THU0363 Position 97 (P97) of HLA-B, A Residue Implicated in Ankylosing Spondylitis Pathogenesis, Plays A Key Role in Cell Surface Free Heavy Chain Expression via Interactions with β2-Microglobulin
Authors: L. Chen, H. Shi, J. Yuan, P. Bowness
Journal: Annals of the Rheumatic Diseases (2016)

<h3>Background</h3> Polymorphisms of position 97 (P97) residue in HLA-B are strongly associated with Ankylosing Spondylitis (AS).<sup>1</sup> 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.<sup>2</sup> Mutation of Asparagine (N) at P97 to Aspartic acid (D) has been reported to decrease HLA-B*27:04 cell surface expression.<sup>3</sup> We hypothesized that variations of residues at P97 might contribute to AS pathogenesis through altering cell surface HLA-B*27 expression. <h3>Objectives</h3> In this study, we studied the effect of P97 residue variants on cell surface expression of HLA-B*27. <h3>Methods</h3> 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. <h3>Results</h3> 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. <h3>Conclusions</h3> 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. <h3>References</h3> 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 Madden DR, Gorga JC, Strominger JL, Wiley DC. The three-dimensional structure of HLA-B27 at 2.1 A resolution suggests a general mechanism for tight peptide binding to MHC. Cell 1992;70(6):1035–48 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 <h3>Acknowledgement</h3> We sincerely thank Arthritis Research UK (20235, LC&amp;HS), Oxford NIHR Biomedical Research Unit (PB) for funding. <h3>Disclosure of Interest</h3> None declared