 7 4 ) .

8.

Von Boehmer H . , 322 ( 1 9 7 5 ) .

9.

Zinkernagel R.M.Jr.: J.

Feig J.,

S . , Opelz G . , Cline K.J.:

Sprent J . ,

Dicke K.A., (1971).

11.

Rich R.R., Kirkpatrick, 5 , 190 ( 1 9 7 2 ) .

R.,

Nathan

van

Eekkum

D.W.,

Stutman

Spooner

D.W.: C.H.,

12.

Dexter

Reisner Y. , Itzicovitch L. , P r o c . N a t l . A c a d . S e i . USA 7 5 ,

14.

L i s H. , S h a r o n N. : of Plants Vol. 6 p. 371.

15.

Reisner (1980).

16.

R e i s n e r Y. , L i n k e r - I s r a e l i 2 5 , 129 ( 1 9 7 6 ) .

17.

Reisner Y., Commun. 7 2 ,

18.

R e i s n e r Y . , Kapoor ( 2 ) , 1320 (1980) .

Sharon

E.:

M.:

0.: J.

N.,

O'Reilly

Natl.

Med. G.,

141, Welsh

3,

666

Immunol.

(1978).

Biochem.

Sharon N . : N.:

Proc.

Sharon

N.:

(ed.): The B i o c h e m i s t r y Academic Press, 1981),

Trends M.,

L.J.:

Cell.

Meshorer A., 2933 ( 1 9 7 8 ) .

i n l^arcus A. (New Y o r k ,

Ravid A . , S h a r o n 1585 ( 1 9 7 6 ) .

135

Johnson Buckner

Dooren

Proc.

Smith T . K . : 275,

A., H. ,

Exp.

Transplant.

Nature

N.:

D.G. :

T e r r i t o M . , Young L. , S a r n a Transplant. Proc. 8, 611

Nabholz

13.

Y.,

Parkman

R.M., Althage A., Callahan, Immunol. 1 2 4 , 2 3 5 6 ( 1 9 8 0 ) .

10.

T.M.,

J.M.,

Biochem. R.J.,

Sei. Cell.

5,

Immunol.

Biophys.

Good R . A . :

29

Res. Lancet

697

1920. 21. 22. 23. 24. 25.

R e i s n e r Y. , Kapoor N . , K i r k p a t r i c k D. , P o l l a c k fc.S., Dupont B . , Good R . A . , O ' R e i l l y R . J . : Lancet ( 2 ) , 327 (1981) . R e i s n e r Y. , Kapoor N . , Hodes M.Z., O ' R e i l l y R . J . , Good R . A . : Blood 5 9 , 360 ( 1 9 8 2 ) . R e i s n e r Y . , Pahwa S . , Chiao J . W . , Sharon N . , Evans R . L . , Good R . A . : P r o c . N a t l . Acad. S c i . USA 77, 6778 ( 1 9 8 0 ) . F r i e d r i c h W., O ' R e i l l y R . J . , Koryiner B . , Gebhard D . F . , Good R . A . , Evans R . L . : Blood 5 9 , 696 ( 1 9 8 2 ) . Korngold R. , S p r e n t J . : J . Exp. fied. 148, 1687 ( 1 9 7 8 ) . Woodruff J . f t . , E l t r i n g h a m J . R . , Casey H.W.: Lab. I n v e s t . 2 0 , 499 ( 1 9 6 9 ) . Good R . A . : New E n g l . J . Med. 306, 421 ( 1 9 8 2 ) .

REPORT ON THE THIRD SESSION OF THE INTERNATIONAL WORKING PART? ON STANDARDIZATION OF LECTINS FOR DIAGNOSIS.

T.C. B0g-Hansen (secretary) The Protein Laboratory, University of Copenhagen, Denmark

The International Working Party on Standardization of Lectins for Diagnosis at the Fourth Lectin Meeting in Copenhagen, June 1981, was outlined in the first report (1). The ultimate goal would be to draw up guidelines for lectin production, the first phase of the program being a well-controlled test scheme for a few widely used lectins. It was voted to test various commercial and non-commercial con A preparations in as many chemical and biological assays as possible, and most major lectin producers responded to my request to provide con A. The preliminary test program is listed in (1). The Third Session was held during Interlec 5 and coded con A samples were distributed for testing.

There was a motion for

a more thorough test program and the following additional groups volunteered to take part in the test program to make it very comprehensive: M.J.Waxdal and B.J.Mathieson (Bethesda), J.Y.Lin (Taiwan), J.A.Kint (Gent), A.M.Wu (College Station), I.Lorenc-Kubis (Wroclaw), B.Ersson (Uppsala), J.Kohn (London) and M.Caron (Bobigny), and G. A. Spengler (Bern). A number of participants wished to provide non-commercial con A for inclusion into the test program.

This was wellcomed,

especially since this would mean a controlled comparison of a lectin provided as a solution and as a freeze dried powder. It is anticipated that all con A samples should be distributed during September 1982.

© Walter de Gruyter & Co. 1983, Berlin • New York Lectins, Vol. Ill

700

It was decided that commercial companies could take part in the program on the same terms as other partners, i.e. promarily that the highest scientific standards should be applied and that all test results of the coded samples should be made available for the Working Party. After the Third Session of the Working Party a letter was received from R.E. Ritts, chairman of International Union of Immunological Societies Standardization Committee Communicating Minutes of the IUIS/WHO Standards Executive Committee, WHO, Geneva, 27-28 May, 1982. The Minutes contain the following paragraph under the heading New Subcommittees: "Several comments or requests have been received concerning standardization of lectins. Correspondence has also been transmitted by WHO from the International Working Party in Standardization of Lectins. After reading Secretary B0g-Hansen's report of this group, the committee members voted to ask Profs. J. Breborowicz and B0g-Hansen if they would be willing to have their group act as an official subcommittee of the WHO/IUIS Standardization Program with the assurance that we would assist them with review of protocols to assure biostatistical acceptibility for eventual WHO submission for the candidate preparations, annual budget support of $2,000 ~ 3,000 if available and assistance in raising funds for this effort.'' On behalf of the International Working Party for Standardization of Lectins for Diagnosis, I have accepted to serve as an IUIS/WHO subcommittee.

1. T.C.B0g-Hansen, J.Breborowicz, H.Franz: A report from the International Working Party on Standardization of Lectins for Diagnosis. On the necessity of standardized products. Lectins - Biology, Biochemistry, Clinical Biochemistry, Vol. 2, pp. 789-791.

A U T H O R

Andersen, M. M. 361 Àngelisova, P. 9 Animashaun, T. 639 Azim, M. 63 Barbieri, L. 293 Beeckmans, S. 629 Berke, G. 55 Bessler, W. G. 45 Bielka, H. 645 Bierbuchen, M. 167 Bjerrum, 0. J. 387 Bladier, D. 319 Bobrow, L. 105 B0g-Hansen, T. C. 27, 699 Bonoldi, E. 87 Bonnafous, J. C. 283 Bonnard, G. D. 37 Bratthall, D. 653 Brooks, D. E. 479 Busing, D. 45

I N D E X

Decker, J. M. 443 Debray, H. 323 De Gennaro, V. 199 Dejaegere, R. 604, 629 Dell'Orto, P. 87, 199 Delrieu, J. 661 Deugnier, M. A. 319 Diaz, C. 521, 531 Doi, P. 87 Dornand, J. 283 Drakeford, D. 275 Driessche, E. van 549, 604, 629 Ersson, B. 503, 667 Eshdat, Y. 667 Falasca, A. 293 Faris, A. 503 Farrar, G. 157 Fauci, A. 235 Faure, A. 661 Favero, J. 283 Fischer, J. 145, 157, 167 Fischer, R. 167 Fowlkes, B. J. 235, 251, Franceschi, C. 293 Franco, M. V. 435 Franz, H. 179, 645 Fujimoto, M. 397

Calafat, J. 119, 131 Campadelli-Fiume, G. 351 Carlier, A. R. 583, 593 Carlsson, P. 653 Caron, M. 319, 623, 661 Causse, H. 559 Chiricolo, M. 293 Christensen, U. 387 Clemetson, K. J. 303 Coggi, G. 87, 199 Colombi, R. 199 Comi, A. 199 Crouther, D. 225

Gallagher, J. T. 225, 311 Gerecke, D. 213 Gilboa-Garber, N. 495 Groeger, B. 179

Dale, R. E. 311 Dall'Olio, F. 351 Debbage, P. L. 179

Hageman, P. C. 105, 131 Harding, M. J. 225, 311 Haynes, B. F. 235, 251

702 Heegaard, N. H. H. 387 Hermann, G. 73, 213 Hilgert, I. 9 Hilkins, J. 105 Htfiby, N. 27 Holthofer, H. 205 Hörejsi, V. 3, 9 Horisberger, M. 189 Hosselet, M. 549 Iren, F. van 521, 531 Iwasa, M. 371 Janssen, H. 119, 131 Jensen, C. 27 Kanarek, L. 549, 604, 629 Karjalainen, K. 327 Kieda, C. 427 Kijne, J. W. 521, 531 Kimber, S. J. 63 Kindt, A. 415, 645 Klein, P. J. 145, 157, 167 Koch, C. 27 Kocourek, J 3, 453 Kohn, J. 405 Krajhanzl, A. 453 Kraut, H. 45 Kristofova, H. 9 Kuwahara, A. 397 Lauwreys, M. 604 Leireson, W. 275 Lembke, J. 213 Lempfried, K. W 73 Liechti, J. 37 Licastro, F. 293 Lindahl, M. 503 Lonnerdahl, B. 503 Loof, A. De 469 Lorenc-Kubis, I. 379 Ltfwenstein, H. 27 Luscher, E. F. 303 Malmi, R. 327 Manen, J. F. 611 Mani, J. C. 283 Marchalonis, J. J. 461 Mathieson, B. J. 235, 251, 263, 275 Mattiasson, B. 653 McGregor, J. L. 303 Miettinen, A. 205 Misdorp, W. 105

Monsigny, M. 427 Montreuil, J. 323 Muller, E. 435 Muller-Hermes, W. J. P. 45 Murre, C. 243 Nelken, D. 17 Noach, S. 361 Norn, S. 27 Nosek, J. 453 O'Brien, R. L. 283 0'Dell, D. S. 179 Ohanessian, J. 623 Permin, H. 27 Peters, J. H. 45 Peumans, W. J. 583, 593 Pierce-Cretel, A. 323 Pigott, J. 179 Poucke, M. van 549 Pueppke, S. G. 513 Pusztai, A. 611 Ramstorp, M. 653 Raymond, J. 405 Reisner, Y. 677 Renndorf, R. 645 Rossel, S. 145 Rossi, C. A. 293 Rouge, P. 559 Sagisaka, K. 371 Scannavini, M. 351 Schaal, I. van der 521, 531 Schauer, R. 435 Schröder, C. 435 Serafini-Cessi, F. 351 Shannon, L. M. 573 Sharon, N. 667 Sharrow, S. 0. 251, 275 Shukla, A. K. 435 Skov, P. S. 27 Soderstrom, K. 0. 327 Solheim, B. 538 Spik, G. 323 Stadler, B. M. 37 Stinissen, H. M. 583, 593 Stirpe, F. 293 Strosberg, A. 604 Stynen, D. 469 Surani, M. A. H. 63 Tacchini, M.

189

Takeo, K. 397 Thomas, C. A. 251 Thomas, C. T. 235 Trust, T. J. 479 Turp, P. 405 Uhlenbrock, G. 145, 157, Valk, M. A. van der 105 Vasta, G. R. 461 Venker, L. 645 Viale, G. 87, 199 Vierbuchen, M. 145, 157 Virtanen, I. 205 Voller, A. 405 Wadstrom, T. 479, 503 Waxdal, M. J. 235, 243, 263, 427 Weil, H. P. 73, 213 Williams, D. 3, 179 Williams, L. G. 179 Ziska, P. 179, 645

S U B J E C T

Abrin 263, 405, 479 Abrus precatorius agglutinin 263, 293 N-Acetyl D-glucosamine 479 Acid phosphatases 371, 379 Acrosome 327 Acute leukaemia 225 Adenia digitata 293 Adenocarcinoma 131 Adherence 479, 495, 667 Adhesins 479 Affinity chromatography 45 167, 243, 303, 335, 371, 379, 387, 443, 503, 513, 539, 559, 573, 593, 623, 629, 639 Affinity electrophoresis 361, 379, 387, 397, 405 Affinity fixation 405 Affinity Immunoelectrophoresis 361, 379, 387 Afzelia africana 639 Ageing 319 Agglutination 3, 263, 283, 303, 427, 531 Agropyrum repens 583 Amino acid composition 461 Antibodies 371, 521, 539, 573, 611 Antioxidant 629 Antiserum 549, 583 ^ - a n t i t r y p s i n defiency 361 Arachis hypogaea lectin 293, 405, 573 Asthma, histamine release 27 Autoaggregate 479 Avidin-biotin-peroxidase complex 87

I N D E X

Bacterial lectins 27, 479, 495, 503, 667 Bandeiraea Simplicifolia lectin 573 Barley lectin 583 Bauhinia purpurea alba lectin 573 Bauhinin 573 Beetle lectin 469 Berberis variegata lectin 405 Binding constants 387, 623 Binding curve 311 Binding sites, lectin 3, 145 Binding sites, multiple 479 Binding specificity 623 Biotinylated lectins 87, 199 Blotting 179 Brachypodium 583 Breast cancer 105, 119, 131, 145, 157 Canavalia ensiformis lectin, see con-A Canavalia gladiata DC lectin 371 Carcinomas 105, 131 Calcium independent interaction 63 Cell culture 179 Cell cycle 37 Cell membranes, cerebellar 179 Cell membranes, liver 179, Cell suspensions 179 Cell sorting 225, 235, 243, 251, 263, 275 Cell-spreading 63 Cells, affinity chromatography 243

706 Cellular location 611 Cereal lectins 583, 593 Cerebellum (chick) 179 Chick embryo cerebella 179 Chitin 629 Chitobiose 311 Cholera lectin 479 Chromatofocusing 521 Colloidal particles 189 Colorado potato beetles 469 Compactin 63 Con-A 17, 27, 63, 73, 87, 131, 167, 189, 293, 303, 327, 335, 361, 379, 397, 405, 415, 573 Coregonus lavaretus M. 293 Cotoneaster horizontalis lectin 405 Cotyledons 559 Countercurrent electrophoresis 405 C-reactive proteins 461 Crotalaria juncea lectin 293, 503 Crossed affinoimmunoelectrophoresis 361, 379, 387 CTL-cytotoxic T lymphocytes 55 Cupressus lectin 405 Cyclostome lectin 461 Cydonia oblonga lectin 405 Cytisus scoparius link lectin 371 Cytofluorimetry 427 Cytolytic activity 17 Cytotoxic T lymphocytes 55 Cytotoxicity lectin-dependent 55 Datura stramonium 293 DBA 87, 105, 327, 573 Definition 3 Diarrhoea 503 Differentiation 145 Differentiation antigens of the breast 105 Difference spectrophometry 623 Differentiation markers 105 Dissociation constant of Concanavalin A 397 DNA 235 DNA synthesis 427 Didemnum candidum 461

Dolichos biflorus 293 Dolichos biflorus lectin, see DBA Double diffusion 521, 611, 639 E. coli 503 ECA 435 Elastase 361 Electron microscopy 131, 189, 199 Embryo 559 Embryonic cells 63 Endocrine therapy 145 Endocrine treatment 157 Endogeneous lectins 427 E-PHA 611 Erythrina crystagalli agglutinin 435 Erythrina indica 293 Erythrocytes 435 Erythrocytes, binding to macrophages 435 Erythrocytes, phagocytosis by macrophages 435 Erythrocytes, sialidase treatment 435 Estrogen 145 Estrogen receptor 131, 145 Evolution 461, 573 Evolutionary relationship 461 Favin 603 Ferritin 87 Fetuin 443 Fetuin-binding material 443 Fimbriae 479, 503, 521 Fish oocyte lectin 453 FITC-lectins 145, 157, 167, 179, 235, 263, 311, 327, 513, 539 Flagellae 521 Flow cytometry 235, 245, 251, 263, 275, 311, 427 Flow microfluormetry 235, 243, 275 Fluorescein labelled lectins see FITC-lectins Fluorescence 205, 225 Fluorescence activated cell sorter 263 Fluorescence technique 319 Fluorescent labelled antibodies 611

707 Fluorochromes 87 Frozen sections 179 Galactomannan 639 Galactose-specific lectins 573 ^-Gal-specific lectin 435 of-Galactosidases 573 Galactosides 623 Gastric cancers 167 Gastric mucosa, normal 167 Gastrointestinal glycoproteins 167 Germinal cells, labeling of 327 Germination 583 Glomerulus 205 Glycine max 293, 513, 573 Glycine soya 513 Glycine soya lectin 513 Glycocalyx 311 Glycoconjugates 205 Glycophorin 387, 479 Goblet cells 167 Gold label 611 Gold markers 189 Gold staining 189 Grass family lectins 583 Grass seeds 379 Grass seed acid phosphatase 379 GSA 335 Host specificity 531 H-2 17 HA 479 Haemadsorption 415 Haemadsorption solid phase technique 415 Haemagglutinating 559 Haemagglutination 469, 495, 503, 513, 521, 549, 573, 639 Haemagglutinin 573 Halocynthia Pyriformis 461 Hemadsorption lectin test 415 HDA 415 Helix pomatia 293 Helix pomatia lectin 73, 213 Helper T cells 427 Herpes virus glycoproteins 351 Histamine release 27

Histochemistry 87, 105, 119, 131, 145, 157, 167, 179, 189, 199, 205 Histocompatibility antigens 55 HL-60 cells 361 Hormone dependence 145, 157 Hormone receptor 145 Hp 167, 213 HPA 131 Hp-peroxidase conjugate 73 Human erythrocyte 319 Hura crepitans 293 Hyperplastic gastric mucosa 167 IL see Interleukin Iodine-125-labelled lectins 303 Immunochemistry 573 Immunocytochemistry 189, 611 Immunodiffusion 549 Immuno double diffusion 573 Immunoelectrophoresis 371, 559 Immunofluorescence label 611 Immunological cross reactions 513, 573 Immune lysis 55 Immunoglobulin 573 Immunoglobulins 27 Immunoglobulin secreting cells 235 Immunoprecipitation 283 Induced tumor 443 Infection threads 513 Infectivity 479, 495, 513, 521, 539 Inhibitors of protein synthesis 293 In vivo synthesis 593 Insect lectin 469 Interleukin 2 (IL 2) 37, 45 Internalisation 179 Intestinal metaplasia 167 Intrinsic asthma 27 Isolectins 45, 539, 583, 593, 611 Iuglans regia lectin 405 Kidneys 205 Kinetic constants 387 Lactose, immobilized 513

708 Labeling lectins 87 L a m p r e y lectin 461 LCA 17, 55, 63, 303, 355, 573, 603 Leaf lectins 583 L e c t i n assay 415 L e c t i n - b l o t t i n g 179 Lectin-carbohydrate i n t e r a c t i o n 623 L e c t i n - d e p e n d e n t lympholysis 55 L e c t i n h i s t o c h e m i s t r y 167 L e c t i n - i m m u n o c h e m i s t r y 167 L e c t i n l e s s soybean lines 513 L e c t i n - r e c e p t o r - c o m p l e x e s 583 Legume lectins 573 Lens c u l i n a r i s lectin 63, 303, 573 Lentil lectin, see LCA L e u c o c y t e s 361 L e u k a e m i a 213 Liver (rat) 179 Limulus polyphemus lectin 63 Lima bean lectin 45 Linear contact assay 63 L i p o p o l y s a c c h a r i d e (LPS) 539 L o c a l i z a t i o n 611 Lotus lectin 573 Lotus tetragonolobus a g g l u t i n i n 335, 573 L - P H A 611 LTA see Lotus T e t r a g o n o l o b u s agglutinin L e u k a e m i a 225 L y m p h o c y t e a c t i v a t i o n 235 L y m p h o c y t e d i s o r d e r s 225 L y m p h o c y t e lectin 443 L y m p o c y t e - m e d i a t e d cytotoxic lysis 55 L y m p h o c y t e - t a r g e t cell c o n j u g a t i o n 55 L y m p h o c y t e s 45, 55, 231, 235, 443 L y m p h o k i n e s 37 Lysis 55 L y s o s o m o t r o p i c 73 M o d e c c i n 293 M a d u r a a u r a n t i a c a 303 M a c r o p h a g e assay 435 M a c r o p h a g e lectin 435 M a c r o p h a g e s 427, 435 Major h i s t o c o m p a t i b i l i t y complex (MHC) 55

M a m m a r y c a r c i n o m a s 145 M a m m a r y gland 105 M a m m a r y tumors 105, 119, 131, 157 Mannose r e s i s t a n t h a e m a g g l u tination 479 Mannose sensitive h a e m a g g l u tination 479 M a r m o s e t l y m p h o c y t e s 443 Membrane g l y c o p r o t e i n s 303, 387 Membrane lectins 427, 435, 443 M i c r o c l u s t e r i n g 55 M i c r o g l i a l 179 M i c r o h e t e r o g e n e i t y 361, 387 M i c r o p i n o c y t o s i s 469 Milk fat g l o b u l e s 105, 157 M i s t e l t o e lectin 179, 645 M i t o g e n m e c h a n i s m 37 M i t o g e n i c lectins 55, 293 M o m o r d i c a c h a r a n t i n 293 Monkey lymphocyte lectin 443 M o n o c l o n a l antibody 243, 275 M o n o c y t e s , human 243 M o n o c y t e s , m a r k e r 235 M o n o s a c c h a r i d e s 63 Motility 179 Mouse embryo 63 MPL 263 MRHA, see m a n n o s e resistant haemagglutination MSHA, see m a n n o s e sensitive haemagglutination M u c i n - l i k e 479 M u c u s 479 N e p h e l o m e t r y 405 N e p h r o n 205 Nervous tissue 179 N e u r a m i n i c acid specific lectin 443 N e u r a m i n i d a s e , see sialidase N i t r o g e n - f i x i n g 513 Nodulating 513 N o n - m i t o g e n i c lectins 55 N o n - s p e c i f i c esterase stain 243 O n o n i s spinosa 293 Oocyte lectin 453

709 P r o c e s s i n g , l e c t i n 539, 603 Postsynthetic modification 603 PA-1 235 PA-2 235, 263, 427 P A - 3 235, 263 PA-4 235, 243 PA-5 235 P e a l e c t i n 521, 531, 603 P e a root l e c t i n 549 P e a seed l e c t i n 549 P e a n u t a g g l u t i n i n , see PNA P e r c a f l u v i a t i l i s 293 P e r c a f l u v i a t i l i s l e c t i n 453 Peroxidase labelled rabbit a n t i b o d i e s 145 Peroxidase labelled lectins 87, 119, 131, 157, 179, 213, 405 P e r s e a g r a t i s s i m a l e c t i n 405 P e t r o m y z o n m a r i n u s 461 PHA 17, 27, 55, 293, 405, 573 P h a g o c y t o s i s 435 P h a s e o l u s 611 P h a s e o l u s c o c c i n e u s 293, 371 P h a s e o l u s l u n a t u s 293 P h a s e o l u s l u n a t u s l e c t i n 573 P h a s e o l u s v u l g a r i s 303, 559 Phaseolus vulgaris agglutinin see PHA P h a s e o l u s v a l p a n i s e e d s 611 P h e n o l o x i d a s e s 629 P h o s p h o l i p i d m e t a b o l i s m 45 P h y s i o l o g i c a l f u n c t i o n 583 P h y t o h a e m a g g l u t i n i n 405 Phytolacca americana polys a c c h a r i d e 243, 427 Phytolacca americana lectins 235, 243, 263, 427 Pili 479, 521 P i n o c y t o s i s 73 P i s u m - r o o t s 521 P i s u m s a t i v u m 293 P i s u m s a t i v u m lectin 573 P l a s m a m e m b r a n e liver cell 179 Plasmid mediated a g g l u t i n a t i o n 479 P l a t e l e t g l y c o p r o t e i n s 303 P l a t e l e t s 303 PNA 55, 63, 87, 105, 119, 131, 145, 157, 167, 205, 243, 263, 275, 283, 303, 327, 415, 623

Pokeweed lectin 235, 243, 263, 427 Pokeweed mitogen 235, 415, 427 P o l y s a c c h a r i d e 427, 639 Post-translational cleavage 603 P o t a t o l e c t i n 5 8 3 , 629 P r e c u r s o r 461, 593, 603 P r e c i p i t i n r e a c t i o n 639 P r o g e s t e r o n e r e c e p t o r 131 P r o s t a t e 371 P r o t e a s e s 361 P r o t e i n b o d i e s 559, 593, 611 P r o t e i n A 27, 189, 283 PSA 335 Pseudomonas aeruginosa l e c t i n s 495 PWM, see p o k e w e e d m i t o g e n Q u a n t i t a t i o n of cell s p r e a d i n g 63 R a d i a l i m m u n o d i f f u s i o n 559 Rat 327 Rat m a m m a r y c a r c i n o m a s 145 RCA-1 63, 131, 167, 335, 405, 415 Receptor characterisation 55, 179 R e c e p t o r - c o m p l e x 583 R e c e p t o r - m e d i a t e d 73 R e c e p t o r m e g a t h e r a p y 479 R e c o g n i t i o n 3, 521, 583 R e c o g n i t i o n m o l e c u l e s 461 Red b l o o d c e l l s 189 Red cell g h o s t s 623 R e j e c t i o n 17 Reversible binding 3 Rh. l e g u m i n o s a r u m , m a n n o s e s p e c i f i c a g g l u t i n i n 521 R h i z o b i a l a g g l u t i n i n 521 R h i z o b i u m 513, 539 R h i z o b i u m l e g u m i n o s a r u m 531, 521 R h i z o b i u m p h a s e o l i 531 R h i z o b i u m r e c o g n i t i o n 513 R h o d a m i n e l a b e l l e d 519, 213 R i c i n 17, 120, 179, 189, 263, 293, 405, 479 R i c i n u s c o m m u n i s 593, 611 Ricinus communis agglutinin 189 R i c i n u s c o m m u n i s l e c t i n 573, 303

710

Robinia pseudoacacia 293 Rutilus rutilus 293 Rye lectin S Q parameter 461 Sarothamnus scoparius 293 SBA 55, 63, 87, 105, 205, 263, 327, 405, 415, 559 Scanning electron microscopy 539 Scatchard analysis 73, 213 Screening for lectins 513 Sea lamprey 461 Seed coat 559 Seminal plasma 371 Serum factor 435 Sialic acid 479, 435, 311 Sialic acid specific lectin 443 Sialidase 73, 145, 157, 167, 179, 189, 205, 213, 225, 283, 303, 311, 435 SJ-11 263 Skin allografts 17 Solanaceous lectins 583 Solid phase lectin sandwich enzymossay 405 Sophora japónica lectin 573 Sophora lectin 573 Soybean agglutinin, see SBA Specificity 623 Spectral properties 629 Spermatid differentiation 327 Spermatogenesis 327 Sphenostylis stenocarpa 639 STA 583 Standardization of electrophoresis 397 Stimulation index 293 Stingray 461 Storage proteins 593 Structural homology 461 Subpopulations 263 Subset 275 Subunits 611 Subunits, leguminous lectins 603 Succinyl concanavalin A 397 Sugar-binding 3 Sugar-specific enzymes 3 Surface-associated lectin 311 Surface glycoprotein 225 Surface receptors 55 Surfaces cell 311

Symbiosis 513, 521, 539 Synthesis, lectin 539, 593, 603 Synthesis 235 Tamoxifen 145 Target cells 55 T cell growth factor 37 TCGF 37 Thomsen-Friedenrich antigen 105 Thymocytes 263 Thymocytes subsets 263 Thymus development 275 Tissue extracts 405 T lymphocyte differentiation 283 T lymphocytes 37, 55, 293 Tolerance 17 Toxic activity 293 Toxicity 293 Transplantation 17 Transport 593 Trifoliin A 521 Trifolium repens 539 Triticeae 583 Triticum vulgare 293 Trypsin 361 Tunicate lectin 461 Tunicates Tumor induction 145 Tumors 105 Two-chain lectins 603 UEA 87, 131, 167, 205, 335, 415 UEA-1 63 Ulex europaeus 293, 573 Ulex lectin 573 Urinary tract infection 503 Vaccines 479 Vascular endothelia 131 Vectorial translation 593 VFA 335 Vicia cracca 293 Vicia hirsuta seed 539 Vicia sativa 293 Vicia villose 293 Vigna catiang endl. varis sinensis 371 Vigna lectin 573 Vigna radiate lectin 573

Viscum album 179 Vitellogenesis 469 von Willebrand factor 303 WGA 55, 63, 73, 87, 131, 167, 189, 205, 263, 303, 311, 319, 327, 335, 387, 405, 415, 479, 539, 583, 593 Wheat germ agglutinin, see WGA Wistaria floribunda 293 Wistaria floribunda agglutinin 73, 573 Wistaris brachbotrys sied, et zucc. 371 Yolk formation 469 Zajdela hepatoma 73

G

Walter de Gruyter Berlin-New York

Bog-Hansen

Lectins

w DE

(Editor)

Biology, Biochemistry, Clinical Biochemistry Volume 1 Proceedings of the Third Lectin Meeting, Copenhagen, June 1980 1981.17 cm x 24 cm. XII, 418 pages with figures and tables. Hardcover. DM 120,-; approx. US $60.00 ISBN 311 008483 X

Bog-Hansen (Editor)

Lectins Biology, Biochemistry, Clinical Biochemistry Volume 2 Proceedings of the Fourth Lectin Meeting, Copenhagen, June 8-12,1981 1982.17 cm x 24 cm. XVI, 801 pages. Numerous figures and tables. Hardcover. DM 248,-; approx. US $124.00 ISBN 3110086808

Scott Brewer (Edltors)

Concise Encyclopedia Q f Biochemistry 1983.14,0 cm x 21,5 cm. VI, 519 pages. Approx. 650 illustrations. Hardcover DM 59,-; US $29.90 ISBN 3110078600 Prices are s u b j e c t t o c h a n g e w i t h o u t n o t i c e