- Email: [email protected]
[11] Structure—function analysis of protein active sites with anti-idiotypic antibody
[11]
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1 mg HSA/ml is mixed with 50/xl of the same buffer containing 0.4/zg of biotinylated anti-Id antibodies immediately before being added to the antiDpt IgG-coated plates. Control experiments include inhibition with pooled human gamma globulins and Fab fragments prepared by papain digestion of the same pool. I N H I B I T I O N O F B I N D I N G TO Dpt ALLERGENS. Polystyrene plates are incubated with 100/zl of 40/zg Dpt/ml in GBS, pH 9.2, for 48 hr at 4°, then the plates are washed 3 times with PBS-Tween and twice with PBS. Residual reactive sites are saturated with 100 gl of PBS containing 5 mg BSA/ml for 4 hr at 37°. After a further wash, 100/zl of biotinylated antiDpt antibodies diluted to 0.5/zg/ml in PBS-BSA is added to the plate, which is incubated overnight at 4°. Washing, addition of avidin-peroxidase, and of OPD are performed as above. For inhibition assays, 50/.d of a dilution of anti-Id antibodies in PBS containing 5 mg BSA/ml is mixed for 2 hr at 37° and 2 hr at 21 ° with 50/zl of 1/~g/ml biotinylated anti-Dpt antibodies before addition to the plate. Comments. The quantity of anti-Id antibodies purified by such procedures is small since it represents on average 1-2 /zg antibodies/ml of serum. However, such amounts can be analyzed when the substrate conversion in ELISA is amplified, as in the case of the biotin-avidin complex. Acknowledgments The authors thank Prof. P. M a s s o n for helpful discussions and review of the manuscript and Mrs. I. Gohy for preparing the typescript.
[11] S t r u c t u r e - F u n c t i o n A n a l y s i s o f P r o t e i n A c t i v e Sites with Anti-idiotypic Antibody
By DAVID S. LUDWIG and GARY K. SCHOOLNIK Introduction Jerne, in 1974, proposed that the immune system is regulated by a "network" of idiotypes and anti-idiotypes wherein antibody elicited to antigen may itself engender an immune response) These anti-idiotypic antibodies (Ab2) are believed to bear an internal image of the original ' N. K. Jerne, Ann. Immunol. (Paris) 125C, 373 (1974).
METHODS IN ENZYMOLOGY,VOL. 178
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[1 1]
antigen since both bind and are complementary to the same idiotypic antibody molecule. Initial experimental support for this theory arose from several observations: Sege and Peterson showed that Ab2 against antibodies to insulin or retinol-binding protein could bind insulin or retinolbinding protein receptors, respectively, and thereby mimic the biological effects of these ligands2; Schreiber et al. found that Ab2 raised against alprenolol (a fl-adrenergic antagonist) specifically agglutinate turkey erythrocytes containing the /3-adrenergic receptor and stimulate basal adenylate cyclase activity3; further, Ab2, produced by Marasco and Becker, to the neutrophil chemoattractant factor N-formyl-Met-Leu-Phe cross-reacted with the peptide's receptor on rabbit neutrophils.4 Indeed, the numerous examples to date in which Ab2 functionally resemble antigen underscore the significance of the concept of internal imagery (for reviews, see Refs. 5 and 6). Recently, studies on anti-idiotypic antibody have extended beyond theoretic confirmation of Jerne's "network" to novel applications in the fields of biochemistry, microbiology, and tumor immunology. In studies of incompletely characterized receptors, Ab2 against reovirus type 3 hemagglutinin7 or flirt-globulin: a complement regulatory factor, facilitated the identification of specific cell membrane-associated binding sites. Along different lines, anti-idiotypic antibody methodology may prove useful in the immunoprophylaxis of infectious diseases. Injection of Ab2 raised to antigen present on Trypanosoma rhodesiense, 9 Schistosoma mansoni, l° or Streptococcus pneumoniae 11 confer protective immunity against these pathogens in experimental animals. Antireceptor antibody designed to elicit internal image-bearing Ab2 in oivo has been proposed as vaccine candidates against antigenically variable pathogens including the acquired immune deficiency syndrome (AIDS) virus. 12Furthermore, Ab2 2 K. Sege and P. A. Peterson, Proc. Natl. Acad. Sci. U.S.A. 75, 2443 (1978). 3 A. B. Schreiber, P. O. Couraud, C. Andre, B. Vray, and A. D. Strosberg. Proc. Natl. Acad. Sci. U.S.A. 77, 7385 (1980). 4 W. A. Marasco and E. L. Becker, J. Immunol. 128, 963 (1982). 5 N. R. Farid and T. C. Y. Lo, Endocr. Reo. 6, 1 (1985). 6 j. C. Venter, J. A. Berzofsky, J. Lindstrom, S. Jacobs, C. M. Fraser, L. D. Kohn, W. J. Schneider, G. L. Greene, A. D. Strosberg, and B. F. Erlanger, Fed. Proc., Fed. Am. Soc. Exp. Biol. 43, 2532 (1984). 7 j. H. Noseworthy, B. N. Fields, M. A. Dichter, C. Sobotka, E. Pizer, L. L. Perry, J. T. Nepon, and M. I. Greene, J. lmmunol. 131, 2533 (1983). 8 j. D. Lambris and G. D. Ross, J. Exp. Med. 155, 1400 (1982). 9 D. L. Sacks, K. M. Esser, and A. Sher, J. Exp. Med. 155, 1108 (1982). ~0j. M. Grzych, M. Capron, P. H. Lambert, C. Dissous, S. Torres, and A. Capron, Nature (London) 316, 74 (1985). i1 M. K. McNamara, R. E. Ward, and H. K6hler, Science 22,6, 1325 (1984). 12 D. S. Ludwig and G. K. Schoolnik, Med. Hypotheses 23, 303 (1987).
[11]
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165
may find utility in oncology by, for example, promotion of delayed-type hypersensitivity to tumors. 13 Concept of "Dissociability" The studies discussed above have sought, in general, to produce antibody with binding properties similar to the antigens against which they were engendered, creating, in effect, immunoglobulin analogs of epitopes from ligands or pathogenic microorganisms. In vaccine development, the advantages of immunogens consisting of antibody instead of components from, or modifications of, pathogens are clear: absence of a potentially infectious agent, availability, and purity. When employed in structurefunction analysis, by contrast, anti-idiotypic antibody with the same binding characteristics as ligand cannot provide more information about a receptor than would the ligand itself. However, ligand and the Ab2 they elicit, owing to potential differences in size and chemical nature, need not necessarily exhibit identical pharmacologic specificity, a phenomenon which may be termed "dissociability." Thus, Couraud et al. showed that antisera containing Ab2 to substance P, a neuropeptide, strongly inhibited its spasmogenic action in guinea pig ileum but specifically activated receptors from rat parotid gland cells) 4 A fundamental question was posed by their investigation: Did the polyclonal antibody examined actually distinguish between two substance P receptors with different structures or was antibody of at least two specificities present? We have attempted to address the concept of dissociability using monoclonal Ab2 to probe the interaction between cholera toxin B subunit and ganglioside GM~. Ligand Binding Domain of Cholera Toxin B Subunit 15 The clinical manifestations of cholera are attributable to the physiological effects of a toxin produced by Vibrio cholerae. 16,~7 Cholera toxin, a protein of 85,000 MW, contains two subunits, A and B. 18The A subunit is t3 j. W. Forstrom, K. A. Nelson, G. T. Nepom, I. Hellstr6m, and K. E. Hellstr6m, Nature (London) 303, 627 (1983). t4 j . . y . Couraud, E. Escher, D. Regoli, V. Imhoff, B. Rossignof, and P. Pradelles, J. Biol. Chem. 260, 9461 (1985). 15 D. S. Ludwig, R. A. Finkelstein, A. E. Karu, U. S. Dallas, E. R. Ashby, and G. K. Schoolnik, Proc. Natl. Acad. Sci. U.S.A. 84, 3673 (1987). t6 S. N. De, Nature (London) 183, 1533 (1959). 17 N. K. Dutta, M. V. Panse, and D. R. Kulkarni, J. Bacteriol. 78, 594 (1959). 18 R. A. Finkelstein, M. Boseman, S. H. Neoh, M. K. LaRue, and R. Delaney, J. lmmunol. 113, 145 (1974).
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an ADP-ribosylating enzyme which can penetrate the plasma membrane of susceptible cells and activate adenylate cyclase. 19"2° The B subunit (CT-B), which mediates binding to cell surfaces, consists of five identical noncovalently associated polypeptide chains 21 whose primary structure has been determined. 2x23 Each polypeptide (l 1,600 MW) is composed of 103 amino acids; an intrachain disulfide bridge links Cys-9 and Cys-86. The B subunit specifically binds the glycosphingolipid ganglioside GMj ( g d = l 0 -9 M ) , present in the membranes of many mammalian cells. 24The oligosaccharide moiety of GMI devoid of ceramide (OS-GM0 carries the determinants for interaction with CT-B. 25 Five OS-GM~ molecules can simultaneously bind the B pentamer. 26 Previous studies have indicated that several amino acids (Try-88, 27-29 Gly-33, 3° the cystine residue, 29 and possibly lysine, 29,31 arginine? 9,32 and histidine residues 33) are critical to ligand recognition. However, these chemical and biophysical investigations were, for the most part, unable to distinguish between amino acid residues interacting directly with GM~ and those which contribute indirectly to function as, for example, in maintaining structure. To circumvent this ambiguity, we sought to produce Ab2 against GMI which would cross-react with CT-B and, therefore, bind amino acids located within the ligand-binding domain. Monoclonal Ab2 were prepared according to a novel "one-step" method, presupposing the existence of a functioning endogenous network, as demonstrated by Cleveland et al. in work on the acetylcholine 19 D. Cassel and T. Pfeuffer, Proc. Natl. Acad. Sci. U.S.A. 75, 2669 (1978). 2o D. M. Gill and R. Meren, Proc. Natl. Acad. Sci. U.S.A. 75, 3050 (1978). 21 D. M. Gill, Biochemistry 15, 1242 (1976). 22 C.-Y. Lai, J. Biol. Chem. 252, 7249 0977). 23 A. Kurosky, D. E. Markel, and J. W. Peterson, J. Biol. Chem. 252, 7257 (1977). 24 p. Cuatrecasas, Biochemistry 12, 3547 (1973). 25 j. Sattler, G. Schwarzmann, J. Staerk, W. Ziegler, and H. Wiegandt, Hoppe-Seyler's Z. Physiol. Chem. 358, 159 (1977). 26 p. H. Fishman, J. Moss, and J. C. Osborne, Jr., Biochemistry 17, 711 (1978). 27 M. J. S. De Wolf, M. Fridkin, and L. D. Kohn, J. Biol. Chem. 256, 5489 (1981). M. J. S. De Wolf, M. Fridkin, M. Epstein, and L. D. Kohn, J. Biol. Chem. 256, 5481
(1981). 29 D. S. Ludwig, R. K. Holmes, and G. K. Schoolnik, J. Biol. Chem. 2,60, 12528 (1985). 3o T. Tsuji, T. Honda, T. Miwatani, S. Wakabayashi, and H. Matsubara, J. Biol. Chem. 260, 8552 (1985). 31 D. E. Markel, K. E. Hejtmancik, J. W. Peterson, and A. Kurosky, J. Supramol. Struct. 10, 137 (1979). 32 L. K. Duffy and C.-Y. Lai, Biochem. Biophys. Res. Commun. 91, 1005 (1979). 33 M. De Wolf, G. Van Dessel, A. Lagrou, H. J. Hilderson, and W. Dierick, Biochim. Biophys. Acta 832, 165 (1985).
[11]
STRUCTURE-FUNCTION
40,000
l
)
I
'1
30.000
m
167
A N A L Y S I S O F P R O T E I N A C T I V E SITES
I
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I
B
-
,,,MAXIMUM BINDING
20,000
10.000
0 10
I o--o--c..o--c--c-=o 100 1000 10,000 ANTIBODY CONCENTRATION (ng/ml)
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Fro. I. Characterization of Ab9B6. (A) Binding to CT-B. Polyvinyl microtiter wells were incubated overnight with 100/~1 CT-B (O) or bovine serum albumin at 7/~g/ml, gelatin at 7 /~g/ml, thyroglobulin at 7 p.g/ml, or Gm at 7 p.g/ml (O), in PBS, pH 7.4, and then washed with PBS containing 0.05% gelatin. Subsequently, serial dilutions of Ab9B6 were added. After 2 hr, the wells were washed, and rabbit anti-mouse antibodies (1 : 2000 dilution of antisera) were introduced. One hour later, the wells were washed, and approximately 75,000 cpm 125I-labeled staphylococcal protein A was added. Following a 1-hr incubation, bound radioactivity was determined by gamma counting. (B) Competition with OS-GMI. Serial dilutions of OS-Gm were mixed with 100 ng Ab9B6, and the resulting solution was added to microtiter wells containing CT-B in the solid phase. After 2 hr, the wells were washed and then incubated with rabbit anti-mouse antibodies and ~25I-labeled protein A as described above.
receptor) 4 In this approach, antigen is presumed to elicit both idiotypic and subsequently anti-idiotypic responses in the same animal. For the present study, 8-week-old female BALB/c mice were immunized i.p. with 50/zg Gm coated on " n a k e d " Salmonella 35 in Freund's complete adjuvant (0.2 ml), boosted with a similar mixture in incomplete adjuvant approximately 1 month later, and then allowed to rest for at least 8 weeks. Mice were hyperimmunized with Gin~Salmonella in saline i.v. on days 4, 3, and 2 prior to fusion, and splenocytes were fused with P3-X63-Ag8.653 myeloma cells according to established methods. 36 Two weeks later, hybridoma supernatants were assayed for the presence of Ab2 which bound CT-B. Of 13 clones which initially tested positive, antibody from one line (Ab9B6) was purified and found to bind CT-B specifically with an avidity constant of 8 x 109 M -l (Fig. 1A). This interaction was completely and 34 W. L. Cleveland, N. H. Wassermann, R. Sarangarajan, A. S. Penn, and B. F. Erlanger, Nature (London) 305, 56 (1983). ~5 C. Galanos, O. Luderitz, and O. Westphal, Eur. J. Biochem. 24, 116 (1971). R. J. Sugasawara, C. M. Prato, and J. E. Sippel, J. Clin. Microbiol. 19, 230 (1984).
168
IDIOTYPES, ANTI-IDIOTYPES, AND MOLECULAR MIMICRY
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specifically inhibited by nanomolar concentrations of OS-GMI, confirming the anti-idiotypic nature of the clone (Fig. 1B). To identify the epitope of Ab9B6, we screened the clone against B subunits of human labile toxin (hLT-B) and porcine labile toxin (pLT-B). These proteins, produced by enterotoxigenic strains of Escherichia coli, bear very close resemblance to CT-B in amino acid sequence (about 80% homology), physiochemical properties, antigenicity, and ligand-binding function. 37-42Ab9B6 bound hLT-B but not pLT-B in solid-phase radioimmunoassays and Western blots. Since the sequences of the two LT-B molecules differ only at positions 4, 13, 46, and 102 (Fig. 2), it follows that the monoclonal antibody binds at least one of these four residues (assuming the B subunits share similar secondary and tertiary structures). Next, Ab9B6 was tested against a family of chimeric proteins produced by recombinant methods43 and containing various combinations of hLT-B and pLT-B residues (Fig. 2). Cross-reaction was obtained with chimera pDL7, containing hLT-B residues at positions 4 and 13, but not with pDL2, pDL3, or pDLS. Therefore, the epitope recognized by Ab9B6 appears to encompass residues 4 and/or 13. Finally, we examined the cross-reactivity of the B subunit from a recently isolated human labile toxin variant (hLT2-B), containing the pLT-B residue at position 13.4o Ab9B6 binds hLT2-B, indicating that the Arg residue at position 13 from the amino terminus does not interact with the antibody. Instead, this finding suggests that Asn-4 constitutes part of the epitope of the antiidiotypic antibody and, by implication, also resides within the GM1binding domain of proteins in the CT/LT family. Of particular significance, the structural detail revealed above could not be deduced by studies on ligand alone but, rather, followed from the unique binding specificity of Ab9B6 (which cross-reacts with hLT-B but not pLT-B) as compared to GMI (affinity for the two proteins approximately equal). This difference in specificity between antigen (ligand) and internal image-bearing anti-idiotypic antibody, termed "dissociability," may arise from subtle stereochemical dissimilarities, as depicted in Fig. 3. 37 W. S. Dallas and S. Falkow, Nature (London) 288, 499 (1980). 3s L. Lindholm, J. Hoimgren, M. Wikstrom, U. Karlsson, K. Andersson, and N. Lycke, Infect. Immun. 40, 570 (1983). 39 y . Takeda, T. Honda, H. Sima, T. Tsuji, and T. Miwatani, Infect. lmmun. 41, 50 (1983). 4o T. Yamamoto and T. Yokota, J. Bacteriol. 155, 728 (1983). 4t j. D. Clements and R. A. Finkelstein, Infect. Immun. 24, 760 (1979). 42 S. J. Geary, B. A. Marchlewicz, and R. A. Finkelstein, Infect. Immun. 36, 215 (1982). 43 R. A. Finkelstein, M. F. Burks, L. C. Rieke, R. J. McDonald, S. K. Browne, and W. S. Dallas, Dev. Biol. Stand. 59, 51 (1985).
[11]
STRUCTURE-FUNCTION
ANALYSIS
OF
PROTEIN
ACTIVE
169
SITES "O
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~
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170
IDIOTYPES, ANTI-IDIOTYPES, AND MOLECULAR MIMICRY ligand
[11]
anti-id ab
Class I
Class II
FIG. 3. Dissociation of binding specificity between ligand and internal image-bearing antiidiotypic antibody. Ligand and antibody, owing to differences in size and chemical nature, may exhibit subtle stereochemical dissimilarities (striped area of antibody). Here, the ligand binds both receptor classes, but the antibody interacts with class I only.
S u c h d i s s o c i a b i l i t y m i g h t b e e x p l o i t e d f o r s t r u c t u r a l i n v e s t i g a t i o n at t h e molecular level.
Future Studies Monoclonal anti-idiotypicantibodies are, potentially,complementary analogs of any biologicallyinteresting,immunogcnic molecule, and could be selected for novel activities.By screening hybridoma banks appropri-
[12]
Id-ANTl-Id IMMUNOASSAYFOR ABP
171
ately, for example, it might be possible to isolate antiligand Ab2 that define receptor classes as muscarine identifies a subset of acetylcholine receptors. In addition, Ab2 may be selected for agonist or antagonist properties, possibly comprising a method for the directed production of pharmacologically active therapeutic agents. Further, these antibodies may constitute probes capable of distinguishing receptor conformation, channel state, or enzyme isotype. Summary Antigen and internal image-bearing anti-idiotypic antibody, owing to potential differences in size and chemical nature, need not necessarily demonstrate identical binding specificities. Such differences, termed "dissociability," may be exploited in structure-function analysis of receptor-ligand interaction to identify functionally important amino acid residues, define receptor class, or distinguish receptor conformation. In this sense, ligand and the anti-idiotypes they elicit constitute alternative and complementary probes of protein active sites.
[12] C o m p e t i t i v e I d i o t y p e - A n t i - i d i o t y p e E n z y m e I m m u n o a s s a y for A d e n o s i n e D e a m i n a s e B i n d i n g P r o t e i n B y M I T C H E L L J. NELLES
Anti-idiotype antibodies (anti-Id) have been used widely in studies of immune regulation and, more recently, as vaccines in a number of animal models.t.2 In addition, they can be used as a replacement for antigen in immunoassays) Although this last application of anti-Id has not been exploited fully, these reagents appear particularly well suited for use in immunoassays in cases where the antigen is not readily available or easily labeled, in cases where the antigen is a small molecule and as such is not amenable to a conventional sandwich assay, and in cases where only one antibody reactive with the antigen is available. Recently, an Id-anti-Id competitive immunoassay was developed which quantitatively detects l K. Rajewsky and T. Takemori, Annu. Rev. Immunol. 1, 569 (1983). 2 F. G. C. M. UytdeHaag, H. Bunschoten, K. Weijer, and A. D. M. E. Osterhaus, Immunol. Rev. 90, 93 (1986). 3 p. Potocnjak, F. Zavala, R. Nussenzweig, and V. Nussenzweig, Science 215, 1637 (1982).
METHODS IN ENZYMOLOGY, VOL. 178
Copyright © 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
STRUCTURE--FUNCTION ANALYSIS OF PROTEIN ACTIVE SITES
163
1 mg HSA/ml is mixed with 50/xl of the same buffer containing 0.4/zg of biotinylated anti-Id antibodies immediately before being added to the antiDpt IgG-coated plates. Control experiments include inhibition with pooled human gamma globulins and Fab fragments prepared by papain digestion of the same pool. I N H I B I T I O N O F B I N D I N G TO Dpt ALLERGENS. Polystyrene plates are incubated with 100/zl of 40/zg Dpt/ml in GBS, pH 9.2, for 48 hr at 4°, then the plates are washed 3 times with PBS-Tween and twice with PBS. Residual reactive sites are saturated with 100 gl of PBS containing 5 mg BSA/ml for 4 hr at 37°. After a further wash, 100/zl of biotinylated antiDpt antibodies diluted to 0.5/zg/ml in PBS-BSA is added to the plate, which is incubated overnight at 4°. Washing, addition of avidin-peroxidase, and of OPD are performed as above. For inhibition assays, 50/.d of a dilution of anti-Id antibodies in PBS containing 5 mg BSA/ml is mixed for 2 hr at 37° and 2 hr at 21 ° with 50/zl of 1/~g/ml biotinylated anti-Dpt antibodies before addition to the plate. Comments. The quantity of anti-Id antibodies purified by such procedures is small since it represents on average 1-2 /zg antibodies/ml of serum. However, such amounts can be analyzed when the substrate conversion in ELISA is amplified, as in the case of the biotin-avidin complex. Acknowledgments The authors thank Prof. P. M a s s o n for helpful discussions and review of the manuscript and Mrs. I. Gohy for preparing the typescript.
[11] S t r u c t u r e - F u n c t i o n A n a l y s i s o f P r o t e i n A c t i v e Sites with Anti-idiotypic Antibody
By DAVID S. LUDWIG and GARY K. SCHOOLNIK Introduction Jerne, in 1974, proposed that the immune system is regulated by a "network" of idiotypes and anti-idiotypes wherein antibody elicited to antigen may itself engender an immune response) These anti-idiotypic antibodies (Ab2) are believed to bear an internal image of the original ' N. K. Jerne, Ann. Immunol. (Paris) 125C, 373 (1974).
METHODS IN ENZYMOLOGY,VOL. 178
Copyright © 1989by Academic Press, Inc. All rights of reproduction in any form reserved.
164
IDIOTYPES, ANTI-IDIOTYPES, AND MOLECULAR MIMICRY
[1 1]
antigen since both bind and are complementary to the same idiotypic antibody molecule. Initial experimental support for this theory arose from several observations: Sege and Peterson showed that Ab2 against antibodies to insulin or retinol-binding protein could bind insulin or retinolbinding protein receptors, respectively, and thereby mimic the biological effects of these ligands2; Schreiber et al. found that Ab2 raised against alprenolol (a fl-adrenergic antagonist) specifically agglutinate turkey erythrocytes containing the /3-adrenergic receptor and stimulate basal adenylate cyclase activity3; further, Ab2, produced by Marasco and Becker, to the neutrophil chemoattractant factor N-formyl-Met-Leu-Phe cross-reacted with the peptide's receptor on rabbit neutrophils.4 Indeed, the numerous examples to date in which Ab2 functionally resemble antigen underscore the significance of the concept of internal imagery (for reviews, see Refs. 5 and 6). Recently, studies on anti-idiotypic antibody have extended beyond theoretic confirmation of Jerne's "network" to novel applications in the fields of biochemistry, microbiology, and tumor immunology. In studies of incompletely characterized receptors, Ab2 against reovirus type 3 hemagglutinin7 or flirt-globulin: a complement regulatory factor, facilitated the identification of specific cell membrane-associated binding sites. Along different lines, anti-idiotypic antibody methodology may prove useful in the immunoprophylaxis of infectious diseases. Injection of Ab2 raised to antigen present on Trypanosoma rhodesiense, 9 Schistosoma mansoni, l° or Streptococcus pneumoniae 11 confer protective immunity against these pathogens in experimental animals. Antireceptor antibody designed to elicit internal image-bearing Ab2 in oivo has been proposed as vaccine candidates against antigenically variable pathogens including the acquired immune deficiency syndrome (AIDS) virus. 12Furthermore, Ab2 2 K. Sege and P. A. Peterson, Proc. Natl. Acad. Sci. U.S.A. 75, 2443 (1978). 3 A. B. Schreiber, P. O. Couraud, C. Andre, B. Vray, and A. D. Strosberg. Proc. Natl. Acad. Sci. U.S.A. 77, 7385 (1980). 4 W. A. Marasco and E. L. Becker, J. Immunol. 128, 963 (1982). 5 N. R. Farid and T. C. Y. Lo, Endocr. Reo. 6, 1 (1985). 6 j. C. Venter, J. A. Berzofsky, J. Lindstrom, S. Jacobs, C. M. Fraser, L. D. Kohn, W. J. Schneider, G. L. Greene, A. D. Strosberg, and B. F. Erlanger, Fed. Proc., Fed. Am. Soc. Exp. Biol. 43, 2532 (1984). 7 j. H. Noseworthy, B. N. Fields, M. A. Dichter, C. Sobotka, E. Pizer, L. L. Perry, J. T. Nepon, and M. I. Greene, J. lmmunol. 131, 2533 (1983). 8 j. D. Lambris and G. D. Ross, J. Exp. Med. 155, 1400 (1982). 9 D. L. Sacks, K. M. Esser, and A. Sher, J. Exp. Med. 155, 1108 (1982). ~0j. M. Grzych, M. Capron, P. H. Lambert, C. Dissous, S. Torres, and A. Capron, Nature (London) 316, 74 (1985). i1 M. K. McNamara, R. E. Ward, and H. K6hler, Science 22,6, 1325 (1984). 12 D. S. Ludwig and G. K. Schoolnik, Med. Hypotheses 23, 303 (1987).
[11]
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165
may find utility in oncology by, for example, promotion of delayed-type hypersensitivity to tumors. 13 Concept of "Dissociability" The studies discussed above have sought, in general, to produce antibody with binding properties similar to the antigens against which they were engendered, creating, in effect, immunoglobulin analogs of epitopes from ligands or pathogenic microorganisms. In vaccine development, the advantages of immunogens consisting of antibody instead of components from, or modifications of, pathogens are clear: absence of a potentially infectious agent, availability, and purity. When employed in structurefunction analysis, by contrast, anti-idiotypic antibody with the same binding characteristics as ligand cannot provide more information about a receptor than would the ligand itself. However, ligand and the Ab2 they elicit, owing to potential differences in size and chemical nature, need not necessarily exhibit identical pharmacologic specificity, a phenomenon which may be termed "dissociability." Thus, Couraud et al. showed that antisera containing Ab2 to substance P, a neuropeptide, strongly inhibited its spasmogenic action in guinea pig ileum but specifically activated receptors from rat parotid gland cells) 4 A fundamental question was posed by their investigation: Did the polyclonal antibody examined actually distinguish between two substance P receptors with different structures or was antibody of at least two specificities present? We have attempted to address the concept of dissociability using monoclonal Ab2 to probe the interaction between cholera toxin B subunit and ganglioside GM~. Ligand Binding Domain of Cholera Toxin B Subunit 15 The clinical manifestations of cholera are attributable to the physiological effects of a toxin produced by Vibrio cholerae. 16,~7 Cholera toxin, a protein of 85,000 MW, contains two subunits, A and B. 18The A subunit is t3 j. W. Forstrom, K. A. Nelson, G. T. Nepom, I. Hellstr6m, and K. E. Hellstr6m, Nature (London) 303, 627 (1983). t4 j . . y . Couraud, E. Escher, D. Regoli, V. Imhoff, B. Rossignof, and P. Pradelles, J. Biol. Chem. 260, 9461 (1985). 15 D. S. Ludwig, R. A. Finkelstein, A. E. Karu, U. S. Dallas, E. R. Ashby, and G. K. Schoolnik, Proc. Natl. Acad. Sci. U.S.A. 84, 3673 (1987). t6 S. N. De, Nature (London) 183, 1533 (1959). 17 N. K. Dutta, M. V. Panse, and D. R. Kulkarni, J. Bacteriol. 78, 594 (1959). 18 R. A. Finkelstein, M. Boseman, S. H. Neoh, M. K. LaRue, and R. Delaney, J. lmmunol. 113, 145 (1974).
166
IDIOTYPES, ANTI-IDIOTYPES, AND MOLECULAR MIMICRY
[11]
an ADP-ribosylating enzyme which can penetrate the plasma membrane of susceptible cells and activate adenylate cyclase. 19"2° The B subunit (CT-B), which mediates binding to cell surfaces, consists of five identical noncovalently associated polypeptide chains 21 whose primary structure has been determined. 2x23 Each polypeptide (l 1,600 MW) is composed of 103 amino acids; an intrachain disulfide bridge links Cys-9 and Cys-86. The B subunit specifically binds the glycosphingolipid ganglioside GMj ( g d = l 0 -9 M ) , present in the membranes of many mammalian cells. 24The oligosaccharide moiety of GMI devoid of ceramide (OS-GM0 carries the determinants for interaction with CT-B. 25 Five OS-GM~ molecules can simultaneously bind the B pentamer. 26 Previous studies have indicated that several amino acids (Try-88, 27-29 Gly-33, 3° the cystine residue, 29 and possibly lysine, 29,31 arginine? 9,32 and histidine residues 33) are critical to ligand recognition. However, these chemical and biophysical investigations were, for the most part, unable to distinguish between amino acid residues interacting directly with GM~ and those which contribute indirectly to function as, for example, in maintaining structure. To circumvent this ambiguity, we sought to produce Ab2 against GMI which would cross-react with CT-B and, therefore, bind amino acids located within the ligand-binding domain. Monoclonal Ab2 were prepared according to a novel "one-step" method, presupposing the existence of a functioning endogenous network, as demonstrated by Cleveland et al. in work on the acetylcholine 19 D. Cassel and T. Pfeuffer, Proc. Natl. Acad. Sci. U.S.A. 75, 2669 (1978). 2o D. M. Gill and R. Meren, Proc. Natl. Acad. Sci. U.S.A. 75, 3050 (1978). 21 D. M. Gill, Biochemistry 15, 1242 (1976). 22 C.-Y. Lai, J. Biol. Chem. 252, 7249 0977). 23 A. Kurosky, D. E. Markel, and J. W. Peterson, J. Biol. Chem. 252, 7257 (1977). 24 p. Cuatrecasas, Biochemistry 12, 3547 (1973). 25 j. Sattler, G. Schwarzmann, J. Staerk, W. Ziegler, and H. Wiegandt, Hoppe-Seyler's Z. Physiol. Chem. 358, 159 (1977). 26 p. H. Fishman, J. Moss, and J. C. Osborne, Jr., Biochemistry 17, 711 (1978). 27 M. J. S. De Wolf, M. Fridkin, and L. D. Kohn, J. Biol. Chem. 256, 5489 (1981). M. J. S. De Wolf, M. Fridkin, M. Epstein, and L. D. Kohn, J. Biol. Chem. 256, 5481
(1981). 29 D. S. Ludwig, R. K. Holmes, and G. K. Schoolnik, J. Biol. Chem. 2,60, 12528 (1985). 3o T. Tsuji, T. Honda, T. Miwatani, S. Wakabayashi, and H. Matsubara, J. Biol. Chem. 260, 8552 (1985). 31 D. E. Markel, K. E. Hejtmancik, J. W. Peterson, and A. Kurosky, J. Supramol. Struct. 10, 137 (1979). 32 L. K. Duffy and C.-Y. Lai, Biochem. Biophys. Res. Commun. 91, 1005 (1979). 33 M. De Wolf, G. Van Dessel, A. Lagrou, H. J. Hilderson, and W. Dierick, Biochim. Biophys. Acta 832, 165 (1985).
[11]
STRUCTURE-FUNCTION
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A N A L Y S I S O F P R O T E I N A C T I V E SITES
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Fro. I. Characterization of Ab9B6. (A) Binding to CT-B. Polyvinyl microtiter wells were incubated overnight with 100/~1 CT-B (O) or bovine serum albumin at 7/~g/ml, gelatin at 7 /~g/ml, thyroglobulin at 7 p.g/ml, or Gm at 7 p.g/ml (O), in PBS, pH 7.4, and then washed with PBS containing 0.05% gelatin. Subsequently, serial dilutions of Ab9B6 were added. After 2 hr, the wells were washed, and rabbit anti-mouse antibodies (1 : 2000 dilution of antisera) were introduced. One hour later, the wells were washed, and approximately 75,000 cpm 125I-labeled staphylococcal protein A was added. Following a 1-hr incubation, bound radioactivity was determined by gamma counting. (B) Competition with OS-GMI. Serial dilutions of OS-Gm were mixed with 100 ng Ab9B6, and the resulting solution was added to microtiter wells containing CT-B in the solid phase. After 2 hr, the wells were washed and then incubated with rabbit anti-mouse antibodies and ~25I-labeled protein A as described above.
receptor) 4 In this approach, antigen is presumed to elicit both idiotypic and subsequently anti-idiotypic responses in the same animal. For the present study, 8-week-old female BALB/c mice were immunized i.p. with 50/zg Gm coated on " n a k e d " Salmonella 35 in Freund's complete adjuvant (0.2 ml), boosted with a similar mixture in incomplete adjuvant approximately 1 month later, and then allowed to rest for at least 8 weeks. Mice were hyperimmunized with Gin~Salmonella in saline i.v. on days 4, 3, and 2 prior to fusion, and splenocytes were fused with P3-X63-Ag8.653 myeloma cells according to established methods. 36 Two weeks later, hybridoma supernatants were assayed for the presence of Ab2 which bound CT-B. Of 13 clones which initially tested positive, antibody from one line (Ab9B6) was purified and found to bind CT-B specifically with an avidity constant of 8 x 109 M -l (Fig. 1A). This interaction was completely and 34 W. L. Cleveland, N. H. Wassermann, R. Sarangarajan, A. S. Penn, and B. F. Erlanger, Nature (London) 305, 56 (1983). ~5 C. Galanos, O. Luderitz, and O. Westphal, Eur. J. Biochem. 24, 116 (1971). R. J. Sugasawara, C. M. Prato, and J. E. Sippel, J. Clin. Microbiol. 19, 230 (1984).
168
IDIOTYPES, ANTI-IDIOTYPES, AND MOLECULAR MIMICRY
[11]
specifically inhibited by nanomolar concentrations of OS-GMI, confirming the anti-idiotypic nature of the clone (Fig. 1B). To identify the epitope of Ab9B6, we screened the clone against B subunits of human labile toxin (hLT-B) and porcine labile toxin (pLT-B). These proteins, produced by enterotoxigenic strains of Escherichia coli, bear very close resemblance to CT-B in amino acid sequence (about 80% homology), physiochemical properties, antigenicity, and ligand-binding function. 37-42Ab9B6 bound hLT-B but not pLT-B in solid-phase radioimmunoassays and Western blots. Since the sequences of the two LT-B molecules differ only at positions 4, 13, 46, and 102 (Fig. 2), it follows that the monoclonal antibody binds at least one of these four residues (assuming the B subunits share similar secondary and tertiary structures). Next, Ab9B6 was tested against a family of chimeric proteins produced by recombinant methods43 and containing various combinations of hLT-B and pLT-B residues (Fig. 2). Cross-reaction was obtained with chimera pDL7, containing hLT-B residues at positions 4 and 13, but not with pDL2, pDL3, or pDLS. Therefore, the epitope recognized by Ab9B6 appears to encompass residues 4 and/or 13. Finally, we examined the cross-reactivity of the B subunit from a recently isolated human labile toxin variant (hLT2-B), containing the pLT-B residue at position 13.4o Ab9B6 binds hLT2-B, indicating that the Arg residue at position 13 from the amino terminus does not interact with the antibody. Instead, this finding suggests that Asn-4 constitutes part of the epitope of the antiidiotypic antibody and, by implication, also resides within the GM1binding domain of proteins in the CT/LT family. Of particular significance, the structural detail revealed above could not be deduced by studies on ligand alone but, rather, followed from the unique binding specificity of Ab9B6 (which cross-reacts with hLT-B but not pLT-B) as compared to GMI (affinity for the two proteins approximately equal). This difference in specificity between antigen (ligand) and internal image-bearing anti-idiotypic antibody, termed "dissociability," may arise from subtle stereochemical dissimilarities, as depicted in Fig. 3. 37 W. S. Dallas and S. Falkow, Nature (London) 288, 499 (1980). 3s L. Lindholm, J. Hoimgren, M. Wikstrom, U. Karlsson, K. Andersson, and N. Lycke, Infect. Immun. 40, 570 (1983). 39 y . Takeda, T. Honda, H. Sima, T. Tsuji, and T. Miwatani, Infect. lmmun. 41, 50 (1983). 4o T. Yamamoto and T. Yokota, J. Bacteriol. 155, 728 (1983). 4t j. D. Clements and R. A. Finkelstein, Infect. Immun. 24, 760 (1979). 42 S. J. Geary, B. A. Marchlewicz, and R. A. Finkelstein, Infect. Immun. 36, 215 (1982). 43 R. A. Finkelstein, M. F. Burks, L. C. Rieke, R. J. McDonald, S. K. Browne, and W. S. Dallas, Dev. Biol. Stand. 59, 51 (1985).
[11]
STRUCTURE-FUNCTION
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170
IDIOTYPES, ANTI-IDIOTYPES, AND MOLECULAR MIMICRY ligand
[11]
anti-id ab
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FIG. 3. Dissociation of binding specificity between ligand and internal image-bearing antiidiotypic antibody. Ligand and antibody, owing to differences in size and chemical nature, may exhibit subtle stereochemical dissimilarities (striped area of antibody). Here, the ligand binds both receptor classes, but the antibody interacts with class I only.
S u c h d i s s o c i a b i l i t y m i g h t b e e x p l o i t e d f o r s t r u c t u r a l i n v e s t i g a t i o n at t h e molecular level.
Future Studies Monoclonal anti-idiotypicantibodies are, potentially,complementary analogs of any biologicallyinteresting,immunogcnic molecule, and could be selected for novel activities.By screening hybridoma banks appropri-
[12]
Id-ANTl-Id IMMUNOASSAYFOR ABP
171
ately, for example, it might be possible to isolate antiligand Ab2 that define receptor classes as muscarine identifies a subset of acetylcholine receptors. In addition, Ab2 may be selected for agonist or antagonist properties, possibly comprising a method for the directed production of pharmacologically active therapeutic agents. Further, these antibodies may constitute probes capable of distinguishing receptor conformation, channel state, or enzyme isotype. Summary Antigen and internal image-bearing anti-idiotypic antibody, owing to potential differences in size and chemical nature, need not necessarily demonstrate identical binding specificities. Such differences, termed "dissociability," may be exploited in structure-function analysis of receptor-ligand interaction to identify functionally important amino acid residues, define receptor class, or distinguish receptor conformation. In this sense, ligand and the anti-idiotypes they elicit constitute alternative and complementary probes of protein active sites.
[12] C o m p e t i t i v e I d i o t y p e - A n t i - i d i o t y p e E n z y m e I m m u n o a s s a y for A d e n o s i n e D e a m i n a s e B i n d i n g P r o t e i n B y M I T C H E L L J. NELLES
Anti-idiotype antibodies (anti-Id) have been used widely in studies of immune regulation and, more recently, as vaccines in a number of animal models.t.2 In addition, they can be used as a replacement for antigen in immunoassays) Although this last application of anti-Id has not been exploited fully, these reagents appear particularly well suited for use in immunoassays in cases where the antigen is not readily available or easily labeled, in cases where the antigen is a small molecule and as such is not amenable to a conventional sandwich assay, and in cases where only one antibody reactive with the antigen is available. Recently, an Id-anti-Id competitive immunoassay was developed which quantitatively detects l K. Rajewsky and T. Takemori, Annu. Rev. Immunol. 1, 569 (1983). 2 F. G. C. M. UytdeHaag, H. Bunschoten, K. Weijer, and A. D. M. E. Osterhaus, Immunol. Rev. 90, 93 (1986). 3 p. Potocnjak, F. Zavala, R. Nussenzweig, and V. Nussenzweig, Science 215, 1637 (1982).
METHODS IN ENZYMOLOGY, VOL. 178
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