Analysis of anchor residues in a naturally processed HLA-DR53 ligand

Abstract

The peptide motif of the HLA-DR53 (DRB4^*0101) molecule, which is associated with autoimmune diseases including Vogt-Koyanagi-Harada's syndrome, 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 (DRB1^*0901/DRB4^*0101) molecules. The binding affinity of each peptide to the HLA-DR53 molecule was measured by fluorescence intensity of biotinylate peptides to L cell transfectants expressing HLA-DR53 molecules, followed by treatment with avidin-fluorescence. Binding of biotinylated peptides to HLA-DR53 molecules was not inhibited by all single-alanine-substituted nonbiotinylated peptides, indicating that the replaced position was important for binding to the HLA-DR53 moleule. The inhibitory motif is considered to be an HLA-DR53-specific binding motif, composed of a positively charged residue (K) at position 1, a hydrophobic residue (I) at position 4, positively charged residue (R or K) at position 8 or 9, and another hydrophobic residue (I) at position 10. This predicted motif is different from the binding motifs of other HLA-DR molecules. binding peptides in combination with functional analyses, by alignment of sequenced endogeneous peptides, and by the use of an M13 display library (Rammensee et al. 1995; Hammer et al. 1993, 1992). No sequence information has been reported for naturally occurring HLA-DR53 (DRB4^*0101)-associated peptides partly because their expression on the cell surface is relatively low for sequencing endogeneous self-peptides (Kinouchi et al. 1995). It has been shown that HLA-DR53 is positively associated with Vogt-Koyanagi-Harada's Syndrome in Japanese subjects (Moriuchi et al. 1979). The identification of a peptide motif for HLA-DR53 may help in understanding the mechanisms of this disease. We have previously reported that naturally processed peptides bound to HLA-DR9/DR53 molecules (Futaki et al. 1995). In this report, we determined the peptide which could bind to the HLA-DR53 molecule and we identified a putative HLA-DR53-specific binding motif by a peptide binding assay using L-cell transfectants expressing HLA-DR molecules.