Lectins: Vol. 2 Proceedings of the Fourth Lectin Meeting: Copenhagen, June 1981 [Reprint 2019 ed.] 9783111555928, 9783111185804

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Lectins Biology, Biochemistry, Clinical Biochemistry Volume 2

Lectins

Biology, Biochemistry, Clinical Biochemistry Volume 2 Proceedings of the Fourth Lectin Meeting Copenhagen, June 1981 Editor T. C. Bog-Hansen

W G DE

Walter de Gruyter • Berlin • New York 1982

Editor T h o r k i l d C h r i s t i a n B o g - H a n s e n , c a n d . scient., lie. t e c h n . The Protein Laboratory University of C o p e n h a g e n S i g u r d s g a d e 34 DK-2200 C o p e n h a g e n N

Library of Congress Cataloging in Publication

Data

Lectin Meeting (4th: 1981: Copenhagen, Denmark) Proceedings of the Fourth Lectin Meeting, Copenhagen, June 1981. (Lectins-biology, biochemistry, clinical biochemistry; v. 2) Bibliography: p. Includes indexes. 1. Lectins-Congresses. I. Bog-Hansen, T. C. (Thorkild Christian), 1939. II. Title. III. Series. QP552.L42L4 1981 574.19'2454 82-9763 ISBN 3-11-008680-8 AACR2

CIP-Kurztitelaufnahme

der Deutschen

Bibliothek

Lectins, biology, biochemistry, clinical biochemistry: proceedings of the ... Lectin Meeting. - Berlin; New York: de Gruyter. NE: Lectin Meeting Vol. 2. Proceedings of the Fourth Lectin Meeting: Copenhagen, June 1981.-1982. ISBN 3-11-008680-8

Copyright © 1982 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 Third Lectin Meeting in Copenhagen, June 1980, Dr. H. Franz, Berlin, urged me to repeat the event the following year. Having done so, this volume number 2 contains the proceeding papers from the Fourth Lectin Meeting, June 8-12, held at the University of Copenhagen, as in the case of the previous meetings. New lectins are being discovered almost every day on mammalian cell surfaces, on microorganisms, in plants etc., and lectins are found to be involved in intricate biological and biochemical processes because of their specific binding of carbohydrate . Therefore , it is not surprising to learn that lectins, long since a useful tool for the immunologist, are now becoming an extremely valuable tool for the study of fundamental cellular and molecular properties. The spectrum of topics in this book is wide, from biological approaches revealing the mysteries of lectin functions to the techniques of fine analysis of proteins. About 100 persons participated in the following sessions: Biological function and biological effects; Cells; Glycoconjugates; Lectin-reactions and methodology; Diagnosis; Lectins from microorganisms and animals; and Plant lectins. Altogether 73 communications were made, the majority of which appears in this volume, organized into five parts. In addition to the sessions, several discussions were organized. One discussion was inspired by the presence of so many people working with affinity electrophoresis

(see pp 779) and several

discussions led to the formation of an international working party for the standardization of lectins for diagnosis PP 791).

(see

VI

The meeting was generously supported by the Peter F. Heering Co. who donated the famous cherry liqueur 'Peter Heering' for the welcome reception. Apart from this 'spiritual' support, I did not manage to obtain any other support for the meeting, partly because of the immense practical work involved in writing applications. Also because of the practical work involved in the actual organization of such a meeting, no matter how informally and leisurely approached, it was found highly desirable to have future meetings organized on a collaborative basis. Also there was much talk about forming an international lectin society. Amongst the foremost advocates for a society was Dr. D.L.J. Freed, Manchester. For the next meeting we decided to go to Bern in Switzerland with Dr. G. Spengler as host of INTERLEC 5, May 31-June 5, 1982. The three main themes will be: 1. 2. 3.

Detection of cancer markers. Biological funtion of lectins. Lectins as specific tools.

Copenhagen, April 1982

T.C. B0g-Hansen

Contents

Part I

Biological Effects and Biological Functions of Lectins

Histophathological Changes Induced in Mice by the Plant Toxin Ricin and its Highly Purified Subunits A-Chain and B-Chain G. Richer, D. Carriere, H.E. Blythman, H. Vidal

3

Suppression of Experimental Allergic Encephalomyelitis in Guinea Pigs and Rats by Concanavalin A R. Mori, Y. Kuroda, T. Aoki, A. Takenaka, T. Nakamura, T. Namikawa

23

Lectins, Allergens and Mucus D.L.J. Freed

33

Effect of Viscum Lectin on Histamine Release from Human Leukocytes I. Sehrt, P. Luther

45

Analysis of the Recycling Behaviour of Galactose-Specific Lectins on Rat Liver Cells D.Vogt, H. Kolb

57

Interaction of Soybean Agglutinin with Human Peripheral Blood Lymphocyte Subpopulations: Evidence for the Existence of a Lectin-Like Substance on the Lymphocyte Surface M. Barzilay, M. Monsigny, N. Sharon

67

Vili A Recognition Mechanism in the Adhesion of Nematodes to Nematode-Trapping Fungi B. Nordbring-Hertz, E. Friman, B. Mattiasson

83

The Lectin-Producing Cells in the Sponge Tissue H. Bretting, G. Jacobs, C. Donadey, J. Vacelet

91

The Non-Random Distribution of Lectin in the Azolla caroliniana-Anabaena azollae Symbiosis R.B. Mellor, P. Rowell, W.D.P. Stewart

105

Distribution, Specificity and in vivo Significance of Phytolectins R.S. Sandhu, R.S. Reen

113

Occurrence of Lectin During the Life Cycle of Lathyrus Species P. Rouge, D. Pere

13 7

Part II

Cell Receptors and Cell Reactions

Monosaccharides and Tamm-Horsefal1 Glycopeptide Inhibit Allogeneic Antigen-Induced Lymphocyte Blàstogenesis in One-Way Mixed Lymphocyte Reaction C. Franceschi, F. Licastro, M. Chiricolo, F. SerafiniCessi, P. Tabacchi

153

Murine 3T3 Cell Deformability and Concanavalin A Agglutination -- Correlation with a Physical Model System P.Y. Wang, D.W. Evans, M.J. Bazos, S.K. Yu, A. Parravano . 167

IX Lectin Binding to and Lectin Induced Agglutination of Haemopoietic Cells M. Harding, J. Gallagher

179

Lectin Binding by Malaria Infected Erythrocytes R.F. Adebiyi, G. Parnell, A. Forrester, A.J.S. Davies .... 197

A Tumor Specific WGA-Receptor on a Chemically Induced Carcinoma H. Hauenstein, G. Hermann, H.P. Weil

211

Distribution of Lectin Receptors in Neonatal and Embryonic Mouse Tissue M.A. van der Valk, P. Hageman

221

Interaction of Peroxidase-Coupled Helix pomatia Lectin with Rat Hepatoma Cells H.P. Weil, K.W. Lempfried, G. Hermann

241

Use of Lectins for the Characterization of the Insulin Receptor Glycosidic Moiety in Rat Adipocytes and Hepatocytes G. Cherqui, J. Capeau, M. Caron, J. Picard

257

Ricin 120 as a Selective Marker in the Nervous System P.L. Debbage, D.S. O'Dell, D.W. James

273

The Role of the Cell Coat in Palatal Shelves Adhesion. The Action of Neuraminidase and Limulus polyphemus Lectin on Shelves Cultivated in vitro E. Heinen, E. Baeckeland, A.M. Renard

285

X Mobility of Con A Receptors at the Surface of Palatal Shelves before Closure of the Secondary Palate E. Baeckeland, E. Heinen, A.M. Renard

295

Increased Alpha-Galactosidase Activity in Normal and Fabry Fibroblasts Treated with Con A and Purified Enzyme L. Hasholt, S.A. S^rensen

305

Lectin-Induced Surface Changes in Lymphocytes of Gnotobiotic Pigs H. Kovaru, F. Kovaru, J. Plasek, Z. Fisar, L. Mandel

315

Mitogenic Lectins and Alpha-Fetoprotein as Modulators of Lymphocyte Surface Enzyme Activities H. Kovaru, F. Kovaru, M. Pospisil

325

Part III

Macromolecules and Lectins

Specific Interaction of R-Type Lipopolysaccharides with Lectins Versus their Non-Specific Reactivity with Basic and Hydrophobic Proteins N.M. Ahamed, H.M. Kuhn, C. Widemann, J. RadziejewskaLebrecht, H. Mayer

341

Lectin Receptors in Proteogalactans from the Albumin Gland of Achatina fulica R.O. Okotore, G. Uhlenbruck

351

Affinity of Leucoagglutinin (L-PHA), Compared with Dolichos biflorus Lectin and Con A, for Human TammHorsefall Glycopeptide F. Serafini-Cessi, F. Dall'Olio

367

XI Interaction of Haptoglobin with Lectin W. Dobryszycka, I. Katnik

381

Interaction of Polyclonal and Monoclonal Human Immunoglobulins G with Various Lectins (Concanavalin A, Lentil and Pea Lectins) C. Chatelain, A. Lemoine, J.M. Dugoujon, M. Blanc, P. Rouge

393

Changes in the Relative Amount of Individual Microheterogeneous Eorms of Serum Alpha-l-Antichymotrypsin in Disease M. Bowen, J.G. Raynes, E.H. Cooper

403

Study of the ConA Binding of Human Alpha-1 Acid Glycoprotein during the Acute Inflammatory Process. Evidence for Alpha-1 Acid Glycoprotein Populations of Different pi Values after ConA Affinity Chromatography in Normal and Inflammatory Sera I. Nicollet, J.P. Lebreton, M. Fontaine, M. Hiron

413

Crossed Affinity Immunoelectrophoresis of Factor VIIIRelated Antigen in van Willebrand's Disease J.S. Krauss, M.H. Sheard

423

Comparability of Carbohydrate Heterogeneity Patterns of Human Alpha-Fetoprotein in Different Biological Fluids K. Toftager-Larsen

433

Demonstration and Quantification of Microheterogeneity Forms of Human Alpha-Fetoprotein by Lectin Affinity Electrophoresis J. Breborowicz, T.C. B^g-Hansen

445

XII

Estimation of ConA-Binding and Non-Binding Forms of Human Alpha-Fetoprotein (AFP) by Rocket Line Affino Immuno Electrophoresis J. Hau, J.G. Westergaard, L. Ipsen, B. Teisner, T.C. B0gHansen, K. S^ndergaard

457

Crossed Charge-Shift Affino-Immunoelectrophoresis of Human and Murine Alpha-Fetoprotein (AFP) J. Hau, B. Teisner, T.C. B^g-Hansen

467

Heterogeneity of Human Ferritins Studied by Crossed Lectin-Affinity-Radioimmunoelectrophoresis M.M. Andersen, A. Lihme, T.C. B^g-Hansen

475

Quantitation of Non-Binding and ConA-Binding Serum Ferritin by Lectin-Affinity-Radioimmunoelectrophoresis M.M. Andersen, A. Lihme, T.C. B0g-Hansen

487

Sequential Lectin Affinity Chromatography for Purification of the MHC-A1loantigens on Chicken Erythrocytes L.B. Nielsen, S. Bisati, L. Mikkelsen, C.H. Brogren

497

A Note on the Heterogeneity of Plant Enzyme Preparations. Acid Phosphatases from Grass Seeds I. Lorenc-Kubis, B. Morawiecka, T.C. B^g-Hansen

Part IV

509

Methods Based Upon Reactions of Lectins

A New Technique for Studying the Early Events of Lymphocyte Activation by Mitogens P. Balding, L. Hughes, P.A. Light, A.W. Preece

519

XIII Lectin Typing of Leishmania-Strains from the New and Old World J. Schottelius

531

The Lectin-Neuraminidase-Assay (LN-Test). A Clinical Use of the Arachis Lectin for Diagnosis P. Luther, S. Weber, K.C. Bergmann

543

Lectin-Antibody and Lectin-Lectin Conjugates H. Franz, J. Mohr, P. Ziska

553

Lectin-Carbohydrate Interactions Studied in a Two-Phase System T.G.I. Ling, B. Mattiasson

563

Lectin-Carbohydrate Interactions as a Tool to Quantify Microbial Cells B. Mattiasson, T.G.I. Ling, J. Nilsson, M. Durholt

573

Studies on the Thermodynamic Evaluation of Concanavalin A-Carbohydrate Interaction by Means of Affinity Electrophoresis K. Takeo

583

Computational Evaluation of Molecular Forms Separated by Crossed Affino Immunoelectrophoresis P. Hindersson, C. Munk, J. Hau

595

Further Control Experiments with Lectins to Assess Microheterogeneity of Glycoproteins L. Faye, J.P. Salier

605

XIV Isoelectric Focusing as a Method for Electrophoretic Desorption in Lectin Affinity Chromatography P. Rautenberg, E. Reinwald, E. Kaufmann, H.J. Risse

619

Rate Nephelometric Measurement of Wheat Germ Lectins A. Bracciali, G. Antoni, P. Tarli, P. Neri

633

Quality Control of Commercial Phytohemagglutinins (PHA) from Red Kidney Beans (Phaseolus vulgaris) L. Riikola, T.H. Weber

Part V

643

Distribution, Isolation,and Characterization of Lectins

A Polyagglutinating, Non-Mitogenic Lectin from Arion empiricorum (Ael) G. Hermann, B. Schmidt-Horstmann, L. Habets, I. Karalus, L. Gurtler, W. Bessler

657

Occurrence and Characterization of Phytolectins in Indian Plants R.S. Sandhu, R.S. Reen, S. Singh, J. Singh, S.K. Chopra .. 679

The Search for New Lectin Sources on the Baja California peninsula E.M. Herrera, C. Montano, J.L. Ochoa, F. Cordoba

693

Some New Phytohemagglutinins: Their Interaction with Red Blood Cells and Serum Proteins A.K. Khanna

701

XV The Lectin from Garden Cress (Lepidium sativum). Isolation and Characterization P. Ziska, H. Franz

711

Isolation of two Lectins from the Cactus Macharocereus eruca E. Zenteno, F. Cordoba

721

The Isolation, further Characterization and Localization of Pea Seed Lectin (Pisum sativum L.) E. van Driessche, G. Smets, R. Dejaegere, L. Kanarek

729

The Isolation and Characterization of an Unusual Seed Lectin from a 'Lectin-Free' Cultivar of Phaseolus vulgaris, Pinto III, and its Relationship to Lectins Synthesize'd by Root Cells A. Pusztai, G. Grant, J.C. Stewart

743

Further Studies on the Mitogenic Activity, Sugar Specificity and Affinity Properties of Vicia sativa Lectin A. Falasca, S. Hrelia, C.A. Rossi, F. Licastro, M. Chiricolo, F. Stirpe, C. Franceschi

759

A Simple Method for the Preparation of the two Different Chains of the Misteltoe Lectin 1 H. Franz, P. Ziska, A. Kindt

771

Appendix

Discussion M.M. Andersen, J. Hau, T.C. B0g-Hansen

779

XVI A Report from the International Working Party on Standardization of Lectins for Diagnosis. On the Necessity of Standardized Products T.C. B0g-Hansen, J. Breborowicz, H. Franz

791

Erratum to Vol. 2

794

Author Index

795

Subject Index

797

PART I BIOLOGICAL EFFECTS AND BIOLOGICAL FUNCTIONS OF LECTINS

HISTOPATHOLOGICAL CHANGES INDUCED IN MICE BY THE PLANT TOXIN RICIN AND ITS HIGHLY PURIFIED SUBUNITS A-CHAIN AND B-CHAIN Gilbert Richer, Dominique Carriere, Hildur E. Blythman et Hubert Vidal Centre de Recherches Clin Midy, rue du Pr. Blayac, 34082 Montpellier-France

The use of the toxin ricin for cancer therapy (1,2,3,4,) encounters difficulties, because of the extremely high toxicity of this protein. It is well known that ricin is constitued by two subunits, an A-chain which exerts the cytotoxic activity by means of its inhibitory effect on protein synthesis, and a B-chain which has the properties of a lectin which binds to galactose residues present on cell membranes (5). When separated and purified, both subunits are considerably less toxic than whole ricin. This is one of the reasons why the A-chain of ricin was chosen as the cytotoxic effector of an immunotoxin, (6,7), where it is coupled to an antibody with antitumoral specificity. It was necessary, in the future prospects of a therapeutical use, to explore the effects of the A-chain in the body and to determine whether the mechanism of action in vivo is similar to these described in vitro by others (8,9). The aim of this work was to complete our knowledge about the effects of ricin in vivo and to report new observations which we made in organs and tissues of. normal animals treated with its purified subunits, A-chain and B-chain. Materials and methods. Ricin was prepared as follows : Castor beans were defatted and ground ; ricin was extracted and then purified by affinity

Lectins - Biology, Biochemistry, Clinical Biochemistry, Vol. II © Walter de Gruyter & Co., Berlin • New York 1982

4 chromatography using stepwise elution with galactose on an agarose column. After the disulphide bridge had been reduced with mercapto-ethanol, the A- and B-subunits were separated by combining ion exchange and affinity chromatography on a DEAE-CL Sepharose 6 B column. The A-chain is then concentrated on a CM-CL Sepharose 6 B column, to approximately 10 mg/ml. The A-chain, which crystallised after the last chromatography, was pure, as shown by electrophoresis, and its isoelectric point 7.6 (10). The B-chain was eluted from DEAE-Sepharose with a buffer of high ionic strength in the presence of galactose, and dialysed and concentrated by ultrafiltration. This protein did not present any inhibitory activity on protein synthesis and was free from detectable traces of ricin. The three proteins thus obtained, ricin, A-chain and B-chain, were injected, separately, in a single dose, via the tail vein, into normal female mice, CD1 (Charles Rivers) weighing approximately 25 g. The LD50, determined in the same mouse strain, at day 7 after one single i.p. injection, was 7.5 yg/kg for ricin, 16 mg/kg for A-chain and 8 mg/kg for B-chain. Due to the different LD50 values, the doses chosen for each protein were proportional to their LD50. Ricin was injected at 10 or 20 yg/kg and the very high dose of 2 mg/kg ; A-chain was injected at doses ranging from 0.8 to 16 mg/kg, and B-chain was injected at 1, 2, 4 and 8 mg/kg. Groups of 3-4 animals/dose were killed under anesthesia (Ketamine i.p. 150 mg/kg) at variable times, between I and 96 hours after injection. Control animals, having received PBS i.v. were sacrified at each time. Tissue fragments from different organes were fixed, embedded

5 and sectioned according to the usual histological technique ; bone-marrow was examined on sternum sections as well as on femoral bone-marrow smears ; liver and small intestine samples were also fixed and prepared for electron microscopic studies.

Lesions produced by intravenous injection of pure ricin The lesions were observed in liver, spleen, thymus, adrenal, heart and bone-marrow, and only with the high dose (2 mg/kg) were lesions also seen in the small intestine : Kidneys, lungs, brain, ovaries and uterine horns were histologically normal.

Sinusoidal cells (Kupffer cells, endothelial cells) from liver appeared hypertrophied as early as the 8th hour after injectioh, and several cells had pycnotic nuclei, sometimes fragmented. The EM study showed that their hyaloplasm was clear, and many organelles were destroyed, with only membrane debris remaining : Platelet clusters were seen occupying the Disse spaces. Hepatocytes, on the other hand, only showed functional alterations : diminution and disappearance of glycogen, increased nucleus size and binucleated cells without degenerating alterations.

After 24 hours, a large proportion of Kupffer and endothelial cells were necrosed, swollen sinusoids were charged with debris ,' acidophilic fragments and fibrinous depots (fig. 1). Disse spaces were occupied by microthrombi which insinuated themselves between hepatocyte microvilli. Hepatocytes from the periportal and mediolobular areas were hypertrophied, the cytoplasm containing vitreous-like granulations, with some of these granules in a state of complete or partial acidophilic homogeneous degeneration ("Councilman bodies"). The EM study showed some hepatocytes whose hyaloplasm was empty and endo-

6

plasmic reticulum was vacuolated, but the most of them showed only tumefied mitochondria and atrophied Golgi apparatus, with rarefaction or disappearance of secretion products.

After 48 hours and 72 hours, necrotic sinusoidal cells were still observed, but enlarged Kupffer cells contained numerous phagolysosomes ; the majority of hepatocytes showed clarified and balloon-like mitochondria.

Fig. 1. Liver - 24 hours after 10 \ig/kg riain i.v. Sinusoid. Endothelial lesions, fibrinous depots in Disse space, x 20.000.

On day 4, regeneration signs appeared in the mice which received the lowest doses ; mitochondria were still moderately hypertrophied, and the Kupffer cells from the sinusoids contained phagocytosed organelle debris.

In summary, hepatic lesions caused by ricin first concerned the sinusoids and the Kupffer-endothelial system ; hepatic necrosis appeared afterwards, but the majority of parenchymal cells presented only reversible alterations of mitochondria and endoplasmic reticulum.

7

Hemolymphoid tissues of animals treated with ricin presented lesions of cytoclasia, indicated by karyorrhexis of the cells in the thymus cortex, of the Malpighi bodies in the spleen, of cortical and paracortical areas in lymph nodes and in the bone marrow. These cytoclasia signs were found in impoverished tissues, more or less numerous according to the toxin dose given.

The white pulp showed lesions 24 hours after an injection of 10 yg/kg, and these lesions increased up to 4 days, together with a decrease of lymphocytes. After administration of 2 mg/kg cytoclasia lesions were abundant as early as 6 hours, and included cells from the red pulp. Thymus showed a complete acute involution 24 hours after an injection of 20 yg/kg and 48 hours after a dose of 10 yg/kg. This involution was still observed in animals killed on days 3 :.and 4. Mice which received the high dose of 2 mg/kg were killed 6 hours later, since their survivaL was compromised beyond the 10 to 12 hours. The bone marrow smears and sections were sparsely populated, with few granulocytes, and non identifiable naked nuclei, nuclei fragments and debris from cell lysis mixed with fibrin.

The cells from the zona fasciculata of the adrenal cortex lost their lipid content, and their cytoplasm became compact and homogenous 24 hours after the injection of 10 y g/kg of ricin. On the 3rd day the zona fasciculata was atrophied and numerous cells were necrosed, and capillary damage with hemorrhagic suffusion were visible. The same lesions appeared after 6 hours with the highest ricin dose.

Myocardial capillaries presented pycnotic and caryorrhectic lesions of the endothelial cells, 24 hours after 10 yg/kg ricin injection. After 48 hours, these lesions were accompanied by lymphocyte and polynucleated neutrophil infiltration, and

8 on the 3rd day, an acute interstitial myocarditis was constituted : myocardial fibers contacting infiltrated capillaries showed degenerative alterations and coagulation necrosis. Examination of digestive tract mucosae did not reveal visible lesions after administration 10 or 20 ug/kg of ricin. On the contrary, mice killed after injection of 2 mg/kg presented alterations of the epithelium of the crypts of Lieberkiihn of the small intestine. At 2 hours, a marked diminution of mitosis could be seen, and at 6 hours, numerous cells presented a pycnonecrotic state. Mitotic counts were performed according to the following simplified protocole. Four crypts from each of 4 sections taken at 1 cm interval from the pylore were analysed ; mitosis and necrosis from 16 crypts per animal were counted and the mean for each experimental group calculated. Control animals presented between 25 and 50 mitoses and less than 5 pycnonecrotic cells. Table

1 - Effects

of a high

dose

of ricin

Untreated mice

on

crypts

Ricin-treated mice 2 hrs

Mitoses Pycnonecroses

(2 mg/kg) 6 hrs

33

5

2

2

3

35

Villous epithelium remanied normal. All lesions observed after i.v. injection of 2 mg/kg of ricin can be summarized as follows : 1. Changes in blood vessel endothelium, evident in liver, myocardium, red pulp of spleen and bone marrow, 2. Adrenal stress and thymic and splenic white pulp involution. Parenchymal degenerative alterations, notably in liver and myocardium, were delayed and consecutive to vascular lesions. The minor lesions in intestinal crypt epithelium, were observed after a very high ricin dose (250 times the LD50) and a very short delay. Intestine of mice treated with lower doses.

9 even if higher than the LD50, did not show noticeable modifications, even after a prolonged time (up to 4 days).

Lesions induced by pure A-chain.

Histological alterations were observed in liver, kidneys, adrenal cortex, thymus and small intestine. Brain, heart, lungs, bone marrow and the genital tract appeared normal.

Hepatic lesions appeared 8 hours after the injection of doses of 4 mg/kg. Hepatocyte lesions were proportional to the quantity of toxin injected. Under the light microscope, altered cells showed partial or total cytoplasm acidophilic coagulation, mostly with the aspect of homogeneous round fragments, similar to Councilman bodies, included in the cytoplasm or free in the sinusoidal spaces. Necrotic hepatocytes were dispersed in the lobes, without apparent organisation. Kupffer cells and sinusoid cells did not show noticeable modifications. By EM, the most frequent lesion was found to be at the level of the rough endoplasmic reticulum cisterns, where the membranes were partially degranulated due to loss of ribosomes. These membranes constituted a mass resembling myelinic structures. The unbound polyribosomes were dispersed and dissociated as free ribosomes.

After 24 hours, the number of necrotic hepatocytes increased, degranulated ergastoplasmic cisterns were grouped giving the aspect of "finger-prints", formed by the assembly of membranes into concentric lamellar bodies (fig. 2). At the interior of these lamellar bodies were seen cytoplasm fragments with mitochondria, lipid vesicles, and more sparsely, cytolysosomes. Golgi apparatus cisterns were often atrophied, and lipoprotein

10 secretions absent. Damaged cells presented lipid vesicles. Kupffer cells appeared loaded with cell debris collected in polymorphic phagolysosomes.

Fig 2. Liver - 48 th after 12 mg/kg A-ehain i.v. Concentric lamellar body in the hepatocyte. x20 000.

After 48 hours, cells presenting acidophilic coagulation had barely disappeared. Under the EM, lipid vesicles increased and some sinusoidal cells were necrotic. On day 3, hepatic periportal cells and their cytoplasm were filled with large granules of vitreous aspect, sometimes microvacuoles. The alterations correspond to a significant tumefaction of mitochondria. On day 4, the liver presented a normal appearance, with a few cytolysosomes formed inside some hepatocytes and the activity of Golgi apparatus and glycogen had reappeared.

To summarize, hepatic lesions provoked by A-chain affected primarily the hepatocytes. The lesions were characterized by the disarray of the ribosomal apparatus, appearance of concentric lamellar bodies formed by altered reticulum, and by cytolysosomes containing cell organelles. On the contrary, Kupffer

11

and endothelial cells showed no degenerating alterations, even if they were loaded with phagolysosomes.

Kidneys of mice killed on days 2 and 3 after injection of 12 mg/kg of A-chain presented acute tubular necrosis, concerning mainly certain proximal tubular segments in the cortex corticis. A few isolated necrotic cells were visible earlier, at 6 hours in mice receiving 16 mg/kg. With lower doses, no notable modification was observed.

The thymus cortex showed cell loss between 6 and 24 hours after the injection of 12 mg/kg, with numerous images of karyorrhexis. On day 2, the thymus presented a complete acute involution, which persisted up to day 4. Lower doses produced only a few karyorrhexes without notable diminution of thymocytes.

The adrenal cortex presented evolutive lesions between 6 hours and 2 days after injection of 12 mg/kg. Cells from zona fasciculata became compact and homogeneous, and some cells from zona reticulata were pycnotic. With lower doses, adrenals showed a normal histology.

Lesions from intestinal epithelium, in Lieberkuhn crypts, were seen at all doses given. An important reduction of mitoses and an increase of pycnonecrotic cells appeared simultaneously. Cellular lesions were characterized by the association of an acidophilic coagulation of the cytoplasm and fragmentation and pycnosis of the nucleus. Altered cells were round and fragmented into acidophilic round bodies containing chromatin debris, frequently having a crescent aspect. Necrosis affected first the cells from the lower and middle third of the crypts ; villous epithelium and Paneth cells remained intact (fig. 3).

12 EM observations showed that the effect of fl-chain was visible one hour after injection of 4 mg/kg. The first signs were a

Fig. 3. Intestinal crypts. Semi-thin sections x 400 -A

- normal aspect

in a control - B - A-chain treated 6 mg/kg, 6 hrs after i.v. injection

deaggregation of free polyribosomes, which are normally very abundant in those cells (fig. 4) and at 2 hours, the presence of nucleous fragmentations in cells exempt of polyribosomes. At the same time, phagosomes bounded by a bilayer membrane and containing altered cells organelles were observed. Cell lesions were visible by light microscopy in the crypts 2 hours after injection of 1.6 mg/kg

(1/10 of the LD50)(fig.5A)

and 6 hours after the injection of 0.8 mg/kg

(1/20 of LD50).

Regardless of the toxin dose, pycnonecrotic lesions were maximal between 4 and 8 hours. At this time, if a dose greater than 2 mg/kg was injected, the high number of necrotic cells observed made it impossible to count

(> 200/16 crypts) (fig.5B).

Ultrastructural alterations were extended to most cells, and the light of the crypts was filled with debris and dead cell fragments.

13

Fig. 4. Cell of a Lieberkhun crypt. A - Control - Normal aspect of polyribosomes. x 70 000. B - 1 hour after 6 mg/kg A-chain i.v. Loss of polyribosomal configuration, disaggregation of ribosomes. x 70 000.

After 24 hours, mitotic images reappeared and some cells in mitosis still contained phagosomes. Regerenating cells were voluminous, with enormous nuclei and nucleolus, and basophilic cytoplasm. After 2 days the number of mitosis was comparable to the controls.

The modification of the chorion were always very minor, limited to a moderate hypercellularity, with eosinophils slightly more elevated than in normal controls.

14

40.

1

I Mitoses

P^xj

Pycnonecroses

100 20 -

60

20

M

P

Ij

0,8

1,6

0,8

1,6

200

m

6

mg/kg

6

rng/ kg

30100

60

5-

20

B

l r

Fig. 5. Mitoses and Pycnonecroses in Crypts-Mice treated, with pure A-ahatn A = 2 hrs after i.v. injection - B = 6 hrs after i.v. injection

In summary, lesions provoked by i.v. injection of pure appeared mainly at the level of liver, kidney and

A-chain

intestinal

epithelial cells. The earliest m o d i f i c a t i o n s w e r e seen in the intestinal crypts rather than in the hepatocytes and later on, with higher doses,

in the kidney tubule cells.

Regenerative

cell lesions w e r e preceded by ultrastructural m o d i f i c a t i o n s

of

15

the ergastoplasm and the polyribosomes.

Lesions induced by injection i.v. of pure B-chain. Lesions appeared in liver, heart, spleen, thymus, adrenals and bone marrow. Hepatic lesions were present 6 hours after injection of 1 mg/ kg of B-chain, in sinusoidal cells. Kupffer cell necrosis and endothelial destruction were more evident following administration of higher doses, and were increased after 24 and 48 hours. On the contrary, hepatocytes showed visible lesions only 48 hours after the injection of the highest dose (8 mg/ kg), and the modifications observed (mitochondrial tumefaction, lipid vesicles) did not induce necrosis.

The first alterations in the heart were visible 24 hours after the injection of 1 mg/kg, at the site of capillary endothelial cells (fig. 6). At 48 hours, dose equal to or higher than 2 mg/kg provoked important and diffused alterations of capillaries and necrosis of numerous myocardial fibers.

The white pulp of the spleen presented caryorrhexis and lymphocyte diminution 24 hours after administration of 1 mg/kg. With 4 and 8 mg/kg, important lesions of the red pulp were seen (necrosis, cytoclasia, fibrinous depots).

Thymus involution was observed from 6 hours onwards ; it was acute at 48 hours, even with 1 mg/kg. The zona fasciculata from adrenals was altered with the same

16

Fig. 6. Myocardium X 200 - A = Aspect normal in a control - B = lesions of capillaries 24 hrs after i.v. 4 mg/kg B-chain.

doses and with the same time schedule. After 48 hours, hemorrhagic necrotic foci affected the inner 1/3 of this area and part of the zona reticulata. In bone marrow, disappearance of polynuclear cells was observed 6 hours after injection of 4 mg/kg. After 24 hours, even with 1 mg/kg, numerous cytoclasia and an important cell loss were evident(fig. 7).

The lesions provoked by the injection of B-chain were identical to those observed after the injection of ricin : blood vessel endothelial lesions and the syndrome of adrenocortical hyperstimulation.

17

Fig. 7. Femoral bone-marrow. Giemsa smear x 1000 A - normal aspect in a control - B - B-chain treated 2 mg/kg i.v. Discussion Lesions provoked by ricin have been studied quite extensively, and all reports mention the necrosing effects on blood vessels and on organ parenchyma tissues

(11), mainly in liver and lymphoid

(12).

Recently, an ultramicroscopio study of liver showed that lesions of sinusoids and Kupffer cells preceded hepatocyte alterations (13). Our results confirm these findings. The experiments performed with the purified subunits permit a better analysis of the physiopathological effects of the whole toxin. The patterns of lesions observed with ricin and its B-chain are identical, with one exception, the absence of coagulation necrosis of hepatocytes after injection of B-chain. The comparative analysis suggests that this necrosis results from the effect of the A-chain on the protein synthesis system. It has been demonstrated, by means of radiolabelled ricin, that

18

50 % of the injected dose is retained in the liver during the first 10 hours (14, 15). However, the characteristics of the hepatic lesions we observed do not correlate with the quantitative information, since other organs, such as lung, where ricin is also present in appreciable quantities, presented no alteration. The particular sensitivity of liver could be due to the high level of synthesis and metabolic activities.

The depletion of the white pulp of the spleen, the thymus involution and the exhaustion of the adrenal cortex appeared earlier with ricin and B-chain than with A-chain. These alterations are not specific ; they have been described already for ricin (16) as a result of the rapid massive mobilization of the adrenocortical steroids (17) and are observed in stress syndromes of the most diverses etiology.

Examination of the bone marrow of a dog treated with a sublethal dose of ricin (18) showed only a transient reduction of cell populations and erythroblasts. In our work with mice,, significant cytoclastic lesions were only obtained with doses very superior to the LD5 0. Nevertheless, the Brchain provoked the same lesions with only 1 mg/kg, which is 8 times less than the LD50, and this suggests that cytoclasia seen in bone marrow are the consequence of a property of the B-chain, injected alone or associated with the A-chain in the whole toxin. Contrary to other groups (19, 2 0) we have not observed significant renal lesions, except for a mild congestion, with ricin, even at very high doses, but under these conditions the life expectancy of mice is of less than 24 hours. With A-chain, however, at dose approaching the LD50, systematic tubular necrosis was produced, albeit rather late (2-4 days after injection). We have not been able to observe any visible brain damage, which suggests that injected proteins did not pass through the hematomeningeal barrier. This observation correlates with the

19 results obtained after intracranial injection of ricin The histopathological observations from mice which

(21).

received

purified A-chain were quite different, in several points,

from

those seen with ricin and B-chain : 1) degenerative hepatocyte lesions appeared earlier and concerned the endoplasmic reticulum and polyribosomes, 2) myocardium and bone marrow remained

normal,

3) the stress syndrome of the adrenal cortex was less marked and appeared much later, 4) the effects on intestinal epithelium are remarkable by their early appearance after doses much lower than the LD50. These lesions appeared with pure A-chain at concentrations and time-schedules at which no other cellular or visceral

lesion

was visible. From the morphological point of view, the pycnonecrosis

(22)

did not differ in aspect from that seen in a few epithelial cells in the crypts, that is, phagocytosis of cell debris by active cells giving images of "apoptosis" in the normal intestine

(23). This phenomenon is the consequence of the physiolo-

gical process which regulates the homeostasis of the cell population of the crypts ; the generation time of cells is very short and the mitotic activity very high, in this epithelium in constant renewal. In these cells, 80 % of which are in S phase A-chain induced the appearance very early

(24, 25), the

(1 hour) of polyri-

bosome dissociation and degranulation of the ergastoplasm.

In

a few hours, a great number of pycnonecrosis were visible in direct relationship with the toxin dose. Regeneration is very rapid, which suggests that the pool of clonogenic cells remained intact and could reconstitute the epithelium in 2 to 3 days.

It is well known that morphologically identical lesions are provoked by ionizing radiation as well as by a great number of cytostatic and antimitotic substances. Protein synthesis

inhi-

20

bitors, such as puromycin (2 6) induce the same effects, or, when injected in low doses, like cycloheximide, can inhibit the radiomimetic activity of antimetabolites or alkylating agents on crypt cells (27, 28). A deficit of "DNA-packaging" proteins could be the cause for chromosomic lesions induced in cells on S phase by inhibitors of protein synthesis (29). It is also known that ricin has an antitumoral activity (1, 2, 3, 4), therefore, the analogies observed between the effects of A-chain and anti-tumoral substances on the epithelium of intestinal crypts suggest that this activity of the toxin results from a quality proper to the A-chain.

Conclusions It is evident that the association of the two subunits, in ricin, considerably amplifies the pathogenic activity, seeing that the whole toxin is 2 000 and 1 000 times more toxic than A-chain and B-chain respectively. However one can find, qualitatively, in the effects of each subunit injected independently, all the elements of the lesions induced by ricin. The mecanism of action of both chains, which has been elucidated by in vitro studies (5, 8, 9, 30), explains the nature of the induced lesions. The B-chain exerts a destructive effect on endothelia and blood cells ; this localization can be related to specific lectin receptors, as Van der Valk and Hageman have shown in this volume, more particularly in myocardial endothelia and sinusoids of the liver. In addition, some pathological events can modify the lectin binding by cells ; Adebiyi et al., in this meeting, have shown that the agglutination of mouse erythrocytes by Lima lectin was enhanced when the cells were malaria infected, whereas the agglutination by lectin from Ricinus was not. However some cells which react in vitro with lec-

21

tins have no detectable lesion in vivo, as lung alveolus, for example. Most likely, the binding alone is not enough, and other factors, probably linked to the physiology of cells and tissues, participate in the cytotoxic effects. These observations suggest that it will be useful, in the future prospect of clinical diagnosis or therapeutical use, to compare the results of in vitro experiments with in vivo effects, for all available lectins, in normal animals and in experimental diseases . The A-chain has no lectin property, its cytotoxic activity appears to be linked directly to its effects on the ribosomal system. In immunotoxin molecules, whose specific binding belongs to the antibody, the A-chain of ricin is the effector. It was chosen on account of its low toxicity, but our findings have shown that, at low doses and short times, its cytotoxic effects appear highly important for a cell population with a high mitotic index, as intestinal crypt cells. This evidence suggests that it could be particularly effective in malignant cells, which will be the target for immunotoxins.

References 1. 2. 3. 4. 5. 6. 7. 8.

Lin, J.T., Tserng, K.Y., Chen, C.C., Lin, L.T., Tung, T. C. : Nature 2j27, 292-293 (1970) . Fodstad, 0., Olsnes, S., Pihl, A. : Cancer Research 37, 4559-4567 (1977). Fodstad, 0., Pihl, A. : Int. J. Cancer 22, 558-563 (1978). Fodstad, 0., Pihl, A. : Cancer Research 40, 3735-3739 (1980). Funatsu, M., Funatsu, G., Ishiguro, M., Nanuo, S. : Jap. J. Med. Sci. Biol. 2Z_, 264 (1970) . Blythman, H.E., Casellas, P. et al. : Nature 290, 145146 (1981) . Jansen, F.K., Blythman, H.E. et al. : Immunology Letters 2, 97-102 (1980) . Olsnes, S., Refnes, K., Pihl, A. : Nature 249, 627-631 (1974) .

9. 10.

Benson, S., Olsnes, S., Pihl, A. : Eur. J. Biochem. 59, 573-580 (1975). Vidal, H., Gros, P., Hennequin, J.R., Jansen, F.K., Paolucci, F. : Proc. 4th Intern. Sympos. Affinity chromatography and related techniques. Nijmegen 1981 Elsevier.

11. 12.

Mosinger, M. : C.R. Soc. Biol. 144, 412-415 (1950). Waller, G.R., Ebner, K.E., Scroggs, R.A. , Das Gupta, B.R., Corcoran, J.B. : Proc. Soc. Exp. Biol. Med. 121, 685-691 (1966) .

13.

Derenzini, M., Bonetti, E., Marinözzi, V. , Stirpe, F. : Virchows Arch. B Cell Path. 20, 15-28 (1976) . Fodstad, 0., Olsnes, S., Pihl, A. : Br. J. Cancer 34, 418-425 (1976).

14. 15.

Miguel, C., Sone, H., Kojima, M., Funatsu, G. : Int. J. Appi. Rad. Isotopes 30, 574-575 (1979).

16.

Mosinger, M. : C.R. Soc. Biol. 145, 738-740 (1951).

17. 18.

Balint, G.A. : Toxicology 1, 329-336 (1973). Fodstad, 0., Johannessen, J.V., Schjerven, L., Pihl, A. : J. Toxicol. Environm. Health 5^ 1073-1084 (1979). Dirheimer, G., Haas, F., Metais, P. : C.R. Soc. Biol. 160, 2458-2461 (1966).

19. 20. 21. 22.

Lugnier, A.A.J., Creppy, E.E., Dirheimer, G. : Path. Biol. 28, 127-139 (1980). Strocchi, P., Novello, F., Montanaro, N., Stirpe, F. : Neurochem. Research 259-268 (1979). Dustin, P. : Pathologie de la croissance mitotique. Leçons d'Anatomie Pathologique Générale - 2nd édition Maloine-Paris.

23.

Kerr, J.F.R., Wyllie, A.H., Currie, A.R. : Br. J. Cancer 26, 239-257 (1972).

24.

Bertalanffy, F.D. : Acta Anat. 40, 130-148 (1960).

25.

Lesher, S., Walburg, H.E., Sacher, G.A. : Nature 202, 884-886 (1964).

26.

Estensen, R.D., Baserga, R. : J. Cell Biol. ^0, 13-22 (1966) . Verbin, R.S., Färber, E. : J. Cell Biol. 35, 649-658 (1967) .

27. 28.

Lieberman, M.W., Verbin, R.S. et al : Cancer Research 30, 942-951 (1970).

29.

Woodcock, D.M., Adams, J.K., Cooper, I.A. : Eur. J. Cancer r7, 173-177 (1981).

30.

Sperti, S., Montanaro, N:, Mattioli, A., Testoni, G., Stirpe, F. : Biochem. J. 156, 7-13 (1976) .

SUPPRESSION OF EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS IN GUINEA PIGS AND RATS BY CONCANAVALIN A

Ryoichi Mori, Yasuo Kuroda, Tomonobu Aoki, Akira Takenaka, Tatsufumi Nakamura and Tadashi Namikawa Department of Microbiology, School of Medicine, Kyushu University, Fukuoka, 812 Japan

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of central nervous system produced in genetically susceptible animals by injection of central nervous tissue or myelin basic protein in complete Freund's adjuvant. The predominant evidence suggests that the disease is mediated by T lymphocytes sensitized to myelin basic protein. In our previous papers, we reported that pretreatment of guinea pigs with bacterial lipopolysaccharide, a B cell stimulator, three days prior to the sensitization with bovine spinal cord in complete Freund's adjuvant at the same sites resulted in marked attenuation of both clinical and histologic EAE with the stimulated anti-myelin basic protein antibody production (1, 2). In the present experiments, we have investigated the effect of Con A, a polyclonal T cell mitogen, on EAE. The aim of the present experiments is to clarify the role of T cells in the pathogenesis of EAE.

Materials and Methods Animals and Antigens. Female Hartley guinea pigs and inbred Lewis rats were used. Bovine spinal cord (for guinea pigs) or guinea pig central nervous tissue (for rats) was used as an antigen for induction of EAE. Bovine myelin basic protein was used as an antigen for antibody assay, skin tests and lymphocyte proliferative responses in guinea pigs. The preparation

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 . II © W a l t e r d e G r u y t e r & C o . , B e r l i n • N e w Y o r k 1982

24 of bovine spinal cord and bovine myelin basic protein, and methods of serum anti-basic protein antibody titer assays, skin tests and lymphocyte proliferative responses were described in detail in previous reports (1, 3). Lewis rats were used only in the experiments of adoptive transfer of unresponsiveness to EAE. General Experimental Schedule. Animals were separated into control and experimental group. Animals from both groups were sensitized with bovine spinal cord (20 mg/guinea pig) in complete Freund's adjuvant. Con A was injected into experimental animals in a various time, dose and route schedule as described in the Results. For the evaluation of effects of Con A on EAE of guinea pigs, the severity of EAE was graded on a scale of 0 to 3. Clinically EAE was graded as follows: 0, no signs; 1, paresis of hind limbs; 2, paralysis and/or incontinence; 3, moribund state or death. For the studies of antibody titers, lymphocyte subpopulations and lymphocyte proliferative responses, some of both groups were sacrificed simultaneously.

Results 1) Suppression of EAE in guinea pigs by Con A First, generalized and local reactions of Hartley guinea pigs to Con A were studied. The largest dose of Con A used in the present experiment was 5 mg per animal and this was well tolerated by the guinea pigs when injected intraperitoneally or subcutaneously. In the case of footpad injection of 5 mg of Con A, a local swelling appeared on the second day, reached a maximum on the third or fourth day but then subsided rapidly. No obvious generalized reactions were apparent and no mortality was noted by Con A treatment. The effect of timing, route and dose of Con A treatment was studied.

All but two control guinea pigs receiving bovine

spinal cord in complete Freund's adjuvant alone exhibited severe clinical manifestation of EAE and all animals examined

25 histologically had moderate to severe histopathologic changes of EAE. Guinea pigs in 6 groups were injected into hind footpads with 5 mg of Con A prior to (day -5 or -3), at the time of (day 0) or after (day +3, +5, or days +6 and +8) sensitization with bovine spinal cord in complete Freund's adjuvant. In addition, one group received an intraperitoneal injection of 5 mg of Con A on day 0. As shown in Table I, EAE was significantly suppressed when Con A was injected in the hind footpads (the sensitizing injection sites) on day -3, 0, or +3. The EAE-suppressive activity of Con A was shown by significant reductions in incidence and severity of EAE, a delayed onset of EAE and low mortality. Other times for footpad injections and an intraperitoneal injection of Con A had no obvious effect on the course and outcome of EAE. When a range of doses from 0.2 to 5 mg of Con A was examined, some dose Table I. Effect of route and timing of Con A treatment on EAE Treatment materials, dose and site3 none Con A 5mg fp Con A 5mg f p

Time

No of

No of

Mean

Clinical

in days

guinea pigs

sick animals'3

incubation time(days)

score of EAE d

10.9

2.7 2.3

-5

Con A 5mg fp

-3 0

Con A 5mg fp Con A 5mg f p

+3 +5

Con A lOmg fp Con A 5mg ip a

c

40 15 14 37 14

+6 ,+8

6 5

0

5

38 (33) 12 (11) 10 f (8) 15 f (8) 7 f (6) 5 (5) 4 (3) 5 (5)

11.3 13.3e 14. 9 e 13. 0 e

2.0 l.l e 1. 4 e

11.0

2.5

12.1 11.4

2.0 3.0

fp, hind footpads; ip, intraperitoneal!. Numbers in parentheses indicate animals died in 2 5 days. Mean days of onset in EAE-positive animals.

Clinical score of EAE was graded from 0 to 3 (see text). Significantly different from controls (pH o 37

37

Fig. 2 Reexpression of lectin activity on hepatocytes after trypsin treatment. C = Control, freshly isolated cells before trypsin treatment. T = Hepatocytes were incubated with trypsin (2 mg/ml) for 5 min at 0 C. S = Sham tratment, hepatocytes were incubated with buffer instead of trypsin. T „ = After trypsin treatment and washing hepatocytes were incubated at 3 7°C for 5 min. S^-y = Sham treatment,Qhepatocytes treated as in S Q , and afterwards incubated at 37 C for 15 min. The percentage of hepatocytes showing lectin activity was determined in the erythrocyte binding assay.

pearance in the Kupffer cell membrane is blocked.

D iscussion The experiments reported here describe the rapid reappearance of lectin activity on liver cells after the destruction of mem-

62

( rain )

Fig. 3 Galactose-specific lectin activity on the surface of rat Kupffer cells before and after tryosin treatment. Cells were treated with 2 mg/ml trypsin at 0 C, washed three times and tested for residual lectin activity in the erythrocyte binding assay. X = trypsin treatment 0 = sham treatment The conditions are the same as in Fig. 1.

brane-bound receptors by trypsin. De novo biosynthesis of receptors does not appear to be involved. This finding is valid for the galactose-specific lectin on rat hepatocytes as well as for that on rat Kupffer cells. In the latter case it was observed that Kupffer cells can enter a physiological state (after phagocytosis of carbon particles) where receptor replacement in the cell membrane does not take place. New membrane receptors probably stem for an intracellular pool although the existence of a trypsin resistent and inactive form of the receptor on the plasma membrane cannot be excluded.

63 100 •p

•>H -H 80 •P U ra c •P £8 U V £ •P 40 4)

O M 20 4) vuJLga/uj, L. lectin (12) appear when the seed reachs 6 mm in diameter. Quantitative radial immunodiffusion (RID) and Immunoelectrophoresis with specific antibodies against both the lectin and total soluble protein of seeds were subsequently applied to various Leguminous plants including lentil (13), garden pea (P^csum icutivum

L.)(14) and VIcJjx icutiva. L. (15). The-

se techniques showed that lectin appear lately during the ripening stage of the seeds after which their accumulation proceeds at the same rate as the total soluble protein to reach a maximum level in the dry seed. Similar results have been obtained with the three Lath.yn.iju> species studied. Recently, Talbot & Etzler (16) have used a more sophisticated and sensitive radioimmunoassay (RIA) to measure the development of lectin turation of VoLLckoi

during the ma-

b-cfitosuib L. seeds. Lectin was not detected during the

first 26 days after flowering and appeared at day 27 in large quantities, reaching a maximum level one day later. However, using a hemagglutination assay, Marty (17) reported a decreasing lectin content during the maturation of UZtx paJiv^loiuA

PouJiA. seeds,

which is in contrast with the above results. Lectin during seed germination : In most of Leguminous plants lectins are present in all parts of the seed: cotyledons, embryo axis and seed coat. Except for soybean (Glycine,

max IL. j

MQAA.) in which the seed coat contains appreciable levels of lectin (18), the highest amounts of these proteins are found in the cotyledons and embryo axis while there is negligible quantities of lectin in the seed coat (8,10,19,21,27). However, recent results obtained with a enzyme-linked immunosorbent assay (ELISA) indicate that soybean seed coat contains a very low amount of lectin (34). In view of this distribution within the seed, the presence of lectins was followed during the gemination process and the early stages of development

145 both in the cotyledons and in the young plant issued from the embryo axis. Previous studies using hemagglutination techniques as in the case of seed maturation, were carried out on the seeds of various Phcueolui P. vulgaAlA

species :

L. (10, 22),P. ¿¿m&yu>.Li MacFad. (23) and P. lunatiu,

L. (9).

They showed that the lectin content of cotyledons decreases gradually until they are shed while other parts of the young plant including leaves, stems and roots ehibit a small decreasing activity during the first two weeks following the beginning of seed germination. Immunochemical techniques gave similar results with Phaseolui (12), lentil (11,19), garden pea (21), I/¿cla iativa and Votichoi

vuZgcuuA L.

L. (15), soybean (18)

blfaloKuA L. (16). The three Lath.yn.iM species give identical

results and shows, in addition, that as in the case of seed maturation, the lectin behaviour both in cotyledons and in the young plant do not differ markedly from that of the soluble protein bulk of the seed. Lectin in the vegetative part of plants : Lectins have been detected in other parts than seeds or organs of the legumes at their early developmental stages, and notably in leaves (35,36,37, 38,39), stems (7,17), roots (7,17,40), flowers (35), pods (16,39), bark (41) and cell sap (42). All the above results have been obtained by agglutination of erythrocytes and should be interpretated with caution since tanins and phenolic compounds present in plant extracts can promote non specific agglutination and give rise to misleading results. However, using antibodies against seed lectin Talbot & Etzler (16) have recently found a cross-reacting substance in leaves and stems of

Votichoi

bifalohiu, L.. In addition, they have shown that this cross-reacting substance, which is present in the organs of the plant at all stages of its development, increases markedly when buds appear. More recently, Gatehouse & Boulter (43) have isolated a lectin from the roots of garden pea seedlings (PLium ¿aJu.vu.rn L.) and have shown that this lectin is located on the surface of root hairs and in the root cortical cells. However, the reported properties of this lectin seems to be very similar to that of the pea seed isolectin II previously isolated from dry seeds (44) and present in the embryo axis and, subsequently, in the young plant during the first week following the beginning of seed germination(21).

146 Since the isolectin I disappears at day 4 following the onset of germination, the pea root lectin identified by these authors coul be in fact the isolectin II present in the dry seed (27). The above reported results are concerned with lectin occurrence during the life cycle of only a few species of three tribes of the Leguminosae which are, according to the Hutchinson cluding the genera Phiuzolai Glycine.)

classification (45): the Phaseoleae (in-

and VoLLchoi),

the Glycineae (with the genus

and the Vicieae (including the genera Lathy/uii,

Lzni,

Pi&um and

VicMi). They indicate that seed lectins accumulate lately during the maturation process and disappear quickly during seed germination, both in the cotyledons and in the young plant issued from the embryo axis. In all cases the total soluble proteins, especially the storage proteins, have a similar behaviour during these developmental stages. Altogether these findings raise many additional questions, namely : 1° Are the kinetics of appearance and disappearance of seed lectins identical in all the tribes of the Leguminoseae ? The results reported by Marty (17) on the genus L\tzx

from the tribe of Cytiseae are in contrast with

those obtained with the other above mentioned tribes. 2° When two or more isolectins are present in the seed, do the isolectins appear and disappear at the same rate ? Conflicting results have been obtained on this point. In garden pea the PS I isolectin disappear more quickly than the PS II isolectin during the seed germination process both in the cotyledons and in the young plant (21), whereas in LcutkysiuA ochuu,

L.

the two isolectins seems to appear and disappear at the same rate (see Fig. 3,5 and 6). 3° Does lectin synthesis occur dz novo

in the seed itself during seed ma-

turation or is it due to the conversion of a precursor synthesized in another part of the plant, the leaves for instance, as suggested by Susplugas & Coulet (22). In garden pea ^C-labeled amino-acids are incorporated into lectin both in the cotyledons and embryo axis during seed maturation suggesting do. novo

synthesis of lectin in the seed itself (14,27). In contrast

Talbot & Etzler (16) have reported the occurrence of a protein cross-reacting with antibodies against the seed lectin, in the leaves and stems of VoLLckoi

bi^loALU,

L.. The fact that seeds evolve high amounts of lectin in

147 a very short time during maturation, suggests that such a protein could be a lectin precursor transported from the leaves to the seeds. 4° Do the lectins present in the the tissues of the seedlings are the lectins initially present in the embryo axis, derive from the cotyledons or are they synthesized within each tissue ? In lentil (20)> garden pea (27) and VicMx. icutiva

L.

(15) seeds it seems that lectins occurring in the young

plant are essentially present initially in the embryo axis. However, PontLezica & al. (46) have reported on the synthesis of a lectin in the pea epicotyl. In soybean (18) the lectin content of the embryo axis cannot account for the much higher lectin content of the different tissues of the young seedling, suggesting a transport of lectin from cotyledons or dz novo synthesis of these molecules in the tissues (34). These findings raise the interesting question of the regulation of genes controlling lectin synthesis (47)» i n the various parts of the plant during its development. In addition, it should be mentioned that only little research has been carried out on the cellular and subcellular location of lectins in seeds and in the different tissues of the plant (for review see 48 ). Recent results clearly indicate that lectins are essentially located in protein bodies but additional work is needed to establish this point.(49,50,51,52). Another very interesting question is the discovery of lectins which seems to be cell wall components (53,54) or membrane components of plant tissues including membranes of the endoplasmic reticulum, of the Golgi apparatus, of the mitochondria and in plasmalemna (55,56,57,58). These results should be interpretated with caution since they may be an artifact during the isolation of membranes (59). However, recent results obtained by Bowles & al. (60) indicate the occurrence of two classes of lectins in plants. One class are freely-soluble or loosely-attached to membranes, exhibiting the same properties as seed lectins. The other class contains lectins which are membrane components, the properties of which seem to differ markedly from the seed lectins. Thus, it appears that lectins are not merely seed components since these proteins may be synthesized in all parts of plants during the life cycle. At the present time, additional data is needed to establish the'relationships between them. In particular further purification of the membrane-

148 associateci lectins is necessary for a proper conparison with the lectins present in seeds (61,62). Finaly, one may conclude that further research with more specific and sensitive techniques is necessary to have a clear picture of lectin occurrence and location in the various plant parts during its life cycle. In this respect, the development of highly specific immunochemical techniques such as RIA and ELISA is very promissing. These techniques, which allow the acurate detection of lectins at concentrations as low as 100 ng/g fresh wt of plant tissue, should be a very valuable tool in the investigation of lectins in tissues. Similarly, lectin location could be studied at the cellular and subcellular levels using highly purified antibodies against lectins REFERENCES 1. Boyd, W.C. : Vox Sang. 8, 1-32 (1963). 2. Liener, I.E. : Annu. Rev. Plant Physiol. 11_, 291-319 (1976). 3. Callow, J.A. : Current Advan. Plant Sci. 7, 181-193 (1975). 4. Kauss, H. : Progress in Bot. 3,8, 58-70 (1976). 5. Pusztai, A., Grant, G. and Stewart, J.C. : these proceedings. 6. Eisler, M. and Portheim, L. : Z. ImmunForsch. 47, 59-82 (1926). 7. Krupe, M. and Ensgraber, A. : Planta 50, 371-378 (1957). 8. Renkonen, K.O. : Ann. Med. Exp. Biol. Fen. 38, 26-29 (1960). 9. Martin, F.W., Waszczenko-Zacharczenko, E., Boyd, W.C. and Schertz, K.F. Ann. Bot. 28, 321-324 (1964). 10. Manen, J.F. : Contribution à la caractérisation et à la biologie des vuZgtwu L. var. Contender, Thèse lectines dans la graine de Phaizolui Doct. ès-Sc., n° 1866, Genève (1978). 11. Howard, I.K., Sage, H.J. and Horton, C.B. : Arch. Biochem. Biophys. 149, 323-326 (1972). 12. Mialonier, G., Privât, J.P., Monsigny, M., Kahlem, G. and Durand, R. : Physiol, vég. JJ_, 519-537 (1973). 13. Rougé, P. and Chatelain, C. : Bull. Soc. bot. Fr. 14. Rougé, P. : C. R. Acad. Se. Paris

421-424 (1978).

282, 621-623 (1976).

15. Gracis, J.P. and Rougé, P. : Bull. Soc. bot. Fr. J_24, 301-306 (1977). 16. Talbot, C.F. and Etzler, M.E. : Plant Physiol. 61_, 847-850 (1978). 17. Marty, B. : Recherches sur les phytoagglutinines d'Ulex VOUMI.

, Thèse Doct. Pharm., n° 102, Montpellier (1974).

paAv^^loAiió

149 18. Pueppke, S.G., Bauer, W.D., Keegstra, K. and Ferguson, A.L. : Plant Physiol. 61_, 779-784 (1978). 19. Rouge, P. : C. R. Acad. Sc. Paris 278, 449-452 (1974). 20. Rougé, P. : C. R. Acad. Sc. Paris 278, 3083-3086 (1974). 21. Rougé, P. : C. R. Acad. Sc. Paris 280, 2105-2108 (1975). 22. Susplugas, J. and Coulet, M. : Tr. Soc. Pharm. Nfontpellier U , 81-85 (1954). 23. Krupe, M. : Blutgruppenspezifische pflanzliche Eiweisskörper (Phytagglutinine), Enke, F. ed., Verlag, Stuttgart (1956). 24. Toms, G.C. and Western, A. : in Chemo taxonomy of the Leguminosae, Harborne, J.B., Boulter, D. and Turner, B.L. ed., Academic Press, Londres, 367-462 (1971). 25. Sandhu, R.S. and Reen, R.S. : these proceedings. 26. Chouard, P. : in L'Aquiculture, Homes, M.V. ed., Bruxelles, 78 (1953). 27. Rougé, P. : Biologie des hémagglutinines chez le Pois (PiAum Thèse Doct. ès-Sc., n° 746, Toulouse (1977).

iativumL.)

28. Scheidegger, J.J. : Int. Arch. Allergy ]_, 103-110 (1955). 29. Uriel, J. : in Immuno-electrophoretic analysis, Grabar, P. and Burtin, P. ed., Elsevier Publ. Co., Amsterdam, 30-57 (1964). 30. Mancini, G., Vaerman, J.P., Carbonara, A.O. and Heremans, J.F. : in Protides Biological Fluids, Peeters, H. ed., Pergamon Press, Oxford, 370-373 (1964). 31. Goa, J. : Scand. J. Clin. Lab. Invest. 5, 218-222 (1953). 32. Davis, B.J. : Ann. N. Y. Acad. Sc. _121_> 404-427 (1964). 33. Chrambach, A., Reisfeld, R.A., Wyckoff, M. and Zaccari, J. : Analyt. Biochem. 20, 1-15 (1967). 34. Causse, H., Lemoine, A. and Rougé, P. : International Symposium on Seed Proteins, Phytochemical Society of Europe, Versailles, September 22-24, France (1981). 35. Cazal, P. and Lalaurie, M. : Acta haemat. 8, 73-80 (1952). 36. Coulet, M. : Contribution à l'étude des hémo-agglutinines végétales, essentiellement chez PhcuzoluA \>UJLQCVUJ> L., Thèse Doct. Pharm. Montpellier (1954). 37. Tétry, A. : C. R. Acad. Se. Paris 240, 434-436 (1955). 38. Tiggelman-van Krugten, V.A.H., Ostendorf-Doyer, C.M. and Collier, W.A. : Ant. Leeu. J. Micro. Serol. 22, 289-303 (1956). 39. Rougé, P. : Ann. Pharm. Fr. 35, 287-294 (1977). 40. Herzog, P. : Ceskosl. Biol. 7, 444-445 (1958). 41. Horejsi, V., Haskovec, C. and Kocourek, J. : Biochim. Biophys. Acta 532, 98-104 (1978).

150 42. Kauss, H. and Ziegler, H. : Planta 121, 197-200 (1974). 43. Gatehouse, J.A. and Boulter, D. : Physiol. Plant. 49, 437-442 (1980). 44. Entlicher, G., Kostir, J.V. and Kocourek, J. : Biochim. Biophys. Acta 221 , 272-281 (1970). 45. Hutchinson, J. : The Genera of Flowering Plants, Clarendon Press, Oxford, vol. 1 (1964). 46. Pont-Lezica, R., Romero, P.A. and Hopp, H.E. : Planta 140, 177-183 (1978). 47. Shannon, L.M., Hankins, C.N. and Strosberg, A.D. : in Lectins, Biology, Biochemistry, Clinical Biochemistry, BjzSg-Hansen, T.C. ed., de Gruyter, Berlin, vol. 1, 81-91 (1981). 48. Rouge, P. and Chatelain, C. : in Lectins, Biology, Biochemistry, Clinical Biochemistry, B^g-Hansen, T.C. ed., de Gruyter, Berlin, vol. 1, 145-150 (1981). 49. Horisberger, M. and Vonlanthen, M. : Histochem. 65, 181-186 (1980). 50. Van Driessche, E., Smets, G., Dejaegere, R., Kanarek, L. : these proceedings . 51. Manteuffel, R., Nieden, U.Z. and Weber, E. : International Symposium on Seed Proteins, Phytochemical Society of Europe, Versailles, September 22-24, France (1981). 52. Manen, J.F. : International Symposium on Seed Proteins, Phytochemical Society of Europe, Versailles, September 22-24, France (1981). 53. Kauss, H. and Bowles, D.J. : Planta 130, 169-174 (1976). 54. Haass, D., Frey, R., Thiesen, M. and Kauss, H. : Planta 151, 490-496 (1981). 55. Bowles, D.J. and Kauss, H. : Plant Sei. Lett. 4, 411-418 (1975). 56. Bowles, D.J. and Kauss, H. : Biochim. Biophys. Acta 443, 360-374 (1976). 57. Bowles, D.J., Schnarrenberger, C. and Kauss, H. : Biochem. J. 160, 375-382 (1976). 58. Yoshida, K. : Plant Cell. Physiol. _19, 1301-1305 (1978). 59. Köhle, H. and Kauss, H. : Biochem. J. J84, 721-723 (1979). 60. Bowles, D.J., Lis, H. and Sharon, N. : Planta 145, 193-198 (1979). 61. Rüdiger, H., Gansera, R., Gebauer, G. and Schurz, H. : in Lectins, Biology, Biochemistry, Clinical Biochemistry, Bjiig-Hansen T.C. ed., de Gruyter, Berlin, vol. 1, 135-144 (1981). 62. Guldager, P. : in Lectins, Biology, Biochemistry, Clinical Biochemistry, B0g-Hansen, T.C. ed., de Gruyter, Berlin, vol. 1, 151-156 (1981).

PART C E L L RECEPTORS AND C E L L

II

REACTIONS

MONOSACCHARIDES AND TAMM-HORSFALL GLYCOPEPTIDE INHIBIT ALLOGENEIC ANTIGEN-INDUCED LYMPHOCYTE BLASTOGENESIS IN ONE-WAY MIXED LYMPHOCYTE REACTION

Claudio Franceschi Institute of General Pathology, University of Padova 35100 Padova, Italy Federico Licastro, Mariella Chiricolo, Franca Serafini-Cessi Institute of General Pathology, University of Bologna 40126 Bologna, Italy Pierluigi Tabacchi Laboratory of Clinical Analysis "M. Malpighi" Hospital 40100 Bologna, Italy

Many effects of monosaccharides on immune responses have been described.They block the expression of spontaneous monocyte mediated cytotoxicity and early events necessary for the expression of antigen specific proliferation (1) and inhibit E-rosette formation (2) . Furthermore monosaccharides interact with chemotactic factors (3) and they are capable of inhibiting lymphokine activity (4). The data we present show that monosaccharides and more complex oligosaccharide structures affect another functional property of T lymphocytes,i.e. their proliferative activity when exposed to allogeneic antigens in one-way mixed lymphocyte reaction (MLR). Controversial data exist in the literature concerning the chemical nature of the antigens which stimulate MLR in the mouse. According to some authors the determinants of la (I-region associated) antigenic system are protein in nature, and consequently a primary gene product (5). Recently, a second set of I-region controlled antigens has been described;

Lectins - B i o l o g y , Biochemistry, C l i n i c a l Biochemistry, V o l . II © W a l t e r d e Gruyter &. Co., Berlin • N e w Y o r k 1982

154 this seems to constitute a totally separate antigen class (6). The data suggest that the oligosaccharide portion of these la antigens determines their specificity (7). Ia-like antigens are present also on human lymphocytes, under the genetic control of the D or the DR region of the human Major Histocompatibility Complex (HLA). They are involved in cell recognition and anti-la-like sera are able to inhibit allogeneic MLR in humans (8). These antigens seems to be protein in nature and they probably represent the human counterpart of the first class of murine la antigens. It is not known if a carbohydrate defined class of Ia-like antigens exist in humans. We descibe here hapten inhibition studies on human allogeneic MLR by using a variety of sugars and a glycopeptide (m.w.4800) purified from human Tanun-Horsfall glycoprotein (TH)(9, 10). The data obtained represent an indirect evidence in favour of the hypothesis that carbohydrate antigens may play a role in this immune response.

Materials and methods Allogeneic MLR employing peripheral blood lymphocytes from human volunteers were established as previously described (11) with slight modifications. Briefly, MLR were established in quadruplicate in 3040 microtest plates (Falcon Plastic, Los Angeles, CA) using RPMI medium containing 2 mM glutamine, 100 U/ml of penicillin, 100 pg/ml streptomycin, and 10 % inctivated pooled human AB serum. 50 ul of responding cells (5 x 4 10 cells) were added to 50 pi of irradiated stimulator cells 4 (5 x 10 cells). In experimental control 50 pi of medium alone was added. Cell irradiation with 3600 rads was performed using a Co source. Allogeneic MLR was incubated for 6 days. Twentyfour hours 3 before the termination of the incubation period, 0.5 yiCi of H-TdR (The Radiochemical Center, Amersham, spec, act. 5 Ci/mmol) was added to each culture well. Cells were har3 vested and H-TdR incorporation was measured as described (12).

155 PHA stimulation was performed as previously described (11). More than 20 sugars (see reference 12 for details) and TH glycopeptide isolated and characterized as previously described (9) were used to inhibit allogeneic MLR, at the final concentration of 50 mM for the sugars and of 2 x 10~7 - 4 x 10 - 5 M for TH glycopeptide. Sugars and TH glycopeptide were usually added at the initiation of the cultures, dissolved in complete medium, in a volume of 2 0 jil.

Results Table 1 shows the control values of eleven allogeneic MLR experiments in which hapten inhibition studies were performed. In many cases we deliberately used the same combinations of responder-stimulator cells in order to measure the experimental variability of our cultures.

Table 1. Control values of allogeneic MLR. a Exp. n. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11 .

Responder cells

Stimulator cells

Responder cells plus Stimulator cells

C.F C.F F.N F.N G.G D.P P.T C.F S.R L.C P.G

F.N. F.N. C.F. C.F. D.P. G.G. C.F. P.T. L.R. P.G. L.S.

26000 33400 7760 1 4000 9640 15100 1 1 600 48400 3 5 300 35100 25500

Results are expressed as mean cpm of quadruplicated determinations rounded off to three significant figures. C.F.,F.N. ^tc. refer to single volunteers who gave their blood. Mean H-TdR incorporation of responder and stimulator cells alone was 1300 + 350 and 171 + 3 0 respectively. Six out of the eight donors were HLA typed.

156 Representative experiments of MLR inhibition by sugars are reported in Table 2.

Table 2. Representative experiments showing inhibition of allogeneic MLR by different sugars. Sugars 1 L-arabinose Cellobiose

Evaluation3

Exp. n.

58 b 81

2

3

4

20

29 46

34

113

1 05

+ -

Fructose

91

52

93

65

-

D-fucose

100

41

89

49

+

D-galactose

83

98

67

99

-

N-acetylgalactosamine

60

55

60

57

+

N-acetyl-glucosamine

71

6

62

28

+

D-glucose

62

1 02

62

79

+

Lactose

67

69

66

81

+

Methyl- 80%

Plastic,

Oxnard,

with

fetal

10%

enriched confluent

CA) bovine

atmosphere. monolayers

serum Cell

3x

in 75-mm style

containing at

Dulbecco's 37"C

in

an

suspensions were 2+ 2+ with Ca , Mg free

169 PBS, followed

by 1 ml

of 0.25%

trypsin at 21"C

trypsin solution was then decanted for

10 min.

inactivate gentle

A

the

volume

of % ml

remaining

shaking

of

and the flask

was

and

flask

to

to

suspend

dislodge

the

the

The

incubated

of the growth medium was

trypsin

the

for 2 min.

at

37'C

then added

cells

cell

populations per ml were counted on a hemocytometer.

excess

by

to

repeated

layer.

Cell

The monolayers were

also dissociated by treatment with 0.02% EDTA; this resulting

suspension

was referred to as the 'untrypsinized' cells.

3)

Formalin fixation of cells.

were

centrifuged

serum

was

at

decanted.

low A

speed, volume

3T3 cell

suspensions

and

growth

of

the ml

of the

added and the small pellet was dispersed

prepared

as

above

medium containing 2+ 2+

Ca

, Mg

by the gentle

free

the

PBS

syphoning

was

of the

suspension with a 1-ml plastic pipet. These washings were 2repeated + ? + twice more

and

PBS.

the

cells

were

again

suspended

in 1 ml

of

Ca

Approximately 2 ml of a 10% formalin solution was

to the cell

suspension with continuous

shaking

, Mg

added

gradually

to prevent clustering

the free cells upon sudden contact with the aldehyde solution. suspension

in formalin

was

which it was centrifuged was discarded. added

and

A volume

the cell

allowed

as 3T3 cell

formalin, resuspended

were in

pellet

after

added.

The

under The

extent

was

again

min

after

solution

dispersed.

The

final

fixing

then was

intervals.

The trypsin treated and untreated 3T3 cells as

were

3x

with

buffer.

, ml

Mg

free

of

PBS

and

approximately

2

x

10^

were transferred to 35 mm x 10 mm style Petri

0.5 ml

conditions

Ca

One-half

of

a 50 to

mixtures were

1,000

agglutination

was

scored

(5).

pg

agitated

as those described

scheme under lOOx magnification thrice.

The cell

30

washed this

Con A - cell

of

for

then

which,

identical

4'C

of

suspensions that were first treated with trypsin, 2+ 2+

cells/ml cell suspensions dishes,

at

of i ml of the 10%3formalin solution was

Agglutination assay.

well

stand

(low speed) and the supernatent fixing

repeated for 1, 6, and 25 hr time 4)

to

. free

Con A/ml on

for

visually

solution was

a metabolic

the

gel

using

sphere the

shaker case.

previous

All assays were conducted at

least

170 5)

Cell

tne

deformability

filtrability

polycarbonate Corp., was of

the

poured Tne

method

memDranes

Pleasanton,

first

evaluation.

3T3

cell the

silicone

(6)

with

deformabi1ity was evaluated

using

8,

10,

25 and

mm 12

diameter

u

by D

Nuclepore

porosities

(Nuclepore

CA,

U.S.A.). Typically, the polycarbonate membrane 2+ 2+ with Ca , Mg free PBS, after which, 4 to 6 ml 5

moistened

into

Cell

suspension plastic

oil

containing

syringe

lubricated

barrel

syringe

about

3

attached

plunger

x

10

to

was

cells/ml

the

filter

positioned

was

holder.

at

a

45"

angle over the upper end of the syringe opening, and oscillated over the opening

in

order

magnitude

in

suspension

co

the

create

syringe

through

a so

slight

intermittent

as

initiate

to

the microporous

memDrane

pressure

the

flow

filter.

of

of

uniform

the

cell

Occasionally,

the

cell suspension in the syringe barrel was mixed by syphoning with a 1-ml plastic

pipet

collected

in

population counted.

to

prevent

a

of

15-ml

cells

the

settling

conical

passing

end

of

the cells.

plastic

through

the

The filtrate

centrifuge

microporous

tube

filter

and was

Cell deformability, as eval uated by filtrability was

was the

again

expressed

in terms of the percentage of cells recovered after filtration. Results

1)

Factors

suspension, The

controlling the

gel

agglutination

agglutinabi1ity

sphere

of

population

gel

G-200

concentration.

The maximum

at

A/ml.

600

ug

completely incubated

Con

The

innibited with

agglutination

882

the ug

was

or

added

to

agglutinated. apparent reversiDly

effect.

The

temperature

to

of

0.1M

A/ml

in

dependent

The

the NaCl

) was

Con

A

observed

gel

G-200 of

results

spheres

1.0M

were

NaSCN,

were

the

identical

in the assay

solution

already

extensively

(++++)

concentrations

dependent.

on

initially

spheres

agglutination

(

++++

assay

spheres/ml.

methyl-a-D-glucopyranoside

presence

inhibited.

In the

x 10 4

5.8 be

When

the

NaSCN was present

However,

spheres.

found

agglutination.

Con

disperse

gel about

extent of agglutination presence

again

regardless of whether

was

of

was

of

gel

Below 4 " C

even

exceeding

G-200 no

was

4M

found

agglutination

had

no

to

be

occurred

171 and the transition temperature was about 15"C for the maintenance reduction

in maximum agglutination.

was between 6.5 to 7.6. to decrease rapidly.

The optimal

pH for

or

agglutination

Outside this range the agglutination was found

With the exception of the Con A inhibitor methyl-a

-D-glucopyranoside, all other experimental conditions which reduced the tendency of gel G-200 to agglutinate in the presence of Con A was due to the dissociation of the agglutination-active tetramer into the inactive dimer.

Tne molecular transition of Con A was observed by means of an

analytical

ultracentrifuge

wnich

snowed

that,

depending

on

the

temperature, pH, or the presence of NaSCN, varying proportions of the 4.15 S dimer species co-exist with the 5.52 S tetramer species.

For

maximum agglutination, at least 80% or more of the 5.52 S tetramer Con A must be present in the solution.

Studies for lectin binding on surface

sites were conducted within a range of 300-fold Con A concentrations. For both gel G-200 and gel reach

a

steady-state

according

G-75 spheres, about 5 hr was required to

binding

condition.

The

results

were

plotted

2.20

x

14

Scatchard (3), to give 2.06 x 10 and ? binding sites/cm for gels G-200 and G-75, respectively. sphere

to

deformabi1ity

measurements

taken

by the

However, gel

capillary

elastometer

showed that the minimum pressure required to draw an individual of

gel

G-200

with

average

diameter

220y

completely

10 1 4

into

sphere a

130u

micropipet was 4x less than that required for a gel G-75 sphere of the same size.

Viscosity measurements of a 75% suspension of gel G-200 and

G-75 spheres in a Ferranti-Shirley rotating disc viscometer (4), showed that at a shear rate of 150/sec, there was a 1.5x difference apparent viscosities. reduction

in

The gel

viscosity

with

in the

G-200 suspension showed a much greater increasing

shear

rate

typical

of

a

suspension of highly deformable particles {7). 2)

Correlation

of

average diameter

3T3

of

cell

3T3 cells

filtrability

and

agglutinability.

The

is 23 * 5 n, and essentially all the

trypsin treated 3T3 cells were able to pass through the 12 y pores in the

polycarbonate

membrane

reduced to one-third

by

deformation.

(i.e. 8 y) of the cell

When

the

diameter,

porosity over

was

one-half

172 of

the

1).

trypsin

treated

cells was

recovered

fixed

cells

passed

passed

through

filtrability

was

extensively former

was

presence

through

the

10

also

trypsin untreated

the y

significant

Dy

200

non-agglutinable. 30

u filter,

comparable

ug

in

in

filtration,

ug

Con

the

the

values

restored

terms

of

the

virtually

1).

This

comparison

obtained

at 21^C. 2

upon warming

x

200

of the

A/ml

of

only

(Table

74

ug

no

cells

reduction

in

the

or

fixed

The later was

1),

whereas

the

A/ml

in

the

untreated

3T3

Con

(1), similar to the gel G-75 under At

5"C, of

the cells

3T3 cells was

For ug

(Table

or the trypsin

percentage,

treated

even

at

at

fixed

conditions.

for the trypsin

agglutination

the

However,

glycogen/ml,

experimental

expressed

while

(Table

filtration

less than 10% of the

3T3 cells to the trypsin treated cells.

agglutinated

of

12

filter

cells became extensively agglutinated

as

following

After brief exposure to the formalin solution,

the Con

supension

filtrability, recovered

essentially

5"C condition, A/ml.

to

The

21 "C as

after

the

there

same

was

agglutination

already

the physical model system described in the preceding

as

no was

observed

by

section.

Table 1 Filtrability and Agglutinabi1ity of mouse 3T3 cells

Cell treatment

Con A agglutinability at 200 ug/ml

Filtrability (% recovery)

++++

95 52

12 u 8 u

Trypsin

Filter porosity

Trypsin, then formalin

0

95% a g g l u t i n a t i o n of u n t r e a t e d aggregate

size,

at a concentration

of 5pg/ml.

c e l l s , with

massive

When the l e c t i n

con-

c e n t r a t i o n was i n c r e a s e d to 50(ig/ml the d e g r e e of a g g l u t i n a t i o n markedly reduced.

These f i n d i n g s may r e s u l t from the o c c u p a n c y

increasing

numbers of WGA b i n d i n g

precluding

the l e c t i n from b r i d g i n g between c e l l s .

c o v a l e n t l y bound c a r b o h y d r a t e

(7),

As WGA l a c k s

the reduced a g g l u t i n a t i o n

at

high

interaction.

Neuraminidase t r e a t e d c e l l s n e i t h e r formed m a s s i v e a g g r e g a t e s >95% a g g l u t i n a t i o n

of

s i t e s by s i n g l e WGA molecules,

WGA c o n c e n t r a t i o n s could not be a t t r i b u t e d to l e c t i n - l e c t i n

showed

was

nor

i r r e s p e c t i v e of the WGA c o n c e n t r a t i o n .

190 No a g g l u t i n a t i o n was seen in untreated c e l l s in the p r e s e n c e of PNA or HPA.

PNA induced >95% a g g l u t i n a t i o n and massive

size with neuraminidase t r e a t e d c e l l s . trations,

HPA, even at high

aggregate concen-

produced only about 80% a g g l u t i n a t i o n with small

aggregate

size. The s p e c i f i c i t y of the a g g l u t i n a t i o n r e a c t i o n was confirmed by experiments in which l e c t i n s and s a c c h a r i d e i n h i b i t o r s were mixed before c e l l s were added. maximal a g g l u t i n a t i o n .

Lectin c o n c e n t r a t i o n s were selected to give Results a r e shown in T a b l e 5.

It was

notable t h a t 200mM g a l a c t o s e was r e q u i r e d to i n h i b i t maximal PNA agglutination.

I n h i b i t i o n experiments were not performed on u n t r e a t e d

c e l l s with PNA or HPA as the previous r e s u l t s had shown no a g g l u t ination. Agglutination of PBL by l e c t i n s showed small q u a n t i t a t i v e differences between samples from different donors. r e s u l t s a v e r a g e d from five

T a b l e L, shows

samples.

Table A:

L e c t i n - i n d u c e d Agglutination of PBL

Concentration Ug/ml 50 20 10 5 1 0.5

WGA PNA HPA Untreated Neuram. Untreated Neuram. Untreated Neuram. ++++ ++++ +++++* +++ +++++* ++++ +++++* +++ +++++* ++++ ++++* ++ ++++* +++ ++++ ++ +++ + ++++ + ++ + ++

Experimental d e t a i l s as in the legend for T a b l e 3 The pattern of a g g l u t i n a t i o n with different l e c t i n s was to that of K562 c e l l s ,

but maximal a g g l u t i n a t i o n r e q u i r e d

similar

higher

191 c o n c e n t r a t i o n s of WGA and PNA. minimal.

Spontaneous a g g l u t i n a t i o n

Comparable degrees of i n h i b i t i o n were obtained

was ( T a b l e 5)

though r e s u l t s r e f e r r i n g to chitobiose and 200mM g a l a c t o s e were derived from only two donor

inhibition

samples.

T a b l e 5: I n h i b i t i o n of Agglutination by Specific Inhibitor 50mM GlcNAc

PBL

Untreated Neuram.

Untreated Neuram.

Marked

ImM Chitobiose

Saccharides

K562 Marked

Complete Complete

Marked

Lectin Marked

lOmM Gal

No

Minimal

No

Minimal

200mM Gal

No

Complete

No

Complete

No

Complete

No

Complete

lOmM GalNAc

WGA

Complete Complete

PNA HPA

Lectins were used at c o n c e n t r a t i o n s required to give maximal a g g l u t i n a t i o n (see T a b l e s 3 & 4 ) . S a c c h a r i d e i n h i b i t o r s were mixed with l e c t i n s prior to addition of c e l l s and the a g g l u t i n a t i o n experiments c a r r i e d out a s d e s c r i b e d in the legend to T a b l e 3 . S i x donors were used a s sources of PBL. Marked - > 70% i n h i b i t i o n Complete - 100% i n h i b i t i o n Minimal - < 20% i n h i b i t i o n

DISCUSSION These r e s u l t s confirm t h a t binding of a l e c t i n to a c e l l is a precondition for l e c t i n mediated c e l l a g g l u t i n a t i o n ,

surface

since PNA and

HPA neither bound to nor a g g l u t i n a t e d K562 c e l l s or PBL unless t r e a t e d with n e u r a m i n i d a s e .

However, l e c t i n binding h a s been demon-

s t r a t e d at 4°C at which temperature minimal a g g l u t i n a t i o n is observed within 3 0 ' .

The temperature dependence of a g g l u t i n a t i o n h a s been

v a r i o u s l y argued to r e s u l t from receptor mobility r e s t r i c t i o n

(4,8)

to the d i s s o c i a t i o n of C o n c a n a v a l i n A (the most commonly tested

or

lectin)

192 to a dimeric form at low temperatures Schnebli

(see Wang et a l ,

this

volume).

(1) h a s claimed t h a t h a e m a g g l u t i n a t i o n does occur with

Concanavlin A at 4°C but at a much reduced

rate.

It h a s been g e n e r a l l y observed t h a t transformed and m a l i g n a n t c e l l s show i n c r e a s e d a g g l u t i n a b i l i t y by comparison with t h e i r transformed or normal c o u n t e r p a r t s at any given l e c t i n (4,9,11).

non-

concentration

However, c a l c u l a t i o n of the numbers of l e c t i n

molecules

bound r e v e a l minimal d i f f e r e n c e s between the c e l l types

(4,10).

These o b s e r v a t i o n s have given r i s e to suggestions t h a t

receptor

mobility

(3,4,8),

s u r f a c e morphology(11) or deformability

th'is volume) a r e r e s p o n s i b l e for the d i f f e r e n c e s in

(Wang et

al,

agglutinability

between normal and malignant c e l l s b i n d i n g s i m i l a r amounts of l e c t i n . We have compared the K562 c e l l l i n e , myeloblasts,

with normal PBL.

derived p r o b a b l y from m a l i g n a n t

Although we have not c a l c u l a t e d the

absolute number of l e c t i n molecules bound for each c e l l t y p e , differences,

b a s e d on mean c e l l fluorescence with

the

FITC-conjugated

l e c t i n s , were of the order of 2 : 1 which may be accounted for by the i n c r e a s e d s u r f a c e a r e a of K562 c e l l s .

Observed d i f f e r e n c e s in

agglut-

ination between the c e l l types was r e s t r i c t e d to minor v a r i a t i o n s the l e c t i n concentration required for maximal

in

agglutination.

The s p e c i f i c i t y of PNA is directed towards terminal

non-reducing

g a l a c t o s e residues and the a f f i n i t y is p a r t i c u l a r l y strong for the d i s a c c h a r i d e Gal S I —> sialic acid residues.

3GalNAc, which is normally masked by PNA-reactive s a c c h a r i d e s were exposed by

neuraminidase treatment of

both c e l l t y p e s , confirming the o b s e r v -

ations of Reisner (12) with r e g a r d to lymphocytes.

S a t u r a t i o n of K562

193 c e l l s u r f a c e binding s i t e s occurred at about 20y g PNA-F/lSOyl whereas a g g l u t i n a t i o n

( T a b l e 3) of these c e l l s was seen at 5 pg PNA/ml

which is e q u i v a l e n t to 0 . 7 5 y g / 1 5 0 p l , ancy.

(Fig.l)

or only 15% b i n d i n g s i t e o c c u p -

For PBL the d i f f e r e n c e s between PNA c o n c e n t r a t i o n s

for s a t u r a t i o n of s u r f a c e binding s i t e s and maximal were smaller but n e v e r t h e l e s s s t r i k i n g , occurred at 20ug/ml ( T a b l e 4 ) ,

required

agglutination

in that maximal

agglutination

e q u i v a l e n t to 3 . 0 pg/150/ji 1.

Inspection of the curve in F i g . 2 shows t h a t at t h i s concentration PNA-F 50% of the s u r f a c e binding s i t e s a r e occupied by the Furthermore, binding ination

of

lectin.

although lOmM g a l a c t o s e s i g n i f i c a n t l y i n h i b i t e d PNA-F

(Table 2 ) ,

200mM g a l a c t o s e was r e q u i r e d to i n h i b i t

(Table 5 ) .

agglut-

These o b s e r v a t i o n s suggest that PNA binding to

the highest a f f i n i t y receptors a r e r e s p o n s i b l e for > 95% a g g l u t i n a t i o n and massive a g g r e g a t e s at s u b - s a t u r a t i o n lectin HPA binding to c e l l s u r f a c e s i n t e r a c t i n g with N-acetyl

(Figs.l

galactosamine

concentrations.

& 2),

the l e c t i n

probably

(GalNAc) r e s i d u e s ,

and the

r e s u l t i n g a g g l u t i n a t i o n ( T a b l e s 3 , 4 ) n e e d e d neuraminidase treatment of K562 c e l l s and PBL, implying masking of binding sites by s i a l i c acid r e s i d u e s .

Unlike Schwenk ( 1 3 ) , who a s s e s s e d

c o n j u g a t e d l e c t i n binding by direct v i s u a l i s a t i o n with

terminal fluorescein

fluorescence

microscopy, we were u n a b l e to demonstrate binding of HPA-F to K562 c e l l s p r i o r to neuraminidase treatment.

With

this lectin

>95% a g g l u t i n a t i o n nor massive a g g r e g a t e s occurred at e q u i v a l e n t to s a t u r a t i o n b i n d i n g .

concentrations

WGA binding to untreated K562

c e l l s induced massive a g g r e g a t e s and 100% a g g l u t i n a t i o n , only small a g g r e g a t e s developed a f t e r removal of s i a l i c (Tables 3 & 4).

neither

whereas acid

194 B h a v a n a n d a n (1979) showed

h a e m a g g l u t i n a t i o n in the p r e s e n c e

of WGA can be i n h i b i t e d by s i a l i c a c i d , p a r t i c u l a r l y when present complex glycopeptides

(14).

This s u g g e s t s t h a t the a g g l u t i n a t i o n

untreated c e l l s by WGA is mediated through terminal s i a l i c residues,

in of

acid

which may account for the observed d i f f e r e n c e s in

agglut-

ination p a t t e r n s between c e l l s with or without nueraminidase

treatment.

If t h i s is so, it s u g g e s t s t h a t the p a t t e r n of >95% a g g l u t i n a t i o n

and

massive a g g r e g a t e s may be mediated by l e c t i n b i n d i n g to r e s i d u e s the non-reducing termini of c e l l s u r f a c e s a c c h a r i d e c h a n g e s .

at

This

would imply t h a t the g a l a c t o s e r e s i d u e s recognised by PNA occupy sites in the c h a i n subterminal to s i a l i c a c i d .

Conversely,

and GluNAc recognised by HPA and WGA r e s p e c t i v e l y , t r e a t e d with n e u r a m i n i d a s e , acid h a s been removed.

the GalNAc

in c e l l s

a r e sited s u b t e r m i n a l l y even when

Alternatively,

sialic

such binding s i t e s may be

located deeply within the s u r f a c e m a t r i x of the c e l l and r e p r e s e n t poor

site for c r o s s - l i n k i n g with neighbouring

cells.

Human lymphocytes a r e d i v i s i b l e into numerous s u b s e t s on the method used. experimentally

by

coupled to WGA ( 1 5 ) , lymphocytes

a

Different f r a c t i o n s have been

depending

separated

t h e i r l e c t i n a f f i n i t y using Sepharose columns PNA (16) and HPA ( 6 ) .

Fractionat.ion of human

on the b a s i s of d i f f e r e n t i a l a g g l u t i n a b i l i t y

performed using SBA (Soybean Agglutinin)

by B a r z i l a y

h a s been

(this

volume).

Reisner ( 1 7 , 1 8 ) h a s u t i l i s e d the a b i l i t y of SBA to a g g l u t i n a t e s u b f r a c t i o n s of bone marrow c e l l s in conjunction with sheep red c e l l rosetting,

to enrich bone marrow samples for haemopoetic stem c e l l s

and deplete them of a l l o r e a c t i v e T lymphocytes r e s p o n s i b l e for Graft

195 v e r s u s Host Disease prior to t r a n s p l a n t a t i o n .

These r e s u l t s

indicate

profound d i f f e r e n c e s between lymphocytes in the structure or conformation of their s u r f a c e s a c c h a r i d e s .

It i s l i k e l y that functional

c h a r a c t e r i s t i c s are correlated with these d i f f e r e n c e s .

Our f i n d i n g s

i n d i c a t e that these d i f f e r e n c e s in a g g l u t i n a b i l i t y may not r e l a t e only to the presence or absence of lectin binding s i t e s , but a l s o to the a f f i n i t y of the lectin for a v a i l a b l e sites and the t o p o g r a p h i c a l location of such s i t e s within the p e r i c e l l u l a r

domain.

In conclusion, it would a p p e a r that q u a n t i t a t i v e e v a l u a t i o n of lectin b i n d i n g and lectin induced cell a g g l u t i n a t i o n a r e complementary techniques for the i n v e s t i g a t i o n of cell s u r f a c e s a c c h a r i d e s :

analysis

of cell s u r f a c e s by both techniques p r o v i d e s information not only on numbers of s i t e s complementary to a l e c t i n , but a l s o the potential role of such s i t e s in the a g g l u t i n a t i o n r e s p o n s e .

Our r e s u l t s

suggest

that p e r i p h e r a l s i a l i c a c i d and g a l a c t o s e r e s i d u e s may represent e s p e c i a l l y potent a g g l u t i n a t i o n s i t e s for l e c t i n s , whereas GalNAc and GlcNAc r e s i d u e s seem to be poor s i t e s for lectin

agglutination.

Abnormalities in malignant c e l l s may be e x p r e s s e d a s d i f f e r e n c e s in arrangement,

sequence or topography of l e c t i n - r e a c t i v e

saccharides

which could l e a d to s t r i k i n g d i f f e r e n c e s in the a g g l u t i n a t i o n whilst e x p r e s s i n g normal numbers of lectin binding Acknowledgement : expert technical

response

sites.

The authors wish to thank Mr. A. Morris for assistance.

196 REFERENCES. 1. S c h n e b l i , H.P. in ' C o n c a n a v l i n A a s a T o o l ' . S c h n e b l i , H.P. John Wiley & Sons (1976)

Eds.Bottiger,

2. Glenney, J . R . , Hixon, D.C. & Walbord, E . G . Exp. Cell Res. 353-364 (1979) 3. Edelman, G . M . , Y a h a r a , 70,

1442-1446

I.

& Wang, J . L .

H. & 118,

Proc.Natl.Acad.Sci.,

(1973)

4. Noonan, K. & B u r g e r , M.M.

J . C e l l Biol. 59,

134-143

(1973)

5. B lIanctk. lJe. d gBiomedical e , G . , Swindell, R . , Hodgson, & Crowther, Computing, U , 41-52 B.W. (1980)

D.

6. Axelsson, A., Kimura, A . , Hammerstrom, S. , Wigzell, H. , Nilsson, K. & Mellstedt, H. Eur . J . Immunol. 8 , 757-764 (1978) 7. Goldstein, 128-340

I.]. & Hayes, C . C . (1978)

8. Hatten, M . E . , ].Biol.Chem.

Scandella, 253,

9. L i s , H. & Sharon,

C.J.,

1972-1977 N.

Adv.Carb.Chem. Horwitz, A . F .

& Biochem.

& Burger,

35,

M.M.

(1978)

Am.Rev.Biochem.

42, 541-574

(1973)

1 0 . S h a r o n , N. & L i s , H. Methods Memb.Biol. 3, 143-200 (1975) 11.Temmine, J . H . M . , C o l l a r d , J . G . , Roosien, J . & Van Den B o s c h , J . F . J . C e l l S c i . 21, 563-578 (1976) 1 2 . R e i s n e r , Y . , Biniaminov, M. , R o s e n t h a l , E . , Ramot, B. P r o c . N a t l . A c a d . S c i . 76, 447-451 13.Schwenk, H.U., S c h n e i d e r , 7-15 (1980) 14.Bhavanandan,

V.P.

Sharon, (1979)

U. & Hertzog, K.H.

& Katlic,

15.Hellstrom, J . , D i l l n e r , M . L . , J . E x p . M e d . , 144, 1381-1385

N. &

Blut,

A.W. J . B i o l . C h e m . ,254,

40, 4000-4008,(1979)

Hammarstrom, S. & P e r l m a n n , (1976)

P.

1 6 . B a l l e t , J . J . , Fellows, M. , S h a r o n , N. , R e i s n e r , Y. & A g r a p a r t , S c a n d . J . Immunol., 1_1, 555-560 (1980) 1 7 - R e i s n e r , Y . , Kapoor, N., O ' R e i l l y , Lancet ( i i ) 1320-1324 (1980)

J.

1 8 . R e i s n e r , Y . , Kapoor, N., K i r k p a t r i c k , Good, R.A. & O ' R e i l l y , R . J . Lancet ( i i ) 327-332 (1980)

& Good,

M.

R.A.

D., Pollack,

M.,

Dupont.B.,

LECTIN BINDING BY MALARIA-INFECTED ERYTHROCYTES

R.F. Adebiyi Immunology Unit, Department of Chemical Pathology, University of Ibadan, Ibadan, Nigeria. G. Parnell, J.A. Forrester and A.J.S. Davies Chester Beatty Research Institute, Royal Cancer Hospital, Fulham Road, London SW3 6JB, U.K.

Malaria (meaning "bad air") is an ancient tropical disease caused by intracellular parasites of the sub-order Haemosporodia and genus Plasmodium and is transmitted by the famle Anopheles mosquito. The disease is closely associated with swamps and marshes. However, it has become widely distributed in recent times due partly to the rapid development of intercontinental transportation. Malaria affects all age groups but children in endemic areas appear to be particularly susceptible. It is estimated that about one million children die each year from this disease. In all, over one billion persons are exposed to the disease world wide (23). Because of the severe debilitation and economic repercussions of the disease, the World Health Organization has been involved in the control and eradication of malaria since the end of World War II (3o). The use of insecticides held much promise initially but the rapid emergence of resistant strains of the mosquito has underscored the need for an elucidation of finer aspect of the parasite's biology and a reformulation of strategies for intervention. The human erythrocyte is susceptible to invasion by four plas-

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 . II © W a l t e r d e G r u y t e r & C o . , B e r l i n • N e w Y o r k 1982

198 moidal species, namely Plasmodium falciparum, P. malariae, P. vivax and P. ovale. The events leading up to internalization of Plasmodia have been studied by Aikawa (1,2), Bannister et al. (3), Dvorak et al. (8) and Miller (18) who showed that initial contact is random and non-specific but that the parasite possesses an organ by which it attaches to the red cell surface prior to penetration. The identification of this organ and the characterization of its active protein has generated considerable interest as it implies that invasion is a specific process which, hopefully, can be prevented by the use of analogous preparations as blocking agents (2,13,14,2o,21). Another implication is that attachment may be to specific surface receptors whose characterization has however been very elusive. The aetiologic agent of benign tertain malaria, Plasmodium vivax, for example, recognizes the Duffy antigen (Fya and/or Fy*3) on red cells. The antigen is required for infection and FyFy cells have been shown to be completely resistant to infection by this parasite (19). Unfortunately, studies designed to discover similar relationships between blood group substances and other plasmodia have not been successful. An obvious limitation in this sort of investigation is the dependence on specific antibody for detection of an antigen. Where an antibody is not immediately available, lectins, which like antibody, interact with specific components of the glycocalyx, are very helpful. Indeed, the lectins from Dolichos biflorus and Ulex europaeus are routinely employed to confirm the presence of A^ and H blood group substances. Other non-blood-group related structures can be readily detected with lectins. It is in this context that lectins have been used in the present study to characterize the erythrocyte surface and to describe in general and specific terms the cell surface changes that my accompany parasite invasion. Seven lectin preparations have been employed in agglutination and binding studies to obtain a profile for normal and malaria-infected mouse erythrocytes and to assess the effect of parasitization

199 on lectin-mediated agglutinability.

Materials and methods Mice: Age matched CBA/Ca female mice from the Chester Beatty Research Institute were used. Some of the mice were rendered T-lymphocyte deficient (to obtain high parasitaemia) by thymectomy and whole body irradiation followed by reconstruction with syngeneic bone marrow cells. Erythrocytes: Red blood cells were obtained by severance of the aorta and irrigation of the thoracic cavity. Clotting was prevented by rapid dilution with phosphate buffered salins pH 7.4. The washed cells were used in haemagglutination and binding assays at concentrations of 2 % and 0.1 % respectively. Parasite: Plasmodium berghei yoelii (P. yoelii) 17X strain, was obtained from the London School of Hygiene and Tropical Medicine. It was maintained at the Chester Beatty Research Institute as a stabilate which was reconstituted as required according to the technique of Lumsden et al. (17). Mean percentage parasitaemia was estimated from giemsa stained thin smears at high dry magnification by the enumeration of the number of parasitized erythrocytes in twenty randomly selected fields containing approximately 4oo erythrocytes each. Lectins:

The lectins used were Dolichos biflorus (DBL), Ara-

chis hypogeae (peanut lectin, PNL), Ricinus communis (RCL), Abrus precatorius (ABL), Glycine max (soyabean lectin, SBL), Phaseolus limensis (limabean lectin, LBL) and Triticum vulgaris (wheatgerm lectin, WGL).

Each lectin was prepared accor-

ding to the method of Lotan et al. (16) which involved grinding and defatting with petroleum ether, extraction with saline, purification by affinity chromatography, dialysis and lyophilization.

Limabean lectin (LBL) and peanut lectin (PNL) were 125 I (iodogen method) and were employed in binding

labelled with

200

assays which were performed as follows; serial two-fold dilutions of iodinated LBL and PNL were prepared starting from 16o mcg/ml and decreasing. To 2oo mcl of each lectin dilution was added 600 mcl of a 0.1 % erythrocyte suspension. Erythrocytes for the PNL assay were pre-treated with neuraminidase. A portion of the mixture was incubated at room temperature for 15 min over half its volume of butylphthalate in plastic vials which were rapidly centrifuged and cut at the interphase to separate the cells from the supernatant. The amounts of bound and unbound label were then determined. Agglutination assay: Agglutination assays were performed in WHO titration plates using two-fold serial dilutions of each lectin and fixed volumes of a 2 % erythrocyte suspension. The degree of agglutination was the last well showing complete agglutination after two hours incubation at room temperature. Thus, a high degree of agglutination corresponds to a low lectin concentration and a high agglutinability of cells. Readings were confirmed after overnight incubation at 4°C.

Results Haemagglutination assays showed that mouse erythrocytes were agglutinated by DBL, ABL, RCL, LBL and WGL. Both SBL and PNL did not cause agglutination unless the cells were pretreated with neuraminidase. No lectin was found to agglutinate only infected or only uninfected cells exclusively. However, infected cells were more readily agglutinated by DBL, LBL and WGL (Fig. 1). With LBL, for example, uninfected cells were agglutinated to the 3rd degree whereas infected cells were agglutinated to the 7th degree, indicating a 16-fold increase in terms of lectin dilution. Cells obtained at different times in the course of a severe infection, exhibited a behaviour which demonstrated a direct

201 relationship between the level of parasitaemia and ease of agglutination (Fig. 2).

The degree of agglutination was vir-

tually unchanged at parasitaemias up to lo % but at 4o % the degree of agglutination by DBL had increased from 1 to 2, for WGL from 4 to 5 and for LBL from 3 to 5.

At 6o % parasitaemia,

the degree of agglutination by LBL had increased to 8, representing a 32-fold increase in the dilution of lectin required to agglutinate the cells completely.

Agglutination by ABL was

unchanged by parasitaemia. Since P. yoelii is believed to prefer young erythrocytes and, indeed, as reticulocytosis regularly accompanies severe murine malaria (12), the agglutinability of reticulocytes and neuraminidase-treated cells were also examined.

•

unparasitized cells

Fig. 3 shows that

g j parasitized cells

Figure 1. Agglutination of CBA mouse erythrocytes by selected lectins. Soya bean (and peanut lectin) does not agglutinate untreated mouse erythrocytes due to masking of D-galactosyl residues by sialic acid. Agglutination regularly occurs after treatment with neuraminidase. Agglutination by other lectins indicate the presence of the respective receptors.

202

reticulocytes were more readily agglutinated than uninfected cells but less than infected cells. Neuraminidase-treated cells, however, were more readily agglutinated by LBL and WGL than both untreated infected and uninfected cells. This was probably due to a reduction of intercellular coulombic repulsion. As usual, PNL agglutinated only neuraminidase-treated cells.

—A Abrin

10-

8-

/

c

o

H

bE b£ rt

Lima

.o Wheatgerm

6-

HI a) u 4ht) 95 %) ascertained the specificity of the lectin effect and the absence of RCA^-induced toxic alterations. Thus, our results indicate the involvement of exposed B-D-galactose residues in the. receptor-insulin interaction. This was further established by the following findings. While B-galactosidase digestion of fat cells ( 1 - 1 0 U/ ml) did not modify insulin binding, simultaneous or sequential digestion of fat cells with neuraminidase (0.25 mU/ml) plus B-galactosidase (1 - 10 U/ml) decreased insulin binding by 43 % (Table 1) . These findings point to the importance of pernultimate B-D-galactose residues for insulin recognition and indicate that sialic acid is present and positioned terminally in the carbohydrate. D-mannose reactive proteins. Effect of Lens culinaris agglutinin (LCA), Con A and a-mannosidase. LCA or Con A (10 - 80 pg/ml) reduced insulin binding considerably (57 or 59 %) and dissociation (90 (br 78 %) (Table 2, Fig. 1). Probably, the lectin-induced decrease was not due to cell agglutination since submaximal effects were observed at concentrations which did not agglutinate fat cells (10 yg/ml). In the presence of a-methyl-D-mannopyranoside ( aMM) (50 mM), the lectins no longer inhibited any of the process, thus arguing for the specificity of their effects. Therefore, the lectin interference with the receptor-insulin interaction could chiefly be attributed to their binding to mannose residues.

263 TABLE 2 : EFFECTS OF VARIOUS LECTINS ON SPECIFIC { 1 2 5 I}-LABELLED INSULIN BINDING TO RAT1 ADIPOCYTES. { 1 2 5 I}-INSULIN BOUND N°of (% of CONTROL ± S.E) Exp.

LECTIN PREINCUBATION

100.0

None RCA

+

4.0

74.8 60.0 59.1 57 .0

+

5.9 8.2 6.4 8.1

100. 4 99 .2 90.5

+

(yg/ml) 10.00 25.00 50.00 80.00

63 .9 43 .4 44.0 43 .3

+

(yg/ml) + aMM(50 mM) 25.00 50.00 80.00

99.8 92.1 84 .5

+

(yg/ml) 10.00 20.00 50.00 80.00

55.5 40.8 40.5 41 .0

+

94 .8 90.5 82.0

+

114.5 40. 6 29.5 15.0 14.5

+

121 .8 124.7 125.0

+

(yg/ml) 10.00 25.00 40.00 80.00

RCA T (yg/ml)+D-Gal (50 mM) 25.00 50.00 80.00 LCA

LCA

Con A

Con A (yg/ml)+ aMM (50 mM) 20.00 40.00 80.00 WGA

(yg/ml) 5 .00 10.00 20.00 50.00 80.00

WGA (yg/ml)+Glc NAc 20.00 50.00 80.00

(50 mM)

+ + +

+ +

+ + +

+ +

+ + +

+ +

+ + + +

+ +

6.0 8.1 9.0 6.2 3 .0 6.1 2.0 8.6 9.0 10.0 5.7 6 .3 4.8 4.2 6.5 6.0 8.8 5.0 10.3 4.0 6.2 5.3 7.4 9.6 10.5

P

25 7

P P P P

<
4DGal

+

I

DGalßl—»6DGal

0

I

DGalßl—4 (?)

+

5) Ricinus communis I 6) Ricinus communis II

P

DGalßl — ( ? )

+

P

DGalßl —» (?)

+

7) Axinella polypoides (I & II) 8) Agaricus bisporus

S P

DGalßl —» 6DGal DGalßl —» 3DGal

0 0

P P

DGalßl—»4 DGalßl —»4

0

P

DGalßl—• (?)

12) Myeloma protein 13) Pseudomonas aeruginosa Wistaria flori14) bunda

V

DGalßl—»6DGal

+

B

DGalßl

0

P

DGalNAcal ->6DGal

+

15) Glycine max 16) Geodia cydonium 17) Cerianthus membranaceus 18) C-reactive protein

P S

DGalNacal—»3DGalßl- 3DGal DGalßl-* 4 (?)

+

0

I

DGalßl—•(?)

+

V

DGalßl—»(?)

0

9) Phaseolus vulgaris lo) Abrus precatorius 11) Viscum album

(?)

+

The precipitation experiments were performed as diffusion in agar (cf. Fig. 2). +, precipitine line; 0, no visible reaction; P, plants; B, bacteria; I, invertebrates; S, sponge; V, vertebrates

360 Discussion The affinity chromatography of the semi-purified fraction from the crude extract, glycosubstance I, on the PNA-agarose matrix provided an unique product from the natural source. According to the analysis it consists only of galactose residues. The isolation procedure (Scheme 1) was not as drastic as the alkaline hydrolysis with phenol-saline extraction as reported by Chatterjee et al. (4). Whereas the preparation from the latter procedure still contained about 8 % protein, the product obtained in this investigation contained less than 0.1 % (Lowry's). The amino acid analysis, which surely provides a better and reliable diagnostic tool, was therefore not carried out. The total carbohydrate content was 91 %. This value was in close agreement with that (92 %) reported by Chatterjee and co-workers (4). However, the presence of hexuronic acid in Achatina fulica galactan has been reported (10). This may account for the rest of 9 %. The galactan was essentially homogeneous as evident by the result of the agar-gel electrophoresis with respect to two lectins (PNA, RCA ). oU The pattern of the precipitin reactions with galactose-binding lectins in agar-gel double diffusion experiments (Table 2) exhibited by this purified material, reconfirms the earlier observation of the heterogeneity with reagents to lectin receptors on the crude mixture of proteogalactans, and of galactans from the gland tissue. The sugar specificity and origin of the lectins that reacted positively in this investigation are highly diverse, but basically they possess in common a combining site with D-galactosyl-(plus x)-residues as shown in Table 3 and 4. There were no precipitin lines (i.e. no precipitin reaction) in agar-gel diffusion tests with Tridacna maxima and Axinella bisporus lectins, both with anti-(3-Gal(1-6)-DGal specificity, although the anti-galactan myeloma protein with an anti-f3-Gal

361 TABLE 3 Precipitation of 3 - g a l a ctosidase-treated Achatina fulica galactan with various galactose-binding lectins

Lectin

Specificity

1) Arachis hypogaea

DGalßl- •3DGalNAc

2) Bauhinia purpurea

DGalßl- »3DGalNAcßl- •3DGal DGalßl ••3DGalNacßl• • 4DGal

3) Choleralectin 4) Tridacnins I. T. maxima T. gigas II. T. crocea T. derasa T. squamosa

Precipitation

(+) 0 0

DGalßl- > 6DGal

0

DGalßl- •4 (?)

0 0

Ricinus communis ] DGalßlRicinus communis DGalßlII

0

Axinella polypoides (I & II) Agaricus bisporus

DGalßl- • 6DGal DGalßl- •3DGal

0 0

9

Phaseolus vulgaris

DGalßl-

0

10

DGalßl- .4

,? DGalßl-

+

11

Abrus precatorius Viscum album

12

Myeloma protein

DGalßl- •6DGal

13

Pseudomonas aeruginosa Wistaria floribunda Glycine max Geodia cydonium

8

14 15 16

17

Cerianthus membranaceus

DGalßl —» ? DGalNñcal- >6DGal DGalNAcal• 3 DGalßl— 3DGal DGalßl—» 4 (?) DGalßl-

18) C-reactive protein DGalßl-

+, strong precipitin line; (+), weak precipitin line; 0, no visible reaction

(+) (+) 0 + (+) 0 (+) 0

362 (1—*6)-DGal, specificity (11) gave visible but weak precipitin lines.

A similar unexpected result was obtained with Cholera-

lectin, which has the specificity for the structure anti-PGal-(1—»3)-DGalNAc(2—»3)-NeuAc.

These latter observations ha-

ve been proven to be not non-specific reactions

(electrostatic,

Schiff base formation etc.), which may occur in diffusion tests as discussed in another context by us (12).

We have excluded

the latter problem by our experiments with purified lectins and other controls. The positive precipitin reactions with the peanut lectin (PNA) and the lectin from the Bauhinia purpurea alba seeds demonstrate another interesting receptor site on this galactan.

The

combining site of the Bauhinia purpurea lectin has been proposed to consist of a structure similar to (3-DGal (1—»3) -DGal (13) while the disaccharide 3-DGalNAc has been equivocably considered as the receptor-dominant structure for the peanut lectin (14).

The peanut lectin, however, does or can react

with 3-Gal(1—»4)-DGalNAc structures (14).

The enzymatic 3~ga-

lactosidase treatment of the galactan resulted in the complete or partial abolishment of the reactivity (i.e. negative precipitin reaction in agar-gel diffusion experiments) with the lectin Bauhinia purpurea, PNA, RCAg Q , RCA^Q» Tridacna squamosa, Tridacna derasa (Table 3) .

Positive precipitin reac^-

tions in agar were still observed for Abrus precatorius which possesses an anti-3-Gal(1—»4)-DGal specificity

(15).

These

observations suggest two facts, namely (a) the presence of 3glycosidic linkage in the galactan and (b) the presence of more than one type of glycosidic linkages in the galactan structure.

These findings await now confirmation from detailed

chemical structural analysis of this galactan. In summarizing our results, we come to the following conclusions : a)

The galactans and glycoconjugates from the albumin gland of Achatina fulica snail contain multiple lectin receptors

363

TABLE 4 Anti-D-galactosyl-(plus x)-specific lectins

1)

Plant lectins a) "Normal" lectins (Bauhinia), lectins from algae b) Lectin-toxin molecules (Ricinus)

2)

Bacterial lectins (Ps. aeruginosa), Choleralectin

3) 4)

Slime mould lectins Sponge lectins

5)

Invertebrate lectins a) Haemolymph: Tridacnins from Tridacna bivalve clams b) Snail eggs or albumin glands (Pomacea, Ampullaria)

6)

Vertebrate lectins a) Fish eggs (Rutilus) b) Membrane-integrated (liver) c) Serum proteins: C-reactive protein (17), 9.5 (^-glycoprotein and Clq

364 for lectins with a closely-related anti-DGal specificity (Table 4). Accordingly they represent excellent models for studying the combining site of lectins. b)

These glycoconjugates may not only be useful tools in detecting lectin-activity but also used to block or to inhibit lectin-receptor sites.

This interesting class of

galactans seems worth to elucidate with respect to their chemical structure in order to establish receptor sites for anti-carbohydrate reagents. c)

These galactans can be used for isolating and characterizing anti-galactan or anti-DGal lectins by affinity chromatography. In fact, partially hydrolysed Sepharose (a galactan, too). In fact, partially hydrolysed Sepharose (a galactan, too) is the best affinity chromatography for isolating and detecting anti—galactosyl lectins (16) .

The future aspect of this investigation is that these galactans, in a labelled form, may represent suitable reagents in detecting vertebrate membrane-integrated lectins of the antigalactosyl type. Galactans have also already been successfully used for demonstrating the fact that certain serum glycoproteins, for instance C-reactive protein, 9.5 a^-glycoprotein and Clq have anti-galactosyl combining sites and may be regarded as having the properties of a lectin (17). The implications of this finding justify further serological investigations on galactans. Acknowledgement. This work was supported by the Deutsche Forschungsgemeinschaft. The authors thank Beatrix Walde for help in preparation of the manuscript.

365 REFERENCES 1.

Uhlenbruck, G., Steinhausen, G., Kareem, A.A. Z. Immun. Forsch. Exp. Ther. 152, 22o-23o.

(1976).

2.

Uhlenbruck, G., Steinhausen, G., Palatnik, M. (1977). Comp. Biochem. Physiol. 57B, 335-339.

3.

Uhlenbruck, G., Steinhausen, G., Geserick, G., Prokop, 0. (1978). Comp. Biochem. Physiol. 59B, 285-288.

4.

Chatterjee, B.P., Chatterjee, S., Prokop, 0., Uhlenbruck, G. (1979). Biol. Zbl. 98, 85-9o.

5.

Uhlenbruck, G., Baldo, B., Steinhausen, G. (1975). Immun. Forsch. 15o, 354-363.

6.

Campbell, D.H., Garvey, F.S., Cremer, N.E., Sussdorf, D.H. (197o). In: Methods in immunochemistry, p. 149, W.A. Benjamin, Inc. New York.

7.

Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951). J. Biol. Chem. 19^3, 265-275.

8.

Dubois, M. , Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F. (1956). Analyt. Chem. 28, 35o-356.

9.

Farrar, G.H., Harrison, R. (1978). 557.

Z.

Biochem. J. 171, 549-

10.

Vaith, P., Uhlenbruck, G., Holz, G. (1979). In: of biological fluids, 27th Colloqium, 455-458.

Protides

11.

Eichmann, K., Uhlenbruck, G., Baldo, B.A. (1976). nochemistry 1^, 1-6.

12.

Uhlenbruck, G., Chatterjee, B.P., Schuldes, U. Z. Naturforsch. 33c, 442-443.

13.

Wu, A.M., Kabat, E.A., Gruezo, F.G., Allen, N.J. Arch. Biochem. Biophys. 2o4, 622-639.

14.

Newman, R.A., Uhlenbruck, G. (1977). 149-155.

15.

Chatterjee, B.P., Chatterjee, S., Uhlenbruck, G. (1978). Experientia 34, 531.

16.

Baldo, B.A., Uhlenbruck, G. (1975). 25-29.

17.

Uhlenbruck, G. , Karduck, D., Haupt, II., Schwick, II. G. (1979). Z. Immun. Forsch. 155, 262-266.

Immu-

(1978). (198o).

Eur. J. Biochem. 76,

FEBS-Letters 55,

AFFINITY OF LEUCOAGGLUTININ

(L-PHA), COMPARED WITH DOLICHOS

BIFLORUS LECTIN AND CON A, FOR HUMAN TAMM-HORSFALL GLYCOPEPTIDE

Franca Serafini-Cessi and Fabio Dall'Olio Istituto di Patologia Generale, Università di Bologna 40126 Bologna, Italy

Leucoagglutinin (also named L-PHA) is the isolectin from Phaseolus vulgaris (red kidney bean) which binds, with high affinity, to receptors on lymphocyte surface inducing agglutination and blastogenesis, whereas the isolectins of H-PHA type interact more specifically with receptors of erythrocytes (1, 2, 3). Leucoagglutinin has been considered practically devoid of haemagglutinating activity; more recently it has been shown that human erythrocytes are agglutinated after neuraminidase treatment and that N-acetylgalactosamine is the only monosaccharide with inhibitory activity (4). The haemagglutination induced by leucoagglutinin is temperature-dependent, increasing at low temperature. The effect seems related to a change in the conformation of lectin, bound to the glycidic receptors, induced by temperature (5). Human Tamm-Horsfall glycoprotein (TH) is the most abundant glycoprotein in urine (6); it is produced by the distal tubules of the kidney and its biological role might be related to electrolyte and water transport in the kidney (7). TH gives a precipitin reaction with leucoagglutinin which is specifically inhibited by high concentration of N-acetylgalactosamine

(8).

Other glycoproteins, such as serum glycoproteins and desialylated mucins, do not precipitate this lectin (8,9). It has been reported that CEA, from malignant tumors of gastrointesti-

Lectins - Biology, Biochemistry, Clinical Biochemistry, Vol. II © Walter de Gruyter &. Co., Berlin • New York 1982

368 nal tract, forms insoluble complexes with leucoagglutinin (10). The major glycopeptide purified after pronase digestion of TH behaves as a powerful hapten of the interaction of leucoagglutinin with specific receptors. At very low concentration it inhibits the precipitin reaction between TH and leucoagglutinin, the haemagglutination of desialylated human erythrocytes and lymphocyte transformation induced by the lectin (4,11). These results indicated that the major TH glycopeptide comprises the binding sites for leucoagglutinin. TH glycopeptide is also able to inhibit lymphocyte proliferation induced by allogeneic antigens (12). Two immunologically distinct forms of TH have been recently described, which are closely related to the human blood group Sd a character (13, 14, 15). Sd a human blood group system has been described independently in 1967 by two groups of English workers (16, 17) who classified the human individuals into two phenotypes, Sd(a+) (more than 90%) and Sd(a-). As in the ABO system the Sd a antigen is also present in most secretions with the greatest concentration in urine (18). The group of Morgan and Watkins (15) demonstrated that the Sd a activity of urine is associated with TH. Individuals with the Sd(a-) red cell phenotype have a TH without Sd a activity, whereas TH preparations from Sd(a+) persons strongly inhibit the Sd a haemagglutination induced by naturally occuring anti-Sda antibodies. According to these Authors the only difference between the two forms of TH concerns the N-acetylgalactosamine content. Values in the range of 1-2% have been found in individual preparations of Sd a active TH, whereas the level is negligible in TH without Sd a activity. Dolichos biflorus lectin, which specifically recognizes the terminal N-acetylgalactosamine residues, precipitates only Sd a active TH (15). In the present report the precipitin reaction between TH of different Sd a activity and leucoagglutinin or Dolichos biflorus lectin is investigated. In contrast to Dolichos biflorus lectin, leucoagglutinin is shown to react in the same way

369 with Sd a active and non-active TH. This result was not expected because considerable evidence indicated that the carbohydrate specificity of leucoagglutinin involves N-acetylgalactosamine. In the attempt to elucidate the structural features of oligosaccharides affecting binding by leucoagglutinin, the affinity of TH glycopeptide has been studied either in the absence or in the presence of N-acetylgalactosamine. The K^ for the binding of TH glycopeptide to Dolichos biflorus lectin and Con A was also determined. Since TH glycopeptide is non diffusible and on gel filtration behaves as a high molecular weight component (11), its binding was determined by a precipitation method based on our observation of a different solubility in ethanol of the glycopeptide and of the lectins. A quantitative assessment of glycopeptide binding has been obtained using a sample of radiolabelled TH glycopeptide.

Precipitin Reaction 14 14 C-labelled lectins were prepared with C-formaldehyde by the method of Means and Feeney (19). TH were separately isolated from urine collected from individuals with Sd(a+) and Sd(a-) red cell phenotypes by a procedure which involves three precipitation steps with 0.58 M NaCl (6). The Sd a activity of TH preparations was determined by the inhibitory haemagglutination test (18), and was the minimum concentration (yg/ml) of protein giving complete inhibition of agglutination of Sd(a+) red cells by human anti-Sda serum. The TH preparations from Sd(a+) individuals could be classified into two classes, strongly active and weakly active. The first had a Sd a activity of about 0.25 yg/ml, the second of about 1 yg/ml. TH from Sd(a-) individuals failed to inhibit at a concentration of 500 yg/ml. The carbohydrate analysis of various TH preparations performed by gas liquid chromatography (20) agreed with the data of Soh et al (15), i. e. Sd a non active TH was practically devoid

370

Fig. 1. Precipitin reaction between TH preparations with different Sd a activity and ^C-leucoagglutinin (a) or ' C-Dolichos biflorus lectin (b). In each determination 25 yg of lectin was present in a final volume of 0.5 ml, phosphate buffer 0.02 M, pH 7. (A) Sd a inactive TH, (A) Sd a weakly active TH, (•) Sd a strongly active TH. of N-acetylgalactosamine, whereas the content of this sugar was in the range of 2-2.5% in the Sd a active TH. The highest values were preferentially found in strongly active TH preparations. The content of N-acétylgalactosamine in Sd a active TH preparations quoted above, higher than that found by Soh et al. (15) is probably related to the different conditions of hydrolysis (see Allen and Neuberger, ref. 21). For the quantitative determination of the precipitin reaction the procedure previously described was used (11). 14 Fig. 1a shows the precipitation of C-leucoagglutinin with individual preparations of TH. No significant difference was found in the amount of leucoagglutinin precipitated by Sd a strongly active and by Sd a non-active TH. The test of double diffusion in agarose gel confirmed this result. The two forms of TH gave strong precipitin lines with unlabelled leucoagglutinin and the lines coalesced to give reaction of identity irrespective of whether the preparations were Sd a active or non-active. At the contrary considerable variations were observed in the pre-

371 cipitation with Dolichos blflorus lectin. As shown in Fig. 1b, Sd a non-active TH did not give any precipitin reaction even at high concentration; moreover the slope of the precipitation curves was less for Sd a weakly active TH than for strongly active TH. These results demonstrated a correlation between Sd a activity of TH and precipitating power of Dolichos biflorus lectin.

Purification and Labelling of Major TH Glycopeptide TH from pooled urine of Sd(a+) individuals was digested for 48 h with pronase and the digest chromatographed on Sephadex G-25. The fractions of the first peak, containing about 70% of neutral sugars of the total digest, were rechromatografed on DEAE-Sephacel. The acidic glycopeptide (fully sialylated) which was eluted as a narrow peak by a gradient of NaCl (0.05-0.5 M) was used for labelling (details of chromatographic steps in ref. 11). As reported elsewhere (22) the carbohydrate composition of this glycopeptide accounts for a very complex structure. The presence of 3 residues of mannose and the high content in N-acetylglucosamine (more than 5 residues) is consistent with a structure "trimannosyl-chitobiose core N-asparagin linked" type, almost invariably found in the high molecular weight glycopeptides (23). Moreover the high content in galactose and in sialic acid (4 residues each) suggested the presence of side chains of sialylgalactosyl-N-acetylglucosamine attached to the mannosyl core. It is worth noting that 1 residue of N-acetylgalactosamine was also present. The labelling of TH glycopeptide was performed by a procedure which consists in a controlled periodate oxidation of the terminal residues of sialic acid, followed by a reduction of al3 dehydes with KB H^ (24). After labelling, TH glycopeptide was further purified by gel filtration on Bio-Gel P-10 column (75x 1 cm) equilibrated and eluted with 0.1 M NH.HCO... As reported

372

3 Fig. 2. Gel filtration of H-TH glycopeptide on Bio-Gel P-10 The void volume is indicated by an arrow. in Fig. 2 a small peak emerged after the void volume. About 85% of total radiolabelled material was collected in a major second peak with a relative elution volume ( V e / V Q ) from 1.7 to 2.0. The difference in the elution volume of the two peaks might depend either on a different size of the residual peptide moiety or on a different degree of sialylation. For the binding studies we used the central fractions of the second peak. The specific activity of the glycopeptide, assuming a molecular weight of 4800 as reported by other Authors (25), was 185 dpm/ 3 pmol. The H-TH glycopeptide showed the same inhibitory activity, in the haemagglutination test, as unlabelled sample.

Binding Studies In preliminary experiments we tried to determine the binding 3 of H-TH glycopeptide to leucoagglutinin by using insoluble lectin bound to agarose. The plot thus obtained indicated a number of molecular binding sites of the insoluble lectin much lower than that predicted by its tetravalency; probably because of a steric hindrance caused by the binding to agarose.

373

Fig. 3. Percentage distribution of leucoagglutinin (•) and of TH glycopeptide (A) in the precipitates collected after addition of increasing amounts of ethanol. The concentration of lectin was 100 yg/ml and that of glycopeptide 10 yg/ml. The use of ammonium sulphate to precipitate the complex TH glycopeptide-leucoagglutinin was also discarded, since very scattered values of TH glycopeptide were recovered in the precipitates collected in the absence of lectin. The selective precipitation of lectin with ethanol gave better results. Fig. 3 shows the precipitation of leucoagglutinin and TH glycopeptide as a function of ethanol concentration. Ethanol at 70% (v/v) precipitated entirely the lectin, whereas nearly all TH glycopeptide remained in solution. Dolichos biflorus lectin and Con A gave the same curve as leucoagglutinin. 70% ethanol was selected for the binding experiments. Constant amounts of lectin (400 pmol) dissolved in 0.02 M phosphate buffer, pH 7.4, containing 0.14 M NaCl, were incubated at room temperature for 1 h with increasing concentrations 3 (1-25 yM) of H-TH glycopeptide in plastic centrifuge tubes. An identical series of tubes without lectin was prepared in parallel. The total volume in each tube was 100 yl. Four volume of ethanol 87% (v/v) were added to obtain 70% final concentration and after 30 min the tubes were centrifuged at

374

Fig. 4. Binding of H-TH glycopeptide for leucoagglutinin. In the absence of N-acetylgalactosamine (•), the equation for the straight line, calculated by the least-square method, was 1/b = 0.061 x 10 + 0.179/c. The equation was not significantly modified in the presence of a constant concentration (5 mM) of N-acetylgalactosamine (o), and was 1/b = 0.061 x 10 6 + 0.182/c. 20000 2 for 30 min at 4° C. Two 200 yl aliquots of each supernatant were separately counted for radioactivity. The pellets were washed with 0.5 ml of ethanol 70% (insignificant radioactivity was measured in these whashing fractions). The pellets were dissolved in 0.5 ml of 0.5 M NaOH, and two 200 yl aliquots were withdrawn and assayed for radioactivity. The radioactivity of the supernatants from the series with lectin was assumed as free glycopeptide (c) and that of the corresponding precipitates as bound glycopeptide (b). Each value of b and c was corrected for the radioactivity recovered in the precipitates of the parallel series without lectin ( each value was subtracted from corresponding b and added to corresponding c). The results of a typical experiment of binding of ^H-TH glycopeptide to leucoagglutinin are shown in Fig. 4. Data are plotted according to the equation 1/b = 1/a+K^/ac (26), where b is the molar concentration of bound glycopeptide, c that of free glycopeptide and a the molarity of the lectin binding

375

3 Fig. 5. Plots of 1/b against 1/c for the binding of H-TH glycopeptide to Dolichos biflorus lectin (a) and to Con A (b). The equation for the straight line was 1/b = 0.083 x10 6 +1.78/c in (a) and 1/b = 0.1 x10 6 +5.2/c in (b). sites. The plot gave a straight line and the abscissa intercept c

(-1/K d ) gave a K d = 3 . 0 x 1 0 M. The ordinate intercept (1/a) allows to calculate the stoicheiometry of the binding, this was 3.5 in good agreement with the suggested tetravalency of the lectin (3). The value of K^ is comparable to that reported for other glycopeptide-lectin interactions (27, 28). The higher affinity obtained by other workers (29) for the binding of iodi_7 nated leucoagglutinin to lymphocytes (K^l .4 x 10 M) may reflect either an oligosaccharide structure differing from that we have examined, or a cooperation between the glycidic binding sites, caused by their specific location on the lymphocyte surface. On the whole the data are consistent with the observation (11) that a large excess of TH glycopeptide is needed to inhibit lymphocyte transformation induced by leucoagglutinin. Fig. 4 shows3 also that N-acetylgalactosamine (5 mM) does not affect the H-TH glycopeptide binding. This result may explain the observation, reported above, that Sd a non-active TH (devoid of N-acetylgalactosamine) interacts with leucoagglutinin in thè same way as Sd a active TH (containing this sugar). It may be

376 proposed that although N-acetylgalactosamine is apparently recognized by leucoagglutinin (4, 8, 11, 29), portion(s) of the glycomoiety of TH other than N-acetylgalactosamine account for the high affinity of the binding. Evidence was reported that Con A (8) interacts with TH from pooled urine; since 96% of the individuals are Sd(a+), pooled urine is theoretically 96% Sd a active. The quantitative precipitin reaction showed that both Con A (see ref. 8) and Dolichos biflorus lectin (Fig. 1b) were precipitated at a lesser extent than leucoagglutinin by Sd a active TH. Fig. 5 shows the plots 3 for the binding of H-TH glycopeptide to Dolichos biflorus lectin and to Con A. The value of K, d with Dolichos biflorus lectin -5 -~5 was 2 x 1 0 M (Fig. 5a) and with Con A 5.6x10 M (Fig. 5b). Both values are lower than that with leucoagglutinin, confirming the high specificity of TH glycopeptide for leucoagglutinin.

Discussion The method worked out for measuring the affinity of TH glycopeptide for lectins has the following advantages: (i) the amount of free and bound ligand can be measured directly; (ii) in the precipitation step the addition of coprecipitating compounds such as albumin, which may interact with the two reactants, is avoided; (iii) ethanol is selective in precipitating lectins. Our procedure for labelling the glycopeptide avoids the handl1 25 ing of more dangerous radionuclides as I, and consents a labelling with high specific activity that can detect concentra-9 tions up to 10 M. On the other hand the transformation of the nonulose chain of sialic acid to the corresponding eptulose chain might interfere with the carbohydrate recognition by 3 lectin. In our case this event did not seem to occur. The H-TH glycopeptide retained the hapten inhibitory activity. In pre-

377 liminary experiments we tried to label TH glycopeptide with dansyl-chloride. The attachment of the dansyl group to the NH 2 terminal moiety of the glycopeptide abolished its inhibitory activity in the haemagglutination induced by leucoagglutinin (unpublished results). In view of this observation also changes in the peptide backbone apparently affect the interaction of leucoagglutinin with glycopeptides. A great deal of evidence indicates that free N-acetylgalactosamine at very high concentration behaves as a specific inhibitor of leucoagglutinin. 0.02 M N-acetylgalactosamine elutes leucoagglutinin from a TH-Sepharose column, whereas isolectins of H-PHA type are retained by the conjugated gel (8). N-acetylgalactosamine is the only monosaccharide able to inhibit both the TH-leucoagglutinin interaction and the haemagglutination of desialylated red cell (4, 8). However its hapten power is 3-4 log lower (in molar terms) than that of TH glycopeptide. It has been suggested that a specific location of this sugar in the oligosaccharide sequence of TH glycopeptide could account for the binding to the lectin. The results reported here that the leucoagglutinin precipitates TH devoid of N-acetylgalactosamine (Sda non-active), and that variation does not occur in the affinity of TH glycopeptide for leucoagglutinin in the presence of 5 mM N-acetylgalactosamine (a molarity 1000 times higher than that of glycopeptide), lead us to suggest an alternative hypothesis: leucoagglutinin binds TH glycopeptide via sialylgalactose-N-acetylglucosamine side chains attached to the mannosyl core, and N-acetylgalactosamine, when present, plays only an additional role in strengthening the binding. It is worth noting that H-PHA isolectins, which have an aminoacid sequence very similar to that of leucoagglutinin (L-PHA) (3), strongly interact with a glycopeptide containing the sialyl-gal-glcNAc-mannosyl sequence (30), and we demonstrated that TH is also precipitated by H-PHA isolectins (8). We concluded that in TH glycopeptide the same glycidic sequence

378

is involved in binding to leucoagglutinin giving the K^ measured in the present studies. Moreover we find that the property of leucoagglutinin to recognize also N-acetylgalactosamine might explain its high affinity for specific receptors, such as those on the lymphocyte surface, and its high mitogenic activity.

The financial support of Consiglio Nazionale delle Ricerche, Rome, and Pallotti's Legacy for Cancer Research is acknowledged.

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3.

Miller, J. B., Hsu, R., Heinrikson, R., Yachnin, S.: Proc. Natl. Acad. Sci. U.S.A. 72, 1388-1391 (1975). Serafini-Cessi, F.: FEBS Lett. 114, 299-301 (1980).

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Serafini-Cessi, F., Montanaro, L., Sperti, S.: FEBS Lett. 120, 115-118 (1980). Tamm, I., Horsfall, F. L.: J. Exp. Med. 95, 71-97 (1952). Sikri, K. L., Foster, C. L., Bloomfield, F. J., Marshall, R. D.: Biochem. J. 181_, 525-532 . (1979). Serafini-Cessi, F., Franceschi, C., Sperti, S.: Biochem. J. 183, 381-388 (1 979) . Yachnin, S.: J. Immunol. 108, 845-847 (1972).

10. Hammarstrom, S., Engvall, E., Johansson, G. B., Svensson, S., Sundblad, G., Goldstein, I. J.: Proc. Natl. Acad. Sci. U.S.A. 72, 1528-1532 (1975). 11. Abbondanza, A., Franceschi, C., Licastro, F., SerafiniCessi, F.: Biochem. J. 187, 525-528 (1980). 12. Franceschi, C., Tabacchi, P., Licastro, F., Chiricolo, M., Serafini-Cessi, F. These proceedings

379 13. Morgan, W. T. J., Soh, C., Watkins, W. M.: Abstr. XV Congr Int. Soc. Blood Trans. Paris, p. 646 (1978). 14. Morgan, W. T. J., Soh, C., Watkins, W. M.: Glycoconjugates (Schauer, B., Boer, P., Buddecke, E., Kramer, M. F., Vliegenthart, J. F. G., Wiegandt, H. Eds.) Thieme, Stuttgart pp. 582-583 (1979). 15. Soh, C., Morgan, W. T. J., Watkins, W. M., Donald, A. S. R Biochem. Biophys. Res. Comm. 93^, 1 1 32-1 1 39 (1 980). 16. Macvie, S. I., Morton, J. A. , Pickles, M. M. : Vox Sang. 1_3 485-492 (1967) 17. Renton, P. H., Howell, P., Ikin, E. W., Giles, C.M., Goldsmith, K. L. G. ; Vox Sang. 1_3 , 1 1 32-1 1 39 (1967). 18. Morton, J. A., Pickles, M. M., Terry, A. M.: Vox Sang. 19, 472-482 (1970). 19. Means, G. E. , Feeney, R. E. : Biochemistry 1_, 2192-2201 (1968). 20. Dunstan, D. R., Grant, A. M. S., Marshall, R. D., Neuberge A.: Proc. R. Soc. London Ser. B. 186, 297-316 (1974). 21. Allen, A. K., Neuberger, A.: FEBS Lett. 60, 76-80 (1975). 22. Abbondanza, A., Franceschi, C., Licastro, F., SerafiniCessi, F.: Atti del XV Congresso della S.I.P. Sorrento1979 (in press). 23. Montreuil, J. : Adv. Carbohydr. Chem. Biochem. 3^7, 1 57-223 (1980) 24. Van Lenten, L., Ashwell, G.: J. Biol. Chem. 246, 1889-1894 (1971) 25. Afonso, A. M. M., Charlwood, P. A., Marshall, R. D.: Carbo hydr. Res. 89, 309-320 (1981). 26. Nisonoff, A., Pressman, D.: J. Immunol. 81_, 126-135 (1958) 27. Young, N. M., Leon, M. A.: Biochim. Biophys. Acta 365, 418 424 (1974). 28. Baenziger, J. U., Fiete, D.: J. Biol. Chem. 254, 9795-9799 (1979). 29. Perles, B., Flanagan, M. T., Auger, J., Crumpton, M. J.: Eur. J. Immunol. 7, 613-619 (1977). 30. Kornfeld, R., Kornfeld, S.: Ann. N. Y. Acad. Sei. 234, 276 -282 (1974).

INTERACTION OF HAPTOGLOBIN WITH LECTIN

Wanda Dobryszycka and Iwona Katnik Department of Biochemistry, Institute of Bioanalysis and Environmental Research, 5o-139 Wroclaw, Poland

Haptoglobin is a genetically determined serum a^-glycoprotein which is known to form with hemoglobin a complex showing catalytic activity of 'true' peroxidase. Human haptoglobin is polymorphic and is found in one of three major phenotypes: Hp 1-1, 2-2, and 2-1. Haptoglobin 1-1 is a tetramer composed of two a-subunits (m.w. 9 loo each) and two |3-subunits (m.w. 4o ooo each), the integrity of the four-chain structure being maintained by interchain disulfide bridges. Haptoglobins 2-2 and 2-1 are polymerized forms of higher molecular weights showing multiple bands in polyacrylamide gel electrophoresis. The.polymerism of haptoglobin was found to be related to a-subunits, whereas the carbohydrate-containing p-subunits are identical in the three haptoglobin types (1). Our previous papers concerned two 'active sites' on the surface of the haptoglobin molecule, namely one hemoglobin-binding, and the other antibody-binding (2,3). Moreover, the reaction of desialylated haptoglobin with specific hepatic receptors (lectins), was examined in vivo (4). The results obtained prompted the present study of haptoglobin interaction with a typical plant lectin concanavalin A (Con A) in vitro. Effect of a genetic type of haptoglobin on the reaction with Con A as well as relationships of hemoglobin- and Con A-binding to haptoglobin, were studied.

Lectins - Biology, Biochemistry, Clinical Biochemistry, Vol. II © Walter de Gruyter & Co., Berlin • New York 1982

382 Materials and methods Human haptoglobins type 1-1, 2-1, and 2-2 were prepared from ascitic fluids as described by Dobryszycka and Lisowska (5). The preparations used for the experiments were of loo % purity as checked by polyacrylamide gel electrophoresis and spectrophotometry. Tryptic glycopeptides of haptoglobin were obtained according to Katnik and Dobryszycka (6). Hemoglobin was isolated from horse blood by the method of McQuarrie and Beniams (7). Glycosylated hemoglobin was prepared from the blood of diabetic patients from the Institute of Internal Diseases of Medical Academy in Wroclaw, according to Saibene et al. (8). Haptoglobin-hemoglobin complex was prepared by mixing equi-molar amounts of both proteins. To form hemoglobin(haptoglobin-Con A) an excess of hemoglobin was added to the preformed haptoglobin-Con A complex and the non-bound hemoglobin was removed from the (haptoglobin-Con A)- hemoglobin precipitate by two-fold washing with saline. Desialylated haptoglobin was prepared by the action of neuraminidase from Vibrio cholerae (Calbiochem, Switzerland), followed by filtration through Sephadex G-25 (Pharmacia, Sweden). Anti-human haptoglobin antiserum was raised in rabbits (6). Con A and Con ASepharose were Pharmacia (Sweden) products, agarose was from Serva (F.R.G.), D (+)-mannose from Sigma (USA), respectively. Other reagents were products of P.O.Ch. (Gliwice, Poland). Haptoglobin concentration was determined by the peroxidase method of Jayle (9), protein concentration was measured by the Hartree (lo) modification of the Lowry 1 s procedure. Coprecipitation of haptoglobin with Con A was carried out according to Young et al. (11) by adding o.2 mg Con A to o.lo.8 mg of haptoglobin. The samples were incubated in PBS, pH 7.3 at 37 C for 3o min; the precipitate was centrifuged at 8ooo rev/min, washed twice with saline and dissolved in o.o2 N NaOH for the protein determination. From the coprecipitation curves, equivalence points were taken into consideration.

383 The amount of haptoglobin-Con A in the equivalence point was taken as loo %. Amounts of the precipitates of haptoglobin glycopeptides in the equivalence points were related to the above value. Electrophoresis in 7.5 % Polyacrylamide gel was carried out by the method of Davies (12), and immuno-affinoelectrophoresis according to B0g-Hansen and Brogren (13).

Results Three main genetic types:

haptoglobin 1-1, 2-2, and 2-1 were

used in the coprecipitation with Con A (Fig. 1). The amount of the precipitate formed was found to be dependent on the degree of polymerization of the haptoglobin molecule. The amount of precipitate for haptoglobin 1-1, 2-1, and 2-2

Figure 1. Coperecipitation of con A with haptoglobin 1-1, 2-1, 2-2, and desialylated haptoglobin 2-1 (asialo-Hp). Asialo-Hp was prepared from Hp 2-1 (neuraminidase removed 88 % of the sialic acid). Coprecipitation curves were obtained by adding 0.2 mg con A to 0.1-0.8 mg of haptoglobin.

384 were at the equivalence points o.24, o.28, and o.36 mg, respectively. The higher molecular weight, the more precipitate was formed. Desialylation of haptoglobin affected neither the shape of the curves nor equivalence points (in Fig. 1 is shown only the curve for desialylated haptoglobin 2-1). It remained to be explained whether all the various polymers of haptoglobin 2-1 and 2-2 reacted to the same extent with Con A or if there was a gradual reaction of particular polymers of these haptoglobin types until complete saturation by the lectin was achieved. Complexes of haptoglobin 2-1 and 2-2 with increasing amounts of Con A were prepared as shown for Hp 2-2 in Fig. 2. Supernatants were submitted to polyacrylamide gel electrophoresis. In the lower range of Con Ahaptoglobin ratios (gels 1-3, Fig. 2) electrophoresis displayed full typical patterns of haptoglobin 2-2 or 2-1. This indi-

i 0,2

OA

0,6

^

3

k

5

6

0.8

MgConA

Figure 2. Precipitation curve and electrophoretic pattern of the supernatant of 0.2 mg haptoglobin 2-2 and conA. The electrophoretic pattern in polyacrylamide is shown for each of the 6 con A quantities used (Gel 1 corresponds to the lowest con A quantity, Gel 6 shows an excess of con A). In Gels 1-3 all bands of Hp 2-2 are visible. The gels were stained with Amido Black 4B. Otherwise as for Fig, 1.

385 cated that specific binding of certain polymers has not occurred as specific binding would result in the selective disappearance of those bands. In the next experiment Con (o.2 mg) was linked to the preformed haptoglobin-hemoglobin complex or hemoglobin was bound to haptoglobin-Con A complex (Fig. 3). It can be seen that the weight of precipitate at the equivalence point of the coprecipitation curve of haptoglobin-hemoglobin complex with Con A was significantly lower than in the curve of haptoglobin-Con A complex with hemoglobin.

U2

OA

Q6

Q®MgHp2-2

Figure 3. Effect of hemoglobin binding to haptoglobin 2-2 on con A precipitation. 0.2 mg con A was added to 0.1-0.8 mg haptoglobin. 0.2 mg con A was added to haptoglobin-hemoglobin complex formed by adding eguimolarr amounts (0.075-0.6 mg) of hemoglobin to 0.1-0.8 mg haptoglobin. 0.2 mg con A was added to 0.1-0.8 mg haptoglobin; to the formed precipitate was added 0.075-0.6 mg hemoglobin, respectively. Otherwise as described in Methods.

386 Peroxidase activity of haptoglobin-Con A complex with hemoglobin as measured by the Jayle's method (9) amounted to 5o % of the activity of the native haptoglobin-hemoglobin complex, submitted to the same procedure as in the preparation of haptoglobin-Con A complex with hemoglobin. Trypsin digestion of haptoglobin resulted in four glycopeptides (6), which were used in coprecipitation reaction with Con A.

In Table 1, haptoglobin-Con A precipitate obtained

at the equivalence point is compared to the precipitates formed with the glycopeptides. The binding of Con A to glycopeptide I was extremely high, exceeding the reaction with native haptoglobin. The lowest binding was exhibited by glycopeptide II.

0.7 a>

|o.5

(Hp-ConA)-Hb

o.

c.0.3

Z

Hb-Hp

0,1 1 2

3 4 5 6 7 8 Hemoglobin glycosyldion ( % )

Figure 4. Effect of carbohydrate content of hemoglobin on the interaction of haptoglobin, hemoglobin and con A. Haptoglobin 2-1 and human hemoglobin with different glucose content were used. To equimolar haptoglobin-hemoglobin complexes (0.2 mg haptoglobin + 0.15 mg hemoglobin with increasing glucose content) was added 0.2 mg con A. To 0.2 mg haptoglobin was added 0.2 mg con A, followed by thorough washing with PBS. To the precipitates was added 0.15 mg hemoglobin with increasing glucose content. Otherwise as for Fig. 1.

387

TABLE 1 Relative reactivity of haptoglobin glycopeptides with Con A

Preparations

Coprecipitation with Con A

Native haptoglobin 1-1

o. 24

Glycopeptide I

o.35

Glycopeptide II

o. o7

Glycopeptide III

o. 2o

Glycopeptide IV

o.242

Glycopeptides I - IV were obtained by submitting haptoglobin, type 1-1 digested by trypsin to DEAE-Sephadex A-5o fractionation as described earlier (6). Coprecipitation reactions were carried out by adding o.2 mg Con A to o.l - 0.8 mg of the respective glycopeptides (11). Otherwise as in Methods.

Haptoglobin interaction with Con A was found to be completely inhibited by mannose concentration of o.o25 % (1.38 mM) in the reaction mixture;

o.o2 % mannose resulted in 36 % inhibition,

whereas o.ol5 % mannose was of no effect. It was of interest if hemoglobin-bound glucose in the blood of diabetics would act as an inhibitor of haptoglobin-Con A interaction when respective complexes with hemoglobin were formed.

In Fig. 4 are shown parallel coprecipitation curves

of glycosylated hemoglobin with haptoglobin-Con A complex or with Con A bound to the preformed haptoglobin-hemoglobin complex.

As can be seen, amount of the precipitate was inverse-

ly proportional to the extent of hemoglobin glycosylation.

388

Characteristic patterns of crossed immuno-affinoelectrophoresis in Sepharose-bound Con A and free Con A containing gels (Fig. 5 - B,C) indicated that haptoglobin must have at least two binding sites for Con A.

Discussion At physiological pH Con A is a tetramer composed of identical subunits of m.w. 26 ooo; de-binding site.

each subunit contains one sacchari-

Con A recognizes a 'mannobiosyl-N-acetyl-

glucosamine' structure which is present in many glycoproteins being a predominant part of the carbohydrate core (14).

The

various biological activities of Con A depend on initial bin-

A

o

n

B

o

c

0

(\

Figure 5. Crossed immuno-affinoelectrophoresis of haptoglobin. A. The reference pattern. The upper gel contained antibodies against haptoglobin (6 |ig/cm2). A blank intermediate gel separated the first dimension gel and the antibody-containing gel. B. The pattern obtained with immobilized conA (conA-Sepharose 100 |j.g/cm2) in the intermediate gel. C. The pattern obtained with free conA (100 ng/cm2) in the intermediate gel. The first dimension electrophoresis was performed for 1.5 h at 10 V/cm and the second dimension electrophoresis for 18 h at 2 V/cm at 4° C. The gels were stained with Amido Black 4B.

389

ding of the lectin to carbohydrate-containing receptors on the cell surface because these activities can be inhibited specifically by simple mono- and oligosaccharides. Interaction with Con A can be used for structural characterization of glycoproteins. It is not necessary that mannose in carbohydrate chains be terminal since the addition of N-acetylglucosamine, galactose and sialic aced distal to mannose did not destroy the binding activity to Con A (15). The outer sequence of the carbohydrate chains of haptoglobin consists of four sugars wherein the terminal sialic acid is bound to galactose, which is linked to N-acetylglucosamine and the latter, in turn to D-mannose (1). The role of the carbohydrate structure of haptoglobin in the formation of insoluble complexes with Con A was shown in the inhibition of coprecipitation by mannose and by hemoglobin-bound glucose. Moreover, desialylation of haptoglobin did not affect haptoglobin-Con A interaction. Coprecipitation of Con A with haptoglobin 1-1, 2-1, and 2-2 (m.w. approximately loo ooo, 2oo ooo, 4oo ooo, respectively) indicated that 1 mole of Con A has bound o.2 mole of haptoglobin independently of type (from data in Fig. 1). This experiment was carried out at pH 7.3, therefore the molecular weight of Con A tetramer was taken for calculations. The value of o.2 mole of haptoglobin per mole of Con A tetramer is rather low in comparison with o.l3 mole of ceruloplasmin per mole of Con A monomer, as found by Dulaney (16). This author suggests that the effective binding capacity would be reduced if the large size of the glycoprotein encouraged multiple binding to more than one lectin tetramer through different carbohydrate chains. This does not seem to be the case with three types of haptoglobin, showing significant differences in molecular weights but exactly the same molecular binding to Con A. Crossed immuno-affinoelectrophoresis allows a distinction between glycoproteins with one and several binding sites to Con A (13). Haptoglobin was found to have at least

390 two binding sites per molecule (Fig. 5). The problem of 'active binding sites' on the surface of haptoglobin molecule was interestingly illustrated in the experiments shown in Fig. 3 as well as in those related to peroxidase activity of haptoglobin-Con A complex with hemoglobin. At the equivalence point o.2 mg Con A + o.2 mg Hp 2-2 yielded o.36 mg of the precipitate. When equimolar haptoglobin-hemoglobin complex was used coprecipitation resuited in o.4 9 mg of the precipitate. On the other hand, when hemoglobin was added to the preformed haptoglobin-Con A complex, the amount of the precipitate increased to o.61 mg. Carbohydrate chains of haptoglobin do not participate in the formation of haptoglobin-hemoglobin complex (17), but the binding of hemoglobin to haptoglobin may establish a steric hindrance for complete Con A binding to the a-D-mannose sites in haptoglobin. If we assume complete binding in the haptoglobin-Con A complex (o.2 mg + o.2 mg = o.36 mg of the precipitate), the content of o.25 mg hemoglobin in the precipitate of (haptoglobin-Con A)-hemoglobin, indicates certain excess of hemoglobin (~o.l mg) , which would be bound to haptoglobin-linked Con A. Such a possibility exists if hemoglobin contained monosaccharide able to form a complex with Con A. When we checked glycosylation of different hemoglobin preparations used in the study of haptoglobin-hemoglobin interaction with Con A, we noticed that the higher glycosylation, the lower coprecipitation (Fig. 4). Independently of the degree of inhibition caused by the glucose content in hemoglobin, hemoglobin-binding to the haptoglobin-Con A complex resulted in higher amounts of precipitates(Fig.,4 upper curve) than in the case of Con Abinding to the preformed haptoglobin-hemoglobin complex (Fig. 4, lower curve). In spite of non participation of carbohydrate moiety of haptoglobin in the formation of an active haptoglobin-hemoglobin complex, it is clear that Con A-binding to haptoglobin interferes with fully active hemoglobin-binding.

The peroxidase

391 activity of the (haptoglobin-hemoglobin)-Con A was half the activity of the native haptoglobin-hemoglobin complex. Subunits of haptoglobin represent the binding site for hemoglobin in the polypeptide part (18), as well as the binding site for Con A in the oligosaccharide chain. These sites probably are located in such a proximity that the binding of one ligand, resulting in conformational changes of the haptoglobin molecule, affects the binding of another one. The reaction of Con A with haptoglobin glycopeptides indicates clearly main site of Con A-binding in the glycopeptide I. All of the four glycopeptides contained characteristic sugars (6), therefore they were able to bind Con A. However, it should be stressed that glycopeptide I was absolutely devoid of peroxidase activity when complexed with hemoglobin and it was inactive in the reaction with anti-haptoglobin antibody i.e. this fragment of haptoglobin molecule may be uniquely the site of Con A-binding. Further characterization of this glycopeptide is being carried out in our laboratory. Acknowledgement. This work was supported by the grant No.II. 1.2.3 from the Polish Academy of Sciences. We thank Mgr. Grzegorz Sawicki for kindly supplying the samples of glycosylated hemoglobins.

REFERENCES 1.

Putnam, F.W. (1975). The Plasma Proteins, Vol. 2 2d Edition, Academic Press, New York, pp. 1-46.

2.

Dobryszycka, W., Bec-Katnik, I. (1975). Polon. 22, 143-153.

3.

Katnik, I., Dobryszycka, W. (1978). 25, 325-332.

4.

Dobryszycka, W., Wozniak, M., Krawczyk, E., Furmaniak, E. (1981). Intern. J. Biochem. 13, 739-743.

Acta Biochim.

Acta Biochim. Polon.

392 5.

Dobryszycka, W., Lisowska, E. (1966). Acta 121, 42-49.

6.

Katnik, I., Dobryszycka, W. (1981). Acta 669.

7.

McQuarrie, E.B., Beniams, H.N. (1954). Biol. Med. 8_6, 627-632.

8.

Saibene, V. , Brembilla, L., Bertoletti, A., Bolognani, L., Pozza, G. (1979). Clin. Chim. Acta 93_, 199-2o5.

9.

Jayle, M.F.

(1951).

10. Hartree, E.F. (1972).

Biochim. Biophys. Biochim. Biophys. Proc. Soc. Exp.

Bull. Soc. Chim. Biol. 33, 876-88o. Anal. Biochem. £8, 422-427.

11. Young, N.M., Leon, M.A., Takahashi, T., Howard, J.K., Sage, H.J. (1971). J. Biol. Chem. 246, 1596-16ol. 12. Davis, B.J. (1964).

Ann. N.Y. Acad. Sei. 121, 4o4-427.

13. B0g-Hansen, T.C., Brogren, C.H. (1975). nol. 4, 135-139. 14. Wang, J.L., Edelman, G.M. (1978). 3ooo-3oo7. 15. Kornfeld, R., Ferris, C. (1975). 2614-2619. 16. Dulaney, J.T. (1979).

Scand. J. Immu-

J. Biol. Chem. 253, J. Biol. Chem. 25o,

Anal. Biochem. 99^ 254-267.

17. Cloarec, L. (1964). Contribution a 1'etude physico-chimique des haptoglobines humaines. Ed. R. Foulon, Paris, pp. 5o-52. 18. Osada, J., Dobryszycka, W. (1975). Acta 412, 3o6-316.

Biochim. Biophys.

INTERACTION OF POLYCLONAL AND MONOCLONAL HUMAN IMMUNOGLOBULINS G WITH VARIOUS LECTINS (CONCANAVALIN A, LENTIL AND PEA LECTINS) C. Chatelain 0 , A. Lemoine 0 0 0 , J.M. Dugoujon 00 , M. Blanc 00 and P. Rouge 0 (°) Laboratoire de Botanique et Biologie cellulaire, Faculté des Sciences Pharmaceutiques, Université Paul Sabatier, 35 chemin des Maraîchers, 31062 Toulouse Cedex, France. ( 0 o ) Centre d'Hémotypologie du C.N.R.S., Hôpital Purpan, 31052 Toulouse Cedex, France. ( o o c ) Laboratoire de Microbiologie et Immunologie, Faculté des Sciences Pharmaceutiques, Université Paul Sabatier, 35 chemin des Maraîchers, 31062 Toulouse Cedex, France. Various lectins interact with human serum glycoproteins including immunoglobulins and especially IgG (1,2,3). This interaction is specific since it can be inhibited by different sugars, which suggests that specific sugar residues occur on the immunoglobulin molecules. Studies on interaction between Con A and polyclonal and monoclonal IgG by means of precipitin tests (4) indicate that less than 51 of these immunoglobulins react with lectin. Similar results have been obtained with lentil (5) and pea (6) lectins. Unlike Ricinwi lectin which reacts with the IgG3 subclass only (7,8,9), the above mentioned lectins and especially the pea lectins (10) seem to interact with a few molecules belonging to several IgG subclasses. In the present work we have tried to verify this hypothesis, using affinity chrmatography techniques and Gm allotypes as specific markers for the IgG1 and IgG3 subclasses, and to compare the reactivity of various lectins (Con A, lentil and pea lectins) towards IgG. MATERIALS AND METHODS Isolation of lectins : Con A, lentil and pea lectins were isolated from the seed proteins precipitated between 301 and 60°ô ammonium sulfate saturation, by affinity chromatography on Sephadex G100 (Pharmacia) according to AGRAWAL & GOLDSTEIN (11). Unretained proteins were eluted with 0.05M tris buffer pH 7.6 containing 0.15M NaCl. Lectins were subsequently eluted with the same buffer containing 0.1M glucose. The pooled active fractions, freed of glucose by

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 . II © W a l t e r d e G r u y t e r &. C o . , B e r l i n • N e w Y o r k 1982

394

extensive dialysis against tris buffer, were concentrated and their purity was checked by disc-electrophoresis on polyacrylamide gel (12). Binding of lectins to CNBr-Sepharose and affinity chromatography : Binding of lectins to cyanogen-bromide-activated Sepharose 4B (100 mg lectin/5 g dry gel) was performed as described by the manufacturer (Pharmacia) . The gel was poured into a column and equilibrated with 0.1M phosphate buffer pH 7.0 and the solutions to be analyzed (polyclonal and monoclonal IgG1 and IgG3 proteins, Fab and Fc fragments of monoclonal IgG1 and IgG3 proteins) were applied to the column. The fractions retained were eluted with phosphate buffer containing 0.1M glucose. Gm allotypes specific for the IgG1 and IgG3 subclasses were determined on the unretained and retained fractions, after dialysis against phosphate buffer and concentration in Minicon (Amicon) cells. Isolation of polyclonal and monoclonal IgG proteins : We have analyzed normal human sera of known Gm phenotypes as a source of polyclonal IgG. The monoclonal IgG proteins were isolated by DEAE-cellulose chromatography (13) or by affinity chromatography on Protein A-Sepharose CL-4B (Pharmacia) according to HJELM (14). The isolated proteins were tested in haemagglutination inhibition experiments and their homogeneity was checked by Immunoelectrophoresis in agarose gel with appropriate rabbit antisera. Preparation of Fab and Fc fragments of IgGI and IgG3 : Monoclonal IgG1 and IgG3 proteins (myeloma sera) were treated with papain without reducing agent according to the procedure of RIVA.T & al. (15) and their Fab and Fc fragments were separated by DEAE-cellulose chromatography. The purity of the fragments was controled by cellulose acetate-strip electrophoresis, immunoelectrophoresis on agarose gel with appropriate rabbit antisera and determination of Gm allotypes. Determination of Gm allotypes : Gm allotypes specific for the IgG1 and IgG3 subclasses were determined by the haemagglutination inhibition technique using fresh 0 Rh+ (R-|R2) human red blood cells sensitized with incomplete (non agglutinating) anti-D antibodies of known specificity (16). All the antisera used were human reference antisera (17,18). Haemagglutination was performed on opalin plate, at room temperature, and the content of On allotypes was estimated by a

395 semi-quantitative method

: the mixtures of polyclonal and monoclonal im-

munoglobulins were diluted and the

greatest dilution producing the wea-

kest agglutination was noted. In all experiments, the antigenic composition of both IgG excluded and retained by affinity columns was studied. For this purpose, the unretained and retained fractions were pooled and concentrated to initial volume in Minicon cells or by lyophilization. Antisera : Rabbit antisera specific for human IgG, IgA, IgM, Fab and Fc fragments, K and L chains, used to check the purity of ours preparations, were purchased from Behring laboratories. RESULTS When polyclonal IgG were filtered on a lectin-Sepharose column, two peaks were obtained : the first one corresponds to the IgG which are not bound to the affinity gel (unretained IgG) and the second one which is eluted by adding glucose to the eluting buffer, represents the IgG molecules reacting with the lectin (retained IgG). In all cases, the retained IgG represents a proportion lower than 2% of the amount of IgG applied to the affinity column. Additional data indicate that lectin-IgG interaction involves the sugar moiety of the Fc fragment of the IgG molecules. Upon chromatography on Con A-, pea- or lentil lectin-Sepharose of either the Fab and Fc fragments prepared from polyclonal IgG, only a proportion smaller than 2% of the Fc fragment is retained and can be eluted by adding 0.1M glucose to the elu-

Fig. a,b : Affinity chromatography of the Fc (a) and Fab (b) fragments on a pea lectin-Sepharose column. Con A- and lentil lectin-Sepharose columns give identical results.

396 The extent to which two different lectins, Con A and pea lectin for example, do or do not react with the same polyclonal IgG was determined as follows : the unretained IgG corresponding to the first peak obtained by chromatography of polyclonal IgG on Con A-Sepharose, in a first step (Fig. c), were applied to a pea lectin-Sepharose column as a second step (Fig. d). The fact that, in this last step, no additional peak was found by adding 0.1M glucose (arrow) to the eluting buffer, means that the polyclonal IgG's which supposedly react with the pea lectin had been previously retained by Con A. The data obtained in a counter experiment (Fig. e,f) where polyclonal IgG which did not bind to a pea lectin-Sepharose column were then applied to a Con A-Sepharose column, were in total agreement with the above data. This indicates that Con A and pea lectin react to the same extent with the same polyclonal IgG's.

Fig. c,d : Affinity chromatography of polyclonal IgG on Con A-Sepharose column (c); the first peak corresponds to IgG unretained by Con A. Affinity chromatography of IgG unretained by Con A on pea lectin-Sepharose column (d); no additional peak corresponding to IgG retained by pea lectin is found by adding glucose (arrow) to the eluting buffer.

Fig. e,f : Affinity chromatography of polyclonal IgG on pea lectin-Sepharose column (e); as above, the first peak corresponds to IgG unretained by the lectin. Affinity chromatography of IgG unretained by pea lectin on Con A-Sepharose column (f); these IgG are totally excluded from the affinity column.

397

Fig, g,h,i,j,k,l,m,n : Affinity chromatography of polyclonal IgG on Con A-Sepharose column (g), lentil lectin-Sepharose column (i and m), pea lectin-Sepharose column (k). Affinity chromatography of IgG unretained by Con A on lentil lectin-Sepharose column (h), of IgG unretained by lentil lectin on Con A-Sepharose column (j), of IgG unretained by pea lectin on lentil lectin-Sepharose column (1) and of IgG unretained by lentil lectin on pea lectin-Sepharose column (n). Lentil lectin only (h and 1) retains polyclonal IgG fractions which are not retained by other lectins, Con A and pea lectin.

398 Figures g,h,i,j, and k,l,m,n, respectively are a similar comparison of Con A and lentil lectin, of pea and lentil lectins. In both cases, lentil lectin shows an additional specificity towards polyclonal IgG by comparison with Con A and pea lectin since a small additional retained peak is found in the second step of the experiment (Fig. h and 1). The specificity of lentil lectin: towards the IgG subclasses was studied by passing polyclonal and monoclonal IgG1 and IgG3 of known Gm phenotypes through a lentil lectin-Sepharose column. In this case, Gm allotypes are used as specific markers for the IgG1 and IgG3 subclasses and are determined both on the unretained and retained IgG fractions. The results (Table) obtained show that lentil lectin reacts with all of the polyclonal and monoclonal IgG1. In contrast, only certain polyclonal and monoclonal IgG3 interact with lentil lectin : practically all of the IgG3 Gm(5) proteins react with the lectin while most of the IgG3 Gm(21) proteins are not retained by filtering through the lentil lectin-Sepharose column. In all cases, only a very small proportion of IgG are retained : less than 21 of the IgG applied to the column, as stated above with polyclonal IgG and Fc fragment. DISCUSSION We have shown that Con A, lentil and pea lectins interact with polyclonal and monoclonal IgG and that the reactive group within the IgG molecules is localized to the Fc fragment. Similar results have been already obtained on IgG3 (9) and IgM (8) interacting with Rldlnvu, lectin. These results together with inhibition experiments using well defined sugars (19) indicate that interaction between immunoglobulins and lectins involves a specific recognition of the carbohydrate moiety of immunoglobulins by the binding sites of lectins. However, lectins exhibit different patterns concerning their interaction with polyclonal IgG. For instance, Con A and pea lectin react to the same extent with the same polyclonal IgG's while lentil lectin has a distinct additional specificity since it also reacts with other IgG fractions. These results suggest that the three lectins, although inhibited by the same sugars from the group III of MAKELA (20), have in fact slightly different binding sites. This .fact would explain that different lectins don't react

399 Table : Antigenic analysis of polyclonal and monoclonal IgG eluted from the lentil lectin-Sepharose column. The Gm allotypes of the retained fractions alone (eluted by adding glucose to the eluting buffer) are reported. (+ : presence, - : absence, (-) : normal absence) Polyclonal IgG

Test System

Phenotypes

for

Gm(1,2,21,4,5) Per. Bri. Rou. Mah.L.

Gm(1,-2,21,4,5) Lam.

Gm(1,2,21,-4,-5)

Mat.

Sam.

IgG1 Gn(1)

+

+

+

+

Gn(2)

+

+

+

+

Gm(4)

+

+

+

+

+

+

-

+

+

+

+

(-)

IgG3 Gn(5) Gn(21)

-

(-) -

-

+

Polyclonal IgG

Monoclonal IgG

Phenotypes

Subclasses

Test System for

Gm(-1,-2,-21,4,5) Mah.O.

IgG1 Gm(1) Llo.

IgG1Gm(4) Roq.

IgG3Gm(5) Rod. Tec. Bla.

IgG1 Gm(1)

C-)

Gn(2) Gm(4)

(-)

(-)

(-)

(-)

(-)

(-)

(")

(")

(-)

(")

+

(-)

+

(-)

(")

(")

+

(-)

(-)

-

IgG3 Gm(5) Gm(21)

400 with the same extent with various serum glycoproteins (1,21). In all cases, only a very small proportion of polyclonal and monoclonal IgG interact with lectins. These results altogether with other data (4,5, 6) indicating that less than 2% of the polyclonal or monoclonal IgG and their Fc fragment react with the three lectins suggest that unlike RicjjiiU lectin which reacts with the total IgG3 subclass (9), Con A, lentil and pea lectins only react with few IgG molecules belonging to one or several IgG subclasses, since IgG1, IgG2, IgG3 and IgG4 constitute respectively about 731, 141, 8% and 5% of polyclonal IgG. The data obtained with lentil lectin using Gm allotypes as markers of IgG subclasses show that this lectin partially reacts with IgG belonging to the IgG1 and IgG3 subclasses. In addition, such an interaction is evidenced with polyclonal and monoclonal IgG1 while only certain polyclonal and monoclonal IgG3, especially IgG3 Gm(5), react with the lectin. The partial interaction of IgG1 and IgG3 with lentil lectin could be explained on the basis of the complexity and heterogeneity of the carbohydrate moieties of IgG. Three main kinds of structural heterogeneity have been reported for the branched glycopeptides isolated from various monoclonal and polyclonal IgG proteins of different subclasses : - a czntAal heterogeneity based on differences in the core structure (22).

- a pMJjpheAai heterogeneity or microheterogeneity of the outer branches which show small differences in their terminal residues (23), reflecting carbohydrate units in varying stages of completion. - differences in the basic structure of the core units as reported for a glycopeptide B3 separated from a human myeloma IgG protein (24), which could reflect genetic differences. Lentil lectin could discriminate between these heterogeneities and only the IgG molecules exhibiting the convenient heterogeneity could interact with the lectin whatever their subclass. Nevertheless, ours results imply that the heterogeneity discriminated by lentil lectin would be inequally distributed among IgG subclasses, since unlike polyclonal and monoclonal IgG1, only certain polyclonal and monoclonal IgG3 and especially IgG3Gm(5) react with the lectin. R-lcXnai lectin (9) has a quite different behaviour towards IgG since it

401 reacts only with the polyclonal and monoclonal IgG3 proteins. SALTVEDT & NATVIG explain this specific interaction in the light of the open structure of IgG3, due to a more expended region of about 50 aminoacids between the C1 and C2 homology regions reported by MICHAELSEN & NATVIG (25 J. This open structure, allowing a better availability of the carbohydrate moieties of the IgG3 proteins for the binding sites of R-idniu, lectin, would explain the above mentioned result. In addition to the already described heterogeneities, the open structure of the IgG3 molecules could be taken into account to explain the different reactivity of IgG1 and IgG3 towards lentil lectin. In conclusion, the results obtained with Con A, lentil and pea lectins, together with the data concerning the Rj.cJ.niu lectin, indicate that phytolectins could be usefull for the study of the sugar moiety of immunoglobulins. REFERENCES 1. Père, M., Père, D. and Rougé, P. : Planta med. 41, 344-350 (1981) 2. Nilsson, M. and Birfg-Hansen, T.C. : Prot. Biol. Fluids 37, 599-602 (1979) 3. LjzSwenstein, H., B0g-Hansen, T.C. and Weeke, B. : Prot. Biol. Fluids 37, 611-614 (1979). 4. Leon, M.A. : Science 158, 1325-1326 (1967). 5. Young, N.M., Leon, M.A., Takahaschi, T., Howard, I.K. and Sage, H.J.: J. Biol. Chem. 246, 1596-1601 (1971). 6. Rougé, P. : C. R. Acad. Sc. Paris 283, Ser. D, 1823-1825 (1976). 7. Harboe, M., Saltvedt, E., Closs, 0. and Olsnes, S. : Scand. J. Immunol. 4, Suppl. 2, 125-129 (1975). 8. Saltvedt, E., Harboe, M., Foiling, I. and Olsnes, S. : Scand. J. Immunol. 4, 287-292 (1975). 9. Saltvedt, E. and Natvig, J.B. : Scand. J. Immunol. 6, 595-600 (1977). 10. Rougê, P., Chatelain, C., Lemoine, A. and Père, D. : C. R. Acad. Sc. Paris 285, Ser. D, 471-473 (1977). 11. Agrawal, B.B.L. and Goldstein, I.J. : Biochim. biophys. Acta 147, 262-271 (1967). 12. Davis, B.J. : Ann. N. Y. Acad. Sc. 121, 404-427 (1964). 13. Filitti-Wurmser, S. : Biochimie 58, 1345-1353 (1976). 14. Hjelm, H. : Scand. J. Immunol. 4, 633-637 (1975).

402 15. Rivât, C., Rivât, L., Lebreton, J.P., Rousseau, P.Y. and Ropartz, C.: Bull. Soc. Chim. Biol. 50, 997-1011 (1968). 16. Ropartz, C., Rivât, L. : Immun. Tech. 32, 1-15 (1967). 17. W.H.O. Committee on Human Immunoglobulin Allotypes. Review of the notation for the allotypic and related markers of human immunoglobulins: J. Immunol. 117, 1056-1058 (1976). 18. Workshop de l'O.M.S. sur les marqueurs génétiques des immunoglobulines humaines, Rouen (1974). 19. Chatelain, C., Rougé, P. and Lascombes, S. : Ann. pharm, fr. 36, 625630 (1978). 20. Makela, 0. : Ann. Med. Exp. Biol. Fen. 35, Suppl. 11, 1-133 (1957). 21. Rougé, P., Chatelain, C. and Père, D. : Planta med. 31, 141-145 (1977). 22. Spragg, B.P. and Clamp, J.R. : Biochem. J. 114, 57-63 (1969). 23. Kornfeld, R., Keller, J., Baenziger, J. and Korneld, S. : J. biol. Chem. 246, 3259-3267 (1971). 24. Kornfeld, R. and Kornfeld, S. : Ann. Rev. Biochem. 45, 217-237 (1976). 25. Michaelsen, T.E. and Natvig, J.B. : J. biol. Chem. 249, 2778-2783 (1974).

CHANGES IN THE RELATIVE AMOUNT OF INDIVIDUAL MICROHETEROGENEOUS FORMS OF SERUM c^-ANTICHYMOTRYPSIN IN DISEASE

Margaret Bowen, John G. Raynes, Edward H. Cooper Unit for Cancer Research, University of Leeds Leeds, Yorkshire, England

a^-Antichymotrypsin

(ACT) is a serum protein with a molecular

weight of approximately 68,000, about which comparatively little is known.

It has a carbohydrate content of 26%

although the structure and composition of the carbohydrate has not been accurately determined (1,2).

ACT has been purified

and its binding properties examined (3).

It has been shown

in vitro to bind chymotrypsin-like enzymes specifically (3). ACT is found in serum at a concentration between 0.2 - 0.6 g/1 and this is known to show a slight elevation during late pregnancy.

However, during an acute phase reaction to tissue

injury the levels of ACT may increase up to 5 times the normal value. The microheterogeneity of ACT has been demonstrated using affinity chromatography on concanavalin A sepharose (4). Lectin affinity crossed Immunoelectrophoresis, however, is a technique which combines the biospecific interaction between lectin and glycoprotein with crossed Immunoelectrophoresis (5).

It is a powerful method for demonstration of micro-

heterogeneity.

The lectin has different affinities for the

various carbohydrate structures of the microheterogeneous forms.

In preliminary experiments we found that the best

separations were obtained using free concanavalin A (con A) in the first dimension (as opposed to sepharose bound con A or intermediate gels).

Lectins - Biology, Biochemistry, C l i n i c a l Biochemistry, V o l . II © W a l t e r d e G r u y t e r &. C o . , Berlin • N e w Y o r k 1982

404 Following recent work on changes in orosomucoid microheterogeneity

(6,7), the aim of this study was to examine the

changes of protein pattern on lectin affinity crossed Immunoelectrophoresis of ACT in serum samples from different diseases, to see if the changes were consistent and related to the nature of the disease.

Materials Normal human sera and pregnancy sera were supplied by the Regional Transfusion Centre, Leeds, Yorkshire, U.K., and sera from patients with acute pancreatitis and following cholecystectomy were supplied by the Department of Surgery, Leects General Infirmary, Leeds, Yorkshire.

Septicaemia sera was

supplied by the Department of Microbiology, University of Leeds.

Samples of rheumatoid arthritis sera were supplied by

the Leeds General Infirmary and cancer sera were obtained from the serum bank at the Unit for Cancer Research, Leeds, Yorkshire.

Antiserum to human ACT was a gift from Behringwerke

AG, Marburg, Germany.

Con A and agarose (type II) were

obtained from the Sigma Chemical Co. Ltd., Poole, Dorset, U.K.

Methods Lectin affinity crossed Immunoelectrophoresis was carried out as described by Weeke (8) and modified with lectin in the first dimension as described by Bizig-Hansen and Brogen (5). Concanavalin A (50 g/1) was dissolved in 1 mmol/1 MgCl 2 , 1 mmol/1 MnCl 2 , 1 mmol/1 CaCl 2 and added to agarose gel in the first dimension to give 100 yg/cm^. run for approximately Veronal buffer

(pH 8.6).

The first dimension was

hours at 8 - 10 V/cm, with Tris Antiserum to human ACT was added to

the second dimension at between 6 - 1 6

vl/ml in order to

ensure that the whole pattern was resolved on the gel.

The

405 second dimension was run at 1 - 2 V/cm for 18 hours.

Total

ACT measurements were determined by radial immunodiffusion (9) against specific ACT antisera when required.

Results Lectin affinity crossed immunoelectrophoresis of ACT with con A on normal serum In normal crossed immunoelectrophoresis ACT appeared as a homogeneous protein with a bell-shaped precipitate (not shown). When con A was included in the first dimension the precipitate was composed of 3 protein components (Fig. 1).

This protein

precipitate pattern was found to be consistent for most normal human serum samples.

The mobility of protein component 1 was

found to be unretarded with con A by comparison with nonglycosylated standards (e.g. albumin).

2

1

3

1 cm

j

Fig. 1 Lectin affinity crossed immunoelectrophoresis of normal human serum (2 yl) against antiserum to ACT'(1 ul/cm 2 ) in the second dimension. The first dimension contained con A (100 vg/cm2).

Specific antiserum to ACT was used rather than anti-whole human serum for clarity.

The relative proportions of the

three peaks are shown in Table 2.

It was found that preg-

nancy serum samples produced a similar protein precipitate.

406 Changes in the protein pattern of ACT during disease processes The levels of ACT increase during disease (as typical for acute phase proteins).

As an example of an acute phase res-

ponse to surgical wounding, samples following cholecystectomy were examined and the pattern in Fig. 2 was obtained.

There

was a variation between patients and the relative proportions of the protein components are given in Table 2.

In some of

the samples (Fig. 2b) there was a shoulder to peak 3 which may indicate a fourth component.

Fig.2 Lectin affinity crossed Immunoelectrophoresis of two serum samples following cholecystectomy at the peak ACT level 3-4 days after surgery with con A (100 yg/cm^) and antisera against ACT (2 ul/cm2). Acute pancreatitis is an example of an acute inflammation of varying severity. The disease involves a release of large quantities of pancreatic enzymes, proteases, amylase and lipase into the extra-cellular fluid, resulting in an acute phase protein response as well as other biochemical changes. The ACT patterns obtained from serum samples of patients with acute pancreatitis can be seen in Fig. 3. Four patients were examined and in all cases the quantity of protein components 2 and 3 were raised. It was found that a comparison could be made between the level of components 2 and 3 and the severity of the attack. Protein component 1 was not increased significantly during the attack. The relative proportions of the component areas during an attack of acute pancreatitis for two patients is shown in Table 1.

407

A

B

3

2

1 \ I cm

Fig. 3 Lectin affinity crossed Immunoelectrophoresis of sera samples from a patient with acute pancreatitis a) at day 3 and b) §t day 5 after admission to hospital following a severe attack, with con A 100 yg/cm2 and antiserum against ACT (2 y 1/cm2). Septicaemia (pattern not shown) was found to show an increase in the components 2 and 3 but the increase was not as pronounced as in acute pancreatitis (Table 2). Advanced cancer and rheumatoid arthritis are both conditions in which ACT may remain high for many months. Examination of the patterns in advanced cancer showed that the level of component 1 was elevated as were component 2 and to a lesser extent, component 3. Four types of cancer (lung, kidney, prostatic and ovarian) were examined and the protein precipitate pattern is shown in Fig. 4. The relative protein component areas were calculated and are included in Table 2. Rheumatoid arthritis and ankylosing spondylitis are two chronic conditions in which ACT levels are elevated.

These

diseases produced patterns (not shown) with an increased peak 1 similar to the protein precipitates of cancer serum samples.

The relative peak areas are given in Table 2.

408 Table

1

Relative areas of peaks during a severe attack of acute pancreatitis

% of total precipitate area

Patient 1 Day 1 2 3 5 11

Patient 2 Day 1 2

4 7

Peak 3

Peak 2

Peak 1

30 42 51 57 30

46 40 37 33 52

24

20 33 46 33

44 42 39

35 24 13

45

18

/

18

12 9 17

• *,cfr\ vA

1cm

Fig. 4 Lectin affinity crossed Immunoelectrophoresis of advanced metastatic cancer serum samples (2 yl) with con A (100 yg/cm) in the 1st dimension and antiserum against ACT (2 yl/cm^) in the 2nd dimension: a) lung cancer b) ovarian cancer c) kidney cancer & d) prostatic cancer.

409 Table

2

Relative areas of peaks in sera from various normal and disease states % of total precipitate area No. of samples

Peak 3

Peak 2

Peak 1

Normal

2

8±5

43±7

49±11

Pregnancy

2

6±1

40±3

54±2

Post-operative cholecystectomy

10

28 ±13

45±5

21±9

50±2

23±4

Septicaemia

4

27±4

Rheumatoid arthritis

4

26±6

42 ± 1

31±13

40±4

29 ±9

24±7

42±1

34±6

Ankylosing spondylitis

4

Lung cancer

4

Kidney cancer

4

Prostatic cancer

4

Ovarian cancer

4

32 ± 5

20±3

44±2

36±6

23±4

45±4

32±6

22±7

49±7

29±3

Discussion a^-Antichymotrypsin forms three peaks on lectin affinity crossed Immunoelectrophoresis with free concanavalin A in the first dimension.

This suggests a degree of microheterogeneity

in the carbohydrate portion of the glycoprotein. experiments

Further

(not shown) involving the use of sugars have

shown the presence of a fourth component of ACT in the affinity precipitate.

The proportions of each protein com-

ponent (1, 2 and 3) are altered in disease.

It is possible

that some of these changes such as the increase in protein

410 component 3 may be due to the presence of proteases bound to the ACT. The presence of two ACT peaks in ordinary crossed Immunoelectrophoresis in amniotic fluid has been thought to be due to complex formation (10). In our experiments ACT did not show two peaks in ordinary crossed Immunoelectrophoresis, but addition of chymotrypsin enhanced a small retarded base line to the ACT precipitate. We are continuing to investigate the possible causes of the microheterogeneity. The changes in proportions of protein components of ACT are largest in acute diseases when there is a rapid release of ACT from the liver into the circulation. In these acute diseases examined there was an increase in the relative proportion of protein components 2 and 3. This was especially true of sera from acute pancreatitis patients where in severe cases the proportion of component 1 dropped below 15%. In certain serum samples from acute diseases a fourth component was evident as a shoulder to component 3 (see Fig. 2b & 3b). In cancer and in rheumatoid arthritis the proportions of the protein components are slightly different to those seen in an acute phase response. The pattern in chronic conditions appears more like the normal pattern but with each peak increased. The protein component 3 appears to increase in all disease states but only to a small extent in chronic states such as cancer. It is unlikely that these findings have an immediate clinical application. However, they raise a number of interesting guestions as to whether these changes in the proportion of microheterogeneous forms of ACT are associated with the function of the protein as an antiprotease after tissue damage, whether it be the result of surgery, acute pancreatitis, cancer or rheumatoid arthritis.

411 References 1.

Travis, J., Garner, D. , Bowen, J.: ..Biochemistry 5647-5651 (1978).

17,

2.

Heimburger, N., Haupt, H.: Clin. Chim. Acta. (1965).

3.

Travis, J., Bowen, J., Baugh, R-: Biochemistry 5651-5656 (1978).

4.

Nilsson, M., B^g-Hansen, T.C.: In: Protides of the Biological Fluids. Proceedings of the 27th Colloquium, Brussels, 1979 (H. Peeters, ed.), Pergamon Press, Oxford (pp. 599-602).

5.

Bjrfg-Hansen, T.C., Bjerrum, O.J., Ramlau, J.: Scand. J. Immunol. 4., suppl. 2, 141-147 (1975).

6.

Wells, C., Bizig-Hansen, T.C., Cooper, E.H., Glass, M.R. : Clin. Chim. Acta. 109, 59-67 (1981).

7.

Wells, C., Cooper, E.H., Glass, M.R., Bjzig-Hansen, T.C.: In: Lectins - Biology, Biochemistry, Clinical Biochemistry, Vol. I (T.C. BgSg-Hansen, ed.), de Gruyter, Berlin (pp. 339-346) .

8.

Weeke, B.: Scand. J. Immunol.

9.

Mancini, G., Carbonara, A.O., Heremans, J.F.: 2, 235-254 (1965).

12., 116-118 17,

2, suppl. 1, 47-56

10. Burnett, D. , Bradwell, A.R. : Biol. Neonate (1980).

(1973).

Immunochem.

37., 302-307

STUDY O F THE CON-A BINDING O F HUMAN ALPHA] ACID GLYCOPROTEIN DURING THE ACUTE INFLAMMATORY PROCESS. EVIDENCE FOR ALPHA] ACID GLYCOPROTEIN POPULATIONS O F DIFFERENT pi VALUES AFTER CON-A AFFINITY CHROMATOGRAPHY

IN

NORMAL AND INFLAMMATORY SERA.

Isabelle Nicollet, Jean-Pierre Lebreton, Marc Fontaine, Martine Hiron U-7B INSERM Génétique des Protéines Humaines, 543 chemin de la Bretèque, 76230 Bois-Guillaume, France

Alphai acid glycoprotein has the highest carbohydrate content (1) among the human serum glycoproteins (41 % of total weight) and due to its great number of sialyl residues (12 %) its isoelectric point (2) is the lowest (2.7). When analysed on starch gel electrophoresis at pH 2.9 or by isoelectric focusing, purified pooled native alphai acid glycoprotein and individual sera show a pi heterogeneity with polymorphic patterns (3). This heterogeneity could be attributed either to different types of sialyl-galactose linkage or to a molecular heterogeneity concerning the polypeptide chain (4). However, B0g-Hansen et-al. (5) reported that only part of alphai glycoprotein was retained by Con-A. In the present work, alphai

acic

ac

id

'

coprotein obtained from normal subjects and from patients undergoing an acute inflammatory process was submitted to a Con-A Sepharose chromatography and the non-reactive and reactive fractions were analysed by isoelectric focusing. In addition, the distribution and the evolution of molecular forms of alphai acid glycoprotein were studied in sera of inflammatory patients using crossed-immunoaffinity electrophoresis with free Con-A.

Materials and Methods

Alphai acid glycoprotein was obtained from normal human sera after precipitation with 1.5 M ammonium sulphate, followed by ion exchange chromato-

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 . II © W a l t e r d e G r u y t e r &. C o . , B e r l i n • N e w Y o r k 1982

414 graphy on CM-Sepharose CL-6B (Pharmacia) at pH 4.0 and by gel-filtration with Sephadex G-100. After inmunization with purified alphai acid glycoprotein, the rabbit immuneserum was precipitated with an equal volume of 70 % ammonium sulphate solution. Sera were obtained from healthy donors and from patients with an acute inflammatory process (6). Samples were collected throughout the course of the disease. Serum levels of alpha^ acid glycoprotein were determined by single radial

immunodiffusion.

Concanavalin-A affinity chromatography (Pharmacia Fine Chemicals Co. Sweden) was performed with 0.01 M sodium acetate buffer (pH 6.0) containing 1 mM MnCl2» 0.1 M NaCl, 1 mM MgCl2 and 1.5 %, sodium azide. The Con-A reactive material was desorbed by 0.2 M methyl-a-D-glucopyranoside

(Siqma

Chem. Co. S^ Louis Mo) in the sodium acetate buffer. Concentrated protein fractions were submitted to immuno-electrophoresis and isoelectric focusing. Isoelectric focusing was performed in a 2.5-4 pH gradient using LKB ampholytes (Ampholine, LKB-Produktor, AB, Bromma, Sweden). Polyacrylamide gels were prepared with acrylamide 4.9 % (w/v). N,N'-methylene-bis-(acrylamide) 0.15 % (w/v) (Fluka AG, Chemische Fabrik, CH 9470 Buchs), riboflavin 10 mg/1 (E. Merck, Darmstadt), acetic acid 1

saccharose 13.6 % (w/v), am-

pholytes 5 % (v/v) and distilled water 50 %_ (v/v). The gel dimensions were 12.5 x 26 x 0.1 cm. Pre-electrofocusing was performed for 1 h at a constant wattage (10 W). Then the samples (20 yl) were applied 1 cm from the cathode on Whatman 3 MM filter papers ( 4 x 2 mm) and submitted to electrofocusing for 2 h using the same wattage. After 2 h, the pH gradient was determined using the Ingold surface pH electrode, and the nel was immediately placed in fixing solution (0.7 M trichloroacetic acid) for 15 min. The polyacrylamide gel was stained for protein with Coomassie Brilliant Blue (Eastman G-250) and destained. The protein bands were identified by immunodiffusion analysis using the monosoecific anti-alpha^ acid glyconrotein inmuneserum. Pieces of the gel, sliced after focusing, were applied directly into the wells for protein identification. Crossed-immunoaffinity electrophoresis was carried out as described by R0nHansen et al. (7) and Kerckaert and Bayard (8) in thin-layer agarose gels using free lectin in the first dimension. Concanavalin-A

(Pharmindustrie,

1.B.F., Clichy, France) was included at a concentration of 1.5 mg/ml in \ %

415 agarose A37 (I.B.F.) melted in a 72 mM Tris/24 mM veronal/0.4 mM calcium lactate/0.2 mM sodium azide solution (pH 8.6). The plate (20 x 10 cm) was divided into two parts, one covered with the Concanavalin-A gel (20 x 5 x 0.1 cm) and the other with the reference gel : agarose 1 % without Concanavalin-A (20 x 5 x 0.1 cm). Samples (5 pi) were applied in each gel and electrophoresis was performed for 3 h at a constant voltage (10 V/cm) on a cooling plate. The second dimension was carried out in a 1 % agarose gel (10 x 10 x 0.1 cm) containing antiserum against human alpha^ acid glycoprotein (80 yl in three-quarters of the gel) for 18 h at 2 V/cm. Then the gels were dried on the glass plate, stained with Coomassie Brilliant Blue (Eastman R250) and destained with an ethanol/water/acetic acid (4.5 : 4.5 : 1, v/v) solution. Peak areas were quantified by planimetric evaluation and by weighing a paper photograph of each pattern. Normal individual serum (1 ml) or 6 mg purified alphai acid glycoprotein were incubated with 100 mil neuraminidase from Vibrio oholerae

in 0.05 M

sodium acetate buffer, pH 5.5 (Behringwerke AG, Marburg, F.R.G.) at 37°C for 15 h.

Results Alphai acid glycoprotein was present in both fractions (Con-A non-reactive and Con-A reactive) of the column even after treatment of serum or of purified alphas

aci

d glycoprotein by neuraminidase.

Immunodiffusion controls revealed that alphai

ac

i d glycoprotein in normal

serum focused at pH 3.0-3.5. Purified alpha^ acid glycoprotein equilibrated in 9 % NaCl focused at pH 2.9-3.2. Isoelectric focusing of purified alphas

ac

i d glycoprotein showed nine bands of different intensity within

the pH range of 2.9 to 3.4, but the Concanavalin-A non-reactive material showed a lower pi than the Con-A reactive material. Isoelectric focusing of the sera of the patients indicated that the bands corresponding to nonreactive material focused at pH 2.9 to 3.4 with six major bands between pH 2.9 and 3.1. The retarded fraction focused at pH 3.1 to 3.4 and also showed six bands. The three bands that focused around nH 3.1 were common to the two fractions. Nevertheless, there was a clear distinction between the average pi values of these two fractions. The sum of bands appearing in

416 the first fraction added to that of the second fraction gave an isofocusing pattern identical to that obtained with initial serum (Fig. 1). When affinity chromatography with immobilized Concanavalin-A permitted distinguishing of two molecular populations of alpha 1 acid glycoprotein, crossed immunoaffinity electrophoresis indicated three subpopulations : Concanavalin-A reactive (03), Concanavalin-A weakly reactive (02) and Concanavalin-A non-reactive (01). In order to compare the results obtained by affinity chromatography and crossed immunoaffinity electrophoresis methods, peak 1 (non-reactive) and peak 2 (reactive) obtained from whole serum or from purified alphai acid glycoprotein were submitted to crossed immunoaffinity electrophoresis. It was found that peak 1 included non reactive and the major part of weakly reactive alphai acid glycoprotein, and that peak 2 corresponded to reactive alphai

glycoprotein and a minor

part of the weakly reactive alphai acid glycoprotein. The methods used for

pH

day

•

33

23

41

90

- •J B i I p » -

lifiSn H F V V B w pi jab-

2.9

S

3.1 —

a

b

c

«*»

*5 -i..ais«!."isHi-.:

mm

«p»

.

^

3.4

^

Figure J : Isoelectric focusing patterns of four serum samples from patient 2 (see table III) . For each sample, (a) whole serum ; (b) Con-A non-reactive material ; (c) Con-A reactive material.

417 alphai acid glycoprotein purification did not induce any modification of the carbohydrate residues responsible for the linkage with Concanavalin-A, since whole normal individual serum and purified alphai acid glycoprotein revealed identical precipitation patterns by crossed immunoaffinity electrophoresis. Whole normal serum, when treated with neuraminidase, preserved its heterogeneity with regard to Concanavalin-A, since the precipitation pattern showed two peaks in these conditions, with an obviously higher cathodic electrophoretic mobility due to the release of sialic acid. When crossed immunoaffinity electrophoresis patterns of normal and inflammatory sera were compared, striking differences were noted. Reactive alohai acid glycoprotein (03 + 02) was greatly enhanced with respect to that present in the normal sera

(Fig.

2).

1

Figure 2 : Crossed immunoaffinity electrophoresis of a pool of normal human sera (1) and of a pool of human inflammatory sera (2).

418 The calculation of (03 + 02)/01 for each serum sample showed that the population of alphai acid glycoprotein molecules having an affinity for Concanavalin-A was significantly higher during inflammatory disease than in the normal state. The value of (03 + 02)/01 was below 1 in reference sera (Table I), while in patients' sera it greatly exceeded 1. For one of these patients (patient 3 (T.A.M.), see Table II), blood samples were obtained throughout the course of the disease until recovery. At the end of the disease period, the alpha^ acid glycoprotein level returned to normal, and the alphai

acic

' glycoprotein populations showed a precipitation profile

very similar to that of normal serum. Moreover, (03 + 02)/01 which was elevated at the onset of the disease, decreased to 0.88 at recovery. In

TABLE I DISTRIBUTION OF THE THREE ALPHAj ACID GLYCOPROTEIN SUBPOPULATIONS DETERMINED BY CROSSED IMMUNOAFFINITY ELECTROPHORESIS IN TWO NORMAL INDIVIDUAL SERA AND PURIFIED ALPHA j ACID GLYCOPROTEIN Con-A weakly reactive ajAG

Con-A nonreactive (03+02)/(01) a jAG

a ] AG

Con-A reactive ajAG

g/l

(03)%

(02)%

(01)%

L.J.P.

0.71

3.1

41.2

55.7

0.79

J.F.

1.08

6.6

41.0

52.4

0.90

1.5

41.9

56.6

0.77

Subj ect

Purified a¡AG from normal subject

-

the case of patient 4 (R.R.), the acute-nhase reactants 1 level

(haptoglo-

bin, alpha-l-antitryosin) was moderately elevated (6) and alpha^ acid glycoprotein level was normal throughout the disease (Table II) rum (03 + 02)/01 remained below 1, as for a normal subjet.

; in this se-

419

L O L O C O «d-

iO 00 «^J" C T t L O L O uDr-coLor-cocOLO

OCT»C O O CTI C O C O "vf L CT% «d- C OO C O C O N kO 1-H o

O +

w C / 3 Z O M H


> •J o

Q M o

< H Q
100 %

88 %

48 % 63 %

27 %

lo %

normal range 2.3-9.5 min

RIPA = Ristocetin Induced Platelet Aggregation FVIII:c, Normal Range: 50-15o %

FVIIIR: ag

36 %

427

1a

1b

Figures 1-3. Crossed Immunoelectrophoresis of Factor VIIIrelated antigen. Lectin is in the first dimension gel of patterns at the right (b) only. The anode is at the right for each electrophoretic pattern. Figure 1.

Single donor plasma.

Note the anodal bend in b.

Figure 2. Propositus' plasma. Figure 3. Maternal grandfathers' plasma. Note the increased precipitin area and the altered precipitin profile in b.

428 TABLE 2 The interaction of F VIII R:ag with con A in crossed Immunoelectrophoresis

Plasma Pool Single donor Maternal grandfather Mother Son

CAEP-area (mm2)

CIEP-area (mm2)

CAEP-area CIEP-area

78

lo2

0.76

12o 84 25 56

162 42 21 42

0.74 2.o 1.2 1.3

CAEP = Crossed affinity Immunoelectrophoresis CIEP = Crossed Immunoelectrophoresis

Discussion These results were consistent with an increased interaction con A and F VIII R:ag in this family, particularly the maternal grandfather. This study, then, conflicts with the conclusions of Bloom and Peake (5) and Howard et al. (6). The relatively high F VIII:RCF and the high F VIII R:ag in the maternal grandfather suggested this family might have variant von Willebrand disease. The mother's low F VIII R:ag lessened the likelihood of her being a hemophilia A carrier. Moreover, the high F VIII:RCF implied this family might have the Type II B variant having heightened interaction between factor VIII and platelets (16). The concept of variants in von Willebrand's disease has recently been questioned, however (15).

429 The finding of the increased precipitin area with lectin in the first dimension for the maternal grandfather's F VIII R: ag was unexpected. This increased precipitin area was reversed by placing 0.2M a.-methyl-D-glucopyranoside in the first dimension with lectin. A similar finding has been reported by Hau et al. for alpha a-fetoprotein (17). These data were consistent with the abnormal F VIII R:ag forming soluble complexes with con A which were electrophoresed into the second dimension and then precipitated by antibody to factor VIII. The con A may have enhanced the antigenicity of F VIII R:ag through steric effects, increased F VIII R:ag electrophoretic mobility, or decreased effective antibody concentration in the second dimension. Moreover, this pattern, suggesting an increased carbohydrate content of F VIII R:ag, may explain the apparent heightened interaction between F VIII and platelets in von Willebrand's disease Type II B. The anodal bend in Fig. lb is consistent with differential con A reactivity in F VIII R:ag identified by Howard et al. (6). They showed that cryoprecipitate of normal plasma has both slow and fast moving factor VIII related antigen with high reactivity to con A while cryosupernate of normal plasma contained fast moving F VIII R:ag with decreased con A reactivity. In order to complete this work additional experiments should be performed including: (a) crossed Immunoelectrophoresis of normal plasma and the maternal grandfather's plasma to identify fast-moving, variant F VIII R:ag; (b) a higher concentration (12 x 10 _6 M) of con A; (c) immobilized con A (18); (d) low mobility con A (Pharmacia) (19); (e) plasmas from patients with classical von Willebrand's disease and hemophilia A; and (f) F VIII:RCF with varying ristocetin concentrations. Finally, our inability to confirm previous data (5,6) probably was related to the use of a von Willebrand variant family.

430 These results, however, are compatible with an abnormal factor VIII carbohydrate content causing a heightened tion with con A.

interac-

This abnormal interaction should be diagno-

stically important since it may be consistently present in certain subgroups of von Willebrand's disease patients, thus helping to differentiate variant von Willebrand disease from classical von Willebrand disease, hemophilia A, and normal. Acknowledgement.

This work was supported in part by grants

from Sigma Xi and the Southern Medical Association. Preliminary work related to this paper was presented in abstract at the VIII^"^1 International Congress on Thrombosis and Haemostasis, Ontario, Canada, July, 1981.

REFERENCES 1.

Anonymous

(1975).

Lancet 2, 516.

2.

Kass, L., Ratnoff O.D., Leon, M.A. vest. 48, 351-58.

3.

Gralnick, H.R., Coller, B.S., Sultan, Y. (1976). ce 192' 56-59.

4.

Zimmerman, T.S., Voss, R., Edgington, T.S. Clin. Invest. 6_4, 1298-13o2.

5.

Peake, I.R., Bloom, A.L. (Stuttg.) 3_Z' 361-62.

6.

Howard, M.A., Perkin, J., Koutts, J., Firkin, B.G. Br. J. Haematol. 4J7, 6o7-15.

7.

Krauss, J.S., Sheard, M.H. (1982). (Stuttg.) (in press) (Abstract).

Thrombos. Haemostas.

8.

Krauss, J.S., Sheard, M.H. (in press) .

Br. J. Haematol.

9.

Langdell, R.D., Wagner, R.H., Brinkhous, K.M. ¿X. Lab. Clin. Med. 41, 637-47 .

(1977).

(1982).

(1969).

J. Clin. InScien-

(1979).

J.

Thrombos. Haemostas. (1981).

(1953).

431 10.

Zimmerman, T.S., Ratnoff, O.F., Powell, A.E. (1971). J. Clin. Invest. 5o, 244-254.

11.

Brinkhous, K.M., Graham, J.E., Cooper, H.A., Allain, J.P., Wagner, R.H. (1975). Thromb. Res. 6, 267-72

12.

Howard, M.A., Firkin, B.G. (1971). Haemorrh. (Stuttg.) 26, 362-69.

13.

B0g-Hansen, T.C., Bjerrum, O.J., Ramlau, J. (1975). Scand. J. Immunol. j4, suppl. 2, 141-47.

14.

Weeke, B. (1973).

15.

Abildgaard, C.F., Suzuki, Z., Harrison, J., Jefcoat, K., Zimmerman, T.S. (198o) . Blood 56^, 712-16.

16.

Ruggeri, Z.M., Pareti, F.I., Manucci, P.M., Ciavarella, N., Zimmerman, T.S. (198o). N. Engl. J. Med. 3o2, lo4751.

17.

Hau, J., Westergaard, J.G., Ipsen, L., Teisner, B., B0gHansen, T.C., S0ndergaard, K. (1982). These Proceedings.

18.

B0g-Hansen, T.C. (1979). In Egly J. Med. Proceedings of the third international symposium on affinity chromatography and molecular interactions. Inserm: Paris, 86, 399-416.

19.

B0g-Hansen, T.C., Takeo, K. (198o). 67-71.

Thrombos. Diathes.

Scand. J. Immunol. 2, 47-46.

Electrophoresis 1,

COMPARABILITY OF CARBOHYDRATE HETEROGENEITY PATTERNS OF HUMAN ALPHA-FETOPROTEIN IN DIFFERENT BIOLOGICAL FLUIDS. Kim Toftager-Larsen Department of Clinical Chemistry, S0nderborg Hospital, DK-6400 S0nderborg, Denmark.

With the introduction and development of crossed affinity-immunoelectrophoresis (4,5) a very useful tool for investigation of glycoprotein heterogeneity became available. Several different glycoproteins have been studied in health and disease, examples being ferritin (1), apha-l-acid glycoprotein (2o), inter-alpha-trypsin inhibitor (9) and alpha-fetoprotein (AFP) (2,8,12,13,15-18) . Human AFP has been extensively studied by the technique and determination of heterogeneous fractions of AFP has proven to be a valuable tool in the prenatal diagnosis of fetal neural tube defects (15-17). Furthermore, certain oncodevelopmental aspects have been verified by the studies of fetal AFP and AFP from AFP-producing tumours (18), and the clinical role of AFP carbohydrate heterogeneity determinations in the management of AFP-producing tumours is presently under investigation (2,18). Due to a large variation in the AFP concentration, the specimens investigated range from undiluted serum samples to highly diluted amniotic fluid, and the concentration of other glycoproteins also reacting with the lectins in question will therefore inevitably show a large variation. Also, slight changes in analytical circumstances such as pH, agarose gel concentration and lectin concentration are likely to occur. The purpose of this study has therefore been to investigate the reliability and comparability of results obtained from va-

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 . II © W a l t e r d e Gruyter & C o . , Berlin • N e w Y o r k 1982

434 rious biological fluids under such differing analytical circumstances .

Material & methods Two lectins with a known affinity for AFP was used. Concanavalin A (con A) was obtained commercially (Pharmacia Fine Chemicals, Uppsala, Sweden), whereas lens culinaris agglutinin (LCA) was purified from lens seeds (19)Unless otherwise stated, crossed affinity Immunoelectrophoresis was performed in a 1.5 mm 1% agarose gel, pH 8.6, samples were diluted to a final concentration of AFP of 2 5 mg/1 and 10 |il (corresponding to 0.25 p.g of AFP) was applied. The first dimension electrophoresis was run at 5 V/cm in a gel containing free lectin until an albumin marker had migrated 4 cm (con A) or 7 cm (LCA). The concentration of free lectin in the gel was 3.81 x 10 ^ M con A or 0.64 x 10 - 5 M LCA. The second dimension electrophoresis was run at 2 V/cm at a right angle to the first for at least 16 hours into.a gel containing 3.75 jil/ml (0.56 M-l/cm2) of anti-AFP antibody (DAKO Immunoglobulins Ltd., Copenhagen, Denmark) and 2.25 )il/ml (0.34 nl/cm 2 ) of anti-prealbumin antibody (DAKO Immunoglobulins) . After the electrophoresis the plates were pressed, dried and stained with Coomassie Brilliant Blue. The migration of AFP fractions was determined relative to prealbumin, and the size of fractions was measured. The following parameters were studied: 1) Analytical variation: 7 amniotic fluid samples containing 2.5, 4.0, 6.5, 10.0, 15,0, 20,0 and 50,0% con A non-reactive AFP were each analysed 25 times with con A. The standard deviation for the value of non-reactive AFP was calculated for each of the samples, and the coefficient of variation was determined from the curve thus found. 2) Concentration of free lectin in the gel: An amniotic fluid sample was studied at 5 different concentrations of free lectin in the gel: 0.95 - 15.24 x 10~ 5 M con A and 0.08 - 5.13 x lo - 5 M LCA (concentration of bindingsites). 3) Concentration of other glycoproteins: An amniotic fluid sample from very early pregnancy (8 weeks of gestation) with a high concentration of AFP (243 mg/1) was chosen, because the LCA weakly-reactive and the con A non-reactive fraction at this stage is relatively high.

435 Aliquots of the sample were diluted 1:10 with a) human AFPfree serum obtained by affinity chromatography on a column of anti-AFP antibody coupled to CNBr-activated Sepharose (Pharmacia Fine Chemicals), b) human AFP-free serum prediluted 1:4 and c) 1:32 with (electrophoresis-)buffer and d) with buffer. The concentration of anti-prealbumin antibody in the second dimension gel was adjusted accordingly. 4) Amount of AFP applied: The same sample was applied a) undiluted, b) diluted 1:3, c) 1:9, d) 1:27, e) 1:81 and f) 1:243 with AFP-free human serum (prediluted 1:12 by buffer) in order to obtain the same total protein concentration in the 6 samples. The anti-AFP antibody was adjusted to obtain precipitates of the same height in all experiments. In the two analyses with the highest dilution visualization was obtained by including isotopelabelled AFP in an intermediate gel as described by N0rgaard-Pedersen (14). 5) pH, agarose gel concentration: An amniotic fluid sample was analysed at pH 8.2, 8.6 and 9.2, and in gels of a concentration of 0.5, 1.0 and 2.0%. Results Analytical variation: The standard deviation increased gradually from Q.3% at a con A non-reactive fraction of AFP of 2.5% to 2.0% at a nonreactive fraction of 50% (figure 1). The corresponding coefficient of variation was very high at low non-reactive fractions, but at 5% non-reactive AFP it decreased to values below 10%, further decreasing to approximately 4% at high non-reactive fractions. The experiments were performed for con A only. However, the events taking place in the second dimension electrophoresis are largely independent of the first dimension electrophoresis, and the variation found is likely to be general to measurements of precipitates in crossed Immunoelectrophoresis . Lectinconcentration: At a concentration of free con A of 0.95 x 10 M the first indication of heterogeneity of AFP appeared as a hump on the cathodal side of the precipitate. At 1.91 x 10" 5 M two distinct precipitates were seen, and with increasing concentra-

436 S D. C.V.

percent c o n A non-reactive A F P

Figure 1. The standard deviation of the con A non-reactive fraction of AFP calculated from 25 electrophoreses of each of 7 indicated samples. The coefficient of variation was calculated from the standard deviations.

tions these precipitates gradually separated more (mot shown). At no concentration was more than two precipitates seen. Low concentrations of LCA in the gel apparently separated AFP in two fractions (figure 2), however at concentrations from 0.32 -5 -5 x 10 to 1.28 x 10 M LCA 3 definite precipitates could be distinguished. At 5.13 x 10 ^ M the weakly and strongly reactive fractions were equally retarded and fused into one precipitate (figure 2). By plotting the inverse of the relative migration as a function of the inverse of the lectin concentration according to B0g-Hansen and Takeo (7) , dissociation constants of 5.6 x 10

M

437

0.08*10~5M

0.64X10-5M

0.16*10

-M

5

1.28*10" ® M

0.32X1O-5M M

u-J^xiO

2.56* 1 0 _ 5 M

Figure 2. The heterogeneity pattern of AFP with increasing concentrations of LCA in the first dimension gel. The first and last 2 cms of the individual plates have been omitted (including the prealbumin precipitate). Anode to the right in first dimension electrophoresis, upward in second dimension.

5.13*10~5M for con A reactive AFP, and 0.46 x 10

-5

M and 0.13 x 10

5

M for

LCA weakly and strongly reactive AFP respectively was found (figure 3). The described fusion of the LCA weakly and strongly reactive AFP precipitates could be predicted from the plot. The distribution of AFP fractions is seen in figure 4. Within the range of lectin concentrations where 2 (con A) or 3 (LCA)

438 1

Figure 3. The plotting of the inverse relative mobility as a function of the inverse lectin concentration, is the mobility of the glycoprotein fraction without lectin in the gel, R,^ the mobility in the presence of lectin. Con A-S is the (strongly) reactive fraction of AFP, LCA-S and LCA-W the strongly and weakly reactive fraction of AFP.

distinct precipitates could be seen, a small but definite increase in the strongly reactive fraction was observed with increasing lectin concentration

(figure 4). This was most pro-

nounced for con A and was of a magnitude that could not be explained by analytical variation only. Other glycoproteins: By the described dilution of a sample with a very high concentration of AFP a 140-fold difference in the total proteincontent was achieved. Although the relative content of glycoproteins in amniotic fluid may be slightly different from serum (the amniotic fluid/serum ratio of serum proteins is dependent on the molecular size of the protein

(11), at least a 100-fold

difference in the glycoprotein content between- the least and

439

Figure 4. Distribution of AFP on fractions with increasing concentration of lectin in the first dimension gel. N, W and S are non-, weakly- and strongly-reactive fractions respectively. coo A

Tee tee 27 &e Tee iee 27 u M MOTilN APPLIED

7ee tee 27 &e pg

tss ies 27 &s pftOTEIN AmiED

Figure 5. Relative migration and distribution of AFP with increasing protein content in applied sample. The total AFP concentration was 25 mg/1 corresponding to 0.25 ßq applied in all samples. Abbreviations as in figure 4.

4 40

Figure 6. Relative migration and distribution of AFP with increasing AFP content in applied sample. The total protein content was approximately the same in all samples. Abbreviations as in figure 4. LCA

2.43 ug A F P

0.81 ug A F P

0.27(iBAFP

Figure 7. Appearance of AFP precipitates with increasing amount of applied AFP in LCA crossed affinity Immunoelectrophoresis. The first 3 cms of the individual plates have been omitted. Anode to the right (1. dimension electrophoresis) . most dilute sample is likely to be present. Even so, no difference in the relative migration of or distribution on fractions was found whether con A of LCA was used

(figure 5).

Amount of AFP: By the dilution of the same amniotic fluid sample by AFP-free human serum prediluted 1:12 with buffer roughly the same total protein concentration in all experiments in this series was

441

achieved, whereas the concentration ef AFP increased. No difference in the relative migration was found, and the distribution on fractions was unchanged (figure 6). However, with very high amounts of AFP in the gel the precipitate became blunt and the interpretation and measurement indeed very difficult or impossible (figure 7) . pH, agarose gel concentration: A pH of 8.2 or 9.0 and gel concentration of 0.5 or 2.0% probably are beyond the extremes likely to be encountered in routine analyses. However, even at these circumstances no change in relative migration or in fractional distribution was found for con A and for LCA.

Discussion In addition to AFP several other glycoproteins present in the biological fluids investigated react with con A and LCA, but no significant change in the mobility or heterogeneity pattern of human AFP was found, even with very large differences in the content of other glycoproteins in the applied samples. . This is in good accordance with B0g-Hansen et al. who found the same heterogeneity pattern for human alpha-l-acid glycoprotein and for mouse serum alpha-l-carboxyl esterase in serum and in a purified preparation (6) . However, Faye and Sailer (9) have shown that the observed pattern for a purified preparation of inter-alpha-trypsin-inhibitor differs from the same protein when present in serum. When inter-alpha-trypsin-inhibitor-free serum was added to the purified preparation, the serum pattern was restored. Therefore, it may be necessary to investigate the influence of other glycoproteins on the carbohydrate heterogeneity pattern of each individual glycoprotein examined by crossed affinity Immunoelectrophoresis.

442 Earlier reports have concluded that human AFP consists of two fractions only, when examined with LCA (3). This may be explained by the use of too high a concentration of LCA as seen from figure 2. Likewise, human AFP has been described as being totally reactive with Ricinus Communis Agglutinin, showing only one precipitate (12). However, experiments with a broad range of Ricinus Communis Agglutinin concentration have revealed as many as 5 fractions of human AFP with different mobilities (Toftager-Larsen, unpublished). From our experiments with increasing lectin concentrations it seems evident that a careful evaluation of the proper lectin concentration should be undertaken before any conclusions concerning the heterogeneity of glycoprotein are drawn. The findings of an increasing strongly-reactive fraction of AFP with increasing amounts of lectin in the gel was somewhat puzzling, but as recently described by Hau et al. (1Q) it may be explained by the occurence of a shielding of some antigenic sites on the protein by the binding of lectin. This would give a higher strongly-reactive precipitate leading to the interpretation of a larger proportion of AFP being strongly-reactive. The problem may be overcome by adding the specific oligosaccharide to the second dimension gel (10), however, the changes in distribution encountered are unlikely to be of any importance in routine clinical use of the method, when care is taken in the preparation of the gels. With high concentrations of AFP in the applied sample interpretation became difficult or impossible because precipitates became blunt and tended to fuse. This presumably is caused by an 'overloading' of the gel with the excess of lectin being diminished. The amount of AFP usually applied is at least ten times less than the amount causing such a blunting, and well within the range where distinct precipitates will be seen. With a careful preparation of the gels for crossed affinity Immunoelectrophoresis small variations in the pH and in the

443

agarose concentration will still be found. In agreement with B0g-Hansen & Takeo (7) it was found that at values of pH and of agarose gel concentration at what can be considered as extremes, the results are not disturbed. The major objective of the present study was to show, whether any one factor of a theoretical potential to distort the results would indeed do so. This is an important objective as it is essential that conclusions drawn from studies of carbohydrate heterogeneity of human AFP are highly reliable. In the field of prenatal diagnosis of neural tube defects crucial decisions may be taken based on the results of determination of the heterogeneity pattern of AFP, and incorrect results may distort the oncodevelopmental interpretation and the conclusions drawn from the comparison of results on AFP from fetal specimens and from AFP-producing tumours. This study has shown that within the limits outlined - and these limits are well outside what is likely to be encountered in clinical materials -, the crossed affinity Immunoelectrophoresis as a method for determination of carbohydrate heterogeneity of human AFP is very stable and reliable with a good reproducibility irrespective of changing analytical circumstances .

Acknowledgements This study has been supported by 'Fonden for laegevidenskabelig forskning m.v. ved sygehusene i Ringk0bing, Ribe og S0nderjyllands amter 1 . The expert technical assistance of Mrs. Jette Rasmussen and secretarial assistance of Mrs. Karin Hansen is gratefully acknowledged.

444 References 1.

Andersen, M.M., Lihme, A., B0g-Hansen, T.C.: These proceedings .

2.

Breborowicz, J., Mackiewicz, A., Breborowicz, D.; Scand. J. Immunol, (in press 1981).

3. 4.

Brock, D.J.H.: Biochem. Soc. Transact. 1179-1195 (1979). B0g-Hansen, T.C.: Anal. Biochem. 56, 48o-488 (1973).

5.

B0g-Hansen, T.C.: In: Proceedings of the Third International Symposium on Affinity Chromatography and Molecular Interaction, Inserm Symposia Series 8_6, 399-416 (1979) (Ed. J.M. Engly).

6.

B0g-Hansen, T.C., Jensen, P., Hinnerfeldt, F,, Takeo, K.: In: Lectins: Biology-Biochemistry-Clinical Biochemistry. W. de Gruyter 1, 241-258 (1981) (Ed. T.C. B0g-Hansen). B0g-Hansen, T.C., Takeo, K.: Electrophoresis 1,67-71(198o)

7. 8. 9. 10.

Endo, Y., Miyazaki, J., Oda, T.: These proceedings. Faye, L., Salier, J.-P.: Electrophoresis 1, 193-197 (198o) Hau, J., Westergaard, J.G., Teisner, B., B0g-Hansen, T.C., S0ndergaard, K. These proceedings.

11.

Johnson, A.M., Umansky, I., Alper, C.A., Everett, C., Greenspan, G.: J. Pediatrics 84, 588-593 (1974),

12.

Kerckaert, J.-P., Bayard, B., Biserte, G.; Biochim. Biophys. Acta 576, 99-lo8 (1979).

13.

Mackiewicz, A., Breborowicz, J.: Oncodevelop. Biol, Med. 1, 251-261 (198o).

14.

N0rgaard-Pedersen, B.: Scand. J. Immunol. ^ suppl. 4, 1-45 (1976).

15.

N0rgaard-Pedersen, B., Toftager-Larsen, K., Philip, J«, Hindersson, P.: Clin. Genet. I7_, 355-361 (198oJ .

16.

Toftager-Larsen, K., Kjaersgaard, E., Jacobsen, J.C., N0rgaard-Pedersen, B.: Clin. Chem. 26., 1656-1659 (198o) .

17.

Toftager-Larsen, K., N0rgaard-Pedersen, B.: In: Lectins; Biology-Biochemistry-Clinical Biochemistry. W. de Gruyter 1, 293-3o2 (1981) (Ed. T.C. B0g-Hansen).

18.

Toftager-Larsen, K., Petersen, P.L., N0rgaard-pedersen, B, In: Lectins: Biology-Biochemistry-Clinical Biochemistry. W. de Gruyter 1, 283-292 (1981) (Ed. T.C. B0g-Hansen). Toyoshima, S., Osawa, T., Tonomura, A.: Biochim. Biophys. Acta 221, 514-521 (197o).

19. 20.

Wells, C., B0g-Hansen, T.C., Cooper, E.H., Glass, M.R.: Clin. Chim. Acta lo9, 59-67 (1981).,

DEMONSTRATION AND QUANTIFICATION OF MICROHETEROGENEITY FORMS OF HUMAN a-FETOPROTEIN BY LECTIN AFFINITY ELECTROPHORESIS

J.Breborowicz and T.C.B0g-Hansen Department of Pathological Anatomy, Academy of Medicine, Poznan, Poland, and The Protein Laboratory, University of Copenhagen, Denmark

The microheterogeneity of human a-fetoprotein (AFP) was first demonstrated by Smith and Kelleher (1) with con A affinity chromatography and then by Kerckaert et al. (2) with lectin affinity Immunoelectrophoresis. AFP is found to be microheterogeneous with both con A and LCA (lens culinaris agglutinin). With both lectins the ratio of the microheterogeneity forms differ in normal and in pathological amniotic fluids (3-6) and also in sera of patients with various pathological disorders (7-10). The results obtained by various groups are consistent and the quantitative assessment of the AFP microheterogeneity forms is proposed as an adjunct diagnostic method for detection of malformations of the central nervous system and for differential diagnosis of cancer. In this work we report some attempts to study the microheterogeneity of AFP qualitatively and quantitatively by electrophoresis. We tested various parameters and confirmed the microheterogeneity of AFP as demonstrated by affinity Immunoelectrophoresis to be due to specific lectin-glycoprotein interaction, and we could also demonstrate additional microheterogeneity forms of human AFP. We suggest some optimal experimental conditions and some technical modifications which might be useful for the clinical evaluation of AFP.

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 . II © W a l t e r d e G r u y t e r &. C o . , B e r l i n • N e w Y o r k 1 9 8 2

446 Materials and Methods Eleven AFP-positive samples were used in this study: Two samples of amniotic fluid obtained by amniocentesis of normal pregnancies in the 12th week of gestation (kindly supplied by dr. Westergaard, Department of Obstetrics and Gynecology, University of Odense, Denmark); A pool of cord serum collected at term (kindly supplied by dr. Jakubek, Institute of Gynecology and Obstetrics, Academy of Medicine, Poznan, Poland); Purified AFP from this cord serum pool (40 jig/ml) ; Serum from three hepatoma patients; Ascites fluid from one hepatoma patient (30 p.g/ml) ; Purified AFP from this ascites fluid (40 jj.g/ml) ; Serum from two patients with gonadal yolk sac tumour. AFP was purified according to Nishi (10) omitting the denaturing conditions for desorbtion of the immunosorbent. All samples, except the first four samples, were previously studied with con A and LCA affinity Immunoelectrophoresis and the microheterogeneity forms have been described (7,8). The samples contained more than 80 ng AFP/ml as estimated by double antibody radioimmunoassay (Swierk, Poland). In rocket Immunoelectrophoresis the AFP concentration ranged from 0.5 to 40 p.g/ml (Dakopatts, Denmark). Antisera: Monospecific antiserum raised in horse against human AFP (kindly supplied by Prof. Hirai, Department of Biochemistry, University of Hokkaido, Sapporo, Japan). Monospecific rabbit antiserum against human prealbumin was purchased from Dakopatts, Denmark. Depending on the concentration of AFP and prealbumin in the samples, 10 to 200 jil anti-AFP and 5 to 10 anti-prealbumin was used for Immunoelectrophoresis. Lectins and Chemicals:

Purified con A (lot FK 18607) was

purchased from Pharmacia Fine Chemicals, Sweden.

A 1% solution

of LCA was obtained by courtesy of dr. B. Ersson, Uppsala, Sweden.

Con A-Sepharose (10 mg con A/ml) and LCA-Sepharose (2 mg

447 LCA/ml) were purchased from Pharmacia Fine Chemicals. a-MethylD-glucopyranoside was purchased from Sigma, U.S.A. Agarose gel (HSA) was from Litex, Denmark. Reaction of AFP with con A before electrophoresis: sample was mixed with 100

A 200 |il

of a 1% solution of con A.

mixture was incubated overnight at 4°C and centrifuged.

The With

immobilized con A, equal volumes of sample and gel was shaken for 10 h a t room temperature and centrifuged.

The supernatant

fluids were used for electrophoresis. Crossed Immunoelectrophoresis with lectin in the first dimension gel was performed essentially as described in (7,12,13). The 5% stock solution of con A in water or 1% saline solution of LCA was mixed with 1% agarose warmed to 60°C. The concentration of con A varied from 8 jig/ml to 8 mg/ml; LCA from 12.5 p.g/ml to 3.5 mg/ml. In control experiments, 0.25 to 50 mg a-methyl-D-glucopyranoside was included per ml of gel. Sepharose gels with lectin was first washed twice with 20 volumes of electrophoresis buffer, then heated to 60°C before mixing with an equal volume of agarose gel (5 mg con A/ml and 1 mg LCA/ml). Lectin included in the intermediate gel: These experiments were performed either as crossed Immunoelectrophoresis or as rocket Immunoelectrophoresis with lectin in the intermediate gel prepared in the same way as for the first dimension technique. In most experiments were included an extra intermediate gel containing sugar as above. Washing, pressing, and staining was according to standard procedures (14). Results When we analyzed the AFP samples by crossed Immunoelectrophoresis with con A in the first dimension, AFP was separated into the two well-known variants. With LCA in the first dimension, three peaks were observed (Fig. 1). The middle peak exhibited an unexpected behaviour; With increasing concentration of LCA

448 this peak shifted from close to the non-retarded component to close to the strongly retarded component. This is similar to the findings of Toftager-Larsen (Fig. 2, p 437, these proceedings) . We varied the LCA concentration from 0.05 to 0.75 mg/ml to observe the three peaks, and the optimal concentration of LCA for separation of the three AFP variants was judged to be about 0.25 mg/ml, with an albumin migration of at least 7 cm. These microheterogeneity forms of AFP were also demonstrated in the other samples (including cord serum), except crude and purified ascites fluid AFP (Fig. 2).

The relative concentration

A

I

A

1

A

A

Figure 1. Lectin affinity crossed Immunoelectrophoresis of human a-fetoprotein in 10 )il of amniotic fluid fromt the 12th week of gestation. LCA was incorporated into the first dimension gel (a: 0.21-10~5 M, b: 0.41-10"5 M, c: 0.79-10 -5 M). Anode is to the right, anodic peak is prealbumin as migration standard. The intermediate gel contained 2.5 %a-methyl-D-glucopyranoside. Arrow indicates component with unexpected migration.

449

of variants differed from sample to sample.

The patterns de-

scribed here were identical with those described for 12 week amniotic fluid (7,15) and in good agreement with our previous estimates (8). The ascites fluid AFP differed from the other samples. However, both crude and purified AFP contained forms not encountered before. The normal two forms were observed with con A at lower concentration (0.2 to 0.5 mg/ml), but when the con A concentration was increased (0.6 to 1.8 mg/ml), the non-retarded component was separated into two peaks. When the con A concentration was increased to 8 mg/ml, the fastest form divided into two further components. Further increase of the con A concentration led to artefacts which made it impossible to evaluate the plates. With LCA in the first dimension only 2 components were seen. The intermediate form with the unexpected behaviour was not seen. Experiments with immobilized lectin in the first dimension gel seemed to indicate further microheterogeneity of AFP as ascites

Figure 2. Crossed Immunoelectrophoresis of AFP with high concentration of LCA in the first dimension gel (6*10~5 m) . a, AFP in 12th week amniotic fluid is divided into only two peaks. Left peak contains the component which is strongly retarded and the component with unexpected migration (cf. Fig. 1). AFP-N-L signifies the non-reacting fraction. b, AFP in ascites fluid of a hepatoma patient is divided into three peaks. Two peaks correspond to those seen in a, the third peak (arrow) is atypical. Details as in Fig. 1.

450 fluid AFP was separated into five components with immobilized LCA. In comparison with experiments with soluble LCA the precipitate peaks were wider, but without impairing the resolution. Control experiments were performed with a specific sugar inhibitor, a-methyl-D-glucopyranoside, incorporated into the lectin gel. Complete inhibitions of the AFP interaction with both LCA and con A was observed with a a-methyl-D-glucopyranoside concentration of 2.5% or higher. Under such conditions the AFP precipitate was identical to that in crossed Immunoelectrophoresis without lectin in the first dimension gel. An a-methyl-D-glucopyranoside concentration of less than 0.025 % in the lectin gel did not have any inhibitory effect. Intermediate concentrations of the sugar shifted the pattern to be comparable to that with a lower concentration of lectin, i.e. the sugar had the effect to lower the effective concentration of lectin. Sugar in the intermediate gel had no effect on the AFP patterns, but resulted in the disappearance of the affinity precipitate

Figure 3. Pretreatment of ascites fluid with soluble conA before electrophoretic analysis. a, control experiment: crossed Immunoelectrophoresis of untreated sample. b, crossed Immunoelectrophoresis of pretreated sample. c, crossed Immunoelectrophoresis with conA in the first dimension gel (1.6 10~5 M). Compare well to distribution obtained with untreated sample.

451 in the first dimension gel. This effect was observed only in experiments where the distance from the affinity precipitate to the sugar-containing gel was less than 1 cm. Pretreatment of the sample with soluble and insolubilized lectin was also tested. Pretreatment with soluble con A resulted in a copious precipitate which was easily spinned down. When the resulting supernatant fluid was analyzed by crossed Immunoelectrophoresis, a tailing of the AFP precipitate was observed (Fig. 3), but the individual microheterogeneity forms could not be distinguished without con A in the first dimension gel. However, with lectin in the first dimension gel this was possible. Moreover, the pattern was identical to that of the untreated samples. In these experiments with pretreated samples the affinity precipitate normally seen in the first dimension gel with lectin was not observed. Shaking the samples with con A-Sepharose for 10 h did not result in any decrease of the area under the precipitates, i.e. in spite of the fact that most samples contained mainly the strongly binding AFP form it was not bound to the lectin during the incubation. The effects of glycoprotein excess were investigated in a series of experiments with increasing amounts of glycoprotein in the sample. Purified AFP was mixed with 5 to 50 jil of normal human serum and analyzed. In all experiments AFP separated into the same microheterogeneity forms as observed before. With up to 25 p.1 serum, the pattern was identical to the control without serum. Admixture of bigger volumes led to broadening of the precipitate arcs. However, in spite of this, the quantitative estimate was in complete agreement. In order to find an easier method for clinical routine estimation of microheterogeneity forms, we performed rocket Immunoelectrophoresis with a lectin-containing intermediate gel. Depending on the lectin concentration and the width of the lectin gel, the AFP was split into several precipitates. The splitting reflected the number and amount of microheterogeneity forms in the sample when the lectin concentration was 1.8 mg

452 con A/ml or 0.25 mg LCA/ml and when the lectin gel was at least 3 cm wide. The precipitates could be separated enough with an intermediate gel of 6 to 7 cm to permit semiquantitative determination of each microheterogeneity form. Each component was identified by its fusion with precipitates from samples with known content of each microheterogeneity form and through correlation to the relative content of each form. The same splitting of the AFP precipitate into several lines was also obtained with immobilized lectins in the intermediate gel. In these experiments a high concentration of lectin-gel was used (5 mg con A and 1 mg LCA per ml) and splitting into microheterogeneity forms was observed even with 1 cm wide intermediate lectin gels.

Discussion The amount of individual microheterogeneity forms of a glycoprotein such as AFP seems to be biologically determined (1-10). Therefore a reliable and simple methodology that permits qualitative as well as quantitative determination of microheterogeneity forms seems attractive not only for prenatal diagnostics but maybe even more for differential diagnosis in oncology (7, 8).

Separation of AFP into two or three components with LCA

will indicate whether it was produced by the liver or by the yolk sac (7-9).

Therefore we investigated various parameters

to improve the methodology available for testing microheterogeneity forms. The lectin concentration was found to be of primary importance for the disolution of the microheterogeneity forms.

With LCA

in the first dimension technique, the separation into three variants was possible only in a narrow concentration interval. We recommend 0.25 mg LCA/ml for the best separation of the three variants.

Also a certain length of the first dimension

migration is necessary. LCA gels 7 to 9 cm.

For con A gels we recommend 6 cm, for

453 Immobilized lectins could give the same resolution as soluble lectins as could be expected (24). However, there are some complicating factors with immobilized lectins. One of the disadvantages is the difficulty of exact calculation of the lectin concentration. Experimentally, differences in electroendosmosis may give uneven water-flow over the plate, giving rise to drying out and water protrusion, if care is not taken to balance the gels. Because of technical difficulties we prefer to use soluble lectins. The specificity of the lectin-AFP interaction during the electrophoresis was proved by the complete blocking of the interaction by inclusion into the lectin-containing gel of sugar with high inhibitory effect. This blocking was dependent upon the sugar concentration. Total inhibition of con A and LCA was found with 2.5% a-methyl-D-glucopyranoside in the first dimension gel. The use of sugar inhibitor in the intermediate gel is similar to the 'electroendosmotic elution' described by Salier and Faye (16,17). In experiments with 2.5 % a-methyl-Dglucopyranoside in the intermediate gel, we observed complete dissolving of the affinity precipitate formed in the first dimension gel under the electrophoresis. Dissolving of the affinity precipitate did not change the AFP immunoprecipitation profile, indicating that AFP components did not precipitate in the affinity precipitate. Salier and Faye recommended (16,17) to use agarose of high electroendosmosis to get a more efficient dissoving of the affinity precipitate. We used an agarose of intermediate electroendosmosis (Mr = -0.13) for all gels and we do not advise the use of different gels in the same experiment due to accumulation of water between the gels of different electroendosmosis. In our system the electroendosmotic flow was sufficient to push the sugar up to 1 cm into the first dimension gel. This resulted in complete dissolving of the affinity precipitate. In order to increase the sensitivity of the method we used up to 50 (il sample in some of our previous studies. From other

454 experiments (18) we could expect that other glycoproteins would influence the lectin-AFP interaction. The results obtained here confirm our expectation, as admixture of 25 to 50 p.1 of normal human serum to a sample of purified AFP led to a broadening of the precipitation peaks. The separation into individual components was impaired only to a minor extent, as we obtained the same multiple peak pattern with crude as well as with purified AFP. The influence of lectin-binding glycoproteins may be minimized by pretreatment of samples with soluble or immobilized lectin before electrophoretic analysis. For AFP neither pretreatment with soluble nor with immobilized lectin resulted in a loss of any of the microheterogeneity forms. However, this could not be expected to be a general phenomenon. With rocket Immunoelectrophoresis with an intermediate gel with lectin, we obtained separate precipitates corresponding to the individual microheterogeneity forms. This was somewhat surprising since the microheterogeneity forms of AFP fuse to form a coherent precipitate in other analyses. In all immunological precipitation techniques, a single precipitate is expected from the reaction of a monospecific antiserum with its respective antigen. However, splitting of precipitation lines can sometimes be observed as an artefact, e.g. in double immunodiffusion. Two lines may be formed if the antigen is applied twice to the same well, provided that there is a significant delay between the applications. Similar experiments with repeated application of the same antigen show double precipitates in rocket Immunoelectrophoresis (14). In our experiments the lectin may have a similar effect as AFP variants migrate with a different speed through the lectin-containing intermediate gel and divide into a number of zones corresponding to the microheterogeneity forms detected by this lectin. First the unretarded AFP variant meets the antibody front and the first precipitate is formed. Due to the electroendosmosis the antibodies may pass the precipitate after it is saturated; then the antibodies will mee the next AFP variant to precipitate either be-

455 low, with, or above the first precipitate, depending upon the ratio of antigen to antibody. In our experiments we observed up to three lines and in every case, the individual lines corresponded to the peaks in crossed Immunoelectrophoresis with lectin in the first dimension gel. The precipitate of the reacting form could form either below or above the precipitate of the non-reacting form. A satisfactory separation of AFP microheterogeneity forms in this was was obtained with both soluble and insolubilized lectins. Similar approaches to measure quantitatively individual microheterogeneity forms are reported by Hau et al. (19) and Toftager-Larsen (20) for AFP; by Andersen et al. (21) for ferritin; and by Kauss et al. (22) for antithrombin, cf, the discussion by Andersen et al. (23). Fundamental aspects and further possibilities have been described recently in (24,25). Acknowledgements. Drs B.Ersson, Uppsala, J.Westergaard, Odense, and Jakubak, Poznan, are thanked for supplying samples. The studies were supported by the Danish Medical Research Council. REFERENCES 1. Smith, C.J., Kelleher, P.C.:Biochem. Biophys. Acta 317, 231 (1973) 2. Kerckaert, J.P., Bayard, B., Biserte, G.: Biochem. Biophys. Acta 576, 99 (1979) 3. Smith, C.J., Kelleher, P.C., Belanger, L., Ballaire, R.: Br. Med. J. 1, 920 (1979) 4. Hindersson, P., Toftager-Larsen, K., N0rgaard-Pedersen, B.: Lancet ii, 906, 1979 5. Ruoslahti, E., Pekkala, A., Comings, D., Seppala, M.: Br. Med. J. 2, 768 (1979) 6. Brock, D.J.H.: Br. Med. J. 2, 1402 (1979) 7. Breborowicz, J., Mackiewicz, A.: These proceedings, Vol.1, p. 315 8. Breborowicz, J., Mackiewicz, A., Breborowicz, D.: Scand. J. Immunol. 14, 15 (1981) 9. Toftager-Larsen, K., N0rgaard-Pedersen, B.: These proceedings, Vol.1, 303

456

10. Endo, Y., Miyazaki, J., Oda, T. Fourth Lectin Meeting, Copenhagen, abstract 28 (1981). 11. Nishi, S.: Cancer Res. 30, 2507 (1970) 12. B0g-Hansen, T.C., Bjerrum, O.J., Ramlau, J.: Scand. J. Immunol. Suppl. 2, 141 (1975) 13. B0g-Hansen, T.C.: Scand. J. Immunol. Suppl. 10 (in press) 14. Axelsen, N.H. (ed.): Scand. J. Immunol. Suppl. 10 (in press) 15. Mackiewicz, A., Breborowicz, J.: Oncodev. Biol. Med. 1, 251 (1980) 16. Salier, J.P., Faye, L., Vercaigne, D., Martin, J.P. Electrophoresis 1, 19 3 (1980) 17. Faye, L., Salier, J.P.: These proceedings, pp. 605 18. B0g-Hansen, T.C., Jensen, P., Hinnerfeldt, F., Takeo, K.: These proceedings, Vol.1, 241 (1981) 19. Hau, J., Westergaard, J., Ipsen, L., Teisner, B., B0g-Hansen, T.C., S0ndergaard, K.: These proceedings, 457 (1982) 20. Toftager-Larsen, K.: These proceedings, 433 (1982) 21. Andersen, M.M., Lihme, A., B0g-Hansen, T.C.: These proceedings, 487 (1982) 22. Krauss, J.S., Sheard, M.H.: These Proceedings, 423 (1982) 23. Andersen, M.M., Hau, J. and B0g-Hansen, T.C. These proceedings, pp. 777. 24. B0g-Hansen, T.C., Hau, J.: J. Chrom. Library, 18B (in press) 25. Horejsi, V.

Anal. Biochem. 112, 1 (1981).

ESTIMATION

OF

CON

A-BINDING

ALPHA-FETOPROTEIN

(AFP)BY

AND

ROCKET

NON-BINDING

FORMS

LINE

IMMUNO

AFFINO

OF

HUMAN

ELECTRO-

PHORESIS

J.

Hau,

J.

G.

ulestergaard,

L.

Ipsen

Laboratory Animal Unit and Department Gynaecology, Odense University 523o Odense M, D e n m a r k

B.

Obstetrics

and

Teisner

Department University

T.

of

C.

of of

Obstetrics and Gynaecology Sydney, Australia

Bag-Hansen,

Kirsten

Sondergaard

The 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 2 2 o o C o p e n h a g e n N, D e n m a r k a n d D e p a r t m e n t of O b s t e t r i c s and G y n a e c o l o g y , H v i d o v r e 265o H v i d o v r e , D e n m a r k

The m e a s u r e m e n t (AFP)

in

prenatal

report

tein

in

Human

AFP

can

the

may

open K.

of

suggested

concentration

(3).

Tube

in

tube

alpha-fetoprotein test

defects Study

Defects

(1,

on

(1979)

stated

a con

The of

that

for

2).

the

The

se-

Alp h a - f e t o p r o described

up

AFP

has

A-reactive

measurement

human

additional of

of

biochemical

to

a

5 ?i o f

af-

diagnosis.

fractions an

neural

o . 4 8 ?o, a n d

miss

separated

as

the main

Collaborative

Neural

fraction two

is

of

to

be

concentration

U.

rate

fetuses

between

vated

the

Relation

non-reactive

been

of

positive

fected

the fluid

diagnosis

cond

false

of

amniotic

Hospital

AFP

in

of

the

measured

Lectins - Biology, Biochemistry, Clinical Biochemistry, Vol. II © Walter de Gruyter &. Co., Berlin • New York 1982

test (4).

a con

A

proportion

amniotic

biochemical been

and

fluid

where

an

During

has elethe

458

fetal tive At

development AFP

the

variant

time

station) tic cy,

is in

fluid

a significant

ding

AFP

tection active tests fino

has of

obtained

observed

ratio

i.e.

con

A-affinity

and

res

sample

large

The p r a s e n t

report

describes

electrophoresis

the

this method can

interassay

Materials Amniotic of

age. nic This

in

and

pregnanA

con

and con

non-bin-

A

the

de-

non-re-

as

clinical

A-crossed

methods

are

af-

time

the c h r o m a t o g r a p h y

some

experiments a rocket

can

be

to

line

of

the

be a n a l y z e d

requi-

find

an

affino two

in

the q u a n t i f i c a t i o n ,

can

samples.

abnormality disorder

AFP

patho-

methodsfor

suggested

two

quantification

AFP-forms.

the

and

easi-

Immuno-

same

run,

intra-

and

controlled.

Amniotic

amniocentesis

3) A b n o r m a l

serum

of c o n

two

ge-

amnio-

of

of

of

Methods

fluid

fetal

and

using

samples

be u s e d

transabdominal mosomal

for many

variations

and

These

18

volumes.

method

standards

(7).

to

(5).

in n o r m a l

level

been

14

stage

A-reactive

have

semiquantitative,

er a n d q u a n t i t a t i v e By

same

chromatography

Immunoelectrophoresis

consuming

the

A-reac-

fluid

In s a m p l e s

Recently

con

fluids

AFP

(5).

decreased

(6).

between

in a m n i o t i c

at

the c o n

(week

A-binding

75-85%

relatively

been

the

AFP

of

of

in a m n i o t i c

amniocentesis

of con

the range

amniotic

proportion

steadily

diagnostic

the p r o p o r t i o n

fluid

logical

for

the r e l a t i v e increases

level.

because

or n e u r a l ultrasound

fluids

(AF) w e r e

from p a t i e n t s of:

tube

1) P r e v i o u s defects.

examination.

Amniocentesis

were

obtained

considered history

2) A d v a n c e d 4)

Elevated

performed

under

at of

by

risk chro-

maternal maternal ultraso-

control. study

normal

consists

outcome.

o f AFP

Aliquots

of

samples AFP

from

samples

14 p r e g n a n c i e s

with

were

-2o° C

stored

at

a

459 until

assayed.

Figure

wells

received

5

Well

1 shows

of

No.

the

mg/1 total AFP concentration

not

determined

2

12

not

determined

13

not

determined

4

37

0.1

5

16

19

6

17

29

2o

(serum

o . o3

samp le)

12

not

determined

9

37

not

determined

lo

29

not

determined

11

4o

not

determined

12

18

13

11

not

determined

14

4o

not

determined

AFP

Crossed to

concentration as

described

line the

buffer,

affino

first

ionic

was

by

27

measured

Hay

et

al.

by

rocket

strength

gel

o.o2o,

was pH

immunoe1ectro -

(8).

Immunoelectrophoresis

dimension

The

samples.

3

phoresis

with

performed

8.5

as

free

in

con

TRIS

previously

A

ad-

barbital

described

lo).

Rocket

line

was

buffer

affino

performed as

voltage Five

plates.

8

Total

sis

following

19

7

(9,

electrophoresis

W e e k of amniocentesis

1

ded

the

gel

cathodic

for

was

Immunoelectrophoresis. on

crossed

11

x

o.9

V/cm

for

strips

were

cast

side:

Gel

A,

2o.5

line

cm

glass

affinity

18 on

a gel

This

plates

electrophoreusing

the

same

Immunoelectrophoresis.

hours. the (4

x

plates

(Fig.

2o.5

0.15

x

1). cm)

From with

the wells

The

460 for

sample

(o.5 con

application

x 2o.5

x o.l5

A-Sepharose

experiments

gel

1 cm

apart,

cm) containing

(Pharmacia)

diluted

B was

gel.

Gel

of

human

empty

x o.l5 cm) containing

a pool

l:loo

D, a g e l

the with

in a g a r o s e .

antibody-gel or

without

gel

Gel

containing 1.5/o

(1 x

DAKO

B, a n

either 1:2 C,

3oo in

intermediate

agarose.

a line

gel

amniotic

2o.5

rabbit

In

anti-AFP

cm). 1.5

(w/v) a-methyl -U-glucopyranoside

^

A

A

t

.

A a

A

A

or

control

(o.5

fluid

x o,15

gel

A/cm2

ug c o n

x

2o.5

diluted Gel

E,

yl/cm2, (HG)»

/N

rvE

D o

b /•s

c

Jit

*

SIP

i - \ ^-v

•:••-•,»

Fig.

1.:

»

0

O

See

text

for

O explanation.

**

..... .-.v- ..... •

O

^

i e -P 0 •H -P > 0

01 01 0

•H rH

(H

d) id 01 o

T3 d)

>iK -p a

H

>1 T3 rH d) H

g g •H

o xi -a -H

01

m a) 0 en

o

01 H

•

01

•P ko

01 •(H ai

d)

tn -H B

a> e

G -r|

S

M

•rl -P id -P (d •C ft oi 0 A a

id

JC

(d

G •H

x: -p •H

M

c o o

c •H

rH

X) rH

e a

m

CD

hl m

Cn

C •H

-P 0) -a

W

S

•H E p

G •H

-o d) -p o

RH

O

G

J

X> CO

CO CO

c CO •o 3 CO

•o G

0 = no agglutination reaction

•O C

a o •H •C -p w

538 Five different Type

agglutination

I includes

pica m a j o r

t y p e s can be o b s e r v e d

the s t r a i n s of L . m e x i c a n a

from

Saudi A r a b i a

and

Israel. These

react w i t h A . p a p i l l a t a

II but w i t h

U.europaeus,

and

L.alpinum

T h e New W o r l d ca

from

mexicana,

to type

not r e a c t n e i t h e r w i t h

A.papillata

alpinum,

A.hypogaea.

and

The s t r a i n s of L . t r o p i c a III. T h e s e Type ni

the

major

as w e l l

L.alpinum

as

II and minor

LV-125 which

and

LRC L-51

from

reactions with

India. These

from

S.

Sudan

strains react

With

tion

t e s t s w e r e not

carried

with

S.hispida,

c o u l d be

the e x t r a c t

A.papillata

the s p e c i f i c

for

only

autoagglutination. inhibited with

w i t h R . c o m m u n i s - 1 2 0 c o u l d be

ted by D - g a l a c t o s e .

sible w i t h L - f u c o s e

a tendency

C.ensiformis

nose. The agglutination

L.alpinum.

L.donova-

lectin.

N o n e of the s t r a i n s s h o w e d

and

L.

type

react with

and L . d o n o v a n i

ted by

do

A.hypogaea.

the s t r a i n s of L . d o n o v a n i

All

to

and

includes

the p e a n u t

strains

S.hispida.

Type V finally with

amazo-

L.aethiopi-

these

the U S S R b e l o n g

s t r a i n s of L . t r o p i c a

U.europaeus,

not

S.hispida,

II nor w i t h U . e u r o p a e u s ,

from

infantum L V - 1 4 0 and L.donovani

hispida,

from

L.mexicana

II. All

strains react with A.papillata

IV i n c l u d e s

1).

L.tro-

s t r a i n s do

the l e c t i n s

braziliensis

the O l d W o r l d b e l o n g S.hispida

(TABLE and

A.hypogaea.

¿trains L.mexicana

nensis and L.braziliensis

pifanoi

of A . p o l y p o i d e s

out. The a g g l u t i n a t i o n II and A . h y p o g a e a

sugars. Opposite to i n h i b i t

to this,

D-man-

inhibiinhibi-

reactions

c o u l d be

inhibi-

it w a s not

the r e a c t i o n s w i t h

pos-

U.europaeus

539 Discussion The results demonstrate that lectins can be used for a differentiation of Leishmania. The cutaneous leishmaniasis of the Old World can be divided into two forms (12,16). Three genic agents are responsible Old World

patho-

for this clinical picture in the

(18): L.tropica minor, L.tropica major,

ca. In consistence with other methods

L.aethiopi-

(6,11) these three

spe-

cies can be differentiated with lectins, too (TABLE 1). In accordance with the geographical origin - USSR, Middle East two agglutination types of L.tropica major were found. This demonstrates that lectins can be used for the

differentiation

of intraspecific variants. The L.tropica major strains

from

Middle East and L.mexicana pifanoi from Venezuela

identi-

show

cal agglutination reactions. This consistence between L.tropica major and L.mexicana

pifanoi was also observed by other

authors (6,8,11,19) with different

methods.

With lectins it is possible to differentiate between L.mexicana mexicana and L.tropica major. The same result was also found using densitygradient

centrifugation

dehydrogenase electrophoresis

(6) while by malate-

(11) no differences were

detec-

ted. Opposite to (6,11) it was not possible to distinguish between L.aethiopica and L.mexicana mexicana

(TABLE 1). The

L.donovani strains from Sudan and L - 5 1 from India show tical reactions and can be differentiated

from L.tropica

nor, L.tropica major and L.aethiopica. Moreover the gated L.donovani L.donovani

iden-

strains can be distinguished

mi-

investi-

from the

strains

infantum LV-140 and L.donovani LV-125 because

these

two strains reacted like L.tropica minor strains (TABLE 1). Identical observations were made by densitygradient gation

(6) and disc electrophoresis

minor have a latent viscerotropism

(10). Strains of L.tropica (15). Consequently

strains L V - 1 4 0 and LV-125 could be L.tropica minor too. The L.aethiopica could be distinguished

centrifuthe

strains,

strains showed uniform reactions and from L.tropica minor, L.tropica

major

540 and L.donovani. This result supports the separation of L.aethiopica

from L.tropica.

It is advantageous to use

for differentiating Leishmania

lectins

strains from the Old World

agglutination tests because no technical big efforts are Although

it is possible to differentiate the Leishmania

in needed.

spe-

cies from the Old World with lectins it is impossible to distinguish the species and subspecies of Leishmania

from the

New World - L.mexicana mexicana, L.mexicana amazonensis, braziliensis braziliensis - with lectins used here Other authors (2,6,9,11,14,17

L.

(TABLE 1).

) were able to distinguish

be-

between L.mexicana and L.braziliensis. Using lectins it is only possible to distinguish L.mexicana

pifanoi

(TABLE 1).

As the agent for diffuse cutaneous leishmaniasis in Venezuela (L.mexicana

pifanoi) and in Brazil

(L.mexicana

amazonensis)

(14) can be distinguished by lectins this result the finding

supports

(7) that this clinical picture is not caused by

any particular parasite.

It is striking that in the New World

no other agglutination type exist than in the Old World (TABLE 1). More experiments with lectins have to whether it is possible to differentiate Leishmania

further

demonstrate between

strains from the New World.

1.

Adams & Maegraith.: Clinical Tropical Diseases. Blackwell Sci. Publ. Oxford, London, Edinburgh, Melbourne; Seventh Edition 1980, p.185

2.

Adler, S.: Rev. Inst. Salubr. Enferm. trop. 139-152 (1963)

(Mex.) XXIII.

3.

Bray, R. S.: Ethiopian Medical Journal 8, 207-212

4.

Bray, R. S., Bryceson, A. D. M.: Trans. R. Soc. Trop. Med. Hyg. 63, 524-527 (1969)

(1970)

5.

Bray, R. S., Rahim, G. A. F.: Trans. R. Soc. Trop. Med. Hyg. 63, 383-397 (1969)

6.

Chance, M. L., Peters, W., Shchory, L.: Ann. Trop. Med. Parasit. 68, 307-316 (1974)

541 7.

C o n v i t , I., P i n a r d i , M. E., R o n d o n , A. J.: T r a n s . R . T r o p . M e d . H y g . 66, 6 0 3 - 6 1 0 (1972)

8.

Ebert, F.: Tropenmed.

Parasit.

25, 4 9 - 5 3

9.

E b e r t , F.: T r o p e n m e d .

Parasit.

25, 2 5 9 - 2 6 6

10. E b e r t , F.: T r o p e n m e d .

Parasit.

28, 279

(1974)

W.: Ann. Trop.

C. A . : T r o p . D i s . B u l l . 41, 3 3 1 - 3 4 5

13. K r a m p i t z , H. E . : B u n d e s b l a t t 14. L a i n s o n , R . ,

(1974)

(1977)

11. G a r d e n e r , P. J., C h a n c e , M. L . , P e t e r s , P a r a s i t . 68, 3 1 7 - 3 2 5 (1974) 12. H o a r e ,

22, 4 6 1 - 4 6 6

Med.

(1944)

(1979)

Shaw, J. J.: N a t u r e 272, 5 9 5 - 6 0 3

(1978)

15. L a t y s c h e v , N. J., K r y n k o v a , A. P.: T r o p . D i s . B u l l . 522-523 (1955) 16. L y s e n k o , A. J.: B u l l . W l d . H l t h . O r g .

Soc.

44, 5 1 5 - 5 2 0

52,

(1971)

17. M o m e n , H., G r i m a l d i , G. F., S o a r e s , M. J.: P e s q u i s a B a s i c a e m D o e n c a de C h a g a s V I I R e u n i ä o A n u a l , H o t e l G l o r i a - C a x a m b u , M. G . , B r a z i l , 3 - 5 de n o v e m b r o 1 9 8 0 R e s u m o s BI 9 18. P e t e r s ,

W.: Tropenmed.

Parasit.

28, 2 7 1 - 2 7 2

(1977)

19. P i f a n o , C. F., S c o r c a , J. V . : A r c h . V e n e z . M e d . T r o p . r a s i t . 3, 1 5 - 3 0 ( 1 9 5 9 / 6 0 )

Pa-

20. S p e c i a l P r o g r a m m e for R e s e a r c h a n d T r a i n i n g in T r o p i c a l D i s e a s e s . T h i r d A n n u a l R e p o r t , 1 J u l y 1 9 7 8 - 30 j u n e 1 9 7 9 . Leishmaniases, p.105 21. W o r l d H e a l t h O r g a n i z a t i o n T e c h n . R e p o r t (1979)

Series 637,

41-42

THE LECTIN-NEURAMINIDASE-ASSAY

(LN-TEST)

CLINICAL USE OF ARACHIS LECTIN IN DIAGNOSIS

Peter Luther, Sylvia Weber, Karl-Christian Bergmann Forschungsinstitut fur Lungenkrankheiten und Tuberkulose 1115 Berlin, GDR

Lectins are proteins which react specifically with certain carbohydrate structures located, for example, on cell membranes. But some structures (receptors) are only exposed under the influence of bacterial or viral neuraminidases by splitting off N-acetyl-neuraminic acid. These receptors are called Thomseij-Friedenreich antigens and can be specifically demonstrated using lectins from Arachis hypogaea (peanut agglutinin, PNA) or Helix pomatia (snail agglutinin, anti-A^p) (4,5,14). The lectin assay is basing on the detection of erythrocyte agglutination after contact with living virus's (neuraminidase treatment) by Arachis lectin (lo,ll), as described in the following paper. Both, the detection of the activity of the virus neuraminidase and the determination of antibodies against the virus neuraminidases (human or animal) are of great epidemiological importance. Standard methods (3) have the disadvantage of employing much manual labour and equipment, also for larger epidemiological investigations they are not very suitable. However, from epidemiological view the permanent control of the immune status of the population regarding neuraminidase-inhibiting antibodies is important. Therefore simplified methods for the detection of neuraminidase and anti-neuraminidase IgG have been published by different authors in the past (2,9,13,16).

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 . II © W a l t e r d e G r u y t e r & C o . , B e r l i n • N e w Y o r k 1982

544 ever, from an epidemiological view the permanent control of the immune status of the population regarding neuraminidaseinhibiting antibodies is important. Therefore, simplified methods for the detection of neuraminidase and anti-neuraminidase IgG have been published by different authors in the past (2,9,13,16). Materials and Methods Arachis-lectin: Arachis-lectin was prepared as described (17). Arachis lectin purity was not an essential criterion for the test. Non-pure extracts, prepared as usual but ommitting the affinity chromatography step, gave results which were practically indistinguishable from those obtained using pure protein (11). Antigens: We used recombinant Virus NIB-4 (H3N2) A/England/ 321/77 x A/PR/8/34 and NIB-6 (H1N1) A/USSR/92/77 x A/PR/8/34 from the WHO International Influenza Center London; dialysisantigens of the following strains and recombinant viruses from the Institut für Angewandte Virologie Berlin; NIB-4 (H3N2) and NIB-6 (H1N1) and the strains A/Victoria /3/75 (H3N2) and B/Victoria/98926/7o; dialysis-antigens of the following strains from the SKISI: Recombinant Virus NIB-4 (H3N2), NIB-6 (H1N1), A/Singapore/6/57 (H2N2), A/Port Chalmers/1/73 (H3N2), B/Hong Kong/5/72 and B/Wellington/1/75. Not infected dialyzed allantoic fluid from 12-13 days old embryonal hen's eggs served as negative control. Erythrocytes: Human erythrocytes (group 0) kept in A-C-D test blood stabilizier were washed three times and resuspended in test medium (PBS, pH a 7.2). Sera: Immune sera were prepared in rabbits by immunizing seronegative males. Human serum was taken before a one-time immunization with influenza virus adsórbate vaccine from the SSW (recombinants NIB-4 and NIB-6) and 4 weeks, 8 weeks and 6 months after it. The LN-test was done as described (10,11).

545 Results Detection of Neuraminidase Activity:

Twenty p.1 of a 5 % human

erythrocyte suspension were added to 20 nl of each dilution to be followed by an incubation of 18 hours (over night) at 31o°K After that the agglutinability of the erythrocytes was tested by adding 2o |il arachis-lectin.

For control 2o |il of a 5 i

human erythrocyte suspension were incubated with 2o jil test

TABLE 1 Reproducibility and repeatability of the detection of biological activity of two virus-antigens (influenza-virus-recombinant NIB-6 (H1N1) from A/USSR/92/77 x A/PR/8/34) in the LN-test

Reproducibility (repeated determination on different days)

Repeatability (repeated determination on one plate)

Day

Determination

titre

titre

1 4

64 64

1 2

64 128

6 6 24

32 64

3

128 128

32 64 64

5 6 7

25 27

The activity is expressed as reciprocal titre.

4

128 64 128

546

medium instead of the virus for 18 hours at 31o K and likewise tested by adding 2o p.1 arachis-lectin.

Macroscopical determi-

nation was done 60 min later (Figs. 1 and 2).

The reproduci-

bility and repeatability of the LN-test in the detection of the neuraminidase activity are shown in Table 1.

\

w

Virus viru HA

cell

>1h 37°C

Figure 1. Model of the contact between receptors on the cell surface with virus and splitting off of the N-acetyl neuraminic acid (NANA) by the viral neuraminidase. ND = neuraminidase; 3-Gal = 3-galactoside; HA = hemagglutinin.

Figure 2. The Arachis lectin agglutinates cells only after virus contact with splitting off of the N-acetyl neuraminic acid. Cells containing N-acetyl neuraminic acid are not agglutinated.

547 TABLE 2 Detection of anti-neuraminidase in a ferret immun-serum (antiPort Chalmers) against different viruses with the LN- test.

virus antigen

antiserum titer against viral neuraminidase 20

80

320

1280

B-Singapore

2

3

3

3

A/Port Chalmers H3N2

0

0

0

A/Victoria H3N2

0

0

A/Texas H3N2

0

NIB-6 H1N1

3

5120

10000

20000

80000

3

3

3

3

0

0

0

0

2

0

0

0

0

1

3

0

0

0

1/0

1/0

1

3

3

3

3

3

3

3

3

0 = complete inhibition, 1 = weak agglutination, 3 = complete agglutination (no inhibition). patient serum with

antineuraminidase

Virus

+

* cell

Figure 3. The reaction of specific antibodies against viral neuraminidase: prevention of splitting off of NANA and inhibition of the agglutination by arachis lectin.

548 Detection of Neuraminidase-Inhibiting Antibodies: Twenty p.1 virus were added to 2o p.1 serum and incubated at room temperature for one hour. The virus was used in a dilution with a titre of 1:16. Then 2o jil of a 5 % human erythrocyte suspension were added and incubated for 18 h at 31o°K. Subsequently 2o n-1 arachis lectin were added to each serum dilution. Reading was done 6o min later at room temperature (see Fig. 3). Some results are shown in Table 2. Generally erythrocytes of most mammals are suitable in the LN-test (Table 3).

TABLE 3 Suitability of different erythrocytes in the LN-test for detection of viral neuraminidase activities and anti-viral neuraminidase in human serum. erythrocytes from

suitability

man (blood groups 0, A or B) guinea pig

very suitable, especially for longer incubation (16-30 h) very suitable, especially for shorter incubation (6 h) or longer incubation using cell medium

sheep

suitable

rabbit

suitable

hamster rat

suitable suitable not suitable without cell medium and short incubation

mouse dog

for detection of antibodies against mumps virus neuraminidase

549 Discussion The biochemical detection of neuraminidase-activity is based on the determination of N-acetyl neuraminic acid released by the viral enzyme acting on a given substrate and the inhibition of the reaction by specific antibodies. The high labour input makes it considerably difficult to use this method in large serological investigations. In the past years there have been made many attempts to replace the method for the detection of neuraminidase-inhibiting antibodies applied and recommended by WHO (3). These replacement methods should ideally show high correlation, and have a low labour equipment demand. In 1973, Holston and Dowdle (9) published a neuraminidase indirect hemagglutination test using sensitized sheep erythrocytes fixed with glutaraldehyde for the detection of neuraminidase.

Spano and Dardanoni (13) described a '3-step' method

enabling them to detect hemagglutination-inhibiting and neuraminidase-inhibiting antibodies in a serum-dilution using fowlerythrocytes. Both methods used erythrocytes as indicators.

Considering

their good correlation with the method applied by WHO, and their higher sensitivity, both tests are described as technically being more simple and more efficient. In 1977, Appleyard and Oram (2) published the elution-inhibition-method. This method used the elution of influenza-viruses from red blood cells catalyzed by neuraminidase for measuring neuraminidase-activity. The Single Radial Hemolysis Test proposed by Callow and Beare (6) and Farrohi et al. (7) employs antigenic hybrid viruses containing an animal hemagglutinin not found in man.

But in

this case, the distinct formation of plaques is, to an even greater extent, related to the adsorption of enough virus when compared with the anti-hemagglutinin antibody-method.

550

In 1979, Takatsy et al. (16) also alluded to the complications of the NI-test as conducted according to Aymard-Henry et al. (3):

'... being less applicable for mass investigations'.

In contrast, they described a simplified method carried out in the microtitrator-system. is the same.

The principle of both methods

The microtitrator-system has also the advantage

of being cheaper, more practicable and less time-consuming. The trend of using lectins and erythrocytes for the specific detection of antigens in methods with high sensitivity and simplicity is furthermore confirmed by the work of Gueston et al. (8) who described an 1erythro-immunoassay'. Here, Con-A-AntiSRBC with SRBC were used. The investigations made with the LN-test showed results comparable to those of the NI-test (r=o.67, a=l %). As described by other authors (Holsten, Spano), the higher sensitivity of the biological indicator-system proved to be an advantage. Another advantage is that this test-system is considerably less subject to external influence. The simple apparatus and the low amount of materials provide the possibility of carrying out larger series of tests, thus rendering a valuable supplement to the NI-test according to Aymard-Henry et al. (3). In contrast to the method described by Appleyard and Oram, determinations in the LN-test have become much more easy and elegant by the use of arachis-lectin for the detection of released neuraminic acid. Concerning LN-test/NI-test comparisons, investigations performed using different micromethods in different laboratories turned out to be favourable.

98 % of all scores showed devia-

tions by 0-2 titres, and thus being within the error limit for micromethods. Arachis lectin can be easily prepared in large quantities. A crude extract from un-roasted peanuts met all criteria for the test, apart from that micropipettes and microtitre-plates or a microtitrator according to Takatsy (15,16) are needed.

551 Determination is done macroscopically. It can be done after 60 min, but is still possible up to 12 hours later. However, a standard determination after an hour is recommended for getting comparable results. Differentiation between agglutination by the viral-hemagglutinin (as membrane-bound lectin) and agglutination by the Arachis lectin is unambiguous. Finally we may say that the LN-test shows results comparable to those of the NI-test, but employs considerably less materials and manual labour. Sensitivity of the LN-test is higher than that of the NI-test. These are excellent reasons why the LN-test is especially suitable for comprehensive field investigations or epidemiological investigations (1) concerning anti-neuraminidase distribution within the population.

REFERENCES 1.

Adamczyk, B., Luther, P., Siegel, J., Bergmann, K.-Ch. (1979). Dt. Gesundh.-Wesen 34.» 2532-2534.

2.

Appleyard, G., Oram, J.D. (1977). rology 34, 137-144.

3.

Aymard-Henry, M., Coleman, M.T., Dowdle, W.R., Laver, W.G., Schild, G.C., Webster, R.G. (1973). Bulletin of the World Health Organization £8, 199-2o2.

4.

Bird, G.W.G. (1964).

5.

Burnet, F.M., McCrea, J.F., Stone, J.D. (1946). exp. Pathol. 21_, 228.

6.

Callow, J.A., Beare, A.S. (1976). 13, 1-8.

7.

Farrohi, K., Farrohi, F.K., Noble, G.R., Kaye, N.S., Kendal, A.P. (1977) . Journal of Clinical Microbiology _5, 353-36o.

8.

Guesdon, J.L., Avrameas, S. (198o). (Institut Pasteur) 131 C, 389-396.

Annales d'Immunologie

9.

Holston, J.L., Dowdle, W.R. (1973). 25, 97-lo2.

Applied Microbiology

Journal of General Vi-

Vox sang. 9, 748. Brit. J.

Infection and Immunity

552 10. Luther, P., Adamczyk, B., Bergmann, K.-Ch. (1979). Gesundh.-Wesen 34, 1858-1862.

Dt.

11. Luther, P., Adamczyk, B., Bergmann, K.-Ch. (198o). Zentralblatt für Bakteriologie, Mikrobiologie und Hygiene, I. Abt. Originale A 248, 281-285. 12. Luther, P., Klett, G., Weber, S., Pechmann, H., Bergmann, K.-Ch. 13. Spano, C., Dardanoni, L. (1973). Milanese 52, 247-255.

Bollettino dell'Istituto

14. Prokop, 0., Uhlenbruck, G., Köhler, W. (1968). (Basel) 14, 321-325.

Vox Sang.

15. Takàtsy,G. (1955). Acta Microbiologica Academiae Scientiarum Hungaricae 2' 191-194. 16. Takatsy, G., Barb, K. (1979). Acta Microbiologica Academiae Scientiarum Hungaricae 2_6, 63-69 . 17. Terao, T., Irimura, T., Osawa, T. (1975). Z. ^Physiol.-Chem. 356, 1685-1689.

Hoppe-Seylers

LECTIN-ANTIBODY AND LECTIN-LECTIN CONJUGATES

H. Franz, J. Mohr and P. Ziska Staatliches Institut fur Immunpraparate und Nahrmedien DDR-112o Berlin

Two years ago we proposed the term 'affinitin' as a collective term for bio-macromolecules containing combining sites, such as immunoglobulins, enzymes and lectins (1). In introducing this term, we aimed to emphasize, among other things, some relationships between the different groups of affinitins (2). The definition of 'affinitin' is in good agreement with the lectin definition of Goldstein et al. (3) and is in still cloV V ser accordance with the proposal of Kocourek and Horejsi (4). Accepting the term 'affinitin', one can consider the following aspects: I. 2.

3.

Comparison of groups of affinitins ('comparative affinitinology" in analogy to comparative anatomy). Interactions between affinitins belonging to different groups (e.g. between lectins and antibodies via the epitops of the lectin and the combining sites of the antibodies, or between the combining sites of the lectins and the carbohydrate moieties of the antibodies). Coupling of affinitins. At present we know not only of conjugates of affinitins belonging to the same group (e.g. hybrid antibodies, enzyme-enzyme conjugates) but also of conjugates of affinitins belonging to different groups (e.g. antibody-enzyme conjugates, antibody-lectin conjugates, and enzyme-lectin conjugates). Only the lectin-lectin combination remain undescribed.

Lectins - Biology, Biochemistry, Clinical Biochemistry, V o l . II © W a l t e r d e Gruyter &. C o . , Berlin • N e w York 1982

554 Here we present results concerning the conjugation of Concanavalin A (Con A) to immunoglobulins, and also our first investigations concerning lectin-lectin conjugates.

Material and methods Preparation of anti-rabbit IgG-Con A conjugates: From goat antiserum against rabbit IgG we isolated an IgG fraction by ammonium sulphate precipitation and subsequent fractionation on DEAE Sephadex A 5o. The conjugation of this IgG fraction with Con A was performed using the technique described by Guesdon and Avrameas (5). The same method was used to prepare a conjugate of human serum albumin and Con A. Modified enzyme immunoassay: The anti-rabbit IgG-Con A conjugates were tested by means of a modified enzyme immunoassay using the IgG anti-IgG system. Plates (polystyrene or polyvinylchloride) and tubes (polystyrene) were coated with an IgG fraction (o.2 ml and o.4 ml respectively) from goat antirabbit IgG, containing 5o ug protein/ml in o.o5 carbonate buffer, pH 9.5. After incubation for at least 24 h at 4°C the plates and tubes were emptied, and washed 3 times with phosphate-buffered saline, pH 7.4, containing o.o5 % Tween-2o (PBS-Tween), and stored at 4°C until used. The IgG-Con A conjugate was quantitated by successive incubation in the wells of 1) serial dilutions of rabbit IgG (acting as antigen) in PBSTween + 1 % bovine serum albumin, 18 - 2o h at room temperature, then 2) the anti-rabbit IgG-Con A conjugate in PBS-Tween + 3 % bovine serum albumin + o.l M a-methyl-D-mannoside (MM), 3 h at 3 7°C, following which the wells were washed five times in PBS-Tween, and finally 3) horseradish peroxidase (POD) in PBS-Tween (o.2 ml per plate, or o.4 ml per tube), 2 h at 37°C. The bound peroxidase was quantitated using o-phenylene diamine and H~0 9 as substrate, 3o min at room temperature.

555 Preparation of lectin-lectin conjugates: Con A was conjugated to peanut agglutinin (PNA) and to wheat germ agglutinin (WGA) as follows. The lectin solutions were dialysed overnight against o.l M phosphate buffer, pH 6.8, at 4°C. 5o mg quantities of Con A were then mixed with either WGA (14 mg) or PNA (5o mg), in 16 ml of phosphate buffer, pH 6.8. Conjugation of the mixed lectins was performed by adding 45o ul of 1 % glutaraldehyde solution, then gently stirring for 3 h at room temperature. At this point, 5o mg glycine were added to each mixture, and these were then left for a further 2 h at room temperature. Finally the solutions, now containing lectin conjugates, were dialysed against phosphate buffer. To remove unbound WGA or PNA, the conjugates were put onto columns of Sephadex G-75 (45 x 1.5 cm), which were washed with phosphate buffer until the effluent was protein-free. The conjugates (Con A-JVGA or Con A-PNA), together with uncoupled Con A, were then eluted with o.l M D-glucose solution. Finally, the eluted conjugates were dialysed against phosphate buffer to remove the D-glucose. Hemagglutination inhibition test: o.o5 ml of a serial dilution of the inhibiting carbohydrate (D-glucose for Con A, Nacetyl-D-glucosamine for WGA, D-galactose for PNA) and o.o25 ml of the conjugate solution containing 4 hemagglutinating units were mixed. Four hemagglutinating units were defined as the conjugate dilution showing a hemagglutination titer of 1:4 on microtitrator plates (6). After incubation for 3o min at 37°C o.o25 ml of a 2 % suspension of human erythrocytes were added. The degree of agglutination was read after 1 h at 37°C.

Results 1.

Lectin-antibody conjugates:

The first IgG-Con A conjugate was prepared using the IgG fraction of an anti-rabbit IgG antiserum.

At a protein concentra-

556 tion of 18 mg/ml this fraction contained 5.5 mg antibody/ml. We conjugated the lectin and antibody by the method of Guesdon and Avrameas (5), but omitted dialysis of the conjugate as we found that up to 4o % of the protein was lost by precipitation, caused by reaction of Con A with the carbohydrate moiety of the IgG. We tried to remove unbound IgG from the IgG-Con A conjugate by affinity chromatography on Sephadex G-75, but found that the total amount of protein passed the column; coupled Con A nor uncoupled Con A was bound.

neither IgGThe conjugate

behaved similarly on Sephadex G-loo, G-15o, and G-2oo, also. We repeated this experiment with a conjugate of human serum albumin and Con A and found, as expected, that in the fraction which passed the Sephadex G-7 5 column we could identify only human serum albumin. The fraction obtained by elution of the column with a o.l M solution of D-glucose contained human serum albumin and Con A. For the modified enzyme immunoassay the anti-rabbit IgG-Con A conjugate was used without any purification. We compared the modified enzyme immunoassay with the usual enzyme immunoassay. From Table 1 it is obvious that both methods produce nearly identical results. The titer of the anti-rabbit IgG-Con A conjugates could be increased by conjugation of the Con A to pure antibodies, isolated by affinity chromatography on rabbit IgG coupled to Sepharose, instead of the IgG fraction. This increase in titre corresponded to the increased antibody content of the conjugate. The sensitivity and specificity of this test were not influenced by use of crude POD (R.Z. = o.3) instead of pure POD (R.Z. = 3.o). The optimal concentration of the enzyme was 5 ug/ml. Our results are in close correspondence to the findings of Guesdon and Avrameas (7).

557 2.

Lectin-lectin conjugates:

The Con A-WGA conjugate showed the properties of both Con A and WGA. The agglutination of human red blood cells by this conjugate could be completely inhibited only by a mixture containing both o.l M D-glucose and o.l M N-acetyl-D-glucosamine. Similar results were obtained with the Con A-PNA conjugate. For complete hemagglutination inhibition of neuraminidase treated erythrocytes it was necessary to use a mixture of o.l M D-glucose and o.l M D-galactose.

TABLE 1 Comparison of different anti-rabbit IgG conjugates

POD Ab

1)

Con AIgG

2)

Con AAb 1 )

Antibody concentration of the conjugate (mg/ml)

o.2

o.l

2.o

Working dilution

l:5o

l:2oo

1:5oo

4.o

3.5

4.o

lo

2o

lo

Antibody concentration in the working dilution (ug/ml) Sensitivity for the detection of rabbit IgG (ng/ml) 1)

Antibody isolated by affinity chromatography

2)

IgG fraction

558 Discussion 1.

Lectin-antibody conjugates:

Inspired by the publication of Guesdon and Avrameas (5) describing an erythro-immunoassay, we prepared Con A-IgG conjugates in order to develop a special kind of enzyme immunoassay. The principle of the method is shown in Figs. 1-5. Fig. 1 gives the explanations of the symbols for the different reaction components.

antigen

carbohydrate (reaction with Con A)

\

IgG

J

O °

O

Figure 1. Explanation of symbols. depends on sterical conditions.

( K / VL/

O

L,

Con A

crude horseradish peroxidase

The valency of Con A

GA -h

Figure 2. Coupling of IgG and Con A by means of glutaraldehyde (GA) in presence of MM.

559

Fig. 2 shows the coupling of antibody and Con A by means of glutaraldehyde. This process is performed in the presence of sugar, in this case a-methyl-D-mannoside (MM). The mannoside blocks the binding sites of Con A in order to prevent reaction with the carbohydrate moiety of the IgG-molecule. In the next step (Fig. 3) the conjugate reacts with antigen bound to a solid carrier, also in presence of MM, which does not influence the immune reaction. The washing procedure shown in Fig. 4 simultaneously removes the unbound conjugate and MM. The combining sites of the lectin are active again.

f

«

Figure 3. Immune reaction in MM-containing solution. The interaction between the carbohydrate moiety of IgG and Con A is inhibited by MM.

4

Figure 4. Washing procedure. MM is removed. sites of Con A are reactive again.

The combining

560 After addition of crude horseradish peroxidase (POD) the enzyme is bound via his carbohydrate moiety (Fig. 5)• The result is a complex containing antigen, antibody, lectin and enzyme.

Between the antigen and antibody and between the

Con A and POD, respectively, there exist typical affinitin bonds, whereas the linkage between antibody and Con A is covalent. Our own results and the findings of Guesdon and Avrameas, reported recently (7), show that it is possible to detect both antigens and antibodies by means of this modified enzyme immunoassay. For the determination of antigens it is necessary to fix the corresponding antibody to the carrier (in addition to Figs. 3-5 which show the main principle of the method). The main advantage of this method is that relatively impure POD preparations can be used. The test can be modified to a large extent by application of other enzymes reacting with Con A or by use of lectins with other carbohydrate specificity and enzymes binding to these lectins. We have not succeeded in purifying the IgG- Con A conjugates. It is well known that Con A reacts with all types of Sephadex which are crosslinked to a lower degree than is Sephadex G-25. This reaction is not only a property of free Con A. Shier (8) used affinity chromatography on Sephadex G-2oo to purify a Con A-trypsin conjugate. Thus it might be expected that free IgG could be separated from an IgG-Con A conjugate by this method. However, it is not possible. On the other hand, the separation of non-bound human serum albumin, WGA, and PNA from their respective conjugates with Con A could be performed on Sephadex G-75. A conjugate of Con A and mistletoe lectin I (ML I) showed the same behaviour as IgG-Con A conjugates. We suppose that the inhibition of the binding of Con A to Sephadex in some conjugates is caused by formation of intramolecular complexes between Con A and the carbohydrates moiety of the conjugation partner.

561 2.

Lectin-lectin conjugates:

There exist two possibilities concerning the conjugation of lectins between each others: 1.

Both lectin molecules contain no carbohydrate.

cannot react to form an affinitin bond. free lectins are Con A, PNA and WGA. easily in the absence of sugar.

Thus they

Such carbohydrate-

They can be conjugated

Likewise, lectins which con-

tain carbohydrates can be coupled if they do not react with each other. This is mostly true of lectins having the same specificity. 2. The two lectins interact, as do, e.g., mistletoe lectin I (ML I) and Con A. In this case additional manipulations are necessary: - conjugation in presence of the appropriate sugars (in the case of Con A-ML I, D-mannose) - destruction or elimination of the carbohydrate moiety of the lectin(s) by periodate oxidation or by enzymatic cleavage. The two lectin-lectin conjugates described here belong to the first group, and could be prepared in the absence of sugar. As mentioned above, a partial purification of Con A-WGA and Con A-PNA conjugates was performed by means of affinity chromatography on Sephadex.

Further purification to remove un-

bound Con A should be possible by adsorption of the Con A-WGA conjugate on a column of chitin, followed by elution with o.l M N-acetyl-D-glucosamine.

The Con A-PNA conjugate should re-

o Figure 5. When a crude horseradish peroxidase solution is added, only the enzyme is bound.

562 act with Sepharose, from which it can be eluted with o.l M Dgalactose solution. These experiments we wish to undertake next. Lectin-lectin conjugates may be useful tools for the following reasons"* - In a mixture non-conjugated lectins react independently of each other, whereas after conjugation one lectin can be fixed indirectly to a receptor which corresponds to another lectin. In a second step all the reactions due to the combining sites of the first lectin remain available. - A special interest attaches to conjugates which react in an antagonistic manner, like the mitogenic lectin Con A and the highly toxic lectin ML I. Such conjugates show a 'janusfaced' behaviour. We would expect the Con A-ML I conjugate to react as a mitogen in the presence of D-galactose (ML I is inhibited) and as a toxin in the presence of D-mannose (Con A is inhibited) . In conclusion conjugates of lectins with other affinitins can provide useful tools in molecular biology. REFERENCES 1.

Franz, H. (1979). 'Affinity chromatography and Molecular Interactions' Colloque Strasbourg 1979, Editor, J.E. Egly, Editions INSERM, Paris.

2.

Frans, H., Bergmann, P., Ziska, P. (1979). 59, 335-342.

3.

Goldstein, I.J., Hughes, R.C., Monsigny, M., Osawa, T. and Sharon, N. (198o). Nature 285, 66.

4.

Kocourek, J., Horejäi, V. (1981).

5.

Guesdon, J.-L., Avrameas, S. (198o). Pasteur) 131 C, 389-396.

6.

Takatsy, G. (1967).

7.

Guesdon, J.-L., Avrameas, S. (198o). J. Immunol. Meth. 39, 1-13. Shier, W.T. (1976). In 'Concanavalin A as a Tool', Edited by H. Bittiger and H.P. Schnebli, John Wiley and Sons, London, New York, Sydney, Toronto, 6o5-611.

8.

Histochemistry

Nature 29o, 188. Ann. Immunol. (Inst.

Symp. Ser. Immunbiol. Stand. 4,

275.

LECTIN - CARBOHYDRATE

INTERACTIONS

STUDIED

IN A

TWO-PHASE

SYSTEM L i n g , T . G . I. a n d M a t t i a s s o n ,

B.

D e p a r t m e n t of P u r e & A p p l i e d B i o c h e m i s t r y , C h e m i c a l C e n t e r , U n i v e r s i t y of L u n d , P . 0 . Box 7 4 0 , S - 2 2 0 07 Lund, Sweden.

When studying

interactions

between

p r e s e n t as f r e e m o n o s a c c h a r i d e s cell surface binding

carbohydrates,

assay which

regardless

lectins and sugar

it w o u l d be a d v a n t a g e o u s

is s u i t a b l e

for d i f f e r e n t

of the s i z e of the l i g a n d

separation

a r e o b t a i n e d by m i x i n g

in a q u e o u s aqueous

one polymer and one salt systems

used

in t w o - p h a s e

two-phase

(1). T h e r e

systems

of a q u e o u s

solutions

c a t i o n , be a s c r i b e d between phase

in b i n d i n g

-

98%)

of p o l y m e r s

to s m a l l d i f f e r e n c e s

the d i f f e r e n t p o l y m e r s . T h u s ,

separation

is the s a m e as

tems w i t h o r g a n i c

solvents.

Due

assays

thus

are

in

which

in c o n v e n t i o n a l the r a n g e of

the p h a s e

or such

Separation and

has

(2,3). separate

simplifi-

hydrophobicity

the d r i v i n g

force

for

two-phase content

sys(85

hydrophobicity

systems are very m i l d

biomolecules. When compared with organic polymer phases

c a n , as a

to the h i g h w a t e r

in a q u e o u s p o l y m e r p h a s e s

is v e r y n a r r o w a n d

toler-

a r e m a n y e x a m p l e s of

is a s i m p l e a n d q u i c k p r o c e d u r e for use

a

step.

s o l u t i o n s of two p o l y m e r s

P h a s e s y s t e m s . T h e r e a s o n for the f o r m a t i o n of two phases

Such

systems,

or

ligands

separation

for s e p a r a t i o n of b i o l o g i c a l m a t e r i a l .

b e e n s h o w n to be s u i t a b l e

to u s e

t y p e s of

to be s t u d i e d .

a n t s y s t e m s m u s t be b a s e d o n a v e r y v e r s a t i l e We have used

residues,

o r as p a r t of g l y c o p r o t e i n s

solvents, all

to aqueous

hydrophilic.

T h e d i s t r i b u t i o n of a m o l e c u l a r

species

in a t w o - p h a s e

Lectins - Biology, Biochemistry, Clinical Biochemistry, V o l . II © W a l t e r d e Gruyter &. C o . , Berlin • N e w Y o r k 1982

system

564 depends on both the physical properties of the substance partitioned

and

to be

the composition of the polymer system. The par-

titioning of a substance

in a two-phase system

by the partition coefficient, K p a r t , defined the concentrations

is characterised

as the ratio of

in the top and bottom phases,

K

respectively:

C, 'top '"bottom

part

Principle for binding assay in two-phase systems. In binding assays there are basically reactant other

two reactants. G e n e r a l l y , one

is a binding protein, in our case a lectin, while

is a ligand. One of the reactants

binding step, the free ligand

is labelled. After

is separated

ligand and the amount of free or bound

free labelled labelled

ligand

label

is determined.

ligand

in the

the

two

to different phases, so that

is found

in one phase and

other.

by using a suitable composition of the phase system. it partition preferentially

the phases. Thus, if a lectin and a labelled

carbohydrate

observed by the displacement of the label when the to the

Materials and Separation

par-

can be

carbohydrate

lectin.

Methods

in phase systems. 900

tem consisting of 13.5% and 13.5%

chemi-

to one of

tition to different phases when free, the interaction is bound

achieved

However,

if this is not possible, one of the reactants could be to make

the

the bound

For a given pair of reactants, such a situation may be

cally modified

the

from the bound

When two-phase systems are used for separation, reactants should partition

the

of a well-mixed

(w/v) poly(ethylene

(w/w) MgSC>4 • 7 H2O was added

glycol),

phase

sys-

PEG-4000

to a test tube

contain-

565 ing 100 nl incubation mixture. The test tube was thoroughly shaken and within 5 min two distinct phases were formed, the upper phase consisting mainly of PEG and the lower phase of salt (3). Assay system. The glucoenzyme horseradish peroxidase (EC 1.11. 1.7) was used as a naturally occurring labelled carbohydrate. Both conA and peroxidase where found to partition to the bottom phase, however, conA could be made to partition to the top phase by covalent attachment of monomethoxy-PEG (3). Attachment of M-PEG to conA. Monomethoxy-poly-(ethylene glycol) (M-PEG), molecular weight 5000, was activated with triazine and washed free of unreacted triazine (4). The activated M-PEG was then allowed to bind to amino groups on conA. When derivatizing a lectin some precautions must be observed. The degree of M-PEG-modification and the molecular weight of the polymer was found to be important. At a high degree of modification

2

10

20

50

100

mg MPEG/ml

Fig. 1 The partition coeffecient (log Kp a r t) of horseradish peroxidase bound to M-PEG-conA plotted against the amount of activated M-PEG added in the coupling step. The molecular weight of M-PEG was 2000 A and 5000 • respectively.

566 the lectin partitioned to the desired phase but the binding capacity was very low, so the ability of conA to transport the bound ligand across the phase boundary was low. Fig 1 shows the transport capacity of modified conA at different degrees of modification. When peroxidase was bound to the M-PEG-modified conA (M-PEGconA) the complex partitioned to the top phase, so that the enzyme activity in the top phase represented bound peroxidase and the activity in the bottom phase free peroxidase. Assay procedure. M-PEG-conA, peroxidase and the molecules or cells to be quantitated were mixed to give a final volume of 100 fil. After incubation at room temperature for 10 or 30 min, 900 nl of a well-mixed two-phase system was added and the tubes were shaken vigorously for 5 sec before phase separation took place. After 10 minutes of separation an aliquot of 200 |il was taken from each phase for determination of enzyme activity.

• O

G

Peroxidase

M PEG-conA

-

Carbohydrate

Fig. 2 Schematic representation of the assay, a: M-PEG-modified conA partitions to the top phase even when horseradish peroxidase is bound to it. Free peroxidase partitions to the bottom phase, b: The presence of a carbohydrate gives a competitive situation where the amount of free enzyme is increased.

567 The reaction mixture had a final composition of 100 mM TrisHC1, pH 7.00, 14 mM phenol, 4 mM amino-antipyrine and 1 mM hydrogen peroxide. The enzyme activity was determined at 510 nm spectrophotometrically. Alternatively, the enzyme substrates were mixed with the phase system so that the enzyme activity could be determined directly in the phase system. A schematic representation of the assay is shown in Fig 2. In a competitive binding assay the binding of peroxidase to conA was influenced by the presence of low molecular weight

—i

10

1

100

i__

1000 UM

methyl a-D-mannopyrarioside

Fig. 3 Enzyme activty in the top phase as a function of the concentration of methyl-a-D-mannopyranoside in the system. On the activity scale 0% is the enzyme activity in the top phase when only the peroxidase is present in the phase system, 100% is the activity when the M-PEG-conA is included.

568

Time

Fig. 4 T i m e c o u r s e for the q u a n t i t a t i o n of m e t h y l - a - D - m a n n o p y r a n o s i d e . The e n z y m e s u b s t r a t e s are included in the p h a s e s y s t e m and the a b s o r b a n c e is read in the b o t t o m p h a s e . The c o n c e n t r a t i o n of the c a r b o h y d r a t e is i n d i c a t e d at the c u r v e s (in nM).

Dextran T 40

Fig. 5 Q u a n t i t a t i o n of d e x t r a n (mol w e i g h t 40,000) u s i n g the same s y s t e m as in F i g . 3. The c o n c e n t r a t i o n of the p o l y m e r is c a l c u l a t e d for the m o n o m e r i c u n i t s .

569 carbohydrates. Fig 3 shows a calibration curve for quantitation of methyl-a-D-mannopyranoside. If the enzyme substrates are included in the phase system it is possible to make a semiquantitative test by comparing the colour intensity in the two phases and in this way perform a quick screening test. When higher accuracy is wanted, the absorbance can be read in a spectrophotometer, using the same experimental procedure. The time course for such an experiment is shown in Fig 4.

Native

conA

Fig. 6 Competitive binding assay of native conA where M-PEGconA and native conA compete on binding to peroxidase. The activity scale is the same as in fig. 3.

570 Macromolecular carbohydrates can also be assayed using the same experimental procedure. Fig 5 shows the calibration curve for dextran

(poly-glucose).

The binding structure can also be quantitated using this experimental setup. Thus, native conA was quantitated using the basic assay system, by including the native conA in the incubation mixture, so that modified and native conA competed for binding peroxidase. Since conA was present in 100-fold excess over peroxidase, the partitioning of the enzyme reflected the ratio between the two forms of conA present in the assay. A calibration curve is shown in Fig. 6.

logKpart

0.5

1.0

2.0 K

5.0

10j0

20j0

ass"«3

Fig. 7 The partitioning coefficient of peroxidase as a function of the association constants between M-PEG-conA and four carbohydrates (3.3mM): • sucrose, A D-fructose, • methy1a-D-glucopyranoside and • methyl-a-D-mannopyranoside.

571 In o r d e r to i l l u s t r a t e

the r e l a t i o n b e t w e e n the

c o n s t a n t and the o u t c o m e of an a n a l y s i s , the

association

interaction

b e t w e e n conA and four c a r b o h y d r a t e s w i t h d i f f e r e n t constants was studied

(Fig. 7). The m e a s u r e d

association

partitioning

c o e f f i c i e n t w a s found to be a linear f u n c t i o n of the a t i o n c o n s t a n t d o w n to the d e t e c t i o n limit for the tion

associ-

concentra-

used.

B i n d i n g a s s a y s can be p e r f o r m e d

in a q u e o u s t w o - p h a s e

for a n y type of l i g a n d and for m i x t u r e s of c e l l s m o l e c u l e s of d i f f e r e n t sizes, and the m e t h o d q u i c k and

systems

(5) and

bio-

is p o t e n t i a l l y

sensitive.

T h e s e n s i t i v i t y of the a s s a y s m e n t i o n e d above

is lower

w h a t is r e p o r t e d for a n t i g e n - a n t i b o d y - i n t e r a c t i o n s . the a s s o c i a t i o n c o n s t a n t for c o n A - c a r b o h y d r a t e a r o u n d 10^ i/mol as c o m p a r e d

to 10^ 1/mol

for

than

However,

reactions

is

antigen-

antibody-reactions . T h i s w o r k w a s s u p p o r t e d by the N a t i o n a l S w e d i s h B o a r d T e c h n i c a l D e v e l o p m e n t and P h a r m a c i a D i a g n o s t i c s

for

AB.

References 1. A l b e r t s s o n , P - A . : P a r t i t i o n of C e l l P a r t i c l e s and M a c r o m o l e c u l e s , S e c o n d E d i t i o n (1971), A l m q v i s t & W i k s e l l , Stockholm. 2. M a t t i a s s o n , B . : J. Immunol. M e t h . 35, 1 3 7 - 1 4 6

(1980).

3. M a t t i a s s o n , B., L i n g , T. G. I.: J. I m m u n o l . M e t h . 3j5, 2 1 7 223 (1980). 4. A b u c h o w s k i , A . , v a n E s , T., P a l c z u k , N. C., D a v i s , F.: J. B i o l . C h e m . 252, 3 5 7 8 - 3 5 8 1 (1976). 5. M a t t i a s s o n , B., L i n g , T . G . I . , N i l s s o n , J . , Diirholt, M. : These proceedings.

LECTIN - CARBOHYDRATE MICROBIAL

I N T E R A C T I O N S AS A T O O L TO

QUANTIFY

CELLS

B. M a t t i a s s o n , T. G. I. Ling, J. N i l s s o n and M.

Dürholt

D e p a r t m e n t of P u r e and A p p l i e d B i o c h e m i s t r y , C h e m i c a l C e n t e r , U n i v e r s i t y of L u n d , P. O. Box 740, S-220 07 L u n d , S w e d e n

I n t e r a c t i o n s b e t w e e n y e a s t cells and the lectin c o n c a n a v a l i n A were studied

in an a q u e o u s t w o - p h a s e s y s t e m . S u c h phase

tems are c h a r a c t e r i z e d by b e i n g b i o c o m p a t i b l e

and in some

cases even stabilizing biological macromolecules.

It has

s h o w n e a r l i e r that small m o l e c u l e s o f t e n p a r t i t i o n e q u a l l y to the two p h a s e s , w h e r e a s m a c r o m o l e c u l e s p r o t e i n s o f t e n p r e f e r one of the two p h a s e s

been

almost such as

(1). C e l l s

o t h e r p a r t i c l e s u s u a l l y p a r t i t i o n b e t w e e n the p h a s e and one of the

sys-

and

boundary

phases.

The s y s t e m s t u d i e d m a y be r e g a r d e d as a m o d e l s y s t e m trating a g e n e r a l p r i n c i p l e . Q u i c k d i a g n o s t i c m e t h o d s g r e a t i n t e r e s t in m i c r o b i o l o g y . During m a n y y e a r s c a t i o n and q u a n t i t a t i o n of m i c r o o r g a n i s m s w e r e

illusare of

identifi-

exclusively

b o u n d to plate c o u n t i n g . T o d a y o t h e r a l t e r n a t i v e m e t h o d s b e e n p r e s e n t e d , b u t still the o l d , t i m e - c o n s u m i n g are w i d e l y used. In m a n y cases the r e s u l t of an a n a l y s i s Immunochemical

it m a y take s e v e r a l days

By using a q u e o u s

to speed up m i c r o -

(2). S e v e r a l new p r o c e d u r e s are now

b u t m o s t of them r e q u i r e a p r e c u l t u r i n g r a t h e r l a b o u r - and

before

is ready.

t e c h n i q u e s have b e e n a d o p t e d

biological assays

have

procedures

step and are

used, therefore

time-consuming.

two-phase

systems

in the s e p a r a t i o n step

Lectins - B i o l o g y , Biochemistry, C l i n i c a l Biochemistry, V o l . II © W a l t e r de Gruyter &. C o . , Berlin • N e w Y o r k 1982

it

574 is possible to quantify cells in a manner analogous to the principles of radio immunoassay and enzyme immunoassay (3). Because of the high sensitivity in these methods it is possible to perform a sensitive analysis with no needs for preculturing of the microorganism. Provided the lectin molecules and the yeast cells were partitioned to different phases it would be possible to study the binding of lectin to the cells and eventually also to use this binding for quantifying one of the biological entities in the binding reaction. We have chosen to quantify yeast cells in this manner. In a preliminary test it was shown that yeast cells as well as conA are partitioned to the bottom phase. Thus a direct binding assay would not be possible without altering the partition behaviour of one of the entities. In an earlier report (4) we described the use of conA with covalently attached monomethoxypoly-(ethylene glycol) molecules (M-PEG-conA) for quantifying small carbohydrates as well as macromolecules. However, since those studies show that the modified lectin loses part of its binding capacity, modified yeast cells were used to compete 125

with native cells for

i-labelled conA in this study.

Materials and Methods Poly-(ethylene glycol) raw 6000 (PEG-6000) and cyanuric chloride were purchased from BDH, England. Monomethoxy-PEG mw 5000 (M-PEG-5000) was a generous gift Union Carbide, Belgium. 125 I was obtained as Nal from Radiochemical Centre, Amersham, 125 UK. ConA came from Miles-Yeda, Israel. Labelling with I was performed according to the lactoperoxidase method

(5).

Yeast (Saccharomyces cerevisiae) was from Jastbolaget, Sweden. All other chemicals used were of analytical grade. Separation in phase system; 3.0 ml of a well-mixed phase system

575 consisting of PEG 6000 15% (w/w) and MgS0 4 -7H 2 0 15% (w/w) was added to a test tube with 500 jil incubation mixture. The test tube was thoroughly shaken and within 5 min two distinct phases were formed, the upper phase consisting mainly of PEG and the lower phase of salt (6). Activation of monomethoxy-PEG. Monomethoxy-PEG was activated according to the following procedure (7): 5.5 g cyanuric chloride was dissolved in 400 ml of water-free benzene containing 10 g of sodium carbonate (water-free). 50 g of M-PEG 5000 was added and the mixture was stirred in a sealed vessel over night at room temperature. After filtration, the clear solution was treated by slowly, under gentle stirring, adding 600 ml of petroleum ether. The precipitate was filtered off and redissolved in 400 ml of benzene. This washing procedure was repeated four times. Finally the precipitate was kept under vacuum in a desiccator. Modification of yeast cells. To 4 ml of yeast cell suspension in 0.1 M triethanolamine buffer pH 9.4 containing 0.1 M NaCl (containing 9*106 cells per ml) was added 10 or 100 mg of the activated M-PEG. Coupling proceeded for 1 h at room temperature. To stop the reaction a five fold molar excess of glycine in the above buffer was added to each reaction vessel. Binding studies. 50 nl of M-PEG-yeast cell-suspension was 125 incubated under gentle mixing with 50 |il of i-conA in 500 \il 0.1 M Tris-HCl, 1 mM in MgCl 2 , CaCl 2 and MnCl 2 , pH 7.2 for 30 min at room temperature. Separation was achieved by addition of the phase system (1.5 ml MgSC>4 and 1.5 ml PEG6000). Phase separation took place at 20° C for 10 min. After phase separation had taken place aliquots of 200 nl were taken from top and bottom phase respectively and the radioactivity was measured using an LKB minigamma counter. Competitive binding assay. M-PEG-modified yeast cells and

576 n a t i v e c e l l s were a l l o w e d to c o m p e t e for the conA before phase separation

took

125

1-labelled

place.

T w o - s t e p b i n d i n g a s s a y . In the f i r s t step n a t i v e c e l l s and labelled

lectin were

a large n u m b e r

i n c u b a t e d for 30 m i n .

In the s e c o n d

the

step

(250,000) of M - P E G - y e a s t c e l l s w a s a d d e d ,

so

that all free l e c t i n should bind to these c e l l s . A f t e r 3 m i n i n c u b a t i o n the phase s y s t e m w a s added and p h a s e s e p a r a t i o n took 125 p l a c e for 10 m i n at 20° C. In this w a y , the a m o u n t of IconA in the b o t t o m phase w a s d e p e n d i n g

on the n u m b e r of

y e a s t c e l l s p r e s e n t since the l e c t i n b o u n d to the cells was transported

R e s u l t s and

to the

studies

is k n o w n that b o t h the n a t i v e c e l l s

the l e c t i n are e n r i c h e d used. To e v a l u a t e

cells,

Table

Number cells 0

interface or the top p h a s e .

Discussion

From earlier tem

in the b o t t o m p h a s e of the p h a s e

the e f f e c t of M - P E G - m o d i f i c a t i o n of

two d i f f e r e n t d e r i v a t i z a t i o n s were m a d e

1.

of

native

modified

Partitioning

A m o u n t of M-PEG added

-

of M - P E G - m o d i f i e d y e a s t

(Table

and systhe

1).

cells

R a d i o a c t i v i t y (cpm) top p h a s e bottom phase interface 5100

55800

0

9-106

10 m g

5600

43400

11900

6

100 mg

8900

6300

45700

9-10

P a r t i t i o n i n g of M - P E G - m o d i f i e d y e a s t c e l l s , s h o w n i n d i r e c t l y by the d i s t r i b u t i o n of 2 5 i - c o n A in the p h a s e s y s t e m . The d e g r e e of m o d i f i c a t i o n is i n d i c a t e d by the a m o u n t of a d d e d a c t i v a t e d M - P E G in the c o u p l i n g step.

577 From

this

tion of

effect,

from

adsorb

to the

interface

and

phase.

partition included

uted mainly b e t w e e n

n a t i v e cells and

due

furthermore

be

figure

concluded but

and

mainly modified

in the system, conA will be

assay

a constant

yeast

in the bottom using

a high v a r i a t i o n limit of around

the

phase. amounts

n u m b e r , 3 0 0 , 0 0 0 , of

curve was obtained

distrib-

cells at

different

of

M-PEG-modified

(Fig. 1). As

is

in the analyses was

to the problem of r e p r o d u c i b l e

quots. A detection this

to obtain the 125 of the bound i-labelled

the M - P E G - m o d i f i e d

cells, a calibration this

It can

derivatiza-

in order

to none of the phases

the native cells

In a c o m p e t i t i v e binding

from

the degree of

interface. Thus, when both native

cells are

partly

that

i.e. translocation

the bottom

that M - P E G - c e l l s yeast

it may be seen

the cells must be rather high

expected conA

table

pipetting

of

seen

obtained, ali-

105 cells was obtained

using

procedure.

cpm tl 10 9

8 7 6 5 4 3 > F

2 1

60

120

180

240

300 « 103

NUMBER OF CELLS Fig. 1 C a l i b r a t i o n curve for native yeast cells in a c o m p e t i tive assay. N a t i v e cells and 3 0 0 , 0 0 0 M - P E G - m o d i f i e d cells w e r e mixed with I - 1 2 5 - l a b e l l e d conA. The r a d i o a c t i v i t y in the bottom phase w a s determined after s e p a r a t i o n .

578

To further improve the sensitivity a two-step incubation was planned. First native cells were allowed to bind labelled conA and after a fixed period of incubation, the second

incubation

was initiated by the addition of an excess of M-PEG-modified yeast cells to bind all free labelled lectin.

cpm

1500 ® (0 (0

E

2

o

.o 0)

1000

c >>

3

500

Mh

_L

2

3

«10

Cell number

Fig. 2 Influence of the amount of M-PEG-modified yeast cells used in the assay and the incubation time in the second step ( # 3 min and • 30 min). After separation the amount of 1-125-labelled con A present in the bottom phase was determined •

579 A crucial point at this stage is the time needed for binding to take place in the second step. Too short a period will not allow the derivatized cells to trap all the labelled lectin. On the other hand too long a period will introduce the risk that dissociation of lectin already bound to the native cells will result in a transfer of label from the native to the modified cells. A time study was undertaken to investigate the time needed for the second step to take place. Fig. 2 shows that, small differences between the results of 3 and 30 min incubation, are observed. This observation is fully in line with earlier observations on the interaction between conA and yeast cells. Fig. 3 shows a calibration curve for yeast cells using this two-step incubation procedure. In the first step the native cells were incubated with labelled conA for 20 minutes before 225,000 M-PEG-modified cells were added. After three minutes the incubation was terminated by the addition of an aqueous two-phase system (total volume 4 ml). The phases were allowed to separate for 10 minutes before 500 jil from each phase was taken for counting. This study clearly demonstrates that lectin-carbohydrate

inter-

actions can be used in binding assays where the separation of bound from free ligand is performed using partition in aqueous two-phase systems. This separation method makes it possible to run assays of whole cells, fragments of cells and free antigens, using basically the same experimental procedure, since the partition characteristics is not primarily due to the size but to the surface properties of the molecule or particle. Furthermore, when the reactants partition to the same phase, thereby ruining the presumption for a successful direct binding assay, the introduction of a fixed amount of modified reactant of one of the two species participating in the binding assay will make it possible to run a competitive binding assay. In

580 addition, a two-step

incubation procedure will markedly

the s e n s i t i v i t y of such a b i n d i n g a s s a y . T h i s is useful where binding

improve

particularly

is q u i c k - as in the s y s t e m s t u d i e d

here.

cpm

800

-

600

-

400

-

200

-

CO

£ C >»

3

12

24

36

48

60

72

«10

Cell number Fig. 3 C a l i b r a t i o n curve for y e a s t cells using the t w o - s t e p i n c u b a t i o n p r o c e d u r e . F i r s t n a t i v e c e l l s were i n c u b a t e d w i t h I - 1 2 5 - l a b e l l e d c o n A and in the s e c o n d s t e p 2 2 5 , 0 0 0 M - P E G - m o d i fied c e l l s were a d d e d . A f t e r s e p a r a t i o n the r a d i o a c t i v i t y in the top # and b o t t o m B p h a s e s , r e s p e c t i v e l y , w a s d e t e r m i n e d .

581 References 1. Albertsson, P-A. : Partition of Cell Particles and Macromolecules, Second edition (1971), Almqvist & Wiksell, Stockholm. 2. Rytel, M. W. (ed): "Rapid Diagnosis in Infectious Disease", CRC Press Inc., Boca Raton, FL, USA. (1979). 3. Mattiasson, B., Ling T.G.I., Ramstorp, M.: J. Immunol. Meth. 41, 105-114 (1981). 4. Mattiasson, B., Ling, T.G.I.: J. Immunol. Meth. 38, 217-223 (1980). 5. Thorell, J.I., Johansson, B.G.: Biochim. Biophys. Acta 251, 363-369 (1971). 6. Ling, T.G.I., Mattiasson, B.: These proceedings. 7. Abuchowski, A., van Es, T., Palczuk, N.C., Davis, F.: J. Biol. Chem. 252, 3578-3581 (1976).

STUDIES ON THE THERMODYNAMIC EVALUATION OF CONCANAVALIN A CARBOHYDRATE INTERACTION BY MEANS OF AFFINITY ELECTROPHORESIS

Kazusuke Takeo Department of Biochemistry Yamaguchi University School of Medicine Kogushi 1144, 755-Ube, Japan

Affinity electrophoresis is a technique combining the principle of electrophoresis with chromatography. Originally it was developed in 1972 in the study of the interaction of phosphorylase with glycogen (1). When electrophoresis was carried out in a gel containing glycogen, the mobility of phosphorylase was retarded. This retardation was reversed by addition of a competitive inhibitor such as maltotriose or cyclodextrin to the gel (2). From the variation in mobility as a function of the substrate or inhibitor concentrations, dissociation constants were calculated. In the related principle, B0g-Hansen (3, 4) developed two-dimensional affinity electrophoresis combining quantitative Immunoelectrophoresis and affinity electrophoresis to study the interaction of Con A with serum glycoproteins. Kocourek and Horejsi (5) studied the interactions of various lectins with glycoside-acrylamide copolymers. Horejsi (6) developed theoretical aspects of affinity electrophoresis. B0g-Hansen and Takeo (7) gave the general equation for determination of dissociation constants by affinity electrophoresis. The technique of affinity electrophoresis is very simple. It required only a small amount of protein, and it is not necessary to purify the protein. When a thermostatic electrophoresis apparatus is available, thermodynamic parameter can

Lectins - Biology, Biochemistry, Clinical Biochemistry, Vol. II © Walter de Gruyter & Co., Berlin • New York 1982

584

be determined from the van't Hoff plot. The thermostatic electrophoresis apparatus. In Fig. 1, photograph (A) and diagram (B) of a simple thermostatic Polyacrylamide gel disc electrophoresis apparatus is presented. Electrophoresis tubes(G) are immersed completely in the buffer solution in the lower electrode vessel(V^). The temperature of the buffer solution is regulated by circulating water at a constant temperature through a Fig. 1. Photograph and diagram of a thermostatic electrophoresis apparatus.

coiled condensor(C) and by simultaneous stirring with a magnetic stirror(M). The plot seen in Fig. 2 is the temperature difference between the gel and the buffer solution during electrophoresis at varying strength of electricity. The temperature is monitored by use of Copper-Constantan thermocouple. When 3 mA per tube or more electricity is applied, the temperature of the gel rises and in these conditions the temperature difference between the gel and the buffer solution exceeds 0.2°C. At electricity under 2.5

585 mA per tube, the temperture difference is maintained under 0.2°C. In this study, 2.0 mA per tube was applied throughout the experiment. Affinity electrophoresis Electrophoresis was carried out with the acidic buffer system (pH 4.3/4.5) according to the method of Reisfeld et al. (8) at constant temperature. The separating gel was Fig. 2. The temperature difference between gel and buffer soltuion during eleetrophoresis

1.0

1.5

Electricity

2 0

2.5

3.0

( mA per tube )

prepared 5 cm in height as a 5.0% gel and the spacer gel 1 cm in height. For the affinity electrophoresis of Con A polysaccharide interactions, polysaccharide was added to the separating gel. For determination of dissociation constant (K^), one set of electrophoresis was run with 12 tubes, duplicated gels of 6 different concentrations of polysaccharide in the separating gels. For determination of dissociation constant (Ki) of Con A - oligosaccharide interactions, electrophoresis was run with 12 tubes; 2 contained neither polysaccharide nor oligosaccharide, 8 tubes con-

586

tained a constant concentration of polysaccharide and 4 different concentrations of oligosaccharide, and the other 2 contained only oligosaccharide. Con A was applied in 10% sucrose solution containing buffer at the same concentration as the spacer gel. As the references for calculation of the relative migration distance of Con A, methylene blue and egg white lysozyme were added to the Con A solution. For electrophoresis 0.5 - 1.5 ug of Con A and 1 - 2 ug of lysozyme were used per tube. Fig. 3. Concanavalin A - dextran interaction A

AFFINITY

PATTERN

AFFINITY

Conen.

PLOT

of

D«»tra"

xi

Electrophoresis was carried out at 120 volts and 2.0 mA per tube until the tracking methylene blue band has migrated 4.5 cm from the origin in the separating gel. After electrophoresis, the gel was removed from the glass tube and stained overnight in 0.02% Coomassie Brilliant Blue R 250 in 7% acetic acid. In this condition, lysozyme migrated to almost the same position as that of the tracking methylene blue band. The relative migtation distance is the ratio of the distance of migration of the Con A to that of the lysozyme.

587

Calculation of dissociation constants for Con A - carbohydrate interaction. Dissociation constants of polysaccharides to Con A (K^) were calculated according to the following equation (1): 1/Riru = l/Rmo( 1 + c/Kj )

(1) ,

where Rm Q and Ritu are the relative migration distances of Con A in the absence or the presence of polysaccharide. C was the concentration of polysaccharide in the separating gel. When 1/Rnu is ploted against c, a straight line is Fig. 4. Coneanavalin A - maltotriose interaction INHIBITION AFFINITY

PATTERN

D e x t r a n 0.5 —

(»)

0.5 0.5 — —

^

INHIBITION AFFINITY PLOT

«V 0 . 0 5 ) and the regression coefficient (O.96) does not differ significantly from 1 (P > 0.05).

642 kinds of lectin (SBA and Con A) when investigated by both immunological methods. The single step procedure used (Fig.2) avoided the possible artefactual absence of immunoprecipitate with antiserum and SBA or Con A due to non-equivalence of antibody and antigen concentrations. In summary, rate nephelometry is a rapid and reproducible method for the quantitative determination of WGA determination and can be used for large numbers of samples. The results may" be printed out and it is not necessary to cast gels or measure areas enclosed by immunoprecipitates - both of which can be time-consuming when many samples must be investigated.

References 1.

Baldwin, J.R., Hendrixson, M. , Derry, C.: Protides Biol. Fluids. Proc. Colloq. 27, 853 (1979).

2.

Laurell, C.B.: Anal. Biochem. 10, 358 (1966).

3.

Mancini, G., Vaerman, J.P., Carbonara, A.O., Heremans, J.F., Protides Biol. Fluids, 11th Colloq. Bruges, 1963, Ed. Elsevier, Amsterdam, p. 3 7 0 (1964). Bracciali, A., Cantagalli, P., Pompucci, G., Tarli, P.: Cereal Chem. 57, 3 6 7 (1980).

5.

Pompucci, G., Cantagalli, P., Piazzi, S.E., Fiaschi, A., Bracciali, A., Bovalini, L.: Boll. Soc. Ital. Biol. Sper. 51, 401 (1978).

6.

Hartree, E.F.: Anal. Biochem. £8, 422 (1972).

7-

Harboe, N., Ingild, A.: Scand. J. Immunol. 2 (Suppl'. 1)

161 (1973).

8.

Ouchterlony 0.: Handbook of Immunodiffusion and Immunoelectrophoresis. Ann Arbor Sci. Publ. Inc., Ann Arbor, Michigan ( 1 9 6 8 ) .

9.

Piazzi, S.E.: Anal. Biochem. 27, 28l

.0.

Cantagalli, P., Bracciali, A., Pompucci, G., Tassi-Micco, C.: Riv. Soc. Ital. Sci. Alim. 2, 69 (1979).

(1969).

QUALITY CONTROL OF COMMERCIAL PHYTOHEMAGGLUTININS (PHA) FROM RED KIDNEY BEANS (Phaseolus vulgaris)

Liisa Riikola and Theodor H. Weber Minerva Institute for Medical Research, P.O.B. 819, SF-00101 Helsinki 10, Finland

In 1959 Hungerford and coworkers discovered, that the red kidney bean extracts were capable of stimulating human blood leucocytes in vitro to cell division (1). Subsequently it was demonstrated, that it is the lymphocyte, which is stimulated in vitro by the kidney bean extract (2). Later on several other lectins have been shown to be capable of stimulating lymphocytes in vitro, but kidney bean extracts are still commonly used for lymphocyte studies in cell culture. Kidney bean extracts contain several lectins, some of which react only with leucocytes, some of which react with leucocytes and erythrocytes and some of which react only with erythrocytes (3, 4, 5). They are used for chromosome analysis of peripheral blood cell mitoses, for studies of lymphocyte function in vitro and for various other purposes, such as isolating glycoproteins by affinity chromatography or for studies of glycoproteins and cell membrane constituents (6). Today several commercial preparations of kidney bean lectins are available. The present investigation was undertaken in order to evaluate the quality and biological activities of different commercially available kidney bean PHAs.

Materials and methods The following PHA preparations were studied: PHA Wellcome, lots K 6359 and 7318; PHA Gibco, lots A 191004 and C 994404,

Lectins - Biology, Biochemistry, Clinical Biochemistry, Vol. II © Walter de Gruyter & Co., Berlin • New York 1982

644

PHA-P Difco, lots 657315, 667375 and 652200; PHA Medix, lots 9001, 9002, 9003; PHA-L* Medix, lots 9101, 9102 and 9103; PHA-L* Sigma, lot 75C-3928. All preparations were examined for the following properties: visual solubility check, sterility, protein content, protein heterogeneity by disc electrophore^ sis, leucoagglutinating, erythroagglutinating and lymphocytestimulating activity. The solubility was checked visually after dissolving the lyophilized preparations according to the instructions of the manufacturer. The protein content of the ampoulles was determined by measuring the absorption at 280 nm and assuming a specific absorption of 1.14/cm for a solution containing 1 g/1 protein (7) . Disc electrophoresis was performed according to Reisfeld et al. at pH 4.5 (8). Sterility was checked by incubating samples of the dissolved phytohemagglutinin extracts for 48 h at 3 7°C in thioglykolate tubes and 24 h at 3 7°C on chocolate agar. Leucoagglutination was determined as described by Weber et al. (9) and erythroagglutination according to Salk (10). Lymphocyte activation was determined by culturing human peripheral blood lymphocytes isolated by Ficoll-Hypaque density centrifugation. The cultures were either set up in tubes containing 0.5 x 10 lymphocytes in 2 ml RPMI 1640 medium supplemented with 10 % pooled human serum or on microtitre plates, each well containing 10 lymphocytes in 0.2 ml medium. * PHA-L = PHA purified for leucoagglutinating activity

645 Table I. Protein content in different PHA-preparations Preparation, lot nr. PHA Wellcome K 63 5 9 PHA Wellcome K 7318 PHA Gibco A 191004 PHA Gibco A 281910 PHA Gibco C 994404 PHA Difco 657315 PHA Difco 667375 PHA Difco 652200 PHA Medix 9002 PHA Medix 9003 PHA-L Medix 9101 PHA-L Medix 9102 PHA-L Medix 9103 PHA-L Sigma 75C-3928

Protein amount mg/vial measured 21 .5 32.2 18.1 35.0 44.8 20.8 25.2 15.2 1 .08 1 .09 5.17 4.96 5.23 2.0

given -

-

-

-

1 .10 1 .10 5. 00 5.00 5. 00

+ 0. 06 + 0. 06 + 0. 25 + 0. 25 + 0. 25 2.0

The cells were incubated for 72 h in a humidified atmosphere containing 5 % CO- in air. The degree of lymphocyte activation 125 was assessed by measuring the incorporation of I -deoxyuridine into the cells during the last 18 h of culture (8). Triplicate cultures were used. Results All preparations dissolved quite easily, but only the PHA from Wellcome, Sigma and Medix gave clear solutions, while the PHA from Difco and Gibco gave notably turbid solutions. All prepiarations were sterile in the culturing systems used.

646

Fig. 1. Disc electrophoresis of different PHA preparations, migration from top. 1, PHA Wellcome; 2, PHA Gibco; 3, PHA Difco; 4, PHA-L Sigma; 5, PHA-L Medix; 6, PHA Medix. Only Sigma and Medix state the protein content of their PHA-L vials on the label. The protein content in lots 9002 and 9003 of the Medix PHA varied about 1 %. The protein content in different lots from Wellcome, Difco and Gibco varied up to 50 % as measured by the absorbance at 280 nm. Only one lot of Sigma PHA-L was tested and its protein content was in agreement with the statement on the label. The results of the protein determinations are shown in Table I. The results of the electrophoretic analyses for protein heterogeneity are shown in Figs. 1 and 2. As can be seen, Difco PHA's contain 6-8 bands, Gibco PHA 5-8 bands, Wellcome PHA 4

647

Fig. 2. Densitograms of disc electrophoretic patterns of different PHA preparations, a) PHA-L Medix and PHA-L Sigma , b) PHA Medix and PHA Wellcome , c) PHA Gibco and d) PHA Difco. bands, Medix PHA 4 bands, Sigma PHA-L 4 bands and Medix PHA-L 2 bands. The second band in Medix PHA-L is very faint and amounts to only 2-3 % of the total proteins. Thus the active main peak is over 95 % pure in Medix PHA-L. All other preparations contain impurities in amounts well over 10 %.

648 Table II. Agglutinating activity of different PHA-preparations Preparation PHA Wellcome K 6359 PHA Wellcome K 7318 PHA Gibco A 191004 PHA Gibco A 281910 PHA Gibco C 994404 PHA Difco 657315 PHA Difco 667375 PHA Difco 652200 PHA Medix 9001 PHA Medix 9002 PHA Medix 9003 PHA-L Medix 9101 PHA-L Medix 9102 PHA-L Medix 9103 PHA-L Sigma 75C-3928

Leucoagglutinating activity yg/ml 134 63 47 32 32 33 33 9 4 9 7 3 2 2 2

Erythroagglutinating activity yg/ml 17 31 24 17 22 41 25 15 284 568 138 > 1000 > 1000 > 1000 > 1000

Leucoagglutinating activity was highest in Sigma and Medix PHA-L of which the latter caused leucoagglutination at a concentration of 2 yg/ml and these preparations did not contain any detectable erythroagglutinating activity at all. The results of the agglutination tests are shown in Table II. The lymphocyte activating capacity in different lots of Difco and Gibco PHA varied considerably as judged by the variation in the minimum amount phytohemagglutinin giving maximum cell growth. As can be seen in Fig. 3, lymphocytes from different 125 individuals differ in the amount of I -deoxy-uridine incorporated, but the maximum incorporation generally occurs at the same lectin concentrations. In Fig. 4 capacity of five differ-

649

Protein

Fig. 3. Activation of lymphocytes from 3 different individuals with Medix • and Difco PHA's A . ent PHA preparations to activate lymphocytes in vitro is compared. As can be seen, the Medix PHA-L is the most efficient activator as judged by the amount of protein giving maximum uridine incorporation, i.e. 1-2 yg PHA-L/ml (Table III).

Discussion The quality requirements and properties of commercially available PHA from kidney beans may vary depending on the intended use of the PHA. However, the use of reproducible and well characterized reagents is certainly advantageous for any type of work. All the tested preparations were sterile, which of course is an essential property for tissue culture work, but also for other purposes.The incomplete - solubility of the Difco and Gibco PHA preparations may not always constitute a drawback, but incomplete solubility may easily lead to concentra-

650

Protein

Fig. 4. Lymphocyte activation with different PHA preparations. Medix PHA-L • , Medix PHA , Wellcome P H A — — — , Difco PHA • • • • , Gibco PHA (cultures on microtitre plates) . tion differences in certain experimental systems, and lot to lot variations in solubility may have consequences for the results . It has been firmly established that the lectins of kidney beans are glycoproteins and thus different activities of kidney bean phytohemagglutinins should be related to the protein content of the extract (4). Non-protein components in kidney bean lectin preparations ought to be regarded as impurities. With this in mind it is surprising, that only Sigma and Medix state the protein content of their PHA-L vials on the labels. The very gross variations in protein content of different lots of PHA from Wellcome, Gibco and Difco reflects varying amounts of impurities in the preparations,but also varying amounts of

651 Table III. Lymphocyte activating efficiency of different PHA-prepara^ tions. Protein amount needed for maximum stimulation (cultures in tubes). Preparation PHA

Wellcome K 6359

PHA

Wellcome K 7318 Gibco A 191004

PHA PHA

Protein amount |ig/ml 11 9

Gibco

A 281910

> 25 26

PHA

Gibco

C 994404

56

PHA

Difco

657315

PHA

Difco

667375

6 14

PHA

Difco

PHA PHA

Medix Medix

652200 9001

PHA

Medix

9002 9003

18 3 3

PHA-•L Medix PHA--L Medix PHA--L Medix

9101 9102 9103

5 1 1 2

PHA--L Sigma

75C-3928

4

lectins as judged by the variations in biological activities and in

protein heterogeneity.

Since different

impurities and contaminants of the lectins

may or may not have biological or chemical effects in various experimental systems, it seems that small lot to lot variations in protein content would be desirable. Also a constant and reproducible protein pattern would be advantageous. Of the semipurified PHA preparations only Medix PHA seems to fulfil these requirements. If PHA is to be used for the removal of erythrocytes by agglutination, a high erythroagglutinating titre is essential.

Pu-

rely erythroagglutinating kidney bean PHA is not commercially

652 available and it cially available erythrocytes. At found effects on

seems doubtful, whether any of the commerPHA preparations are suitable for removing least this cannot be achieved without proother cell types in the suspension.

In work employing lymphocyte activation in vitro either for cytogenetic studies or for lymphocyte function studies, a reproducible and effective activation capacity is essential. In addition it seems that at least in lymphocyte studies erythroagglutination is not a very desirable feature, although its effect is small in culturing systems using relatively pure lymphocytes. In cell culturing systems using whole blood strong erythroagglutination is definitely a draw-back.Semipurified PHA is per se suitable for studies where the aim is to cause as many mitotic divisions in vitro as possible for cytogenetic analysis. In these instances the main requirement is a reproducible and effective lymphocyte-stimulating capacity. However, when lymphocyte function is studied in vitro the requirements for well-characterized and controlled systems grow and in these instances only very well standardized semipurified PHA can be used, but the use of the more extensively purified PHA-L is highly recommendable. When kidney bean lectins are used for more sophisticated studies of lymphocyte function, membrane events or protein characterization only pure lectin is suitable. According to the product information some commercially available kidney bean PHA preparations are still manufactured and processed by rather crude methods. However, the methods for protein purification have greatly developed during the last decades and one would expect manufacturers of commercial protein preparations to take advantage of this in order to obtain qualitatively better and more exactly defined products. This seems to be the case only for a part of the manufacturers of kidney bean PHA.

653 Several commercially available kidney bean PHA preparations have highly different properties depending on the manufacturer. In addition some of the preparations show a very great lot to lot variation. When selecting a PHA for scientific or other purposes, one has to evaluate carefully the properties desired i.e. leucoagglutination, erythroagglutination, lymphocytestimulating. capacity etc. Today very few commercially available PHA preparations fulfil even moderate quality requirements in respect of batch to batch variation and content of impurities. However, as well as the medical doctor today treats his patients with pure digitalis compounds instead of using the leaves of the foxglove plant as William Withering during the 18th century, it would seem rational for the cell biologist and scientist to use a purified, well characterized PHA in his work, rather than unpurified extracts of kidney beans.

Acknowledgements. This work was supported by grants from the Sigrid JusSlius Foundation, Helsinki, Finland.

References 1. Hungerford, D.A., Donally, A.J., Nowell, P.C., Beck, S.: Am. J. hum. Genet. 11, 215-236 (1959). 2. Carstairs, K.: Lancet ii, 984 (1961). 3. Weber, T.H.: Scand. J. clin. Lab. Invest. Suppl. 111(1969). 4. Weber, T.H., Aro, H., Nordman, C.T.: Biochim. Biophys. Acta 263, 94-105 (1972) . 5. Egorin, M.J., Bachur, S.M., Felsted, R.L., Leavitt, R.D., Bachur, N.R.: J. biol. Chem. 25f, 894-898 (1979). 6. Sharon, N., Lis, H.: Science 177' 949-959 (1972). 7. Rasanen, V., Weber, T.H., Grasbeck, R.: Eur. J. Biochem. 38, 193-200 (1973) .

654 8.

Reisfeld, R.A., Leius, U.J., Williams, D.E.: Nature 195, 281-283 (1962).

9.

Weber, T.H., Nordman, C.T., Gräsbeck, R.: Scand. J. Haemat. 4, 77-80 (1967).

10. Salk, J.E.A.: J. Immunol. 49, 87-88 (1944).

PART V DISTRIBUTION, ISOLATION AND CHARACTERIZATION OF LECTINS

A POLYAGGLUTINATING,NON

MITOGENIC

LECTIN

FROM ARION IMPERICORUM

(Ael) DR. M A R T I N IN

MEMORIAM

Günther Hermann, Bernhard Ingrid Karalus Immunologische Abteilung 5ooo Köln 4 1 , G e r m a n y Lutz

KRÜPE

Schmidt-Horstmann, der C h i r u r g i s e h e n

Gürtler

Wolfgang

Approximately

4oooo

molusca

- mussles

f o u n d a r o u n d the w o r l d "and h a v e

been

d e s c r i p t i o n of eel 1 a g g l u t i n a t i n g lished

(I), a l a r g e

discovered

(2,3,4).

in M a r b u r g / L a h n pioneers piece

Universität

Especially

and Fulda

at l e a s t

hemagglutinins

s t a n c e s ,whi ch w e natural lectins

during call

more

in p l a n t s

During

the

than

reported

that Lima bean

could

been

has

pubbeen

worked

lectins.

Already

3oyearsto today.

in

1971

of the f ami 1 y CI ausi 1 i idae

scientific interest was

of l e c t i n s

lectin

lectins

KrUpe, who

a n d di s t r b u t i o n of A B H bl o o d

"lectins"

stances.

- have

as P r o f e s s o r w a s o n e of the

species

(5).His

polymorphism

snail

Dr. M a r t i n

of snail

par p i e c e for p r e s e n c e

ted and devoted

and snails

s u b s t a n c e s in s n a i 1 s w a s

(Germany)

13 snail

Tübingen

d e s c r i b e d . S i n c e the f i r s t

s p e c t r u m of o t h e r

in the e x a m i n a t i o n

he e x a m i n e d specific

der

Bessler

I n s i t u t für B i o l o g i e II der U n i v e r s i t ä t 74oo T ü b i n g e n , G e r m a n y

that

Habets,

Universitätsklinik,

I n s t i t u t für A n t h r o p o l o g i e und H u m a n g e n e t i k München, 8000 München, Germany

the

Leo

this

He w a s

group direc-

k i n d of

interested

and d e v e l o p p e d

the

subin

theory

be c a r b o h y d r a t e - t r a n s p o r t i n g

colloquium

at C o l o g n e

seeds, collected

in 1979

in M e x i c o

Lectins - Biology, Biochemistry, Clinical Biochemistry, V o l . II © W a l t e r d e Gruyter & C o . , Berlin • N e w Y o r k 1982

in

subhe

1925

658 by Prof. N e u m a n n

(Hygiene M u s e u m U n i v e r s i t y of Hamburg), still

a f t e r 55 y e a r s s h o w e d h e m a g g l u t i n a t i n g

activity

m e n t i o n this in honor of our c o l l e a g u e

Dr. M a r t i n

died in M a r c h

(6). We Krlipe who

1981.

Here we show that this p r o m i s i n g has been f a s c i n a t i n g

field of r e s e a r c h on

for many y e a r s ,

lectins

i s fasci nati ng today

w o u l d p r o b a b l y be so t o m o r r o w . We also w a n t to point out already

in 184o

(7) the

"Chimic c o m p o s i t i o n " of the mucus

Helix p o m a t i a and p h a r m a c e u t i c a l and d e s c r i b e d

in detail

in 1953

p r e p a r a t i o n s were (8). H o w e v e r , the

of the a c t i v i t y was not found at that The use of A r i o n e m p i r i c o r u m , for medical knowledge ful

reported

time.

the well

known red road

Krlipe e t a l . (9) made an

in 1966 to find l e c t i n - l i k e a c t i v i t y

by P e m b e r t o n

(11) in the same y e a r .

corum lectin afterwards

Using

popular

in

saline hemagglu-

(lo),

then

hemagglutination

a n a l y s i s we found the Arion

(Ael) first in w h o l e body e x t r a c t s

in the mucus of this snail

snail,

unsuccess-

body e x t r a c t s of this snai1 . The o c c u r e n c e of

t i n i n s was d e s c r i b e d by S c h n i t z l e r e t a l . in 197o and i m m u n o e l e c t r o p h o r e t i c

of

principle

is o c c u l t and the o r i g i n of this

c a n n o t be dated.

search

whole

purposes

and that

and

empirishortly

(12). P r e s e n c e or

sence of the lectin can p o s s i b l y be e x p l a i n e d by the

ab-

existence

of d i f f e r e n t s p e c i e s of "red road s n a i l s " w h i c h can only

be

distinguished

or-

gans

(13,

after anatomical

of the sexual

14).

The a l b u m i n

gland of snails has often been zsed as the

of lectins as well periments

preparation

as whole body e x t r a c t s .

But in our

source ex-

and in the case of A.e. the m u c u s of this snail

r a t h e r easy to p r e p a r e and c o n t a i n s m o r e active 3 road snails are known to c o n t a i n syn. A r i o n e m p i r i c o r u m

substance.

l e c t i n s : A r i o n rufus

(Fer.), A r i o n

lusitanicus

is

(MAB)

(1.) (12),

659 and Arion

subfuscus

(15).

established

if A r i o n

cal

the

because

brown.

Also

Even

rufus

snails

today

it is n o t

and Arion

show

mimetisme

Arion

subfuscus

may

is n o t c o n s i d e r e d

to r e a l l y

belong

exactly

empiricorum in c o l o r

be a v a r i a n t

are

from

o f A.

to a n o t h e r

identired

to

rufus

family

of

the m u c u s

of

distinguish

A.

and

snails. When

starting

different ricorum nation tumor (13)

road

snails.

and A. of

red

cells

selected

and

blood

with

Lectins cells,

as

and

terminal

sugars

distinguished

As

there

lectins positive

active

is s t i l l Zajdela

from

the

rufus

found

nothing

in t h e

biotops,

with

to

the

melacologists.

hemagglutination, cells

cell

i.e.

cancer

chemotactic

property

is

reaction

Lectins

can

be

and

should

considered be

as a s p e c i a l

group

(18). tumor-cell

cells

of

an

existing

isohistogenic activity

The

agglutinating ©¿-foeto in o u r

rats.

saline

properties aim of

this

protein

laboratory

Holtzman

of crude

other

literature.

the

receptors

and antibodies

about

have

stimulation

hepatoma in

hemagglutinating

from Arion tin was

line

was

(16), m i t o g e n i c

for new

ascitic

as t r a n s p l a n t a t i o n

Ant

of g l y c o p r o t e i n s ,

substances

tumor

as w e

comlicated

of t r a n s f o r m e d

proteins.

a need

animal

o f H.

againstdifferent

In p r i n c i p l e , and

specific

(19) w e e x a m i n e d

and

A. e m p i r i c o r u m

activity

empi-

aggluti-

advices

in c o o p e r a t i o n

A common

from enzymes

biologically

snail

activities:

of

possessing

of

the

flagellates

for

species

helpful

precipitation (2,17).

titers

the

substances defined

agglutination

etc.

proteins

(14)

of

could

of d i f f e r e n t

demonstrated.

different

bacteria

reactions

on we

Different

polyagglutinating

be w e l l

lymphocytes,

with

cells

biological

agglutination, of

Later

Jungbluth the

have

had mixtures

found. With

of c e l l s w a s

snails must

we

lusitanicus.

were

and J.H.

types deal

this work

Apart

extracts

of this study

lecwas

660 therefore

the c h a r a c t e r i z a t i o n ,

agglutinating

the i s o l a t i o n , the study

p r o p e r t i e s with normal

cer cells as well

n u c l e a t e d cells and

as the m i t o g e n i c a c t i v i t y of this

to d i f f e r e n t i a t e

The c h a r a c t e r i z a t i o n

certain

of Ael was a c h i e v e d by a b s o r p t i o n

ion-exchange

was tested w i t h animal c e l l s , human

and RBC 3

t a i n e d and m o n o s p e c i f i c

in the

antisera

polyvalant agglutination normal

(21,22). The

cancer

mitogenic mi-

^ - r e g i o n ; pure Ael was r a i s e d in r a b b i t s ; Ael

of d i f f e r e n t c e l l s , but does

liver cells. The lectin

is

ob-

shows

not

practically

mitogenic.

M a t e r i a l s and

Methods

Snails: A r i o n e m p i r i c o r u m ubiquitous

(Fer) syn. Arion rufus

in G e r m a n y . We d e s c o v e r e d

a well

(1.) is

defined

of these s n a i l s in S p i t z e near B e n s b e r g , G e m e i n d e Arion lusitanicus

Immunoelectrophoretic and W i l l i a m s degger

in Murg a.d.

a n a l y s i s was done a c c o r d i n g

(23, 24) in the m i c o r m o d i f i c a t i o n

(25). E x t r a c t s

conditions

colony

K'urten.

(MAB) is only p r e s e n t s o u t h of the

river. The snails have been c o l l e c t e d

mal

the

Agglutination

H - t h y m i d i n e - i n c o r p o r a t i o n ; Ael

grates e l e c t r o p h o r e t i c a l l y

not

chromatography.

of

analysis,

tumor c e l l s , i s o l a t e d human

lymphocytes

assay was p e r f o r m e d with

agglutinate

inter-

cells.

l e c t i o n w i t h r a b b i t RBC and i m m u n o e l e c t r o p h o r e t i c the i s o l a t i o n w i t h

can-

lectin

because the a g g l u t i n a t i o n of c a n c e r cells could be an esting tool

of

and mucus

of

Main

Murg. to

Grabar

Schei-

could be e x a m i n e d under

(26): A g a r o s e o.8%

(Difco);

barbital-Na-

buffer pH 8 . 2 , o.o5 M; 4o-5o V and lo-2o mA per p l a t e ; cubation

time a p p r o x i m a t e l y

Absorption

experiments.

16 h at room

norin-

temperature.

R a b b i t - R B C were w a s h e d 3 times

with

661 PBS and c e n t r i f u g e d

at 3ooo rpm after each w a s h i n g . The w a s h e d

RBC w e r e r e s u s p e n d e d

in PBS c o n t a i n i n g

(2 ml p a c k e d RBC in loo ml

fixation

1% g l u t a r a l d e h y d e

s o l u t i o n ) and

incubated

w i t h c o n t i n u o u s s t i r r i n g during 3o min at 4°C. Then the were spun down and 2 vol of cells m i x e d w i t h empiricorum mucus

(4-5 mg/ml

total

protein

1 vol

content),

ed for 3o min at 37°C and 9o min at 4°C u n d e r s l i g h t ring. Finally diments

the cells were c e n t r i f u g e d

s t o r e d at 4°C and used as soon as

mechanical

methods

lian cells o r i g i n a l l y Misra

et al.

t h r o u g h steel

through defined

injection

and/or nylon m e s h e s ,

needles

and g r a d i e n t

w e r e used in d i f f e r e n t c o m b i n a t i o n s lymphocytes

(12,29); purification

cells from tat and mouse cancers

mammaand and

Potterizaaspiration

centrifucation

for i s o l a t i o n of

f r o m human beings and r o d e n t s , cells

liver and s p l e e n

organs:

(27)

cells and w o r k e d out in our l a b o r a t o r y :

tion, seeving

blood

from

of a s c i t i c

kidney,

tumor

(12) and of cells from s o l i d

human

(21).

H e m a g g l u t i n a t i o n was p e r f o r m e d micorti.ter s y s t e m

in serial

(Cook E n g e n e e r i n g

V i r g i n i a , USA) and s c o r e d as Cell

and

for i s o l a t i o n of human and

i n d i c a t e d by Biozzi

se-

possible.

(28) have been m o d i f i e d for i s o l a t i o n of human

mammalian

incubatstir-

at 45oo rpm, the

I s o l a t i o n of n u c l e a t e d cells from b l o o d , a s c i t e s Several

RBC

of A r i o n

d i l u t i o n with

Company,

usual.

a g g l u t i n a t i o n was done in p l a s t i c tubes

croscopically

on a m i c r o s c o p e

agglutinates were

slide

and reas

mi-

(12, 21). M u l t i p l e

c o n s i d e r e d to be +; several

bigger

nates ++ and few i m p o r t a n t and lage a g g l u t i n a t e s (see fig.

the

Alexabdria,

small

aggluti-

to be ++++

3).

Immunofluorescence.

The m e t h o d s

d e s c r i b e d by Nairn

(3o) were

662

employed. Ael

Fluorescein

- and o t h e r

i s.othiocyanate

lectins

( F I T C ) was c o u p l e d

- w i t h the same methods d e s c r i b e d

IgG or

antibodies.

Either

the l e c t i n was c o a t e d to the c e l l s

monospecific fraction

and v i s u a l i z e d

FITC-coupled anti-1 ectin antiserum

from r a b b i t s ;

anti-lection

i n c u b a t e d w i t h Ael

IgG a n t i s e r u m .

s e r v e d as c o n t r o l s

with

the

IgG

a n t i s e r u m added. Immuno-

f l u o r e s c e n c e was then o b t a i n e d w i t h c o m m e r c i a l l y goat a n t i - r a b b i t

for

(IgG-fraction )

or the l e c t i n was c o u p l e d to the c e l l s ,

of m o n o s p e c i f i c

FITC-coupled

to

available

Cells

not

and never gave

being

immuno-

f 1 u o r e s cence. The method f o r m i t o g e n i c s t i m u l a t i o n o f mouse s p l e e n c e l l s 3 w i t h l e c t i n by, H - t h y m i d i n e i n c o r p o r a t i o n was d e s c r i b e d by Bessler

(31).

Description

of animals

description

of the t r a n s p l a n t a t i o n

cells

s h i c h were used as d o n o r s o f

and o f i m m u n i z a t i o n

as methods f o r

o f animal

ascitic

tumor

(32)

well

have been p u b l i s h e d

i m m u n i z a t i o n and column

RBC, as

chromatography.

Results Arion empiricorum l e c t i n

( A e l ) was found i n s a l i n e

and i n the mucus o f t h i s

red r o a d s n a i l

r a b b i t and r a t e r y t h r o c y t e s . positive

Zajdela

Also

with a g g l u t i n a t i o n o f an

hepatoma grown i n a s c i t i c

the i n b r e d Holtzman s t r a i n were The l e c t i n was c h a r a c t e r i z e d (Fig.

cells

l a and l b ) .

tify

the l e c t i n

by i m m u n o e l e c t r o p h o r e t i c

fixed rabbit it

of

agglutinated.

As we had a l r e a d y

by a b s o r b i n g

of

-foetoprotein

form i n r a t s

analysis

found t h a t the l e c t i n

the crude e x t r a c t or i n the mucus a g g l u t i n a t e d RBC and g l u t a r a l d e h y d e

extracts

in

strongly rabbit

RBC, we attempted to i d e n -

to r a b b i t

RBC and

analyzed

663 the

supernatant

lectin cus

coated

(upper

trough) mucus.

pitation lacking

Fig.

line

Fig.

trough:

against

lb. and

which

serum

from

represent

the

from Ae.

absorbed

mucus

^-region

supernatant

rabbit

except in the

mu(lower

against

for one

Ae

preci-

Ae m u c u s

precipitation

Upper

but

line

trough:

indicates

analysis Well

lectin.

demonstrates

the

crude

was

mucus

from

from Ae. Well: antiserum

The arrow

Ae

the

Ae-lectin.

of mucus

against

from

This

of

the

is p r e s e n t

t r o u g h Ae m u c u s .

rabbit

of

be s e e n

mucus.

Ae m u c u s .

lower

antigens

antiserum

could

Immunoelectrophoretic

Upper in the

antigens

the

absorbed

la. A n a l y s i s

rabbit

and

(arrow) to

by c e n t r i f u g a t i on of

la the

pattern

taken

lower

elimination

In f i g .

revealed with same

in the

therefore

Ae,

trough)

are The

after

RBC.

the

of mucus

IgG One

from

lectin.

from

fraction

Ael.

of

anti-

precipitation

the m o n o s p e c i f i ci ty of

line

the

serum. The

Ae m u c u s

rabbit

RBC

as w e l l

and

as

the

glutaraldehyd

purified fixed

lectin

rabbit

agglutinated

RBC.

anti-

664

When gl u t a r a l dehyde f i x e d r a b b i t

RBC were i n c u b a t e d w i t h

Ae mucus, c a r e f u l l y washed and i n j e c t e d specific

a n t i s e r u m was o b t a i n e d .

the same p r e c i p i t a t i o n

line

the p r e c i p i t a t i o n

(arrow)

line

It

into

sites

of

i n the ¿ ' - r e g i o n fig.

la

f l o w r a t e 2o ml/h)

This

demonstrates

the

antigenic

against

rabbit

RBC ( 3 4 ) .

a n t i s e r a were o b t a i n e d . dent a _ f t e r

buffer

isolation

(Tris-HCl

o . o 5 M, pH 7 . o ,

peak o f the " f a l l

through"

This

fraction

contains

immunization of r a b b i t s

respectively

pure

was o n l y

containing after

actiAel.

monospecific

But the m o n o s p e c i f i c i t y

o f the a n t i b o d y

by Na 2 S0 / j p r e c i p i t a t i o n

Sepha-

up to 80% o f the a g g l u t i n a t i n g

T h i s was seen when a f t e r

IgG

evi-

fraction

chromatography

on

cellulose. precipitates

rat, lines

i n f a c t two serum p r o t e i n s

and human serum. With human serum t h i s appear i n the ^j. and

because the p r e c i p i t a t i o n g antibodies teins

is

different

and the A e l .

obtained.

the O u c h t e r l o n y

technic

r e a c t i o n of i d e n t i t y

p u r e " Ael

revealed in

2 bands c o r r e s p o n d i n g

and

a n o t h e r e x p e r i m e n t we found 2 bands c o r r e s p o n d i n g c u l a r w e i g h t o f 13ooo ds and 63ooo ds w h i c h may K. F e l g e n h a u e r f o r t h i s

was the

to a m o l e -

c u l a r w e i g h t o f 43ooo d s + + and 9oooo ds r e s p e c t i v e l y .

++ ds = B a l t o n s

mobili-

from the complex formed by serum p r o -

S o , no c l e a r c u t

We w i s h to t h a n k Dr.

mouse,

precipitation

complex between serum p r o t e i n s

Polyacrylamidegel-electrophoresis

"immunologically

in rabbit,

1 -p •H O •H m -H o d) a Ul

G O 0) Cu •H

+> u

ta -p g w td G •H s 4-> £ y •H 3 U pí •H a m m •H O Ul •p til -p a g ta a> T) H 0 a td o o •H

>

«

tó

0 iH O -H m

•H XI X) td

«

c (0 o I m 3 < K

P

o

«! (tí

o ití

o

o

<


-H •rl 01 -P 3 •H

rH 01 U O —"

ft

01 •H -P Cn 0 C 0 fi 3 XI ai -d 1

•H

m -p

fi

i—l rd 3 fi u rd U] •H (U U C 0] •H C 0 CM

0 rH CN m •H rH rH rH

1) rd ai u rd

rd ai

ai

05 3 M 0 rH MH -rl -P rH 3 g

SH ft ai 01 rd Oi g m •H 01 > c 3 • a) H fi X XI 0 •r| rd rd ai U g rH ta XI id >1 • • rH rd .fi U U J Pi X

3 H

,—. •d

rd xi n) M-l

0 O

§ o 01

c rd 01 01 •H •H 3 G 01 (d fi •rl -H -H u 1 Ü U M •rH rd -d « U C rd r*rH rH X

689

most sensitive having affinity for 16 different lympho-agglutinins. The number of lectins capable of agglutinating sperms were further reduced to lo, that is, 5 lympho-agglutinins could bind with both human and bull sperms, whereas 5 reacted with human sperms only (Table IV). As already reported for other lectins (14,15) the pattern of agglutination of sperms was generally of mixed type involving random tail-to-tail, head-to-tail and head-to-head agglutination of sperms (Fig. Ic). However, 3 lectins, namely, Erythrina arborescens, Glycine max and Phaseolus multiflorus brought about a tailto-tail agglutination only (Fig. Id).

TABLE IV Biological and sugar specificities of spermagglutinins from Indian plants

S.No. Plant species

1 .Parkia biglandulosa 2 •Abrus fruticulosus 3 .A. precatorius 4 .Canavalia obtusifolia 5 -Erythrina arborescens

Biological specificity Human Bull

Sugar specificity

+ m + m

+ m

U Gai,Lac

-

Meb,Lac

7 .Glycine max

+ m + m + tt + m + tt

8 .Lablab niger 9 •Mucuna nigricans

+ m + m

6 .E.subrosa

lo •Phaseolus multiflorus

+ tt

-

+ m

+ tt -

+ tt -

-

+ tt

Glc,Man,Frux Gai,galNac Ribx,galNAc,Lac Mez Glc,a-methyl D-Glc Frux,galNAc,Mez Gai

m and tt denote mixed and tail-to-tail agglutination of sperms respectively x Fru = D-fructose, Rib = D(-)ribose

690

TABLE V Distribution of 5o phytolectins according to their differential agglutinating properties

Cell type

RBCs

Lymphocytes

5o

17

14

lo

Human

26

16

14

lo (5)

Guinea pig

23

16

14

NT

Sheep

lo

11

2

NT

5

11

1

NT

Total

Goat

RBCs + RBCs+lympholymphocytes cytes+spermsx

x 5 lectins reacted with bull sperms also; these animals not tested

NT - sperms of

Among the 26 lectins reacting with human cells, lo agglutinated only RBCs, 2 only the lymphocytes and 14 both the RBCs and lymphocytes, while the remaining lo agglutinated all the three types of cells tested. For the two ruminant species, however, the hemagglutinins and lympho-agglutinins were by and large of independent origin (Table V). Apparently, the cells of the immune system in ruminants are much more receptive to a variety of phytolectins than are the erythrocytes. The similarity of receptor sites in three entirely unrelated cell types is suggested by the specificity of Parkia biglandulosa lectin for human sperms, rabbit RBCs and guinea pig lymphocytes exclusively. The molecular basis of such a similarity needs to be worked out. The sugar inhibition tests have revealed the affinity of 27 agglutinins for 9 different sugars and their derivatives. In

691 accordance with the already observed trend, most of the lectins were inhibited by galactose series of sugars (16). As many as 19 seed extracts were inhibited by galactose, and of these, 14 were also inhibited by lactose and 8 each by N-acetylgalactosamine and melibiose. Glucose and mannose were found to inhibit only 7 lectins. As expected there was no lectin which responded to both galactose and mannose/glucose. About half the number of phytoagglutinins discovered in this study did not respond to any of the test sugars. These included all the agglutinins binding to ruminant RBCs. Presumably, such lectins may be having binding sites for complex oligosaccharides .

Acknowledgements This study has been supported in part by the Univeristy Grants Commission, New Delhi. One of us (RSR) is a recipient of CSIR Research Fellowship. The authors are thankful to Dr. B.D. Sanwal, Department of Biochemistry, University of Western Ontario, London, Canada, for his keen interest and gift of some sugar derivatives required in this study.

Note added in proof: A complete list of all the plant species surveyed is available with the authors.

692 REFERENCES 1.

Gold, E.R., Balding, P. (1975). Receptor-Specific Proteins - Plant and Animal Lectins, American Elsevier Publishing Company Inc., New York pp. 151-236.

2.

Sandhu, R.S., Reen, R.S.(1982).

3.

Bhalla, V. , Gaur, V., Bhatia, K. (1978). ¿5, 241-247

4.

Bhalla, V. , Roy, S. (198o).

5.

Pull, S.P., Pueppke, S.G., Hymowitz, T., Orf, J.H. (1978). Science 2oo, 1977-1979.

6.

B0yum, A. (1968). Scand. J. Clin. Lab. Invest. 21, Suppl. o 9_7 ' 1~29 .

7.

Toms, G.C., Western, A. (1971). Chemotaxonomy fof Leguminosae, Eds. J.B. Harborne, D. Boulter and B.L. Turner, London and New York, pp. 367-462.

8.

Tobiska, J. (1964). Verlag Berlin.

9.

Boyd, W.C., Waszczenko-Zacharczenko, E., Goldwasser, S.M. (1961). Transfusion 1, 374-382 (1961).

These proceedings. Vox Sang.

IRCS Med. Sei. 8, 53o-531.

Die Phythamagglutinine, Akademie-

10.

Boyd, W.C. (1963).

Vox Sang. 8, 1-32.

11.

Hossaini, A.A. (1968).

12.

Boyd, W.C. (195o).

13.

Allen, N.K., Brilliantine, L. (1969). 1295-1299.

14.

Uhlenbruck, G., Herrmann, W.P. (1972). 444-491.

15.

Nicolson, G.L., Yanagimachi, R. (1974). 276-279.

16.

Hankins, C.N., Kindinger, J.I., Shannon, L.M. Plant Physiol. £4, lo4-lo7.

Vox Sang. 15, 41o-417.

J. Immunol. 65, 281-284. J. Immunol. lo2, Vox Sang. 23, Science 177, (1979).

T H E S E A R C H FOR NEW LECTIN SOURCES ON T H E BAJA PENINSULA

CALIFORNIA

Herrera, E.M., Montano, C . , Ochoa, J . L . and Córdoba, F. Biological

Research Center, Box 128, La Paz, Baja C a l i f o r n i a S . , México

The r a p i d l y growing world-wide i n t e r e s t in l e c t i n s i s e a s i l y explained in terms o f ' t h e i r a p p l i c a t i o n s in a number of f i e l d s of medicine and b i o l o g y . B i o c h e m i s t s , eel 1 - b i o l o g i s t s , immunologists, e t c . , are sooner or l a t e r faced with research problems that somehow i n v o l v e the use of a p a r t i c u l a r l e c t i n e i t h e r f o r p u r i f i c a t i o n purposes ( 1 , 2 , 3 ) , a n a l y s i s structure characterizations

( 5 - 9 ) , etc.

(4) or molecular

Thus, the need f o r

a v a i l a b l e and cheap sources of l e c t i n s i s s t r o n g .

easily

We have embarked upon

a program dealing with the search of new l e c t i n s present in the t y p i c a l components of the Baja C a l i f o r n i a f l o r a .

Since the r e g i o n i s

characteriz-

ed by a semidesertic c l i m a t e , t h i s i s the f i r s t systematic study of l e c t i n s from desert p l a n t s o r i g i n reported so f a r .

M a t e r i a l s and Methods All

seeds, or p l a n t s s t u d i e d were c o l l e c t e d in the southern region of the

Baja C a l i f o r n i a Peninsula and c l a s s i f i e d at the B i o l o g i c a l

Research Center

of Baja C a l i f o r n i a , Mexico. Sugars and p r o t e i n s were purchased from Sigma Chemical Company ( S t . Mo., U . S . A . ) .

A l l other chemicals came from v a r i o u s commercial

Lectins - Biology, Biochemistry, Clinical Biochemistry, Vol. II © W a l t e r d e Gruyter &. Co., Berlin • N e w York 1982

Louis,

sources.

694 Extraction Procedure Seeds, stems, leaves, or barks, were f i n e l y ground in a morter or with the aid of a mill (Arthur, H., Thomas, Co., P h i l . Pa., U.S.A.) and then suspended in phosphate s a l i n e buffer, PBS, (0.02M I^HPO^, pH 7.4 containing 0.9M NaCl and 0.02% Na Nj) in 1:10 W/V proportion.

After an overnight

s t i r r i n g period at 20° C, defating was c a r r i e d out according to (10)

if

necessary, or the suspension was d i r e c t l y centrifuged (3000 Xg x 15 min.) at 20° C to eliminate i n s o l u b l e materials.

The supernatants were then

assayed for a g g l u t i n a t i o n a c t i v i t y before and a f t e r extensive d i a l y s i s against PBS.

The dialyzates showing detectable a g g l u t i n a t i o n capacity

were further investigated with regard to t h e i r blood group and carbohydrate s p e c i f i c i t y at 20® C. A g g l u t i n a t i o n Test The double s e r i a l d i l u t i o n procedure using m i c r o t i t e r plates was employed with a 2% erythrocyte suspension of blood groups ABO from healthy donors in a l l cases.

The t r y p s i n treatment was done by mixing 50 ug of the enzyme

(ED 3.4.21.4. 11,800 BAEE units/mg) per 100 ml of 2% r e d - c e l l s in PBS.

suspension

After incubation at 37° C, 1 h r , under gentle s t i r r i n g , the

erythrocytes were washed once again with PBS and used for the a g g l u t i n a t i o n test as indicated.

S p e c i f i c i t y of the d i f f e r e n t extracts under study was

determined by mixing the dialyzates with various carbohydrate or protein s o l u t i o n s (0.1 M or lmg/ml r e s p e c t i v e l y ) , and then t e s t i n g t h e i r a g g l u t i n ation a c t i v i t y as above.

Results and D i s c u s s i o n

As mentioned before, the present i s a complementary study of the f l o r a on the Baja C a l i f o r n i a peninsula.

For h i s t o r i c a l

reasons, t h i s portion of

Mexico's t e r r i t o r y has remained p r a c t i c a l l y unexplored.

Geologically,

i s believed that the peninsula suffered from a quite d i f f e r e n t

it

evolutionary

695 process as regards to the mainland American Continent.

Therefore, some

animal and plant species are typical and seldom found in other regions around the world.

This preliminary communication describes the presence

of agglutinins in 25 extracts from different plants (Table I).

As shown

in Table I, in some cases, there is a considerable blood group specificity but many extracts agglutinate the different blood groups equally well. Moreover, some of them cause the cell lysis and few show no effect at all when mixed with the erythrocytes (Table I).

We also investigated the susceptibility of trypsin treated red cells to agglutination by some extracts selected from those currently under study and found important increments on their agglutinability or agglutination titers in most cases (Table lib).

Although this phenomenon has been

interpreted before as being due to an increase in the number of agglutinin receptors made available after the trypsin treatment, we lack experimental evidence to support this hypothesis (11-12).

It should be mentioned that most of the extracts dealt with are strongly coloured.

Therefore, it has been suspected that the agglutination

capacity of the various extracts may be simply due to tannic acid or its derivatives.

However, extensive dialysis using a membrane pore size of

about 12000-14000 Daltons does not result in any loss of agglutination activity.

Furthermore, since tannins are known to react with proteins

preferentially, it is difficult to explain the increase on the hemagglutination titer after trypsin digestion of the red cells as being due to tannins effect alone.

We attempted to identify the chemical nature of the cell receptors involved in the agglutination caused by the extracts under study by inhibition assays.

For this purpose, we used carbohydrate, protein and

glycoprotein solutions.

As shown in Table II it is readily clear in the

first place that the blood group specificity of the various extracts is not necessarily associated to the blood group carbohydrate determinant since the corresponding sugars were unable to inhibit the induced cell agglutination.

Therefore, we believe that there may be other components

696

697

o oo so

o

o

o

O

«i-

o

E ^


>

o

o

VI

cu

4->

ZD

a> e E sai 4-> aj

ai

to E 1— 1—

ZD

ai e E i. ai •i-j ai

Z3

=3

X) C

-O C

TJ a)

XJ

aj

XJ

XI

c.

C

C

E

E t ai XJ c

E saj -M ai

E s_ ai +-> aj T3

C

S0J -M

aj c

XJ

cu

XJ C

Z3

aj

C

C 3

T3 ai c E sai cu

XJ

a)

ai 4-> cu XI c ZD x> aj c: E sa> +-> cu

X) C

13

XI

a> e ^

ai +-> ai •o c Z3

698 of the cell membrane that are characteristic of the various blood groups in addition to the already known carbohydrate determinants (13).

As

indicated in Table II, albumin, which is not a glycoprotein, inhibits also the agglutination phenomenon induced by such extracts.

This fact suggests

that other structures characteristic of the peptide chain of the cell receptors might also be involved in the binding of the agglutinins in question.

The tannins are known to interact with proteins in a reversible fashion. The mechanism of this interaction is very complex and remains unclear.

largely

When ovalbumin is adsorbed on immobilized tannin, for instance

(14), elution is best accomplished either by changing the pH, or by using a mixture of 1M NaCl and 50°/ ethylene glycol or 1M NaCl and 50% sucrose. The sugar solution alone is as good

eluant

as the ethylene glycol

and

1M NaCl solutions used separately, but not as effective as the mixture already mentioned.

Therefore, it might be concluded that the adsorption

process of ovalbumin on immobilized tannin is the result of both electrostatic and hydrogen-bonding

interactions (14), though a hydrophobically

driven mechanism cannot be excluded (15).

Thus, the interaction of tannins

with proteins may be considered a m o r e or less selective process.

If the substances responsible for the hemagglutinating activity in our extracts are tannins, it is interesting to note first, that under the experimental conditions employed in our assay there is an apparent blood-group specificity, and second, that this effect is not abolished by carbohydrate solutions of the corresponding blood-group determinant.

In summary, in this paper, special

emphasis on the

blood-group-specificity

and carbohydrate-specificity of a number of plant extracts has been made. The observed hemagglutination phenomenon has been ascribed to lectins, but the possibility of dealing with other substances such as tannins cannot be overlooked.

Further purification and characterization of the extracts

under study is necessary to clarify this point.

699

TABLE II a EFFECT OF SUGARS AND PROTEINS ON THE AGGLUTINATING ACTIVITY OF FOUR PLANT EXTRACTS TOWARDS NORMAL HUMAN ERYTHROCYTES Torote B 0

A

2 1 1 2 2 2 3 2 2

6 5 6 4 6 6 5 6 6

4 3 4 4 4 4 5 4 4

2 0 1 0 0 1 1 1 1

4 2 2 2 2 2 2 2 2

0 0 0 0 0 0 0 0 0

3 2

2 1

2 1

1 1

1 1

0 0

Blood group:

A

Control Fuc Gal GalN GalNAc Glc GlcN GlcNAc Man Gamma globulin BSA

Copal B 0

S.Miguelito A B 0 9 9 9 9 9 9 9 9 9 9 9 9 10 9 10 9 9 10 5 3

9 3

Ciruelo A B 0

9 9 9 9 9 9 9 8 8

5 4 5 5 4 5 5 5 5

3 3 3 3 3 3 3 3 3

3 3 3 2 2 3 2 2 3

5 3

2 0

1 0

1 0

Table II b EFFECT OF SUGARS AND PROTEINS ON THE AGGLUTINATING ACTIVITY OF FOUR PLANT EXTRACTS TOWARDS TRYPSINIZED HUMAN ERYTHROCYTES Torote B 0

Blood group:

A

Control Fuc Gal GalN GalNAc Glc GlcN GlcNAc Man

4 4 3 3 4 4 3 3 4

7 7 6 7 7 7 7 7 6

Gamma globulin BSA

4 4

6 2

A

Copal B 0

S. Miguelito A B 0

7 7 7 7 7 7 7 7 7

2 1 0 0 0 0 1 1 0

3 3 3 3 3 3 3 3 3

0 0 0 0 0 0 0 0 0

9 9 9 9 9 9 9 9 9 10 9 9 9 9 9 10 9 9 9 9 8 10 10 10 10 9 9

5 3

1 1

1 1

0 0

9 0

8 0

9 0

Ciruelo A B 0 6 6 6 6 6 6 6 6 6

4 4 4 5 4 4 4 4 4

5 5 5 5 5 5 5 5 6

2 0

2 0

3 0

700

References 1.

Goldstein, I.J., Hayes, C.E.: In: Adv. in Carbohydrate Chemistry and Biochemistry, 35, 127 (Tipson, R.S., Horton, D., Ed.) Academic Press, New York, (1978).

2.

Lis, H., Sharon, N.: In: The Antigens, Vol. IV, 429, (Sela, M., Ed.) Academic Press, New York, (1977).

3.

Bittiger, H., Schnebli, H.D., (Eds.): In: Concanavalin A as a Tool Wiley, New York, (1976).

4.

Borrebaeck, C., Mattiasson, B., Anal. Biochem., 107, 466, (1980).

5.

Monsigny, M., In: Les Colloques De L'Inserm, Affinity Chromatography, INSERM, (J.M. Egly, Ed.), 86, 207, (1979).

6.

B0g-Hansen, T.C., In: Coll. de L'Inserm, Affinity Chromatography, (J.M. Egly, Ed.), 86, 399, (1979).

7.

Duleney, J.T., Molecular and Cellular Biochem. 21, 43, (1979).

8.

Poliquin, L., Shore, G.C., Anal. Biochem., 109, 460, (1980).

9.

Etzler, M.E., Borrebaeck, E., Biochem. and Biophys. Res. Commun., 96, 92, (1980).

10.

Lotan, R., Skultelsky, E., Danon, D., Sharon, N., J. Biol. Chem, 250, 8818, (1975).

11.

Cline, M.J., Livingstone, D.C., Nature, New Biol. (London), 232, 156, (1971).

12.

Makel a, 0., Act. Fenn. Scand. 35 Supl. 11, 1, (1957).

13.

Lemieux, R.U., Chem. Soc. Rev. 1_, 423 (1978).

14.

Watanabe, To, Mori, T., Tosa, T., and Chibata, I., J. of Chromatogr. 207, 13, (1981).

15.

Oh, H.L., Hoff, J.E., Armstrong, G.S., and Haff, L.A., J. Agric. Food. Chem. 28, 394, (1980).

SOME NEW PHYTOHEMAGGLUTININS:

THEIR INTERACTION WITH RED

BLOOD CELLS AND SERUM PROTEINS

Ashwani K. Khanna Department of Cardiology, Postgraduate Institute of Medical Education and Research, Chandigarh-16ool2 India.

The haemagglutinating activity of plant extracts was the earliest interest of researchers in lectins (1-6). This activity was shown to have a potential for typing human blood cells and marking differences in a specie or among species, as well as it helped in understanding the chemical structure of carbohydrate polymers. Later lectins have been applied as specific reagents for isolation and characterization of polysaccharides and glycoproteins by affinity chromatography (7-9) and affinity electrophoresis (lo). The hemagglutinating activity of plant lectins show species specificity or individual specificity. • Among the blood group specific lectins are Dolichos biflorus as Anti A1, Iberis amara as Anti M, Vicia gramineae as Anti N, Bondeirala simplicifolia as Anti B and Ulex europeus as Anti H. Stillmark (11) who discovered the lectin from Ricinus communis thought the agglutinating activity to reside in the toxic proteins of the plant. This theory was discarded due to the fact that many plant species possessed agglutinating proteins without being toxic. Recently we have suggested that the property of agglutination is under genetic control and resides in the seeds to get distributed to all parts of the plant (12). Of a few plants we observed a serological activity of proteins of all plant parts viz stem, leaves, roots, floral parts, milky juices, embryos and growing buds. However, a small difference in

L e c t i n s - B i o l o g y , Biochemistry, C l i n i c a l Biochemistry, V o l . II © W a l t e r d e Gruyter & Co., Berlin • N e w Y o r k 1982

702 hemagglutination titre was found. Lectins also exhibit affinity for some antigens.

Two lectins

were studied by Ikemato et al. (13) and Khanna and Sehaj Pal (14) with affinity for Australia antigen. centration of these investigations.

This work is a con-

Plant extracts were stu-

died for hemagglutination activity, hemolysing activity and interaction with human serum proteins.

Material and methods Plant material: Plant seeds and different plant parts were from commonly occurring Indian plants and were obtained from local botanical gardens and their identity was confirmed by a botanist. Crude extracts from seeds and hard parts like stem, root etc were prepared according to the method of Boyd and Reguera (16) and from leaves and soft part like floral parts etc by the method of Ikemoto et al. (13). Briefly the leaves were dried well at room temperature and were ground into powder. Three gram of powder was dissolved in lo ml of PBS pH 7.2 and was allowed to stand at 4°C for 2 hrs after which the solution was centrifuged at 2ooo rpm for 3o minutes in the cold. The supernatant fluids were used as crude extracts. A, B, 0, and AB blood cells were taken from healthy donors. Bombay phenotype red cells, which did not possess antigen H on their surface, were from a rare case to be reported by us (15). Agarose, sugars and other chemicals were from Sigma, U.S.A. or Sisco, India. Sugar inhibition studies were done by the method of Makela (6). Polyacrylamide disk gel electrophoresis was done by the method of Davis (17).

Polyacrylamide gels were 7.5 % and 15o (ig/2o

crude lectin was applied per gel and stained with Amido black lo B.

703 Affinity electrophoresis analysis was done analogously to the method of immunoelectrophorectic analysis a.m. Grabar. Counter Immunoelectrophoresis was done using barbital - HC1 buffer pH 8.2 1 % agarose in 1:1 buffer was used throughout. The hemagglutination titre was determined in IHA titre U well plates (obtained from Laxbro India). Serial two-fold dilutions of lectins were made and a 2 % suspension of red cells were added. The end points of red cell agglutination were recorded after incubation at room temperature . In case of hemolysis, the titre was not recorded at the lectin was classified as hemolysin.

Results Lectins were extracted from various parts of commonly occurring Indian plants. The crude extract was tested for hemagglutinating activity with human red blood cells of A, B, O and AB types. Table 1 gives the titre of a number of plant extracts against human O red cells. Notable is the high activity found in extracts from seeds, leaves and stems of Ricinus Communis. Roots from this plant also possessed considerable activity. Extracts from seeds, leaves and stems of Bauhinia tomentosa were found to agglutinate all types of human red cells viz A, B, 0 and AB, but highest activity was found against 0 red cells (Table 2). The lectin did not agglutinate Bombay phenotype red cells. The hemagglutinating activity was inhibited by o.l M lactose and o.2 M galactose but not by salicin or glucose. Thus results show that extracts of Bauhinia tomentosa have anti H-specificity. Table 3 shows the hemagglutinating activity of extracts from leaves of Clerodendron inerme.

This crude extract agglutina-

ted some human red cells of A, B, 0, and AB types.

The lectin

was tested with 7 7 healthy blood donors and 4 7 were found to

704

be agglutinated and 3o were found not to be agglutinated.

Re-

sults, when seen in terms of different blood groups, show that whereas individuals with 0 and B blood groups were more readily agglutinated, the number of positive and negative individuals of A and AB blood groups were the same (Table 3) .

TABLE 1 Hemagglutination titre of extracts of plants

Plant

Plant part

Titre of extract

Crotolaria mediciginea

Seed, leaves, stem root Seeds, leaves, stem root

1: 16 1: 4 1: lo24 1: 256 1: 32 1: 16 1: 4 1: 4 1: 2

Ricinus commuinis Vicia sativa

Seeds peaves, stem

Calatropis procera Jatropha andriacfolia

Milky juice of stem Floral parts

Sonchus oleraceaus

Milky juice of stem and branches

Hibiscus rosa sinensis Canavalia ensiformis

Anthers Radical and plumule

Solanum tuberosum

Growing buds

1: 4 1: 4 1: 8

Milletia ovalifolia

Seeds

1: 16

Phybrida ulor

Seeds

1: 16

Vicia faba Saliva officianialis

Seeds, leaves Leaves, seeds

1: 32

Eruca sativa

Seeds, leaves Succulent leaves

Agava whightii Thuja orientalis Glycine max

Seeds, leaves Seeds Leaves

1: 4 1: 8 1: 32 1: 8 1: 32 1: 16

Plant parts were homogenized and extracted as described in Material and Methods. Agglutination was performed with human red cells of 0 type and the reaction studied at 22°C

705 A few of the crude extracts showed hemolysing activity (Verbena bipinatifida, Dodonea ciscosa, Convolvulus arvensis and

TABLE 2 Hemagglutinating activity of extracts from Bauhinia tomentosa

Blood group

Titre

0

++++

0 with Bombay phenotype B

+++

AB

++

A

+

Monkey Mice Antigen H in saliva inhibitor + signifies agglutination, - signifies no agglutination

TABLE 3 Hemagglutination of different blood group types with extracts of leaves of Clerodendron inerme

Number

Percent of total

0

B

AB

A

Agglutination

47

61

15

15

4

13

No agglutination

3o

39

8

6

3

13

Figures specify the number of individuals studied

706

Asparagns spp). A considerable hemolysing activity was shown by Verbeur pipinatifid and Dodonea vis Cosa. Interactions of plant proteins in crude extracts with human serum proteins were studied in counter Immunoelectrophoresis. The serum was loaded into anodal well and lectin in the cathodal well. Crude extract of Acacia ferenesiana was found to contain protein that gives a line of precipitation with human serum proteins. The serum, when heated to 56° for 3o min,failed to react with the lectin. Thus the lectin precipitated human sera but the nature of the precipitating component was not worked out. Crude lectin from leaves of Ricinus Communis formed an arc with human serum only when mixed with hemoglobin.

This analy-

sis was performed analogously to immunoelectrophoretic analysis a.m. Grabar, where the lectin was loaded into the trough and the serum sample into the well (Fig. 1).

Thus the extract

seemed to possess an affinity for haptoglobin, the hemoglobin-

1 2 3 A 5

6

Figure 1. Affinity electrophoretic analysis of huitian serum with and without hemoglobin against Crude extracts from leaves of Ricinus Communis. (1) Lysed serum sample. (2) Sample without hemoglobin. (3) Sample without hemoglobin. (4) Same sample Hp2~2phenotype with hemoglobin.

(5) Serum sample without hemoglobin. (6) Same sample Hp2-1 phenotype with hemoglobin.

707 binding protein is serum. The different genetic types Hp 2-2, Hp 2-1 and Hp 1-1 (18) did not show any differences with respect to the position of the affinity precipitate. Interaction of the Australia antigen was studied with different lectins in counter Immunoelectrophoresis and hemagglutinating assay. The crude extract from Crotolaria mediciginea agglutinated human red cell of all types viz A, B, 0, and AB with titre 1:16. When the extract was absorbed with a serum containing Australia antigen, the titre was reduced to 1:1, where

o Figure 2. Counter affinity electrophoresis of lectin from Crotolaria medicigineae var.Luxarinus against Australia antigen, arrows mark the precipitation line between lectin and Australia antigen. Anode is on left, Australia antigen was applied in left wells.

Figure 3. Diagrammatic representation of PAGE patterns of some lectins (a) from seeds of Clerodendron inerme,(b) lectin from leaves of Clerodendron inerme, (c) lectin from seeds of Bauhinia tomentosa, (d) lectin from seeds of Crotolaria medicigineae, (e) lectin from leaves of Crotolaria medicigineae.

708 as when absorbed with normal sera, the titre was reduced to only 1:8.

The lectin gave a line of precipitation in counter

Immunoelectrophoresis with Australia antigen sera.

Thus the

Crotolaria mediciginea lectin showed affinity for the Australia antigen (Fig. 2). To see the difference in proteins in crude extracts of leaves and seeds of different plants, the extracts were subjected to polyacrylamide gelelectrophoresis. The protein patterns are shown in Fig. 3. The extract from seeds of Clerodendron inerme and leaves of same plant showed almost identical patterns except for the 5th band which was absent in extracts of leaves. Similarly in the case of Crotolaria mediciginea and Bauhinia tomentosa, the patterns were very similar although the differences in intensity of staining was observed. The band at position 8 was absent in Crotolaria mediciginea leaves as compared to seeds.

Discussion Our screening of lectin activities in some Indian plants showed that screening for Australia antigen can be performed with extracts from Crotolaria mediciginea and that a new blood group can be distinguished with extracts from Clerodendon inerme. A common origin from all parts of the plant can be suggested for the proteins that show interaction with red cells and serum proteins, though they differ quantitatively in each part. Talbot and Etzler (19) have shown that there exists a protein in stem and leaves of Dolichos biflous which cross reacts with antibodies against the seed lectin. Etzler (2o) has shown that many structural properties are common for proteins in seeds and in stem and leaves. This common property was believed to be under regulatory control (2o). We suggested the control to be genetic.

709

Some of the lectins studied by us may have potential use in the biological, therapeutical and clinical research. not study lymphocyte stimulation.

We did

If these lectins possess a

mitogenic capacity they may be used for studying congenital or acquired immunological deficiency, for detection of sensitization by infections agents or in auto-immune diseases, and to monitor the effect of .various immunosuppressive and immunotherapeutic agents. REFERENCES 1.

Krupe, M.

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G.W.G.

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(Doroat)

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I Kemato, S., Yashido, H., Shinizu, M., Mayumi, M., Tomita, K. (1978). Vox. Sang. 34, 22.

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Khanna, A.K., Kaur, H., Sehaj Pal, P.K., Shrivastava, P.K. (1978). Acta Anthropogenetica, 2, 57.

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Talbot, Etzler, M.E.

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Etzler, M.E. (Jan. 1981). Paper presented at International Symposium on lectins as tools in Biology and Medicine Calcutta (India).

(in oreparation). (1964). Ann. New York Acad. Sc. 121, 404.

(1978). Biochemistry 17, 1474.

THE LECTIN FROM GARDEN CRESS (LEPIDIUM SATIVUM). ISOLATION AND CHARACTERIZATION

P. Ziska and H. Franz Staatliches Institut fur Immunprâparate und Nàhrmedien, DDR-1120 Berlin

We considered that seeds of garden cress (Lepidium sativum) should contain a lectin, because we assumed that there is a relationship between the rapidity of germination of plant seeds and their lectin content. We assume that lectins can stimulate germination of plant seeds, and garden cress is well known to be a fast growing plant. Preliminary experiments showed that germination of its seeds could be inhibited by addition" of a 1 % solution of bovine mucin or human serum of blood group AB in distilled water (unpublished data). It seemed likely that a reaction between the lectin and the glycoproteins had taken place and therefore the lectin could not stimulate germination. Adamkova and Tobiska reported in 196o that they did not find any lectin activity in the seeds of Lepidium sativum (1). In contrast to this report however, we have found lectin activity in crude extracts of garden cress seeds. The apparent contradiction can be explained by the following facts. First, it is well known that the content of lectins in plants depends on the conditions of cultivation, ageing of the seeds and genetic variation of the plants (2) . Second, for the demonstration of lectins it is important to know details of the method of extraction, the kind of hemagglutination technique and the type of erythrocytes used. In this paper we describe the first results of experiments designed to isolate and characterize the lectin from garden cress.

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 . II © W a l t e r d e G r u y t e r & C o . , B e r l i n • N e w Y o r k 1982

712 Materials and methods 5o gr ground seeds of Lepldium sativum were ml petroleum-ether, then extracted with 5oo solution. The suspension was stirred for 3 rature. The slimy extract then centrifuged g and the supernatant filtered.

defatted in 3oo ml o.l5 M NaCl h at room tempefor 3o min at 3ooo

The clear filtrate was applied to a column (2o x 1.5 cm) of immunoglobulin-Sepharose and the column was washed with o.l5 M NaCl solution to remove unbound proteins. The affinity adsorbent had been prepared by coupling human immunoglobin, mainly immunoglobin G, to BrCN-activated Sepharose 4 B (2o mg/ ml gel). The bound lectin was eluted with 3oo ml 1 M KSCN solution. The fractions containing the hemagglutinating protein were pooled, extensively dialysed against distilled water to remove the KSCN, and freeze-dried. (The yield was about 2o mg). Hemagglutinating activity was determined with a 1 % suspension of washed human erythrocytes, using the microtitrator described by Takatsy (3). Hemagglutination-inhibition tests were performed as follows: to o.o5 ml of a 2-fold serial dilution of carbohydrate was added o.o25 ml of lectin solution with hemagglutinating activity of 4 units. (Four units were defined as that amount of lectin giving a titer of 1:4 using microtitrator plates). After incubation at 37°C for 3o min, o.o25 ml of a 2 % suspension of human erythrocytes was added. After 1 h at 37°C the degree of agglutination was estimated. Stock solutions of o.2 M carbohydrate in o.l5 M NaCl were prepared. Inhibition assays of glycoproteins were performed using only o.o25 ml glycoconjugate and o.o25 ml lectin solution adjusted to 2 hemagglutinating units. Gel chromatography for determination of the molecular weight was carried out with a column (1.5 x 9o cm) of 'Acrylex P 15o' (polyacrylamide purchased from Reanal, Hungary).

The molecu-

713 lar weights of the lectin chains were obtained by disc electrophoresis in lo % polyacrylamide gels in the presence of 1 % sodium dodecyl sulphate. Proteins with known molecular weights were used as markers (cytochrome c 12 4oo; chymotrypsinogen 25 7oo; ovalbumin 45 ooo; and bovine serum albumin 68 ooo). The carbohydrate content of the lectin was determined using the method of Dubois (4) with glucose as reference. The thermal sensitivity of the lectin was determined by incubating the protein in o.l5 M NaCl solution for lo, 2o and 3o min at 3o, 4o, 5o, 6o and 7o°C, and then assaying its hemagglutinating activity at room temperature (5). Its sensitivity to pH was determined by dialysing the lectin (o.5 mg/ml) against 3 changes of buffers (5o ml) of various pH values during an interval of 1 h, and then dialysing back into 3 changes of o.l5 M NaCl solution, after which its hemagglutinating activity was determined (5). The requirement for divalent cations for lectin activity was determined by extensive dialysis of the lectin (1.5 mg/ml) against 5 changes of 25o ml of o.o5 M EDTA solution for 48 h. Removal of sialic acid from the glycoproteins of some samples was performed by acid hydrolysis using o.o5 M f^SO^ for 6o min at 8o°C.

Samples were then dialysed against o.l5 M NaCl so-

lution.

Desialylation of immunoglobulin-Sepharose was carried

out by enzymatic hydrolysis using lo U neuraminidase per ml gel for 1 h at 37°C in o.o5 M sodium acetate, pH 5.5. was then washed with o.l5 M NaCl solution.

The gel

Acid-treated Se-

pharose was prepared by hydrolysis of the gel with o.2 N HC1 2 h at 5o°C (6).

Results Preliminary experiments showed that the lectin from garden cress does not react with insolubilized polysaccharides such

714 as Sephadex G-25, G-5o, G-loo, G-15o and G-2oo or Sepharose 2B, 4B, 6B, or HCl-treated Sepharose. During recent years we have experienced considerable success in using immunoglobulin-Sepharose to isolate lectins having different carbohydrate specificities. For this purpose a fraction of human immunoglobulins, mainly IgG, was coupled to BrCN-activated Sepharose (2o mg immunoglobulin per ml gl). We therefore used this affinity gel for isolation of the lectin from garden cress. The molecular weight of the lectin is about 12o ooo, as determined by gel filtration on polyacrylamide and using proteins with known molecular weights as markers. The lectin consists of 4 subunits with molecular weights of about 3o ooo as estimated by polyacrylamide gel disc electrophoresis in the presence of SDS. A carbohydrate content of 2.2 % was ascertained. The specific adsorption coefficient 1 % Eno was found to be 11.o. 28o nm The lectin does not possess specificity for any individual human blood group, but agglutinates human erythrocytes of blood groups A, B and 0 equally well.

Red blood cells of

different origin were also agglutinated, with the exception of cow erythrocytes.

The results of the hemagglutination

tests are summarized in Table 1. To examine the possibility that the agglutination activity of the lectin depends on divalent cations, as is the case for Con A (7), a solution of the purified lectin was extensively dialysed against 5 changes of o.o5 M EDTA solutin for 48 h.

Loss of activity was not observed.

The lectin activity

was also unchanged by warming a solution, of purified lectin on a water-bath to 5o°C for 3o min, but heating to about 7o°C destroyed the agglutinating activity (Table 2).

To estimate

the stability of the lectin with respect to pH, solutions of lectin were dialysed against buffers of different pH values, and then dialysed back against o,15 M NaCl solution.

After

dialysis against strong acid buffers the activity of the lec-

715 tin was abolished.

The results of hemagglutination tests af-

ter various treatments are summarized in Table 3.

Various

carbohydrates and glycoproteins were employed in a hemagglutination inhibition test (5) to estimate the sugar specificity of the lectin. Inhibition of hemagglutination of human erythrocytes could not be found using o.2 M solutions of any of the following carbohydrates: D-glucose, D-mannose, D-galactose, L-fucose, L-arabinose, D-glucosamine, D-galactosamine, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, N-acetyl-D-mannosamine, D-glucuronic acid, D-galacturonic acid, melibiose, lactose, lactulose, turanose, melezitose, maltose and raffinose. At a concentration of lo mg/ml in o.l5 M NaCl neither dextran, glycogen, mannan TABLE 1 Hemagglutinating activity of the lectin from Lepidium sativum against erythrocytes of different origin

Source

Titer

Human (A) Dog

1:32

Rabbit Mouse Guinea-pig

1:16 1:16 1:32

Horse

1:8 1:8

Sheep

1:32

Goat Pig

1:16 1:128 0

Cow

For hemagglutination a solution of o.5 mg lectin in o.l5 M NaCl and suspensions of 1 % washed erythrocytes were used. Determination was carried out with the microtitrator of Takatsy (3).

716 TABLE 2 Thermal sensitivity. The titer of the lectin from Lepidium sativum after incubation at various temperatures for various times

Temperature

Incubation interval 2o'

3o1

°C

lo'

3o 4o

1:32

1:32

1:32

1:32

1:32

5o 6o

1:32 1:32

1:32 1:32

1:32 1:32 1:16

7o

0

0

0

Solutions of o.5 mg lectin per ml o.l5 M NaCl were warmed for different times on a water bath. Samples were cooled and the hemagglutinating activity ascertained.

TABLE 3 pH sensitivity. The titer of the lectin from Lepidium sativum after dialysis against buffers of various pH values

pH value 1. 7o 2. lo 4 .oo 5. lo 7.2o 8. lo 9 . 2o

titer 0 0 1:32 1:32 1:32 1:32 1:32

Solutions of o.5 mg lectin per ml were dialysed against 3 x 5o ml buffer for 1 h. Dialysis was then performed against o.l5 M NaCl solution, and the hemagglutinating activity determined.

717 from yeast nor galactan from Luplnus albus showed any inhibitory activity.

Although the lectin did not react with D-glu-

cose, it was lightly retarded by Sephadex (cross linked dextran).

For the molecular weight of the lectin, a value of

about 4o ooo was obtained, using columns of Sephadex G-15o. However, the same experiment performed in the presence of o.l M D-glucose resulted in an elution volume corresponding to a molecular weight of 12o ooo. Using lectin concentrations of only 2 hemagglutinating units, we found inhibition of hemagglutination with N-acetylneuraminic acid (NeuNAc) and also with N-acetyl-D-galactosamine

TABLE 4 Agents inhibiting hemagglutination by the lectin from Lepidium sativum

polysaccharides and glycoconjugates

minimal concentration producing complete inhibition

N-acetyl-D-galactosamine

35 mg/ml

N-acetyl-neuraminic acid Vi antigen, Poly-N-acetylgalacturonic acid

30 mg/ml

blood group substance A

0.2 mg/ml

blood group substance B

0.2 mg/ml

bovine mucin desialylated bovine mucin

0.1 mg/ml 0.1 mg/ml

oxacid glycoprotein

0.1 mg/ml

desialylated a^acid glycoprotein

0.1 mg/ml

25 p.g lectin solution containing 2 hemagglutinating units were incubated with 25 p.1 carbohydrate or glycoprotein solution at 37°C for 30 min. 25 (il of 1% erythrocyte suspension was then assed

718 (GalNAc). Both are rather weak inhibitors. Minimal inhibitory concentration amounted to 35 mg GalNAc/ml and 3o mg NeuNAc/ ml. Under the same conditions N-acetyl-D-glucosamine (GlcNAc) did not act as an inhibitor. An excellent inhibitor was the Vi antigen isolated from Salmonella typhi. It is a polysaccharide which consists of N-acetyl-D-galacturonic acid. Details of the inhibitory activity of the Vi antigen and of the other polysaccharides are summarized in Table 4. Hemagglutination was inhibited by blood grpup substance A and B, bovine mucin and a^-acid glycoprotein (orosomucoid), but also by desialylated glycoproteins.

In contrast to this fin-

ding, the lectin reacted only with untreated immunoglobulinSepharose.

After enzymatic treatment with neuraminidase the

lectin did not bind to the immunoglobulin.

Discussion At present we do not know the exact carbohydrate specificity of this lectin. Mucin contains mainly terminal disaccharide units of NeuNAc-GalNAc, whereas both a^-acid glycoprotein and immunoglobulin G contain NeuNAc-Gal disaccharide units. The carbohydrate specificity of most lectins is well established, but for some lectins monosaccharides inhibiting the hemagglutination reaction are not known. This is the case for the lectins from Agaricus bisporus (8), Agaricus campestris (9), Agaricus edulis (lo) and Vicia graminea (11). These lectins probably require more complex carbohydrate structures for efficient interaction. Recently it has been reported that the lectin from Triticum vulgaris reacts not only with GlcNAc but also with GalNAc, NeuNAc, NeuNAc and sialoglycoproteins. Models for the carbohydrate interaction of the wheat germ agglutinin have been presented (12,13,14). The results are interpreted in terms of the configurational similarity of the three carbohydrates at the equatorial C-2 acetamido group and the equatorial C-3 hydroxyl group of the pyranose ring. These are

719 the positions critical to effective contact with the wheat germ lectin.

We assume there are similarities in carbohydrate-

lectin interaction between the lectin from Triticum vulgaris and the lectin from Lepidium sativum. Further examination of the lectin-carbohydrate interaction with NeuAc derivates will be necessary to clarify the exact carbohydrate specificity of this lectin. REFERENCES 1.

Adamkova, B., Tobiska, J. (196o). 9o-95.

2.

Tobiska, J. (1959).

Z. Immunfschg. 117, 197-212.

3.

Takatsy, G. (1967). 275-28o.

Symp. Series Immunbiol. Standar.

4.

Dubois, M., Gilles, K.A., Hamilton J.K., Rebers, P.A., Smith, E. (1959). Analyt. Chem. 28_, 35o-356.

5.

Nicolson, G.L., Blaustein, J., Etzler, M.E. (1974). chemistry 13^ 196-2o4.

6.

Ersson, B., Aspberg, K., Porath, J. (1973). phys. Acta 31o, 446-452.

7.

Hardmann, K.D., Goldstein, I.J. (1977). teins 2, 373-416.

8.

Kaifu, R., Osawa, T. (1979).

9.

Sage, H., Vazquez, J.J. (1967). 125.

10. Eifler, R., Ziska, P. (198o).

Z. Immunfschg. 12o,

Bio-

Biochim. Bio-

Immunochem. Pro-

Carbohydr. Res. j39, 79-88. J. biol. Chem. 242, 12oExperientia 36, 1285-1286.

11. Prignent, M.J., Bourillon, P. (1976). Acta 42o, 112-121. 12. Bhavanandan, V.P., Katlic, A.W. (1979). 254, 4ooo-4oo8.

Biochim. Biophys. J. biol. Chem.

13. Peters, B.P., Ebisu, S., Goldstein, I.J. Flashner, M. (1979). Biochemistry ¿8, 55o5-5511. 14. Monsigny, M., Roche, A.C., Sene, C., Maget-Dana, R., Delmotte, R. (198o). Eur. J. Biochem. lo4, 147-153.

ISOLATION OF TWO LECTINS FROM THE CACTUS Machaerocereus eruca.

Edgar Zenteno and F e l i x Cordoba Dept. of Experimental B i o l o g y , Faculty of Medicine, Universidad Nacional Autonoma de Mexico, and B i o l o g i c a l Research Center, Box 128, La Paz, Baja C a l i f o r n i a Sur, Mexico.

Our work on l e c t i n s has been focussed mainly on c a c t i i and other desert plants (see also Herrera et a l . , these proceedings), because of the widespread i n t e r e s t in developing the a r i d and semi-arid areas in Mexico. Studies of t h i s kind may become s i g n i f i c a n t as the prices for commercially a v a i l a b l e l e c t i n s increase as a consequence of the world wide economic c r i s i s o r , simply, r i s e s in cost production for unpredictable crop y i e l d s . As l e c t i n s have been found in almost a l l kinds of organisms i t may also be expected that new l e c t i n s which are b i o l o g i c a l l y and economically valuable can be found in these kind of unexplored sources. M a t e r i a l s and Methods

The cactus Machaerocerus eruca (1) was collected near San Carlos Harbour, Baja C a l i f o r n i a Sur.

Erythrocytes were obtained from health human

donors, as well as from burro (donkey), r a b b i t , sheep, r a t , mouse, hamster, guinea pig and chicken.

Sephadex G-200 was purchased from

Pharmacia Fine Chemicals (Uppsala, Sweden).

D - g l u c o s e , D-mannose,

N-acetyl-D-glucosami ne, N-acetyl-D-galactosamine, N-acety1-D-mannosami ne, saccharose, D-galactose and L-fucose were obtained from Sigma Fine Chemicals ( S t . L o u i s , M i s s o u r i , U . S . A . ) .

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 . II © W a l t e r d e G r u y t e r &. C o . , B e r l i n • N e w Y o r k 1982

722 Preparation of crude extract and ammonium sulphate fractionation:

The

juice from 1 Kg. of plant stems was obtained with a food processor (Philips) and centrifugated at 3,000 x g for 15 min.

The clear super-

natant was collected and solid ammonium sulphate was added to a final concentration of 66%.

The precipitate, Fraction P, was resuspended

and dialysed against 0.1 M sodium citrate, pH 5.5.

Gel Filtration Chromatography:

The dialyzate was filtered through a

Sephadex G-200 column (1.6 x 50 cm), equilibrated with citrate buffer. Fractions with hemagglutinating activity were pooled and concentrated using Amicon X-10 membranes (Amicon Corp., Lexington, Massachusetts, U.S.A.) before rechromatography on the same column.

Agglutination tests:

Agglutination activity was assayed with 1.5% red

cell suspension using the double dilution procedure.

The hemaggluti-

nation titer, obtained after 1 hour, is expressed as the highest dilution showing detectable agglutination.

The hemagglutinating units are

expressed as the minimum concentration of protein necessary to obtain agglutination (HA units), and the specific activity is the number of HA units per mg of protein.

Lymphocytes were obtained from whole sera

by density gradient centrifugation using histopaque (Sigma).

After

washing in Hank's balanced salt solution (DIFC0), Detroit, Michigan, g U.S.A.) the number of cells was adjusted to 1.4 x 10

cell per ml.

This suspension was incubated with a lectin solution of 128 HA units. The aggregation of the cells was directly observed in a microscope after 5 min.

.Inhibition of hemagglutination with various sugars was

performed using a lectin solution of 128 HA units.

The lectin was two

fold serially diluted and mixed with 0.1 M of sugar solution, and the mixture allowed to stand 30 min. at room temperature before the red cells were added.

723 Immunosuppression assay:

Twelve mice were divided into four equal

groups and injected intraperitoneally.

One group was injected with

0.1 ml of crude extract protein from M. eruca.

Two other groups were

injected with the two hemagglutinating active fractions obtained after the rechromatography of the crude extract.

The control group was

injected with 0.1 ml of saline phosphate buffer (PBS).

All injections

were done before sensitizing the mice with similar doses of sheep redblood cells, and the presence of antibodies was checked by direct hemagglutination assay. (2, 3).

Immunodiffusion:

Double diffusion was performed in agar, 1.5% (Bacto-

Agar, DIFCO) in isotonic saline solution according to the procedure of Ouchterlony (4).

Antisera were produced by immunizing rabbits with

TABLE 1 Hemagglutinating effect of Macharocereus eruca extract towards red cells from several species. Erythrocyte

Titer

Specific Activity

Human A Human B Human 0 Burro Rabbit Sheep Rat Mouse Hamster Guinea Pig Chicken

32 4 16 4096 256 8 4 16 64 128 4

13.3 1.6 6.6 1724.1 106.6 3.3 1.6 6.6 26.6 53.3 1.6

Crude extract was used in a concentration of 2.4 mg/ml of protein. The titer represents the highest dilution factor with agglutinating activity. Specific activity is the number of Hemagglutinating units per miligram of protein (for more details see materials and methods).

724

crude extract of M. eruca.

The animals were injected intraperitoneal ly

with 1 mg of l e c t i n extract in Freund's complete adjuvant (DIFCO) once a week for 4 weeks.

The animals were bled for 3 consecutive days, 1

week after the f i n a l i n j e c t i o n , and the sera of these bleedings pooled.

Protein determination:

The protein concentrations of the samples were

determined by the Lowry procedure ( 5 ) .

Results

The s e l e c t i v i t y of M. eruca hemagglutinating extracts for erythrocytes from different species i s shown in Table 1.

This table shows that

there i s no s i g n i f i c a n t difference between hemagglutination t i t e r s of

Gel f i l t r a t i o n of M. eruca j u i c e on Sephadex G-200 (1.6 x 50 cm) e q u i l i b r a t e d with 0.1 M sodium c i t r a t e buffer pH 5.5; applied sample: 1.5 mg of f r a c t i o n P (for d e f i n i t i o n see materials and methods). Flow rate: 16 ml/hr. Fracti on s i z e : 1.8. ml per tube. Temperature: 20° C. Symbols: ( • — • ) , Optical density at 280 nm; (®—®), A g g l u t i n a t i n g a c t i v i t y towards human erythrocytes; (©—©), A g g l u t i n a t i n g a c t i v i t y towards Burro erythrocytes ; Fraction A, corresponds to material eluted in the void volume with no hemagglutinating a c t i v i t y . Fraction B, corresponds to the pooling of a l l hemagglutinating active material. Fraction C, corresponds to small molecular weight material with no hemagglutinating activity.

725 M. eruca extracts for the different human blood groups.

Extracts from

M. eruca show no agglutination activity towards leucocytes.

A partial

purification of the substance responsible for the hemagglutination was obtained by ammonium sulphate precipitation and by gel filtration on Sephadex G-200 (Table 2 and Fig. 1, respectively).

Further ^chromato-

graphy of the hemagglutinating fraction (fraction B) obtained in the first chromatographic step, yields two active fractions with apparently TABLE 2 Hemagglutination activity and purification yields of M. eruca Fractions.

Fraction

Weight (mg)

Crude P B B-I B-II

1200 307.5 126.6 16.6 25.5

Fi

9-

2

Specific Activity Human Erythrocytes Burro Erythrocytes type B 1.6 476 2000 2800 179

1063 46984c 3.2x10' I.6XIO7

3.2x10

FRACTION No.

Rechromatography of fraction B (see Fig. 1) on Sephadex G-200; all experimental conditions and symbols as indicated in Fig. 1. B-I corresponds to fractions 19 to 26; B-II corresponds to fractions 27 to 37.

726 different carbohydrate and erythrocyte species s p e c i f i c i t y :

Fraction

B - I i s best inhibited by L-fucose, N-acetyl-D-galactosamine, and N-acetyl-D-mannosamine, while B - I I i s , in addition, also inhibited by D-glucose, N-acetyl-glucosamine and D-galactose (Table 3).

Fraction

B - I agglutinates both human and burro erythrocytes, whereas B - I I agglutinates only burro red-blood c e l l s .

The fractions which were injected intraperitoneally into mice decreased their immunological response towards sheep red-blood c e l l s as antigens. However, fraction B - I I was an immunosuppressive agent about three times more potent than B - I .

Immunodiffusion experiments showed that both

fractions B - I and B - I I possess antigenic determinants in common.

The

p o s s i b i l i t y that the précipitants bands were non-specific was investigated using the serum from a non-immunised rabbit in a control experiment. In this case, no precipitation bands were seen with the crude extract or the hemagglutinating fractions.

Discussion The work presented here shows that the crude extracts of ^ possess hemagglutinating a c t i v i t y .

eruca

As t h i s a c t i v i t y i s inhibited by

sugars, i t i s apparently due to a l e c t i n .

Two hemagglutinating frac-

t i o n s , B - I and B - I I have been obtained by chromatography of the crude extract Sephadex G-200 showing different sugar s p e c i f i c i t i e s .

Immuno-

precipitation studies indicated common epitopes on both protein fractions B - I and B - I I , although further p u r i f i c a t i o n and physicocnemical characterization i s needed to c l a r i f y i f both fractions are the same.

The results obtained from hemagglutination studies show

that M. eruca crude extract cannot d i s t i n g u i s h between human blood groups A, B and 0, but show different t i t e r s with red-blood c e l l s from several species.

These observations are similar to the results of

other studies on lectins from different sources (6).

The lectins

obtained from M. eruca are thus potentially useful, although the carbo-

727 hydrate binding properties of the two l e c t i n preparations obtained are yet to be c l e a r l y determined.

TABLE 3 I n h i b i t i o n of a g g l u t i n a t i n g a c t i v i t y of M. eruca l e c t i n s by various sugars.

SUGAR D-glucose N-acety1-D-glucosamine D-galactose L-fucose N-acetyl-D-glactosamine N-acetyl-D-manosami ne

Fraction B - I

Fraction B - I I

128 128 128 16 64 64

32 64 32 64 16 64

Sugars ( 0 . 1 M) were assayed with f r a c t i o n s B - I and B - I I showing a hemagglutinating t i t e r of 128 hemagglutinating u n i t s against burro (donkey) red-blood c e l l s .

TABLE 4 I n h i b i t i o n of the Immuno-response in mice towards sheep erythrocytes by M. eruca a g g l u t i n i n s .

Fraction

Control P B-I B-II

Dose (ug/0.1 ml) 0 220 153 40

Titer

64 8 8 8

728 Acknowledgements: and

Many thanks to Dres. M. C. Agundis,

R. Carvajal

J. L. Ochoa for helpful discussions and Karen S. Starr for

help in the preparation of the manuscript.

References

1.

Bravo-Hollis, H., Las cactaceas de Mexico, Vol. 1, pp. 624, ed. Universidad Nacional Autonoma de Mexico, (1978).

2.

Markowitz, H.,

3.

Calderón, R. A. and Cordoba, F., 575, (1976).

4.

Ouchterlony, 0., (1948).

5.

Lowry, H. 0., Rosebrough, N. J., Lewis, Farr, A. and Randall, R. J., J. Biol. C h e m . , 2 9 3 , pp. 265, (1951).

6.

Shandu, R. S., Reen, R. S., Sing, S., Sing, J. and Chopra, S. K., These Proceedings.

Science, ^63, pp. 476, (1968). Eur. J. Immunology, j>, pp. 522-

Acta. Pathol. Microbiol. Scand.,2->, pp. 186,

THE ISOLATION, FURTHER CHARACTERIZATION AND LOCALIZATION OF PEA SEED LECTIN (PISUM SATIVUM L.)

Edilbert Van Dnessche

+8 « . 8 , Gerda Smets 1 R o b e r t Dejaegere0

and Louis Kanarek"®1 t § Laboratorium Chemie der Proteinen, Laboratorium Plantenfysiologie, Paardenstraat 65, B-1640 Sint-Genesius-Rode, and ^Laboratorium Experimentele Pathologie, Laarbeeklaan 103, B-1090 Brüssel.

The lectin from the garden pea has been investigated by several groups (1-20). During our work with this lectin, it was noticed that when pea seeds (variety Wonder van Kelvedon) were extracted with water (1, 4) or phosphate buffered saline up to 50% or more of the initial agglutinating activity was lost after ammoniumsulphate precipitation of the extract. When the lectin had been purified by affinity chromatography on Sephadex according to published procedures (1, 4), dialyzed against bidistilled water and lyophilized, the protein was partially insoluble and unstable; it aggregates and precipitates, especially in media of low ionic strength. Furthermore, these purified preparations were only retarded on Sephadex, rather than being retained on the column. These observations have led us to reinvestigate the purification procedure and storage conditions in order to overcome the problems mentioned. The lectin has been further characterized and localized in 24-hours imbibed seeds.

Lectins - Biology, Biochemistry, Clinical Biochemistry, Vol. II © Walter de Gruyter &. Co., Berlin • New York 1982

730 Materials and Methods 1. Purification of pea seed lectin. In order to compare and evaluate different extraction and purification procedures, pea seeds were collected from "the same seed stock (variety Wonder van Kelvedon). In a typical experiment, 100 grams of dry seeds were allowed to imbibe overnight in glass-distilled water at 4°C. The seeds were then homogenized in a Warring blender in 1 liter 1 M NaCl + 20 mM MnCl2 + 20 mM CaCl2 + 0.02% (w/v) sodium azide at pH 6.5 (further called 'solvent') and extracted twice at 4°C. The slurry was passed through cheesecloth and the extract adjusted to pH 4.6 with HC1. The suspension was clarified by centrifugation. Solid ammoniumsulphate (360 g per liter of the supernatant) was added and the precipitate was left to settle overnight at *4°C. The ammoniumsulphate pellet was collected by centrifugation, redissolved and intensively dialyzed against solvent. Precipitated proteins were removed by centrifugation and the clear solution applied on a Sephadex G-7 5 column equilibrated with solvent. The column was then washed with solvent until the absorption at 280 nm of the effluent had dropped to nearly zero. In these conditions approximately 1 mg lectin could be bound per ml gel. The lectin was then eluted with 0.2 M glucose in the solvent. The purified protein is stored under 60% saturated ammoniumsulphate. Whenever needed, the desired quantity of ammoniumsulphate suspension is collected by centrifugation, the pellet resuspended in solvent, dialyzed against it and then against the buffer of choice. When prepared, stored and handeled as described here, the lectin remains stable for at least three years. 2.

For electrophoresis in the presence of sodium dodecylsul-

phate, the gels were prepared according to Laemmli (21), while the running buffer was that described by Weber and Osborn (22) Subunit molecular weights were estimated from migration dist-

731

ances relative to standard proteins. 3.

Circular dichroism measurements were recorded on a Cary 61

apparatus at 25°C. Ellipticities were calculated using the MRW equation [ 0^] = jog" x ~¡J where MRW (mean residual weight) is 110 and c and d stand for respectively protein concentration in g/ml and optical path length in dm.

Protein concentrations

& E„° were determined using 2 8 0nm = 15.9.

4.

Sequence analyses were performed as described elsewhere

(10) . 5. Localization studies were performed using the peroxidaseantiperoxidase technique described by Sternberger (23). Experimental details are given elsewhere (20).

Results 1. Isolation and characterization. When pea seed lectin was prepared following described procedures (1, 4) 250 to 300 mg could be purified per kg dry seeds. When the extraction was allowed to proceed in 0.15 M NaCl or in 1 M NaCl instead of in water, a substantial increase in the quantity of finally purified lectin was noticed. However, after ammoniumsulphate precipitation of the extract, considerable losses (up to 50% or more of the initial extract activity) were noticed. 2+

These losses could be avoided by adding Mn

2+

and Ca to the dialysis buffer after the precipitation step. Finally, when these two ions are added during the whole purification procedure, including the extraction, 1500 mg lectin can be purified per kg dry seeds. The addition of glucose in the extraction medium had no affect on the quantity of recovered lectin. As can be seen from table 1, lectin prepared and stored according to (4) is partially insoluble. The lyophilized lectin

732 Table 1.

Solubility of lectin prepared and stored according to (4). Further explanation in text.

Solvent

% Solubility

Water

36

Water + 0 . 1 M g l u c o s e

56

1 M NaCl + 20 mM M n C l 2 , 20 mM C a C l 2

79

1 M NaCl + 20 mM M n C l , , 20 mM C a C l 9 + 0 . 1 M g l u c o s e

78

Time (hours)

Fig. 1. (a) (b) (c)

Precipitation of lectin prepared and stored according to (4) in : water water + 0.1 M glucose 1 M NaCl + 20 mM MnCl 2 + 20 mM CaCl2

and of lectin prepared and stored as described in this paper in (e) water (f) 1 M NaCl + 20 mM MnCl 2 + 20 mM CaCl2

733

was dissolved in different media to obtain 1 mg/ml solutions. In all cases, these solutions were turbid and they were filtered through Millipore membranes. When the clear solutions were left at room temperature, turbidity reappeared as can be seen from Fig. 1. These problems were not met when the lectin was prepared and stored as described in this paper. Even after one week standing in 10 mM potassium phosphate buffer, turbidity was not seen. When the structural stability was checked by circular dichroism measurements, no differences were observed when compared with freshly prepared lectin under the same conditions. Circular dichroism measurements of lectin (Fig. 2), prepared and stored as described in (4) and as described in this paper, are compared. Although the general features of the spectra are conserved in both preparations, appreciable differences, especially in the magnitude of > are noticed.

120

-i

CM

e

d* 0 210

230 Wavelength (nm)

Fig. 2.

250

260

280

300

Wavelength (nm)

Circular dichroism spectra of affinity purified pea lectin in far (A) and near (B) UV. *••• Lectin prepared and stored according to (4). Lectin prepared and stored as described in this paper. Solutions were buffered with 0.15 M NaCl + 10 mM potassium phosphate pH 7.2.

734

When affinity purified pea lectin is submitted to electrophoresis in the presence of sodium dodecylsulphate, besides the alpha- and beta-chain, minor polypeptide bands with molecular weight of 6.5, 8.5 and 2 8 kd are seen (Fig. 3). When affinity purified pea lectin is gel filtrated on Biogel P-100 in 6 M guanidine-HCl (Fig. 4) we observed two major fractions, the heavy chain (peak II; MW 18 kd)' and the light chain (peak V; MW 5.8 kd), as well as three extremely minor fractions. In Fig. 5, the N-terminal sequences of fraction III (MW 6.5 kd) and fraction IV (MW 8.5 kd) are compared with the homologous sequence stretches of Pisum sativum (10) and Lens culinaris (24) lectin. From these results it is clear that fraction III is an internal fragment of the beta-chain, while fraction IV corresponds to its N-terminal part.

Fig. 3.

SDS Polyacrylamide gel electrophoresis of affinity purified pea lectin.

Band 1 precursor of ß-chain or precursor polypeptide from which the pea lectin subunits are derived. MW : 28000 daltons Band 2 ß-chain. MW : 18 000 d. Band 3 proteolytic fragments of ß-chain (fractions III and IV from Fig. 4). Respective MW : 65 0 0 and 8 50 0 d. Band 4 a-chain. MW : 5800 d.

735

1.5 F

Fraction number

Fig. 4.

Gel filtration of affinity purified pea lectin on Biogel P-100 in 6 M guanidine-HCl. Fraction II : g-chain; fraction V : a-chain; fractions III and IV : see text.

Le Ps fIV

Le fill Fig. 5.

1

10 20 T E T T S F S I T K F S P D Q Q N L I F Q G B G Y T L L B Z L L B Z 77 80 90 G Y N V A D G F T F F I A P V D T K P Q B

B

()

( ) — ( ) — B —()() Z

Comparison of N-terminal amino acid sequences of fill = fraction III, and fIV = fraction IV (Fig. 4) with homologous sequences of the g-chains of : Lc = Lens culinaris (24) and Ps = Pisum sativum (10) lectins. Parentheses ( ) correspond to unidentified residues.

736

2.

Localization of pea lectin.

From Fig. 6 and 7 it is obvious that the lectin is localized in the protein bodies of both cotyledons and embryo axis cells .

Fig. 6. (above) Semi-thin section of cotyledons (A) and embryo axis (B) of 24-hours imbibed pea seeds. Tissue was fixed in 2.5% glutaraldehyde for 12 hours and embedded in Araldite (enlargement : x 225 ) . RH rhizodermis » protein body Fig. 7. (opposite page) Ultra-thin section of the embryo axis of 24-hours imbibed seeds. Tissue was fixed in 2.5% glutaraldehyde for 12 hours and embedded in Araldite. A. Control experiment in which the specific anti-pea lectin antibodies were replaced by buffer (enlargement : x 11000). B. Control experiment in which the specific anti-pea lectin antibodies were blocked with an excess of soluble lectin. C. Localization of soluble pea seed lectin. Specific antipea lectin antibodies were used as primary antibodies. CW "A" »

cell wall protein body peroxidase-antiperoxidase complexes

737

Special attention has been paid to the specificity of the observed staining. In one set of control experiments, the primary antibodies (anti-lectin) were omitted (Fig. 7A). In these conditions staining was not observed, indicating that neither the goat anti-rabbit globulins nor the peroxidase-anti-peroxidase complexes react with the sections. From these exper-

cw \

cw

7A

*

w «

S

7B

?jt * S m

CW

* If

* a %l if • •'-Si Iis S p • JF W ^HhmH^ ft

/

JV *

7C

738

iments, non-specific staining due to internal peroxidase activity was excluded as well. In another set of control experiments, the primary antibodies were either replaced by non-immune IgG or by anti-lectin antibodies which were saturated with lectin (Fig. 7B). When Fig. 7A and 7B are compared a darkning of the cell walls in 7B relative to 7 A is seen, indicating that non-specific interactions might exist between the IgG molecules and cell wall components. The extent of darkning of the cell walls could be greatly reduced, but not completely avoided, when the Tris-buffered saline was made 0.5 M in NaCl instead of 0.15 M (25). When the specific anti-pea lectin antibodies are used as primary antibodies, the protein bodies of both cotyledons (Fig. 6A) and embryo axis (Fig. 6B and 7C) are the only sites at which peroxidase-anti-peroxidase complexes can be found. The darkning of the cell walls in Fig. 7C does not exceed that seen in the control experiments (Fig. 7B). From these results we conclude that pea seed lectin is only present in the protein bodies. No lectin is associated with cell walls, plasmalemma or other membraneous structures such as the nuclear envelope. Special attention has been paid to the rhizodermis of the embryo axis. Although the protein bodies of these cells are positively stained, the cell walls and plasmalemma are completely negative (Fig. 6B).

Discussion The increase in recovered protein by increasing the ionic strength of the extraction medium could be either due to greater stability of the protein in high ionic strength media or to a more efficient extraction.

The losses in agglutinating .

.

activity after ammomumsulphate precipitation, when Mn Ca

2+

2+

and

are omitted in the dialysis step, can be explained as

follows : Paulova et al. (2) demonstrated that the metal ions 2+ 2+ Mn and Ca are essential for agglutination and carbohydrate

739

precipitation.

For concanavalin A, it was shown that water is 2+

2+

indispensable for binding of Mn and Ca to the protein (26). These essential water molecules could partially be removed by ammoniumsulphate which precipitates proteins by dehydration (27) with a concomitant loss of metal ions; however these 2+ 2+ are added effects are reversible. Indeed, when Mn and Ca in the dialysis buffer, no losses of agglutinating activity 2+

2+

are noticed. The beneficial effect of adding Mn and Ca to the extraction buffer on the quantity of purified lectin suggests that in dry pea seeds the lectin is partially present as an apo-protein or as a lectin that is only partially saturated with metal ions. These results are in agreement with those of Guldager (14). This author found that the addition of metal ions in the extraction buffer had no effect on the quantity of extracted lectin. In his experimental system, apo-lectin or lectin with only partially saturated metal binding sites will not be distinguished from lectin that is completely saturated with metal ions. However, apo-lectin will not be recovered during the purification. As the addition of glucose during the extraction has no effect on the quantity of purified lectin, no lectin seems to be, to a major extent at least, associated with glucoconjugates via the carbohydrate binding site. Although Rouge et al. (18) showed that vicilin and legumin interact specifically with lectin which was immobilized on Sepharose, these interactions seem to be of no importance on the recovery of the lectin in our extraction conditions. Our lectin preparations have more organized structure than those which have been prepared and stored according to (M-) (Fig. 2). The circular dichroism spectrum in the far ultraviolet region is qualitatively in good agreement with that described by Herrmann et al. (28) and Jirgensons (29). The general features of the spectrum in this region are not changed upon lyophilization, but part of the beta-pleated sheets may have become randomized. From Fig. 2B it is clear that the micro-environment of the aromatic residues is slightly diff-

740 erent in our lectin in comparison with that prepared according to (4). The presence of lectin in the protein bodies of both cotyledons and embryo axis might indicate that the soluble pea seed lectin serves as a reserve protein in accordance with the studies of Rouge et al. (11, 12).

These authors found a

parallellism between the synthesis and disappearance of soluble seed lectins and reserve proteins.

The presence of

natural proteolytic fragments of the beta-chain could point in the same direction. Until now, much contradictory information is available about the physiological function of lectins. the lack of localization studies.

This is mainly due to

Indeed, any proposed funct-

ion should be consistent with the physical location of the protein.

References 1.

Entlicher, G., Kostir, J.V. and Kocourek, J. : Biochim. Biophys. Acta 221, 271-281 (1970)

2.

Paulova, M., Entlicher, G., Ticha, M., Kostir, J.V. and Kocourek, J. : Biochim. Biophys. Acta 237, 513-518 (1971)

3.

Bures, L., Entlicher, G. and Kocourek, J. : Biochim. Biophys. Acta 28_5 , 235-242 (1972)

4.

Trowbridge, I.S. : Proc. Nat. Acad. Sci. USA 7_0 , 3650-3654 (1973)

5.

Trowbridge, I.S. : J. Biol. Chem. 249, 6004-6012 (1974)

6.

Harik, T., Entlicher, G. and Kocourek, J. : Biochim. Biophys. Acta 336, 53-61 (1974)

7.

Entlicher, G. and Kocourek, J. : Biochim. Biophys. Acta 393, 165-169 (1975)

8.

Van Wauwe, J.P., Loontiens, F.G. and De Bruyne, C.K. : Biochim. Biophys. Acta 379, 456-461 (1975)

9.

Cermakova, M., Entlicher, G. and Kocourek, J. : Biochim. Biophys. Acta 4j^0_, 236-245 (1976)

10.

Van Driessche, E., Foriers, A., Strosberg, A.D. and Kanarek, L. : F.E.B.S. Lett. 7^, 220-222 (1976)

741

11. 12. 13. 14.

Rouge, M.P. and Plantefol, M.L. : C.R. Acad. Sc. Paris 282, D 621-623 (1976) Rouge, P. and Labroue, L. : C.R. Acad. Sc. Paris 284, D 2423-2426 (1977) Foriers, A., Wuilmart, C., Sharon, N. and Strosberg, A.D.: Biochem. Biophys. Res. Comm. 7_5, 980-986 ( 1977) Guldager, P. : Theor. Appl. Genet. _53, 241-250 (1978)

15.

Richardson, C., Behnke, W.D., Freisheim, J.H. and Blumenthal, K.M. : Biochim. Biophys. Acta 537, 310-319 (1978)

16.

Guldager, P. and BaSg-Hansen, T.C. : Protides of Biological Fluids (Peeters, H. ed.) 27_, 401-404 (1979) (Pergamon Press, Oxford) Guldager, P. : Lectins : Biology, Biochemistry and Clinical Biochemistry (BaSg-Hansen, T.C. ed.) _1, 151-156 (1980) (W. de Gruyter, Berlin)

17. 18. 19.

Rouge, P. and Chatelain, C. : Lectins : Biology, Biochemistry and Clinical Biochemistry (BaSg-Hansen, T.C. ed. ) 145-150 (1980) (W. de Gruyter, Berlin) Rüdiger, H., Gansera, R., Gebauer, G. and Schurz, H. : Lectins : Biology, Biochemistry and Clinical Biochemistry (BaSg-Hansen, T.C. ed.) 1, 135-144 (1980) (W. de Gruyter, Berlin)

20.

Van Driessche, E., Smets, G., Dejaegere, R. and Kanarek, L. : Planta, in press (1981)

21. 22.

Laemmli, U.K. : Nature (London) 2T7, 680-685 (1970) Weber, K. and Osborn, H. : J. Biol. Chem. 2_44, 4406-4412 (1969)

23.

Sternberger, L.A., Hardy, Jr.P.H., Cuculis, J.J. and Meyer, H.G. : J. Histochem. Cytochem. 18_, 315-333 (1970 )

24.

Foriers, A., Lebrun, E., Van Rapenbusch, R., De Neve, R. and Strosberg, A.D. : J. Biol. Chem. 256, 5550-5560 (1981)

25.

Grube, D. : Histochemistry £6, 149-167 (1980)

26.

Cardin, A.D., Behnke, W.D. and Mandel, F. : J. Biol. Chem. 254, 8877-8884 (1979) Dixon, M. and Webb, E.C. : Adv. Prot. Chem. _16, 197-219 (1971)

27. 28.

Herrmann, M.S., Richardson, C.E., Setzier, L.M. and Behnke, W.D. : Biopolymers 17_, 2107-2120 (1978)

29.

Jirgensons, B. : Biochim. Biophys. Acta 623, 69-76 (1980)

THE ISOLATION AND CHARACTERIZATION OF AN UNUSUAL SEED LECTIN FROM A "LECTIN-FREE" CULTIVAR OF PHASEOLUS VULGARIS, PINTO III, AND ITS RELATIONSHIP TO LECTINS SYNTHESIZED BY ROOT CELLS.

Arpad Pusztai, George Grant, James C. Stewart The Rowett Research Institute, Bucksburn, Aberdeen, AB2 9SB, Scotland, U.K.

The seeds of most varieties of Phaseolus vulgaris contain two related families of tetrameric glycoprotein isolectins (PHA) of 119000 molecular weight (1). In addition to a number of soluble glycoproteins, these lectins also react with a wide variety of eukaryotic normal or transformed cells (2,3). They are powerful though non-selective agglutinants of erythrocytes of diverse origin and some of them are also mitogenic for lymphocytes. When ingested, the lectins react with the enterocytes lining the intestine and, by disrupting the brush borders, cause a serious interference with food adsorption (4). The seeds of a small number of Phaseolus vulgaris cultivars, less than 10% of those examined, apparently contain no such erythroagglutinins (5). However, in a more detailed examination of one such cultivar, the widely grown Pinto III, we have shown that these seeds possess a small but genuine agglutination activity towards rabbit erythrocytes and a high reactivity towards pronase-treated rat erythrocytes (6). This agglutinin, in contrast to PHA, is found not to bind to either fetuin-Sepharose or thyroglobulin-Sepharose and to show negligible immunochemical cross-reactivity with rabbit anti-PHA serum. Additionally, roots grown from these Pinto III seeds were also shown to develop high erythroagglutinating activity (7). As this activity could not be due to PHA from the seeds,

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 . II © W a l t e r d e G r u y t e r &. C o . , B e r l i n • N e w Y o r k 1 9 8 2

744

root tissues were fractionated and their haemagglutinin components characterized. The properties of these root lectins (PRL) were compared with those isolated from Pinto III seeds (PSL) and with those of conventional Phaseolus vulgaris seed lectins (PHA).

Results Pinto III seed lectin. Seed lectins were first purified by conventional protein fractionation methods. Finely ground seed meal was extracted with 0.05 M sodium borate, pH 8.0, the extract cleared by centrifugation and the supernatant dialysed against 3 changes of 0.033 N sodium acetate buffer, pH 5.0. All agglutinating activity remained soluble under these conMost of the lectin content was precipitated from ditions. solution between 45 and 50% ammonium sulphate saturation and this was further fractionated by continuous high-voltage, free-flow electrophoresi s at pH 6.5. The final step in the purification procedure was done by molecular sieve chromatography first on Sephadex G 150 and then on a Bio Gel P 100 column. (Details of the purifications will be given elsewhere.) On the basis of the overall activity recovered (Table I) the lectin content of the Pinto III seed was 0.03% (w/w) . This was less than 17» of the lectin content of most Phaseolus vulgaris cultivars (8). The final product had an activity of 2.5 x 10^ H.U./mg when tested with pronase-treated rat erythrocytes. The extent of purification was 3200 x and 32% of the initial activity was recovered. The Pinto III seed lectin was also purified by immunoaffinity chromatography. Pinto III seed lectin purified by conventional methods, mixed with Freund complete adjuvant was injected subcutaneously into rabbits (four fortnightly injections of 25-50 yUg lectin per injection). Immunoglobulins were isolated by precipitation with ammonium sulphate, dialysis and chromatography on DEAE-cellulose (9). The antibody pre-

745

Table 1. Overall recovery for the lectin purified by conventional methods from the seeds of Pinto III beans , Fraction Seed meal Albumin Ammonium sulphate precipitate High-voltage electrophoresis Sephadex G 150 Bio Gel P 100

, H A L M

°f purification

Total 1QJ7

Activity recovered

8.0 x 101 2.5 x 103

0 32

8.0 x 106 8.2 x 106

100 100

4.0 x 10\ 3

rn 50

/. o x..10" i n6 4.9

u TT

8.0 x

103

2.0 x 105 2.5 x 105

100 2500 3200

3.2 x 106 3.5 x 106 2.5 x 106

62 39 44 32

paration obtained was further purified by absorption with appropriate crude fractions from the initial lectin purification procedure and finally about 100 mg of monospecific immunoglobulin was attached to 15 g of CNBr-activated Sepharose-4B. The resulting affinity absorbent had a capacity to bind about 6-7 mg of PSL. The lectin was released from the absorbent by elution with 0.05 M glycine-HCl buffer containing 0.5 M NaCl, pH 2.5 and after thorough dialysis against distilled water was recovered by freeze-drying. Characterization of Pinto III seed lectins. Partial specific volume at several different concentrations was estimated from density measurements in a precision density meter (1). The conventionally purified PSL gave a value of 0.691 ml.g"^ with a sampling variation of + 0.002, while the immunoaffinitypurified PSL gave 0.700 ml.g -1 (+ 0.007). Both preparations showed one main sedimenting component in the ultracentrifuge. With the conventional preparation this amounted to 94%, while in the case of the affinity-purified PSL the main component comprised of 957» of the preparation. All S2Q values for the main component of the two preparations could be fitted with the following equation to express concentration dependence:

746 S

20 w = (± 0.01) [1-0.062 x concentration] Molecular weight for the two lectin preparations were also estimated at pH 5.10 from the results of sedimentation equilibrium experiments carried out at several different concentrations and speeds (1,10). The average of all point weight average molecular weights was calculated to be 52300 + 600 (455)* for the conventional preparation and 54800 + 800 (345)* for the affinity purified material. The difference between the two molecular weights was significant at the 0.17» level. The molecular weight showed negligible concentrationdependence and the proteins were thus non-aggregating in solution. Both preparations of PSL gave one subunit band on SDS-polyacrylamide gel electrophoresis (11) with an apparent subunit weight of 28-29000 (Fig. la). However, by isoelectric focusing in polyacrylamide gels (1,12) both preparations were resolved into several isolectin components (Fig. lb). All had isoelectric points between pH 4.7 and 5.0 (Table 3). Both preparations contained covalently bound carbohydrate; the neutral sugar content of the conventional PSL preparation was, however somewhat higher than that of the affinity-purified material (Table 3). Both had the same specific activity towards pronase-treated rat erythrocytes, 2.5 x 10^ H.U./mg. This agglutination was not inhibited by any of the simple sugars tested; fetuin and thyroglobulin were also very poor inhibitors. Both lectin preparations also had a low but definite agglutinating activity towards rabbit erythrocytes, o 2 x 10 H.U./mg. This activity could not be inhibited by simple sugars, fetuin or thyroglobulin either. PSL preparations gave negligible cross-reaction with rabbit anti-PHA serum. Pinto III root lectin. Haemagglutination activity increased rapidly in developing roots. This was particularly noticeable in roots nodulated with Rhizobium phaseoli or in the nodules themselves (Table 2). A high proportion of this activity,

747

Fig. la

Fig. 1 (a)

(b)

Fig. lb

Patterns of SDS-polyacrylamide gel electrophoresis of Pinto seed lectin preparations. Lane 1, 'Processor' seed extract + cytochrome c (control); 2, immunoaffinity purified PSL, 2 yUg; 3, conventionally purified PSL, 20 JLtg; 4, lower molecular weight impurity separated from the lectin on Bio Gel P 100 chromatography; 5, 'Pinto III' seed extract. Isoelectric focusing patterns (pH 4-6 ampholine) of 1, Pharmacia gel calibration mixture; 2, PSL purified by immunoaffinity chromatography; 3, Pharmacia gel calibration mixture; 4, PSL, conventionally purified.

Table 2. The effect of Rhizobial nodulation on the haemagglutinating activity (H.U./mg material) of materials obtained from five week-old Pinto III roots Material Insoluble Organic solvent soluble Globulins Albumins

Non -nodulated 1 2 .6 7 2

x x x x

1

10 10 2 10 1 10 1

Nodulated 1

1 x 10 1.7 x IO3 3.3 x 103

3.5 x 103

Nodules 1 1.7 1 2.6

x x x x

10 1 103 10 1 103

748 FLOW CHART OF PURIFICATION

NODULATED ROOTS (19370 mg)

Extraction with CHCl3-MeOH (2:1 v/v) then with 607. ETOH 1 INSOLUBLE

SOLUBLE (2771 mg)

Extraction with 0.2 M Borate pH 8

INSOLUBLE (15950 mg)

SOLUBLE Dialysis against 0.033 N acetate pH 5

SOLUBLE (Albumin) (161 mg)

INSOLUBLE (Globulin) (41 mg)

Affinity chromatography on fetuin-Sepharose-4B I

1

INSOLUBLE

NON-RETAINED

(28 mg)

(116 mg)

' pH 3 ELUTED (7.5 mg)

1 6 M UREA ELUTED (2.5 mg)

749

however, was soluble in organic solvents and could not be abolished by boiling. Thus most of this activity was associated with low molecular weight, lipid type substances of nonprotein nature. For the preparation of protein lectins from root tissue the freeze-dried root powder was first repeatedly and exhaustively extracted with chloroform-methanol (2:1; v/v) solvent mixtures. The insoluble part was further extracted with 60% aqueous ethanol before extraction with aqueous buffers as set out in the FLOW CHART. In addition to a strong haemagglutinating activity towards pronase-treated rat erythrocytes, both albumin and globulin proteins from nodulated roots also developed strong cross-reactivity with rabbit anti-PHA antibodies (Fig. 2a). This was particularly striking when it is considered that the extracts of the Pinto III seed were not reactive with this antibody and contained no conventional PHA (Fig. 2b). Root haemagglutinating proteins were further purified by affinity chromatography on fetuin-Sepharose (FLOW CHART). About 23% of the activity was not retained by this column. This fraction however (specific activity: 5 x 10 H.U./mg) also contained over 90% of the material applied to the affinity column. Small amounts of a material which was retained by the affinity column was recovered by elution with 0.05 M glycine-HCl buffer, pH 3.0, also containing 0.5 M NaCl. This fraction had a high specific activity: 2 x 10^ H.U./mg. Small amounts of material were also eluted by washing the column with 6 M urea. This fraction had a specific activity 4 x 103 H.U./mg. The materials eluted with the pH 3.0 buffer or with 6 M urea cross-reacted with rabbit anti-PHA serum, while the non-absorbed fraction gave negligible cross-reaction (Fig. 3). In addition, the active haemagglutinating fractions gave a subunit band in the region of 29 to 30000 subunit weight by SDS-polyacrylamide gel electrophoresis (not shown). The haemagglutinating activity could be inhibited by N-acetyl-Dgalactosamine and very strongly by fetuin or thyroglobulin. Thus over half of the protein-bound haemagglutinating activity synthesized by the roots of the Pinto III plantlet resembled in

750

Fig. 2 (a)

(b)

Fig. 2 (a)

Fused-rocket Immunoelectrophoresis of samples from Pinto III roots against rabbit anti-PHA ('Processor') antiserum. The following materials were examined: 1, PHA; 2, Pinto non-nodulated albumin; 3, nodulated albumin; 4, PHA; 5, nonnodulated globulin; 6, nodulated globulin; 7, PHA; 8, nodule extract; 9, non-nodulated albumin; 10, non-nodulated globulin; 11, nodulated albumin; 12, nodulated globulin; 13, nodule extract; 14, PHA.

(b)

Fused-rocket Immunoelectrophoresis of lectin samples. The first six lanes were run against rabbit anti-PSL serum, while the last six against rabbit anti-PHA ('Processor') antiserum. Sample 7 in the middle was run against both antisera. The following samples were run: 1, PSL; 2, PSL (3x concentrated); 3, PHA (E4); 4, PHA (L4); 5, Pinto nodule albumin lectin; 6, Pinto nodulated root albumin lectin; 7, PSL; 8, Pinto nodulated root albumin lectin; 9, Pinto nodule albumin lectin; 10, PHA (L4) ; 11, PHA (E4); 12, PSL; 13, PSL (3x concentrated).

its properties those of the usual Phaseolus vulgaris seed lectins (PHA). About one quarter of the protein-bound activity, however, had very different properties. A partially

751

Fig. 3

^ 3 4 Fig. 3

5 6 7 8

Fused-rocket Immunoelectrophoresis of lectin samples from the fetuin-Sepharose-4B affinity column against rabbit anti-PHA ('Processor') antiserum. The following materials were examined: 1, Insoluble; 2, pH 8soluble (solution loaded onto the affinity column) ; 3, PHA; 4, non-retained, leading part of the peak; 5, non-retained, trailing part of the peak; 6, PHA; 7, pH 3-eluted; 8, 6 M-urea eluted.

purified active fraction was obtained from the material not bound to fetuin-Sepharose-4B by absorption with £-N-caproylgalactosamine-Sepharose-4B and elution with 0.2 M galactose solution. Another active material was obtained from the fraction not retained by the £-N-caproyl-galactosamineSepharose-4B column by absorption onto a pronase-treated rat stroma column and elution with 0.05 M glycine-HCl buffer containing 0.5 M NaCl, pH 3.0. Neither of these two active fractions were obtained in pure state, though the material eluted from the stroma column had a molecular weight of 50 to 60000 as shown by chromatography on Bio Gel P 100. Neither of these two haemagglutinating fractions cross-reacted with rabbit anti-PHA or anti-PSL serum and their haemagglutinating activity could not be inhibited by any of the simple sugars tested.

752

Discussion The general occurrence of lectins in the seeds of higher plants is now well-established. However the amount of the lectins in cultivars of a given legume species has been reported to vary a great deal and can even be entirely absent. Thus, seed extracts of about ten per cent of the cultivars of Phaseolus vulgaris examined appeared to have no haemagglutinating activity (5). Similarly, out of a total of 102 screened, 5 lines of Glycine max L. appeared to lack the 120000-Dalton seed lectin (13). Screening investigations have also been carried out with other legume seeds with rather similar results (for references see 13). However, these results cannot be generalized. For example, in the screening of soya beans (13) the results have established the absence in 5 cultivars of the 120000-Dalton lectin. The results however do not exclude the possibility of these seeds containing a lectin different from the usual seed lectin. In BQcher's screening of Phaseolus vulgaris cultivars (5) because of the haemagglutination method used and the choice of the erythrocytes, no generally valid conclusion can be drawn from the negative results. Thus it has now been shown that the seeds of Phaseolus vulgaris cv. Pinto III, one of those seeds found lectin-free by BQcher (5), does in fact contain a lectin, PSL, which is quite different from PHA, the usual Phaseolus vulgaris seed lectin (1). Although it too is a glycoprotein, the molecular weight of PSL is less than half of that of PHA and it contains two subunits only in place of the four in PHA. Haemagglutination and haemagglutination-inhibition studies with simple sugars and glycoproteins have also shown up the differences between the two lectins. The almost complete absence of immunochemical cross-reactivity suggests that very few antigenic determinants could be common between PHA and PSL. Indeed the results indicate that there is a more distant structural relationship between these two lectins from the seeds of the same species than that found between several lectins from different species

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