Dominant Role of the ERAP1 Polymorphism R528K in Shaping the HLA–B27 Peptidome Through Differential Processing Determined by Multiple Peptide Residues

Abstract

Objective. To characterize the alterations, as well as their mechanisms, induced in the HLA-B27-bound peptidome expressed in live cells by the natural ERAP1 polymorphisms predisposing to ankylosing spondylitis (AS): R528K and N575D/Q725R.Methods. HLA-B*27:05-bound peptides were isolated from 3 human lymphoid cell lines expressing distinct ERAP1 variants differing at residues 528 and/or 575/725. The high-performance liquid chromatographyfractionated peptide pools were compared by mass spectrometry based on identity of molecular mass and chromatographic retention time. The relative amount of each shared peptide in any given cell line pair was estimated from the respective ion peak intensities. Peptide sequencing was also carried out by mass spectrometry.Results. HLA-B27-bound ligands predominant in the context of the ERAP1 variant with K528 collectively showed higher molecular mass, higher frequency of N-terminal residues resistant to ERAP1, and bulkier residues downstream of the N-terminus, relative to peptides predominant in the R528 context. None of these differences were observed with ERAP1 variants differing at positions 575/725, but not at residue 528. Neither R528K nor N575D/Q725R altered the mean length of B*27:05-bound ligands.Conclusion. The R528K, but not the N575D/ Q725R, polymorphism alters the expression levels of many HLA-B*27:05-bound peptides, depending on the susceptibility of their N-terminal residues to trimming and depending on the size of the amino acid side chains at multiple positions downstream of the N-terminus. The significant alterations in the B*27:05 peptidome and the structural features of the peptides that determine their differential expression in distinct ERAP1 contexts account for the association of the R528K polymorphism with AS.

Crystal Structure of Insulin-Regulated Aminopeptidase with Bound Substrate Analogue Provides Insight on Antigenic Epitope Precursor Recognition and Processing

“…Enzymatic analysis indicated a small, but not statistically significant, reduction in enzymatic activity due to that polymorphism, supporting the poor association of this SNP with ankylosing spondylitis (20). In contrast, polymorphism A609T interacts with the hinge domain III of IRAP, similarly to SNP K528R in ERAP1, which has been repeatedly validated for disease association and effects on activity (15,46). We find that the A609T mutation leads to an almost 2-fold reduction in activity, a finding that provides functional validation for the disease association of this SNP (Fig.…”

“…The most commonly discovered SNP is K528R, a position located in the interface of domains II and III that has been shown to reduce ERAP1 activity and affect intracellular Ag processing as well as the immunopeptidome (18,19,44,46). Two recent studies have implicated two IRAP SNPs in the pathogenesis of autoimmune disease (20,21).…”

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

“…However, such study suffered from low statistical power due to the small cohorts analysed. Seminal work from Lopez de Castro's group has outlined how the B27 peptidome is influenced by ERAP allotypes [89,90]. 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.…”

Endoplasmic Reticulum Aminopeptidase 1 (ERAP1) Polymorphism Relevant to Inflammatory Disease Shapes the Peptidome of the Birdshot Chorioretinopathy-Associated HLA-A*29:02 Antigen*

“…This subdivision was arbitrarily set based on the assumption that quantitative effects of ERAP1 on the expression levels of MHC-I ligands should be best observed among peptides showing the maximal intensity differences among cell lines. It was used in previous studies from our laboratory to reveal the effects of ERAP1 polymorphism on the HLA-B*27 peptidome (16,17). The relationship of the IR subsets to the statistical significance of the intensity differences observed in the pairwise peptidome comparisons among cell lines is shown in Fig.…”

“…Yet, an involvement of functional ERAP1 polymorphisms, not determining protein expression, was not excluded. These polymorphisms have a large influence on the HLA-B*27 peptidome (16,17). In contrast, the effects of ERAP2 on MHC-I peptidomes are poorly understood and are probably dependent on the particular ERAP1 context since ERAP2 cooperates with ERAP1 in peptide processing.…”