Molecular Diversity

, Volume 1, Issue 3, pp 141–148 | Cite as

Mapping protein-protein contact sites using cellulose-bound peptide scans

  • Ulrich Reineke
  • Robert Sabat
  • Achim Kramer
  • Rolf D. Stigler
  • Martina Seifert
  • Thomas Michel
  • Hans D. Volk
  • Jens Schneider-Mergener
Research Papers


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.


TNF IL-10 Discontinuous epitope Antibody Peptide scan Protein-protein interaction Spot synthesis 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Phizicky, E.M. and Fields, S.,Protein-protein interaction: Methods for detection and analysis, Microbiol. Rev., 59 (1995) 94–123.PubMedGoogle Scholar
  2. 2.
    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.PubMedGoogle Scholar
  3. 3.
    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.PubMedGoogle Scholar
  4. 4.
    Frank, R.,Spot synthesis: An easy technique for the positionally addressable, parallel chemical synthesis on a membrane support, Tetrahedron, 48 (1992) 9217–9232.Google Scholar
  5. 5.
    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.PubMedGoogle Scholar
  6. 6.
    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.PubMedGoogle Scholar
  7. 7.
    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.Google Scholar
  8. 8.
    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.Google Scholar
  9. 9.
    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.Google Scholar
  10. 10.
    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.PubMedGoogle Scholar
  11. 11.
    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.Google Scholar
  12. 12.
    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.PubMedGoogle Scholar
  13. 13.
    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.Google Scholar
  14. 14.
    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.PubMedGoogle Scholar
  15. 15.
    Walter, M.R. and Nagabhushan, T.L.,Crystal structure of interleukin-10 reveals an interferon-gamma-like fold, Biochemistry, 34 (1995) 12118–12125.PubMedGoogle Scholar
  16. 16.
    Beutler, B. and Cerami, A.,The biology of cachectin/TNFa primary mediator of the host response, Annu. Rev. Immunol., 7 (1989) 625–655.PubMedGoogle Scholar
  17. 17.
    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.PubMedGoogle Scholar
  18. 18.
    Gausepohl, H., Boulin, C., Kraft, M. and Frank, R.W,Automated multiple peptide synthesis, Pept. Res., 5 (1992) 315–320.PubMedGoogle Scholar
  19. 19.
    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.PubMedGoogle Scholar
  20. 20.
    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.Google Scholar
  21. 21.
    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.Google Scholar
  22. 22.
    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.PubMedGoogle Scholar
  23. 23.
    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.PubMedGoogle Scholar
  24. 24.
    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.PubMedGoogle Scholar
  25. 25.
    Jones, E.Y., Stuart, D.I. and Walker, N.P.C.,Structure of tumor necrosis factor, Nature, 338 (1989) 225–228.PubMedGoogle Scholar
  26. 26.
    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.PubMedGoogle Scholar
  27. 27.
    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.PubMedGoogle Scholar
  28. 28.
    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.PubMedGoogle Scholar
  29. 29.
    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.PubMedGoogle Scholar
  30. 30.
    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.PubMedGoogle Scholar

Copyright information

© ESCOM Science Publishers B.V 1996

Authors and Affiliations

  • Ulrich Reineke
    • 1
  • Robert Sabat
    • 1
  • Achim Kramer
    • 1
  • Rolf D. Stigler
    • 1
  • Martina Seifert
    • 1
  • Thomas Michel
    • 1
  • Hans D. Volk
    • 1
  • Jens Schneider-Mergener
    • 1
  1. 1.Institute for Medicinal ImmunologyUniversity Hospital Charité, Humboldt University at BerlinBerlinGermany

Personalised recommendations