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

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 further decreased ERAP1 activity. Polymorphism at residue 730 showed a significant influence on peptide length, because of distinct effects on trimming of nonamers compared with 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, because of 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.

Molecular and pathogenic effects of endoplasmic reticulum aminopeptidases ERAP1 and ERAP2 in MHC-I-associated inflammatory disorders: Towards a unifying view.

The inflammatory diseases that are most strongly associated with major histocompatibility Complex class I (MHC-I) alleles are also influenced by endoplasmic reticulum aminopeptidase (ERAP) 1 and/or 2, often in epistasis with the susceptibility MHC-I allele. This review will focus on the four major MHC-I-associated inflammatory disorders: ankylosing spondylitis, birdshot chorioretinopathy, Behçet's disease and psoriasis. The genetics of ERAP1/ERAP2 association and the alterations induced by polymorphism of these enzymes on the risk MHC-I allotypes will be examined. A pattern emerges of analogous effects on peptide length, sequence and affinity of disparate peptidomes, suggesting that similar peptide-mediated mechanisms underlie the pathogenesis and the joint contribution of ERAP1/ERAP2 and MHC-I to distinct inflammatory diseases. Processing of specific antigens, peptide-dependent changes in global properties of the MHC-I molecules, such as folding and stability, or both may be pathogenic.

The Peptidome of Behçet's Disease-Associated HLA-B*51:01 Includes Two Subpeptidomes Differentially Shaped by Endoplasmic Reticulum Aminopeptidase 1.

OBJECTIVE: To characterize the peptidome of the Behçet's disease-associated HLA-B*51:01 allotype as well as the differential features of major peptide subsets and their distinct endoplasmic reticulum aminopeptidase 1 (ERAP-1)-mediated processing. METHODS: The endogenous B*51:01-bound peptidome was characterized from 721.221 transfectant cells, after affinity chromatography and acid extraction, by tandem mass spectrometry. Recombinant ERAP-1 variants were used to digest synthetic B*51:01 ligands. HLA and transporter associated with antigen processing (TAP) binding affinities of peptide ligands were calculated with well-established algorithms. ERAP-1 and ERAP-2 from 721.221 cells were characterized by genomic sequencing and Western blotting. RESULTS: The B*51:01 peptidome consisted of 29.5% octamers, 61.7% nonamers, 4.8% decamers, and 4.0% longer peptides. The major peptide motif consisted of Pro and Ala at position 2, aliphatic/aromatic position 3 residues, and Val and Ile at the C-terminal position. The ligands with Pro or Ala at position 2 constituted 2 distinct subpeptidomes. Peptides with Pro at position 2 showed higher affinity for B*51:01 and lower affinity for TAP than those with Ala at position 2. Most important, both peptide subsets differed drastically in the susceptibility of their position 1 residues to ERAP-1, revealing a distinct influence of this enzyme on both subpeptidomes, which may alter their balance, affecting the global affinity of B*51:01-peptide complexes. CONCLUSION: ERAP-1 has a significant influence on the B*51:01 peptidome and its affinity. This influence is based on very distinct effects on the 2 subpeptidomes, whereby only peptides in the subpeptidome with Ala at position 2 are extensively destroyed, except when their position 1 residues are ERAP-1 resistant. This pattern provides a mechanism for the epistatic association of ERAP-1 and B*51:01 in Behçet's disease.

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

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, psoriasis, and Behçet'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.

Peptide handling by HLA-B27 subtypes influences their biological behavior, association with ankylosing spondylitis and susceptibility to endoplasmic reticulum aminopeptidase 1 (ERAP1).

HLA-B27 is strongly associated with ankylosing spondylitis (AS). We analyzed the relationship between structure, peptide specificity, folding, and stability of the seven major HLA-B27 subtypes to determine the role of their constitutive peptidomes in the pathogenicity of this molecule. Identification of large numbers of ligands allowed us to define the differences among subtype-bound peptidomes and to elucidate the peptide features associated with AS and molecular stability. The peptides identified only in AS-associated or high thermostability subtypes with identical A and B pockets were longer and had bulkier and more diverse C-terminal residues than those found only among non-AS-associated/lower-thermostability subtypes. Peptides sequenced from all AS-associated subtypes and not from non-AS-associated ones, thus strictly correlating with disease, were very rare. Residue 116 was critical in determining peptide binding, thermodynamic properties, and folding, thus emerging as a key feature that unified HLA-B27 biology. HLA-B27 ligands were better suited to TAP transport than their N-terminal precursors, and AS-associated subtype ligands were better than those from non-AS-associated subtypes, suggesting a particular capacity of AS-associated subtypes to bind epitopes directly produced in the cytosol. Peptides identified only from AS-associated/high-thermostability subtypes showed a higher frequency of ERAP1-resistant N-terminal residues than ligands found only in non-AS-associated/low-thermostability subtypes, reflecting a more pronounced effect of ERAP1 on the former group. Our results reveal the basis for the relationship between peptide specificity and other features of HLA-B27, provide a unified view of HLA-B27 biology and pathogenicity, and suggest a larger influence of ERAP1 polymorphism on AS-associated than non-AS-associated subtypes.

ERAP1 structure, function and pathogenetic role in ankylosing spondylitis and other MHC-associated diseases.

The endoplasmic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme involved in the final processing of Major Histocompatibility Complex class I (MHC-I) ligands and with a significant influence in the stability and immunological properties of MHC-I proteins. ERAP1 polymorphism is associated with ankylosing spondylitis among HLA-B27-positive individuals and the altered enzymatic activity of natural variants has significant effects on the HLA-B27 peptidome, suggesting a critical pathogenetic role of peptides in this disease. Likewise, the association of ERAP1 with other MHC-I associated disorders and its epistasis with their susceptibility MHC alleles point out to a general role of the MHC-I peptidome in these diseases. The functional interaction between ERAP1 and HLA-B27 or other MHC-I molecules may be related to the processing of specific epitopes, or to a more general peptide-dependent influence on other biological features of the MHC-I proteins. In addition, from a consideration of the reported functions of ERAP1, including its involvement in angiogenesis and macrophage activation, a more complex and multi-level influence in the inflammatory and immune pathways operating in these diseases cannot be ruled out.

Novel HLA-B27-restricted epitopes from Chlamydia trachomatis generated upon endogenous processing of bacterial proteins suggest a role of molecular mimicry in reactive arthritis.

Reactive arthritis (ReA) is an HLA-B27-associated spondyloarthropathy that is triggered by diverse bacteria, including Chlamydia trachomatis, a frequent intracellular parasite. HLA-B27-restricted T-cell responses are elicited against this bacterium in ReA patients, but their pathogenetic significance, autoimmune potential, and relevant epitopes are unknown. High resolution and sensitivity mass spectrometry was used to identify HLA-B27 ligands endogenously processed and presented by HLA-B27 from three chlamydial proteins for which T-cell epitopes were predicted. Fusion protein constructs of ClpC, Na(+)-translocating NADH-quinone reductase subunit A, and DNA primase were expressed in HLA-B27(+) cells, and their HLA-B27-bound peptidomes were searched for endogenous bacterial ligands. A non-predicted peptide, distinct from the predicted T-cell epitope, was identified from ClpC. A peptide recognized by T-cells in vitro, NQRA(330-338), was detected from the reductase subunit. This is the second HLA-B27-restricted T-cell epitope from C. trachomatis with relevance in ReA demonstrated to be processed and presented in live cells. A novel peptide from the DNA primase, DNAP(211-223), was also found. This was a larger variant of a known epitope and was highly homologous to a self-derived natural ligand of HLA-B27. All three bacterial peptides showed high homology with human sequences containing the binding motif of HLA-B27. Molecular dynamics simulations further showed a striking conformational similarity between DNAP(211-223) and its homologous and much more flexible human-derived HLA-B27 ligand. The results suggest that molecular mimicry between HLA-B27-restricted bacterial and self-derived epitopes is frequent and may play a role in ReA.

ERAP1 in ankylosing spondylitis: genetics, biology and pathogenetic role.

PURPOSE OF REVIEW: Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an aminopeptidase of the endoplasmic reticulum involved in trimming of peptides to their optimal size for binding to major histocompatibility complex class I molecules. Natural ERAP1 polymorphism resulting in altered enzymatic activity is associated with ankylosing spondylitis, an inflammatory disorder very strongly linked to HLA-B27. RECENT FINDINGS: This review will summarize recent advances in the genetics of ERAP1 association with this disease, in the molecular basis of ERAP1 function and in the mechanism of functional interaction between ERAP1 and HLA-B27. SUMMARY: The findings suggest that the pathogenetic role of ERAP1 in ankylosing spondylitis is due to allotype-dependent alterations of the HLA-B27 peptidome that affect the immunologic and other features of HLA-B27.

Functional interaction of the ankylosing spondylitis-associated endoplasmic reticulum aminopeptidase 1 polymorphism and HLA-B27 in vivo.

The association of ERAP1 with ankylosing spondylitis (AS)1 among HLA-B27-positive individuals suggests that ERAP1 polymorphism may affect pathogenesis by altering peptide-dependent features of the HLA-B27 molecule. Comparisons of HLA-B*27:04-bound peptidomes from cells expressing different natural variants of ERAP1 revealed significant differences in the size, length, and amount of many ligands, as well as in HLA-B27 stability. Peptide analyses suggested that the mechanism of ERAP1/HLA-B27 interaction is a variant-dependent alteration in the balance between epitope generation and destruction determined by the susceptibility of N-terminal flanking and P1 residues to trimming. ERAP1 polymorphism associated with AS susceptibility ensured efficient peptide trimming and high HLA-B27 stability. Protective polymorphism resulted in diminished ERAP1 activity, less efficient trimming, suboptimal HLA-B27 peptidomes, and decreased molecular stability. This study demonstrates that natural ERAP1 polymorphism affects HLA-B27 antigen presentation and stability in vivo and proposes a mechanism for the interaction between these molecules in AS.