ANALYSIS OF ANCHOR RESIDUES IN A NATURALLY PROCESSED HCA-DR53 LIGAND

The peptide motif of the HLA-DR53 (DRB40101) molecule, which is assoc iated with autoimmune diseases including Vogt-Koyanagi-Harada's syndro me, was determined by peptide binding assay using human L plastin p581 -595 peptide and its substituted analogues. L plastin p581-595 peptide is one of the naturally processed peptides bound to HLA-DR9/DR53 (DRB 10901/DRB4*0101) molecules. The binding affinity of each peptide to t he HLA-DR53 molecule was measured by fluorescence intensity of biotiny lated peptides to L cell transfectants expressing HLA-DR53 molecules, followed by treatment with avidin-fluorescence. Binding of biotinylate d peptides to HLA-DR53 molecules was not inhibited by all single-alani ne-substituted nonbiotinylated peptides, indicating that the replaced position was important for binding to the HLA-DR53 moleule. The inhibi tory motif is considered to be an HLA-DR53-specific binding motif, com posed of a positively charged residue (Kj at position 1, a hydrophobic residue (T) at position 4, positively charged residue (R or K) at pos ition 8 or 9, and another hydrophobic residue (Ij at position 10. This predicted motif is different from the binding motifs of other HLA-DR molecules. binding peptides in combination with functional analyses, b y alignment of sequenced endogeneous peptides, and by the use of an M1 3 display library (Rammensee et al. 1995; Hammer rt al. 1993, 1993). N o sequence information has been reported for naturally occurring HLA-D R53 (DRB40101)-associated peptides partly because their expression on the cell surface is relatively low for sequencing endogeneous self-pe ptides (Kinouchi et al. 1995). Et has been shown that HLA-DR53 is posi tively associated with Vogt-Koyanagi-Harada's Syndrome in Japanese sub jects (Moriuchi et at. 1979). The identification of a peptide motif fo r HLA-DR53 may help in understanding the mechanisms of this disease. W e have previously reported that naturally processed peptides bound to HLA-DR9/DR53 molecules (Futaki et al. 1995). In this report, we determ ined the peptide which could bind to the HLA-DR53 molecule and we iden tified a putative HLA-DR53-specific binding motif by a peptide binding assay using L-cell transfectants expressing HLA-DR molecules.