Fine-specificity analysis of antibodies directed to the C-terminal peptides of cytochrome c recognized by T-lymphocytes

Abstract

Recognition of cytochrome c by T-lymphocytes seems to involve the amino acid residues in the C-terminal region of the molecule. Lys-99 has particularly been identified as one of the critical residues in the recognition process. We have now raised antibodies against the C-terminal region of the cytochrome c molecule to map the residues that may be recognized by B-lymphocytes. These antibodies were generated in high-responder B10.A mice against either the 81–104 CNBr fragment of pigeon cytochrome c or against the synthetic spliced fragment (86–90)-(94–103) of the tobacco hornworm moth cytochrome c. A good antibody response was obtained for both fragments as measured by solid-phase radioimmunoassay. A series of peptides related to these fragments were synthesized for competitive inhibition to assess the antigenic sites on these molecules. In spite of substantial homology between the moth (86–90)-(94–103) and pigeon (81–104) fragments, the antibody populations raised against each fragment differed in their recognition patterns. Residues 99 (Lys), 103 (Ala) and 104 (Lys) were found to be crucial for binding of the anti-pigeon antibody to the pigeon 81–104 fragment. The fine specificity mapping of the antigenic sites on the moth (86–90)-(94–103) fragment indicated that along with some of the residues in the N-terminus (86–90), residue 99 (Lys) was involved in recognition of the moth (86–90)-(94–103) fragment by its antibody. This residue (Lys-99) also acts as a T-cell receptor contact site for both pigeon and moth cytochrome c. We therefore conclude that common patterns of recognition must exist between T and B-eells that recognize the C-terminal region of cytochrome c. Since Lys-99 is also present in mouse cytochrome c, the antigenic site must involve both self and non-self residues.