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Ranking the contribution of ankylosing spondylitis-associated ERAP1 polymorphisms to shaping the HLA-B*27 peptidome

ABSTRACT: The Endoplasmic reticulum aminopeptidase I (ERAP1) trims peptides to their optimal size for binding to Major Histocompatibility Complex class I proteins. The natural polymorphism of this enzyme is associated with ankylosing spondylitis (AS) in epistasis with the major risk factor for this disease, HLA-B*27, suggesting a direct relationship between AS and HLA-B*27-bound peptides. Three polymorphisms that affect peptide trimming protect from AS: K528R, D575N/R725Q, and Q730E. We characterized and ranked the effects of each mutation, and their various combinations, by quantitative comparisons of the HLA-B*27 peptidomes from cells expressing distinct ERAP1 variants. Five features were examined: peptide length, N-terminal flanking residues, N-terminal residues of the natural ligands, internal sequences and affinity for B*27:05. Polymorphism at residue 528 showed the largest influence, affecting all five features regardless of peptide length. D575N/R725Q showed a much smaller effect. Yet, when co-occurring with K528R, it added to this latter change in decreasing ERAP1 activity. Polymorphism at residue 730 showed a significant influence on peptide length, reflecting differential trimming of nonamers and longer peptides. Accordingly, multiple features were affected by the Q730E mutation in a length-dependent way. The alterations induced in the B*27:05 peptidome by natural ERAP1 variants with different K528R/Q730E combinations reflected separate and additive effects of both mutations. Thus, the influence of ERAP1 on HLA-B*27 is very diverse at the population level, due to the multiplicity and complexity of ERAP1 variants, and to the distinct effects of their co-occurring polymorphisms, leading to significant modulation of disease risk among HLA-B*27-positive individuals.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Arie Admon

LAB HEAD: Arie Admon

PROVIDER: PXD008500 | Pride | 2018-04-10

REPOSITORIES: Pride

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Ranking the Contribution of Ankylosing Spondylitis-associated Endoplasmic Reticulum Aminopeptidase 1 (ERAP1) Polymorphisms to Shaping the HLA-B*27 Peptidome.

Sanz-Bravo Alejandro A Alvarez-Navarro Carlos C Martín-Esteban Adrian A Barnea Eilon E Admon Arie A López de Castro José A JA

Molecular & cellular proteomics : MCP 20180409 7

The Endoplasmic reticulum aminopeptidase I (ERAP1) trims peptides to their optimal size for binding to Major Histocompatibility Complex class I proteins. The natural polymorphism of this enzyme is associated with ankylosing spondylitis (AS) in epistasis with the major risk factor for this disease, HLA-B*27, suggesting a direct relationship between AS and HLA-B*27-bound peptides. Three polymorphisms that affect peptide trimming protect from AS: K528R, D575N/R725Q, and Q730E. We characterized and ...[more]

PMID: 29632046

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Project description:Human leukocyte antigen (HLA) - B*51:01 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are two strong predisposing genetic factors to Behçet's disease (BD). Previous studies have focused on two subgroups of HLA-B*51 peptidome containing Proline (Pro) or Alanine (Ala) at position 2 (P2). Little is known about the unconventional non-Pro/Ala2 HLA-B*51-bound peptides. We aimed to study the features of this novel sub-peptidome, and investigate its regulation by ERAP1. CRISPR-Cas9 was used to generate an HLA-ABC-knockout HeLa cell line (HeLa.ABC-KO), which was subsequently transduced to express HLA-B*51:01 (HeLa.ABC-KO.B51). ERAP1 was silenced using lentiviral shRNA. Peptides bound to HLA-B*51:01 were eluted and analyzed by Mass Spectrometry. The characteristics of non-Pro/Ala2, Pro2 and Ala2 peptides, and their alteration by ERAP1 silencing were investigated. Effects of ERAP1 silencing on cell surface expression of HLA-B*51:01 were studied using flow cytometry. More than 20% of peptides eluted from HLA-B*51:01 lacked Proline or Alanine at P2. This unconventional group of HLA-B*51:01-bound peptides was enriched for 8-mers (with fewer 9-mers) compared to Pro2 and Ala2 sub-peptidomes, and had similar N-terminal and C-terminal residue usages to Ala2 peptides with the exception of the less abundant Leucine at position Ω. Knockdown of ERAP1 increased the percentage of non-Pro/Ala2 to approximately 40%, increased the percentage of longer (10-mer and 11-mer) peptides eluted from HLA-B*51:01 complexes, and abrogated the predominance of Leucine at P1. Interestingly knockdown of ERAP1 altered the length and N-terminal residue usage of non-Ala2&Pro2 and Ala2 but not the Pro2 peptides. Finally, ERAP1 silencing regulated the expression levels of cell surface HLA-B*51 in a cell type - dependent manner. The HLA-B*51:01 peptidome includes a surprisingly high proportion of unconventional non-Pro/Ala2 peptides which are increased by ERAP1 silencing, mimicking the loss-of-function BD risk variant.

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