Carbohydrate Antigens - American Chemical Society

Chapter 11 Molecular Biology of Anti-α-(1-->6)dextrans Antibody Responses to a Single-Site-Filling Antigenic Determinant

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Elvin A. Kabat Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, NY 10032

α(1->6)dextrans are homopolymers of glucose with chains of α(1->6) linked glucoses generally connected by short branches withα(1->2),α(1->3)andα(1->4)linkages (1). During the 1980's extensive improvements in determining dextran structures have been made in analytical periodate structural analysis (POSA), methylation structural analysis (MSA), non-reducing end group analysis (NREG) which have made possible more exact correlations with immunochemical studies and led to an improved notation (1) as well as to the elimination of ambiguities resulting from the use of(1->6)-likeinstead of (1-> ) for a terminal non-reducing end and(1->6)for two possibilities. The second residue may be α(1->2)-, α(1->3)-,α(1->4)-,orα(1->6)-linked in various dextrans (cf 1). Dextran B512F which was generally given as having 95%α(1->6)and 5%α(1->3)linkages actually has 90% α(1->6), 5% α(1-> ) and 5%α(1->3;b)with b denoting a branch point. Oligosaccharide chains containing 2-10 or moreα(1->6)linked glucose oligosaccharides have been isolated (2,3). α(1->6) linked dextrans with molecular weights of over 90,000 have been shown to be antigenic in humans (4) giving rise to precipitating antibodies and to wheal and erythema type skin sensitivity which seriously restrict its use as a plasma volume expander. Partially hydrolyzed fractions of dextran have been shown to be less antigenic and products of molecular weights

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11.

KABAT

Molecular Biology of Anti-a-(l->6)dextrans


of about 50,000 o r below responses i n humans (4).

did

not induce

147

antibody

α ( l->6) d e x t r a n itself and α ( l->6 ) 1 i n k e d o l i g o s a c c h a r i d e s o f dextran coupled t o stearylamine t o give synthetic glycolipids a r e both T-independent antigens i n mice (5-9) g i v i n g r i s e t o p r e c i p i t a t i n g monoclonal hybridoma a n t i b o d i e s o f t h e IgM and IgA v a r i e t y p l u s s e v e r a l IgG3 hybridomas (10). a (l->6) l i n k e d o l i g o s a c c h a r i d e s coupled t o BSA and KLH have been found t o be Τ dependent antigens i n mice (11). Moreover, the s t u d i e s on mouse monoclonal myeloma p r o t e i n s i n i t i a t e d by Michael P o t t e r (5), have augmented t h e a v a i l a b l e r e p e r t o i r e of anti-a(l->6) s p e c i f i c monoclonals and have made i t d e s i r a b l e t o use these t o study the scope and nature o f the r e p e r t o i r e o f hybridoma a n t i b o d i e s t o a w e l l d e f i n e d and c h a r a c t e r i z e d s i n g l e epitope - a c h a i n of α (l->6) l i n k e d glucoses which f i l l the combining s i t e s of v a r i o u s myeloma and hybridoma anti-α (l->6) dextrans. I t must be emphasized t h a t the methodology developed f o r these s t u d i e s i s immunochemical and i s u n r e l a t e d t o and independent o f x-ray c r y s t a l l o g r a p h i c methods o f e x p l o r i n g antibody combining s i t e s . S i z e of Anti-a(l-»6) dextran combining s i t e s The s i z e o f anti-α (1-+ 6) dextran combining s i t e s i s d e f i n e d by t h e l a r g e s t o l i g o s a c c h a r i d e which will s a t u r a t e the s i t e so t h a t l a r g e r o l i g o s a c c h a r i d e s are equal i n i n h i b i t i n g a c t i v i t y on a molar b a s i s (12, c f 13). This i s a quite reproducible f i n d i n g with monoclonals b u t i s n o t seen with heterogeneous p o p u l a t i o n s of a n t i b o d i e s produced by d i r e c t immunization with dextran. F i g u r e 1 shows the r e s u l t o f immunization of humans with two i n j e c t i o n s o f 0.5 mg o f n a t i v e a (l-*6) dextran a day apart (13). T h i s was done before such s t u d i e s were p r o h i b i t e d . These data are o f g r e a t i n t e r e s t . The s i x i n d i v i d u a l s of whom I was s u b j e c t No. 1, s i n c e I always b e l i e v e d t h a t I should i n j e c t myself with any m a t e r i a l before i t was used on other persons, show d e c i d e d l y d i f f e r e n t p a t t e r n s of anti-a(l-»6)dextran responses. Thus comparing the r e l a t i v e q u a n t i t i e s o f isomaltotriose (IM3), isomaltotetraose (IM4), isomaltopentaose (IM5), isomaltohexaose (IM6) g i v i n g 50 percent i n h i b i t i o n of p r e c i p i t a t i o n , i t can be seen t h a t the r a t i o s o f IM6 : IM5 : IM4 : IM3 d i f f e r e d from one human antiserum t o another. Thus the r a t i o of IM6:IM3 f o r the s i x s e r a s t u d i e d v a r i e d from 30:1 t o 2.5:1. For each antiserum t h e same amount o f antibody and t h e same concentration o f i n h i b i t o r on a molar b a s i s were compared. Thus t h e i n h i b i t i o n data were d i r e c t l y comparable i n terms o f molecules. These s t u d i e s were c a r r i e d out by q u a n t i t a t i v e p r e c i p i t i n i n h i b i t i o n assays u s i n g the q u a n t i t a t i v e p r e c i p i t i n methodology developed

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CARBOHYDRATE ANTIGENS

148

by Michael H e i d e l b e r g e r and F o r r e s t Ε. K e n d a l l i n 1929 (15).


Were one d e a l i n g with i d e n t i c a l p o p u l a t i o n s o f a n t i b o d i e s with r e s p e c t t o s i t e s i z e and b i n d i n g constant, such as i s seen with myeloma p r o t e i n s o r hybridomas, t h e s i x s e t s o f curves should have been superimposable. Thus F i g u r e 1 e s t a b l i s h e s t h a t each o f the s i x a n t i s e r a were p o l y c l o n a l mixtures o f a n t i ex (1-+6) d e x t r ans with combining s i t e s o f d i f f e r e n t s i z e s and o f d i f f e r e n t Ka. Subsequently, Schlossman (16) and G e l z e r (17) f r a c t i o n a t e d my anti-a(l-*6) dextran i n t o p o r t i o n s w i t h predominantly s m a l l e r and l a r g e r s i z e s i t e s by absorbing s u b s t a n t i a l q u a n t i t i e s (ca 2 l i t e r samples) o f my serum on Sephadex G25, washing out extraneous a n t i b o d i e s and serum p r o t e i n s and e l u t i n g a n t i b o d i e s with s m a l l e r s i z e s i t e s w i t h IM3 and then l a r g e r s i z e s i t e s with IM6. They confirmed t h a t the antiserum contained mixtures o f ant ί α (1-* 6) dextran s with s m a l l e r and l a r g e r s i z e d combining s i t e s as had been i n f e r r e d from F i g u r e 1. The next important development came from two monoclonal myeloma c e l l l i n e s , W3129 (18,20) and QUPC52 (19) which s e c r e t e d anti-α (l->6) dextrans. These were s t u d i e d by C i s a r e t a l . (20,21). Measurement o f s i t e sizes o f these two a n t i b o d i e s r e v e a l e d important differences i n s i t e structure. W3129 had a s i t e s a t u r a t e d by isomaltopentaose, whereas t h a t o f QUPC52 accommodated isomaltohexaose. T h e i r b i n d i n g constants were 1x10 f o r W3129 and 5 x l 0 f o r QUPC52. Since p r e v i o u s s t u d i e s had shown t h a t an antibody w i t h a l a r g e r s i t e s i z e had a h i g h e r Ka, we measured t h e r e l a t i v e c o n t r i b u t i o n o f each glucose t o t h e t o t a l b i n d i n g by i n h i b i t i o n using equilibrium d i a l y s i s . s

3

The r e s u l t was e x t r a o r d i n a r y . F o r W3129 with t h e s m a l l e r s i t e , methyl α-D-glucoside and isomaltose (IM) c o n t r i b u t e d 50 percent o f t h e t o t a l b i n d i n g o f t h e IM5 which s a t u r a t e d t h e s i t e , whereas with QUP52 with t h e larger site, methyl α-D-glucoside and isomaltose c o n t r i b u t e d l e s s than f i v e percent o f t h e t o t a l b i n d i n g of the s i t e - f i l l i n g IM6 and only with IM3 d i d s i g n i f i c a n t b i n d i n g occur. The s t r u c t u r a l b a s i s o f these f i n d i n g s became c l e a r when Ruckel and Schuerch (22) generously p r o v i d e d a s y n t h e t i c l i n e a r α (l-> 6) dextran with 200 glucoses. With W3129 i t i n h i b i t e d p r e c i p i t a t i o n by dextran on a molar b a s i s e q u i v a l e n t t o IM5. With QUPC52, the s t r i k i n g f i n d i n g was t h a t i t p r e c i p i t a t e d t h e antί α (1-+6) dextran. These

results

indicated

that

t h e W3129

site


was


i a

anti-native

0 4

0 6

0 8

10

12

14

16

1.8

2 2 2 4

2 6 2 8

3 0

2 8 3 0

Oligosaccharide added (/6) dextran s i t e s may shed new l i g h t on t h e r e p e r t o i r e o f antibody combining s i t e s d i r e c t e d toward a s i n g l e a n t i g e n i c determinant (40) . Wang e t a l (40) have examined t h e number o f (V D J ) χ (V J ) combinations o f sequenced anti-cr(l-+6) monoclonal hybridoma and myeloma p r o t e i n s and have d e f i n e d 18 combinations, 15 with groove-type and three with c a v i t y - t y p e s i t e s . 11 V genes o f 7 V subgroups (I, IIA, IIB, IIIC, and V f o r groove-type s i t e s , IB and IIIB f o r c a v i t y - t y p e s i t e s ) ; 7 V^ genes o f subgroups ( I I , I I I , V and VI f o r the groove-type, I I f o r t h e c a v i t y type) ; 9 D minigenes and a l l a c t i v e J minigenes a r e used. The s e t s o f d i v e r s e mAb were f u r t h e r expanded by j u n c t i o n a l d i v e r s i t y , Ν element i n s e r t i o n s , D-D f u s i o n and somatic p o i n t mutation (40). Ρ i n s e r t i o n s and gene conversions are not yet observed i n anti-α(l->6)dextrans. The p o p u l a t i o n o f antibody combining s i t e s t o t h e s i n g l e a n t i g e n i c determinant become q u i t e l a r g e and comparable those seen t o other more complicated antigen systems. H

H

L

L

H

H

H

The f i n d i n g t h a t i n forming anti-α (l->6) dextrans, so many germ-line genes a r e used t o generate s i m i l a r - t y p e combining s i t e s i n d i c a t e s t h a t t h e antibody forming system i s h i g h l y p r o t e c t e d against c a t a s t r o p h i c germ l i n e gene l o s s s i n c e even i f one o r more o f the nine germ l i n e gene f a m i l i e s were t o disappear the i n d i v i d u a l could


156


s t i l l make a reasonably complete ex (l-> 6) dextran combining s i t e s .

repertoire

of anti-

The l a b o r a t o r y has a l s o (41) i d e n t i f i e d two major families o f monoclonal anti-a(l->6)dextrans, with prototypes designated V 19.1.2 and V 9.14.7 which show d i s t i n c t patterns of J and J minigene usage w i t h d i f f e r e n t amino a c i d s u b s t i t u t i o n s i n CDR3 a l l have groove-type s i t e s ; t h e r e a r e V 10 19.1.2 and 11 V 9.14.7 mAb. Both f a m i l i e s use t h e same D minigene (DFL16.1). 19.1.2 used only J 2 and J 3 whereas 9.14.7 used J 1 , J 2 , and J 3 and a l l f o u r a c t i v e J , J 1, J 2, J 4 , ^ 5 ) . A l l used t h e V -Oxl germ-line genê (Réf. 21 and 22) . These data r e v e a l some o f the i n f l u e n c e s o f genes and minigenes i n c o n t r o l l i n g t h e types o f antibody combining s i t e s t o a s i n g l e epitope. The nature o f these i n f l u e n c e s r e q u i r e s f u r t h e r study and may w e l l p l a y a dominant r o l e i n t h e g e n e r a t i o n o f t h e other carbohydrate and non carbohydrate determinants. H

H

K

H

H


H

H

H

K

H

H

K

K

H

K

K

Acknowledgments The author thanks Drs. Denong Wang Glaudemans f o r going over t h e manuscript.

and

Niel

Aided by grants from t h e N a t i o n a l I n s t i t u t e o f A l l e r g y and I n f e c t i o u s Diseases, 1R01 AI-19042, 5R01 AI-125616, 3R01 AI-127508 and t h e N a t i o n a l Science Foundation DMB 8600778 t o EAK o f Columbia U n i v e r s i t y , and t h e N a t i o n a l Cancer I n s t i t u t e NCI-CA13696 t o Columbia U n i v e r s i t y . Work with t h e PROPHET computer system i s supported by a grant t o Columbia U n i v e r s i t y from the N a t i o n a l I n s t i t u t e of A l l e r g y and I n f e c t i o u s Diseases, t h e N a t i o n a l Cancer I n s t i t u t e , the N a t i o n a l I n s t i t u t e o f Diabetes, D i g e s t i v e and Kidney Diseases, t h e N a t i o n a l I n s t i t u t e o f General Medical Sciences and t h e N a t i o n a l L i b r a r y o f Medicine with a subcontract from Columbia U n i v e r s i t y t o B o l t Beranek and Newman.

References 1.

Jeanes, A. Mol. Immunol. 23:999-1028, 1986 and papers of F. Seymour cited therein. See also for an improved notation for representing dextran structures which had led to improved understanding of antiα(1->6) dextrans and their combining sites. See also Rees, D.A., Richardson, N.G., Wright, N.J. and Hirst,


11. KABAT Molecular Biology of Anti-α-(1->6)dextrans 157

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.

E. Carb. Res. 9:451-462, 1969 for distinguishing between linear and branched oligosaccharides. Turvey, J.R. and Whelan, W.J. Biochem. J . 67:49-52, 1957. Lai, E. andKabat,E.A. Mol. Immunol. 22:1021-1037, 1985. Kabat, E.A. and Bezer, A.E. Arch Biochem. Biophys. 78:306-318, 1958. Potter, M. Physiol Revs. 52:631-719, 1972. Sharon, J., Kabat, E.A. and Morrison, S.L. Mol. Immunol. 18:831-846, 1981. Wood, C. and Kabat, E.A. J . Exp. Med. 154:432-449, 1981. Wood, C. and Kabat, E.A. Arch. Biochem. Biophys. 212:262-276; 277-289, 1981. Lai, E . , Kabat, E.A. and Mobraaten, L. Cellular Immunol. 92:172-183, 1985. Chen, H.T., Makover, S.D. and Kabat, E.A. Mol. Immunol. 24:333-338, 1987. Matsuda, T. and Kabat, E.A. J . Immunol. 142:863870, 1989. Kabat, E.A. J . Immunol. 84:82-85, 1960. Kabat, E.A. Structural Concepts in Immunology and Immunochemistry, Second Edition Holt, Rinehart and Winston, 1976. Kabat, E.A. J . Immunol., 97:1-11, 1966. Heidelberger, M. and Kendall, F.E. J . Exp. Med. 50:809-823, 1929. Schlossman, S.F. and Kabat, E.A. J . Exp. Med. 116:535-552, 1962. Gelzer, J . and Kabat, E.A. Immunochemistry 1:303316, 1964. Weigert, M., Raschke, W.C., Carson, D. and Cohn, M. J. Exp. Med. 139:127-146, 1974. QUPC52: Obtained from the NIH collection of Michael Potter. Cisar, J.O., Kabat, E.A., Liao, J . and Potter, M. J. Exp. Med. 139:159-179, 1974. Cisar, J., Kabat, E.A., Dorner, M.M. and Liao, J . J. Exp. Med. 142:435-459, 1975. Ruckel, E.R. and Schuerch, C. J . Am. Chem. Soc. 88:2605-2606, 1966. Davies, D.R. and Metzger, H. Ann. Rev. Immunol. 1:87-117, 1983. Bennett, L. and Glaudemans, C.P.J. Carbohy. Res. 72:315-319, 1979. Borden, P. and Kabat, E.A. Mol. Immunol. 25:251-262, 1988. Padlan, E.A. andKabat,E.A. Proc. Natl. Acad. Sci. 85:6885-6889, 1988. Padlan, E.A. andKabat,E.A. Methods in Enzymology, Academic Press NY 203:3-21, 1991. Borden, P. and Kabat, E.A. Proc. Natl. Acad. Sci. 84:2440-2443, 1987. Sikder, S.R., Akolkar, P.N., Kaladas, P.M., Morrison, S.L. and Kabat, E.A. J . Immunol. 135:4215-4221, 1985 Garegg and Lindberg; Carbohydrate Antigens ACS Symposium Series; American Chemical Society: Washington, DC, 1993.



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30. Padlan, E.A., Cohen, G.H. and Davies, D.R. Protein Data Bank, File No. 2, MCP. 31. Bhat, T.N., Padlan, E.A. and Davies, D.R. Protein Data Bank, File 2FBJ. 32. Jones, T.A. J . Applied Crystallography 11:268-272, 1978. 33. Glaudemans, C.P.J. Chem. Rev. 91:25-33, 1991. 34. Nashed, E.M., Perdomo, G.R., Padlan, E.A., Kovac, P., Matsuda, T., Kabat, E.A. and Glaudemans, C.P.J. J . Biol. Chem. 265:20699-20707, 1990. 35. Matsuda, T. and Kabat, E.A. J . Immunol. 142:863-870, 1989. 36. Lemieux, R.U., Wang, T.C., Liao, J . and Kabat, E.A. Mol. Immunol. 21:751-759, 1989. 37. Quicho, F.A. and Vyas, N.K. Nature 310:381-386, 1984 38. Vyas, N.K., Vyas, M.N. and Quicho, F.A. Science 242:1290-1295, 1988. 39. Wang, D. and Kabat, E.A. unpublished. 40. Wang, D., Liao, J., Mitra, D., Akolkar, Ρ.Ν., Gruezo, F. and Kabat, E.A. Mol. Immunol. 28:1387-1397,1991. 41. Wang, D., Chen, H-T., Liao, J., Akolkar, Ρ.Ν., Sikder, S., Gruezo, F. and Kabat, E.A. J . Immunol. 145:3002-3010, 1990. 42. Kabat, E.A. The Physico-chemical Biology (Japan) 34:11-24, 1990. RECEIVED

August 10, 1992


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