The antigen processing machinery (APM) plays an important role in immune recognition of virally infected and transformed cells. Defective expression of the APM component ERAP1 is associated with progression and poor clinical outcome in cervical carcinoma. Howev
er, the underlying mechanisms of ERAP1 protein downregulation remain to be established. We investigated ERAP1 mRNA expression levels in 14 patients with established ERAP1 protein downregulation. To further examine the possible pretranscriptional mechanisms of ERAP1 downregulation, ERAP1 DNA mutation status was analyzed alongside existing data on various single nucleotide polymorphisms. Moreover, loss of heterozygosity at various loci in the ERAP1 gene was investigated. In cases with ERAP1 protein downregulation, ERAP1 mRNA quantities were found to be significantly lower than in a cohort with normal ERAP1 protein expression (P = 0.001). Loss of heterozygosity was demonstrated to occur in up to 50% of tumors with ERAP1 downregulation. Our data indicate that ERAP1 downregulation is associated with loss of heterozygosity. These data provide the first insight into in vivo mechanisms of ERAP1 downregulation in cervical carcinoma.
OBJECTIVE: The results of a recent genome-wide association study have shown that ERAP1 and IL23R are associated with ankylosing spondylitis (AS) in Caucasian populations from North America and the UK. Based on these findings, we undertook the current study to
investigate whether single-nucleotide polymorphisms (SNPs) covering the genes ERAP1 and IL23R are associated with AS in a Han Chinese population. METHODS: A case-control study was performed in Han Chinese patients with AS (n = 527) and controls (n = 945) from Shanghai and Nanjing. All patients met the modified New York criteria for AS. The Sequenom iPlex platform was used to genotype cases and controls for 21 tag SNPs covering IL23R and 38 tag SNPs covering ERAP1. Statistical analysis was performed using the Cochran-Armitage test for trend. RESULTS: Multiple SNPs in ERAP1 were significantly associated with AS (for rs27980, P = 0.0048; for rs7711564, P = 0.0081). However, no association was observed between IL23R and AS (for all SNPs, P > 0.1). The nonsynonymous SNP in IL23R, rs11209026, widely thought to be the primary AS-associated SNP in IL23R in Europeans, was found not to be polymorphic in Chinese. CONCLUSION: Our results demonstrate that genetic polymorphisms in ERAP1 are associated with AS in Han Chinese, suggesting a common pathogenic mechanism for the disease in Chinese and Caucasian populations, and that IL23R is not associated with AS in Chinese, indicating a difference in the mechanism of disease pathogenesis between Chinese and Caucasian populations. This may result from the fact that rs11209026, the nonsynonymous SNP in IL23R, is not polymorphic in Chinese patients, providing further evidence that rs11209026 is the key polymorphism associated with AS (and likely inflammatory bowel disease and psoriasis) in this gene.
Precursors to major histocompatibility complex (MHC) class I-presented peptides with extra NH2-terminal residues can be efficiently trimmed to mature epitopes in the endoplasmic reticulum (ER). Here, we purified from liver microsomes a lumenal, soluble aminopeptidase that removes NH2-terminal residu
es from many antigenic precursors. It was identified as a metallopeptidase named "adipocyte-derived leucine" or "puromycin-insensitive leucine-specific" aminopeptidase. However, because we localized it to the ER, we propose it be renamed ER-aminopeptidase 1 (ERAP1). ERAP1 is inhibited by agents that block precursor trimming in ER vesicles and although it trimmed NH2-extended precursors, it spared presented peptides of 8 amino acid and less. Like other proteins involved in antigen presentation, ERAP1 is induced by interferon-gamma. When overexpressed in vivo, we found that ERAP1 stimulates the processing and presentation of an antigenic precursor in the ER.
Birdshot chorioretinopathy is a rare ocular inflammation whose genetic association with HLA-A*29:02 is the highest between a disease and a major histocompatibility complex (MHC) molecule. It belongs to a group of MHC-I-associated inflammatory disorders, also including ankylosing spondylitis, psorias
is, and Behcet's disease, for which endoplasmic reticulum aminopeptidases (ERAP) 1 and/or 2 have been identified as genetic risk factors. Since both enzymes are involved in the processing of MHC-I ligands, it seems reasonable that common peptide-mediated mechanisms may underlie the pathogenesis of these diseases. In this study, comparative immunopeptidomics was used to characterize >5000 A*29:02 ligands and quantify the effects of ERAP1 polymorphism and expression on the A*29:02 peptidome in human cells. The peptides predominant in an active ERAP1 context showed a higher frequency of nonamers and bulkier amino acid side chains at multiple positions, compared with the peptides predominant in a less active ERAP1 background. Thus, ERAP1 polymorphism has a large influence, shaping the A*29:02 peptidome through length-dependent and length-independent effects. These changes resulted in increased affinity and hydrophobicity of A*29:02 ligands in an active ERAP1 context. The results reveal the nature of the functional interaction between A*29:02 and ERAP1 and suggest that this enzyme may affect the susceptibility to birdshot chorioretinopathy by altering the A*29:02 peptidome. The complexity of these alterations is such that not only peptide presentation but also other potentially pathogenic features could be affected.
Ankylosing spondylitis is a common form of inflammatory arthritis predominantly affecting the spine and pelvis that occurs in approximately 5 out of 1,000 adults of European descent. Here we report the identification of three variants in the RUNX3, LTBR-TNFRSF1A and IL12B regions convincingly associ
ated with ankylosing spondylitis (P < 5 x 10(-8) in the combined discovery and replication datasets) and a further four loci at PTGER4, TBKBP1, ANTXR2 and CARD9 that show strong association across all our datasets (P < 5 x 10(-6) overall, with support in each of the three datasets studied). We also show that polymorphisms of ERAP1, which encodes an endoplasmic reticulum aminopeptidase involved in peptide trimming before HLA class I presentation, only affect ankylosing spondylitis risk in HLA-B27-positive individuals. These findings provide strong evidence that HLA-B27 operates in ankylosing spondylitis through a mechanism involving aberrant processing of antigenic peptides.
Abdullah H, etal., Discov Med. 2015 Jul-Aug;20(108):79-89.
Ankylosing spondylitis (AS) is a chronic, inflammatory arthritis of the spine and peripheral joints linked to the antigen presenting molecule HLA-B27. The risk of AS is increased in patients possessing endoplasmic reticulum aminopeptidase-1 (ERAP1) polymorphism
s rs30187 and rs27044 encoding amino acid changes K528R and Q730E, respectively. Dysfunction of ERAP1 is hypothesized to cause changes in expression of HLA-B27 classical (pHLA) and non-classical (FHC) conformers on antigen presenting cells (APCs), which interact with the natural killer (NK) cell receptor KIR3DL1. Dysregulation of this pathway may be pathogenic in AS. APC cell lines expressing HLA-B27 were found to inhibit cytokine production in KIR3DL1+ NK cells due to decreased APC-NK cell adhesion, and possibly activation of receptor down-regulation. Blocking pHLA and FHC reveals that both conformers inhibit cytokine production through KIR3DL1. KIR3DL1 affinity and HLA-B27 surface expression studies suggest that ERAP1 R528 and E730 expression protects from AS by generating sub-optimal pHLA, causing reduced KIR3DL1 affinity and weaker cytokine inhibition. Secondarily we observed that KIR3DL1 binding to C1R-B27 APCs is enhanced by blocking pHLA, but not FHC, raising the possibility that antibody mediated HLA-B27 cross-linking may be important in enhancing KIR3DL1+ NK cell function. This study establishes the role of both FHC and pHLA in modulating NK cell cytokine secretion and adhesion functions by interacting with KIR3DL1. This interaction varies depending on the AS association status of the ERAP1 variant expressed in APCs. Additionally antibody cross-linking of HLA-B27 enhances KIR3DL1 binding and as such could be an important pathogenic mechanism in AS.
Cortes A, etal., Nat Commun. 2015 May 21;6:7146. doi: 10.1038/ncomms8146.
Ankylosing spondylitis (AS) is a common, highly heritable, inflammatory arthritis for which HLA-B*27 is the major genetic risk factor, although its role in the aetiology of AS remains elusive. To better understand the genetic basis of the MHC susceptibility loci, we genotyped 7,264 MHC SNPs in 22,64
7 AS cases and controls of European descent. We impute SNPs, classical HLA alleles and amino-acid residues within HLA proteins, and tested these for association to AS status. Here we show that in addition to effects due to HLA-B*27 alleles, several other HLA-B alleles also affect susceptibility. After controlling for the associated haplotypes in HLA-B, we observe independent associations with variants in the HLA-A, HLA-DPB1 and HLA-DRB1 loci. We also demonstrate that the ERAP1 SNP rs30187 association is not restricted only to carriers of HLA-B*27 but also found in HLA-B*40:01 carriers independently of HLA-B*27 genotype.
Chen L, etal., Ann Rheum Dis. 2016 May;75(5):916-23. doi: 10.1136/annrheumdis-2014-206996. Epub 2015 Jun 30.
OBJECTIVE: Human leucocyte antigen (HLA)-B27 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are strongly associated with ankylosing spondylitis (AS). ERAP1 is a key aminopeptidase in HLA class I presentation and can pote
ntially alter surface expression of HLA-B27 free heavy chains (FHCs). We studied the effects of ERAP1 silencing/inhibition/variations on HLA-B27 FHC expression and Th17 responses in AS. METHODS: Flow cytometry was used to measure surface expression of HLA class I in peripheral blood mononuclear cells (PBMCs) from patients with AS carrying different ERAP1 genotypes (rs2287987, rs30187 and rs27044) and in ERAP1-silenced/inhibited/mutated HLA-B27-expressing antigen presenting cells (APCs). ERAP1-silenced/inhibited APCs were cocultured with KIR3DL2CD3epsilon-reporter cells or AS CD4+ T cells. Th17 responses of AS CD4+ T cells were measured by interleukin (IL)-17A ELISA and Th17 intracellular cytokine staining. FHC cell surface expression and Th17 responses were also measured in AS PBMCs following ERAP1 inhibition. RESULTS: The AS-protective ERAP1 variants, K528R and Q730E, were associated with reduced surface FHC expression by monocytes from patients with AS and HLA-B27-expressing APCs. ERAP1 silencing or inhibition in APCs downregulated HLA-B27 FHC surface expression, reduced IL-2 production by KIR3DL2CD3epsilon-reporter cells and suppressed the Th17 expansion and IL-17A secretion by AS CD4+ T cells. ERAP1 inhibition of AS PBMCs reduced HLA class I FHC surface expression by monocytes and B cells, and suppressed Th17 expansion. CONCLUSIONS: ERAP1 activity determines surface expression of HLA-B27 FHCs and potentially promotes Th17 responses in AS through binding of HLA-B27 FHCs to KIR3DL2. Our data suggest that ERAP1 inhibition has potential for AS treatment.
Genetic variation of the antigen processing machinery (APM) components TAP2, LMP7, and ERAP1 is related to cervical carcinoma risk, although the relation with expression and clinical outcome remains unknown. We have investigated the occurrence of APM component s
ingle nucleotide polymorphisms (SNPs) in cervical carcinoma. Twelve nonsynonymous, coding SNPs in the TAP1, TAP2, LMP2, LMP7, and ERAP1 genes were genotyped in 75 cervical carcinoma patients with known APM component and HLA class I expression levels. Individual genotype distributions were assessed for association with APM component expression, various histopathological parameters and survival. Genotype distributions at the ERAP1-56 and ERAP1-127 loci were significantly associated with overall survival (OS); haplotype construction spanning these two SNPs revealed that the combination of a major allele at ERAP1-56 and a minor allele at ERAP1-127 was significantly associated with survival, homozygosity for this haplotype being associated with decreased OS (5-year survival 50% vs. 70 and 81% for complete absence or heterozygosity for this haplotype, respectively; P = 0.021). Heterozygosity for this haplotype was an independent predictor for better OS in multivariate analysis (HR = 0.219; P = 0.014). These data indicate that genetic variation in APM component genes, particularly ERAP1, is an important contributing factor in cervical carcinogenesis, progressive tumor growth and survival. The location of the ERAP1-127 SNP in the peptidase M1 domain of the ERAP1 aminopeptidase suggests the possibility of direct functional consequences of variation at this locus.
York IA, etal., Nat Immunol. 2002 Dec;3(12):1177-84. Epub 2002 Nov 18.
Endoplasmic reticulum (ER) aminopeptidase 1 (ERAP1) appears to be specialized to produce peptides presented on class I major histocompatibility complex molecules. We found that purified ERAP1 trimmed peptides that were ten
residues or longer, but spared eight-residue peptides. In vivo, ERAP1 enhanced production of an eight-residue ovalbumin epitope from precursors extended on the NH2 terminus that were generated either in the ER or cytosol. Purified ERAP1 also trimmed nearly half the nine-residue peptides tested. By destroying such nine-residue peptides in normal human cells, ERAP1 reduced the overall supply of antigenic peptides. However, after interferon-gamma treatment, which causes proteasomes to produce more NH2-extended antigenic precursors, ERAP1 increased the supply of peptides for MHC class I antigen presentation.
Barnea E, etal., Mol Cell Proteomics. 2017 Apr;16(4):642-662. doi: 10.1074/mcp.M116.066241. Epub 2017 Feb 10.
HLA-B27 is a class I major histocompatibility (MHC-I) allele that confers susceptibility to the rheumatic disease ankylosing spondylitis (AS) by an unknown mechanism. ERAP1 is an aminopeptidase that trims peptides in the endoplasmic reticulum for binding to MHC-
I molecules. ERAP1 shows genetic epistasis with HLA-B27 in conferring susceptibility to AS. Male HLA-B27 transgenic rats develop arthritis and serve as an animal model of AS, whereas female B27 transgenic rats remain healthy. We used large scale quantitative mass spectrometry to identify over 15,000 unique HLA-B27 peptide ligands, isolated after immunoaffinity purification of the B27 molecules from the spleens of HLA-B27 transgenic rats. Heterozygous deletion of Erap1, which reduced the Erap1 level to less than half, had no qualitative or quantitative effects on the B27 peptidome. Homozygous deletion of Erap1 affected approximately one-third of the B27 peptidome but left most of the B27 peptidome unchanged, suggesting the possibility that some of the HLA-B27 immunopeptidome is not processed in the presence of Erap1. Deletion of Erap1 was permissive for the AS-like phenotype, increased mean peptide length and increased the frequency of C-terminal hydrophobic residues and of N-terminal Ala, Ser, or Lys. The presence of Erap1 increased the frequency of C-terminal Lys and Arg, of Glu and Asp at intermediate residues, and of N-terminal Gly. Several peptides of potential interest in AS pathogenesis, previously identified in human cell lines, were isolated. However, rats susceptible to arthritis had B27 peptidomes similar to those of non-susceptible rats, and no peptides were found to be uniquely associated with arthritis. Whether specific B27-bound peptides are required for AS pathogenesis remains to be determined. Data are available via ProteomeXchange with identifier PXD005502.
