Critical Role of Endoplasmic Reticulum Aminopeptidase 1 in Determining the Length and Sequence of Peptides Bound and Presented by HLA–B27

Abstract

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.The human leukocyte antigen HLA-B27 is very strongly associated with development of the common inflammatory rheumatic disease ankylosing spondylitis (AS). However, the pathogenic mechanism remains unclear. Recent genome-wide association studies have identified additional AS-associated genes, of which the strongest association is with endoplasmic reticulum aminopeptidase 1 (ERAP1) (1). The strong genetic association of AS with ERAP1 single-nucleotide polymorphisms has been confirmed in different populations (2-7).

The interplay between HLA-B27 and ERAP1/ERAP2 aminopeptidases: from anti-viral protection to spondyloarthritis

“…The effect impacts mainly the P1 residue and, to a lesser extent, the remaining peptide sequence, the peptide length, the amount of specific ligands and the B27 affinity and thermostability of the overall peptide/B27 complexes. It is noteworthy that another study has shown that ERAP1 silencing, as expected, decreased the amount of B27-bound nonamer peptides and, interestingly, increased the number of longer ligands, especially with extended C-terminus [91].…”

“…Interestingly, the decreased CTL recognition of the cells expressing the Arg528 ERAP1 variant or the minigenes containing KK10 precursors was reversed by the combination Arg528/730Glu, supporting the concept that the global ERAP1 haplotype modulates the fine enzymatic specificity [91].…”

“…Another study has reported that the silencing of ERAP1 as well as the AS-protective allelic variant Arg528 reduced the presentation of the HIV-Gag immunodominant HLA-B27 epitope, KK10 [91]. Interestingly, the decreased CTL recognition of the cells expressing the Arg528 ERAP1 variant or the minigenes containing KK10 precursors was reversed by the combination Arg528/730Glu, supporting the concept that the global ERAP1 haplotype modulates the fine enzymatic specificity [91].…”

Functionally distinct ERAP1 allotype combinations distinguish individuals with Ankylosing Spondylitis

“…Differences in trimming properties may predispose an individual to the formation of ER B27 2 and UPR. In addition, the generation of suboptimal HLA-B27 ligands created by aberrant ERAP1 activity that bind with sufficient affinity to pass intracellular quality control (16,29) may dissociate rapidly at the cell surface, leading to increased aberrant forms of B27. These mechanisms are not necessarily mutually exclusive, nor do they preclude other possible mechanisms such as the ability of different ERAP1 variants to generate specific arthritogenic peptides.…”

HLA-B*51 the primary risk in Behçet disease

“…Heterozygous ERAP-1 +/− mice showed an intermediate phenotype that suggest polymorphisms that affect ERAP-1 function could affect antigen presentation (17). ERAP-1 has recently been shown to be associated with anklyosing spondylitis, a condition strongly linked to another HLA class I molecule: HLA-B*27 (18). The relevance of these findings to BD includes the possibility that different peptides binding in the groove could either activate a different population of CD8 +ve cells or stabilize HLA molecules on the surface, potentially increasing the inhibitory signal to NK cells.…”