The Cys-67 Residue of HLA-B27 Influences Cell Surface Stability, Peptide Specificity, and T-cell Antigen Presentation

Abstract

Cys-67 of HLA-B27 is located in the B pocket, which determines peptide-binding specificity. We analyzed effects of the Cys-67 3 Ser mutation on cell surface expression, peptide specificity, and T-cell recognition of HLA-B*2705. Surface expression was assessed with antibodies recognizing either native or unfolded HLA proteins. Whereas native B*2705 molecules predominated over unfolded ones, this ratio was reversed in the mutant, suggesting lower stability. Comparison of B*2705-and Cys-67 3 Ser-bound peptides revealed that the mutant failed to bind ϳ15% of the B*2705 ligands, while binding as many novel ones. Two peptides with Gln-2 found in both B*2705 and Cys-67 3 Ser are the first demonstration of natural B*2705 ligands lacking Arg-2. Other effects of the mutation on peptide specificity were: 1) average molecular mass of natural ligands higher than for B*2705, 2) bias against small residues at peptide position (P) 1, and 3) increased P2 permissiveness. The results suggest that the Cys-67 3 Ser mutation weakens B pocket interactions, leading to decreased stability of the mutant-peptide complexes. This may be partially compensated by interactions involving bulky P1 residues. The effect of the mutation on allorecognition was consistent with that on peptide specificity. Our results may aid understanding of the pathogenetic role of HLA-B27 in spondyloarthropathy.

Similar cell surface expression of β2‐microglobulin–free heavy chains by HLA–B27 subtypes differentially associated with ankylosing spondylitis

“…The S67 mutant, which has significantly lower cell surface stability and higher dissociation capacity than B*2705 (38), was used as a control in these experiments. It showed significantly higher HC10:ME1, HC10:W6/32, and HC10:BBM.1 fluorescence ratios relative to any of the natural subtypes tested ( Figure 1C).…”

“…Our approach was inspired by previous results from our laboratory (38) with the S67 mutant of B*2705. This mutant bound many B27 ligands with lower affinity than the wild type, due to suboptimal anchoring of the peptidic R2 motif in the B pocket, and showed significantly increased cell surface dissociation of the peptide-MHC complexes.…”

Lymphoblastoid cells express HLA‐B27 homodimers both intracellularly and at the cell surface following endosomal recycling

“…However, Dangoria et al [20] recently showed that B7 heavy chains can dimerize. Recent reports indicate that C67S mutation decreases the stability of B27 molecules, and promotes unfolding to high molecular weight species [43,44]. Although cells with the C67S mutation express HC-10-reactive heavy chain monomers and high molecular weight multimers, these mutant heavy chains were unable to form stable homodimers at the cell surface.…”

The peptide-binding motif of HLA-DR8 shares important structural features with other type 1 diabetes-associated alleles

“…Immunoaffinity purification of HLA-DR-peptide complexes Peptide-HLA-DR complexes were purified as described, 38,39 with some modifications. Briefly, 10 (ref.…”