The ER Aminopeptidases, ERAP1 and ERAP2, synergize to self-modulate their respective activities.

Introduction: Critical steps in Major Histocompatibility Complex Class I (MHC-I) antigen presentation occur in the endoplasmic reticulum (ER). In general, peptides that enter the ER are longer than the optimal length for MHC-I binding. The final trimming of MHC-I epitopes is performed by two related aminopeptidases, ERAP1 and ERAP2 in humans that possess unique and complementary substrate trimming specificities. While ERAP1 efficiently trims peptides longer than 9 residues, ERAP2 preferentially trims peptides shorter than 9 residues. Materials and Methods: Using a combination of biochemical and proteomic studies followed by biological verification. Results: We demonstrate that the optimal ligands for either enzyme act as inhibitors of the other enzyme. Specifically, the presence of octamers reduced the trimming of long peptides by ERAP1, while peptides longer than nonomers inhibit ERAP2 activity. Discussion: We propose a mechanism for how ERAP1 and ERAP2 synergize to modulate their respective activities and shape the MHC-I peptidome by generating optimal peptides for presentation.

Substantial Influence of ERAP2 on the HLA-B*40:02 Peptidome: Implications for HLA-B*27-Negative Ankylosing Spondylitis.

HLA-B*40:02 is one of a few major histocompatibility complex class I (MHC-I) molecules associated with ankylosing spondylitis (AS) independently of HLA-B*27. The endoplasmic reticulum aminopeptidase 2 (ERAP2), an enzyme that process MHC-I ligands and preferentially trims N-terminal basic residues, is also a risk factor for this disease. Like HLA-B*27 and other AS-associated MHC-I molecules, HLA-B*40:02 binds a relatively high percentage of peptides with ERAP2-susceptible residues. In this study, the effects of ERAP2 depletion on the HLA-B*40:02 peptidome were analyzed. ERAP2 protein expression was knocked out by CRISPR in the transfectant cell line C1R-B*40:02, and the differences between the peptidomes from the wild-type and ERAP2-KO cells were determined by label-free quantitative comparisons. The qualitative changes dependent on ERAP2 affected about 5% of the peptidome, but quantitative changes in peptide amounts were much more substantial, reflecting a significant influence of this enzyme on the generation/destruction balance of HLA-B*40:02 ligands. As in HLA-B*27, a major effect was on the frequencies of N-terminal residues. In this position, basic and small residues were increased, and aliphatic/aromatic ones decreased in the ERAP2 knockout. Other peptide positions were also affected. Because most of the non-B*27 MHC-I molecules associated with AS risk bind a relatively high percentage of peptides with N-terminal basic residues, we hypothesize that the non-epistatic association of ERAP2 with AS might be related to the processing of peptides with these residues, thus affecting the peptidomes of AS-associated MHC-I molecules.

Editing the immunopeptidome of melanoma cells using a potent inhibitor of endoplasmic reticulum aminopeptidase 1 (ERAP1).

The efficacy of cancer immunotherapy, including treatment with immune-checkpoint inhibitors, often is limited by ineffective presentation of antigenic peptides that elicit T-cell-mediated anti-tumor cytotoxic responses. Manipulation of antigen presentation pathways is an emerging approach for enhancing the immunogenicity of tumors in immunotherapy settings. ER aminopeptidase 1 (ERAP1) is an intracellular enzyme that trims peptides as part of the system that generates peptides for binding to MHC class I molecules (MHC-I). We hypothesized that pharmacological inhibition of ERAP1 in cells could regulate the cellular immunopeptidome. To test this hypothesis, we treated A375 melanoma cells with a recently developed potent ERAP1 inhibitor and analyzed the presented MHC-I peptide repertoire by isolating MHC-I, eluting bound peptides, and identifying them using capillary chromatography and tandem mass spectrometry (LC-MS/MS). Although the inhibitor did not reduce cell-surface MHC-I expression, it induced qualitative and quantitative changes in the presented peptidomes. Specifically, inhibitor treatment altered presentation of about half of the total 3204 identified peptides, including about one third of the peptides predicted to bind tightly to MHC-I. Inhibitor treatment altered the length distribution of eluted peptides without change in the basic binding motifs. Surprisingly, inhibitor treatment enhanced the average predicted MHC-I binding affinity, by reducing presentation of sub-optimal long peptides and increasing presentation of many high-affinity 9-12mers, suggesting that baseline ERAP1 activity in this cell line is destructive for many potential epitopes. Our results suggest that chemical inhibition of ERAP1 may be a viable approach for manipulating the immunopeptidome of cancer.

Redundancy and Complementarity between ERAP1 and ERAP2 Revealed by their Effects on the Behcet's Disease-associated HLA-B*51 Peptidome.

The endoplasmic reticulum aminopeptidases ERAP1 and ERAP2 trim peptides to be loaded onto HLA molecules, including the main risk factor for Behçet's disease HLA-B*51. ERAP1 is also a risk factor among HLA-B*51-positive individuals, whereas no association is known with ERAP2. This study addressed the mutual relationships between both enzymes in the processing of an HLA-bound peptidome, interrogating their differential association with Behçet's disease. CRISPR/Cas9 was used to generate knock outs of ERAP1, ERAP2 or both from transfectant 721.221-HLA-B*51:01 cells. The surface expression of HLA-B*51 was reduced in all cases. The effects of depleting each or both enzymes on the B*51:01 peptidome were analyzed by quantitative label-free mass spectrometry. Substantial quantitative alterations of peptide length, subpeptidome balance, N-terminal residue usage, affinity and presentation of noncanonical ligands were observed. These effects were often different in the presence or absence of the other enzyme, revealing their mutual dependence. In the absence of ERAP1, ERAP2 showed similar and significant processing of B*51:01 ligands, indicating functional redundancy. The high overlap between the peptidomes of wildtype and double KO cells indicates that a large majority of B*51:01 ligands are present in the ER even in the absence of ERAP1/ERAP2. These results indicate that both enzymes have distinct, but complementary and partially redundant effects on the B*51:01 peptidome, leading to its optimization and maximal surface expression. The distinct effects of both enzymes on the HLA-B*51 peptidome provide a basis for their differential association with Behçet's disease and suggest a pathogenetic role of the B*51:01 peptidome.

Allele-specific Alterations in the Peptidome Underlie the Joint Association of HLA-A*29:02 and Endoplasmic Reticulum Aminopeptidase 2 (ERAP2) with Birdshot Chorioretinopathy.

Virtually all patients of the rare inflammatory eye disease birdshot chorioretinopathy (BSCR) carry the HLA-A*29:02 allele. BSCR is also associated with endoplasmic reticulum aminopeptidase 2 (ERAP2), an enzyme involved in processing HLA class I ligands, thus implicating the A*29:02 peptidome in this disease. To investigate the relationship between both risk factors we employed label-free quantitative mass spectrometry to characterize the effects of ERAP2 on the A*29:02-bound peptidome. An ERAP2-negative cell line was transduced with lentiviral constructs containing GFP-ERAP2 or GFP alone, and the A*29:02 peptidomes from both transduced cells were compared. A similar analysis was performed with two additional A*29:02-positive, ERAP1-concordant, cell lines expressing or not ERAP2. In both comparisons the presence of ERAP2 affected the following features of the A*29:02 peptidome: 1) Length, with increased amounts of peptides >9-mers, and 2) N-terminal residues, with less ERAP2-susceptible and more hydrophobic ones. The paradoxical effects on peptide length suggest that unproductive binding to ERAP2 might protect some peptides from ERAP1 over-trimming. The influence on N-terminal residues can be explained by a direct effect of ERAP2 on trimming, without ruling out and improved processing in concert with ERAP1. The alterations in the A*29:02 peptidome suggest that the association of ERAP2 with BSCR is through its effects on peptide processing. These differ from those on the ankylosing spondylitis-associated HLA-B*27. Thus, ERAP2 alters the peptidome of distinct HLA molecules as a function of their specific binding preferences, influencing different pathological outcomes in an allele-dependent way.

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.

Separate effects of the ankylosing spondylitis associated ERAP1 and ERAP2 aminopeptidases determine the influence of their combined phenotype on the HLA-B*27 peptidome.

Ankylosing spondylitis (AS) is an inflammatory disease strongly associated with the Major Histocompatibility Complex class I (MHC-I) allotype HLA-B*27. The endoplasmic reticulum aminopeptidases (ERAP)1 and 2, which trim peptides to their optimal length for MHC-I binding, are also susceptibility factors for this disease. Both highly active ERAP1 variants and ERAP2 expression favor AS, whereas loss-of-function ERAP1 and loss-of-expression ERAP2 variants are protective. Yet, only ERAP1 is in epistasis with HLA-B*27. We addressed two issues concerning the functional interaction of ERAP1 and ERAP2 with the HLA-B*27 peptidome in human cells: 1) distinguishing the effects of ERAP1 from those of ERAP2, and 2) determining the influence of ERAP2 in distinct ERAP1 contexts. Quantitative comparisons of the HLA-B*27:05 peptidomes from cells with various ERAP1/ERAP2 phenotypes were carried out. When cells expressing ERAP2 and either high or low activity ERAP1 variants were compared, increased amounts of nonamers, relative to longer ligands, and decreased amounts of peptides with Ala1, were observed in the more active ERAP1 context. When cells expressing ERAP2 in a low activity ERAP1 context or lacking ERAP2 but expressing a highly active ERAP1 variant were compared, the same effects on peptide length and Ala1, but also significantly lower amounts of peptides with N-terminal basic residues and lower affinity of the peptidome, were observed in the ERAP2-positive context. Thus, ERAP1 and ERAP2 have significant and distinct effects on the HLA-B*27 peptidome, suggesting that both enzymes largely act as separate entities in vivo. This may explain their different patterns of association with AS.

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.

Functional Interaction of the Ankylosing Spondylitis-Associated Endoplasmic Reticulum Aminopeptidase 2 With the HLA-B*27 Peptidome in Human Cells.

OBJECTIVE: To determine the influence of endoplasmic reticulum aminopeptidase 2 (ERAP-2) expression on the HLA-B*27 peptidome in live cells. METHODS: Using immunoaffinity chromatography and acid extraction, HLA-B*27:05-bound peptides were isolated from 2 ERAP-2-negative lymphoblastoid cell lines and 1 ERAP-2-positive lymphoblastoid cell line expressing functionally indistinguishable ERAP-1 variants. More than 2,000-4,000 B*27:05 ligands were identified from each cell line, and their relative abundance was established by quantitative tandem mass spectrometry and MaxQuant-based peptide analyses. Pairwise comparisons were used to determine the structural features of peptides whose relative abundance was dependent on the presence of ERAP-2. Synthetic peptide digestions were performed with recombinant ERAP-1 and ERAP-2. Peptide affinity was estimated with standard algorithms. RESULTS: The B*27:05 peptidome from ERAP-2-positive cells showed 3-4% fewer peptides with N-terminal basic residues than did the peptidome from ERAP-2-negative cells. Among the shared peptides, those most abundant in the presence of ERAP-2 included more nonamers, fewer decamers, and fewer N-terminal basic residues than the peptides predominant in ERAP-2-negative cells. These ERAP-2-dependent changes did not alter the global affinity of the B*27:05 peptidome. CONCLUSION: ERAP-2 significantly influences the B*27:05-bound peptidome by destroying some ligands and decreasing the abundance of many more ligands with N-terminal basic residues, while increasing the abundance of nonamers. The former effects are best explained by direct ERAP-2 trimming. The effects on peptide length might be attributed to ERAP-2-induced activation of ERAP-1 trimming. These data support the notion of a peptide-mediated mechanism as the basis for the association of ERAP-2 with ankylosing spondylitis. Analogous effects on other major histocompatibility complex class I peptidomes might explain the involvement of ERAP-2 in HLA-B27-negative spondyloarthritis.

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.

Combined effects of ankylosing spondylitis-associated ERAP1 polymorphisms outside the catalytic and peptide-binding sites on the processing of natural HLA-B27 ligands.

ERAP1 polymorphism involving residues 528 and 575/725 is associated with ankylosing spondylitis among HLA-B27-positive individuals. We used four recombinant variants to address the combined effects of the K528R and D575N polymorphism on the processing of HLA-B27 ligands. The hydrolysis of a fluorogenic substrate, Arg-528/Asp-575 < Lys-528/Asp-575 < Arg-528/Asn-575 < Lys-528/Asn-575, indicated that the relative activity of variants carrying Arg-528 or Lys-528 depends on residue 575. Asp-575 conferred lower activity than Asn-575, but the difference depended on residue 528. The same hierarchy was observed with synthetic precursors of HLA-B27 ligands, but the effects were peptide-dependent. Sometimes the epitope yields were variant-specific at all times. For other peptides, concomitant generation and destruction led to similar epitope amounts with all the variants at long, but not at short, digestion times. The generation/destruction balance of two related HLA-B27 ligands was analyzed in vitro and in live cells. Their relative yields at long digestion times were comparable with those from HLA-B27-positive cells, suggesting that ERAP1 was a major determinant of the abundance of these peptides in vivo. The hydrolysis of fluorogenic and peptide substrates by an HLA-B27 ligand or a shorter peptide, respectively, was increasingly inhibited as a function of ERAP1 activity, indicating that residues 528 and 575 affect substrate inhibition of ERAP1 trimming. The significant and complex effects of co-occurring ERAP1 polymorphisms on multiple HLA-B27 ligands, and their potential to alter the immunological and pathogenetic features of HLA-B27 as a function of the ERAP1 context, explain the epistatic association of both molecules in ankylosing spondylitis.

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.