Determination of the allele-specific antigen-binding site on I-Ak and I-Ab molecules.

Residues 46 and 54 on a pigeon cytochrome c 43–58 analog, 50E, function as major histocompatibility complex class II contact sites. A peptide, 46F50E54A, with phenylalanine (F) at position 46 and alanine (A) at 54 on 50E bound to Ab and a peptide, 46D50E54A, with aspartic acid (D) at 46 and alanine at 54, bound to Ak. To determine the allele-specific peptide contact sites on I-A molecules corresponding to the I-A contact sites of the peptides, we analyzed responses of Ak- and/or Ab-restricted T cell hybridomas to 46F50E54A or 46D50E54A using L cell transfectants expressing recombinant I-A molecules between Ak and Ab or point mutants of Ak as antigen presenting cells. It was shown that the N-terminal half of the α helix of the Aα chain determined the allele-specific T cell responses. Furthermore, with arginine (k type amino acid) or alanine (b type amino acid) at position 56 of the Ak α chain, these T cell hybridomas were stimulated predominantly by 46D50E54A (Ak binding peptide) or 46F50E54A (Ab binding peptide), respectively. Thus, the amino acid at position 56 of the Aα chain determines allele-specific antigen presentation. This postulate was confirmed by direct binding analysis of 50E analogs of various I-A molecules. A single amino acid change (arginine to alanine) at position 56 of the Ak α chain altered the peptide binding specificity (46D50E54A to 46F50E54A).