Background Polymorphisms of position 97 (P97) residue in HLA-B are strongly associated with Ankylosing Spondylitis (AS).1 Position 97 residue locates in the α2 domain of HLA-B*27 and contributes to the C/F pocket that accommodates the C-terminal of peptide ligands.2 Mutation of Asparagine (N) at P97 to Aspartic acid (D) has been reported to decrease HLA-B*27:04 cell surface expression.3 We hypothesized that variations of residues at P97 might contribute to AS pathogenesis through altering cell surface HLA-B*27 expression.
Objectives In this study, we studied the effect of P97 residue variants on cell surface expression of HLA-B*27.
Methods Flow cytometry was used to measure surface expression of HLA-B*27 in C1R and HeLa cells expressing wildtype HLA-B*27 (N97) and six mutants at position 97 (N97T, N97S, N97V, N97R, N97W and N97D). TAP-deficient T2, Tapasin-deficient 220 cells, ERAP1-silenced and β2m-silenced C1R cells were used to identify protein interactions. β2m-deficient HCT15 cells and β2m-knockout HeLa cells were used to study the effect of P97 residues on HLA-B*27 expression in the absence of β2m. Surface expression of HLA-B*7/ HLA-B*51 and their P97 mutants in C1R and HeLa cells was also studied.
Results Mutation of P97 to AS risk residue Threonine, but not to the protective residues (Serine and Valine) or the non-associated residues (Arginine and Tryptophan), increased surface free heavy chain (FHC) expression in C1R and HeLa cells. The HLA-B*27-N97D mutation significantly reduced both of HLA-B*27 FHC and classical complex expression on the surface of C1R and HeLa cells. Only β2m, not TAP, Tapasin or ERAP1, was essential for the reduction of cell surface HLA-B*27 FHC expression in HLA-B*27-N97D mutant. The expression of HLA-B*7 and HLA-B*51 was also affected by P97 residue mutations.
Conclusions The P97 residue affects HLA-B*27 free heavy chain and classical complex cell surface expression, in which β2m plays a key role. Cell surface expression of HLA-B*7 and HLA-B*51 is also affected by residue variations at P97. The association of P97 amino acid polymorphisms with AS is, at least in part, explained by its effect on HLA-B*27 free heavy chain cell surface expression.
Cortes A, Pulit SL, Leo PJ, et al. Major histocompatibility complex associations of ankylosing spondylitis are complex and involve further epistasis with ERAP1. Nat Commun 2015;6
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Blanco-Gelaz MA, Suarez-Alvarez B, Gonzalez S, Lopez-Vazquez A, Martinez-Borra J, Lopez-Larrea C. The amino acid at position 97 is involved in folding and surface expression of HLA-B27. International immunology 2006;18(1):211–20
Acknowledgement We sincerely thank Arthritis Research UK (20235, LC&HS), Oxford NIHR Biomedical Research Unit (PB) for funding.
Disclosure of Interest None declared