Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1983 Aug;80(16):4949–4953. doi: 10.1073/pnas.80.16.4949

Generation of protein-reactive antibodies by short peptides is an event of high frequency: implications for the structural basis of immune recognition.

H L Niman, R A Houghten, L E Walker, R A Reisfeld, I A Wilson, J M Hogle, R A Lerner
PMCID: PMC384165  PMID: 6192445

Abstract

Recent studies have shown that chemically synthesized small peptides can induce antibodies that often react with intact proteins regardless of their position in the folded molecule. These findings are difficult to explain in view of the experimental and theoretical data which suggest that in the absence of forces provided by the folded protein, small peptides in aqueous solution do not readily adopt stable structures. In order to rationalize the two findings, there has been general acceptance of a stochastic model which suggests that the multiple conformers of a peptide in solution induce sets of antibodies with a small percentage reactive with conformations shared by the folded protein. This stochastic model has become less tenable as the success rate for the generation of protein-reactive anti-peptide antibodies has grown. To test the stochastic model, we have used monoclonal anti-peptide antibodies as a way of estimating the frequency with which small peptides induce antibodies that react with folded proteins. We have made monoclonal antibodies to six chemically synthesized peptides from three proteins. The frequency with which the peptides induce protein-reactive antibodies is at least 4 orders of magnitude greater than expected from previous experimental work and vastly different from what would be predicted by calculating the possible number of peptide conformers in solution. These findings make the stochastic model less likely and lead to consideration of other models. Aside from their practical significance for generation of highly specific reagents, these findings may have important implications for the protein folding problem.

Full text

PDF
4949

Images in this article

4950Image
on p.4950
4951Image
on p.4951
4951Image
on p.4951

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anfinsen C. B., Scheraga H. A. Experimental and theoretical aspects of protein folding. Adv Protein Chem. 1975;29:205–300. doi: 10.1016/s0065-3233(08)60413-1. [DOI] [PubMed] [Google Scholar]
  2. Arnheiter H., Thomas R. M., Leist T., Fountoulakis M., Gutte B. Physicochemical and antigenic properties of synthetic fragments of human leukocyte interferon. Nature. 1981 Nov 19;294(5838):278–280. doi: 10.1038/294278a0. [DOI] [PubMed] [Google Scholar]
  3. Baron M. H., Baltimore D. Antibodies against a synthetic peptide of the poliovirus replicase protein: reaction with native, virus-encoded proteins and inhibition of virus-specific polymerase activities in vitro. J Virol. 1982 Sep;43(3):969–978. doi: 10.1128/jvi.43.3.969-978.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bierzynski A., Baldwin R. L. Local secondary structure in ribonuclease A denatured by guanidine . HCl near 1 degree C. J Mol Biol. 1982 Nov 25;162(1):173–186. doi: 10.1016/0022-2836(82)90167-x. [DOI] [PubMed] [Google Scholar]
  5. Bierzynski A., Kim P. S., Baldwin R. L. A salt bridge stabilizes the helix formed by isolated C-peptide of RNase A. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2470–2474. doi: 10.1073/pnas.79.8.2470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bittle J. L., Houghten R. A., Alexander H., Shinnick T. M., Sutcliffe J. G., Lerner R. A., Rowlands D. J., Brown F. Protection against foot-and-mouth disease by immunization with a chemically synthesized peptide predicted from the viral nucleotide sequence. Nature. 1982 Jul 1;298(5869):30–33. doi: 10.1038/298030a0. [DOI] [PubMed] [Google Scholar]
  7. Brown J. E., Klee W. A. Helix-coil transition of the isolated amino terminus of ribonuclease. Biochemistry. 1971 Feb 2;10(3):470–476. doi: 10.1021/bi00779a019. [DOI] [PubMed] [Google Scholar]
  8. Creighton T. E. Experimental studies of protein folding and unfolding. Prog Biophys Mol Biol. 1978;33(3):231–297. doi: 10.1016/0079-6107(79)90030-0. [DOI] [PubMed] [Google Scholar]
  9. Elder J. H., Jensen F. C., Bryant M. L., Lerner R. A. Polymorphism of the major envelope glycoprotein (gp70) of murine C-type viruses: virion associated and differentiation antigens encoded by a multi-gene family. Nature. 1977 May 5;267(5606):23–28. doi: 10.1038/267023a0. [DOI] [PubMed] [Google Scholar]
  10. Furie B., Schechter A. N., Sachs D. H., Anfinsen C. B. Antibodies to the unfolded form of a helix-rich region in staphylococcal nuclease. Biochemistry. 1974 Apr 9;13(8):1561–1566. doi: 10.1021/bi00705a001. [DOI] [PubMed] [Google Scholar]
  11. Green N., Alexander H., Olson A., Alexander S., Shinnick T. M., Sutcliffe J. G., Lerner R. A. Immunogenic structure of the influenza virus hemagglutinin. Cell. 1982 Mar;28(3):477–487. doi: 10.1016/0092-8674(82)90202-1. [DOI] [PubMed] [Google Scholar]
  12. Hampe A., Laprevotte I., Galibert F., Fedele L. A., Sherr C. J. Nucleotide sequences of feline retroviral oncogenes (v-fes) provide evidence for a family of tyrosine-specific protein kinase genes. Cell. 1982 Oct;30(3):775–785. doi: 10.1016/0092-8674(82)90282-3. [DOI] [PubMed] [Google Scholar]
  13. Jou W. M., Verhoeyen M., Devos R., Saman E., Fang R., Huylebroeck D., Fiers W., Threlfall G., Barber C., Carey N. Complete structure of the hemagglutinin gene from the human influenza A/Victoria/3/75 (H3N2) strain as determined from cloned DNA. Cell. 1980 Mar;19(3):683–696. doi: 10.1016/s0092-8674(80)80045-6. [DOI] [PubMed] [Google Scholar]
  14. KABAT E. A. Size and heterogeneity of the combining sites on an antibody molecule. J Cell Physiol Suppl. 1957 Dec;50(Suppl 1):79–102. doi: 10.1002/jcp.1030500406. [DOI] [PubMed] [Google Scholar]
  15. Kim P. S., Baldwin R. L. Specific intermediates in the folding reactions of small proteins and the mechanism of protein folding. Annu Rev Biochem. 1982;51:459–489. doi: 10.1146/annurev.bi.51.070182.002331. [DOI] [PubMed] [Google Scholar]
  16. Kim P. S., Bierzynski A., Baldwin R. L. A competing salt-bridge suppresses helix formation by the isolated C-peptide carboxylate of ribonuclease A. J Mol Biol. 1982 Nov 25;162(1):187–199. doi: 10.1016/0022-2836(82)90168-1. [DOI] [PubMed] [Google Scholar]
  17. Kratzin H., Yang C. Y., Götz H., Pauly E., Kölbel S., Egert G., Thinnes F. P., Wernet P., Altevogt P., Hilschmann N. Primärstruktur menschlicher Histokompatibilitätsantigene der Klasse II. 1. Mitteilung: Aminosäuresequenz der N-terminalen 198 Reste der beta-Kette des HLA-Dw2,2;DR2,2-Alloantigens. Hoppe Seylers Z Physiol Chem. 1981 Dec;362(12):1665–1669. [PubMed] [Google Scholar]
  18. Lerner R. A., Sutcliffe J. G., Shinnick T. M. Antibodies to chemically synthesized peptides predicted from DNA sequences as probes of gene expression. Cell. 1981 Feb;23(2):309–310. doi: 10.1016/0092-8674(81)90126-4. [DOI] [PubMed] [Google Scholar]
  19. Lerner R. A. Tapping the immunological repertoire to produce antibodies of predetermined specificity. Nature. 1982 Oct 14;299(5884):593–596. doi: 10.1038/299592a0. [DOI] [PubMed] [Google Scholar]
  20. Marglin A., Merrifield R. B. Chemical synthesis of peptides and proteins. Annu Rev Biochem. 1970;39:841–866. doi: 10.1146/annurev.bi.39.070170.004205. [DOI] [PubMed] [Google Scholar]
  21. Niman H. L., Elder J. H. Molecular dissection of Rauscher virus gp70 by using monoclonal antibodies: localization of acquired sequences of related envelope gene recombinants. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4524–4528. doi: 10.1073/pnas.77.8.4524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Niman H. L., Elder J. H. Structural analysis of Rauscher virus Gp70 using monoclonal antibodies: sites of antigenicity and P15(E) linkage. Virology. 1982 Nov;123(1):187–205. doi: 10.1016/0042-6822(82)90305-1. [DOI] [PubMed] [Google Scholar]
  23. Némethy G., Scheraga H. A. Protein folding. Q Rev Biophys. 1977 Aug;10(3):239–252. doi: 10.1017/s0033583500002936. [DOI] [PubMed] [Google Scholar]
  24. Privalov P. L. Stability of proteins: small globular proteins. Adv Protein Chem. 1979;33:167–241. doi: 10.1016/s0065-3233(08)60460-x. [DOI] [PubMed] [Google Scholar]
  25. Ptitsyn O. B., Finkelstein A. V. Similarities of protein topologies: evolutionary divergence, functional convergence or principles of folding? Q Rev Biophys. 1980 Aug;13(3):339–386. doi: 10.1017/s0033583500001724. [DOI] [PubMed] [Google Scholar]
  26. Péterfy F., Kuusela P., Mäkelä O. Affinity requirements for antibody assays mapped by monoclonal antibodies. J Immunol. 1983 Apr;130(4):1809–1813. [PubMed] [Google Scholar]
  27. Richardson J. S. The anatomy and taxonomy of protein structure. Adv Protein Chem. 1981;34:167–339. doi: 10.1016/s0065-3233(08)60520-3. [DOI] [PubMed] [Google Scholar]
  28. Rossmann M. G., Argos P. Protein folding. Annu Rev Biochem. 1981;50:497–532. doi: 10.1146/annurev.bi.50.070181.002433. [DOI] [PubMed] [Google Scholar]
  29. Schaffhausen B., Benjamin T. L., Pike L., Casnellie J., Krebs E. Antibody to the nonapeptide Glu-Glu-Glu-Glu-Tyr-Met-Pro-Met-Glu is specific for polyoma middle T antigen and inhibits in vitro kinase activity. J Biol Chem. 1982 Nov 10;257(21):12467–12470. [PubMed] [Google Scholar]
  30. Sen S., Houghten R. A., Sherr C. J., Sen A. Antibodies of predetermined specificity detect two retroviral oncogene products and inhibit their kinase activities. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1246–1250. doi: 10.1073/pnas.80.5.1246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sutcliffe J. G., Shinnick T. M., Green N., Lerner R. A. Antibodies that react with predetermined sites on proteins. Science. 1983 Feb 11;219(4585):660–666. doi: 10.1126/science.6186024. [DOI] [PubMed] [Google Scholar]
  32. Tanford C. Protein denaturation. C. Theoretical models for the mechanism of denaturation. Adv Protein Chem. 1970;24:1–95. [PubMed] [Google Scholar]
  33. Tanford C. Protein denaturation. Adv Protein Chem. 1968;23:121–282. doi: 10.1016/s0065-3233(08)60401-5. [DOI] [PubMed] [Google Scholar]
  34. Walker L. E., Hewick R., Hunkapiller M. W., Hood L. E., Dreyer W. J., Reisfeld R. A. N-terminal amino acid sequences of the alpha and beta chains of HLA-DR1 and HLA-DR2 antigens. Biochemistry. 1983 Jan 4;22(1):185–188. doi: 10.1021/bi00270a027. [DOI] [PubMed] [Google Scholar]
  35. Wetlaufer D. B. Folding of protein fragments. Adv Protein Chem. 1981;34:61–92. doi: 10.1016/s0065-3233(08)60518-5. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES