Summary
We have characterized the interaction of two monoclonal antibodies with their respective antigens using cellulose-bound sets of overlapping peptides (peptide scans). Both antibodies CB/RS/5 and CB/MT/1 recognize discontinuous epitopes present in human interleukin-10 (IL-10) and tumor necrosis factor alpha (TNF-α). In addition, the interaction between TNF-a and its 55-kDa receptor (TNF-R) was investigated by the same approach. Both antibodies, as well as TNF-α, interacted with two or more regions of the peptide scans. Antibody-binding competition studies between the native antigens and peptides, covering single parts of the binding regions, enabled us to distinguish between binding to the paratope or other regions of the antibody. The combination of these experimental approaches allowed the identification of short antigen-derived sequences that are separated on the primary sequence but close in space on the surface of IL-10 and TNF-α, thus representing putative discontinuous epitopes. In the case of the TNF-R-derived peptide scans, two of the identified regions interact with the structurally similar TNF-β in the TNF-β-TNF-R complex. These data indicate that this approach should be generally applicable for mapping nonlinear protein-protein contact sites.
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References
Phizicky, E.M. and Fields, S.,Protein-protein interaction: Methods for detection and analysis, Microbiol. Rev., 59 (1995) 94–123.
Geysen, H.M., Meloen, R.H. and Barteling, S.J.,Use of peptide synthesis to probe viral antigens for epitopes to resolution of a single amino acid, Proc. Natl. Acad. Sci. USA, 81 (1984) 3998–4002.
Geysen, H.M., Rodda, St.J., Mason, T.J., Tribbick, G. and Schoofs, P.G.,Strategies for epitope analysis using peptide synthesis, J. Immunol. Methods, 102 (1987) 259–274.
Frank, R.,Spot synthesis: An easy technique for the positionally addressable, parallel chemical synthesis on a membrane support, Tetrahedron, 48 (1992) 9217–9232.
Houghten, R.A.,General method for the rapid solid-phase synthesis of large numbers of peptides: Specificity of antigen-antibody interaction at the level of individual amino acids, Proc. Natl. Acad. Sci. USA, 82(1985)5131–5135.
Kramer, A., Volkmer-Engert, R., Malin, R., Reineke, U. and Schneider-Mergener, J.,Simultaneous synthesis of peptide libraries on single resin and continuous membrane supports: Identification of protein-, metal- and DNA-binding peptide mixtures, Pept. Res., 6 (1993) 314–319.
Kramer, A., Schuster, A., Reineke, U., Malin, R., Volkmer-Engert, R., Landgraf, C. and Schneider-Mergener, J.,Combinatorial cellulose-bound peptide libraries: screening tool for the identification of peptides that bind ligands with predefined specificity, METHODS (Comp. Meth. Enzymol.), 6 (1994) 912–921.
Stigler, R., Kramer, A., Henklein, P., Nugel, E., Porstmann, T., Rüker, F. and Schneider-Mergener, J.,Characterisation of the interaction between a Fab fragment against gp41 of HIV-1 and its peptide epitope using a peptide epitope library and molecular modelling, Protein Eng., 8 (1994) 471–480.
Schneider-Mergener, J., Kramer, A. and Reineke, U.,Cellulosebound peptide libraries as a tool to study molecular recognition, In Cortese, R. (Ed.) Combinatorial Libraries, W. de Gruyter, Berlin, Germany, 1996, pp. 53–68.
Weiergräber, O., Schneider-Mergener, J., Grötzinger, J., Wollmer, A., Küster, A., Exner, M. and Heinrich, P.C.,Use of immobilized synthetic peptides for the identification of contact sites between human interleukin-6 and its receptor, Febs Lett., 379 (1996) 122–126.
McCarty, J., Rüdiger, S., Schönfeld, H.-J., Schneider-Mergener, J., Nakahigashi, K., Yura, T. and Buckau, B.,Regulatory region C of the E. coli heat-shock transcription factor, σ32,constitutes a DnaK binding site and is conserved among eubacteria, J. Mol. Biol., 256 (1995) 829–837.
Moore, K.W., Vieira, P., Fiorentino, D.F., Trounstine, M.L., Khan, T.A. and Mosmann, T.R.,Homology of cytokine synthesis inhibitory factor (IL-10) to the Epstein Barr Virus gene BCRF1, Science, 248 (1990) 1230–1234.
Moore, K.W., O'Garra, A., De Waal Malefyt, R., Vieira, P. and Mosmann, T.R.,Interleukin-10, Ann. Rev. Immunol., 11 (1993) 165–190.
Zdanov, A., Schalk-Hihi, C., Gustchina, A., Tsang, M., Weatherbee, J. and Wlodawer, A.,Crystal structure of interleukin-10 reveals the functional dimer with an unexpected topological similarity to interferon-γ, Structure, 3 (1995) 591–601.
Walter, M.R. and Nagabhushan, T.L.,Crystal structure of interleukin-10 reveals an interferon-gamma-like fold, Biochemistry, 34 (1995) 12118–12125.
Beutler, B. and Cerami, A.,The biology of cachectin/TNF —a primary mediator of the host response, Annu. Rev. Immunol., 7 (1989) 625–655.
Seckinger, P., Isaaz, S. and Dayer, J.-M.,Purification and biologic characterization of the specific tumor-necrosis-factor-α inhibitor, J. Biol. Chem., 264 (1989) 11966–11973.
Gausepohl, H., Boulin, C., Kraft, M. and Frank, R.W,Automated multiple peptide synthesis, Pept. Res., 5 (1992) 315–320.
Friguet, B., Chaffotte, A.F., Djavadi-Ohaniance, L. and Goldberg, M.E.,Measurement of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay, J. Immunol. Methods, 77 (1985) 305–319.
Porstmann, T., Porstmann, B., Witschke, R., von Baehr, R. and Egger, E.,Stabilization of the substrate reaction of horseradish peroxidase with o-phenylenediamine in the enzyme immunoassay, J. Clin. Chem.: Clin. Biochem., 23 (1984) 41–44.
Glaser, R.W.,Determination of antibody affinity by ELISA with a nonlinear regression program: Evaluation of linearized approximations, X Immunol. Methods, 160 (1993) 129–133.
Geysen, H.M., Rodda, S.J. and Mason, T.J.,A priori delineation of a peptide which mimics a discontinous antigenic determinant, Mol. Immunol., 23 (1986) 709–715.
Houghten, R.A., Pinilla, C., Blondelle, S.E., Appel, J.R., Dooley, C.T. and Cuervo, J.H.,Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery, Nature, 354 (1991) 84–85.
Eck, M.J. and Sprang, S.R.,The structure of tumor necrosis factor alpha at 2.6 Ängstrøms resolution: Implications for receptor binding, J. Biol. Chem., 264 (1989) 17595–17605.
Jones, E.Y., Stuart, D.I. and Walker, N.P.C.,Structure of tumor necrosis factor, Nature, 338 (1989) 225–228.
Banner, D.W., D'Arcy, A., Janes, W, Gentz, R., Schoenfeld, H. J., Broger, C., Loetscher, H. and Lesslauer, W.,Crystal structure of the soluble human 55-kDa TNF-receptor-human-TNF-β complex: Implications for TNF receptor activation, Cell, 73 (1993) 431–445.
Yamagishi, J., Kawashima, H., Matsuo, N., Ohue, M., Yamayoshi, M., Fukui, T., Kotani, H., Furuta, R., Nakano, K. and Yamada, M.,Mutational analysis of structure-activity relationships in human tumor necrosis factor alpha, Protein Eng., 3 (1990) 713–719.
Van Ostade, X., Tavernier, J., Prange, T. and Fiers, W,Localization of the active site of human tumor necrosis factor (hTNF) by mutational analysis, Embo J., 10 (1991) 827–836.
Loetscher, H., Pan, Y.-C.E., Lahm, H.-W, Gentz, R., Brockhaus, M., Tabuchi, H. and Lesslauer, W,Molecular cloning and expression of the human 55-kDa tumor necrosis factor receptor, Cell, 61 (1990) 351–359.
Lie, B.-L., Tunemoto, D., Hemmi, H., Mizukami, Y, Fukuda, H., Kikuci, H., Kato, S. and Numao, N.,Identification of the binding site of the 55-kDa tumor necrosis factor receptor by synthetic peptides, Biochem. Biophys. Res. Commun., 188 (1992) 503–509.
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Reineke, U., Sabat, R., Kramer, A. et al. Mapping protein-protein contact sites using cellulose-bound peptide scans. Mol Divers 1, 141–148 (1996). https://doi.org/10.1007/BF01544952
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DOI: https://doi.org/10.1007/BF01544952