Abstract
Monoclonal antibodies against an alpha-helical region of the iron-containing, oxygen-binding protein myohemerythrin were isolated following immunization of mice with either the whole protein or a peptide homolog of the helix. Three distinct epitopes within the myohemerythrin helix were identified. The individual residues within two of these epitopes that were essential for antibody binding were determined by measuring antibody binding to a set of peptides in which each amino acid of the epitope was replaced in turn by each of the other 19 amino acids. Hydrophilic residues that are exposed in the native conformation and buried, hydrophobic residues were both shown to be irreplaceable, suggesting their direct involvement in antibody binding. The influence of antigen conformation on antibody binding to these amphipathic epitopes was assessed by measuring the relative affinities of the antibodies for peptides, intact protein, and apoprotein. All of the antibodies bound to apoprotein better than to native protein, indicating that relaxation of the native structure by removal of the iron center increases antibody affinity for myohemerythrin. However, not all of the antibodies tested bound to peptides better than to protein, suggesting that increased antigen flexibility is not always sufficient to maximize antibody binding. Antibody binding to peptides appeared to also be influenced by the ability of the peptides to attain secondary structure at the epitopes, either alone or due to carrier influences.
Full text
PDFImages in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- 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]
- Bahraoui E. M., Granier C., Van Rietschoten J., Rochat H., el Ayeb M. Specificity and neutralizing capacity of antibodies elicited by a synthetic peptide of scorpion toxin. J Immunol. 1986 May 1;136(9):3371–3377. [PubMed] [Google Scholar]
- Berzofsky J. A. Intrinsic and extrinsic factors in protein antigenic structure. Science. 1985 Sep 6;229(4717):932–940. doi: 10.1126/science.2410982. [DOI] [PubMed] [Google Scholar]
- Chang C. T., Wu C. S., Yang J. T. Circular dichroic analysis of protein conformation: inclusion of the beta-turns. Anal Biochem. 1978 Nov;91(1):13–31. doi: 10.1016/0003-2697(78)90812-6. [DOI] [PubMed] [Google Scholar]
- Cooper H. M., Todd P. E., Leach S. J. Antibody response to the C-terminal peptide sequence in beef myoglobin. Mol Immunol. 1986 Dec;23(12):1289–1299. doi: 10.1016/0161-5890(86)90014-3. [DOI] [PubMed] [Google Scholar]
- Dyrberg T., Oldstone M. B. Peptides as antigens. Importance of orientation. J Exp Med. 1986 Oct 1;164(4):1344–1349. doi: 10.1084/jem.164.4.1344. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dyson H. J., Cross K. J., Houghten R. A., Wilson I. A., Wright P. E., Lerner R. A. The immunodominant site of a synthetic immunogen has a conformational preference in water for a type-II reverse turn. Nature. 1985 Dec 5;318(6045):480–483. doi: 10.1038/318480a0. [DOI] [PubMed] [Google Scholar]
- Engvall E. Enzyme immunoassay ELISA and EMIT. Methods Enzymol. 1980;70(A):419–439. doi: 10.1016/s0076-6879(80)70067-8. [DOI] [PubMed] [Google Scholar]
- GREENWOOD F. C., HUNTER W. M., GLOVER J. S. THE PREPARATION OF I-131-LABELLED HUMAN GROWTH HORMONE OF HIGH SPECIFIC RADIOACTIVITY. Biochem J. 1963 Oct;89:114–123. doi: 10.1042/bj0890114. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Galfrè G., Milstein C. Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol. 1981;73(Pt B):3–46. doi: 10.1016/0076-6879(81)73054-4. [DOI] [PubMed] [Google Scholar]
- Getzoff E. D., Geysen H. M., Rodda S. J., Alexander H., Tainer J. A., Lerner R. A. Mechanisms of antibody binding to a protein. Science. 1987 Mar 6;235(4793):1191–1196. doi: 10.1126/science.3823879. [DOI] [PubMed] [Google Scholar]
- Geysen H. M., Barteling S. J., Meloen R. H. Small peptides induce antibodies with a sequence and structural requirement for binding antigen comparable to antibodies raised against the native protein. Proc Natl Acad Sci U S A. 1985 Jan;82(1):178–182. doi: 10.1073/pnas.82.1.178. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Geysen H. M., Tainer J. A., Rodda S. J., Mason T. J., Alexander H., Getzoff E. D., Lerner R. A. Chemistry of antibody binding to a protein. Science. 1987 Mar 6;235(4793):1184–1190. doi: 10.1126/science.3823878. [DOI] [PubMed] [Google Scholar]
- Harrington P. C., Muhoberac B. B., Wharton D. C., Wilkins R. G. Some redox properties of myohemerythrin from retractor muscle of Themiste zostericola. Biochemistry. 1981 Oct 13;20(21):6134–6139. doi: 10.1021/bi00524a034. [DOI] [PubMed] [Google Scholar]
- Hopp T. P., Woods K. R. Prediction of protein antigenic determinants from amino acid sequences. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3824–3828. doi: 10.1073/pnas.78.6.3824. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Horejsí V., Matousek V. Equilibrium in the protein-immobilized-ligand-soluble-ligand system: estimation of dissociation constants of protein-soluble-ligand complexes from binding-inhibition data. Mol Immunol. 1985 Feb;22(2):125–133. doi: 10.1016/s0161-5890(85)80006-7. [DOI] [PubMed] [Google Scholar]
- 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 U S A. 1985 Aug;82(15):5131–5135. doi: 10.1073/pnas.82.15.5131. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kim P. S., Baldwin R. L. A helix stop signal in the isolated S-peptide of ribonuclease A. 1984 Jan 26-Feb 1Nature. 307(5949):329–334. doi: 10.1038/307329a0. [DOI] [PubMed] [Google Scholar]
- Klippenstein G. L., Cote J. L., Ludlam S. E. The primary structure of myohemerythrin. Biochemistry. 1976 Mar 9;15(5):1128–1136. doi: 10.1021/bi00650a027. [DOI] [PubMed] [Google Scholar]
- Klippenstein G. L., Van Riper D. A., Oosterom E. A. A comparative study of the oxygen transport proteins of Dendrostomum pyroides. Isolation and characterization of hemerythrins from muscle, the vascular system, and the coelom. J Biol Chem. 1972 Sep 25;247(18):5959–5963. [PubMed] [Google Scholar]
- Lando G., Reichlin M. Antigenic structure of sperm whale myoglobin. II. Characterization of antibodies preferentially reactive with peptides arising in response to immunization with the native protein. J Immunol. 1982 Jul;129(1):212–216. [PubMed] [Google Scholar]
- Liu F. T., Zinnecker M., Hamaoka T., Katz D. H. New procedures for preparation and isolation of conjugates of proteins and a synthetic copolymer of D-amino acids and immunochemical characterization of such conjugates. Biochemistry. 1979 Feb 20;18(4):690–693. doi: 10.1021/bi00571a022. [DOI] [PubMed] [Google Scholar]
- Novotný J., Handschumacher M., Haber E., Bruccoleri R. E., Carlson W. B., Fanning D. W., Smith J. A., Rose G. D. Antigenic determinants in proteins coincide with surface regions accessible to large probes (antibody domains). Proc Natl Acad Sci U S A. 1986 Jan;83(2):226–230. doi: 10.1073/pnas.83.2.226. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rivier J., McClintock R., Galyean R., Anderson H. Reversed-phase high-performance liquid chromatography: preparative purification of synthetic peptides. J Chromatogr. 1984 Apr 24;288(2):303–328. doi: 10.1016/s0021-9673(01)93709-4. [DOI] [PubMed] [Google Scholar]
- Schulze-Gahmen U., Prinz H., Glatter U., Beyreuther K. Towards assignment of secondary structures by anti-peptide antibodies. Specificity of the immune response to a beta-turn. EMBO J. 1985 Jul;4(7):1731–1737. doi: 10.1002/j.1460-2075.1985.tb03843.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sheriff S., Hendrickson W. A., Smith J. L. Structure of myohemerythrin in the azidomet state at 1.7/1.3 A resolution. J Mol Biol. 1987 Sep 20;197(2):273–296. doi: 10.1016/0022-2836(87)90124-0. [DOI] [PubMed] [Google Scholar]
- Tainer J. A., Getzoff E. D., Alexander H., Houghten R. A., Olson A. J., Lerner R. A., Hendrickson W. A. The reactivity of anti-peptide antibodies is a function of the atomic mobility of sites in a protein. Nature. 1984 Nov 8;312(5990):127–134. doi: 10.1038/312127a0. [DOI] [PubMed] [Google Scholar]
- Thornton J. M., Edwards M. S., Taylor W. R., Barlow D. J. Location of 'continuous' antigenic determinants in the protruding regions of proteins. EMBO J. 1986 Feb;5(2):409–413. doi: 10.1002/j.1460-2075.1986.tb04226.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Westhof E., Altschuh D., Moras D., Bloomer A. C., Mondragon A., Klug A., Van Regenmortel M. H. Correlation between segmental mobility and the location of antigenic determinants in proteins. Nature. 1984 Sep 13;311(5982):123–126. doi: 10.1038/311123a0. [DOI] [PubMed] [Google Scholar]
- Young J. D., Leung C. Y. Immunochemical studies on lysozyme and carboxymethylated lysozyme. Biochemistry. 1970 Jul 7;9(14):2755–2762. doi: 10.1021/bi00816a001. [DOI] [PubMed] [Google Scholar]