Carbohydrate-Protein Interaction 9780841204669, 9780841206007, 0-8412-0466-7

Table of contents :
Title Page......Page 1
Half Title Page......Page 3
Copyright......Page 4
ACS Symposium Series......Page 5
FOREWORD......Page 6
PdftkEmptyString......Page 0
PREFACE......Page 7
I. Properties of Lectins......Page 10
II. Soybean Agglutinin and Peanut Agglutinin......Page 12
III. Mitogenic Stimulation......Page 13
IV. Cell Surface Sugars on Lymphocyte Subpopulations......Page 17
Literature Cited......Page 19
2 The Carbohydrate Binding Site of Concanavalin A......Page 21
The Three-Dimensional Structure......Page 22
ABSTRACT......Page 33
LITERATURE CITED......Page 34
3 Binding of Mono- and Oligosaccharides to Concanavalin A as Studied by Solvent Proton Magnetic Relaxation Dispersion......Page 36
Results......Page 37
Discussion......Page 42
Abstract......Page 50
Literature Cited......Page 51
4 Binding of p-Nitrophenyl 2-O-α-D-Mannopyranosyl-α-D-Mannopyranoside to Concanavalin A......Page 53
Literature Cited......Page 64
H-2D Antigens......Page 65
Lectin Induced Accumulation of Lysosomes in Fibroblasts......Page 70
References......Page 73
Materials and Methods......Page 76
Results and Discussion......Page 77
Literature Cited......Page 82
7 Dependence of Agglutination on Concanavalin A Molecular Transition......Page 85
Materials and Methods......Page 86
Results......Page 87
Discussion......Page 93
Literature Cited......Page 97
8 Antibodies Against Oligosaccharides......Page 99
LITERATURE CITED......Page 110
9 Interaction of Glycosyl Immunogens with Immunocyte Receptor Sites in the Synthesis of Anti-glycosyl Isoantibodies......Page 111
Literature Cited......Page 123
Method......Page 125
Results......Page 126
Discussion......Page 134
Abstract......Page 137
Literature Cited......Page 138
11 The Coordinated Action of the Two Glycogen Debranching Enzyme Activities on Phosphorylase Limit Dextrin......Page 140
Literature Cited......Page 169
Acknowledgements......Page 171
12 The Role of Glycosidically-Bound Mannose in the Cellular Assimilation of β-D-Galactosidase......Page 172
Discussion......Page 186
Literature Cited......Page 187
13 The Absence of Carbohydrate Specific Hepatic Receptors for Serum Glycoproteins in Fish......Page 190
Results and Discussion......Page 191
Literature Cited......Page 194
14 Carbohydrate-Protein Interactions in Proteoglycans......Page 195
Cartilage Proteoglycans......Page 196
Blood Vessel Proteoglycans......Page 215
LITERATURE CITED......Page 222
C......Page 226
I......Page 227
O......Page 228
W......Page 229

Citation preview

Carbohydrate-Protein Interaction

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

Carbohydrate-Protein Interaction

I r w i n J . Goldstein, EDITOR University

of

Michigan

A symposium sponsored by the Division of Carbohydrate Chemistry at the 174th Meeting of the American Chemical Society, Chicago, Illinois, August 31— September 1,

1977.

ACS SYMPOSIUM SERIES 88

AMERICAN CHEMICAL SOCIETY WASHINGTON,

D.C.

1979

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

L i b r a r y o f Congress CIP D a t Carbohydrate-protein interaction ( A C S symposium series; 88 I S S N 0097-6156) "Based on a symposium sponsored by the D i v i s i o n of Carbohydrate Chemistry at the 174th meeting of the American Chemical Society, Chicago, Illinois, August 31-September 1, 1977." Includes bibliographies and index. 1. Lectins—Congresses. 2. Carbohydrates—Congresses. 3. Proteins—Congresses. 4. Concanavalin A — Congresses. I. Goldstein, Irwin Joseph. II. American Chemical Society. D i v i s i o n of Carbohydrate Chemistry. III. Series: American Chemical Society. A C S symposium series; 88. QP552.L42C37 I S B N 0-8412-0466-7

574.1'9248 ASCMC 8

78-25788 88 1-222 1979

Copyright © 1979 American Chemical Society All Rights Reserved. T h e appearance of the code at the bottom of the first page of each article i n this volume indicates the copyright owner's consent that reprographic copies of the article may be made for personal or internal use or for the personal or internal use of specific clients. T h i s consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. T h i s consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating new collective works, for resale, or for information storage and retrieval systems. The citation of trade names and/or names of manufacturers i n this publication is not to be construed as an endorsement or as approval by A C S of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may i n any way be related thereto. PRINTED IN T H E UNITED STATES O F AMERICA

American Chemical Society Library

1155 16th St.. N.W. Washington, D.C. 20036

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

ACS Symposium Series Rober

Advisory Board Kenneth B. Bischoff

James P. Lodge

Donald G . Crosby

John L. Margrave

Robert E. Feeney

Leon Petrakis

Jeremiah P. Freeman

F. Sherwood Rowland

E. Desmond Goddard

Alan C. Sartorelli

Jack Halpern

Raymond B. Seymour

Robert A . Hofstader

Aaron Wold

James D . Idol, Jr.

Gunter Zweig

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

FOREWORD The ACS SYMPOSIUM SERIES was founded in 1974 to provide

a medium for publishing symposia quickly in book form. The format of the SERIES parallels that of the continuing ADVANCES IN CHEMISTRY SERIES except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. As a further means of saving time, the papers are not edited or reviewed except by the symposium chairman, who becomes editor of the book. Papers published in the ACS SYMPOSIUM SERIES are original contributions not published elsewhere in whole or major part and include reports of research as well as reviews since symposia may embrace both types of presentation.

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

PREFACE À lthough the physiological importance of sugar units in complex carbohydrates was long ignored, studies on carbohydrates as recognition markers in biological processes have blossomed in recent years. Even biologists who did not acknowledge the existence of carbohydrates in biological membranes have accepted the concept of an active role for sugar units in a variety of cellular phenomena which include cellular adhesion, cellular recognition growth. Enhanced interest in the function of carbohydrate residues derives from the fact that all plant and animal cells are "sugar coated. Because of their strategic position, cell-surface carbohydrates have been implicated in cell-cell communication and in the interaction of cells with their environment. The possibility that complex carbohydrates like nucleic acids and proteins might also serve as informational molecules has created intensive interest and research on the structure, metabolism, and function of glycoconjugates. One discovery of fundamental importance is that the ordered sequence of sugar units in human cell-surface glycolipids and glycoproteins specify blood-group type. Evidence is also rapidly accumulating to suggest that specific carbohydrate residues on many plasma proteins designate these molecules for uptake by specific cells and tissues. The mechanism involves recognition by membrane-bound receptor (glyco) proteins. This volume contains the papers presented in a symposium on carbohydrate-protein interaction. The symposium was devoted to an exploration of protein-glycoconjugate interaction in a wide range of biological phenomena: the interaction of enzymes, antibodies, and lectins with complementary carbohydrate molecules; the recognition of carbohydrate-containing structures by chemoreceptors such as taste and other plasma membrane proteins; and the role of carbohydrates in the organization of connective tissue. Although the physiological function of plant and animal lectins is unknown, these ubiquitous carbohydrate-binding (glyco)proteins can recognize and bind to complex carbohydrates as they occur in solution and on membranes and cell surfaces. A series of papers (by Hardman; Brewer and Brown; Williams and coworkers; Thomas and colleagues; and Evans and Wang) deal with the fundamental chemistry of lectin ,>

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

binding sites. The application of this versatile group of substances as structural and biological probes is described in a paper by Sharon and colleagues, and in a paper by Poretz. Similarly, papers by Pazur and Dreher and by Zopf and colleagues deal with the preparation and carbohydrate-binding specificity of immune antibodies raised in rabbits against sequences of carbohydrate units as they occur in polysaccharides and in synthetic carbohydrate-protein conjugates, respectively. The stereochemical specificity of taste receptor proteins capable of combining with carbohydrate molecules and of translating the message into sweet sensation is investigated by Jakinovich using a sophisticated neurophysiological technique. The glycogen debranching enzyme is the first bifunctional eukaryotic enzyme to be reported that consists of a single polypeptide chain. It catalyzes two distinct activities: an oligosaccharide frans-glycosylation followed by hydrolysis o free glucose. Physical-chemical and kinetic characterization of this novel bifunctional enzyme is described by Nelson. The molecular mechanism whereby cells recognize, interact with, and internalize glycoprotein molecules is discussed in two papers. Distler and colleagues describe the much studied but controversial role of glycosidically bound D-mannose-6-phosphate in the cellular assimilation of the enzyme β-D-galactosidase; Ashwell and Morgan study the meta­ bolic fate of plasma glycoproteins in fish. Finally the organization of connective tissue proteoglycans is described in terms of melocular interactions between hyaluronic acid, link protein, and proteoglycan monomers by Rosenberg and colleagues. Support for the molecular structure of proteoglycans is presented in a series of outstanding electron micrographs. University of Michigan Received September 8, 1978.

IRWIN J . GOLDSTEIN

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

Carbohydrate-Protein Interaction

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

1

Studies o n the Interaction of Lectins w i t h Saccharides o n L y m p h o c y t e Cell Surfaces

NATHAN SHARON, YAIR REISNER, AMIRAM RAVID, and AYA PRUJANSKY Department of Biophysics, The Weizmann Institute of Science, Rehovoth, Israel

Research c a r r i e d out mainly during the l a s t decade has attested to the importanc f th carbohydrat moieties of glycoprotein recognition between c e l l s or between molecules and c e l l s ( 1 - 4 ) . Concomitantly, there has been increased a c t i v i t y in the study of carbohydrate-binding proteins such as glycosidases (5), l e c t i n s (6-9), a n t i - c a r b o hydrate antibodies (10), toxins (such as the cholera and botulinum toxins) and glycoprotein hormones ( e . g . , thyrotropin and human chorionic gonadotropin) (11,12). Investigations of these carbohydrate-binding proteins and t h e i r i n t e r a c t i o n with c e l l s are providing e s s e n t i a l clues to the structure of c e l l - s u r f a c e sugars and t h e i r possible roles i n growth, d i f f e r e n t i a t i o n and development, and i n malignant transformation. I.

Properties of Lectins

Lectins are cell agglutinating proteins of nonimmune o r i g i n that are widely d i s t r i b u t e d i n nature, being found i n p l a n t s , microorganisms and animals. They bind mono- or oligo-saccharides with remarkable s p e c i f i c i t y , i n the same way as enzymes bind substrates and antibodies bind antigens. Binding may involve several forces, mostly hydrophobic and hydrogen bonds, and i s competitively i n h i b i t e d by s p e c i f i c sugars. Many l e c t i n s combine p r e f e r e n t i a l l y with a single sugar s t r u c t u r e , for example Ç - g a l a c t o s e or L-fucose. For some l e c t i n s the s p e c i f i c i t y i s broader and includes several c l o s e l y related sugars, e . g . , D-mannose, g-glucose and g-arabinose; other l e c t i n s i n t e r a c t only with complex carbohydrate structures such as those that occur i n glycoproteins, g l y c o l i p i d s , 0-8412-0466-7/79/47-088-001$05.00/0 © 1979 American Chemical Society

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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CARBOHYDRATE-PROTEIN

INTERACTION

o r on c e l l s u r f a c e s . The sugars w i t h which l e c t i n s combine b e s t a r e those t h a t a r e t y p i c a l c o n s t i t u e n t s of glycoproteins or g l y c o l i p i d s . Perhaps t h i s i s a r e f l e c t i o n o f t h e way i n which l e c t i n s a r e d e t e c t e d (namely, by h e m a g g l u t i n a t i o n ) , as a r e s u l t o f which l e c t i n s s p e c i f i c f o r s u g a r s o t h e r than t h o s e p r e s e n t on a n i m a l c e l l s u r f a c e s , might be o v e r l o o k e d . To date o v e r 5 0 l e c t i n s have been o b t a i n e d i n p u r i f i e d form (1.'2) · They v a r y c o n s i d e r a b l y i n amino a c i d c o m p o s i t i o n , sugar c o n t e n t , m o l e c u l a r w e i g h t , s u b u n i t s t r u c t u r e , number o f c a r b o h y d r a t e b i n d i n g s i t e s p e r m o l e c u l e , and m e t a l r e q u i r e m e n t . Many l e c t i n s c o n t a i n c o v a l e n t l y bound sugar and a r e t h e r e f o r e glycoproteins. However, c o n c a n a v a l i n A, wheat germ a g g l u t i n i n and peanu best characterized protein of sugar. Soybean a g g l u t i n i n , t h e most t h o r o u g h l y investigated glycoprotein l e c t i n , contains s i x percent sugar comprised o f D-mannose and N - a c e t y l - D - g l u cosamine, and c o n s i s t s o f f o u r s u b u n i t s ( M ^ W . 3 0 , 0 0 0 ) each o f which c a r r i e s one c a r b o h y d r a t e c h a i n , D-Manq ( D - G l c N A c ^ / l i n k e d t o t h e p r o t e i n v i a an N - a c e t y l - D glucosaminyl-asparagine linkage. TTïë"~structure o f t h e c a r b o h y d r a t e s i d e c h a i n o f soybean a g g l u t i n i n has been r e c e n t l y e l u c i d a t e d i n o u r l a b o r a t o r y (13)· I t cont a i n s t h e branched c o r e a-D-ManjD- (1+3Ma-D-ManjD( 1 + 6 ) ] -e-g-Man£- ( 1 + 4 ) - 6 - D - 5 l c N A c £ - ( 1 + 4 ) -β-β-GlcNAc, p r e v i o u s l y found i n many a n i m a l g l y c o p r o t e i n s as w e l l as i n t h o s e from f u n g i and y e a s t s and now shown f o r the f i r s t time t o o c c u r i n a p l a n t g l y c o p r o t e i n . The same b a s i c c a r b o h y d r a t e s t r u c t u r e has a l s o been found i n t h e l i m a bean l e c t i n ( 1 4 ) . A wide range o f s p e d " ? i c i t i e s and b i o l o g i c a l a c t i v i t i e s has been o b s e r v e d i n t h e i n t e r a c t i o n o f l e c t i n s with c e l l s . L e c t i n s show s e l e c t i v i t y i n t h e i r a g g l u t i n a t i o n o f e r y t h r o c y t e s o f d i f f e r e n t animal s p e c i e s , w i t h human e r y t h r o c y t e s ; some o f them a r e even b l o o d type s p e c i f i c . Thus, c e r t a i n l e c t i n s a g g l u ­ t i n a t e o n l y human b l o o d type A e r y t h r o c y t e s , w h i l e o t h e r s a g g l u t i n a t e o n l y type 0(H) e r y t h r o c y t e s . Such l e c t i n s a r e used as an a i d i n b l o o d t y p i n g , e s p e c i a l l y s i n c e n a t u r a l anti-O(H) i s o h e m a g g l u t i n i n i n humans i s very rare. Both s p e c i e s and c l a s s (T o r B) s p e c i ­ f i c i t y have a l s o been demonstrated i n t h e i n t e r a c t i o n o f l e c t i n s w i t h lymphocytes; moreover, c e r t a i n l e c t i n s d i s t i n g u i s h between lymphocyte s u b p o p u l a t i o n s from t h e same a n i m a l o r organ (see l a t e r ) . Another i n t r i g u i n g property of l e c t i n s i s t h e i r a b i l i t y to a g g l u t i n a t e m a l i g n a n t l y t r a n s f o r m e d c e l l s much b e t t e r than normal c e l l s .

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

1.

SHARON ET AL.

Interaction of Lectins with Saccharides

3

A l t h o u g h the r o l e o f l e c t i n s i n n a t u r e i s not known, t h e r e are i n c r e a s i n g i n d i c a t i o n s t h a t they f u n c t i o n i n r e c o g n i t i o n phenomena o f m i c r o o r g a n i s m s , p l a n t s and a n i m a l s , b o t h i n t e r c e l l u l a r and i n t r a c e l l u ­ l a r ( f o r a r e c e n t d i s c u s s i o n , see 15> ) . They may be responsible f o r a v a r i e t y of i n t e r c e l l u l a r i n t e r ­ a c t i o n s , from the a d h e s i o n of b a c t e r i a t o a n i m a l c e l l s , t o the attachment of sperm t o egg. Since r e c o g n i t i o n a l s o i m p l i e s d i s t i n c t i o n between s e l f and n o n s e l f and between f r i e n d and enemy, t h e s e i d e a s are i n l i n e w i t h e a r l i e r s u g g e s t i o n s t h a t l e c t i n s p l a y an important r o l e i n h o s t - p a r a s i t e r e l a t i o n s h i p s , both i n animals and p l a n t s . L e c t i n s may, t h u s , s e r v e as p a r t o f the defense mechanisms o f p l a n t s a g a i n s t p a t h o g e n i c m i c r o o r g a n i s m s , whethe by l e c t i n s may a l s o between legumes and t h e i r s y m b i o t i c n i t r o g e n - f i x i n g bacteria. The ready a v a i l a b i l i t y o f l e c t i n s , t h e i r ease o f p r e p a r a t i o n i n p u r i f i e d form, the f a c t t h a t they are amenable t o c h e m i c a l m a n i p u l a t i o n and t h a t many o f them are i n h i b i t e d by s i m p l e s u g a r s , has made them a most a t t r a c t i v e t o o l i n b i o l o g i c research. The f o l l o w i n g i s a review o f r e c e n t s t u d i e s i n our l a b o r a t o r y on the i n t e r a c t i o n o f soybean a g g l u t i n i n and o f peanut a g g l u t i n i n w i t h lymphocytes from d i f f e r e n t sources. II.

Soybean A g g l u t i n i n and

Peanut A g g l u t i n i n

These two l e c t i n s have been p u r i f i e d and e x t e n ­ s i v e l y c h a r a c t e r i z e d i n our l a b o r a t o r y (reviewed i n 7). For the purpose o f t h i s p r e s e n t a t i o n , o n l y some o f t h e i r p r o p e r t i e s are r e l e v a n t , the most i m p o r t a n t o f which i s t h e i r sugar s p e c i f i c i t y . This s p e c i f i c i t y i s d e f i n e d i n terms o f the mono- o r o l i g o - s a c c h a r i d e which i n h i b i t s a t the l o w e s t c o n c e n t r a t i o n the hemagg l u t i n a t i n g o r p r e c i p i t a t i n g a c t i v i t y o f the l e c t i n . Thus, soybean a g g l u t i n i n i s s p e c i f i c f o r N - a c e t y l - D g a l a c t o s a m i n e , and t o a l e s s e r e x t e n t (at l e a s t 20 times l e s s ) f o r D - g a l a c t o s e (16-18). Peanut a g g l u t i n i n , on the o t h e r hand, i s s p e c i f i c f o r the d i s a c c h a r i d e β-D-Galrj-(l-*3)-D-GalNAc, which i s some 50 times b e t t e r an i n h i b i t o r o f " t h e l e c t i n than D - g a l a c ­ tose (Ι^,^Ο). The sequence i3-D-Gal]3—(1+3)-D-GalNAc i s p r e s e n t i n many a n i m a l g l y c o p r o t e i n s , i n c e r t a i n g l y c o l i p i d s , as w e l l as on c e l l s u r f a c e s , e.g., on human e r y t h r o c y t e s and p i g lymphocytes. However, t h i s d i s a c c h a r i d e i s commonly s u b s t i t u t e d by one o r two s i a l i c a c i d r e s i d u e s , and as a r e s u l t most

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

4

CARBOHYDRATE-PROTEIN INTERACTION

g l y c o p r o t e i n s and c e l l s do not i n t e r a c t w i t h peanut agglutinin. Thus, human e r y t h r o c y t e s o r p e r i p h e r a l lymphocytes do not b i n d peanut a g g l u t i n i n t o any con­ s i d e r a b l e e x t e n t , nor a r e they a g g l u t i n a t e d by t h i s l e c t i n u n l e s s the c e l l s have been t r e a t e d w i t h neuraminidase ( 1 9 . * £ 1 * 2 2 ) . T h i s i s a l s o true f o r mouse s p l e e n lymphocytes. As t o g l y c o p r o t e i n s , peanut a g g l u t i n i n does not i n t e r a c t w i t h f e t u i n o r o ^ - a c i d g l y c o p r o t e i n (orosomucoid) b u t i n t e r a c t s r e a d i l y w i t h a s i a l o f e t u i n and asialo-ατ-acid g l y c o p r o t e i n . A n o t a b l e e x c e p t i o n a r e the a n t i f r e e z e g l y c o p r o t e i n s o f a n t a r c t i c f i s h , i n which u n s u b s t i t u t e d β-g-Galrj- ( 1 + 3 ) D-GaINAc r e s i d u e s i s a t t a c h e d t o the p o l y p e p t i d e Backbone; t h e s e g l y c o p r o t e i n s r e a c t w i t h peanut a g g l u t i n i n without neuraminidas (23). There are a l s o c e r t a i w i t h peanut a g g l u t i n i n , as w i l l be shown l a t e r on. A n o t h e r p r o p e r t y o f b o t h soybean a g g l u t i n i n and peanut a g g l u t i n i n , which i s r e l e v a n t t o t h i s p r e s e n t a ­ t i o n , i s the number o f sugar b i n d i n g s i t e s : these l e c t i n s have two sugar b i n d i n g s i t e s each, p e r "monomer" o f 1 2 0 , 0 0 0 o r 1 1 0 , 0 0 0 d a l t o n s , r e s p e c t i v e l y ; i n o t h e r words, they a r e d i v a l e n t ( 2 £ , 2 5 J . In t h i s r e s p e c t they d i f f e r , f o r example, from wheat germ a g g l u t i n i n and c o n c a n a v a l i n A, b o t h o f which a r e t e t r a v a l e n t a t p h y s i o l o g i c a l pH. III.

Mitogenic

Stimulation

One o f the most s t r i k i n g p r o p e r t i e s o f l e c t i n s i s the t r i g g e r i n g o f q u i e s c e n t , n o n d i v i d i n g lymphocytes t o grow and p r o l i f e r a t e , an e f f e c t known as m i t o g e n i c stimulation. The g r o s s m o r p h o l o g i c a l changes and b i o c h e m i c a l events o c c u r r i n g i n l e c t i n - s t i m u l a t e d lymphocytes i n v i t r o resemble many o f the a n t i g e n - i n ­ duced immune r e a c t i o n s t h a t o c c u r In v i v o . Lectins are t h e r e f o r e an i m p o r t a n t a i d i n immunology. They a r e a l s o used e x t e n s i v e l y i n attempts t o u n d e r s t a n d the mechanisms by which s i g n a l s a r e t r a n s m i t t e d from the o u t s i d e o f the c e l l t o i t s i n t e r i o r . S t i m u l a t i o n o f lymphocytes by m i t o g e n i c l e c t i n s , such as p h y t o h e m a g g l u t i n i n (PHA) o r c o n c a n a v a l i n A, does n o t r e q u i r e any p r e t r e a t m e n t o f the c e l l s . This i s n o t the case w i t h soybean a g g l u t i n i n , which, as f i r s t demonstrated by Novogrodsky and K a t c h a l s k i ( 2 6 ) s t i m u l a t e s mouse s p l e e n c e l l s o r human lymphocytes o n l y a f t e r the c e l l s had been t r e a t e d by n e u r a m i n i d a s e . They s u g g e s t e d t h a t n e u r a m i n i d a s e removes from the c e l l surface s i a l i c a c i d residues attached to g-galactose and N - a c e t y l - g - g a l a c t o s a m i n e . The l a t t e r sugars are

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

1.

SHARON ET AL.

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5

thus exposed and become a c c e s s i b l e t o soybean agglutinin; b i n d i n g to these s i t e s r e s u l t s i n stimu­ lation. As e x p e c t e d , t h i s s t i m u l a t i o n i s i n h i b i t e d b e s t by N - a c e t y l - D - g a l a c t o s a m i n e : a t about Ι Ο " M some 50 p e r c e n t i n h i b i t i o n i s o b s e r v e d (27). D-Galact o s e i s a much p o o r e r i n h i b i t o r , 10-20 times more o f t h i s sugar b e i n g r e q u i r e d f o r t h e same i n h i b i t i o n . F u r t h e r s t u d i e s o f the m i t o g e n i c p r o p e r t i e s o f soybean a g g l u t i n i n r e v e a l e d t h a t d i f f e r e n t p r e p a r a ­ t i o n s o f the l e c t i n e x h i b i t e d d i f f e r e n t m i t o g e n i c a c t i v i t i e s , as measured by the c o n c e n t r a t i o n o f l e c t i n r e q u i r e d t o g i v e maximal s t i m u l a t i o n . The d i f f e r e n c e s o b s e r v e d were o f t e n v e r y l a r g e , c e r t a i n p r e p a r a t i o n s b e i n g o n l y a c t i v e a t 1-1.5 mg/ml, w h i l e o t h e r s gave a peak o f m i t o g e n i c s t i m u l a t i o reason f o r t h e s e anomalou i t was found t h a t the l e c t i n undergoes a g g r e g a t i o n upon s t o r a g e ( 2 8 , 2 9 K Upon g e l f i l t r a t i o n o f s t o r e d (aggregated) soybean a g g l u t i n i n on Sephadex G-150, t h r e e f r a c t i o n s were o b t a i n e d : the soybean a g g l u t i n i n monomer (M.W. 120,000) had two sugar b i n d i n g s i t e s and was n o t m i t o g e n i c up t o 1 mg/ml; the t e t r a v a l e n t dimer (M.W. 240,000) e x h i b i t e d maximal m i t o g e n i c a c t i v i t y a t 10 yg/ml; and the p o l y v a l e n t polymer (M.W. > 360,000) had the same m i t o g e n i c a c t i v i t y as t h e dimer (27). Soybean a g g l u t i n i n p o l y m e r i z e d by c r o s s l i n k i n g with glutaraldehyde i s also mitogenic at low c o n c e n t r a t i o n s (10-15 yg/ml). More r e c e n t l y , we have found t h a t whereas peanut a g g l u t i n i n i s n o t m i t o ­ g e n i c f o r n e u r a m i n i d a s e - t r e a t e d mouse lymphocytes (30), the p o l y m e r i z e d l e c t i n o b t a i n e d by c r o s s l i n k i n g witn~~ g l u t a r a l d e h y d e i s m i t o g e n i c (31). Our f i n d i n g s a r e i n agreement w i t h those o f G o l d s t e i n , B e s s l e r and t h e i r co-workers w i t h the l i m a bean l e c t i n s which have a sugar s p e c i f i c i t y s i m i l a r t o t h a t o f soybean a g g l u t i n i n (32./33). From l i m a beans s e v e r a l i s o l e c t i n s w i t h d i f f e r e n t numbers o f b i n d i n g s i t e s f o r methyl 2-acetamido-2-deoxy-a-D-galactop y r a n o s i d e have been i s o l a t e d : a d i v a l e n t tetramer (M.W. 124,000), a t e t r a v a l e n t octamer (M.W. 247,000) and s e v e r a l h i g h e r polymers. The m i t o g e n i c a c t i v i t y o f the i s o l e c t i n s was r e l a t e d t o t h e i r v a l e n c y : whereas the d i v a l e n t l e c t i n was a poor mitogen, the t e t r a v a l e n t l e c t i n was a s t r o n g one. These f i n d i n g s can be b e s t e x p l a i n e d by assuming t h a t i n o r d e r t o i n d u c e m i t o g e n i c s t i m u l a t i o n , the l e c t i n must not o n l y b i n d t o c e l l s u r f a c e s u g a r s , b u t cause c r o s s l i n k i n g o f c e l l s u r f a c e r e c e p t o r s . Such c r o s s l i n k i n g may cause l a t e r a l movement o f transmem­ b r a n e g l y c o p r o t e i n s r e s u l t i n g perhaps i n the opening 4

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o f a c h a n n e l f o r c a l c i u m i o n s . Enhanced movement o f c a l c i u m i o n s a c r o s s t h e membrane i s , a c c o r d i n g t o one view, t h e i n i t i a l b i o c h e m i c a l o r m e t a b o l i c event i n the m i t o g e n i c p r o c e s s subsequent t o b i n d i n g o f t h e l e c t i n t o t h e c e l l s u r f a c e (3£; f o r a discussion, see r e f . 7 ) . There i s i n d e e d e v i d e n c e t h a t b i n d i n g o f m i t o ­ g e n i c l e c t i n s causes c o n f o r m a t i o n a l changes i n membrane structure. We have r e c e n t l y found (31) t h a t when t h e Scatchard p l o t s o f the b i n d i n g data of l e c t i n s t o lymphocytes were l i n e a r , no s t i m u l a t i o n o f t h e c e l l s was o b s e r v e d . However, when the p l o t s were n o n l i n e a r , t h e r e was s t i m u l a t i o n . The n o n l i n e a r p l o t s were c h a r a c t e r i s t i c of binding with p o s i t i v e c o o p e r a t i v i t y . In b i n d i n g phenomena that the b i n d i n g constan p l e x i n c r e a s e s as t h e e x t e n t o f occupancy o f r e c e p t o r s i t e increases. P o s i t i v e c o o p e r a t i v i t y i n the i n t e r ­ a c t i o n o f l e c t i n s w i t h c e l l s u r f a c e s can be e x p l a i n e d by e i t h e r an i n c r e a s e i n t h e a f f i n i t y o f t h e r e c e p t o r s t o t h e l e c t i n , o r by an i n c r e a s e i n t h e number o f a v a i l ­ a b l e b i n d i n g s i t e s caused by unmasking o f c r y p t i c receptors. Both types o f change may be t h e r e s u l t o f c o n f o r m a t i o n a l changes i n membrane components, p a r t i c u l a r l y membrane g l y c o p r o t e i n s and g l y c o l i p i d s , the s a c c h a r i d e m o i e t i e s o f which b i n d t o l e c t i n s . Such changes may a l s o be t h e r e s u l t o f r e d i s t r i b u t i o n o f t h e s e components i n t h e membrane, f a c i l i t a t e d by t h e f l u i d character o f the l a t t e r . In o r d e r t o induce t h e r e q u i r e d c o n f o r m a t i o n a l changes, d i v a l e n c e i s i n s u f f i c i e n t , a t l e a s t f o r l e c t i n s s p e c i f i c f o r g - g a l a c t o s e and g - g a l a c t o s e - l i k e r e s i d u e s i n t e r a c t i n g w i t h mouse lymphocytes, and t h e s e l e c t i n s must be t e t r a v a l e n t (or o f h i g h e r v a l e n c e ) t o cause t h e n e c e s s a r y c o n f o r m a t i o n a l changes i n t h e membrane. Only such a l e c t i n w i l l p u l l t o g e t h e r t h e r e c e p t o r s (with t h e i r p o l y p e p t i d e c h a i n s and t h e transmembrane p r o t e i n s t o which they a r e a t t a c h e d ) , c r o s s l i n k them and i n d u c e c l u s t e r i n g o f t h e r e c e p t o r s , and thus cause t h e changes i n membrane s t r u c t u r e t h a t are p r e r e q u i s i t e f o r mitogenic s t i m u l a t i o n . To o b t a i n f u r t h e r i n s i g h t i n t o the s t r u c t u r e o f the s u g a r s on t h e c e l l s u r f a c e which b i n d soybean a g g l u t i n i n and peanut a g g l u t i n i n , we used i n a d d i t i o n t o n e u r a m i n i d a s e t h e enzyme 3 - g a l a c t o s i d a s e from D i p l o c o c c u s pneumoniae which removes g - g a l a c t o s e r e s i d u e s from g l y c o p r o t e i n s and from c e l l s u r f a c e s . Whereas t h e n e u r a m i n i d a s e - t r e a t e d mouse s p l e e n c e l l s were s t i m u l a t e d by soybean a g g l u t i n i n , s e q u e n t i a l t r e a t m e n t w i t h n e u r a m i n i d a s e and β-galactosidase

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a b o l i s h e d almost c o m p l e t e l y t h e s t i m u l a t i o n by t h e l e c t i n (30). T h i s s u g g e s t s t h e presence on t h e lympho­ c y t e s u r f a c e o f g - g a l a c t o s y l r e s i d u e s t o which soybean a g g l u t i n i n b i n d s w i t h e n s u i n g s t i m u l a t i o n and makes i t u n l i k e l y that N-acetyl-D-galactosamine residues (to which soybean a g g l u t i n i n a l s o b i n d s ) a r e i n v o l v e d i n the m i t o g e n i c e f f e c t . I n c o n t r o l experiments w i t h c o n c a n a v a l i n A, s t i m u l a t i o n o c c u r r e d b o t h a f t e r i n c u b a ­ t i o n w i t h neuraminidase a l o n e and a f t e r s e q u e n t i a l t r e a t m e n t w i t h neuraminidase and 3 - g a l a c t o s i d a s e . The above f i n d i n g s on t h e e f f e c t o f g l y c o s i d a s e s on lymphocyte s t i m u l a t i o n can be r e a d i l y r a t i o n a l i z e d by assuming t h a t t h e " m i t o g e n i c s i t e s " on t h e c e l l s u r f a c e a r e p a r t o f t h e commonly o c c u r r i n g a s p a r a g i n y l - l i n k e d sequenc B-D-GlcNAc£- (1+2)-a-g-Man£ β-Β-GlcNAcp-(1+4)-β-D-GlcNAc. As l o n g as t h e s i a l i c a c i d (NANA) r e s i d u e s a r e p r e s e n t , b i n d i n g o f soybean a g g l u t i n i n o r peanut a g g l u t i n i n cannot o c c u r . After removal o f t h e s i a l i c a c i d by n e u r a m i n i d a s e , soybean a g g l u t i n i n and peanut a g g l u t i n i n b i n d t o t h e c e l l surface, with resultant stimulation. Removal o f t h e D - g a l a c t o s e r e s i d u e s w i t h β-galactosidase a b o l i s h e s t h e Binding to the mitogenic s t i e s . B i n d i n g t o such s i t e s of c o n c a n a v a l i n A, however, o c c u r s i r r e s p e c t i v e o f t h e p r e s e n c e o f s i a l i c a c i d o r D - g a l a c t o s e on t h e c a r b o ­ hydrate side chain s i n c e t h i s l e c t i n binds t o i n t e r n a l D-mannose r e s i d u e s . Another p o s s i b i l i t y i s t h a t t h e D - g a l a c t o s e - s p e c i f i c mitogens i n t e r a c t w i t h t h e O - g l y c o s i d i c a l l y l i n k e d sequence a-NANA-(2+6)-e-D-Gal£-(1+3)-a-gGalNAc£- ( 1+0^)-Ser (Thr) · I f t h i s i s t h e c a s e , then concanavalin A binds t o a d i f f e r e n t o l i g o s a c c h a r i d e c h a i n , a t t a c h e d e i t h e r t o t h e same p o l y p e p t i d e which c a r r i e s t h e O - g l y c o s i d i c a l l y l i n k e d moiety (as has been found, f o r example, i n g l y c o p h o r i n ) , o r t o another p o l y p e p t i d e c h a i n . A t p r e s e n t we do n o t know which i s the sequence i n v o l v e d , n o r can we say whether i t i s a t t a c h e d t o membrane g l y c o p r o t e i n ( s ) o r g l y c o l i p i d s . One approach t o t h i s problem would be t o i s o l a t e the i n t a c t l e c t i n - r e c e p t o r m o l e c u l e s from t h e c e l l membranes and c h a r a c t e r i z e them. Work i n t h i s d i r e c ­ t i o n has been i n i t i a t e d i n o u r l a b o r a t o r y , and a method f o r t h e i s o l a t i o n o f t h e peanut a g g l u t i n i n r e c e p t o r from membranes o f n e u r a m i n i d a s e - t r e a t e d human e r y t h r o ­ c y t e s on a column o f peanut a g g l u t i n i n - p o l y a c r y l h y d r a z i d o - S e p h a r o s e has been developed (21). The amino a c i d c o m p o s i t i o n , D-glucosamine ancHD-galactosamine c o n t e n t , and the e l e c t r o p h o r e t i c m o b i l i t y on p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i n sodium

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d o d e c y l s u l f a t e o f t h e peanut a g g l u t i n i n r e c e p t o r , were s i m i l a r t o those o f a s i a l o g l y c o p h o r i n . Experiments u s i n g t h e same approach f o r t h e i s o l a t i o n o f l e c t i n r e c e p t o r s from lymphocytes a r e i n p r o g r e s s . IV.

C e l l S u r f a c e Sugars

on Lymphocyte

Subpopulations

D u r i n g r e c e n t y e a r s i t has been e s t a b l i s h e d t h a t lymphocytes a r e comprised o f many s u b p o p u l a t i o n s , such as Τ and B, mature and immature c e l l s , e t c . Although v a r i o u s t e c h n i q u e s f o r lymphocyte s e p a r a t i o n have been d e v i s e d , based m a i n l y on c e l l s i z e , d e n s i t y , e l e c t r i c a l charge and s p e c i f i c s u r f a c e a n t i g e n s , none o f t h e s e i s satisfactory. Studies c a r r i e d out r e c e n t l y i n our l a b o r a t o r y have demonstrate o f l e c t i n s t o sugar can s e r v e as a b a s i s f o r a s i m p l e and e f f e c t i v e method for c e l l fractionation. The f i r s t a p p l i c a t i o n o f t h i s approach was a method f o r f r a c t i o n a t i o n , by t h e use o f peanut a g g l u t i n i n , o f mouse thymocytes i n t o m e d u l l a r y (immuno­ l o g i c a l l y mature) and c o r t i c a l ( i m m u n o l o g i c a l l y immature) c e l l s which d i f f e r i n many o f t h e i r s u r f a c e p r o p e r t i e s and b i o l o g i c a l a c t i v i t i e s (3_5) . As mentioned e a r l i e r , t h i s l e c t i n does n o t as a r u l e i n t e r a c t w i t h c e l l s u n l e s s they have been t r e a t e d w i t h neuraminidase. However, e x a m i n a t i o n under t h e m i c r o ­ scope o f t h e b i n d i n g o f f l u o r e s c e i n - l a b e l e d peanut a g g l u t i n i n t o mouse thymocytes r e v e a l e d t h a t t h e m a j o r i t y o f t h e c e l l s were s t a i n e d , whereas a s m a l l p r o p o r t i o n (some 10 p e r c e n t ) were n o t . Moreover, i n c o n t r a s t t o most c e l l s which a r e n o t a g g l u t i n a t e d by peanut a g g l u t i n i n , t h e b u l k o f t h e thymus c e l l s were a g g l u t i n a t e d by t h e l e c t i n . Separation o f the c e l l clumps from t h e s i n g l e c e l l s was a c h i e v e d by l a y e r i n g the a g g l u t i n a t e d c e l l m i x t u r e on f e t a l c a l f serum (20%) whereupon t h e c e l l clumps s e t t l e d a t t h e bottom o f t h e tube and t h e s i n g l e , u n a g g l u t i n a t e d c e l l s remained a t the t o p . The c e l l f r a c t i o n s were then c o l l e c t e d s e p a r a t e l y , suspended i n a s o l u t i o n o f D - g a l a c t o s e t o d i s s o c i a t e t h e clumps and remove t h e l e c t i n , and washed s e v e r a l times i n p h o s p h a t e - b u f f e r e d s a l i n e . E x a m i n a t i o n o f t h e s e p a r a t e d c e l l s , which were o b t a i n e d i n good y i e l d (up t o 80 p e r c e n t ) , showed t h a t they were f u l l y v i a b l e (>95 p e r c e n t i n each f r a c t i o n ) . In a l l t h e p r o p e r t i e s t e s t e d ( s u r f a c e markers, response t o mitogens and immunological a c t i v i t i e s ) , t h e c e l l s a g g l u t i n a t e d by peanut a g g l u t i n i n were e s s e n t i a l l y i d e n t i c a l w i t h t h e i m m u n o l o g i c a l l y immature c e l l s , whereas t h e n o n a g g l u t i n a t e d f r a c t i o n c o n s i s t e d o f

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c e l l s which were s i m i l a r t o t h e h y d r o c o r t i s o n e - r e s i s t ant mature thymocytes as w e l l as t o s p l e e n Τ c e l l s (3J5,36) . T h i s method has now been used s u c c e s s f u l l y in several other laboratories. F u r t h e r experiments have l e d t o t h e development o f a s i m i l a r method f o r t h e s e p a r a t i o n o f mouse s p l e e n Τ and Β c e l l s , a l t h o u g h i n t h i s case peanut a g g l u t i n i n c o u l d n o t be used s i n c e n e i t h e r lymphocyte subpopula­ t i o n b i n d s t h e l e c t i n , n o r a r e they a g g l u t i n a t e d by i t . I t was found, however, t h a t t h e Β and Τ c e l l s d i f f e r i n t h e i r a b i l i t y t o b i n d soybean a g g l u t i n i n : the Β c e l l s were s t a i n e d by f l u o r e s c e i n - l a b e l e d soybean a g g l u t i n i n , whereas t h e Τ c e l l s were not. The s p l e e n lymphocytes c o u l d be r e a d i l y s e p a r a t e d by soybean a g g l u t i n i n , which a g g l u t i n a t e agglutinate the Τ c e l l On t h e b a s i s o f t h e r e s u l t s on t h e b i n d i n g o f peanut a g g l u t i n i n and soybean a g g l u t i n i n t o t h e d i f ­ f e r e n t lymphocyte s u b p o p u l a t i o n s (both b e f o r e and a f t e r n e u r a m i n i d a s e t r e a t m e n t ) , we p o s t u l a t e t h a t t h e s e c e l l s c a r r y D - g a l a c t o s e r e s i d u e s ( r e c e p t o r s f o r peanut a g g l u t i n i n and p o s s i b l y a l s o f o r soybean a g g l u t i n i n ) and N - a c e t y l - g - g a l a c t o s a m i n e ( r e c e p t o r s f o r soybean a g g l u t i n i n ) , which a r e p a r t i a l l y o r f u l l y s i a l y l a t e d ; the e x t e n t o f s i a l y l a t i o n i n c r e a s e s w i t h c e l l d i f f e r ­ e n t i a t i o n and m a t u r a t i o n . Moreover, i t i s v e r y l i k e l y t h a t peanut a g g l u t i n i n , which r e a c t s p r e f e r e n t i a l l y w i t h e a r l y , p r i m i t i v e c e l l s , may s e r v e as a marker f o r the r e c o g n i t i o n and i d e n t i f i c a t i o n o f such c e l l s . Indeed, peanut a g g l u t i n i n was found t o i n t e r a c t w i t h embryonal carcinoma c e l l s b u t n o t w i t h t h e i r d i f f e r ­ entiated derivatives; separation of undifferentiated c e l l s from t h e d i f f e r e n t i a t e d ones, by s e l e c t i v e a g g l u t i n a t i o n o f t h e former w i t h peanut a g g l u t i n i n , was a c h i e v e d (3J3) . Another i m p o r t a n t assumption from t h e l e c t i n b i n d i n g s t u d i e s was t h a t h e m o p o i e t i c stem c e l l s i n t h e mouse may c a r r y r e c e p t o r s f o r b o t h peanut a g g l u t i n i n and soybean a g g l u t i n i n . Evidence i n support o f t h i s assumption was o b t a i n e d when, w i t h t h e a i d o f peanut a g g l u t i n i n and soybean a g g l u t i n i n a c e l l f r a c t i o n was i s o l a t e d from mouse bone marrow and s p l e e n , which was e n r i c h e d w i t h h e m o p o i e t i c stem c e l l s and d e p l e t e d o f "graft-versus-host" a c t i v i t y . F u r t h e r m o r e , such a c e l l f r a c t i o n c o u l d be used f o r s u c c e s s f u l r e c o n s t r u c t i o n o f i r r a d i a t e d a l l o g e n e i c mice {39) .

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

CARBOHYDRATE-PROTEIN INTERACTION

10 Acknowledgments

Studies from the authors' laboratory were sup­ ported in part by grants from the Leukemia Research Foundation (Chicago, I l l i n o i s ) , from the Foundation for the Advancement of Mankind (Jerusalem) and by a contribution from a friend of the Weizmann Institute in Argentina. Nathan Sharon is an Established Investigator of the Chief Scientist's Bureau, Israel Ministry of Health. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Sharon, Ν . , Scient. American (1974) 230 (5), 78. Sharon, Ν . , "Comple Chemistry, Biosynthesi Addison-Wesley, Reading, Mass., 1975. Hughes, R. C . , "Membrane Glycoproteins", 367 pp., Butterworths, London and Boston, 1976. Sharon, Ν . , Proceedings of the F i r s t Congress of the Federation of Asian and Oceanic Biochemists, Nagoya, Japan, Oct. 1977 (in press). Flowers, H. M. and Sharon, Ν. , Advan. Enzymol. (in press) 48. Sharon, Ν. and L i s , Η., Science (1972) 177, 949. L i s , H. and Sharon, Ν . , In "The Antigens" (ed. Sela, M . ) , pp. 429, Academic Press. Vol. IV, 1977. Sharon, Ν . , Scient. American (1977) 236 (6), 108. Goldstein, I. J. and Hayes, C. Ε., Advan. Carbohyd. Chem. Biochem. (1978) 35, 127. Glaudemans, C. P. T., Advan. Carbohyd. Chem. Biochem. (1975) 31, 313. Fishman, P. H. and Brady, R. O., Science (1976) 194, 906. Kohn, L., In "Receptors and Recognition" (ed. Cuatrecasas, P. and Greaves, M. F.), p. 134, Chapman and Hall, London, Series A, Vol. 5, 1978. L i s , H. and Sharon, N . , J. B i o l . Chem. (1978) 253, 3468. Misaki, A. and Goldstein, I. J., J. B i o l . Chem. (1977) 252, 6995. Sharon, Ν . , Proceedings of the Fourth Interna­ tional Symposium on Glycoconjugates, Academic Press (in press). L i s , H., Sela, Β. Α . , Sachs, L., and Sharon, Ν . , Biochim. Biophys. Acta (1970) 211, 582. Pereira, Μ. Ε. Α . , Kabat, Ε. Α . , and Sharon, Ν . , Carb. Res. (1974) 37, 89. Hammarström, S., Murphy, L . Α . , Goldstein, I. J., and Etzler, M. E., Biochemistry (1977) 16, 2750.

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

1. sharon et al. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

Interaction of Lectins with Saccharides

11

Lotan, R., Skutelsky, E., Danon, D . , and Sharon, N . , J. B i o l . Chem. (1975) 250, 8518. Pereira, Μ. Ε. Α . , Kabat, Ε. Α . , Lotan, R., and Sharon, Ν . , Carb. Res. (1976) 51, 107. Carter, W. G. and Sharon, Ν . , Arch. Biochem. Biophys. (1977) 180, 570. Skutelsky, Ε . , Lotan, R., Sharon, Ν . , and Danon, D., Biochim. Biophys. Acta (1977) 467, 165. Glockner, W. Μ., Newman, R. Α . , and Uhlenbruck, G . , Biochem. Biophys. Res. Commun. (1975) 66, 701. Lotan, R., Siegelman, H. W., L i s , Η . , and Sharon, N . , J. B i o l . Chem. (1974) 249, 1219. Prujansky, Α . , L i s , Η . , and Sharon, Ν . , Unpub­ lished results Novogrodsky, A Acad. S c i . U.S.A. (1973) 70, 2515. Schechter, Β . , L i s , Η . , Lotan, R., Novogrodsky, Α . , and Sharon, Ν . , Eur. J. Immunol. (1976) 6, 145. Lotan, R., L i s , Η . , and Sharon, N . , Biochem. Biophys. Res. Commun. (1975) 62, 144. Lotan, R. and Sharon, N . , In " P r o t e i n Cross­ -linking" (ed. Friedman, M.), Part A, p. 149, Plenum. Novogrodsky, Α . , Lotan, R., Ravid, Α . , and Sharon, N . , J. Immunol. (1975) 115, 1243. Prujansky, Α . , Ravid, Α . , and Sharon, Ν . , Biochim. Biophys. Acta (1978) 508, 137. Ruddon, R. W., Weisenthal, L . M . , Lundeen, D. Ε . , Bessler, W., and Goldstein, I. J., Proc. Natl. Acad. S c i . U.S.A. (1974) 71, 1848. Bessler, W., Resch, Κ., and Ferber, E., Biochem. Biophys. Res. Commun. (1976) 69, 578. Maino, V. C . , Green, Ν. Μ., and Crumpton, M. J., Nature (1974) 251, 324. Reisner, Υ . , Linker-Israeli, Μ., and Sharon, Ν . , Cellular Immunol. (1976) 25, 129. Umiel, T . , Linker-Israeli, Μ., Itzchaki, Μ., Trainin, Ν . , Reisner, Υ . , and Sharon, Ν . , Cellular Immunol. (1978) 37, 134. Reisner, Υ . , Ravid, Α . , and Sharon, Ν . , Biochem. Biophys. Res. Commun. (1976) 72, 1585. Reisner, Y . , Gachelin, G . , Dubois, P . , Nicolas, J.-F., Sharon, Ν . , and Jacob, F., Developmental B i o l . (1977) 61, 20. Reisner, Y . , Itzicovitch, L., Meshorer, Α . , and Sharon, Ν . , Proc. Natl. Acad. S c i . U.S.A. (1978) (June issue, in press).

RECEIVED September 8, 1978.

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

2

T h e Carbohydrate B i n d i n g Site of Concanavalin A

KARL D. HARDMAN IBM Thomas J. Watson Research Center, Yorktown Heights, NY 10598

Lectins, the class organisms, have been studie commonly quoted for this activity is Stillmark(1) who described the hemagglutinating activity of plant extracts.* Additionally, he later found that Ricinus communis (castor beans) contain a toxic protein,ricin,which likewise agglutinates red blood cells. Such proteins have since been of interest to immunologists and more recently to cell biologists for their carbohydrate-binding activity. They provide a variety of useful functions. In fact, the most frequently studied lectin, concanavalin A (Con A)†, is the subject of a book entitled "Concanavalin A as a Tool"(3). Other discussions of the structure and carbohydrate-binding activity of Con A have recently appeared(4,5). The proteins from the jack bean (Canavala ensiformis) were first studied over 60 years ago by Jones and Johns(6). Several years later, Sumner(7), while studying urease (also from the jack bean), isolated three other proteins, two of which could be crystallized, concanavalin A and B. It should be noted that this report of crystalline Con A by Sumner appeared about seven years before he reported the first crystallization of an enzyme, urease(8). Con A was identified as the hemagglutinin from aqueous extracts of the jack bean by Sumner and Ho well (9). Subsequently, the specificities of various plant agglutinins were shown by Boyd and Reguera(lO) when they demonstrated that certain seeds contained agglutinins that would react only with selected blood group antigens. Con A binds human blood groups very poorly; however, it strongly binds polysaccharides and glycoproteins containing glycosyl residues of the D-arabino configuration(Π). The monosaccharide which binds most strongly is aMeMan whereas crMeGlc, the C2 epimer of aMeMan binds 1/4 as strongly and galactose, the C4 epimer of glucose, does not bind(ll). * For additional references of early work on lectins see Lis and Sharon (ref. 2). t Abbreviations: Con A, concanavalin A (which refers to the protein with a full complement of manganese and calcium , i.e., the holoprotein); aMeMan, methyl «t-D-mannopyranoside; «MeGlc, methyl α-D-glucopyranoside; /SIphGlc, /3-(0-iodophenyl) D-glucopyranoside; /HphGal, /3-(0-iodophenyl) D-galactopyranoside; apo-Con A, metal-free concanavalin A. 0-8412-0466-7/79/47-088-012$05.00/0 © 1979 American Chemical Society

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

2. HARDMAN

Binding Site of Concanavalin A

Due to the vast amount of recent research on lectins, it is quite apparent that proteins which bind to specific cell-surface glycoproteins are involved in modulation of a variety of mitotic and metabolic events within the cell. It is hoped that structural studies of these proteins and their receptors will prove to be informative in determining the mechanisms of these and other such events. In this presentation, I would like to discuss the structural features of Con A, such as the β-sheets, subunit structure, the manganese, calcium and carbohydrate binding sites and close, by mentioning some recent advances in crystallography which are relevant to the studies of proteins and how these should effect such future re­ search. The Three-Dimensional Structure Subunit Composition. The earliest report of molecular weight determina­ tion for Con A was by Sumne of 98,000 g/mole at pH More recent studies have given values between 50 and 120 K(13-15). Experi­ ments below pH 6 gave values near 50 K, whereas data obtained nearer 7 and above gave higher values. The explanation of these disparate results was clear upon the examination of the first low-resolution electron density maps of crystal­ line Con A(16). In crystals of native Con A, pH 6.0, space group 1222, the molecules are packed as four identical subunits of 26 Κ daltons each to form pseudotetrahedral clusters of 104 Κ daltons. However, in solutions below pH 6, these molecules dissociate into dimers. More details of the dimer and tetrameric structures are discussed later. In the native crystals, one asymmetric unit consists of one protomer (chemically identical subunit) which occupies 50% of the volume, the rest consists of solvent. The same subunit structure is found in Con A crystals of the Con A-aMeMan complex at pH 7.4(4), even though in the latter case the space group is different and the tetramers are packed quite differently with respect to their neighbors. The carbohydrate-specific site has been identified in this complex(4) and is discussed later. Although the explanation is not known, preparations of Con A contain fragmented as well as intact polypeptide chains(17). As many as three polypep­ tide chains can be isolated, the intact chain of 237 amino acids (molecular weight 26,500), plus two fragments which result from splitting the intact chain between residues 118 and 119(18). Most preparations contain as much as 30% split chains unless special care is taken during preparation. Crystallographic compari­ son of crystals of purified intact chains and those containing some fragments show no apparent differences in their electron density maps(^9). The amino acid sequence has been reported(20) and notable features are that the 40 amino acids at the N-terminus contain a high content of polar side chains whereas the C terminal half contains an usually high percentage of nonpolar residues, which results in a very low solubility of many proteolytic peptides. The effects of this distribution of amino acid types is quite apparent on examination of the 3dimensional structure of the monomer. The crystallographic structure has been detennined(lj),2p and complete atomic coordinates for both studies can be obtained from the Protein Data Bank at Brookhaven National Laboratory. Monomer. The polypeptide chain of the monomer is outlined in Figures 1, 2A and 2B by vectors linking the α-carbon positions of all 237 amino acids.

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

13

14

CARBOHYDRATE-PROTEIN INTERACTION

Figure 1. a-Carbon stereogram of the Con A monomer. Positions marked are for the manganese (MN), calcium (CAL), carbohydrate binding site (CHO), nonpolar binding site (NP), and a number of amino acids involved with binding the metal ions and the carbohydrate. The standard single-letter code for amino acids is used, β-sheet I is "behind" the rest of the monomer, while β-sheet II is curved through the center and β-sheet III, which is much smaller, is to the upper-right and connects the first two.

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

HARDMAN

Binding Site of Concanavalin A

Figure 2A. Another orientation of the monomer, which includes some of the side chain atoms in the Mn \ Ca \ and carbohydrate binding sites, β-sheet I is to the left (including His 127), β-sheet II is through the center (almost perpendicular to the plane of the paper), and β-sheet III is to the lower-left. 2

Figure 2B.

2

Same as 2A, except every 15th residue is labelled for reference

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CARBOHYDRATE—PROTEIN INTERACTION

Also shown are the sites for M n , C a , the nonpolar binding site and the carbohydrate-specific site, each occurring once per monomer (discussed later). The most unusual feature of the Con A structure is the arrangement of three j8-structure regions in the monomer. The chain folds into a single domain where 50-60% of the amino acids are involved in one of the three β-sheet regions. A high percentage of /3-structure was first predicted by Kay(22) from optical rotato­ ry dispersion and circular dichroism studies. Very few other polypeptide chains longer than Con A fold up into a single domain and these (for example, carbonic anhydrase(23) and carboxypeptidase(24)) also have large amounts of /3-structure (10 or 8 strands, respectively) twisting through the center of the molecule. In Con A , the most prominent /3-sheet (sheet I) contains 54 amino acids and continues across a crystallographic 2-fold rotation axis, into the second monomer, (Figure 3) forming a continuous 12-strand, flat /3-structure "waU (21). This structural "wall" forms a large outer surface for the dimer and provides the contacts for the tetramer formatio II, contains 7 strands whic subunit. The third region, /3-sheet III, is the smallest of the three and is folded across the outer end of the monomer and serves basically as a connection between sheets I and II. This can be seen most readily in Figures 2A and 2B. Sheet I is fairly flat but with a slight twist which is commonly found in other proteins with /3-sheets. However, sheet II appears to be quite unique. The upper-inside region of this semicylinder contains polar side chains involved in binding C a and M n , near the carbohydrate side, whereas the lower region contains nonpolar side chains exclusively, which interact with the C-terminal region through numer­ ous van der Waals contacts. The side chains on the "outer" wall of the cylindrical portion are all nonpolar as well and interact with the nonpolar residues of the "inside" of /3-sheet I. Details of the hydrogen bonding patterns of these /3-structure regions can be found else where (25,26). Con A contains no standard α-helices, however, there is one loop which contains one or two hydrogen bonds of the α-helical type (residues 80-85), but the carbonyl oxygens appear to tip outward forming bifurcated hydrogen bonds to solvent water molecules. Also, it should be noted that the first 40 amino acids, which have a large percentage of polar side chains, contribute all the atoms which bind directly to the M n and C a ions. This includes the loop which folds around this double ion site (amino acids 10-23) and, in the absence of these ions, would be completely solvated. Dimer. The two monomers with the most contacts are related across the crystallographic x-axis to form the continuous 12-strand β-sheet (mentioned earlier) and are shown in Figure 3. The intersubunit contacts involve an area of approximately 40 χ 25 Â. There are approximately 250 atomic contacts (atoms closer than about 4 Â) of which about 14 are hydrogen bonds(26), the rest are van der Waals contacts. No contacts between this set of monomers would suggest a susceptability to pH changes in the 6 to 7 region. However, in contrast, the intersubunit interface between monomers around the y-axis is larger, about 40 χ 40 Â but contains fewer contacts (about 150) and is susceptible to pH changes (see below). Furthermore, monomers around the z-axis have almost no contacts. Across the x-axis (normal to the plane of the paper, through the center of the dimer, Figure 3), 8 amino acids, 124 through 131, form hydrogen bonds to the equivalent strand of the second monomer, by necessity, in an anti-parallel mode (see discussion of the tetramer). A similar feature had been seen earlier in the 2 +

2 +

,f

2 +

2 +

2 +

2 +

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

HARDMAN

Binding Site of Concanavalin A

Figure 3. a-Carbon stereogram of the aimer

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

18

CARBOHYDRATE-PROTEIN INTERACTION

structure of insulin(27) where two Β chains in the dimer hydrogen-bond across a local 2-fold axis, although in this case there are no additional strands. These similar features were first noted by Dorothy Hodgkin and, since Con A had been observed to mimic the effects of insulin on fat cells(28), the question was raised as to whether these similar structural features were involved with binding to cell surfaces. However, this feature has since appeared more frequently in other unrelated structures, and this correlation seems unlikely. Tetramer. The center of the tetramer is the intersection of 3 unique 2-fold rotation axes (point group 222). At this point, the 12 strand β-sheets of two dimers interact as mentioned previously (Figure 4). For the dimer these side chains must be on the surface; however, in the tetramer, they must point inward(25). This mode of association does not appear to be simply a function of lattice packing, because in crystals of the Con A-aMeMan complex (space group C222j), three different tetramers are found and all have this same orientation(4). The amino acid side chains of -sheet I which project to the inside of the monom­ er are entirely hydrophobic (toward the center of 222 symmetry) are mostly polar. Ten of these per monomer are serine and threonine, while histidine, asparagine, and alanine each contribute two. His 127 is the residue of each monomer nearest the point of 222 symmetry, thus producing a cluster of four imidazole side chains which play a role in the dimer-tetramer association. The apparent mid-point of this transition (dissociation around the y-axis), judged by the data of Pflumm and Beychok(29), is about pH 6.5. Lowering the pH from above pH 7 to below 6 would result in protonation of the imidazole groups and charge repulsion would occur. Also His 51, which interacts with Lys 116 of the second monomer, would affect the dimer-tetramer transition in this pH range. It is also apparent that the center of the tetramer is accessible to solvent molecules even in the crystals, since His 127 and Met 129 bind a number of heavy-atom derivatives without disruption of the crystalline lattice(21,30). The Carbohydrate Binding Site. It had been shown that jSIphGlc binds to a site referred to here as the nonpolar binding cavity; Figure 1(M). Since this compound inhibits dextran precipitation by Con A(32), it was assumed to identify the carbohydrate binding site. However, it was then shown that 0IphGal and many other nonpolar molecules which do not inhibit dextran precipitation also bind to this same site(33). Therefore, the binding of /HphGlc in this cavity is due to the iodophenyl moiety and not to the glucosyl residue(33). Diffusing carbohydrates which specifically bind to Con A , such as aMeMan, aMeGlc or D-glucose, into native crystals (1222) causes disruption of the lattice and the crystals dissolve. Therefore, direct identification of the carboh­ ydrate binding site in native crystals is not possible. However, the first suggestion of the correct site was obtained by first covalently crosslinking the crystals with glutaraldehyde and subsequently diffusing in sugars(25). The peaks in the electron density maps were weak since full occupancy could not be obtained and small peaks elsewhere on the surface of the molecule appeared also. Higher concentration of sugars disturbed the lattice of even the crosslinked crystals so these experiments were not considered conclusive. Subsequently, however, the carbohydrate-specific binding site has been identified in crystals grown from the Con A-aMeMan complex(4). Growth of these crystals (space group C222j) was preceded by incubation of Con A with a large excess of aMeMan. The three-dimensional structure was solved at 6 À

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

2.

HARDMAN

Figure 4.

Binding Site of Concanavalin A

19

a-Carbon stereogram of the four β-sheet I sections comprising the tetramer

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

CARBOHYDRATE-PROTEIN INTERACTION

20

resolution and the binding site identified by replacing aMeMan by two iodo sugar derivatives, /HphGlc and methyl 2-deoxy-2 iodoacetamido-a-D-glucopyranoside, in different experiments. The atomic coordinates of native Con A were then fit into this low resolution map and the amino acids around the carbohydrate position identified. They are Tyr 12 and 100, Asp 16 and 208, Asn 14, Leu 99, Ser 168 and Arg 228, Figures 5 and 6. This region has a large number of van der Waals contacts and hydrogen bonds to the side chains of Asn 82, Asp 83, Ser 184 and 185 of the neighboring molecule in the 1222 lattice. The destruction of the native crystals by the addition of carbohydrate(4) is thus easily explained since there is not sufficient space for a monosaccharide to bind without notably altering these contacts. Additionally, Becker et Û/.(34) have recently continued studying the carbohydrate binding to cross-linked 1222 crystals. They diffused methyl 2-deoxy-2-iodo-a-D-mannopyranoside into the modified crystals and collected data to 3.5 Â. The site they identified as the iodine position was in the same region as mentioned above. Details of the M n Figures 5 and 6. The orientation of these figures is the same as in Figure 1. The Mn and C a are about 4.5 Â apart and form what may be considered to be a double site, since Asp 10 and 19 contribute both carboxyl oxygens, one each as ligands to the M n and C a . They are about 12 and 7 À from the carbohydrate binding site, respectively. A strand of /3-sheet II supplies carboxyls from Glu 8 and Asp 10 and the carbonyl oxygen of Tyr 12 to the M n and C a . The chain then loops out around these ions forming the outer surface of the subunit, from residues 12 to 23, donating Asn 14 and Asp 19 as ligands, then continues back into /3-sheet II adding His 24 and Ser 34 (Figure 7). His 24 contributes ligands NE1 to M n and a water molecule appears to bind to both Ser 34 and M n . The sixth coordinate position of the M n appears as a weak region of electron density and must be water of partial occupancy. Therefore, this position is interpreted as the site for the rapidly exchanging water bound to M n found by proton magnetic relaxation dispersion experiments(35). The water between Ser 34 and M n would not be expected to exchange as rapidly and would not have the same symmetry. The distance from the carbohydrate site to the M n in the crystal is in good agreement with several NMR studies(36^38). These values vary between 10 and 14 Â. From NMR experiments with C-enriched α and β anomers of methyl D-glucopyranoside, Brewer et a I.(36) calculated average carbon-to-Mn distances for the six non-methyl carbons to be 10.3 and 11 A, respectively. Similarly, Villafranca and Viola(37) reported an average of 10 À for the nonmethyl carbons of methyl α-D-glucopyranoside and 13.8 Â for the aglycone methyl carbon from natural abundance C - N M R results. Alter and Magnuson(38) reported average F - Mn distances of 12.1 and 14.0 Â for the α and β anomers of N trifluoroacetyl-D-glucosamine at two pH values, 5.1 and 7.0, where Con A is predominantly dimeric and tetrameric, respectively. These results additionally suggest no major differences in carbohydrate binding occur between the dimeric and tetrameric forms(38). Numerous aryl-pyranosides have been shown to bind more strongly to Con A than their alkyl analogs(32,39,40). Also, acetylation studies with N acetylimidazole in the presence and absence of carbohydrate have implicated the involvement of tyrosine residues(41). It is quite clear from the three-dimensional structure that Tyr 12 and 100, which are found in this region and exposed to 2 +

2 +

2 +

2 +

2 +

2 +

2 +

2 +

2 +

2 +

2 +

2 +

2 +

l3

l 5

1 9

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

2.

HARDMAN

Binding Site of Concanavalin A

21

Figure 5. The metal ions Mn and Ca * and their ligands, near the carbohydrate binding site (CHO). (Figures 5-8 have same orientation as Figure 1.) 2+

2

Figure 6. Stereogram of the metal ions and carbohydrate binding regions. The backbone atoms from Glu 8 to Asp 19 and from Ser 203 to Ala 11 plus designated side chains. The cis peptide bond is between Ala 207 and Asp 208, producing a distinct "kink" in this strand of β-sheet.

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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CARBOHYDRATE-PROTEIN INTERACTION

solvent, could easily account for these increased affinities by a network of ir-ir interactions. Chemical modification experiments and hydrogen ion titration studies(42) suggested that two carboxyl groups per monomer are involved in carbohydrate binding. Glu 8, Asp 10 and 19 would appear to remain involved with binding Mn and C a and should not be available for reaction.' However, Asp 208, which has a water molecule bound between its carboxyl and the C a , is placed suitably for direct interaction with the carbohydrate. Additionally, Asp 16 is located close enough for direct interaction. Although the location of the binding site has been identified, details of the interaction between the carbohydrate and Con A at atomic resolution are not yet available. NMR results(36,37) have suggested that the plane of the pyranose ring is nearly normal to a line drawn from the carbohydrate to the M n and that C-6 is closer to M n than C - l . The low resolution map of the Con A-aMeMan complex did not allow the identification or orientation of the ring in the site; however, the maximum differenc suggest that the C - l carbon extends outward from the protein surface, further from the M n and C a than the rest of the sugar molecule(4). Additionally, the involvement of the M n and C a with carbohydrate binding and the stability of various conformations of the polypeptide chain is evolving as a quite complex situation. This is being studied quite extensively with various NMR techniques and is the subject of another article in this volume(43) as well as elsewhere (44). The peptide bond between Ala-207 and Asp 208 has been placed in the cis configuration, Figures 6 and 7, in order to obtain a reasonable fit of the polypeptide chain in this region(25). With this exception and perhaps one or two others, only cis peptides involving proline have been found in proteins to date; however, there appears to be no theoretical reason why they cannot occur elsewhere(45). (Proline occurs at 206, one residue before this peptide bond.) Adjacent to this region is Glu 102, which appears to be the only charged side chain on the "interior" of the monomer. These features appear to destabilize the structure in this region and are prime candidates for involvement in conformation­ al changes which occur upon binding carbohydrate(5). It is clear that higher resolution studies of Con Α-carbohydrate complexes must be completed to address properly these and similar structural questions. Di-, tri-, and tetra-saccharides containing a-D-(l-*2)-mannosidic linkages have been found to bind much more strongly to Con A than corresponding monosaccharides. If there exists a second pyranosyl binding site adjacent to the first, examination of the three-dimensional model indicates the most likely region involved would be two sections of the polypeptide chain from 97 to 103 and 202 to 208 (Figure 8). These sections include three carboxylic acid side chains, three hydroxyls, two prolines, one lysine, and one histidine. More details of the sugar complexes must await further analyses. The Con A carbohydrate binding site is quite clearly different from that found in lysozyme, which is a long cleft in the molecule containing up to six subsites for hexopyranose residues(46). The site in Con A is much smaller, perhaps only involving one sugar residue and is not a "groove" but only a shallow indentation in the surface where the pyranose ring lies parallel or almost parallel to the protein surface. Lysozyme has a number of tryptophan side chains in­ volved in binding carbohydrate and in stabilizing the structure around these sites, 2+

2 +

2 +

2 +

2 +

2 +

2 +

2 +

2 +

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

2.

HARDMAN

Binding Site of Concanavalin A

23

Figure 7. The polypeptide backbone from Lys 200 (top-right) to The 213. The carbohydrate binding position is represented by the stick drawing of aMeMan. The cis peptide bond is the first peptide bond to the left of Pro 206 (see Figure 6 for the residue numbers).

Figure 8. Most probable site for the binding of a second pyranoside ring of a polysaccharide (indicated by aMeMan), adjacent to the principal site (CHO) (see text).

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

CARBOHYDRATE—PROTEIN INTERACTION

24

whereas Con A has none. Lysozyme contains very little 0-structure and Con A has no α-helix and no disulfide crosslinks. Thus, there are no obvious underlying similarities in the construction of the carbohydrate binding regions, only that the binding involves carboxylic acid side chains and carbonyl oxygens. The precipitation of large polysaccharides by Con A was first proposed to be analogous to antibody-antigen reactions by Sumner and Howell(9). Since the determination of the three-dimensional structures of Con A and various immuno­ globulin molecules and fragments (for a list of references, see ref. 47), structural interests in the lectins has shifted to their specific reactions with carbohydrates with respect to cell surface phenomena, metabolic modulation and cell differenti­ ation. However, there are structural features common to both molecules which are the criss-crossed pairs of /3-sheets(21_,48), which have also recently been observed in other moleucles(49). In Con A this occurs at two levels: between the 12-strand sheets between dimers and between sheets I and II (Figures 1 and 4). In Con A , prealbumin(50) and the immunoglobulins, binding regions of two classes are formed by thes hapten binding region of immunoglobulin fragments and the center of the Con A tetramer, which bindings a number of heavy-atom molecules; and the second, a convex cavity, such as the nonpolar binding cavity in Con A and the thyroxine binding cavity in prealbumin. Recent Advances In Crystallography. In closing, I would like to emphasize three areas of advancement in the field which will soon routinely increase the speed of determining structures of large molecules and greatly improve the resolution and accuracy of the final model. These are: first, the adaptation of two-dimensional detectors for diffrac­ tion experiments(51_); second, the successful use of cryogenic temperatures for crystallographic enzyme-substrate complexes(52), and third, the development of very fast computational algorithms for refining atomic coordinates, i.e., optimizing large molecular models such as proteins and nucleic acids to crystallographic data(53). It is expected that such advances will significantly advance studies on the correlation of macromolecular structure and biological function. ABSTRACT The carbohydrate binding site of concanavalin A has been identified in crystals of the concanavalin A — methyl-α-D-mannopyranoside complex (Hardman and Ainsworth (1976), Biochemistry 15; 1120) and is 35 Åfrom the iodophenyl binding site, which had been postulated to be contiguous with the carbohydrate-specific site (Edelman et al. (1972), Proc. Natl. Acad. Sci. U. S. A. 69; 2580 and Becker et al. (1975), J. Biol. Chem. 250; 1513). This resolves the disparity between previous interpretations of crystal data and nuclear magnetic resonance data in solution (Brewer, et al. (1973), Biochemistry 12; 4448). There appear to be no profound conformational changes between the two states. These differences resulted because, in the native crystalline form, o-iodophenyl β-D-glucopyranoside binds as a result of interactions with the iodophenyl moiety, as was shown by the finding that o-iodophenyl β-D-galactopyranoside binds to the identical site (Hardman and Ainsworth (1973), Biochemistry 12; 4442). The carbohydrate-specific site is near Tyr 12 and 100, and Asp 16 and 208, a region about 12-14 Åfrom the manganese ion, in good agreement with NMR studies.

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

2. hardman

Binding Site of Concanavalin A

LITERATURE CITED 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

Stillmark, H., Inaugural Dissertation, Dorpat., 1888. Lis, H., and Sharon, N., Annu. Rev. Biochem. (1973) 42; 541. Bittiger, H., and Schnebli, H. P., "Concanavalin A As A Tool", Wiley, New York, New York, 1976. Hardman, K. D., and Ainsworth, C. F., Biochemistry (1976)15;1120. Hardman, K. D., and Goldstein, I. J., in Immunochemistry of Proteins, Vol. 2, Atassi, M. Z., ed., pp. 373-416, Plenum Press, New York, New York, 1977. Jones, D. B., and Johns, C. O.,J.Biol. Chem. (1916) 28; 67. Sumner, J. B., J. Biol. Chem. (1919) 37; 137. Sumner, J. B., J. Biol. Chem. (1926) 69; 435. Sumner, J. B., and Howell, S. F.,J.Bacteriol. (1936) 32; 227. Boyd, W. C., and Reguera Goldstein, I. J., Hollermann 4; 876. Sumner, J. B., Gralen, N., and Eriksson-Quensel, I. B., J. Biol. Chem. (1938) 125; 45. Olson, M. O. J., and Leiner, I. E., Biochemistry (1967) 6; 3801. Agrawal, B. B. L., and Goldstein, I. J., Biochim. Biophys. Acta (1967) 147; 262. Kalb, A. J., and Lustig, Α., Biochim. Biophys. Acta (1968) 168; 366. Hardman, K. D., Wood, M. K., Schiffer, M., Edmundson, A. B., and Ainsworth, C. F., Proc. Nat. Acad. Sci. U. S. A. (1971) 68; 1393. Wang, J. L., Cunningham, Β. Α., and Edelman, G. M., Proc. Nat. Acad. Sci. U. S. A. (1971) 68; 1130. Wang, J. L., Cunningham, Β. Α., Waxdal, M. J., and Edelman, G. M., J. Biol. Chem. (1975) 250; 1490. Edelman, G. M., Cunningham, Β. Α., Reeke, G. N., Jr., Becker, J. W., Waxdal, M. J., and Wang, J. L., Proc. Nat. Acad. Sci. U. S. A. (1972) 69; 2580. Cunningham, Β. Α., Wang, J. L., Waxdal, M. J., and Edelman, G. M., J. Biol. Chem. (1975) 250; 1503. Hardman, K. D., and Ainsworth, C. F., Biochemistry (1972)11;4910. Kay, C., FEBS Lett. (1970) 9; 78. Kannan, Κ. K., Lijas, Α., Waara, I., Bergsten, P. C. Lörgren, S., Strandberg, B. Bengtsson, U., Carlbom, U., Fridborg, K., Jarup, L., and Petef, M., Cold Spring Harbor Symp. Quant. Biol. (1971) 36; 221. Lipscomb, W. N., Hartsuck, J. A. Reeke, G. N., Jr., Quiocho, F. Α., Bethge, P. H., Ludwig, M. L., Steitz, Τ. Α., Muirhead, H., and Coppola, J. C., Brookhaven Symp. Biol. (1968) 21; 24. Hardman, K. D., Adv. Exp. Med. Biol. (1973) 40; 103. Reeke, G. N., Jr., Becker, J. W., and Edelman, G. M., J. Biol. Chem. (1975) 250; 1525. Blundell, T., Dodson, G., Hodgkin, D., and Mercola, D., Adv. Protein Chem. (1972) 26; 279. Cuatrecasas, P., and Tell, G. P. E., Proc. Nat. Acad. Sci. U. S. A. (1973) 70; 485. Pflumm, M. N., and Beychok, S., Biochemistry (1974) 13; 4982.

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

26 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53.

carboiiYDRATE—PROTEIN interaction Becker, J. W., Reeke, G. N., Jr., Wang, J. L., Cunningham, Β. Α., and Edelman, G. M., J. Biol. Chem. (1975) 250; 1513. Becker, J. W. Reeke, G. N., Jr., and Edelman, G. M., J. Biol. Chem. (1971) 246; 6123. Poretz, R. D., and Goldstein, I. J., Biochemistry (1970) 9; 2890. Hardman, K. D., and Ainsworth, C. F., Biochemistry (1973)12;4442. Becker, J. W., Reeke, G. N., Jr., Cunningham, Β. Α., and Edelman, G. M., Nature (London) (1976) 259; 406. Koenig, S. H., Brown, R. D., and Brewer, C. F., Proc. Nat. Acad. Sci. U. S. A. (1973) 70; 475. Brewer, C. F., Sternlicht, H., Marcus, D. M., and Grollman, A. P., Biochemistry (1973)12;4448. Villafranca, J. J., and Viola, R. E., Arch. Biochem. Biophys. (1974) 160; 465. Alter, G. M., and Magnuson J Α. Biochemistr (1974)13;4038 Loontiens, F. G., VanWauwe Carbohydr. Res. (1973) ; Bessler, W., Shafer, J. Α., and Goldstein, I. J., J. Biol. Chem. (1974) 249; 2819. Doyle, R. J., and Roholt, Ο. Α., Life Sci. (1968) 7; 841. Hassing, G. S. Goldstein, I. J., and Marini, M., Biochim. Biophys. Acta. (1971) 243; 90. Brewer, C. F., Koenig, S. H., and Brown, R. D., (1978) this volume, pp. Brown, R. D., Brewer C. F., and Koenig, S. H., Biochemistry (1977) 16; 3883. Ramachandran, G. N., and Mitra, A. K., J. Mol. Biol. (1976)107;85. Ford, L. O., Johnson, L. N., Machin, P. Α., Phillips, D. C., and Tjian, R., J. Mol. Biol. (1974) 88; 349. Davies, D. R., Padlan, Ε. Α., and Segal, D. M., Annu. Rev. Biochem. (1975) 44,; 639. Poljak, R. J., Amzel, L. M., Avey, H. P., Chen, B. L., Phizackerley, R. P., and Saul, F., Proc. Natl. Acad. Sci. U. S. A. (1973) 70; 3305. Richardson, J. S., Nature (1977) 268; 495. Blake, C. C. F., Geison, M. J., Swan, I. D. Α., Rerat, C., and Rerat, B., J. Mol. Biol. (1974) 88; 1. Charpak, G., Nature (1977) 270; 479. Petsko, G., and Tsernoglu, D., Am Crystallogr. Assn., National Meeting, Abstracts, (Feb.1977) Asilomar, California. Agarwal, R. C., Acta Crystallograph. (1978) in press.

RECEIVED September 8, 1978.

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

3 Binding of Mono- and Oligosaccharides to Concanavalin A as Studied by Solvent Proton Magnetic Relaxation Dispersion C. FRED BREWER Department of Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461 RODNEY D. BROWN, III IBM Thomas J. Watson Research Center, Yorktown Heights, NY 10598 Interest in the protein concanavalin A (Con A) , a lectin isolated from the jack bean (Canavalia ensiformis), derives from its unusual biological properties. In particular, its ability to bind to the surface of both normal and transformed cells has made it a powerful too -surface related biologica (1) Con A with cell-surface membranes is related to the saccharide binding properties of the protein. The saccharide binding specificity of Con A has been shown by Goldstein et a l . (2) to be directed toward the monosaccharides glucose and mannose, which contain similar hydroxyl group configurations at the C-3, 4 , and 6 positions. The protein binds the α-anomers of these glycosides more strongly than the β-anomers. Since cell surface carbohydrate determinants occur as oligosaccharides in the form of glycoproteins and glycolipids, it is important to understand the interaction of Con A with these larger complex molecules. Goldstein (3) has shown that there exist essentially two classes of oligosaccharides that bind to Con A. The first class of oligomers demonstrates no enhanced binding to the protein relative to monsaccharides; the second class shows enhanced binding. Included in the first class area-(l-*-3) , a-(l->4) , a-(l-H>) linked oligosaccharides which contain a non-reducing terminal glucose or mannose residue (2); in the second class are ct-( 1+2)-linked mannose oligomers (4JJ5) . Thea-(l-v2) linked trisaccharide of mannose, for example, has a 20-fold greater affinity constant than methyl a-D-mannopyranoside (4). The enhanced binding of such oligosaccharides has prompted speculation (4) that the carbohydrate combining site of Con A may bind more than one saccharide residue. Thus, Goldstein and others have suggested that the specificity of Con A binding to oligo- and polysaccharides may involve extended interactions of the protein with several carbohydrate residues. ' Abbreviations used: Con A for concanavalin A with unspecified metal content; Ca*-Mn-Con A for concanavalin A containing manganese at the Si site and calcium at the S2 site; Ca-Zn*-Con A for concanavalin A containing zinc at the Si site and calcium at the S2 site; a- and /9-MDG for methyl a- and /3-D-glucopyranoside; a-MDM for methyl a-r>mannopyranoside; /3-IPG for o-iodophenvl /3-D-glucopyranoside; βIPGal for o-iodophenyl /3-D-galactopyranoside; α-( 1-» 2;-mannobioside for O-a-Dmannopyranosyl-( 1 -» 2)-D-mannose; α-( 1 2 )-mannotrioside for O-a-D-mannopyranosyf-(l -* 2)-0-a-r>mannopyranosyl-( 1 -* 2)-r>mannose; NMR for nuclear mag­ netic resonance; and NMRD for nuclear magnetic relaxation dispersion. 0-8412-0466-7/79/47-088-027$05.00/0 © 1979 American Chemical Society 1

2

+ 2

+ 2

2

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

28

CARBOHYDRATE-PROTEIN

INTERACTION

In l i g h t o f the i n t e r e s t i n i s o l a t i n g s o - c a l l e d "Con A r e c e p t o r s " from the s u r f a c e o f a v a r i e t y o f c e l l s , i t i s of considerable importance t o determine the mode of i n t e r a c t i o n of not only simple monosaccharides but a l s o more complex o l i g o ­ saccharides w i t h Con A i n order t o e l u c i d a t e the complete saccharide b i n d i n g s p e c i f i c i t y of the p r o t e i n . The goal of t h i s paper i s to e x p l a i n the mode of i n t e r a c t i o n s of mono- and o l i g o ­ saccharides t o Con A. We report evidence that the carbohydrate combining s i t e o f Con A accommodates only one saccharide residue and t h a t the enhanced b i n d i n g of c e r t a i n o l i g o s a c c h a r i d e s can be explained by a s t a t i s t i c a l argument. M a t e r i a l s and Methods 2+ 2+ P r e p a r a t i o n o f Co obtained from Miles-Yeda p r e v i o u s l y described ( 6 ) . Atomic a b s o r p t i o n a n a l y s i s o f these two Con A preparations showed e s s e n t i a l l y equal amounts o f the t r a n s i t i o n metal i o n and calcium i o n s . Sample s o l u t i o n s (0.6 ml) contained Con A at the appropriate c o n c e n t r a t i o n i n pH 5.60, 0.1N potassium acetate b u f f e r , μ = 1.0 i n potassium c h l o r i d e . The f i n a l p r o t e i n c o n c e n t r a t i o n was determined s p e c t r o p h o t o m e t r i c a l l y u s i n g Α ^ · ^ = 12.4 at 280 nm (7>.8) · Saccharides. Thea-{1^2) mannose o l i g o s a c c h a r i d e s were g i f t s from Dr. I r w i n G o l d s t e i n . The s y n t h e s i s o f o-iodophenyl 3£-glucopyranoside (3-IPG) and ρ,-iodophenyl 3-£-galactopyranoside (3-IPGal) w i l l be reported elsewhere. The r e s t o f the saccharides used i n t h i s study were obtained from commercial sources. R e l a x a t i o n Measurements. Measurements of the magnetic f i e l d " dependence of the s o l v e n t water proton r e l a x a t i o n r a t e ( T j ~ * ) , i . e . , nuclear magnetic r e l a x a t i o n d i s p e r s i o n (NMRD), were made by the f i e l d c y c l i n g method p r e v i o u s l y described (9,10) . R e l a x a t i o n Theory. The theory of magnetic r e l a x a t i o n and the procedure used f o r o b t a i n i n g the r e l e v a n t parameters a r e found i n Koenig jet a l . ( 6 ) . 1

7

Results The NMRD o f solvent water protons i n s o l u t i o n s of C a -Mn -Con A, Ca -Zn +-Con A and f r e e M n i s shown i n F i g . 1. Tlpara""^' ^ P g e t i c c o n t r i b u t i o n of the bound Mn i o n of the p r o t e i n t o T j " , obtained by c o r r e c t i n g the observed NMRD o f the Ca -Mn - C o n A s o l u t i o n f o r the water background (T^ ~^) and the diamagnetic c o n t r i b u t i o n o f the p r o t e i n e x p e r i m e n t a l l y determined from the NMRD of C a - Z n - C o n A, i s shown i n F i g . 2, upper curve. When s u f f i ­ c i e n t α-MDG i s added t o s a t u r a t e the saccharide b i n d i n g s i t e s of Ca -Mn -Con A, Τ ^ ^ " i s reduced by approximately 25% 2

2+

2+

t

ie

2

2 +

a r a m a

n

2+

2+

2 +

1

w

2+

2+

2+

1

*We indicate magnetic field intensity in units of the Larmor precession of fre­ quency of protons in that magnetic field. The conversion is 4.26 kHz = 1 Oersted = 1 Gauss.

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

3.

BREWER

AND

Binding of Mono- and Oligosaccharides

BROWN

JO

MAGNETIC FIELD (Oersteds) 100 IK

29

OK

FREQUENCY (MHz)

Figure 1. The magneticfielddependence of the spin-lattice relaxation rate of solvent-water protons in solutions of: (top curve) Ca *-Mn *-Con A, 1.83 mM (monomer), 1.56 mM bound Mn ; (middle curve) free Mn \ 0.15 mM; (bottom curve) Mn *-Zn *-Con A, 1.83 mM. Thefieldindependent rate for pure water is indicated by W. All measurements were made at 25° in pH 5.60, 0.1 M potassium acetate buffer, μ = 1.0 in KCl. The solid curves are theoreticalfitsto data. 2

2+

2

2

2

2

Figure 2. The paramagnetic contribution of the bound Mn * to the Ca *-Mn *Con A dispersion shown in Figure 1: (top curve) in the absence of sugar; (bottom curve) in the presence of saturating (0.1 M) a-MDG. The solid lines are theoretical fits to the data. 2

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

2

2

CARBOHYDRATE-PROTEIN

30

INTERACTION

( F i g . 2, lower curve). The l i n e s a r e from f i t s t o NMRD theory ( 6 ) . The NMRD o f C a ^ - Z n ^ - C o n A i s uneffected by the a d d i t i o n of the saccharide. When α-MDG i s t i t r a t e d i n t o a s o l u t i o n o f Ca -Mn -Con A, the change i n i ~* given magnetic f i e l d r e f l e c t s the fraction F of Con A w i t h saccharide bound: s 2+

T

a

t

2+

a

p a r a

T

1

S

lpara

(

S

}

T

o " lpara -1 -1 Τ ( S ) - T, (S ) lpara ο lpara s

s

X

m

A

Ί

where T. ~ (S ) , T. "^Jand T " ( S ) a r e the T, ~ lpara ο * lpara r lpara s lpara value i n the absence of α-MDG, i n the presence of a given t o t a l c o n c e n t r a t i o n o f the saccharide s u f f i c i e n t concentratio b i n d i n g s i t e of the p r o t e i n , r e s p e c t i v e l y . Since the water and diamagnetic c o n t r i b u t i o n s a r e e s s e n t i a l l y independent o f sugar c o n c e n t r a t i o n a t the concentrations used, F can be determined ι s from the observed T\~ v a l u e s : X

l

v

T

l~

T

l

1 ( S

o

)

T

"

1

l "

T

2) mannobioside, 83 m M , ( V ) and a-(l -» 2) mannotrioside, 15 m M (+). Measurements were made at 25° in pH 5.60, 0.1 M potas­ sium acetate buffer, μ = 1.0 in KCl. 2

2

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

32

CARBOHYDRATE-PROTEIN

INTERACTION

Table I L i s t of saccharides used to determine the effects of monoand oligosaccharide binding to Ca -Mn -Con A on the NMRD p r o f i l e of the protein. 2+

2+

methyl a-D-glucopyranoside methyl 3-D-glucopyranosid methyl a-g-mannopyranoside pj-iodophenyl 3-D-glycopyranoside J> galactose o-iodophenyl 3-J?-galactopyranoside maltose maltotriose maltotetraose 0-a-£-mannopyranosyl-(l->-2)-p-mannose O-a-D-mannopyranosyl- ( 1+2) -Oa-p-mannopyranosyl- (l->-2) -D-mannose O- α-ρ-mannopy r ano sy 1- (l-*2) -Ο-αD-mannopyranosyl-(l->2)-0-α-D-mannopyr

anosyl-

( l-*2)-D-mannos e melezitose

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

3.

BREWER

2+

AND BROWN

Binding of Mono- and Oligosaccharides

33

2+

of Ca -Mn -Con A i s shown i n Table I I i n terms of the parameters that determine "*lpara* ™ ^ a n a l y s i s f o l l o w e d the procedure of Koenig e t a l . ( 6 ) . The parameter which best describes the changes i n the NMRD o f the p r o t e i n upon b i n d i n g of these saccharides i s τ^, the residence time of the exchanging water molecule(s) on the manganese i o n , which i n c r e a s e s by ~ 40%. A s m a l l e r (-15%) change i n τ„ i s a l s o observed. Changes i n the other parameters o f the f i t ( r , T g Q and T ) are not considered s i g n i f i c a n t ; v a r i a t i o n i n r and T g , a r e c o r r e l a t e d s i n c e these parameters enter i n t o the theory as s/ ^* anomalously s m a l l value f o r r (~ 2.35 S) i s discussed i n Koenig and Brown (to be p u b l i s h e d ) . T

T

h

e

R

0

T

r

T

n

e

r

e

a

s

o

n

f

o

r

t

n

e

Discussion NMRD measurements o f Ca -Mn -Con A. I n an e a r l i e r r e p o r t , Koenig e t al. (6) observed that the NMRD of Ca -Mn -Con A was perturbed upon a d d i t i o n of amounts of α-MDG s u f f i c i e n t t o s a t u r a t e the carbohydrate b i n d i n g s i t e s o f the p r o t e i n . This suggested that changes i n NMRD could be used t o monitor the b i n d i n g of saccharides t o Con A. Measurements of the T j ~ * of solvent water protons have been widely used t o o b t a i n i n f o r m a t i o n on the b i n d i n g o f organic l i g a n d s t o paramagnetic m e t a l l o p r o t e i n s (15). Koenig and coworkers (16) have shown that measurements of T ] ~ l of water protons over a wide range o f magnetic f i e l d values (2 Oe t o 12 kOe) r e v e a l very d i f f e r e n t NMRD p r o f i l e s f o r the b i n d i n g o f v a r i o u s i n h i b i t o r s t o M n - c a r b o x y p e p t i d a s e , suggest­ ing d i f f e r e n t modes o f b i n d i n g i n each case. Measurements over t h i s extended magnetic f i e l d range permits considerable i n f o r m a t i o n t o be c o l l e c t e d on the e f f e c t s of l i g a n d i n t e r ­ a c t i o n s w i t h a manganese m e t a l l o p r o t e i n s i n c e the f i v e parameters that c o n t r i b u t e t o T j " * of water protons ( 6 , Koenig and Brown i n press) can be evaluated i n the absence and presence o f bound l i g a n d . Measurements a t a s i n g l e magnetic f i e l d may be m i s l e a d i n g s i n c e the f i v e parameters cannot be evaluated, and d i f f e r e n t l i g a n d s may a f f e c t these parameters i n d i f f e r e n t ways (16). Figure 1 shows the observed NMRD of Ca -Mn -Con A, C a - Z n - C o n A and f r e e M n ions i n s o l u t i o n over a magnetic f i e l d range corresponding t o proton Larmor p r e c e s s i o n frequencies from 10 kHz t o 50 MHz. The NMRD f o r Ca -Mn -Con A i s observed to be d i s t i n c t from the other two curves. A q u a n t i t a t i v e a n a l y s i s o f the NMRD of Ca -Mn -Con A i n terms o f the para­ meters that enter i n t o the theory o f magnetic r e l a x a t i o n d i s p e r s i o n has been p r e v i o u s l y published ( 6 ) . Values of these parameters determined from a f i t of t h i s theory t o the data are given i n Table I I . Of importance i s that a l l o f the manganese ions i n the Ca -Mn -Con A s o l u t i o n s a r e t i g h t l y bound t o the p r o t e i n , and that a water l i g a n d ( s ) o f the manganese ion i n the p r o t e i n i s exchanging f a i r l y r a p i d l y (τ - 6 χ 10"^sec) 2+

2+

2+

2+

2+

2+

2+

2 +

2+

2+

2+

2+

2+

2+

Μ

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

34

CARBOHYDRATE-PROTEIN

INTERACTION

Table I I 2+ 2+ a, Water-Ca -Mn -Con A i n t e r a c t i o n parameters— from f i t o f d i s p e r s i o n theory t o data; e f f e c t of s a t u r a t i n g c o n c e n t r a t i o n s of sugars. Ca -Mn +-Con A = 1.56 mM; Τ = 25°; pH = 5.6. 2+

2

Sugar

_r

T

_S0 10

(ft

(10 sec)

(10" sec

(+0.02)

(±0.4)

(+0.05)

-1

2.30

9.08

1.51

cKl+2) mannobioside

2.37

7.15

1.75

α-(1+2) mannotrioside

2.43

7.97

1.82

^M

^R

NONE α-MDG

Sugar

8

6

(10" sec)

(10" sec)

(+0.6)

(+0.02)

NONE

4.88

1.43

α-MDG

6.34

1.96

oKl+2) mannobioside

1.91

α-*2) mannose residues appeared t o have i n t e r n a l residues b i n d i n g t o Con A as judged by t h e i r increased a g g l u t i ­ n a t i o n a c t i v i t y w i t h the p r o t e i n . Further s t u d i e s by So and G o l d s t e i n (4) showed that a-il-^2) mannose-oligosaccharides possessed enhanced b i n d i n g r e l a t i v e t o α-MDM: a 5 - f o l d increase i n b i n d i n g a c t i v i t y f o r the d i s a c c h a r i d e , a-il+2) mannobioside; a 2 0 - f o l d increase i n b i n d i n g a c t i v i t y f o r the t r i s a c c h a r i d e , ot"(l->2) mannotrioside. Other r e p o r t s i n d i c a t e d that c e r t a i n complex glycopeptides showed enhanced b i n d i n g a c t i v i t y (3,29). K o r n f e l d and F e r r i s (30) reported that a glycopeptide d e r i v a t i v e i s o l a t e d from an IgE myeloma p r o t e i n showed a 240-fold i n c r e a s e i n b i n d i n g r e l a t i v e t o α-MDM, the monosaccharide w i t h t h e highest known a f f i n i t y constant f o r Con A. To account f o r t h e i n c r e a s e b i n d i n g a c t i v i t y o f these o l i g o s a c c h a r i d e s , i t was suggested that the carbohydrate b i n d i n g s i t e o f Con A may bind to more than one saccharide r e s i d u e . In order t o examine the b i n d i n g s p e c i f i c i t y of Con A, a comparative study o f the b i n d i n g of mono- and o l i g o s a c c h a r i d e s to the p r o t e i n was c a r r i e d out using the NMRD p r o f i l e o f Ca -Mn -Con A as an index o f the conformational change i n the p r o t e i n induced upon saccharide b i n d i n g . The r a t i o n a l e f o r t h e experiments f o l l o w s the observations made by Teichberg and S h i n i t s k y (31) who observed that o l i g o s a c c h a r i d e s o f i n c r e a s i n g

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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length and a f f i n i t y that bind to lysozyme produce d i f f e r e n t conformational changes i n the p r o t e i n , as detected by f l u o r e s ­ cence quenching of aromatic residues near the combining s i t e . As more contacts were made by l a r g e r o l i g o s a c c h a r i d e s i n the combining s i t e c l e f t of lysozyme, which i s b e l i e v e d to accommo­ date up to s i x saccharide r e s i d u e s , the p r o t e i n underwent concomitant s t e r i c adjustments. S i m i l a r observations have been made f o r wheat germ a g g l u t i n i n which a l s o binds o l i g o s a c c h a r i d e s (32). By analogy, i f o l i g o s a c c h a r i d e s with enhanced b i n d i n g a c t i v i t y toward Con A have a d d i t i o n a l b i n d i n g contacts with the carbohydrate combining s i t e , then a d d i t i o n a l conformational changes might occur i n the p r o t e i n . The saccharides t e s t e d f o r t h e i r e f f e c t on the NMRD p r o f i l e of C a - M n -Con A are l i s t e d i n Table I . They i n c l u d e the β-anomer of g l u c o s e , methy binds a f a c t o r of 2 5 - f o l suggested by Brewer et. a l . (22) to have a d i f f e r e n t b i n d i n g o r i e n t a t i o n to Con A than α-MDG. α-MDM was a l s o included s i n c e i t possesses the highest known a f f i n i t y constant f o r Con A f o r a monosaccharide. Of the o l i g o s a c c h a r i d e s t e s t e d , maltose, m a l t o t r i o s e and maltotetraose represent a major c l a s s of oligomers which b i n d to the p r o t e i n w i t h a f f i n i t y constants n e a r l y the same as that of the corresponding monosaccharide, α-MDG. In a d d i t i o n , s e v e r a l 0>(l->-2) mannose oligomers were s t u d i e d s i n c e these o l i g o s a c c h a r i d e s show enhanced b i n d i n g to the p r o t e i n . Melezitose, 0-a-D-glucopyranosyl-(l->3)-o-3-£-fructofuranosyl( 2 < - » i ) - 0 - a - D - g l u c o p y r a n o s i d e , has been demonstrated by G o l d s t e i n et a l . (2) to show enhanced b i n d i n g by a f a c t o r of approximately 3 r e l a t i v e to α-MDG. The a r y l g l u c o s i d e , 3-IPG, has been shown by Brewer et a l . (22) to b i n d to the saccharide b i n d i n g s i t e of Con A i n s o l u t i o n , but the corresponding a r y l g a l a c t o s i d e (3-IPGal) does n o t . Both of these a r y l g l y c o s i d e s bind to the "aromatic" b i n d i n g s i t e i n the p r o t e i n i n the c r y s t a l l i n e s t a t e (33). D-Galactose, which binds weakly to Con A , was a l s o t e s t e d as a control. 2+

Representative r e s u l t s o f the e f f e c t of b i n d i n g s a t u r a t i n g amounts of s e v e r a l of the saccharides l i s t e d i n Table I on the NMRD p r o f i l e of Ca - M n - C o n A are shown i n F i g . 4. As can be observed, the r e s u l t s were e s s e n t i a l l y the same f o r α-MDG, ct-{l-*2) mannobioside, and oKl+2) mannotrioside. The r e s u l t s , i n f a c t , were the same f o r a l l of the saccharides tested i n Table I with the exception o f D-galactose and $-IPGal which bind only very weakly to the p r o t e i n . Table I I i n d i c a t e s that τ i s the parameter which changes s i g n i f i c a n t l y and that t h i s change i s the same when α-MDG, ct-il+2) mannobioside or0l-(l->»2) mannotrioside binds to C a - M n - C o n A . The data i n d i c a t e , t h e r e f o r e , that the saccharides l i s t e d i n Table I which b i n d to C a - M n - C o n A produce the same change i n the residence time of the exchanging water l i g a n d ( s ) of the Mn^ i o n o f the p r o t e i n . Since t h i s change i n τ appears to be 2 +

Μ

2 +

2 +

2 +

2 +

Μ

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a s s o c i a t e d w i t h a conformational t r a n s i t i o n i n the p r o t e i n , t h e argument can be advanced that a l l of the saccharides that bind to Con A i n Table I produce e s s e n t i a l l y the same conformational changes i n the p r o t e i n upon b i n d i n g . An argument against t h i s c o n c l u s i o n i s that our NMRD measurements o f Ca -Mn -Con A are not s e n s i t i v e t o a d d i t i o n a l b i n d i n g i n t e r a c t i o n s that might take place between o l i g o s a c c h a ­ r i d e s such as cKl+2) mannotrioside, which binds a f a c t o r of 20 times b e t t e r than α-MDM, and the p r o t e i n . The very s m a l l changes that take place i n the c i r c u l a r dichroism spectrum o f the p r o t e i n upon saccharide b i n d i n g (17) does not appear to be a promising way to answer t h i s question. However, another c r i t e r i o n can be used concerning extended i n t e r a c t i o n s that may be o c c u r r i n g between the p r o t e i n and c e r t a i n o l i g o s a c c h a r i d e s . In the case of lysozym combining s i t e s , i n h i b i t o r p r o t e i n s a l l possess very s i m i l a r s t e r i c f e a t u r e s regarding t h e i r o v e r a l l conformations. This i s demanded by the s t e r i c c o n s t r a i n t s o f the combining s i t e s o f these p r o t e i n s . Therefore, i f Con A possesses an extended combining s i t e , there should be equal c o n s t r a i n t s on the three dimensional s t r u c t u r e s o f saccharides that b i n d t o the p r o t e i n . However, s p a c e - f i l l i n g models (CPK) o f s e v e r a l o f the o l i g o s a c c h a r i d e s that show enhanced b i n d i n g such as(X-(l-*2) mannotrioside, melezitose and the G l glycopeptide i s o l a t e d by K o m f e l d and F e r r i s (30), which shows enhanced b i n d i n g , i n d i c a t e l i t t l e o r no s i m i l a r i t y i n t h e i r o v e r a l l conformations. I t does not seem l i k e l y , t h e r e f o r e , that these o l i g o s a c c h a r i d e s possess higher K values because of s i m i l a r extended contacts w i t h the p r o t e i n . I n f a c t , t h e s m a l l increments of enhanced b i n d i n g o f f a c t o r s of 5 and 20 f o r oKl+2) mannobioside and a-(l->-2) mannotrioside, r e s p e c t i v e l y , r e l a t i v e t o α-MDM, does not compare w i t h the l a r g e enhancements of 1 0 - 1 0 i n b i n d i n g that are observed f o r d i - and t r i s a c c h a r i d e s , r e s p e c t i v e l y , r e l a t i v e to monosaccharides, that bind t o lysozyme (34) which contains an extending b i n d i n g s i t e . Wheat germ a g g l u t i n i n , which appears to have an extended b i n d i n g s i t e , a l s o shows l a r g e enhancements of 1 0 - 1 0 f o r b i n d i n g d i - and t r i s a c c h a r i d e s r e l a t i v e t o monosaccharides (35). These argu­ ments a l s o weigh against extended i n t e r a c t i o n s between the b i n d i n g s i t e o f Con A and o l i g o s a c c h a r i d e s . Our proposed i n t e r p r e t a t i o n o f the data i s that the monoand o l i g o s a c c h a r i d e s l i s t e d i n Table I that bind t o Con A a l l i n t e r a c t w i t h the p r o t e i n i n the same manner. That i s , s i n c e o l i g o s a c c h a r i d e s produce the same change i n the NMRD p r o f i l e as monosaccharides, t h e i r b i n d i n g modes must be q u i t e s i m i l a r . This would suggest that the o l i g o s a c c h a r i d e s are b i n d i n g through only one of t h e i r residues a t any one time, i n a manner s i m i l a r to monosaccharide i n t e r a c t i o n s w i t h the p r o t e i n . The enhanced b i n d i n g of c e r t a i n o l i g o s a c c h a r i d e s can t h e r e f o r e be explained on a s t a t i s t i c a l b a s i s . Of the o l i g o s a c c h a r i d e s that 2+

2+

a

2

3

2

3

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possess enhanced b i n d i n g constants, the 0t-(l->2) l i n k e d mannose oligomers are most evident. These oligomers d i f f e r from the maltose and isomaltose oligomers, which show no enhanced b i n d i n g , i n the p o s i t i o n o f t h e i r g l y c o s i d i c l i n k a g e s . As p r e v i o u s l y noted, Con A demonstrates b i n d i n g s p e c i f i c i t y toward saccharides w i t h glucose or mannose residues that possess f r e e C-3, 4 and 6 hydroxyl groups. Since o l i g o s a c c h a r i d e s witho>< 1+3) o>(l-*4) o r a-(l-*6) g l y c o s i d i c l i n k a g e s possess modified hydroxyl groups a t these c r i t i c a l b i n d i n g p o s i t i o n s , only the non-reducing t e r m i n a l saccharide o f these oligomers can bind t o the p r o t e i n , a conclusion reached by G o l d s t e i n and coworkers (2). However, where the g l y c o s i d i c l i n k a g e isa-(l-^2) f o r mannose oligomers, the i n t e r n a l residues a l s o possess f r e e C-3, 4> and 6 h y d r o x y l groups. G o l d s t e i n e t a l (3) has shown t h a t when o>( 1+2) manno­ t r i o s i d e was s e l e c t i v e l such that they could no c o n t a i n i n g an unmodified i n t e r n a l mannopyranoside r e s i d u e was observed t o bind as w e l l as α-MDM. Using s i m i l a r dérivâtization techniques, the reducing t e r m i n a l r e s i d u e o f a-(l+2) mannotrioside was a l s o shown t o bind as w e l l as f r e e mannose; G o l d s t e i n thus concluded that i n t e r n a l a-(l-*2) l i n k e d mannose residues could bind t o Con A as w e l l as non-reducing t e r m i n a l r e s i d u e s . Another o l i g o s a c c h a r i d e w i t h enhanced b i n d i n g i s m e l e z i t o s e , which i s s t r u c t u r a l l y d i s s i m i l a r t o the a-(l+2) mannans. However, the two are s i m i l a r i n that melezitose a l s o possesses more than one residue w i t h f r e e C-3, 4, and 6 hydroxyl groups: the f i r s t and t h i r d glucose u n i t s . The enhanced b i n d i n g o f m e l e z i t o s e by a f a c t o r o f seven r e l a t i v e t o m a l t o t r i o s e , which contains only one " b i n d i n g " residue a t the non-reducing t e r m i n a l end, i s s i m i l a r t o the enhancement observed f o r α-2) mannobioside (which a l s o contains two " b i n d i n g " residues per molecule) r e l a t i v e t o α-MDM. Thus, i t appears that a necessary r e q u i r e ­ ment f o r enhanced b i n d i n g i s f o r a molecule t o c o n t a i n m u l t i p l e glucose o r mannose residues which possess f r e e c-3, 4, and 6 hydroxyl groups. We t h e r e f o r e suggest that the enhanced b i n d i n g of c e r t a i n o l i g o s a c c h a r i d e s t o Con A appears t o be due not t o an extended b i n d i n g s i t e on the p r o t e i n , but r a t h e r t o e f f e c t s which r e s u l t from c l u s t e r i n g s e v e r a l " b i n d i n g " residues (glucose o r mannose) i n the same molecule, any one o f which can bind t o the s i n g l e s i t e on the p r o t e i n . To our knowledge, the e f f e c t s o f b i n d i n g p o l y v a l e n t l i g a n d s t o monovalent p r o t e i n b i n d i n g s i t e s has never been s t u d i e d . Nevertheless, we f e e l the enhanced b i n d i n g could come about from an increase i n the forward r a t e constant f o r complex formation which, t o the f i r s t approximation, would be expected t o be p r o p o r t i o n a l t o the number of b i n d i n g residues i n the molecule. However, the observed enhancements for oKl+2) mannobioside and -2)D-mannose c o m p e t i t i v e l y d i s p l a c e the chromogenic l i g a n d £-nitrophenyl 3 -D-mannopyranoside from Con A. These workers concluded that the mono- and o l i g o s a c c h a r i d e s used i n the study competed f o r the same s i t e on the p r o t e i n . I n a d d i t i o n , t h e s p e c i f i c i t y o f the same s i t e on Con A would r e q u i r e that a l l residues i n a saccharide that bind d i r e c t l y t o the p r o t e i n possess the same molecular s p e c i f i c i t y as monosaccharides that bind t o the p r o t e i n . I t i s w e l l known that the monosaccharides glucose and mannose b i n d w e l l , as opposed t o galactose which binds p o o r l y , and that the α-anomers o f these monosaccharides bind b e t t e r than the 3-anomers. Indeed, G o l d s t e i n et_ a l . (3) have shown that when the t e r m i n a l non-reducing residue of a-(l+2) mannotrioside i s converted t o a galactose r e s i d u e , a l o s s i n b i n d i n g i s observed. This r e s u l t i s c o n s i s t e n t w i t h the t e r m i n a l non-reducing mannose residue (X-(l+2) mannotrioside b i n d i n g a t t h e same s i t e as α-MDM. Furthermore, G o l d s t e i n and coworkers (2) have shown that r e d u c t i o n of mannose t o give mannitol e l i m i n a t e s b i n d i n g . When the t e r m i n a l reducing end of α-( 1+2) m a n n o t r i i t o l , a l o s s i n b i n d i n g occurs which i s a l s o c o n s i s t e n t w i t h the t e r m i n a l reducing residue of Ot-^1+2) mannotrioside b i n d i n g t o the same s i t e as α-MDM. S i m i l a r s u b s t i t u t i o n s a f f e c t the b i n d i n g o f

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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another a-( 1+2) l i n k e d o l i g o s a c c h a r i d e , k o j i b i o s e (Q-a-D-gluco­ py r a n o s y l - ( 1+2)-D-glucose) . K o j i b i o s e shows enhanced~~binding by a f a c t o r of three r e l a t i v e to ο - α - D - g a l a c t o p y r a n o s y l - ( 1 + 2 ) D-glucose ( 5 ) . T h i s suggests that the non-reducing residue of k o j i b i o s e c o n t r i b u t e s to b i n d i n g , and the s p e c i f i c i t y of b i n d i n g i s s i m i l a r to that observed f o r monosaccharide b i n d i n g to Con A . I n t e r e s t i n g l y , the α-methyl anomer of k o j i b i o s e shows a f u r t h e r enhancement of b i n d i n g by a f a c t o r of two r e l a t i v e to k o j i b i o s e , which i s presumed to be a mixture of the a- and 3-anomers. This suggests that the reducing end r e s i d u e of the d i s a c c h a r i d e binds to the p r o t e i n at a s i t e which a l s o p r e f e r e n t i a l l y binds the α-anomers of monosaccharides. The above data are a l l c o n s i s t e n t w i t h the p r o t e i n c o n t a i n i n g a s i n g l e b i n d i n g s i t e which can i n t e r a c t w i t h e i t h e r t e r m i n a l non-reducing r e s i d u e s i n t e r n a l r e s i d u e s , or t e r m i n a r i d e s p r o v i d i n g that thes free C - 3 , 4, and 6 h y d r o x y l groups. Thus, from c o n s i d e r a t i o n of a l l of the a v a i l a b l e evidence, i t appears that the saccharide b i n d i n g s p e c i f i c i t y of Con A can be accounted f o r by a b i n d i n g s i t e which accommodates only one saccharide r e s i d u e , and that o l i g o s a c c h a r i d e s c o n t a i n i n g m u l t i p l e glucose or mannose residues which have free C - 3 , 4, and 6 h y d r o x y l groups can demonstrate enhanced b i n d i n g to the p r o t e i n , r e l a t i v e to monosaccharides, due to i n c r e a s e s i n t h e i r p r o b a b i l i t y of b i n d i n g . These r e s u l t s have important i m p l i c a ­ t i o n s regarding the molecular p r o p e r t i e s of " s o - c a l l e d Con A receptors on the surface c e l l s . 11

Acknowledgments This work was supported i n part by grant # CA-16054, awarded by the N a t i o n a l Cancer I n s t i t u t e , Department of H e a l t h , Education and Welfare. C F . Brewer i s a r e c i p i e n t of a Research Career Development Award, grant // 1-K04-CA-00184 from the Dept. of H e a l t h , Education and W e l f a r e . (A p r e l i m i n a r y report of the work was presented at the U n i v e r s i t y of Oklahoma Symposia on Concanavalin A, (Brown, R . D . , Brewer, C F . and Koenig, S . H . (1975), i n Concanavalin A , Adv. Exp. Med. B i o l . , 55, 323). Abstract We have measured the effects of binding of a series of mono­ -and oligosaccharides to Ca -Mn -Con A on the solvent water proton relaxation rate over a range of magnetic f i e l d s from 5 Oe to 12 kOe. We find that the binding of methyl α- and β-D­ -glucopyranoside, methyl α-D-mannopyranoside and o-iodophenyl β­ -D-glucopyranoside produce the same increase i n the residence time of the exchanging water ligand(s) of the Mn ion and therefore the same conformational change in the protein, whereas galactose and o-iodophenyl β-D-galactopyranoside, which do not 2+

2+

2+

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bind under the same conditions, show no effects. The same reduction in relaxation rate as that caused by the above mono­ saccharides was observed with the following oligosaccharides: D-maltose, D-maltotriose, D-maltotetraose, O-α-D-mannopyranosyl(1->2)-D-mannose, Ο-α-D-mannopyranosyl-( 1->2)-O-α-D-mannopyranosyl(1->2)-D-mannose, Ο-α-D-mannopyranosyl-(1->2)-O-α-D-mannopyranosyl(1->2)-Ο-α-D-mannopyranosyl-(1->2)-D-mannose and melezitose. Goldstein and coworkers have shown that the first three oligo­ saccharides have nearly the same affinity as monosaccharides, whereas the α-(1->2) linked mannans show increasing affinity constants with increasing chain length. Melezitose also shows enhanced binding by a factor of three relative to methyl-α-D­ -glucopyranoside. The water relaxation data suggest that the above mono- and oligosaccharides bind to Con A by a similar mechanism involving onl with the protein at on greate y tose and theα-(1->2)mannose oligosaccharides appears to be due to an increase in the probability of binding associated with the presence of more than one binding residue in the chain and not to an extended binding site on the protein. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

L i s , H. and Sharon, N . , Annual Revs. Biochem., (1973), 42, 541. Goldstein, I.J., Hollerman, C.E. and Smith, E.E., Biochemistry, (1965), 4, 876. Goldstein, I.J., Reichert, C.M., Misaki, A. and Gorin, P.A.J. Biochim, Biophys. Acta, (1973), 317, 500. So, L . L . and Goldstein, I.J., Biochim. Biophys. Acta, (1968), 165, 398. Goldstein, I.J., Cifonelli, J.A. and Duke, J., Biochemistry, (1974), 13, 867. Koenig, S.H., Brown, R.D. III and Brewer, C.F., Proc. Nat. Acad. S c i . , (1973), 70, 475. Yariv, J., Kalb, A . J . and Levitzki, Α., Biochim. Biophys. Acta, (1968), 165, 303. Brown, R.D., Brewer, C.F. and Koenig, S.H., Biochemistry, (1977), 16, 3883. Koenig, S.H. and Schillinger, W.E., J. Biol. Chem., (1968), 244, 3283. Hallenga, K. and Koenig, S.H., Biochemistry, (1976), 15, 4255. Steinhardt, J. and Reynolds, J., "Multiple Equilibria in Proteins", Academic Press, New York, (1969). McKenzie, G.H. and Sawyer, W.H., J. Biol. Chem., (1973), 248, 549. Becker, J.W., Reeke, G.N.Jr., Wang, J.L., Cunningham, B.A. and Edelman, G.M., J. Biol. Chem., (1975), 250, 1513.

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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14.

43

Loontiens, F.G., van Wauve, J.P. and DeBruyne, C . K . , Carbohydrate Res., (1975), 44, 150. 15. Mildvan, A. and Cohn, Μ., Advan. Enzymol., (1970), 33, 1. 16. Quiocho, F.A., Bethge, P.H., Lipscomb, W.N., Studebaker, J.F., Brown, R.D. and Koenig, S.H., Cold Springs Harbor Symposia on Quantitative Biology XXXVI, 561, (1971). 17. Pflumm, M.N., Wang, J.L. and Edelman, G.M., J. Biol. Chem., (1971), 246, 4369. 18. Brewer, C.F., Sternlicht, Η., Marcus, D.M. and Grollman, A.P., "Lysozyme", Academic Press, New York, (1974). 19. Becker, J.W., Reeke, G.N. Jr., Cunningham, B.A. and Edelman, G.M., Fed. Proc., (1976b), 35, 1716. 20. Brewer, C.F., Sternlicht H. Marcus D.M and Grollman A.P., Abstracts National Meeting 21. Brewer, C.F., Sternlicht, H., Marcus, D.M. and Grollman, A.P., Proc. Nat. Acad. Sci. USA, (1973a), 70, 1007. 22. Brewer, C.F., Sternlicht, H., Marcus, D.M. and Grollman, A.P., Biochemistry, (1973b), 12, 4448. 23. Villafrance, J.J. and Viola, R.E., Arch. Biochem. Biophys., (1974), 165, 51. 24. Alter, G.M. and Magnuson, J.A., Biochemistry, (1974), 13, 4038. 25. Edelman, G.M., Cunningham, B.A., Reeke, G.N. Jr., Becker, J.W., Waxdal, M.J. and Wang, J . L . , Proc. Nat. Acad. Sci. USA, (1972), 69, 2580. 26. Hardman, K.D. and Ainsworth, C.F., Biochemistry, (1976), 15, 1120. 27. Becker, J.W., Reeke, G.N. Jr., Cunningham, B.A. and Edelman, G.M., Nature, (1976a), 259, 406. 28. Hehre, E . J . , Bull. Soc. Chim. Biol., (1960), 42, 1581. 29. Young, N.M. and Leon, M.A., Biochim, Biophys. Acta, (1974), 365, 418. 30. Kornfeld, R. and Ferris, C., J. Biol. Chem., (1975), 250, 2614. 31. Teichberg, V.I. and Shinitzky, M., J. Mol. Biol., (1973), 74, 519. 32. Privat, J.R., Domotte, F., Mialonier, G., Bouchard, P. and Monsigny, Μ., Eur. J. Biochem., (1974), 47, 5. 33. Hardman, K.D. and Ainsworth, C.F., Biochemistry, (1973), 12, 4442. 34. Banerjee, S.K. and Rupley, J.Α., J. Biol. Chem., (1973), 248, 2117. 35. Allan, A.K., Neuberger, A. and Sharon, Ν., Biochem. J., (1973), 131, 155. 36. Abdullah, Μ., French, D. and Robyt, J.F., Arch. Biochem. Biophys., (1966), 114, 595. 37. Thoma, J.Α., Spradlin, J.E. and Dygert, S., "The Enzymes", p. 115, D. Boyer (Ed.), (1971). RECEIVED September 8, 1978. In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

4 Binding of p-Nitrophenyl 2-O-α-D-Mannopyranosyl-α-D­Mannopyranoside to Concanavalin A TAFFY WILLIAMS, JULES SHAFER, and IRWIN GOLDSTEIN Department of Biological Chemistry, The University of Michigan, Ann Arbor, MI 48109

For many years this laboratory has been studying the inter­ actions of concanavalin A (con A) a carbohydrate binding protein of considerable interes hydrates (5-8). It wa charides, oligosaccharides composed of α-(1->2)-linked D-mannose units exhibited an enhanced affinity for con A (9). This increased affinity for con A was explained both in terms of an extended binding site (5,10) and a s t a t i s t i c a l mode (11). In order to further examine the nature of the binding phe­ nomenon, kinetic and thermodynamic parameters of the binding of p­ -nitrophenyl 2-O-α-D-mannopyranosyl-α-D-mannopyranoside (M ) (see Figure 1 ) were compared to p-nitrophenyl α-D-mannopyranoside (Μ ) (and i t s 2-O-methyl derivative). The above disaccharide contains two α-D-mannopyranosyl residues both of which are capable of interacting with con A. Any enhanced affinity of M toward con A due to a s t a t i s t i c a l effect should be reflected by differences in the entropy of activation for binding M and the corresponding monosaccharide. No difference should be seen in the enthalpy of activation for binding the mono- and disaccharide since only entropy terms are affected in true s t a t i s t i c a l modes of binding. Therefore, the activation parameters for the binding of M 2 to con A were examined i n order to determine whether con A interacts with both mannopyranosyl residues at an extended binding s i t e or the enhanced affinity of M 2 for con A is due to a s t a t i s t i c a l effect whereby the two mannopyranosyl residues of M 2 simply increase the probability of forming a complex with the protein. I n i t i a l observations indicated that con A as isolated by the conventional Sephadex procedure (9) exhibited heterogeneity with respect to binding M . Spectral studies of the interactions of con A with M 2 indicated that con A contains at least two compon­ ents which interact differently with M . The simple reaction, 2

1

2

2

2

2

P+L

^ PL,

(1)

*s

represents the formation of a con A-ligand complex wherein the 0-8412-0466-7/79/47-088-044$05.00/0 © 1979 American Chemical Society

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2-0-a-O-Mannopyranosal-a-O-Mannopyranoside

concentration of PL i s determined by the concentrations of Ρ and L. The assignment of the terms Ρ and L to ligand and protomer i s arbitrary, and symmetrically interchanging the concentrations of protein and ligand should not affect the concentration of the com­ plex. Such behavior i s observed when one studies the interaction of con A with monosaccharides . However, when M 2 i s mixed with con A prepared by the conventional Sephadex procedure the amount of complex formed appears to depend upon which ligand i s in excess. Figure 2 depicts the asymmetric response observed by means of difference spectra for formation of a con A - M 2 complex when solutions of con A and M 2 are mixed. It i s evident that the apparent amount of complex formed as reflected by the magnitude of the difference spectra, depends on which component i s in excess. Such a result could be explained i f M were impure, or i f the con A were heterogeneous with was examined by severa terize the heterogeneity of the con A - M 2 interaction, equilibrium dialysis studies were performed with the concentrations of M 2 equal to twice the concentration of binding sites. The concentra­ tion of both protein and ligand were kept high to ensure occupancy of a l l the binding sites. A plot of M 2 bound versus the concen­ tration of binding sites (Figure 3) gave a straight line with a slope of 0.9 demonstrating that nearly a l l the protein combining sites are capable of binding M 2 . However, a l l of these con A binding sites do not necessarily have the same a f f i n i t y for M 2 as indicated by the nonlinear Scatchard plot i n Figure 4. In this figure, the line drawn through the points i s a computer f i t of the data for a two component system i n which M 2 binds each component with a different a f f i n i t y . Since con A as isolated conventionally (9) exists as a mix­ ture of intact and nicked protomeric subunits (12), a method was developed to purify large quantities of these species so that they might be examined as a source of the heterogeneous-like behavior seen i n the Scatchard plot (Figure 4) and ultraviolet difference spectra (Figure 2). Con A was applied to a Sephadex G-75 column equilibrated at pH 7.2 (0.02 M Tris-HCl) and 4°C. Elution with 19 mM D-glucose, i n 0.02 M Tris-HCl (pH 7.2) resulted i n the dis­ placement of a fraction of protein composed of 70-80% nicked subunits. Further elution with 100 mM D-glucose gave a second peak composed of 100% intact subunits. These results are indicated i n Figure 5. In this procedure, about 40% of the protein remained on the column. A Scatchard plot obtained for the interaction of M and "intact" con A, Figure 6 , was found to be linear as would be ex­ pected for single component systems and a value of 1.9 χ 10 M" was obtained for the association constant. Also, identical d i f ­ ference spectra were obtained when the concentration of ligand and protein were symmetrically interchanged. Both of these results demonstrate the homogeneous behavior of the "intact" con A with respect to M 2 . Interestingly, the "nicked" con A that was obtained 2

5

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

1

46

CARBOHYDRATE-PROTEIN

INTERACTION

/?-Nitrophenyl 2-0-a-o-Mannopyranosyl-a-D-Mannopyranoside Figure 1. Structure of p-nitrophenyl 2-O-a-O-mannopyranosyl-a-O-mannopyranoside

Figure 2. Difference spectra observed on mixing M and con A obtained com­ mercially from Calbiochem or prepared according to Ref. 9. A, 40 μΜ M and 100 Μ con A; B, 100 μΜ M and 40 μΜ con A. g

t

μ

t

J

250

I

I

3 0 0 350

Wavelength (nm)

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

L

400

4.

WILLIAMS

ET

2-0-a-ry~Mannopyranosal-a-O-Mannopyranoside

AL.

47

Figure 3. The dependence of bound ligand ([M ] ) on the total concentration of protomeric units ([P] ) of con A from Calbiochem. The total concentration of M was maintained at twice [P] t

b

t

t

t

12.0 10.0

Figure 4. Scatchard plot for the binding of M to commercially prepared con A as determined by equilibrium dialysis. The total concentration of con A proto­ meric units was 15 μΜ. The solid line represents a computer simufoted fit of the t

dat

itmimxmflcW

tem

Society Library 1155 16th st. N. w. In Carbohydrate-Protein Interaction; Goldstein, I.; Washington, D. C. Society: 20036 ACS Symposium Series; American Chemical Washington, DC, 1979.

48

CARBOHYDRATE-PROTEIN

INTERACTION

1 0 0 % Intact Con A

Mixture of Intact and Nicked Con A 0.019 Μ ο - G l u c o s e

J

^

20

0.1 M o-Glucose

6

V

Fraction Number

Figure 5. Elution profile obtained for Calbiochem con A on Sephadex G-75 in a Tris-HCl pH 7.2 buffer. Elution was effected with 0.019 M glucose followed by 0.1 M glucose.

20.0

Figure 6. Scatchard plot for the bind­ ing of M to intact cou A as determined spectrophotometrically t

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2-0-a-O~Mannopyranosal-a-O-Mannopyranoside

from the Sephadex G-75 chromatography was indistinguishable from the intact con A i n i t s interactions with M . Furthermore, mix­ tures of intact and nicked con A behaved as a single homogeneous protein with respect to M . Therefore, we conclude that the ma­ t e r i a l which remains bound to the Sephadex G-75 column gives rise to the heterogeneous response to M . The elution and characteri­ zation of the protein fraction which remains bound on the column is currently under investigation. The fraction of protein, eluted from Sephadex G-75 which behaves as a single homogeneous protein with respect to i t s interactions with M was also studied with regard to i t s interaction with mono­ saccharides. The association constant of the interaction of M with intact con A i s 19 times greater than that obtained for the interaction of p-nitrophenyl α-D-mannopyranoside with con A. The normalized spectra (Figur charide complexes were foun the interactions between con A and the nitrophenyl group which causes the spectral perturbation are the same for both complexes. The "intact" con A which behaves homogeneously toward M was used i n further kinetic studies. Stopped-flow studies were per­ formed on a structural analog of M , p-nitrophenyl 2- 1000

30

> 1000

4

> 1000

23

> 1000

0

α α Man 1+6 Man 1+6 Man

> 1000

12

Mannose

> 1000

0

Man

1+2 Man α 1+2 Man

α Man 1+3 Man 1*4 GlcNAc

3

Man 1*3 Man 1+4 GlcNAc 2 la Man 1

Immune r a b b i t serum, 5 y l i s mixed with v a r y i n g amounts o f i n h i b i t o r i n b u f f e r i n a f i n a l volume o f 300 y l . A f t e ^ i n c u b a t i o n f o r 2 h a t 2 5 ° , 6 . 7 pmole o f [ H]mannotetraose (10 cpm) i n 100 y l o f b u f f e r i s added and i n c u b a t i o n i s continued f o r 2 h a t 2 5 ° . Binding o f t r i t i a t e d sugar i s determined as described i n Figure 2 . 3

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CARBOHYDRATE-PROTEIN INTERACTION

Figure 9. Hypothetical scheme hexaose I (Anti-LND I) and antibody against lacto-N-tetraose (Anti-LNT) to opposite sides of the oligosaccharide ucto-N-difucohexaose I. Profiles of monosaccharide units were drawn from a molecular model similar to those shown in Figure 8.

Figure 10. Photomicrographs of a suspension of Saccharomyces cerevisiae incubated for 3 hr at 35° with rabbit antimannotetraose serum diluted 1:100 in buffered saline in the absence (A) or presence (B)of 1 mM mannotetraose

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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101

chain by elongation with an additional non-reducing mannosyl residue reduces binding 20-fold and changing the non-reducing terminal linkage from Manal-3 to Manal-2 reduces oinding by a factor of 200. Deletion of non-reducing terminal residues, alteration of the penultimate linkages, and changes in linkage positions result in almost complete loss of binding activity. As the antimannotetraose antibodies in this serum show specific­ ity for the non-reducing trisaccharide sequence Manal-3Manal-2Man they can be used for detection of this sequence in biological material. For example, this antiserum specifically agglutinates whole yeast cells (Figure 10). Agglutination is inhibited as shown on the right by free mannotetraose but not by free mannose nor by other sugars we have tested shown in Table I I . In summary, i t is possible to immunize animals with purified oligosaccharides couple that can be characterize prove useful for studying complex carbohydrates. LITERATURE CITED 1) Kobata, Α., in Methods in Enzymology (Ginsburg, V . , ed.), Vol. 28, pp. 262-271, Academic Press, New York (1972). 2) Zopf, D.A., Ginsburg, Α., and Ginsburg, V . , J. Immunol. (1975) 115 1525-1529. 3) Zopf, D.A., Tsai, C., and Ginsburg, V . , Arch. Biochem. Biophys. (1978) 185 61-71. 4) Arakatsu, Y . , Ashwell, G., and Kabat, E.A., J. Immunol. (1966) 97 858-866. 5) Jeffrey, A.M., Zopf, D.A., and Ginsburg, V . , Biochem. Biophys. Res. Commun. (1975) 62 608-613. 6) Himmelspach, K., and Kleinhammer, G., in Methods in Enzymology (Ginsburg, V . , ed.), Vol. 28, pp. 222-231, Academic Press, New York, (1972). 7) Wiederschain, G.Y., and Rosenfeld, E . L . , Biochem. Biophys. Res. Commun. (1971) 44 1008-1014. 8) Alhadeff, J.J., Miller, A . L . , Wenaas, H . , Vedrick, T., and O'Brien, J.S., J. B i o l . Chem. (1975) 250 7106-7113. 9) Schwarting, G.A., and Marcus, D.M., J. Immunol. (1977) 118 1415-1419. 10) Tsai, C.-M., Zopf, D.A., Wistar, R., and Ginsburg, V . , J. Immunol. (1976) 117 717-721. RECEIVED

September 8, 1978.

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

9 Interaction of Glycosyl Immunogens with Immunocyte Receptor Sites in the Synthesis of Anti-glycosyl Isoantibodies JOHN H. PAZUR and KEVIN L. DREHER Department of Biochemistry and Biophysics, The Pennsylvania State University, University Park, PA 16802 DAVID R. BUNDLE Division of Biological Sciences, National Research Council, Ottawa, Canada, KIA OR6 Anti-glycosyl antibodies are defined as those antibodies which are induced by carbohydrat combine with a specifi (1). In chemical structure, the immunogens may be glycans (2-5), glycoproteins (6-9), glycolipids (10,11) or synthetic carbohydrate-protein conjugates (12-15). The immunogens of the glycan type are important components of the cell walls of pathogenic bacteria and such compounds form the basis of the scheme for the serological classification of these organisms (16). The immunogens of the glycoprotein type are important constituents of mammalian cells and are of great significance in the transformation of such cells into neoplasmic cells, particularly in the transformations brought about by viruses (17,18). The immune system is the primary means of defense against infections by bacterial pathogens and against the proliferation of neoplasmic cells of many types of carcinomas. The immunogens on the surface of these cells stimulate the immune system to produce antibodies which initiate the process that ultimately leads to the destruction of the bacterium or to the retardation of growth and in some cases the elimination of the neoplasmic cells. Basic information on the biochemical reactions of the immune process is highly desirable in order that more effective methods of utilizing the immune system for the diagnosis and treatment of diseases may be developed. Many steps of the process are already reasonably well delineated, but the sequence of reactions and the biological mechanisms for some remain obscure (19). For example, it is well known that only certain structural features of the immunogen, the immunodeterminant groups, are involved in the stimulation of immunocytes to produce antibodies. These immunodeterminant groups are also the structural moieties of the antigens which combine with antibodies to form the antibody-antigen complex. However, it is not known how these groups interact with the receptor substances on the immunocytes or how this interaction initiates the sequence of biochemical reactions resulting in the synthesis of antibodies. 0-8412-0466-7/79/47-088-102$05.00/0 © 1979 American Chemical Society

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In antigens of the glycan and glycoprotein types the immunodeterminant groups are very often terminal carbohydrate moieties. In such cases the antibodies which are elaborated against these antigens are anti-glycosyl antibodies. Studies on the types of anti-glycosyl antibodies induced by different antigens with the same immunodeterminant groups should yield valuable information on the immune process. During the past few years we have been investigating the nature of the anti-glycosyl antibodies induced i n rabbits by immunogens with many types of carbohydrate immunodeterminant groups. In these studies rabbits have been immunized with vaccines of bacterial glycosyl antigens or with synthetic carbohydrate-protein conjugates. Antisera have been obtained from these rabbits after suitable periods of immunization by standard methods of immunology. in the sera have been deduce experiments u t i l i z i n g a variety of carbohydrates. A f f i n i t y chromatography techniques have then been used to prepare antiglycosyl antibodies which are specific for a single type of carbohydrate unit. Some of the chemical and immunological properties of such antibodies have been determined. To date, antibodies of anti-galactose (20), anti-glucose (21), anti-N-acetyl-glucosamine (22) , anti-glucuronic acid (23), anti-lactose (20) and anti-isomaltose (24) types have been obtained. To obtain these antibodies different types of bacterial antigens and carbohydrate-protein conjugates have been used, but only three w i l l be discussed i n this report. One of these antigens i s a diheteroglycan of glucose and galactose in thé c e l l wall of Streptococcus faecalis and the other two are synthetic carbohydrate-protein conjugates, galactosyl bovine serum albumin (GalBSA) and lactosyl bovine serum albumin (Lac-BSA). The methods for the preparation of these antigens are described later. It should be pointed out that a l l of these antigens possess the same types of terminal carbohydrate moieties, galactosyl or lactosyl units, which as w i l l be seen, are the immunodeterminant groups. The diheteroglycan of glucose and galactose i s a type specific carbohydrate in the c e l l wall of Streptococcus faecalis (5). This glycan was extracted by a trichloroacetic acid method and purified to homogeneity by alcohol precipitation and bio-gel f i l t r a t i o n methods. The complete molecular structure of the glycan has been deduced from data of methylation analysis i n combination with enzymic and chemical degradation reactions (25, 26). A diagrammatic representation of the total structure for this glycan i s shown in Figure 1. The molecular weight of the glycan i s 15,000 and a typical molecule i s composed of a main chain of eighteen glucose-glucose-galactose repeating units and eighteen disaccharide side chains attached to the central glucose residue of each repeating unit. The disaccharide side chains are mainly lactose units but a few are cellobiose units. As indicated above the types of glycosidic linkages have been determined by

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

CARBOHYDRATE-PROTEIN INTERACTION

Carbohydrate Research

Figure 1. Diagrammatic representation of the structure of a typical molecule of the diheteroglycan of glucose and galactose from Streptococcus faecalis (26;

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methylation a n a l y s i s and the c o n f i g u r a t i o n o f the l i n k a g e s has been shown to be beta by enzymic t e s t s . The experimental d e t a i l s of the s t r u c t u r a l s t u d i e s have been p u b l i s h e d (25,26). The carbohydrate-protein conjugates t h a t have been employed i n these s t u d i e s are g a l a c t o s y l bovine serum albumin (Gal-BSA) and l a c t o s y l bovine serum albumin (Lac-BSA). The r e a c t i o n sequence f o r the synthesis of these conjugates i n v o l v e s the p r e p a r a t i o n of the p e r - a c e t y l d e r i v a t i v e s o f g a l a c t o s y l bromide or l a c t o s y l bromide, r e a c t i o n of e i t h e r d e r i v a t i v e with 8-ethoxycarbonyl o c t a n o l , d e a c e t y l a t i o n o f the product, formation o f the hydrazide, conversion to the azide and f i n a l l y c o u p l i n g the azide to the bovine serum albumin. Reaction c o n d i t i o n s f o r the v a r i o u s steps of the s y n t h e s i s are d e s c r i b e d i n the l i t e r a t u r e (14,27) and the sequence i s shown diagrammatically i n F i g u r e 2 A n a l y s i s of the conjugates f o r carbohydrat method (28) showed tha bovine serum albumin were bonded to carbohydrate r e s i d u e s . Two types of vaccines and immunization regimes were employed f o r immunizing r a b b i t s with these immunogens. The b a c t e r i a l vaccine was a suspension of non-viable c e l l s of Streptococcus f a e c a l i s with the glycan i n s i t u i n the c e l l w a l l . The d e t a i l s of the p r e p a r a t i o n o f the vaccine have been d e s c r i b e d (25). Immuni­ z a t i o n was intraveneously three times per week f o r periods of up t o f i f t e e n weeks. Blood samples were c o l l e c t e d weekly and a n t i sera were prepared from these samples. Vaccines of Gal-BSA and Lac-BSA were prepared by mixing 0.2% s o l u t i o n of the conjugates i n saline-phosphate b u f f e r of pH 7.2 with an equal volume of complete Freunds adjuant. Immunizations with the l a t t e r vaccines were performed subcutaneously i n the back o f the neck three times i n weekly i n t e r v a l s (27). Blood samples were c o l l e c t e d 30 days a f t e r the beginning o f immunization and a n t i s e r a were prepared from these samples i n the usual manner. Agar d i f f u s i o n patterns f o r the r e a c t i o n s of these a n t i s e r a with the immunogens are shown i n F i g u r e 3, l e f t p l a t e . I t w i l l be noted t h a t a strong p r e c i p i t i n band was obtained with each s e t of antigens and a n t i s e r a . Hapten i n h i b i t i o n experiments with g a l a c ­ tose, l a c t o s e and a v a r i e t y of other carbohydrates showed t h a t galactose and l a c t o s e were potent i n h i b i t o r s o f the p r e c i p i t i n r e a c t i o n s and thus a l l three a n t i s e r a contained a n t i b o d i e s which combined with galactose or l a c t o s e u n i t s . These a n t i b o d i e s have been i s o l a t e d by adsorption on l a c t o s y l - s e p h a r o s e and e l u t i o n of the adsorbed a n t i b o d i e s with galactose and l a c t o s e s o l u t i o n s . T y p i c a l e l u t i o n p a t t e r n s f o r the three a n t i s e r a samples and f o r a pre-immune serum from one o f the r a b b i t s are shown i n Figure 4. I t w i l l be noted i n p a t t e r n A o f F i g u r e 4, t h a t only serum p r o t e i n was present i n the pre-immune serum and a n t i b o d i e s were not e l u t e d by galactose or l a c t o s e . P a t t e r n Β of the Figure shows t h a t , i n a d d i t i o n t o the serum p r o t e i n s , two 280 nm absorbing components were obtained from the anti-iS. f a e c a l i s serum. One component e l u t e d with galactose and the other e l u t e d with l a c t o s e .

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

CARBOHYDRATE—PROTEIN INTERACTION

HO(CH ) C0 CH CH

+

2

R 0 (CH ) C0 CH 2

e

2

8

2

2

3

3

J NH -NH ,EtOH 2

2

R 0 (CH ) CONH-NH 2

8

2

t-BUTYL NITRITE DIMETHYL FORMAMIDE SULFAMIC ACID R Ο (CH ) C0N 2

t

8

3

Buffered BSA soin. pH8.9

R Ο (CH ) CONH-BSA 2

8

(6AL-BSA) ! for the synthesis of gafoctosyl bovine seru min (Gal-BSA)

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Journal of Biochemistry

Figure 3. Agar diffusion plates: left plate as labeled, right plate contains anti-gal or anti-foc antibodies in the center wells and the diheteroglycan in the peripheral wells (29)

Figure 4. Elution patterns for rabbit antisera samples from a lactosyl sepharose column: A = preimmune serum, Β = anti-S -faecalis serum, C = anti-Gal-BSA serum and D = anti-Lac-BSA serum

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That both components were antibodies was established by agar diffusion tests with these preparations and solutions of the glycan performed i n the usual manner. The results of the agar diffusion tests are shown i n Figure 3, right plate. The two preparations of antibodies have been designated as anti-galactose (anti-gal) antibodies and anti-lactose (anti-lac) antibodies and some of the properties of the two sets of antibodies are described in a later section. The a f f i n i t y chromatographic patterns from the antisera from animals immunized with Gal-BSA and Lac-BSA are shown in patterns C and D respectively of Figure 4. It w i l l be noted that only one set of antibodies i s present in the antisera of animals immunized with these immunogens. Thus only anti-gal antibodies were obtained from the anti-Gal BSA serum and anti-lac antibodies from the anti-Lac BSA serum of the immunogens posses galactosyl units and two possess common terminal β-lactosyl units. Therefore i t was very surprising to find that the three immunogens e l i c i t e d different types of immune responses. A number of properties of the anti-gal and anti-lac antibodies obtained from the anti-S. faecalis serum have been determined. Both of the preparations react i n agar diffusion tests with the glycan (Figure 3, right plate) and are therefore antibodies. Data on hapten inhibition with various carbohydrates and the two anti­ body types are shown in Figure 5. It w i l l be noted in the right panel that the anti-lac antibodies are inhibited only by lactose and lactose derivatives and not by galactose or compounds with terminal galactose units. However the anti-gal antibodies are inhibited by a l l compounds with terminal galactose units as seen by the data i n the l e f t panel of Figure 5. Further, i t should be noted that a forty-fold difference i n the concentration of the inhibitors exists with the anti-lac antibodies being inhibited by the lower concentration of inhibitors. These results establish that one set of antibodies combines with galactose and the other with lactose. The combination occurs even when the galactose or lactose are terminal structural units of other compounds. The sedimentation constants and molecular weights calculated from ultracentrifugation data for both types of antibodies were 7s and 150,000 respectively. Agar diffusion tests with goat antisera against rabbit IgA, IgG and IgM showed that both antibody sets are of the IgG immunoglobulin type (29). When the anti-gal and the anti-lac antibodies were subjected to gel electrophoresis an unexpected result was obtained. Such gels showed that each preparation consisted of multi-protein components with the anti-gal antibodies consisting of 6 proteins and the anti-lac antibodies consisting of 12 different proteins. Photographs of the gels for the two preparations are shown in Figure 6. Gels stained with glycoprotein stains (30,31) showed that a l l the components are glycoproteins.

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Journal of Biological Chemistry

Figure 5. Quantitative inhibition curves for the precipitin reactions between the diheteroglycan and the anti-gal antibodies (left panel) or the anti-foe antibodies fright panel) (29)

Biochemical and Biophysical Research Communications

Figure 6. Gel electrophoretic patterns for the preparations of anti-gal and antilac antibodies (1)

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Since the components of each set were eluted by the same hapten group, galactose or lactose, and each protein combines with the same structural unit of the antigen, the individual proteins of each set have been designated as isoantibodies. This defi n i t i o n i s a more restrictive definition of an isoantibody than i s employed by immunologists (32) but i s in line with the terminology employed by enzymologists for multi-molecular forms of enzymes (33K

The isoantibodies of the anti-gal and the anti-lac types have been separated into individual components by electrofocusing techniques. Results with the anti-gal isoantibodies are shown in Figure 7, top pattern. It w i l l be noted that six distinct 280 nm absorbing peaks were obtained by the electrofocusing method. Gel electrophoresis patterns of the peak fractions for the anti-gal antibodies are also show noted that each componen electrophoresis. Since the original preparation of anti-gal isoantibodies on ultracentrifugation yield a symmetrical pattern (20), the isoantibodies are of the same molecular size. On the basis of these findings i t i s reasonable to conclude that the individual components are homogeneous antibodies. The three antisera and the anti-glycosyl antibodies isolated from these antisera have been tested in agar diffusion tests for cross-reactivity with the three immunogens and with bovine serum albumin. These results are shown i n Figure 8. It w i l l be noted i n the top panels of Figure 8 that the original anti-Gal-BSA serum and the purified anti-gal antibodies from this serum reacted only with Gal-BSA but not with the other immunogens tested, Lac-BSA, glycan or BSA. As seen in the middle panels of the Figure, the anti-Lac-BSA serum reacted with Lac-BSA, Gal-BSA and BSA but the anti-lac antibodies from such antiserum reacted only with the Lac-BSA. Evidently this serum contained a population of antibodies which reacted with the BSA portion of the immunogens, thereby accounting for the reaction of this serum with Gal-BSA and BSA. The anti-S. faecalis antiserum reacted with Lac-BSA and the glycan as shown i n the bottom panels of Figure 8. The anti-lac antibodies from this serum also reacted with these two immunogens yielding a similar pattern as the unfractionated antiserum. It should be noted that the antiserum and antibody preparations exhibited p a r t i a l identity with these two antigens. The anti-gal antibodies also reacted with Lac-BSA and the glycan but the prec i p i t i n bands were quite different. The antisera from six rabbits immunized with vaccine of d i heteroglycan i n s i t u i n the c e l l wall, from three rabbits immunized with the Lac-BSA, and from two rabbits immunized with Gal-BSA vaccine have been analyzed by the above method. The results with a l l rabbits collaborate the findings presented i n the foregoing. These findings can be interpreted as a manifestation of the mode of interaction of the immunodeterminant groups of the immunogens

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Figure 7.

Electrofocusing pattern (top panel) and gel ekctrovhoretic patterns (bottom panel) for the anti-gal isoantibodies (20)

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Figure 8.

Agar diffusion plates of various antisera and antibody preparations with the different immunogens

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with the substances that compose the receptor sites of the immunocytes of the host. Two types of interactions w i l l be considered and the consequences of these interactions w i l l be indicated. F i r s t , i n line with the current notion that a single immunocyte synthesizes a single type of protein (19), i t i s l i k e l y that six different immunocytes are stimulated by the terminal galactose units and twelve different immunocytes are stimulated by terminal lactose units. Since each of the immunocytes i s programmed to synthesize a different and unique protein, six anti-gal proteins and twelve anti-lac proteins w i l l be produced. If this interpretation i s correct then the isoantibodies of each set could be significantly different i n the basic structure of the polypeptide chains. Studies are in progress on the determination of the structural features of the isoantibodies with the view of obtaining evidence for or agains Second, the interactio be one i n which the immunodeterminant group of the immunogen combines with a single immunocyte. This single type of immunocyte w i l l multiply to produce a uniform population of the new immunocytes. These immunocytes produce a protein with the same basic polypeptide structure but i n the processing of the protein, different amounts of carbohydrate residues or amide groups are introduced into the protein. As a result the antibodies directed against a single immunodeterminant group occur i n multiple molecular forms, the isoantibodies. If the f i r s t interpretation i s the correct one, then i n the immunization with £. faecalis c e l l s with the diheteroglycan of glucose and galactose, one group of immunocytes recognizes terminal galactose units while another group recognizes terminal lactose units. The result i s the multiplication of these two groups of c e l l s and the synthesis of sets of anti-gal and anti-lac isoantibodies. If the second interpretation i s the correct one, then only one immunocyte recognizes the galactose units and another immunocyte recognizes the lactose units. In this case, one population of a single c e l l type leads to the synthesis of the set of anti-gal isoantibodies and another population of a single c e l l type leads to the synthesis of the set of anti-lac isoantibodies. In the immunizations with the synthetic carbohydrate-protein conjugates the galactosyl unit of Gal-BSA i s responsible for the stimulation of immunocytes leading to the production of anti-gal isoantibodies, while with the Lac-BSA only the lactosyl moiety i s recognized by the immunocytes and only anti-lac isoantibodies are produced. With the latter antigen anti-gal isoantibodies are not produced even though the immunogen possesses terminal galactose units. The differences in the responses to the three immunogens with the same immunodeterminant groups can be interpreted to indicate a role for c e l l surface topography and macromolecular conformation of the immunogen i n directing the synthesis of antibodies. Several recent reports (34-36) have appeared pointing to possible roles for these structural features i n the interaction of

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immunodeterminant groups with the receptor substances on different c e l l surfaces. Literature Cited 1.

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

9.

10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

20. 21.

Pazur, J . H., Miller, K. B., Dreher, K. L. and Forsberg, L. S., Biochem. Biophys. Res. Commun., (1976), 70, 545-550. Avery, O. T., Heidelberger, Μ., and Goebel, W. F., J . Exp. Med., (1925), 42, 709-725. Kabat, Ε. Α., and Berg. D., J . Immunol., (1953), 70, 514-532. Krause, R. M. and McCarty, Μ., J Exp. Med., (1962) 115, 131-140. Pazur, J. Η., Anderson, J . S. and Karakawa, W. W., J . B i o l . Chem., (1971), 246 Gold, P., and Freedman 439-461. Terry, W. D., Henkart, P. Α., Coligan, J . E. and Todd, C. W., Transplant. Rev., (1974), 20, 100-129. Hammarström, S., Engvall, E., Johansson, B. G., Svensson, S., Sunblad, G. and Goldstein, I. J., Proc. Nat. Acad. Sci., USA, (1975), 72, 1528-1532. Kabat, Ε. Α., in H. S. Isbell (Editor), "Carbohydrates i n Solution, Advances i n Carbohydrate Series," 334-361, American Chemical Society, Washington, D.C. (1973). Siddiqui, B. and Hakomori, S., J . Biol. Chem., (1971), 246, 5766-5769. Sung, S. S. J . , Esselman, W. J . and Sweeley, C. C., J . B i o l . Chem., (1973), 248, 6528-6533. Karush, F., J . Am. Chem. Soc., (1957), 79, 3380-3384. Allen, P. Ζ., Goldstein, I. J., and Iyer, R. Ν., Biochemistry, (1967), 6, 3029-3036. Lemieux, R. U., Bundle, D. R. and Baker, D. Α., J. Am. Chem. Soc., (1975), 97, 4076-4083. Zopf, D. Α., Tsai, C. and Ginsburg, V., Arch. Biochem. Biophys., (1978), 185, In Press. Lancefield, R. C., Harvey Lectures Ser., (1940-1941), 36, 251-290. Duff, R. and Rapp, F., J . V i r o l . , (1971), 8, 469-477. H i l l , Μ., and Hillova, J., Adv. Cancer Res., (1976), 23, 237-297. Eisen, Η. N. i n B. D. Davis, R. Dulbecco, H. N. Eisen, H. S. Ginsberg and W. A. Wood (Editors), "Microbiology," Chapters 14 and 17, Harper and Row Publishers, Hagerstown, MD (1973). Pazur, J . Η., and Dreher, K. L., Biochem. Biophys. Res. Commun., (1977), 74, 818-824. Pazur, J . Η., Cepure, Α., Kane, J . A. and Karakawa, W. W., Biochem. Biophys. Res. Commun., (1971), 43, 1421-1428.

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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22.

115

Karakawa, W. W., Wagner, J . E. and Pazur, J . H., J . Immunol., (1971), 107, 554-562. 23. Pazur, J . H., Dropkin, D. J., Dreher, K. L., Forsberg, L. S., and Lowman, C. S., Arch. Biochem. Biophys., (1976), 176, 257-266. 24. Kane, J . Α., Karakawa, W. W. and Pazur, J. H., J . Immunol., (1972), 108, 1218-1226. 25. Pazur, J . H., Cepure, Α., Kane, J. and Hellerquist, C. G., J. Biol. Chem., (1973), 248, 279-284. 26. Pazur, J . H. and Forsberg, L. S., Carbohyd. Res., (1978), 58, In Press. 27. Lemieux, R. U., Baker, D. Α., and Bundle, D. R., Can. J . Biochem., (1977), 55, 507-512. 28. Dubois, M., G i l l e s Κ Α. Hamilton J D. Rebers D A and Smith, F., Anal 29. Pazur, J . H., Dreher Chem., (1978), 253, In Press. 30. Fairbanks, G., Steck, T. L. and Wallach, D. F. H., Biochemistry, (1971), 10, 2606-2617. 31. Eckhardt, Α. Ε., Hayes, C. E. and Goldstein, I. J., Anal. Biochem., (1976), 73, 192-197. 32. Potter, M., Lieberman, R., and Dray, S., J. Mol. Biol., (1966), 334-346. 33. Commission on Biochemical Nomenclature, "Enzyme Nomenclature," pg. 32, Elsevier Scientific Publishing Co., Amsterdam, The Netherlands (1973). 34. Ramasamy, R., Immunochemistry, (1976), 13, 705-708. 35. Ostrand-Rosenberg, S., Immunogenetics, (1976), 3, 53-64. 36. Gurd, J. W., Biochemistry, (1977), 16, 369-374. RECEIVED September 8, 1978.

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

10 The Specificity of Sugar Taste Responses in the Gerbil WILLIAM JAKINOVICH, JR. The Department of Biological Sciences, Herbert Lehman College, The City University of New York, Bronx, NY 10468

S i n c e s u c r o s e is one o f t h e most common s o l u b l e sugars a s s o c i a t e d w i t s in seeds (1,2), t h e ability an a n i m a l ' s ability t o t a s t e s u c r o s e (3,4) - hence, the e v o l u t i o n o f a s u c r o s e r e c e p t o r site. Such an i d e a has s u p p o r t because s u c r o s e is t h e sweetest sugar t o humans (5,6), and by f a r t h e most u n i f o r m l y p r e f e r r e d by mammals (7,8). Moreover, it evokes in mammals a g r e a t e r g u s t a t o r y n e r v e d i s c h a r g e than any o t h e r sugar (3,9,10,11). T h i s is a r e p o r t o f a d e t a i l e d physiological study o f t h e sugar t a s t e response in t h e gerbil (12,13,14). For the gerbil, the best taste s t i m u l i a r e s u g a r s which most c l o s e l y resemble sucrose. Method The M o n g o l i a n gerbil was chosen as t h e e x p e r i mental a n i m a l because among mammals it p o s s e s s e s t h e l a r g e s t t a s t e n e r v e r e s p o n s e and l o w e s t t a s t e t h r e s h old to s u c r o s e (3). The sugar t a s t e response used was the i n t e g r a t e d (average) response o f t h e t a s t e neurons s i n c e this is an i n d e x o f t h e response o f t h e entire t a s t e r e c e p t o r p o p u l a t i o n (15). The electrical activity was o b t a i n e d by t o u c h i n g t h e chorda tympani n e r v e o f an a n e s t h e t i z e d g e r b i l w i t h a nichrome e l e c t r o d e (100 µm diameter) connected t o a d i f f e r e n t i a l a m p l i f i e r . The electrical activity, displayed on an oscilloscope, c o u l d be m o n i t o r e d by a l o u d s p e a k e r ( F i g u r e 1 ) . Stimulus P r e s e n t a t i o n . D i s t i l l e d water was a l l o w e d t o f l o w c o n t i n u o u s l y a t a r a t e o f 0.13-0.17 ml/sec o v e r a g e r b i l ' s tongue, extended w i t h a f i n e fishhook. T e s t s o l u t i o n s (2-4 ml) were a l t e r n a t e d w i t h t h e d i s t i l l e d water r i n s e w i t h o u t i n t e r r u p t i o n o f 0-8412-0466-7/79/47-088-116$05.00/0 © 1979 American Chemical Society

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f l u i d flow. The temperature o f the d i s t i l l e d water r i n s e and o f the t a s t e s o l u t i o n were i d e n t i c a l (25° * 1 ° C ) . Sugar s o l u t i o n s were p r e s e n t e d i n a s e r i e s o f s t e p s i n c r e a s i n g by a p p r o x i m a t e l y 1/2 l o g m o l a r con­ c e n t r a t i o n , i . e . , 0.0001 M, 0.0003 M, 0.001 M...0.1 M, 0.3 M. Each a n i m a l r e c e i v e d a s u c r o s e s e r i e s and a t e s t compound s e r i e s a t l e a s t t w i c e . The mean responses were c a l c u l a t e d and used i n f u r t h e r a n a l y s i s . A s t a n d a r d s o l u t i o n (0.3 M sucrose) was p r e s e n t e d f r e ­ q u e n t l y between t e s t s o l u t i o n s . Whenever the s t a n d a r d s o l u t i o n s e l i c i t e d r e s p o n s e s t h a t v a r i e d more than * 10%, a l l i n t e r j a c e n t r e s p o n s e s were r e j e c t e d . Sugars. Sugars were o b t a i n e d L a b o r a t o r i e s , Waukegan Company, S t . L o u i s 13,14).

from P f a n s t i e h l

Results When any e f f e c t i v e c h e m i c a l flowed onto the tongue, t h e r e was an i n i t i a l r a p i d r i s e o f n e u r a l a c t i v i t y which was dependent upon c o n c e n t r a t i o n . (A response i n t h i s study was d e f i n e d as the d i f f e r e n c e between the i n t e g r a t e d p o t e n t i a l o f the spontaneous a c t i v i t y evoked by the water f l o w and the g r e a t e s t i n t e g r a t e d p o t e n t i a l e l i c i t e d by a g i v e n s o l u t i o n a p p l i e d t o the tongue.) F i g u r e 2 i s a t y p i c a l s e r i e s o f r e c o r d i n g s . On s e m i l o g a r i t h m i c c o o r d i n a t e s , the c o n c e n t r a t i o n - r e s p o n s e f u n c t i o n f o r sugars was always s i g m o i d a l , s i m i l a r t o the s u c r o s e c o n c e n t r a t i o n response c u r v e ( F i g u r e 3 ) . Disaccharides. s u c r o s e was the b e s t g r e a t e s t response a t disaccharides tested 30 times h i g h e r than

Of a l l the d i s a c c h a r i d e s t e s t e d , s t i m u l u s ( T a b l e I ) ; i t gave the a l l concentrations. A l l other had response t h r e s h o l d s 10 o r sucrose.

M e t h y l G l y c o s i d e s o f D - F r u c t o s e and D-Glucose. Reducing sugars m u t a r o t a t e i n aqueous s o l u t i o n s p r o d u c i n g isomers which have d i f f e r e n t t a s t e i n t e n s i t i e s (1^,17,18,19,20^) and t a s t e q u a l i t i e s (21,22) ; such isomers t e n d t o o b s c u r e t a s t e - s t r u c t u r e r e l a t i o n s h i p s . T h e r e f o r e , where p o s s i b l e , m e t h y l g l y c o s i d e s o f D - f r u c t o s e and D-glucose were compared. The two m e t h y l g l y c o s i d e s whicH resemble the m o i e t i e s o f s u c r o s e most c l o s e l y , m e t h y l α-D-glucopyranoside and m e t h y l 3-Df r u c t o f u r a n o s i d e were the most s t i m u l a t o r y ( F i g u r e 4 ) .

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Integrator and

®

Otcflkwcopt

Figure 1.

Schematic drawing of the experimental set up

w

fl/

Out

Ntrvt

10 sec 0.001

0.1

0.003

0.01

0.3

0.03

1.0M Sucrose Brain Research

Figure 2. Integrated neural discharge from the gerbiVs chorda tympani nerve in response to a series of increasing concentrations of sucrose applied to the tongue. The solid bars under the records indicate stimulus duration, R is the measure of response (12).

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CONCENTRATION (Molor)

Figure 3. Plot of neural discharge from Figure 2

Concentration (molar) Figure 4. Mean integrated response of the chorda tympani nerve discharge in the gerbil to sucrose (A), methyl α-Ό-glucopyranoside (A) methyl β-Ό-glucopyranoside methyl β-Ό-fructofuranoside (%), methyl α-Ό-fructofuranoside (O), methyl a-D-fructopyranoside (H), methyl β-Ό-fructopyranoside (if)> l 5~anhydroΏ-mannitol (O), fructose (equilibrium mixture, 25°C) (V). Responses are relative to the maximum sucrose response. y

y

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

Glu Glu Glu Glu Glu Glu Glu Glu Gai Gai Gai Gai Gai

a ( l ~ 2 )ι F ru a ( l - 3 ) Fru a(l-"6) Fru a ( l - 4 ) Glu β ( 1 - 4 ) Glu α ( 1 - 4 ) GluOH β ( 1 - 4 ) GluOH α(1~1) G l u β(1-4) Fru β ( 1 - 4 ) Glu α ( 1 - 6 ) Glu β(1-^) GluOH α ( 1 - 4 ) GluOH

Structure*

0.001 0.03 0.03 0.01 0.01 0.03 0.03 0.03 0.01 0.01 0.03 0.01 0.03

Thresh­ old** (molar) 50

± 0.05

± 0.03 ± 0.02 ± 0.03

0.24

0.21 0.18 0.18

0.042 ± 0.005 0.23 ± 0.02

(molar)

C R

A

0.037 0.30 0.49 0.29 0.33 0.34 0.50 0.26 0.23 0.31 0.37 0.26 0.23

d (molar)

K

0.68

0.83 0.88

0.75

1.0 0.69

± 0.27

± 0.10 ± 0.08

± 0.06

± 0.08

Maximum response

(MEAN VALUES)

* A b b r e v i a t i o n s : G l u , glucose; G a l , g a l a c t o s e ; F r u , f r u c t o s e ; GluOH, g l u c i t o l . ** Threshold i s d e f i n e d as the lowest c o n c e n t r a t i o n t e s t e d which e l i c i t e d a measurable response i n 50% o f the animals. C R ^ Q = the c o n c e n t r a t i o n t h a t evokes a response 50% o f maximum = the d i s s o c i a t i o n constant Ν = no. o f animals a = 95% confidence i n t e r v a l

Sucrose Turanose Palatinose Maltose Cellobiose Maltitol Cellobiitol Trehalose Lactulose β-Lactose Melibiose Lactitol Melibiitol

Sugar

STIMULATING EFFECTIVENESS OF SOME DISACCHARIDES

TABLE I

5 5 5 5

32 5 4 5 5 5 5 5 5

Ν

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Other D - P y r a n o s i d e s . F u r t h e r study o f t h e t a s t e response t o D-pyranose sugars r e v e a l e d t h a t t h e replacement o f t h e anomeric h y d r o x y l groups o f a-gg l u c o p y r a n o s e by a number o f s u b s t i t u e n t groups l e d t o molecules of d i f f e r e n t stimulatory e f f e c t i v e n e s s ( F i g u r e 5 ) : OCH >F>OH>H. I n a d d i t i o n t o replacement o f a s u b s t i t u e n t group, t h e o r i e n t a t i o n o f t h e OCH^ o r OH group i n t h e e q u a t o r i a l p l a n e , as i n m e t h y l β-Dg l y c o p y r a n o s i d e o r β-D-glucopyranose, d r a m a t i c a l l y reduced t h e e f f e c t i v e n e s s o f t h e sugar as a s t i m u l u s ( F i g u r e 6 ) . A s i m i l a r e f f e c t was o b s e r v e d f o r m e t h y l α-g-xylopyranoside and methyl (3-D- ::ylopyranoside. The compounds which d i f f e r e d from methyl a-Dg l u c o p y r a n o s i d e by t h e replacement o r r e o r i e n t a t i o n o f the e q u a t o r i a l h y d r o x y of the g-pyranoside than methyl α-g-glucopyranoside. M e t h y l a-D-mannop y r a n o s i d e , t h e C-2 a x i a l epimer, and m e t h y l a-D-galact o p y r a n o s i d e , t h e C-4 a x i a l epimer, were c o n s i d e r a b l y p o o r e r s t i m u l i compared t o t h e p a r e n t D - g l u c o s i d e ( F i g u r e 7 ) . The response t o t h e 2-deoxy d e r i v a t i v e , methyl 2-deoxy-a-D-arabino-hexopyranoside, was i n f e r i o r t o t h e response o f m e t h y l a - D - g l u c o p y r a n o s i d e . In c o n t r a s t , s t i m u l a t i o n w i t h methyl a-D-xylop y r a n o s i d e , which l a c k s a hydroxy-methyl group a t t h e C-5 p o s i t i o n o f methyl a - D - g l u c o p y r a n o s i d e , e q u a l e d t h e response o f m e t h y l a - D - g l u c o p y r a n o s i d e ( F i g u r e 7A). Polyols. L i n e a r p o l y o l s c o n t a i n i n g two carbon t o seven carbon atoms a l l evoked n e u r a l r e s p o n s e s . Two i m p o r t a n t r e s u l t s were o b s e r v e d ( F i g u r e 8 ) : (1) The e f f e c t i v e n e s s ( C R ^ Q , ) o f t h e p o l y o l s i n c r e a s e d as t h e c h a i n l e n g t h i n c r e a s e d up t o f i v e carbon atoms; (2) I n c o n t r a s t t o monosaccharides, t h e c o n f i g u r a t i o n s o f l i n e a r p o l y o l may n o t p l a y a r o l e i n t h e t a s t e response. T h i s i s i n d i c a t e d by t h e i d e n t i c a l responses t o t h e f o u r p e n t i t o l s : D-arabinitol, L-arabinitol, D-ribitol, or D-xylitol. Competitive I n t e r a c t i o n . The t h e o r e t i c a l c u r v e f o r competitive i n t e r a c t i o n s f i t s the a c t u a l data very c l o s e l y with the f o l l o w i n g discrepancies (Figure 9 ) . The m i x t u r e o f 1.0 M m e t h y l α-D-glucopyranoside and s u c r o s e and t h e m i x t u r e o f 0.1~M m e t h y l a-D-gluco­ p y r a n o s i d e and s u c r o s e evoked r e s p o n s e s s l i g h t l y g r e a t e r and s l i g h t l y lower, r e s p e c t i v e l y , than p r e ­ d i c t e d by t h e c u r v e . I n no case d i d t h e response t o the m i x t u r e e v e r exceed t h e maximum r e s p o n s e evoked by sucrose alone.

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Figure 5. Effect of the substituent group at position C-l on the stimulatory ability of Ώ-glucopyranose. Responses are refotive to the maximum sucrose re­ sponse. Methyl α-Ό-glucopyranoside (Άλ α-Ό-glucopyranosyl fluoride Ν = 5); myo-inositol* (6C, 0 , Ν = 10); perseitol* (7C, Δ, Ν — 6); sucrose (V, Ν «= 28). Asterisk (*) indi­ cates sugars whose insolubility prevented direct determination of maximum re­ sponse. The CR for these compounds was estimated from K . S0

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Figure 9. Concentration-response curve of sucrose in the presence of methyl a-D-glucopyranoside (MaG). The solid lines are theoretical curves obtained from an equation describing the competitive interaction of two substances with a single receptor site (see below). Data points for sucrose alone (%); sucrose -j- O.J M MaG (A); sucrose + 0.3 M M a G , • ; sucrose + 1.0 M M a G , O ; M a G alone (MiDashed line is the theoretical curve drawn from the binding equation for MaG. Κ,ι for sucrose (0.05 M) and K for MaG (0.18 M) were determined from the Beidler plot (see inset). CR for sucrose = 0.052 and for MaG = 0.18 (13). d

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S i m i l a r t o the m i x t u r e s o f m e t h y l a-D-gluco­ p y r a n o s i d e and s u c r o s e , m i x t u r e s o f s u c r o s e and g - s o r b i t o l c l o s e l y f i t the c o m p e t i t i v e i n t e r a c t i o n c u r v e ( F i g u r e 10). Response t o the m i x t u r e gave a less-than-additive e f f e c t at high concentrations. The maximum response f o r the m i x t u r e d i d not exceed the maximum response evoked by s u c r o s e a l o n e . Discussion As a t a s t e s t i m u l u s , t h e e f f e c t i v e n e s s o f s u c r o s e o v e r o t h e r d i s a c c h a r i d e s and the e f f e c t i v e n e s s o f methyl β-D-fructofuranoside and m e t h y l a - g - g l u c o p y r a n o s i d e o v e r o t h e r monosaccharides can be e x p l a i n e d by the p r e s e n c e o f on of s i t e s : sucrose, furanose. ~~ The β-D-Fructofuranose S i t e . I n t h e g e r b i l MBFF (methyl β-D-fructofuranoside) as w e l l as s u c r o s e (α-g-glucopyranosyl - β-D-fructofuranoside)was the most s t i m u l a t o r y D - f r u c t o s e d e r i v a t i v e s t e s t e d . T h i s f i n d i n g s u g g e s t s the p r e s e n c e o f a s p e c i f i c f r u c t o s e site. T h i s i s n o t u n l i k e the f l y ' s g - f r u c t o s e s i t e (23) which responds b e s t t o β-g-fructofuranose (24). In c o n t r a s t man may have a β-D-fructopyranose s i t e s i n c e t h i s compound i s b e l i e v e d t o be the sweetest f r u c t o s e isomer (25^,26). F u r t h e r evidence f o r a D-fructose s i t e i n otKer animals completely d i s t i n c t ïrom a s u c r o s e o r a D-glucose s i t e i s e v i d e n t i n s i n g l e f i b e r responses (27^28) o r i n b i o c h e m i c a l s t u d i e s (29). Some d e t a i l s o f t h e s p e c i f i c D - f r u c t o s e response follow: (1) Those compounds such as 2 - d e o x y - g - f r u c t o s e (1,2-anhydro-g-mannitol) which l a c k a h y d r o x y l group o r c o n t a i n a b u l k y s u b s t i t u e n t a t C-3 ( t u r a n o s e ) , C-4 ( l a c t u l o s e ) o r C-6 ( p a l a t i n o s e ) a r e n o t as e f f e c t i v e s t i m u l i as MBFF o r s u c r o s e (Sucrose can be c o n s i d e r e d t o be a g - f r u c t o s e d e r i v a t i v e w i t h a b u l k y d e r i v a t i v e a t C-2, i . e . a - D - g l u c o p y r a n o s y l - 3 - g - f r u c t o f u r a n o s i d e ) . The t h r e e g - f r u c t o s e - c o n t a i n i n g d i s a c c h a r i d e s a r e r e d u c i n g s u g a r s , u n l i k e s u c r o s e , and e x i s t i n s o l u t i o n as a m i x t u r e o f f u r a n o s e and pyranose i s o m e r s . Should the D - f r u c t o s e s i t e r e q u i r e the β-D-fructofuranose r i n g form, s u c r o s e would be the most s t i m u l a t o r y . (2) The g - f r u c t o s e d e r i v a t i v e s which c o n t a i n p y r a n o i d r i n g s such as m e t h y l α-g and 3 - g - f r u c t o p y r a n o s i d e s were poor s t i m u l i compared t o MBFF. (3) A f u r a n o i d r i n g D - f r u c ­ t o s e d e r i v a t i v e i n i t s e l f does n o t produce a good response as i n d i c a t e d by the reduced response o f m e t h y l α-D-fructofuranoside. (4) The e q u i l i b r a t e d m i x t u r e o f

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Figure 10. Concentration-response curve of sucrose in the presence of D-sorbitol [sorb]. The solid lines are the theoretical curves obtained from the equation describing the competitive interaction of two substances with a single receptor site (see Figure 9). Data points for sucrose alone (%) sorbitol alone (Ά λ sucrose + 0.03 M [sorb] O , sucrose + 0.1 M [sorb] (jg), sucrose + 0.3 M [sorb] (O). Dashed line ( ) is the theoretical curve drawn from the taste equation for sorbitol alone. K for sucrose = 0.015 M, CR for sucrose = 0.016 M, K for sorbitol -= 0.29 M. Dissociation constants were determined from the Beidler plot (inset) (14). y

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Figure 11. Proposed model for the "sucrose site" in the membrane of the gerbil gustatory cell (14)

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g - f r u c t o s e ( F i g u r e 4 ) , which has 31% β-D-fructof u r a n o s e (30), was a l s o a h i g h l y s t i m u l a t o r y monosac­ charide. The α-Ρ-Glucopyranose S i t e . S i n c e methyl ct-gg l u c o p y r a n o s i d e was t h e most s t i m u l a t o r y g l u c o s e d e r i v a t i v e t e s t e d , t h e p r e s e n c e o f an a-D-glucopyranose r e c e p t o r s i t e was s u g g e s t e d . T h i s t y p e o f s i t e has been p o s t u l a t e d i n t h e b l o w f l y and f l e s h f l y (2±,31) · Some o f t h e c h a r a c t e r i s t i c s o f t h e t a s t e response to D-glucopyranosyl d e r i v a t i v e s follow: (1) U n l i k e the f l y ' s t a s t e r e c e p t o r (24,31), t h e r e i s no s t r i c t c o n f i g u r a t i o n a l r e q u i r e m e n t f o r an a - g l u c o s i d e i n t h e g e r b i l ' s t a s t e r e s p o n s e . N e v e r t h e l e s s ot-g-glucopyranosyl derivative the β-g-glucopyranosy the M o n g o l i a n g e r b i l compares f a v o r a b l y t o humans (5^, 16,20), t h e hamster (32.) and o t h e r g e r b i l s p e c i e s (3) I n wKich α-D-glucopyranose d e r i v a t i v e s a r e sweeter o r produce l a r g e r t a s t e r e s p o n s e s than β-D-glucopyranose derivatives. (2) The r e q u i r e m e n t o f e q u a t o r i a l h y d r o x y I s a t C-2 and C-4 o f t h e g - g l u c o p y r a n o s i d e m o l e c u l e f o r a maximum s t i m u l a t i o n i n t h e g e r b i l p a r a l l e l s a s i m i l a r r e q u i r e m e n t f o r i n c r e a s e d sweet t a s t e i n man (33.) and maximum s t i m u l a t i o n i n t h e sugar r e c e p t o r s o f t h e g l y (3JL) . (3) The r e s i d u e s a t t h e C-5 p o s i t i o n a r e l e s s i m p o r t a n t i n b o t h t h e f l y (31) and g e r b i l . (4) The f a i l u r e o f t h e a-D-glucopyranos i d e s , t u r a n o s e , p a l a t i n o s e , m a l t o s e , m a l t i t o l and t r e h a l o s e t o s t i m u l a t e as w e l l as s u c r o s e o r methyl α-g-glucopyranoside c o u l d be a t t r i b u t a b l e t o s t e r i c hinderance i n v o l v i n g the substituents a t p o s i t i o n C - l of the glucopyranoside r i n g . The S u c r o s e S i t e . The p r e v i o u s d i s c u s s i o n s can­ not exclude the presence o f a s u c r o s e - s p e c i f i c s i t e s i n c e s u c r o s e can f i t i n t o a D-glucose s i t e and D-fructose s i t e simultaneously. A sucrose binding s i t e s e p a r a t e from a g l u c o s e o r f r u c t o s e b i n d i n g s i t e has been found i n cow tongue t a s t e p a p i l l a e (29). Based on the f o l l o w i n g r e s u l t s t h e p r e s e n c e o f a " s u c r o s e s i t e " as shown i n F i g u r e 11 i s s u g g e s t e d ; (1) Among t h e d i s a c c h a r i d e s s u c r o s e was t h e most s t i m u l a t o r y s u g a r . (2) M e t h y l ct-D-glucopyranoside and m e t h y l β-D-fructof u r a n o s i d e , which have s t r u c t u r a l f e a t u r e s i n common w i t h s u c r o s e were t h e most e f f e c t i v e monosaccharides f o r e l i c i t i n g a n e u r a l response. (3) The l e n g t h s o f superimposed D r e i d i n g models o f a p e n t i t o l and s u c r o s e c o i n c i d e almost p e r f e c t l y . (This c o u l d a c c o u n t f o r t h e l e v e l i n g o f f o f t h e r e s p o n s e t o t h e p o l y o l s as t h e

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number o f carbon atoms i n c r e a s e ) . (4) The r e s p o n s e s t o m i x t u r e s o f methyl α-g-glucopyranoside and s u c r o s e and m i x t u r e s o f g - s o r b i t o l and s u c r o s e suggest t h a t t h e s e sugars a c t a t a common r e c e p t o r s i t e . The " s u c r o s e s i t e " p r e s e n t e d i n F i g u r e 11 would evoke a response when a sugar m o l e c u l e o c c u p i e d i t as follows: The β-fructofuranosyl p o r t i o n i s t e n t a t i v e l y c o n s i d e r e d t o occupy a "deep" s u b s i t e and t h e hydroxym e t h y l group a t C-5 o f t h e g l u c o p y r a n o s i d e i s e x p e c t e d to p r o j e c t i n t o the s o l u t i o n . The deep s u b s i t e i s a s s o c i a t e d w i t h a h i g h degree o f s p e c i f i c i t y as e v i ­ denced by t h e f a i l u r e o f t h e two f r u c t o s y l g l u c o s i d e s , t u r a n o s e ( 3 - 0 - a - D - g l u c o p y r a n o s y l - D - f r u c t o s e ) and p a l a t i n o s e ( 6 - 0 - a - g - g l u c o p y r a n o s y I - D - f r u c t o s e ) t o be as s t i m u l a t o r y as s u c r o s e data support the propose p o s i t i o n s C - l , C-2, and C-4 o f t h e D-glucose moiety. The D - p y r a n o s i d e s which have e q u a t o r i a l s u b s t i t u e n t s a t C-2 and C-4 and t h e C - l a x i a l s u b s t i t u e n t were t h e most e f f e c t i v e monosaccharides. A C-5 hydroxymethyl b i n d i n g locus i s not required. T h i s s u p p o r t s a model w i t h t h e C-6 hydroxy group p r o t r u d i n g i n t o t h e s u r r o u n d i n g s o l u ­ tion. The enhanced a c t i v i t y o f methyl a-D-gluco­ p y r a n o s i d e o v e r α-D-glucopyranose p o i n t s t o a h y d r o ­ phobic region i n the s i t e . This coincides with the r e l a t i v e l y h y d r o p h o b i c c a r b o n atom o f t h e f r u c t o s e moiety shown i n F i g u r e 11. A l s o , l a r g e b u l k y s u b s t i t u ­ e n t groups a t C - l would b l o c k e f f e c t i v e i n t e r a c t i o n between t h e sugar and t h e s u c r o s e s i t e . Therefore, t h i s model would a c c o u n t f o r t h e r e l a t i v e l y weak r e s p o n s e s o f t h e o t h e r d i s a c c h a r i d e s as g u s t a t o r y stimulants.

Abstract THE SPECIFICITY OF SUGAR TASTE-RESPONSE IN THE GERBIL Solutions of sugars were applied to the tongues of gerbils, and the responses of their taste nerves (chorda tympani) were measured electrophysiologically. Sucrose elicited a nerve response at lower concentra­ tions than any other substance tested. The a b i l i t y of a monosaccharide or polyol to e l i c i t a nerve response depends on the degree to which it resembles sucrose in certain structural details; structures nearly identi­ cal to either half of the sucrose molecule give responses at lower concentrations than molecules that differ considerably from sucrose in conformation and configuration. Responses to mixtures of sucrose with methyl α-D-glucopyranoside or D-sorbitol indicate that

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a c t a t a common r e c e p t o r site. These suggest t h e e x i s t e n c e o f a s u c r o s e r e c e p ­

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September 8, 1978.

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

11 The Coordinated Action of the T w o Glycogen Debranching Enzyme Activities on Phosphorylase Limit Dextrin T. E. NELSON and R. C. WHITE Department of Rehabilitation, Baylor College of Medicine, Houston, TX 77030 B. K. GILLARD Department of Pediatrics, University of California School of Medicine, Los Angeles, CA 90024 Glycogen debranching enzyme ( a m y l o - 1 , 6 - g l u c o s i d a s e / 4 - α – glucanotransferase) fro r a b b i muscl is th classical m u l t i catalytic site enzyme a s s o c i a t e the total degradation o f glycogen ( 1 ) . The enzyme was dis­ covered in 1950 by C o r i and Larner when it was found t h a t h i g h l y purified phosphorylase ( 1 , 4 - α - D - g l u c a n : o r t h o p h o s p h a t e α - g l u c o s y l t r a n s f e r a s e , EC 2 . 4 . 1 . 1 ) would not completely degrade glycogen whereas l e s s purified p r e p a r a t i o n s would ( 2 ) . They showed t h a t phosphorylase could not go past the outer tier branch p o i n t s o f glycogen and produced a limit d e x t r i n s t r u c t u r e which contained the original branches w i t h an average o f four r e s i d u e s l e f t on each c h a i n . At the time it was thought t h a t the limit d e x t r i n produced by phosphorylase (Ø-dextrin, LD) had an asymmetric s t r u c t u r e w i t h seven glucose u n i t s on the main c h a i n and a s i n g l e glucose u n i t remaining as the branch. The debranching enzyme was thought to have a s i n g l e activity, the f u n c t i o n o f which was to remove the glucose s t u b , thus a l l o w ­ ing phosphorylase to f u r t h e r a t t a c k the debranched s t r u c t u r e . The enzyme was c a l l e d a m y l o - l , 6 - g l u ç o s i d a s e , i n r e c o g n i t i o n o f i t s a c t i o n as a s p e c i a l i z e d g l u c o s i d à s e ( 2 ) . About 10 years l a t e r i t was found t h a t the phosphorylase l i m i t d e x t r i n o f glycogen ( 0 - d e x t r i n ) had a symmetrical outer t i e r s t r u c t u r e w i t h four r e s i d u e s on each c h a i n and t h a t a m y l o - l , 6 - g l u c o s i d a s e p r e p a r a ­ t i o n s contained a second a c t i v i t y . T h i s second a c t i v i t y was a t r a n s f e r a s e which was suggested by Whelan and co-workers to d i s ­ p r o p o r t i o n a t e the branch c h a i n by t r a n s f e r r i n g a three u n i t seg­ ment to the main c h a i n , e l o n g a t i n g i t and l e a v i n g a one u n i t stub C 3 , 4 ) . The presence o f t h i s second a c t i v i t y was confirmed by C o r i , I l l i n g w o r t h and Brown and named o l i g o - l , 4 - * l , 4 - g l u c a n t r a n s f e r a s e " (5_,6) . T h i s sequence o f a c t i o n s and s t r u c t u r e s i s shown i n F i g u r e 1. Subsequent work by Brown and I l l i n g w o r t h and t h e i r group, by Whelan and c o l l e a g u e s , and by Hers and co-workers showed t h a t the two a c t i v i t i e s could be measured s e p a r a t e l y and independently o f each other (1>^7>tt

Π

Q

OOOOèO

Legend: Ο: α glucose residues; I: Walker and Whetan structured); Π : Cori and Lamer structure (2); NRT: non-reducing terminal; A: A-chaln; Β: Β-chain; €): glucose residues removed by phosphorylase; · : glucose residues repositioned by the transferase-,^ ο -(l->4Hinlcage; [ll a-(l-*Minlcage; 0-. free glucose residue.

Figure 1.

The action of the dehranching enzyme on phosphoryhse limit dextrin structure

Biochimica et Biophysica Acta

Figure 2. Sodium dodecxjl sulfate-polyacryfomide gel electrophoresis of the purified dehranching enzyme (26)

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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the combined a c t i o n o f both enzymes on l i m i t d e x t r i n which r e ­ q u i r e s both a c t i v i t i e s (1_, 10_, 11) . The g l u c o s i d a s e a c t i v i t y alone can be measured by i t s a b i l i t y to r e i n c o r p o r a t e glucose back i n t o polymer (10). This method was f i r s t developed by Hers and u t i ­ l i z e s l^C-glucose (12,13). T h i s a c t i o n depends on the micror e v e r s i b i l i t y o f the g l u c o s i d a s e r e a c t i o n (14,15,16) . The gluco­ s i d a s e a c t i o n can a l s o be measured by i t s a b i l i t y t o remove a glucose stub from o l i g o s a c c h a r i d e s such as the s i n g l e u n i t branched pentasaccharide " f a s t B5" ( 6 ^ - a - g l u c o s y l m a l t o t e t r a o s e ) produced by α-amylase. T h i s method was developed by Brown and I l l i n g w o r t h (1,]_,17) . The g l u c o s i d a s e w i l l a l s o remove s i n g l e g l u c o s y l stubs from s i n g l y branched α-glucosyl Schardinger d e x t r i n i n an assay method developed by Whelan and h i s group (18). Brown and colleagues have measured the t r a n s f e r a s e chromatographically by i t s a b i l i t y t o d i s p r o p o r t i o n a t In L a m e r s l a b o r a t o r y f e r a s e a c t i v i t y by i t s a b i l i t y t o d i s p r o p o r t i o n a t e o r attenuate the outer chains o f amylopectin was developed (20). The e l o n ­ gated outer chains have more amylose c h a r a c t e r and t h i s can be measured s p e c t r o p h o t o m e t r i c a l l y by the change i n the i o d i n e spec­ trum. T h i s assay f o r t r a n s f e r a s e a c t i v i t y i s independent o f g l u c o s i d a s e a c t i o n (20). We have employed these three k i n e t i c methods o f measuring the dehranching enzyme system t h a t we have developed i n the s t u d i e s t o be d i s c u s s e d : the combined a c t i v i ­ t i e s ( g l u c o s i d a s e - t r a n s f e r a s e ) were measured by the p r o d u c t i o n o f glucose from glycogen phosphorylase l i m i t d e x t r i n o r "LD" (11). The g l u c o s i d a s e a c t i v i t y alone was measured by l^C-glucose i n c o r ­ p o r a t i o n i n t o polymer (16) and the t r a n s f e r a s e a c t i v i t y alone was measured by the change i n the i o d i n e spectrum o f amylopectin (20). Subsequently the enzyme was p u r i f i e d i n L a m e r s l a b o r a t o r y to a s i g n i f i c a n t l y higher degree than p r e v i o u s l y r e p o r t e d . The two a c t i v i t i e s were p h y s i c a l l y i n s e p a r a b l e and r e s i d e d i n a homo­ geneous p r o t e i n . Other groups a l s o were unable t o separate the two a c t i v i t i e s . The molecular weight o f the enzyme was thought t o be 260,000. Because o f i t s l a r g e s i z e , the enzyme was assumed t o be composed o f subunits and t o be double-headed; t h a t i s , two a c t i ­ v i t i e s a s s o c i a t e d w i t h a s i n g l e homogeneous p r o t e i n (7_,23). The p o s s i b i l i t y a l s o remained t h a t i t was a multi-enzyme complex which was d i f f i c u l t t o separate i n t o i t s component a c t i v i t i e s . Addi­ t i o n a l i n f o r m a t i o n p e r t a i n i n g t o the debranching enzyme can be found i n s e v e r a l recent reviews (21,22). The f a c t remained t h a t a l l attempts t o separate the enzyme i n t o subunits o r t o separate the a c t i v i t i e s from each other were u n s u c c e s s f u l (24,25). The reason f o r t h i s i s now c l e a r . We have e s t a b l i s h e d t h a t the enzyme i s a s i n g l e p o l y p e p t i d e molecule under both denaturing and non-denaturing c o n d i t i o n s and t h a t the molecular weight i s 160-170,000 (26,27). The evidence f o r t h i s i s shown i n F i g u r e 2. The f i g u r e shows the homogeneity o f the p r o t e i n u s i n g the method­ ology o f Weber, P r i n g l e and Osborn u s i n g r e d u c t i o n i n the p r e s ­ ence o f 2-mercaptoethanol (MSH), a l k y l a t i o n by i o d o a c e t i c a c i d 1

1

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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(IAA) and dénaturâtion i n sodium dodecyl s u l f a t e (SDS) (28). Band A i s the standard b o i l i n g water bath (BWB) method (Method 1). Band Β i s omission of the BWB treatment, and C i s an extension of the BWB treatment. This i n d i c a t e s t h a t no a r t i f a c t s are produced by contaminating proteases. Band D i s an a l t e r n a t e c o n t r o l t r e a t ­ ment (Method 2) where the enzyme i s f i r s t denatured i n 7 M guanidine»HCl, then reduced i n MSH f o l l o w e d by a l k y l a t i o n by IAA, subsequent d i a l y s i s against 9 M urea and f i n a l l y exposure t o 0.1% SDS. The l a t t e r treatment w i l l d i s s o c i a t e any known p r o t e i n i n t o component polypeptides (28). As can be seen the p r o t e i n i s a s i n ­ gle polypeptide i n a l l cases. Figure 3 shows the molecular weight under denaturing c o n d i t i o n s i n SDS to be 160-170,000. Figure 4 shows the molecular weight to be the same under non-denaturing c o n d i t i o n s . The e l u t i o n p o s i t i o n o f debranching enzyme a c t i v i t y was detected d i r e c t l y withou a c t i v e as the 160,000 molecula The debranching enzyme ( g l u c o s i d a s e - t r a n s f e r a s e ) i s t h e r e f o r e a c t i v e as a monomer and represents the f i r s t m u l t i - c a t a l y t i c s i t e enzyme of e u c a r y o t i c o r i g i n that f u n c t i o n s as a s i n g l e polypeptide molecule (26,27,29). The débrancher i s thus e x c e p t i o n a l i n t h i s regard. I t i s a l s o unique i n terms o f the f u n c t i o n that i t performs. As opposed to multi-enzyme complexes or m u l t i - c a t a l y t i c enzymes, the débrancher does not c a t a l y z e two r e l a t e d chemical r e a c t i o n s . Rather, i t makes p o s s i b l e a d i s c r e t e sequence of changes i n the p h y s i c a l s t r u c t u r e o f the substrate by two d i f f e r e n t r e a c t i o n mechanisms. The s t r u c t u r a l changes i n v o l v e d r e s u l t i n debranching. The two r e a c t i o n s have very d i f f e r e n t chemical mechanisms. The f i r s t i s an o l i g o s a c c h a r i d e t r a n s g l y c o s y l a t i o n ; an α-(1+4) bond o f an o l i g o s a c c h a r i d e moiety i s cleaved and then reformed w i t h another acceptor. There i s no t r a n s f e r to water nor has the formation of another type o f l i n k a g e ever been repor­ ted (21,22). This i s apparently a s t r i c t d i s p r o p o r t i o n a t i o n reac­ t i o n c a t a l y z e d by the t r a n s f e r a s e . The r e a c t i o n proceeds w i t h no change i n f r e e energy s i n c e both the l i n k a g e cleaved and the l i n k a g e formed i s a-(l-*4). In t h i s respect the t r a n s f e r a s e (1,4α-g-glucan:1,4-ot-D-glucan 4 - a - g l y c o s y l t r a n s f e r a s e , EC 2.4.1.25) resembles JE. coli'amylomaltase (1,4-a-g-glucan:D-glucose 4-glucos y l t r a n s f e r a s e , EC 2.4.1.3) and p l a n t D-enzyme,""both o f which are d i s p r o p o r t i o n a t i n g t r a n s f e r a s e s t h a t reform the same type o f l i n k a g e they cleave (30). The second step, the h y d r o l y s i s o f an a-(1+6) l i n k a g e to form f r e e glucose, i s c a t a l y z e d by a s p e c i a l ­ i z e d g l u c o s i d e hydrolase. Although the f r e e energy change i n t h i s case i s c o n s i d e r a b l e , approximately 3-4000 c a l . , the glucosidase i s capable o f c a t a l y z i n g the reverse r e a c t i o n , i n c o r p o r a t i o n o f glucose i n t o polymers such as glycogen, to a s l i g h t extent (10, 13^, 14_, 15_,1_6,22.>32) . The enzyme w i l l a l s o t r a n s f e r glucose to other carbohydrate acceptors such as Schardinger d e x t r i n , or malt o t e t r a o s e t o form an a-(l->6)-linked g l u c o s y l Schardinger d e x t r i n or a branched pentasaccharide, or even to f r e e glucose to form isomaltose (15,33). In t h i s respect the glucosidase ( d e x t r i n 6-

In Carbohydrate-Protein Interaction; Goldstein, I.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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Glycogen Dehranching Enzyme Activities

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