OBJECTIVE: HLA-B27 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are the two strongest genetic factors predisposing to ankylosing spondylitis (AS). A key aminopeptidase in class I major histocompatibility complex presentation, ERAP1 potentially contributes to the pathogenesis of AS by altering HLA-B27 peptide presentation. The aim of this study was to analyze the effects of ERAP1 on the HLA-B27 peptide repertoire and peptide presentation to cytotoxic T lymphocytes (CTLs). METHODS: ERAP1-silenced and -competent HeLa.B27 and C1R.B27 cells were isotope-labeled, mixed, lysed, and then immunoprecipitated using W6/32 or ME1 antibodies. Peptides bound to HLA-B27 were eluted and analyzed by tandem mass spectrometry. Selected peptides were synthesized and tested for HLA-B27 binding ability. The effect of ERAP1 silencing/mutation on presentation of an immunodominant viral HLA-B27 epitope, KK10, to CTLs was also studied. RESULTS: In both HeLa.B27 and C1R.B27 cells, the proportion of 9-mer HLA-B27-bound peptides was decreased by ERAP1 silencing, whereas the percentages of longer peptides (11-13 mer) were increased. Surprisingly, following ERAP1 silencing, C-terminally extended peptides were readily identified. These were better able to bind to HLA-B27 than were N-terminally extended peptides lacking an arginine at position 2. In both HeLa.B27 cells and mouse fibroblasts expressing HLA-B27, the absence of ERAP1 reduced peptide recognition by HLA-B27-restricted KK10-specific CTLs following infection with recombinant vaccinia virus or transfection with minigenes expressing KK10 precursors. Presence of an AS-protective variant of ERAP1, K528R, as compared to wild-type ERAP1, reduced the peptide recognition by KK10 CTLs following transfection with extended KK10 minigenes. CONCLUSION: These results show that ERAP1 directly alters peptide binding and presentation by HLA-B27, thus demonstrating a potential pathogenic mechanism in AS. Inhibition of ERAP1 could potentially be used for treatment of AS and other ERAP1-associated diseases.
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Aminopeptidases, Animals, B-Lymphocytes, Cell Line, Cells, Cultured, Disease Models, Animal, Epitopes, Gene Silencing, HLA-B27 Antigen, HeLa Cells, Humans, Mice, Knockout, Minor Histocompatibility Antigens, Mutation, Peptides, RNA, Small Interfering, Spondylitis, Ankylosing, T-Lymphocytes, Cytotoxic