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. 2015 Apr 14;180(2):289–304. doi: 10.1111/cei.12575

Differences between disease-associated endoplasmic reticulum aminopeptidase 1 (ERAP1) isoforms in cellular expression, interactions with tumour necrosis factor receptor 1 (TNF-R1) and regulation by cytokines

N Yousaf *, W Y Low *,†, A Onipinla †, C Mein ‡, M Caulfield †, P B Munroe †, Y Chernajovsky *,✉
PMCID: PMC4408164  PMID: 25545008

Abstract

Endoplasmic reticulum aminopeptidase 1 (ERAP1) processes peptides for major histocompatibility complex (MHC) class I presentation and promotes cytokine receptor ectodomain shedding. These known functions of ERAP1 may explain its genetic association with several autoimmune inflammatory diseases. In this study, we identified four novel alternatively spliced variants of ERAP1 mRNA, designated as ΔExon-11, ΔExon-13, ΔExon-14 and ΔExon-15. We also observed a rapid and differential modulation of ERAP1 mRNA levels and spliced variants in different cell types pretreated with lipopolysaccharide (LPS). We have studied three full-length allelic forms of ERAP1 (R127-K528, P127-K528, P127-R528) and one spliced variant (ΔExon-11) and assessed their interactions with tumour necrosis factor receptor 1 (TNF-R1) in transfected cells. We observed variation in cellular expression of different ERAP1 isoforms, with R127-K528 being expressed at a much lower level. Furthermore, the cellular expression of full-length P127-K528 and ΔExon-11 spliced variant was enhanced significantly when co-transfected with TNF-R1. Isoforms P127-K528, P127-R528 and ΔExon-11 spliced variant associated with TNF-R1, and this interaction occurred in a region within the first 10 exons of ERAP1. Supernatant-derived vesicles from transfected cells contained the full-length and ectodomain form of soluble TNF-R1, as well as carrying the full-length ERAP1 isoforms. We observed marginal differences between TNF-R1 ectodomain levels when co-expressed with individual ERAP1 isoforms, and treatment of transfected cells with tumour necrosis factor (TNF), interleukin (IL)-1β and IL-10 exerted variable effects on TNF-R1 ectodomain cleavage. Our data suggest that ERAP1 isoforms may exhibit differential biological properties and inflammatory mediators could play critical roles in modulating ERAP1 expression, leading to altered functional activities of this enzyme.

Keywords: alternative splicing, cytokines, ERAP1, exosome, TNF-R1

Introduction

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a multi-functional enzyme belonging to the M1 family of zinc metalloaminopeptidases that contain a conserved catalytic zinc-binding motif (HEXXH(X)18E) 1–4. It is also known as an aminopeptidase regulator of tumour necrosis factor receptor 1 (TNF-R1) shedding (ARTS-1) 5, or adipocyte-derived leucine aminopeptidase (A-LAP) 2–4. The role of ERAP1 (MIM number: 606832) in generating peptides for their presentation by major histocompatibility complex (MHC) class I molecules is now well established 1,6–9. This enzyme was also shown to bind TNF-R1, interleukin (IL)-6 receptor α chain (IL-6R) and IL-1 type II decoy receptor (IL-1RII) and promote cytokine receptor ectodomain shedding 5,10,11. Other studies have suggested that it plays a role in the regulation of blood pressure through inactivation of angiotensin II 12. The ERAP1 gene in humans contains 20 exons, and two alternatively spliced variants of the full-length ERAP1 protein have been reported 3. Isoform 1 of ERAP1 (A-LAP2, GenBank Accession no. NM_016442), containing 948 amino acids, is identical up to residue 939 to isoform 2 (A-LAP1/ARTS-1, GenBank Accession no. AF106037/AF222340), with only the last nine amino acids being encoded by exon 20 due to a differential splice site in exon 19 3. The ERAP1 isoform 2 contains 941 amino acids and its termination codon is located in exon 19 2,3,5.

Residue K528 in ERAP1 is highly conserved among the closely related members of the M1 family of zinc metalloaminopeptidases 2, and in a Japanese study this polymorphic position, K528R, was linked initially to essential hypertension 13. In another report, the R528 allele was associated with left ventricular mass in response to anti-hypertensive treatment in patients with essential hypertension and left ventricular hypertrophy 14. In a genetic association analysis, we detected no association (unpublished observations) between genetic variants at the ERAP1 locus and essential hypertension in a Caucasian cohort of 1700 extreme hypertensives and 1700 normotensive controls, who were part of the MRC British Genetics of Hypertension (BRIGHT) study 15. Meta-analyses of genome-wide association studies (GWAS) of systolic and diastolic blood pressure have not reported association of variants at this locus to date 16. However, GWAS have identified several polymorphisms in ERAP1 that are associated strongly with ankylosing spondylitis (AS) in all populations studied so far 17–20. Genetic polymorphisms in ERAP1 have also been associated with other autoimmune diseases such as spondyloarthritis, psoriasis, multiple sclerosis and type 1 diabetes 18,21–25, as well as with cervical carcinoma 26, suggesting that the relevance of ERAP1 is not restricted to autoimmune diseases.

Because ERAP1 processes peptides for MHC class I presentation and promotes ectodomain shedding of cytokine receptors, these functions may explain its association with a range of human diseases. The genetic association of ERAP1 with AS has been observed only in human leucocyte antigen (HLA)-B27-positive patients 19, and in the case of psoriasis ERAP1 is associated with HLA-C positivity 22,23. These findings seem to emphasize the role of ERAP1 in antigen processing as an important contributory factor in the pathogenesis of these diseases and, consequently, the polymorphic positions affecting the enzymatic activity of this protein have been the major focus of all studies related to AS. In all GWAS, the polymorphic position K/R528 correlates strongly with disease and in addition, Q/E730 also appears to be critical for AS 17–23. Several in-vitro studies using purified recombinant forms of full-length ERAP1 isoforms have reported reduced enzymatic activity and altered peptide processing capacity of R528 and E730 allelic forms 19,27–29. Using cleavage of angiotensin II and kallidin by ERAP1, the first published study by Goto et al. 27 reported only the R528 allele with reduced enzymatic activity, while the polymorphic position P/R127 had no effect. Both R528 and E730 alleles have also been associated with reduced antigen presentation by HLA-B27 molecules 29,30, but the potential role of P/R127 in ERAP1 remains largely unknown. Despite the recent interesting results, the function of ERAP1 in promoting cytokine receptor shedding is still debated. Haroon et al. 31 found no influence of specific AS-associated ERAP1 genotypical polymorphisms on the serum levels of TNF-R1, IL-6R or IL-1RII and their respective cytokines in AS patients, as determined by enzyme-linked immunosorbent assay (ELISA). Another report also failed to detect any difference in the levels of soluble cytokine receptors released from splenocytes of ERAP1−/− mice 19. However, few data for the cellular levels of ERAP1 protein by other analyses such as immunoblotting have been reported in these studies. Whether or not the polymorphic position K/R528 in ERAP1 protein exhibits a differential role for interactions with TNF-R1 is still unclear. Although the genotype for the polymorphic position P/R127 associates strongly with tumour progression and reduced survival in cervical cancer, as well as with a subgroup of cases with psoriasis 29,32, a direct functional contribution of these residues in ERAP1 protein has not been addressed so far.

Given the complexities of several polymorphisms/mutations in ERAP1 associating with a range of different diseases and our additional finding of novel spliced variants, defining functional consequences for each situation is challenging. It is difficult to dissociate allele-dependent forms of ERAP1 when assessing the functional activities of total endogenous protein. Furthermore, different anti-ERAP1 antibodies may exhibit differential recognition of ERAP1 isoforms. Additional factors such as haplotype effects and differential regulation of ERAP1 gene alleles may also contribute to an overall expression and function of this protein within a cellular environment. To overcome some of these issues, we have focused on generating epitope-tagged constructs of ERAP1 harbouring single polymorphic residues in order to study their cellular expression and functional properties. In this study, we addressed the role of polymorphic residues only at positions 127 and 528 of ERAP1 using constructs for three full-length allelic forms of ERAP1 (R127-K528, P127-K528 and P127-R528) and one novel naturally occurring alternatively spliced variant (ΔExon-11), which lacks the residue 528, for transfection studies in mammalian cells. We provide the first comparative description for cellular expression profiles of individual ERAP1 isoforms and we also compare their potential interactions with TNF-R1.

Materials and methods

Reagents

Anti-human TNF-R1/TNFRSF1A monoclonal antibody (mAb 225) and biotinylated anti-human TNF-R1 (BAF225) were purchased from R&D Systems Ltd (Abingdon, UK). Monoclonal anti-tag epitope antibodies, anti-V5 and anti-V5-HRP, were purchased from Invitrogen (Paisley, UK) and anti-FLAG (M2) and anti-β-actin obtained from Sigma-Aldrich Co (Dorset, UK). Horseradish peroxidase (HRP)-conjugated F(ab′)2 goat anti-mouse IgG was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and sheep anti-rabbit immunoglobulin (Ig)G-HRP was purchased from AbD Serotech (Kidlington, Oxford, UK). Rabbit anti-human TNF-R1 polyclonal antibody, recombinant human TNF-R1, recombinant human TNF and recombinant IL-1β were purchased from PeproTech EC Ltd (London, UK). Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (clone 6C5) was obtained from Ambion/Life Technologies (Paisley, UK). All protease inhibitors were purchased from Calbiochem (Merck Biosciences Ltd, Nottingham, UK). Rabbit anti-human ERAP1 immune serum (referred to here as RIKEN antibody) was a kind gift from Dr Masafumi Tsujimoto (RIKEN, Saitama, Japan).

Human subjects

Our unpublished observations of genetic association analysis for essential hypertension at ERAP1 locus involved individuals of white European ancestry, who were part of the MRC BRIGHT study (http://www.brightstudy.ac.uk) [15]. Ethics Committee approval from the multi- and local research committees of the partner institutes had been obtained and all subjects participated as volunteers and gave informed written consent.

Human CD14− cells and cell lines

Human peripheral blood mononuclear cells (PBMC) from normal white Caucasian individuals were prepared from heparinized blood, as described previously 33. Isolated PBMC were washed and resuspended in complete RPMI-1640 medium (Invitrogen) containing 10% fetal bovine serum (FBS) (Invitrogen), 100 units/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine. CD14+ cells were then separated from PBMC using magnetic beads for positive selection columns, according to the manufacturer's instructions (Miltenyi Biotec, Bisley, UK). Residual negatively selected cells (CD14− cells) were washed and cultured in complete RPMI medium for 18 h at 37°C/5% CO2 prior to RNA preparation. U937 (human myelomonocytic leukaemia) cells were maintained in complete RPMI medium at 37°C/5% CO2. When required, cells were treated with lipopolysaccharide (LPS) (Sigma-Aldrich) at a concentration of 1 μg/ml for 1–3 h prior to RNA preparation. Human embryonic kidney 293T cells were maintained in Dulbecco's modified Eagle's minimal essential medium (DMEM) (Lonza Verviers, Verviers, Belgium) containing 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg streptomycin and 2 mM L-glutamine at 37°C/10% CO2, and used for RNA preparation or transfection studies.

RNA isolation and reverse transcription–polymerase chain reaction (RT–PCR)

Total RNA was prepared from cells using an RNeasy kit (Qiagen, Manchester, UK). For human CD14− cells, total RNA had been prepared as described previously 34. Following reverse transcription (RT), the complementary DNA (cDNA) (2 μl) was used for PCR amplification with Pfu DNA polymerase (Promega, Southampton, UK) in a final volume of 50 μl using a range of specific primer sets (Supporting information, Table S1) designed over the coding region of ERAP1 (GenBank Accession no. AF106037, AF222340). For amplification of exons 8–18 region of ERAP1, PCR was carried out for 36 cycles as follows: 95°C for 30 s, 58°C for 30 s and 72°C for 4 min. All other PCR amplifications were performed for 35 cycles at 95°C for 30 s, 60°C for 30 s and 72°C for 2 min, except when using exon 8 sense primer (Supporting information, Table S1), where the annealing temperature was 58°C. For assessing the expression of alternatively spliced ERAP1 variants at the mRNA level, the exon 8 sense primer was used in combination with the variant-specific boundary primer (Supporting information, Table S1) using cDNA as a source of template. Specific primers for β2 microglobulin (β2 m) (Supporting information, Table S1) were used as controls. PCR products were analysed by agarose gel electrophoresis and images visualized with ethidium bromide under ultraviolet (UV) light using an UVItec imaging system (UVitec Ltd, Cambridge, UK). PCR products were either cloned into pCR2·1 TA vector (Invitrogen) or inserted into the EcoRV cloning site of the pGT vector 34, which was used in the current study as a shuttle vector for several subcloning steps. The resulting plasmids were analysed by restriction enzyme digestion and by sequencing both DNA strands using the ABI Prism BigDye terminator sequencing kit and an ABI automated DNA analyser at the Genome Centre of the William Harvey Research Institute. The analyses were performed by DNA and protein databases (GenBank, http://www.ncbi.nlm.nih.gov, ExPASY, http://www.expasy.org and Ensembl, http://www.ensembl.org).

Cloning of ERAP1 isoforms and expression constructs

Briefly, two alleles (P127-K528 and R127-K528) of the full-length ERAP1 cDNA (GenBank Accession no. AF106037/AF222340), lacking the first Met of the cDNA, were cloned initially from CD14− cells using RT–PCR, with the Myc epitope tag (containing the first Met and Kozak sequence) present at their N-termini. All constructs were assembled in modified pcDNA6/V5-His B vector (Invitrogen), and hence will be referred to here as pcDNA6. To generate the initial FLAG-tagged R127-K528 ERAP1 isoform construct, the FLAG coding sequence was introduced at the 3′ end of ERAP1 by PCR. The resulting construct (Myc-R127-K528-FLAG/pcDNA6) was then used to generate other FLAG-tagged isoforms. The 5′ end (exons 2–12) of ERAP1 cDNA cloned from 293T cells expressed P127 and R528 polymorphisms. An EcoRV–KpnI fragment (1·38 kb) encoding P127-R528 was isolated and used to replace an equivalent fragment in R127-K528-FLAG/pcDNA6 construct, thereby generating Myc-P127-R528-FLAG isoform. To produce FLAG-tagged P127-K528 isoform, a BamHI–MfeI fragment (∼2·7kb), encoding Myc-tagged R127-K528, was exchanged with an equivalent fragment coding for Myc-tagged P127-K528, to produce the full-length Myc-P127-K528-FLAG/pcDNA6 construct. Allele frequency of polymorphisms was analysed by single nucleotide polymorphism (SNP)/HapMap databases (http://www.ncbi.nlm.nih.gov/SNP).

Clones isolated from CD14− cells containing the 3′ end (exons 8–19) region of ERAP1 with deletion of exon 11 were identified. One of these clones was used to insert a FLAG tag at the 3′ end of ΔExon-11 variant sequence by PCR. Following subcloning of these PCR products into a shuttle vector (pGT) 34, a fragment [441 base pairs (bp)] encoding the variant sequence and FLAG epitope was isolated and used to replace an equivalent region from the full-length Myc-R127-K528-FLAG construct. This exchange finally generated a fully assembled Myc-ΔExon-11-FLAG/pcDNA6 spliced variant construct.

TNF-R1 expression construct

The V5-His epitope tagged TNF-R1/pcDNA6 construct has been generated in a previous study 34.

Transfections and Western blot analysis

Human embryonic kidney 293T cells were maintained in complete DMEM. Transient transfections were performed in six-well plates using the calcium phosphate precipitation method, as described previously 34. The cells were transfected with plasmid DNA and, when necessary, pcDNA6/V5-His B vector plasmid was used to compensate for the total amount of DNA in each transfection. Cell cultures were terminated 40–42 h post-transfection. To assess the release of soluble receptors from transfected cells, culture medium was replaced by serum-free DMEM 18–20 h after transfection. For cytokine stimulation experiments, transfected cells were treated with IL-1β (10 ng/ml) or IL-10 (10 ng/ml) for 18 h or TNF (10 ng/ml) for 5 h, as required. Conditioned medium was then removed and processed further for assessing soluble cellular proteins. Transfected cells were scraped, washed in ice-cold PBS and then lysed in radioimmunoprecipitation assay (RIPA) buffer, as described previously 34. Total protein concentrations of cytosolic extracts were determined using a bicinchoninic acid protein assay reagent kit (Pierce/Fisher Scientific, Loughborough, UK). All cellular extracts were stored at −80°C until analysed. Equal amounts of lysates (30 μg total protein) were subjected to 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions 34 and analysed by Western blotting.

Immunoprecipitation and immunoblotting

Procedures for immunoprecipitation with nickel-nitrilotriacetic acid (Ni-NTA) agarose, protein A-Sepharose or protein G-Sepharose have been described in an earlier study 34. For immunoprecipitation experiments, 1 μl of RIKEN antibody (anti-ERAP1 serum) or 1 μg of anti-TNF-R1 antibody was added to 200 μg total cytosolic protein, while normal rabbit or mouse serum or protein A-purified normal rabbit IgG was used as a relevant control. Bead-bound complexes boiled in SDS-sample buffer were separated on 10% SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Watford, UK) and then blotted with appropriate primary antibodies. Detection was performed using horseradish peroxide (HRP)-conjugated secondary antibodies, followed by development with an enhanced chemiluminescence (ECL) detection system (Amersham GE Healthcare, Little Chalfont, UK) or a luminal detection reagent (ThermoScientific). Densitometry was performed using NIH ImageJ software version 1·45.

Isolation of vesicles from cultured medium

The procedure used for isolating secreted or shed vesicles was similar to that described by Hawari et al. 35 and was performed at 4°C. Medium (3 ml) from untransfected or transfected cultured cells was collected and centrifuged initially at 800 g for 10 min to remove nuclei and cell debris. The supernatants were then centrifuged at 5000 g for 90 min, and the resulting supernatants used to sediment vesicles at 175 000 g for 16 h in an Optima TL ultracentrifuge (Beckman-Coulter, High Wycombe, UK). The vesicle pellets were resuspended in lysis buffer and subjected to 10% SDS-PAGE under reducing conditions for Western blot analysis.

Results

ERAP1 mRNA is differentially spliced in human cells

We first investigated the expression levels of ERAP1 in human cells using RT–PCR with a range of primers spanning exons 8–18 of ERAP1 mRNA (Supporting information, Fig. S1) for ERAP1 cloning and subsequent functional studies. In the current study, CD14− cells [believed to constitute predominantly T cells, natural killer (NK) cells and some B cells] were used mainly as a source of primary cell population for cloning full-length ERAP1 cDNA from normal white Caucasian subjects, as described in Materials and methods. As ERAP1 may play a role in both innate and adaptive immune responses, we also tested the effect of lipopolysaccharide (LPS) on ERAP1 expression in CD14− cells initially with the aim of facilitating ERAP1 cloning. Surprisingly, we observed a dramatic up-regulation of the ERAP1 mRNA in LPS-treated CD14− cells but not in non-adherent monocyte-like U937 cells, which also lack CD14 expression, suggesting cell type-dependent modulation of ERAP1 mRNA in response to a proinflammatory signal (Supporting information, Fig. S1). Although CD14+ cells are considered to be the major responders of LPS, their complete exclusion from the cellular composition of the CD14− population cannot be confirmed at this stage.

During subsequent analyses of cloned PCR products from exons 8 to 18 of ERAP1, several alternatively spliced variants were isolated from CD14− cells and 293T fibroblast-like cells. Deletion of exon 11 (155 bp) caused a shift in the reading frame and a unique tail of 40 amino acids is introduced by exons 12 and 13 with a termination codon occurring in exon 13. The predicted variant protein, termed ΔExon-11, contains 548 amino acids, of which 508 residues correspond to the full-length ERAP1 (Supporting information, Fig. S2). A second variant sequence, termed ΔExon-13, containing a deletion of 184 bp corresponding to the whole of exon 13 also caused a frame-shift, introducing 16 amino acids and a termination codon in exon 14. The encoded protein for ΔExon-13 variant is predicted to contain 602 amino acids, the first 586 residues being identical to the full-length ERAP1 (Supporting information, Fig. S2). Two further alternatively spliced ERAP1 variants were detected in 293T cells, one lacking all of exon 14 (157 bp) (ΔExon-14) and the other without exon 15 (185 bp) (ΔExon-15) (Supporting information, Fig. S2). The ΔExon-14 variant-predicted protein containing 654 amino acids shares the first 647 residues with the full-length ERAP1. Only seven amino acids were encoded by exon 15 due to a termination codon present in this exon. The potential variant, ΔExon-15, also resulted from a frame-shift that occurred due to the deletion of exon 15, introducing 24 amino acids and a termination codon from exon 16. The predicted protein encoded by this variant contains 724 amino acids, the first 700 residues of which are identical to the full-length ERAP1 (Supporting information, Fig. S2). So far, no spliced variant lacking an exon upstream of exon 11 has been detected. Taken together, these results indicate that ERAP1 is susceptible to alternative splicing after exon 10 in human cells. As ERAP1 mRNA is inducible by LPS in primary CD14− cells but not in U937 cells (Supporting information, Fig. S1), we hypothesized that such cellular responses may be associated with changes in the expression of different spliced variants of this enzyme. Additional experiments revealed that the expression of all spliced variants was up-regulated, albeit to variable levels, in the LPS-treated CD14− cell population but not in U937 cells, suggesting differential modulation of some ERAP1 spliced variants in a cell type-dependent manner (Supporting information, Fig. S3). However, which primary cell type(s) up-regulates ERAP1 spliced variants in response to LPS still remains to be established.

All four of the predicted protein variants identified in our study express very different carboxyl-termini of variable lengths and contain unique sequences that define potential functional differences (Supporting information, Fig. S2). Prominent features associated with the C-termini of all spliced variants include the presence of cysteine residues, and while arginine and histidine residues are also notable in ΔExon-11 and ΔExon-15 variants, the ΔExon-14 variant is rich in serine. The significance of these spliced variants of ERAP1 is as-yet unclear, but we have addressed the cellular expression and biological properties of ΔExon-11 variant in the current study.

Characterization of ERAP1 isoforms

We first tested a range of human cell lines for expression profiles of endogenous ERAP1 protein. We observed differences in the molecular mass of this protein (Supporting information, Fig. S4), which may be due to potential occurrence of the two known full-length ERAP1 isoforms (isoform 1: 948 residues, isoform 2: 941 residues) 2,3,5. We also considered that the relative expression of these ERAP1 isoforms may be subject to differential modulation within the cellular environment. Furthermore, there is a polymorphic d(AC/GT)n repeat microsatellite in the promoter region 3, which may be important in regulating the transcription level of ERAP1 gene. In contrast to most cell lines, a band around 100 kDa corresponding to the full-length ERAP1 was barely detectable in 293T cells by Western blotting with RIKEN (anti-ERAP1) antibody (Supporting information, Fig. S4). During ERAP1 cloning, we had noted that 293T cells harbour polymorphic nucleotides for P127 and R528 residues. We further considered the possibility that some polymorphisms might induce a conformational change in ERAP1 protein affecting its recognition by an anti-ERAP1 antibody. Hence, tagging a defined haplotype with a small epitope would not only dissociate the over-expressed ERAP1 isoform from endogenous protein but also provide multiple antibodies for comparisons. The P/R127 and K/R528 polymorphisms associate strongly with a range of human diseases. For northern and western European ancestry, P127 and R528 are the major alleles (NCBI/SNP/HapMap database), and the presence of minor alleles (R127 and K528) is linked to several clinical situations, with genotype for K528 allele showing the strongest association with AS and other autoimmune disorders 17–25,36. Therefore, to compare the functional properties of individual polymorphic residues, we designed constructs to produce three full-length alleles of ERAP1 (R127-K528, P127-K528 and P127-R528), and one naturally occurring spliced variant, ΔExon-11, for cellular expression under identical promoter elements (Fig. 1a).

Fig 1.

Fig 1

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Schematic representation of endoplasmic reticulum aminopeptidase 1 (ERAP1) and tumour necrosis factor receptor 1 (TNF-R1) expression constructs. (a, top panel) Features of epitope tagged full-length ERAP1 isoforms indicate positions of hydrophobic region (TM), and consensus zinc metalloprotease catalytic motif (HELAH E). Polymorphic residues at position 127 (P/R) and at 528 (K/R), and encoding exons shown by arrows. Three full-length ERAP1 isoforms are listed on the right; first and second isoforms compare residues at position 127 and second and third compare residues at 528, with major alleles (P127, R528) and minor alleles (R127, K528) occurring at these positions in ERAP1. (a, lower panel) Structural features of ΔExon-11 ERAP1 spliced variant indicate position of variant specific unique region by thick arrow. This spliced variant carrying R127 is identical up to amino acid position 508 of the full length and lacks polymorphic residue at position 528. Each ERAP1 construct contains a Myc tag at N-terminus and a FLAG tag at C-terminus. (b) Full-length human TNF-R1 carries V5-His tag at the C-terminus.

The two alleles of full-length ERAP1 cDNA cloned from CD14− cells contained K528 residue but differed only at two other polymorphic positions. One allele bearing residues I12 and P127 was identical to the first published reference sequence (GenBank Accession no. AF106037) 2. The second cloned ERAP1 allele, harbouring T12 and R127 residues, matched the published sequence, GenBank Accession no. AF222340 5. Except for these two polymorphic positions, both published sequences are identical for other polymorphic residues in ERAP1. There are 10 polymorphic residues in human ERAP1 and the two alleles are not expected to be identical for all the polymorphisms. Our aim was to address the role of polymorphic residues only at positions 127 and 528 of ERAP1. Considering the occurrence of possible haplotype effects between these target positions and other regions of ERAP1, the remaining eight polymorphic residues (T12, E56, I276, G346, M349, D575, R725, Q730) were kept consistent for different ERAP1 isoforms used for the study.

Each isoform of ERAP1 carried the Myc-tag at the N-terminus (Fig. 1a). Upon transfections into 293T cells, the Myc epitope could not be detected. These unexpected findings suggested that some processing event is occurring within the N-terminus of ERAP1 that results in the removal of the Myc epitope and, consequently, a FLAG epitope was inserted at the C-terminus of ERAP1. As shown in Fig. 1a, we generated four FLAG-tagged constructs, where ΔExon-11 spliced variant carries residue R127 but lacks the polymorphic residue K/R528. All full-length ERAP1 isoforms contain 941 amino acids, and ΔExon-11 contains 548 amino acids. We used these constructs to define directly how these four forms of ERAP1 differ in their cellular expression and interactions with TNF-R1 that was V5-His tagged at the C-terminal end (Fig. 1b).

Cellular expression of two ERAP1 isoforms is enhanced by TNF-R1 co-expression

In some human cell lines, endogenous ARTS-1 (ERAP1) was shown to bind endogenous TNF-R1 5. However, in parallel with low ERAP1 protein level in 293T cells (Supporting information, Fig. S4), endogenous TNF-R1 was also undetectable by direct immunoblotting of cytosolic extracts, but our previous study had indicated that these cells express low levels of endogenous TNF-R1, as assessed by flow cytometry (unpublished observations), and can activate NF-κB in response to TNF 34. We reasoned, therefore, that potential contribution from endogenous ERAP1 or TNF-R1 to the functional analyses of over-expressed tagged proteins should be minimal in 293T cells. To examine whether our full-length ERAP1 isoforms and ΔExon-11 spliced variant affect the cellular expression of TNF-R1, we transfected 293T cells initially with varying amounts of individual ERAP1 isoforms in the presence or absence of V5-His tagged TNF-R1. Following Western blotting of cytosolic lysates using anti-tag antibodies, protein bands migrating around 55 kDa corresponding to the full-length TNF-R1 were observed with an anti-V5 antibody, even in the presence of a full-length ERAP1 isoform (Fig. 2a,b, top panels). We also noted that low and high levels of co-transfected R127-K528 (R127) or P127-K528 (P127) ERAP1 slightly reduced the TNF-R1 expression, with the former isoform being more effective. When blotted with an anti-FLAG antibody, the epitope-tagged ERAP1 protein with a molecular mass of about 100 kDa was detected for the full-length isoforms, R127-K528 and P127-K528 (Fig. 2a,b, bottom panels), but R127 exhibiting lower expression level than P127. Furthermore, the expression of each of these isoforms, which differ only at residue 127 of ERAP1, was enhanced upon co-transfection with TNF-R1-V5-His (Fig. 2a,b, bottom panels).

Fig 2.

Fig 2

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Cellular expression of endoplasmic reticulum aminopeptidase 1 (ERAP1) isoforms is enhanced in the presence of tumour necrosis factor receptor 1 (TNF-R1). 293T cells transfected with varying amounts of R127-K528-FLAG (R127) (a), P127-K528-FLAG (P127) (b), or ΔExon-11 spliced variant (c) plasmid DNA. For co-transfections, TNF-R1-V5-His (1 μg plasmid DNA) was used, with total amount of DNA transfected being 8 μg. Empty vector (pcDNA6/V5-HisB) plasmid DNA used as control (Vec) and to compensate total amount of DNA. Cell lysates (30 μg total protein) subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and analysed by Western blotting with anti-V5 antibody for TNF-R1 (top panels) or anti-FLAG antibody for ERAP1 isoforms (bottom panels). (d,e) Cells transfected with plasmid DNA (5 μg) of individual ERAP1 isoforms with or without TNF-R1-V5-His and cell lysates analysed by Western blotting as above. (d) Following probing with anti-FLAG, cytosolic expression levels of R127 ±TNF-R1 (n = 4), P127 ± TNF-R1 (n = 6), R528 (P127-R528) ± TNF-R1 (n = 4), ΔExon-11 ± TNF-R1 (n = 3), expressed as ratio to β-actin. Data shows mean ± standard error of the mean (s.e.m.), Student's t-test: *P < 0·01; **P < 0·02. (e) Membranes probed with RIKEN antibody (top panel) or anti-V5 (bottom panel), profiles representative of two separate experiments. Membranes reprobed for β-actin (a–c) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (e). Untransfected (Unt) 293T cells used as control (b). Specific bands for ERAP1 isoforms and TNF-R1 are shown by thick and thin arrows, respectively. Profiles shown are representative of at least three separate experiments (a–c).

We next assessed the cytosolic expression of ΔExon-11 spliced variant in the presence of TNF-R1. Full-length TNF-R1 (55 kDa) was detected with an anti-V5 antibody, but its expression was relatively reduced when co-transfected with ΔExon-11 spliced variant (Fig. 2c, top panel). Upon blotting with anti-FLAG antibody, a specific band around 60 kDa was seen for ΔExon-11 spliced variant and its expression was also enhanced when co-transfected with TNF-R1 (Fig. 2c, bottom panel). For quantitative analysis, as assessed by immunoblotting with anti-FLAG antibody, the expression levels of full-length P127-K528 (P127) and ΔExon-11 spliced variant were enhanced significantly in the presence of TNF-R1, but the apparent increased expression seen for either R127-K528 (R127) or P127-R528 (R528) under the same experimental conditions was not statistically significant (Fig. 2d). Consistently low cellular expression of R127-K528 isoform and greater variability in expression levels of P127-R528 in the presence of TNF-R1 may account for the observed lack of statistical significance for these two allelic forms of ERAP1 (Fig. 2d).

We further determined the expression profiles of all four isoforms using an anti-ERAP1 antibody to assess possible differences between the antibodies used to detect ERAP1 proteins. Following blotting with RIKEN anti-ERAP1 antibody 37, the comparative profiles seen in Fig. 2e suggest that the presence of TNF-R1 increases the stability of ERAP1 proteins, with R127-K528 consistently being the least expressed isoform. We also noticed that the RIKEN antibody recognized ΔExon-11 spliced variant less efficiently than the anti-FLAG antibody in Western blot analysis of cytosolic lysates. The reason for this difference may reside within the availability of the epitopes recognized by individual antibodies.

ERAP1 isoforms interact with TNF-R1

To address further whether ERAP1 isoforms associate physically with TNF-R1, we transfected individual isoforms with or without V5-tagged TNF-R1, and a series of co-immunoprecipitation experiments was performed. We first determined the recognition profiles of ERAP1 isoforms by RIKEN antibody. Following immunoprecipitation of cell lysates with anti-FLAG antibody and blotting with RIKEN antibody, the comparative profiles seen in Fig. 3a correlate well with our data (Fig. 2) that the expression of each ERAP1 isoform is enhanced in the presence of TNF-R1. In contrast, as shown in Fig. 3b (left top panels), immunoprecipitation of cytosolic lysates with RIKEN antibody and blotting with anti-FLAG revealed similar levels of 100 kDa P127-K528 with or without TNF-R1 co-expression, while enhanced expression of P127-K528 were seen when co-transfected with TNF-R1 in the lysates. Blotting with anti-V5 revealed full-length 55 kDa TNF-R1 when co-expressed with P127-K528 (Fig. 3b, left middle panels), suggesting interactions between these proteins. In control immunoprecipitation with normal rabbit IgG, no specific bands were seen around 100 kDa for ERAP1 or 55 kDa for TNF-R1 (Fig. 3b, right top and middle panels), while cytosolic lysates exhibited strong expression for both these proteins.

Fig 3.

Fig 3

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Endoplasmic reticulum aminopeptidase 1 (ERAP1) interacts with tumour necrosis factor receptor 1 (TNF-R1). 293T cells transfected with 5 μg of individual isoforms of ERAP1 plasmid DNA with or without TNF-R1-V5-His (1 μg plasmid DNA), as described in Fig. 2. (a) Cell lysates immunoprecipitated with anti-FLAG antibody/protein G-sepharose and complexes were separated by reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by blotting with RIKEN antibody. Untransfected (Unt) 293T cells used as control. (b–d) Cell lysates immunoprecipitated with either RIKEN antibody or normal rabbit immunoglobulin (Ig)G (b) or normal rabbit serum (c,d) and protein A-sepharose. Complexes resolved as above and analysed by immunobloting with anti-FLAG antibody (top panels) or anti-V5 (bottom panels). (e and f) Cell lysates immunoprecipitated with either anti-TNF-R1 monoclonal antibody (e) or normal mouse serum (f) and protein G-sepharose. Complexes analysed by immunoblotting with RIKEN antibody (e,f, top panels), anti-FLAG (e, lower panel) or anti-V5 (f, bottom panel). Cell lysates and complexes were analysed on the same gels; for clarity, some results shown (b–d) are from different exposure times following enhanced chemiluminescence (ECL) detection. Membranes reprobed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (b–d,f). Specific bands for ERAP1 isoforms and TNF-R1 are shown by thick and thin arrows, respectively. Positions of heavy chain of IgG (H) are indicated. Profiles shown are representative of two to three separate experiments.

For the second full-length ERAP1 isoform, P127-R528 (R528), similar expression levels were observed with or without TNF-R1 co-transfection following immunoprecipitation of cell lysates with RIKEN antibody (Fig. 3c, top panels). The levels of V5-tagged TNF-R1 detected in the absence or presence of co-transfected P127-R528 were also similar. The reason for this difference between K528 (Fig. 3b) and R528 (Fig. 3c) isoforms is unclear, but differential recognition of these two isoforms when associated with TNF-R1 by RIKEN antibody may account for the observed results. It is also possible that different ERAP1 alleles exhibit differential affinity for TNF-R1.

We next determined whether there is any direct association between ΔExon-11 spliced variant and TNF-R1. The results shown in Fig. 3d demonstrate enhanced expression of ΔExon-11 spliced variant (60 kDa) when co-transfected with V5-tagged TNF-R1 in cell lysates and following immunoprecipitation with RIKEN antibody and blotting with anti-FLAG antibody (top panels). Upon blotting with anti-V5 antibody, a specific band corresponding to 55 kDa full-length TNF-R1 was observed when co-expressed with ΔExon-11 spliced variant (lower panels). No band was seen in control immunoprecipitation with normal rabbit serum, while lysates used for this procedure exhibited strong expression of these proteins (Fig. 3d).

The results from immunoprecipitation experiments described above suggested potential interactions between transfected TNF-R1 and endogenous ERAP1, as seen in Fig. 3c. To elucidate further, immunoprecipitation of cytosolic lysates with an anti-TNF-R1 antibody and blotting with RIKEN antibody revealed a specific 100 kDa band corresponding to each full-length ERAP1 isoform that showed enhanced expression level when co-transfected with TNF-R1 (Fig. 3e, top panels). Once again, R127-K528 (R127) exhibited the lowest expression level in this comparative experiment. Furthermore, we noticed that all full-length ERAP1 isoforms were also detectable in the absence of transfected V5-tagged TNF-R1, a finding which reflects binding of ERAP1 isoforms to endogenous TNF-R1. Because the ΔExon-11 spliced variant in this immunoprecipitation procedure was not detected easily by the RIKEN antibody (Supporting information, Fig. S5), we believe that this anti-ERAP1 antibody preferentially recognizes the variant protein in its native form, as seen from the data shown in Fig. 3d. However, an approximate 60 kDa band was observed for ΔExon-11 spliced variant when blotted with an anti-FLAG antibody (Fig. 3e, lower panel). Because both anti-FLAG and anti-TNF-R1 are mouse antibodies, control immunoprecipitation was performed with normal mouse serum, but no specific bands were observed at the expected positions for ERAP1 isoforms or TNF-R1 when compared with the lysates used as blotting controls (Fig. 3f).

From a comparison of several co-immunoprecipitation described here, we noticed that the level of TNF-R1 following immunoprecipitation with RIKEN antibody (anti-ERAP1) was lower (Fig. 3b,c) when compared with the levels of P127-K528 or P127-R528 isoform pulled down in reciprocal immunoprecipitation with anti-TNF-R1 monoclonal antibody (Fig. 3e). Differential accessibility of epitopes involving possible conformational change in ERAP1 within the ERAP1–TNF-R1 complex molecules, as recognized by their respective antibodies, may account for the observed differences. Taken together, our results indicate that ERAP1 interacts with TNF-R1 and this interaction requires a region which resides within the first 10 exons of ERAP1.

Soluble TNF-R1 and secreted ERAP1 are associated with supernatant-derived vesicles

Large levels of soluble TNF-R1 were released in the culture medium by 293T cells transfected with V5-His tagged TNF-R1 as assessed by ELISA (data not shown). The soluble form of TNF-R1 is also known to occur as a 55-kDa full-length protein that is associated with membrane-bound exosome-like vesicles 35. We examined whether the level of soluble TNF-R1 is influenced by the expression of individual ERAP1 isoforms. Supernatants from cultured transfected cells were processed further by sedimentation at 175 000 g, and analysed by Western blotting to assess the levels of full-length and cleaved ectodomain of TNF-R1. Such preparations of soluble receptors are referred here as supernatant-derived vesicles, as they may contain a mixed population of vesicles resulting from both the exosome-like membranous structures and shed vesicles from plasma membranes 38–40. As shown in Fig. 4a (top and middle panels), not only the full-length (55 kDa) TNF-R1 but the cleaved ectodomain (28–34 kDa) and the cytoplasmic domain (30–32 kDa) of this receptor were also detected within the vesicles following ultracentrifugation. In addition, the vesicle-associated full-length TNF-R1 and cleaved cytoplasmic domain could still bind Ni-beads through the His tag (middle panel). We observed somewhat higher levels of the vesicle-associated full-length and cleaved ectodomain of TNF-R1 when co-expressed with P127-R528 (R528) or ΔExon-11 spliced variant (Fig. 4a, top panels). Upon blotting with anti-FLAG antibody, approximately 100 kDa full-length P127-K528 (P127) and P127-R528 (R528) were also detected within the same pool of supernatant-derived vesicles (Fig. 4a, bottom panels). Both these isoforms, differing only at residue 528 in ERAP1, exhibited a relatively lower molecular mass when co-expressed with TNF-R1, but ΔExon-11 spliced variant was hardly detectable in these preparations of supernatant-derived vesicles (Fig. 4a, bottom panel). In contrast, an analysis of the cytosolic proteins from corresponding transfectants confirmed enhanced expression of P127-K528, P127-R528 and ΔExon-11 spliced variant in the presence of TNF-R1 without any effect on their molecular mass migration patterns, whereas TNF-R1 existed predominantly as a full-length 55 kDa protein (Fig. 4b). In additional experiments both β-actin and annexin 1, which are known to be associated with exosome-like vesicles 38–40, were also detected within the supernatant-derived vesicle pool that contained the soluble forms of TNF-R1 as well as P127-K528 ERAP1 (Supporting information, Fig. S6).

Fig 4.

Fig 4

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Secreted tumour necrosis factor receptor 1 (TNF-R1) and endoplasmic reticulum aminopeptidase 1 (ERAP1) isoforms are vesicle-associated. 293T cells transfected with P127-K528-FLAG (P127), P127-R528-FLAG (R528) or ΔExon-11 spliced variant plasmid DNA (5 μg), with or without TNF-R1-V5-His (1 μg plasmid DNA), as described in Fig. 2. Empty vector plasmid DNA used as control (Vec). (a) Supernatants from cultured transfectants sedimented at 175 000 g and resulting vesicle pellets (top and bottom panels) or nickel-nitrilotriacetic acid (Ni-NTA) agarose bead-bound complexes (middle panel) resolved by reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and analysed by blotting with anti-TNF-R1 antibody (top panel), anti-V5 (middle panel) or anti-FLAG (bottom panel). (b) Expression profiles of TNF-R1 and ERAP1 isoforms in cell lysates from the same transfectants analysed by Western blotting using anti-TNF-R1 antibody (top panel) or anti-FLAG antibody (middle panel). Membranes reprobed for β-actin (bottom panel). Band intensities for TNF-R1 ectodomain (a, top panel) and ERAP1 isoforms (a, bottom panel) within supernatant-derived vesicles are shown as densitometry units. Expression levels of ERAP1 isoforms in cell lysates expressed as ratio to β-actin (b, middle panel). Specific bands indicating full-length TNF-R1, ectodomain and cytoplasmic domain are shown by thin arrows and those for ERAP1 isoforms by thick arrows. Data shown are representative of at least three separate analyses.

Cytokines exert differential effects on TNF-R1 ectodomain cleavage

Because proinflammatory cytokines play critical roles in the pathogenesis of chronic inflammatory disorders, we tested the impact of TNF and IL-1β treatment on the release of soluble TNF-R1 and ERAP1 isoforms that differ only at residue position 127. Following transfection of P127-K528 (P127) or R127-K528 (R127) isoforms with or without TNF-R1, the cells were treated with TNF or IL-1β and supernatant-derived vesicles analysed by Western blotting (Fig. 5). For cells transfected with TNF-R1 alone, exposure to TNF somewhat enhanced the cleaved ectodomain levels, whereas IL-1β treatment caused a reduction in the level of TNF-R1 ectodomain cleavage when the profiles of soluble proteins within the supernatant-derived vesicles were analysed (Fig. 5a,c, top panels, and Fig. 5e). However, similar levels of cleaved TNF-R1 ectodomain were observed when the cells had been co-transfected with P127-K528 (Fig. 5a, top panel, Fig. 5e) or R127-K528 (Fig. 5c, top panel, Fig. 5e) following treatment with these cytokines. Furthermore, the cleaved cytoplasmic domain of TNF-R1 was seen clearly in supernatant-derived vesicles (Fig. 5a, middle panel). While both P127-K528 (Fig. 5a, bottom panel) and R127-K528 (Fig. 5c, bottom panel) were also present within these vesicles, they migrated with a slightly lower molecular mass when co-transfected with TNF-R1. Within the cytosolic lysates, TNF-R1 occurred predominantly as a full-length 55 kDa protein, and minor bands around 40–42 kDa were also seen (Fig. 5b,d, top panels), whereas both P127-K528 and R127-K528 appeared as 100 kDa full-length proteins (Fig. 5b,d, middle panels). In additional experiments, no major changes were seen in the levels of TNF-R1 ectodomain cleavage following treatment of transfected cells with IL-10, an anti-inflammatory cytokine, while the profiles observed for secreted P127-K528 (P127) and R127-K528 (R127) isoforms (Supporting information, Fig. S7) were similar to those seen in Fig. 5a,c.

Fig 5.

Fig 5

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Tumour necrosis factor (TNF) and interleukin (IL)-1β exert differential effects on TNF receptor 1 (TNF-R1) ectodomain cleavage. 293T cells transfected with P127-K528-FLAG (P127) (a and b) or R127-K528-FLAG (R127) (c,d) plasmid DNA (5 μg), with or without TNF-R1-V5-His (1 μg plasmid DNA), as described in Fig. 2. Empty vector plasmid DNA used as control (Vec). Transfected cells treated with TNF for 5 h or IL-1β for 18 h and conditioned medium collected. Following sedimentation at 175 000 g, pelleted supernatant-derived vesicles resolved by reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and analysed by immunoblotting with anti-TNF-R1 antibody (a,c, top panels), anti-V5 (a, middle panel) or anti-FLAG (a,c, bottom panels). (b,d) Cell lysates from the same transfectants assessed by Western blotting using anti-TNF-R1 antibody (top panels) or anti-FLAG antibody (middle panels). Membranes reprobed for β-actin (bottom panels). Specific bands indicating full-length TNF-R1, cleaved ectodomain and cytoplasmic domain are shown by thin arrows and those for endoplasmic reticulum aminopeptidase 1 (ERAP1) isoforms by thick arrows. (e) Levels of cleaved TNF-R1 ectodomain within supernatant-derived vesicles expressed as mean densitometry units (a,c, top panels) are shown for TNF-R1 alone (n = 3), P127-K528 + TNF-R1 (n = 2) or R127-K528 + TNF-R1 (n = 2) transfectants. Control transfectants (−) were unstimulated. Data shown are representative of two to three separate experiments.

Discussion

We have described four novel isoforms of ERAP1, which occur as a result of differential splicing of pre-mRNA. We detected sequential deletions of exons 11, 13, 14 or 15, when compared with the two known full-length ERAP1 transcripts 2,3. A rapid up-regulation of the spliced variants by LPS in CD14− cells suggests that they may play a role in modulating ERAP1 activity, particularly within an inflammatory site. We have not yet assessed the cellular composition of CD14− cells, but this cell population is likely to contain mainly T cells, NK cells and some B cells. Human T cells have been reported to express Toll-like receptor-4 (TLR-4), and LPS could directly modulate certain T cell responses such as adherence to fibronectin and migration via TLR-4 signalling 41. Natural killer cells can also express TLR-4 and can respond to LPS in the presence of other accessory cells 42. A recent study has shown that ERAP1 modulates maturation and functional activities of NK cells in innate responses 43. Although the identity of the primary cell type(s) that up-regulates the expression of ERAP1 spliced variants in response to LPS is still unclear, our results presented here are encouraging, as they point to ERAP1 function in innate responses.

A BLAST (Basic Local Alignment Search Tool) search of public databases (GenBank, ExPASY and Ensembl) revealed no single protein sequence that matched the whole of the unique C-termini of the newly identified spliced variants, although short sequence segments were found in several known or predicted proteins. Major features associated with C-termini of these variants include the presence of cysteine, arginine, histidine and serine residues. The location and increased frequency of these amino acids within the C-termini of ΔExon-11 and ΔExon-15 variants suggests that these residues may form zinc-finger-like structures implicated in protein–protein interactions 44. However, recent crystallographic studies show that ERAP1 is enzymatically inactive in its open conformation but becomes catalytically active upon transition to a closed state, indicating that peptide processing activity of ERAP1 resides within the full-length protein 28,45,46. Exon 10 harbours a sequence that mediates the ER localization of ERAP1 47, but whether these spliced variants possessing an intact zinc-binding catalytic domain exhibit the capacity to reside within the ER also remains to be determined. Interestingly, the C-terminus of ΔExon-11 variant also contains a potential atypical N-linked glycosylation motif, N-X-C, where X is a non-P residue. Atypical N-glycosylation site is functionally relevant in a small group of proteins including human CD69, epidermal growth factor receptor and cystatin F 48–50. The function of C-terminal specific motifs in ΔExon-11 variant is yet to be examined, but its association with TNF-R1 has been demonstrated clearly here.

Our results have highlighted differences for the polymorphic position P/R127, with the full-length R127-K528 showing much reduced cellular expression compared with the P127-K528 isoform or even the ΔExon-11 spliced variant, which also carries R127, indicating variation in cellular stability among these isoforms under identical promoter-dependent regulation. Interestingly, 293T cells show very low levels of endogenous ERAP1 that contains P127 and R528 polymorphisms, but the P127-R528 isoform was well expressed in these cells suggesting the involvement of other factors including the promoter region regulating ERAP1 expression. It is possible that ΔExon-11 spliced variant has gained cellular stability through its unique C-terminal sequence. Alternatively, it seems reasonable to speculate that other polymorphisms including the C-terminal region (residues 509–941) of ERAP1 may also contribute to the contrasting effects observed for the polymorphic position P/R127 through haplotype interactions. It is noteworthy that all four isoforms studied here carry the major allele at position 56 (E56) in ERAP1. Our findings may have important implications in some human diseases. Genetic variations in ERAP1 for minor alleles at positions 56 and 127, or a combination of these positions (a major allele at position 56 and a minor allele at position 127) as a haplotype, have been linked to cervical cancer and low ERAP1 expression correlated with tumour progression and reduced survival rate 26. Another recent genetic study in psoriasis has reported the polymorphic position P/R127 associating strongly with a subgroup of cases with the disease 32. Furthermore, our full-length ERAP1 isoforms also contain the minor allele at position 730 (Q730), but the potential contribution of this residue to the observed results described here remains to be assessed, as the genotype for Q730 allele associates strongly with the presence of AS 17–20,36.

Our data suggest that in contrast to R127-K528 and P127-R528 isoforms, the cellular expression of full-length P127-K528 ERAP1 and ΔExon-11 spliced variant is enhanced significantly by the availability of an additional binding partner such as TNF-R1. The reasons for these observed differences are as-yet unclear, but it is conceivable that the C-terminal region of ERAP1 may exert conformational constraints on the full-length protein leading to differential affinity for TNF-R1 within the cytoplasmic compartment. ERAP1 isoforms may also have variable effects on the cytosolic co-expression of TNF-R1. ERAP1 (ARTS-1) associates with the extracellular region of TNF-R1 5. We believe that ERAP1 binds preferentially to the full-length TNF-R1, and this interaction occurs via a region located within the first 10 exons of ERAP1. Interestingly, ΔExon-11 spliced variant caused a reduction in the co-expressed cytosolic TNF-R1 at all expression levels (Fig. 2), which may have occurred through a functional gain by this spliced variant involving an as-yet undefined molecular pathway. In contrast, low and high levels of R127-K528 and P127-K528 full-length isoforms somewhat reduced the TNF-R1 expression, with the former isoform being more effective due possibly to its differential affinity for TNF-R1. Although speculative, this observation may reflect a potential requirement for equilibrium between ERAP1 and TNF-R1 proteins within the cytoplasmic compartment. It is also possible that only a proportion of ERAP1 and TNF-R1 remain associated. Two additional ARTS-1 (ERAP1) associating proteins, nucleobindin 2 and RNA-binding motif protein, X-linked (RBMX)/heterogeneous ribonucleoprotein G, have also been implicated in TNF-R1 intracellular trafficking 51,52, but we have not examined the involvement of these proteins in the current study. However, it is tempting to speculate that an interaction between ERAP1 and TNF-R1 may have functional regulatory consequences for both proteins. For example, ERAP1 may maintain TNF-R1 in an inactive conformational form that would suppress the formation of functional TNF-R1 units, thereby preventing inappropriate activity via this receptor. Such a regulatory role of ERAP1 would be in line with our view held in a previous study, which suggested that TNF-R1 expression on the cell surface may be regulated by an additional cellular component 34.

Our analysis of the soluble proteins has shown that the full-length TNF-R1, its cleaved ectodomain and the cytoplasmic domain were all associated with released vesicles isolated from cultured supernatants. We also detected all full-length ERAP1 isoforms within the same pool of exosome-like vesicles released from transfected cells, whereas the ΔExon-11 spliced variant was largely absent from these vesicular structures, reflecting differential compartmentalization from the full-length ERAP1. Surprisingly, each secreted ERAP1 isoform exhibited a lower molecular mass when co-transfected with TNF-R1, which may reflect changed environment during its passage through the secretory pathway. Overall, these results are in line with a recent study by Goto et al. 37, showing enhanced secretion of endogenous full-length, enzymatically active ERAP1 from murine macrophages in response to LPS/IFN-γ treatment. In our study, we found variable effects on TNF-R1 ectodomain shedding in response to cytokine treatment (Fig. 5). Exposure to TNF increased TNF-R1 ectodomain levels, while IL-1β treatment somewhat reduced the cleavage of the ectodomain, and these profiles were not influenced by the presence of R127-K528 or P127-K528 isoforms. Moreover, cytokine treatment did not exert any major effects on ERAP1 secretion. Our results contrast with those reported by Islam et al. 51, who showed that ARTS-1 increased IL-1β-induced TNF-R1 ectodomain cleavage, but this discrepancy may be due to differences in the cell types and IL-1β exposure time used in the two studies.

We observed only marginal differences between TNF-R1 ectodomain cleavage levels in comparative co-expression experiments performed with individual ERAP1 isoforms. In contrast to P127-K528 (K528 allele), higher levels of soluble TNF-R1 ectodomain were seen in the presence of P127-R528 (R528 allele) or the ΔExon-11 spliced variant that lacks this polymorphic position (Fig. 4). If this spliced variant is catalytically inactive, we would expect that TNF-R1 ectodomain cleavage is independent of ERAP1 enzymatic activity. This view is compatible with other studies, suggesting that endogenous ARTS-1 (ERAP1) itself does not possess sheddase activity for TNF-R1, and ADAM17 (a disintegrin and metalloprotease 17) most probably mediates the ectodomain cleavage of this receptor 5. Although the molecular mechanisms by which full-length P127-R528 (R528) isoform and ΔExon-11 spliced variant that lacks 46% of the C-terminal region of ERAP1 could promote TNF-R1 ectodomain cleavage are unclear at this stage, these functional consequences may favour dampening the inflammation promoting processes within an inflammatory environment. Furthermore, it is worth noting that R528 allele also exhibits reduced enzymatic activity and peptide processing capacity, and this allele associates with protection from autoimmune disorders 19,27–30,36. The functional pathways through which R528 allele could exert its protective effects may vary in different autoimmune diseases, but we speculate that R528 residue might keep the full-length ERAP1 in a conformation that may allow a gain of function via interactions with other cellular proteins. It is also feasible that the functional contribution of polymorphic position K/R528 is influenced by specific combinations of haplotype interactions.

In conclusion, our study has identified that ERAP1 is subject to alternative splicing in human cells and provides new insight into differential cellular expression exhibited by different ERAP1 allelic isoforms. We propose that inflammatory mediators, particularly within the site of inflammation, may play critical roles in modulating ERAP1 expression that could lead to altered functional activities of this aminopeptidase.

Acknowledgments

This work was supported by Arthritis Research UK (grant numbers 18330 and 17559). The BRIGHT study was supported by the Medical Research Council of Great Britain (grant number G9521010D) and the British Heart Foundation (grant number PG02/128). This work forms a part of research themes contributing to translational research portfolio of Barts Cardiovascular Biomedical Research Unit, which is funded by the National Institute of Health Research (M. C. and P. B. M). The Genome Centre is funded by the Barts and The London Charity. We thank Dr Masafumi Tsujimoto (RIKEN, Saitama, Japan) for providing anti-human ERAP1 antibody and Professor Mauro Perretti (Queen Mary University of London, London, UK) for annexin I antibody. We also thank Dr Alexander Annenkov (Queen Mary University of London, London, UK) for helpful suggestions and critical reading of this manuscript.

Disclosure

The authors declare no competing financial interests.

Supporting Information

Additional Supporting information may be found in the online version of this article at the publisher's web-site:

Fig. S1. Endoplasmic reticulum aminopeptidase 1 (ERAP1) mRNA expression is modulated by lipopolysaccharide (LPS) in human CD14− cells and U937 cells. RNA prepared from untreated (Unt) or ligand treated cells and cDNA was used in polymerase chain reaction (PCR) with primers spanning the region between exons 8–18 [product size, 1440 base pairs (bp)] or exons 10–15 (product size, 602 bp) of ERAP1. CD14− cells were treated with LPS for 3 h (a) and U937 cells for 1 and 3 h (b). PCR products for untreated (Unt) and LPS-treated U937 cells were analysed on the same gels (b). PCR products of expected sizes are indicated by arrows. Reactions with β2 m specific primers were used as controls. Negative controls (−) were without template.

Fig. S2. The C-termini of endoplasmic reticulum aminopeptidase 1 (ERAP1) splice variant proteins express unique features. For each variant, position of the last residue shared with full-length ERAP1 shown in bold. C-termini of variant proteins contain cysteine residues. Arginine and histidine are prominent in ΔExon-11 and ΔExon-15, and serine in ΔExon-14 variant. Cysteine and histidine residues underlined. Encoded variant proteins for ΔExon-11, ΔExon-13, ΔExon-14 and ΔExon-15 expected to contain 548, 602, 654 and 724 amino acids, respectively.

Fig. S3. Human CD14− cells and U937 cells exhibit differential expression of alternatively spliced variants of endoplasmic reticulum aminopeptidase 1 (ERAP1) mRNA. Cells were left untreated (Unt) or stimulated with lipopolysaccharide (LPS) for 3 h before RNA preparation and cDNA used as template in polymerase chain reaction (PCR). (a) Expression of ΔExon-11 variant in CD14− cells assessed by PCR using a sense primer (exon 8) in combination with a specific antisense primer designed for exon 10–exon 12 boundary sequences. (b) Expression of ΔExon-13, ΔExon-14 and ΔExon-15 ERAP1 variants in LPS-treated CD14− cells determined using a sense primer (exon 8) in combination with a specific anti-sense primer for exon 12–exon 14 (for ΔExon-13), exon 13–exon 15 (for ΔExon-14) or exon 14–exon 16 (for ΔExon-15) boundary sequences. These variants were undetectable in unstimulated CD14− cells (not shown). (c) Expression of ΔExon-11 and ΔExon-15 ERAP1 variants in U937 cells examined by PCR performed as described above. ΔExon-13 and ΔExon-14 variants not detected in U937 cells (not shown). Positions of expected fragment sizes for ΔExon-11 (346bp), ΔExon-13 (584bp), ΔExon-14 [666base pairs (bp)] and ΔExon-15 (922 bp) ERAP1 variants are shown by arrows. Reactions with β2 m specific primers (300 bp) were used as controls. Negative controls (−) were without template. Data shown is representative of two to three separate experiments.

Fig. S4. Expression profiles of endogenous endoplasmic reticulum aminopeptidase 1 (ERAP1) in human cell lines. Cytosolic lysates (30 μg total protein) prepared from cell lines were analysed by reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with RIKEN anti-ERAP1 antibody. Human umbilical vein endothelial cells (HUVEC, lane 1), oral cell carcinoma (OC, lane 2), melanoma cell line (A375, lane 3), proximal tubular epithelial cells (HK2, lane 4), Hela (lane 5), Ramos (lane 6), THP1 (lane 7), U937 (lane 8) and 293T (lane 9) cells.

Fig. S5. Endoplasmic reticulum aminopeptidase 1 (ERAP1) associates with tumour necrosis factor receptor 1 (TNF-R1). 293T cells transfected with 5 μg of R127-K528-FLAG (R127), P127-K528-FLAG (P127), P127-R528-FLAG (R528) or ΔExon-11 spliced variant of ERAP1 plasmid DNA with or without TNF-R1-V5-His (1 μg plasmid DNA), total amount of DNA transfected being 8 μg. Empty vector plasmid DNA used as control (Vec). Cell lysates immunoprecipitated with mouse anti-TNF-R1 monoclonal antibody and protein G-sepharose. Complexes analysed by immunoblotting with RIKEN antibody. No specific band for ΔExon-11 spliced variant (60 kDa) is apparent. Specific bands for full-length ERAP1 isoforms indicated by thick arrow. Position of heavy chain of immunoglobulin (Ig)G (H) is shown. Data shown is representative of two separate analyses.

Fig. S6. Soluble tumour necrosis factor receptor 1 (TNF-R1), endoplasmic reticulum aminopeptidase 1 (ERAP1), β-actin and annexin 1 are vesicle-associated. 293T cells transfected with P127-K528-FLAG (P127) ERAP1 plasmid DNA (5 μg), with or without TNF-R1-V5-His (1 μg plasmid DNA), total amount of DNA transfected being 8 μg. Empty vector plasmid DNA used as control (Vec). (a) Supernatants from cultured transfectants sedimented at 175 000 g and resulting vesicle pellets resolved by reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and analysed by immunoblotting for TNF-R1 (top panel), ERAP1 (second panel), β-actin (third panel) or annexin 1 (bottom panel). (b) Expression profiles of TNF-R1 and ERAP1 isoforms in cell lysates from the same transfectants were analysed by Western blotting as described above. Membranes reprobed for annexin 1 and β-actin. Specific bands indicating full-length TNF-R1 and ectodomain are shown by thin arrows and those for P127 ERAP1 isoform by thick arrows. Data shown are representative of at least two separate analyses.

Fig. S7. Interleukin (IL)-10 treatment does not exert a major effect on tumour necrosis factor receptor 1 (TNF-R1) ectodomain cleavage or endoplasmic reticulum aminopeptidase 1 (ERAP1) secretion. 293T cells transfected with P127-K528-FLAG (P127) or R127-K528-FLAG (R127) plasmid DNA with or without TNF-R1-V5-His, as described in Supporting information, Fig. 6. Empty vector plasmid DNA used as control (Vec). Transfected cells treated with IL-10 for 18 h, control transfectants (−) left untreated. Supernatant-derived vesicles obtained after sedimentation of conditioned medium at 175 000 g, resolved by reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with anti-TNF-R1 antibody (top panels), or anti-FLAG (bottom panels). Samples for both ERAP1 isoforms were analysed on the same gels but results shown for R127-K528 with anti-FLAG antibody (bottom panel) are from longer exposure time following enhanced chemiluminescence (ECL) detection. Data shown are representative of two separate experiments. Specific bands indicating full-length TNF-R1 and ectodomain are shown by thin arrows and those for ERAP1 isoforms by thick arrows. Band intensities for cleaved TNF-R1 ectodomain within supernatant-derived vesicles are shown as densitometry units.

Table S1. Nucleotide sequences of polymerase chain reaction (PCR) primers.

cei0180-0289-sd1.docx (1MB, docx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1. Endoplasmic reticulum aminopeptidase 1 (ERAP1) mRNA expression is modulated by lipopolysaccharide (LPS) in human CD14− cells and U937 cells. RNA prepared from untreated (Unt) or ligand treated cells and cDNA was used in polymerase chain reaction (PCR) with primers spanning the region between exons 8–18 [product size, 1440 base pairs (bp)] or exons 10–15 (product size, 602 bp) of ERAP1. CD14− cells were treated with LPS for 3 h (a) and U937 cells for 1 and 3 h (b). PCR products for untreated (Unt) and LPS-treated U937 cells were analysed on the same gels (b). PCR products of expected sizes are indicated by arrows. Reactions with β2 m specific primers were used as controls. Negative controls (−) were without template.

Fig. S2. The C-termini of endoplasmic reticulum aminopeptidase 1 (ERAP1) splice variant proteins express unique features. For each variant, position of the last residue shared with full-length ERAP1 shown in bold. C-termini of variant proteins contain cysteine residues. Arginine and histidine are prominent in ΔExon-11 and ΔExon-15, and serine in ΔExon-14 variant. Cysteine and histidine residues underlined. Encoded variant proteins for ΔExon-11, ΔExon-13, ΔExon-14 and ΔExon-15 expected to contain 548, 602, 654 and 724 amino acids, respectively.

Fig. S3. Human CD14− cells and U937 cells exhibit differential expression of alternatively spliced variants of endoplasmic reticulum aminopeptidase 1 (ERAP1) mRNA. Cells were left untreated (Unt) or stimulated with lipopolysaccharide (LPS) for 3 h before RNA preparation and cDNA used as template in polymerase chain reaction (PCR). (a) Expression of ΔExon-11 variant in CD14− cells assessed by PCR using a sense primer (exon 8) in combination with a specific antisense primer designed for exon 10–exon 12 boundary sequences. (b) Expression of ΔExon-13, ΔExon-14 and ΔExon-15 ERAP1 variants in LPS-treated CD14− cells determined using a sense primer (exon 8) in combination with a specific anti-sense primer for exon 12–exon 14 (for ΔExon-13), exon 13–exon 15 (for ΔExon-14) or exon 14–exon 16 (for ΔExon-15) boundary sequences. These variants were undetectable in unstimulated CD14− cells (not shown). (c) Expression of ΔExon-11 and ΔExon-15 ERAP1 variants in U937 cells examined by PCR performed as described above. ΔExon-13 and ΔExon-14 variants not detected in U937 cells (not shown). Positions of expected fragment sizes for ΔExon-11 (346bp), ΔExon-13 (584bp), ΔExon-14 [666base pairs (bp)] and ΔExon-15 (922 bp) ERAP1 variants are shown by arrows. Reactions with β2 m specific primers (300 bp) were used as controls. Negative controls (−) were without template. Data shown is representative of two to three separate experiments.

Fig. S4. Expression profiles of endogenous endoplasmic reticulum aminopeptidase 1 (ERAP1) in human cell lines. Cytosolic lysates (30 μg total protein) prepared from cell lines were analysed by reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with RIKEN anti-ERAP1 antibody. Human umbilical vein endothelial cells (HUVEC, lane 1), oral cell carcinoma (OC, lane 2), melanoma cell line (A375, lane 3), proximal tubular epithelial cells (HK2, lane 4), Hela (lane 5), Ramos (lane 6), THP1 (lane 7), U937 (lane 8) and 293T (lane 9) cells.

Fig. S5. Endoplasmic reticulum aminopeptidase 1 (ERAP1) associates with tumour necrosis factor receptor 1 (TNF-R1). 293T cells transfected with 5 μg of R127-K528-FLAG (R127), P127-K528-FLAG (P127), P127-R528-FLAG (R528) or ΔExon-11 spliced variant of ERAP1 plasmid DNA with or without TNF-R1-V5-His (1 μg plasmid DNA), total amount of DNA transfected being 8 μg. Empty vector plasmid DNA used as control (Vec). Cell lysates immunoprecipitated with mouse anti-TNF-R1 monoclonal antibody and protein G-sepharose. Complexes analysed by immunoblotting with RIKEN antibody. No specific band for ΔExon-11 spliced variant (60 kDa) is apparent. Specific bands for full-length ERAP1 isoforms indicated by thick arrow. Position of heavy chain of immunoglobulin (Ig)G (H) is shown. Data shown is representative of two separate analyses.

Fig. S6. Soluble tumour necrosis factor receptor 1 (TNF-R1), endoplasmic reticulum aminopeptidase 1 (ERAP1), β-actin and annexin 1 are vesicle-associated. 293T cells transfected with P127-K528-FLAG (P127) ERAP1 plasmid DNA (5 μg), with or without TNF-R1-V5-His (1 μg plasmid DNA), total amount of DNA transfected being 8 μg. Empty vector plasmid DNA used as control (Vec). (a) Supernatants from cultured transfectants sedimented at 175 000 g and resulting vesicle pellets resolved by reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and analysed by immunoblotting for TNF-R1 (top panel), ERAP1 (second panel), β-actin (third panel) or annexin 1 (bottom panel). (b) Expression profiles of TNF-R1 and ERAP1 isoforms in cell lysates from the same transfectants were analysed by Western blotting as described above. Membranes reprobed for annexin 1 and β-actin. Specific bands indicating full-length TNF-R1 and ectodomain are shown by thin arrows and those for P127 ERAP1 isoform by thick arrows. Data shown are representative of at least two separate analyses.

Fig. S7. Interleukin (IL)-10 treatment does not exert a major effect on tumour necrosis factor receptor 1 (TNF-R1) ectodomain cleavage or endoplasmic reticulum aminopeptidase 1 (ERAP1) secretion. 293T cells transfected with P127-K528-FLAG (P127) or R127-K528-FLAG (R127) plasmid DNA with or without TNF-R1-V5-His, as described in Supporting information, Fig. 6. Empty vector plasmid DNA used as control (Vec). Transfected cells treated with IL-10 for 18 h, control transfectants (−) left untreated. Supernatant-derived vesicles obtained after sedimentation of conditioned medium at 175 000 g, resolved by reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with anti-TNF-R1 antibody (top panels), or anti-FLAG (bottom panels). Samples for both ERAP1 isoforms were analysed on the same gels but results shown for R127-K528 with anti-FLAG antibody (bottom panel) are from longer exposure time following enhanced chemiluminescence (ECL) detection. Data shown are representative of two separate experiments. Specific bands indicating full-length TNF-R1 and ectodomain are shown by thin arrows and those for ERAP1 isoforms by thick arrows. Band intensities for cleaved TNF-R1 ectodomain within supernatant-derived vesicles are shown as densitometry units.

Table S1. Nucleotide sequences of polymerase chain reaction (PCR) primers.

cei0180-0289-sd1.docx (1MB, docx)

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