Computational peptide dissection of Melan-a/MART-1 oncoprotein antigenicity.
ABSTRACT We have mapped the linear antigenic determinant of a commercial MAb raised in the mouse against the melanoma-associated-antigen Melan-A/MART-1. The B cell epitope on the Melan-A/MART-1 oncoprotein is located in the 15-mer amino acid sequence 101-115 PPAYEKLSAEQSPPP, within residues 102-106. The definition of the antigenic sequence on Melan-A/MART-1 oncoprotein was reached following analyses of MHC II binding potential and similarity level to the mouse proteome, that put into evidence the 15-mer amino acid sequence 101-115 PPAYEKLSAEQSPPP as the top scoring peptide in binding H2-A(d) molecules and the epitopic sequence residues 102-106 (i.e., the peptide sequence PAYEK) as having low-similarity level to the mouse proteome. Dot-blot epitope mapping immunoassay identified proline residue 102 as critical, based on its effect on antibody recognition. The present study adds to previous companion reports in validating the hypothesis that low-similarity to the host's proteome and binding potential to MHC II molecules are essential concurring factors in the modulation of the pool of epitopic sequences.
- SourceAvailable from: oxfordjournals.org[show abstract] [hide abstract]
ABSTRACT: A line of tumor-infiltrating lymphocytes (660TIL) specifically lysed the autologous HLA-A2+ melanoma (660MEL) and also most A2+ melanoma cell lines. We immunoprecipitated A2 from a large number (>10(12)) of 660MEL cells, extracted naturally processed peptides, fractionated them by HPLC, screened the fractions for recognition by 660TIL, and found a single predominant and a minor peak of activity. Although too little was recovered of the major 660MEL peptide to establish its sequence, HPLC fingerprinting showed that it did not correspond to any of the known A2-associated melanoma peptides recognized by T cells, including peptides from tyrosinase, MART-1/Melan-A, gp100 and MAGE-3. The major 660MEL antigenic peptide appears to be derived from MART-1/Melan-A but is neither AAGIGILTV nor ILTVILGVL nor any other MART-1/Melan-A peptide containing the A2 consensus motif. The multiplicity of melanoma peptides recognized by CD8+ T cells, most of which are non-mutated (including most likely the present 660MEL peptide), suggests the existence of unknown mechanisms, perhaps similar to those operating in autoimmune disorders, whereby T cells that recognize normal 'self' sequences become activated.International Immunology 02/1997; 9(2):327-38. · 3.14 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: It has been shown that the minimal-length peptide having full stimulatory activity for pigeon cytochrome c-primed T cells from B10.A mice is composed of residues 88-103 of the moth (or 87-104 of the pigeon) sequence. However, to date, only residues 99-103(104)have been shown to be involved in contacting the T-cell receptor or the macrophage Ia molecule. Because the x-ray structure of tuna cytochrome c, and prior calculations on many homologous cytochrome c proteins, showed that segment 88-103(104) exists in the alpha-helical conformation, we postulate that residues 88-98 are necessary for maintaining the alpha-helical conformation of the COOH-terminal pentapeptide (99-103) involved in receptor recognition. To test this hypothesis, we have examined the conformational preferences of polypeptide segments from known antigenic regions near the carboxyl terminus of cytochrome c (pigeon, moth, and fly sequences) using conformational energy calculations for peptides in a nonpolar environment. We show here that fragments consisting of residues 88-91 and 94-98 of pigeon, moth, and fly cytochrome c have a strong alpha-helical preference, despite differences in sequence at residues 88-89 (Lys-Ala in pigeon, Ala-Asn in moth, and Pro-Asn in fly). In contrast, the tripeptide 91-93 (Arg-Ala-Asp) has a strong nonhelical preference. Furthermore, the COOH-terminal peptide 99-103 exists as a statistical coil. However, addition of residues 94-98 to residues 99-103 results in a peptide that has a strong preference for alpha-helix. From these computational results, we predict (i) that fragment 94-103, existing predominantly as an alpha-helix, should exhibit stimulatory activity and (ii) that the nonhelical peptide 91-93 can be deleted from fragment 88-103 without affecting its antigenicity. Both of these predictions have been borne out by experiments in which the two peptides were synthesized and shown to stimulate a T-cell proliferative response. These results establish a strong correlation between conformation (here, alpha-helix) and biological activity and suggest that T-cell activation is sensitive to the organized backbone structure that the antigen adopts in the nonpolar environment of the macrophage membrane or in the combining site of the T-cell receptor.Proceedings of the National Academy of Sciences 07/1983; 80(11):3297-300. · 9.74 Impact Factor