The class II histocompatibility molecule I-A(k) was photoaffinity labeled by NH2- and COOH-terminal photoreactive conjugates of an immunogenic hen egg white lysozyme (HEL) peptide. The labeled α and β chains were digested with protease from Staphylococcus aureus strain V-8 (protease V-8) and/or trypsin, and the proteolytic fragments were separated by high performance liquid chromatography (HPLC) (peptide mapping). Reproducible peptide maps containing a major labeled component were obtained from the three conjugates reported here whose photoreactive group was attached via short spacers of limited flexibility. The COOH-terminal conjugate N-acetyl HEL-(49-61)-iodo-4-azidosalicyloyl thioester (compound 1) labeled hydrophilic tryptic digest fragments on both chains of I-A(k). The labeled digest fragments were homogeneous in reverse-phase and anion-exchange HPLC, indicating that the photoaffinity labeling was site-specific. Conversely, the NH2-terminal conjugate iodo-4-azidosalicyloyl HEL-(46-61) (compound 2: IASA-(46-61)) labeled exceptionally hydrophobic sequences on both chains of I-A(k). The labeling was also site-specific because reverse-phase HPLC of primary digests with protease V-8 and secondary digests with trypsin showed single major labeled components. The labeling of I-A(k) by IASA-(46-61) was fully inhibitible by HEL-(46-61). In contrast, IASA attached to the smallest immunogenic peptide 52-61 (compound 3) labeled a distinctly different hydrophilic tryptic fragment. The site of the I-A(k) molecule that was photoaffinity labeled by IASA-(46-61) (compound 2) was determined. IASA-(46-61) labeled selectively at Pro-118 of a primary α chain fragment most likely encompassing residues 115-134. It labeled Thr-121 of a primary β chain fragment most likely encompassing residues 109-138. We also obtained evidence that IASA-(46-61) occupied the antigen-specific site; the conjugate stimulated a T-cell hybridoma that recognizes the sequence 52-61 and also competed for the binding of this smaller peptide to I-A(k). Thus, peptides that bind to the allele-specific binding site and are long enough to extend beyond it can interact with a hydrophobic area of class II molecules. This area is formed by sequences of the first halves of the second domain of both α and β chains.
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - Jul 20 1990|