Functional Interaction of the Ankylosing Spondylitis-associated Endoplasmic Reticulum Aminopeptidase 1 Polymorphism and HLA-B27 in Vivo
2012; Elsevier BV; Volume: 11; Issue: 11 Linguagem: Inglês
10.1074/mcp.m112.019588
ISSN1535-9484
AutoresNoel García‐Medel, Alejandro Sanz‐Bravo, Dung Van Nguyen, Begoña Galocha, Patricia Gómez-Molina, Adrian Martín‐Esteban, Carlos Alvarez‐Navarro, José A. Łópez de Castro,
Tópico(s)Inflammasome and immune disorders
ResumoThe association of ERAP1 with ankylosing spondylitis (AS) 1The abbreviations used are:ASankylosing spondylitisERendoplasmic reticulumERAPendoplasmic reticulum aminopeptidaseHCheavy chainIRintensity ratioIRAPinsulin-regulated amino peptidaseLCLlymphoblastoid cell linemAbmonoclonal antibodyMHC-IMajor Histocompatibility Complex class IMwmolecular massSNPsingle nucleotide polymorphismWE-IWEWAK I.1The abbreviations used are:ASankylosing spondylitisERendoplasmic reticulumERAPendoplasmic reticulum aminopeptidaseHCheavy chainIRintensity ratioIRAPinsulin-regulated amino peptidaseLCLlymphoblastoid cell linemAbmonoclonal antibodyMHC-IMajor Histocompatibility Complex class IMwmolecular massSNPsingle nucleotide polymorphismWE-IWEWAK I. 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. The association of ERAP1 with ankylosing spondylitis (AS) 1The abbreviations used are:ASankylosing spondylitisERendoplasmic reticulumERAPendoplasmic reticulum aminopeptidaseHCheavy chainIRintensity ratioIRAPinsulin-regulated amino peptidaseLCLlymphoblastoid cell linemAbmonoclonal antibodyMHC-IMajor Histocompatibility Complex class IMwmolecular massSNPsingle nucleotide polymorphismWE-IWEWAK I.1The abbreviations used are:ASankylosing spondylitisERendoplasmic reticulumERAPendoplasmic reticulum aminopeptidaseHCheavy chainIRintensity ratioIRAPinsulin-regulated amino peptidaseLCLlymphoblastoid cell linemAbmonoclonal antibodyMHC-IMajor Histocompatibility Complex class IMwmolecular massSNPsingle nucleotide polymorphismWE-IWEWAK I. 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. ankylosing spondylitis endoplasmic reticulum endoplasmic reticulum aminopeptidase heavy chain intensity ratio insulin-regulated amino peptidase lymphoblastoid cell line monoclonal antibody Major Histocompatibility Complex class I molecular mass single nucleotide polymorphism WEWAK I. ankylosing spondylitis endoplasmic reticulum endoplasmic reticulum aminopeptidase heavy chain intensity ratio insulin-regulated amino peptidase lymphoblastoid cell line monoclonal antibody Major Histocompatibility Complex class I molecular mass single nucleotide polymorphism WEWAK I. The mechanism underlying the strong association of HLA-B27 with ankylosing spondylitis (AS) remains unknown. Three main possibilities, each one based on a different molecular feature of HLA-B27, are currently being investigated. The arthritogenic peptide hypothesis (1Benjamin R. Parham P. Guilt by association: HLA-B27 and ankylosing spondylitis.Immunol. Today. 1990; 11: 137-142Abstract Full Text PDF PubMed Scopus (350) Google Scholar), based on the canonic antigen-presenting properties of Major Histocompatibility Complex class I (MHC-I) molecules, assumes that a peptide epitope of external origin would activate HLA-B27-restricted T-cells, whose cross-reactivity with a self-derived HLA-B27 ligand would result in autoimmune damage. The misfolding hypothesis (2Colbert R.A. HLA-B27 misfolding: a solution to the spondyloarthropathy conundrum?.Mol. Med. Today. 2000; 6: 224-230Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) is based on the slow folding and tendency to misfold of HLA-B27 (3Mear J.P. Schreiber K.L. Münz C. Zhu X. Stevanovic S. Rammensee H.G. Rowland-Jones S.L. Colbert. R.A. Misfolding of HLA-B27 as a result of its B pocket suggests a novel mechanism for its role in susceptibility to spondyloarthropathies.J. Immunol. 1999; 163: 6665-6670Crossref PubMed Google Scholar, 4Dangoria N.S. DeLay M.L. Kingsbury D.J. Mear J.P. Uchanska-Ziegler B. Ziegler A. Colbert R.A. HLA-B27 misfolding is associated with aberrant intermolecular disulfide bond formation (dimerization) in the endoplasmic reticulum.J. Biol. Chem. 2002; 277: 23459-23468Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). An accumulation of misfolded heavy chains (HCs) in the endoplasmic reticulum (ER) would elicit an unfolded protein response and activate pro-inflammatory pathways. The surface homodimer hypothesis (5Allen R.L. O'Callaghan C.A. McMichael A.J. Bowness P. Cutting edge: HLA-B27 can form a novel β2-microglobulin-free heavy chain homodimer structure.J. Immunol. 1999; 162: 5045-5048PubMed Google Scholar, 6Bowness P. HLA B27 in health and disease: a double-edged sword?.Rheumatology (Oxford). 2002; 41: 857-868Crossref PubMed Scopus (64) Google Scholar) is based on the expression of HLA-B27 HC homodimers at the cell surface and their recognition by leukocyte receptors (7Kollnberger S. Bird L. Sun M.Y. Retiere C. Braud V.M. McMichael A. Bowness P. Cell-surface expression and immune receptor recognition of HLA-B27 homodimers.Arthritis Rheum. 2002; 46: 2972-2982Crossref PubMed Scopus (201) Google Scholar), which leads to immunomodulation of inflammatory responses. Because the constitutive binding of endogenous peptides by MHC-I molecules determines not only their antigen-presenting specificity, but also their folding and stability, it was proposed that the HLA-B27 peptidome, through its global influence on the biological behavior of the molecule, is critical to its pathogenetic role (8Marcilla M. Lopez de Castro J.A. Peptides: the cornerstone of HLA-B27 biology and pathogenetic role in spondyloarthritis.Tissue Antigens. 2008; 71: 495-506Crossref PubMed Scopus (52) Google Scholar). This idea found strong support with the discovery of the association of ER aminopeptidase (ERAP) 1 with AS (9WTCC Consortium Association scan of 14,500 nonsynonymous SNPs in four diseases identifies autoimmunity variants.Nat. Genet. 2007; 39: 1329-1337Crossref PubMed Scopus (1148) Google Scholar) in HLA-B27-positive, but not B27-negative, disease (10TASK and WTCCC2 Consortia Interaction between ERAP1 and HLA-B27 in ankylosing spondylitis implicates peptide handling in the mechanism for HLA-B27 in disease susceptibility.Nat. Genet. 2011; 43: 761-767Crossref PubMed Scopus (649) Google Scholar). With an estimated population attributable risk of 26%, ERAP1 is the non-MHC gene most strongly associated with AS. Given that ERAP1 is involved in the N-terminal trimming of peptides to their optimal size for MHC-I binding (11Serwold T. Gaw S. Shastri N. ER aminopeptidases generate a unique pool of peptides for MHC class I molecules.Nat. Immunol. 2001; 2: 644-651Crossref PubMed Scopus (164) Google Scholar, 12Serwold T. Gonzalez F. Kim J. Jacob R. Shastri N. ERAAP customizes peptides for MHC class I molecules in the endoplasmic reticulum.Nature. 2002; 419: 480-483Crossref PubMed Scopus (482) Google Scholar, 13York I.A. Chang S.C. Saric T. Keys J.A. Favreau J.M. Goldberg A.L. Rock K.L. The ER aminopeptidase ERAP1 enhances or limits antigen presentation by trimming epitopes to 8–9 residues.Nat. Immunol. 2002; 3: 1177-1184Crossref PubMed Scopus (394) Google Scholar), its association with AS suggests a pathogenetic mechanism of functional interaction with HLA-B27 that influences peptide binding and antigen presentation. ERAP1 trimming is limited by peptide size, becoming highly inefficient for 8-mers and shorter peptides (13York I.A. Chang S.C. Saric T. Keys J.A. Favreau J.M. Goldberg A.L. Rock K.L. The ER aminopeptidase ERAP1 enhances or limits antigen presentation by trimming epitopes to 8–9 residues.Nat. Immunol. 2002; 3: 1177-1184Crossref PubMed Scopus (394) Google Scholar, 14Chang S.C. Momburg F. Bhutani N. Goldberg A.L. The ER aminopeptidase, ERAP1, trims precursors to lengths of MHC class I peptides by a "molecular ruler" mechanism.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 17107-17112Crossref PubMed Scopus (241) Google Scholar). This is a seemingly unique feature of ERAP1 that is not even shared by its analog ERAP2 (14Chang S.C. Momburg F. Bhutani N. Goldberg A.L. The ER aminopeptidase, ERAP1, trims precursors to lengths of MHC class I peptides by a "molecular ruler" mechanism.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 17107-17112Crossref PubMed Scopus (241) Google Scholar, 15Birtley J.R. Saridakis E. Stratikos E. Mavridis I.M. The crystal structure of human endoplasmic reticulum aminopeptidase 2 reveals the atomic basis for distinct roles in antigen processing.Biochemistry. 2012; 51: 286-295Crossref PubMed Scopus (73) Google Scholar). The only putative exception, which has not been entirely ruled out, might be insulin-regulated amino peptidase (IRAP), an endosomal analog of ERAP1 involved in cross-presentation, but probably not in processing of constitutive MHC-I ligands (16Saveanu L. Carroll O. Weimershaus M. Guermonprez P. Firat E. Lindo V. Greer F. Davoust J. Kratzer R. Keller S.R. Niedermann G. van Endert P. IRAP identifies an endosomal compartment required for MHC class I cross-presentation.Science. 2009; 325: 213-217Crossref PubMed Scopus (199) Google Scholar, 17Saveanu L. van Endert P. The role of insulin-regulated aminopeptidase in MHC class I antigen presentation.Front. Immunol. 2012; 3: 57Crossref PubMed Scopus (32) Google Scholar). IRAP degrades peptides to smaller products than ERAP1 in vitro (18Georgiadou D. Hearn A. Evnouchidou I. Chroni A. Leondiadis L. York I.A. Rock K.L. Stratikos E. Placental leucine aminopeptidase efficiently generates mature antigenic peptides in vitro but in patterns distinct from endoplasmic reticulum aminopeptidase 1.J. Immunol. 2010; 185: 1584-1592Crossref PubMed Scopus (35) Google Scholar). The three-dimensional structure of ERAP1 reveals a substrate binding cavity close to the catalytic site, as well as four domains; the conformational rearrangement between an open and a closed conformation, presumably induced upon substrate binding, regulates its enzymatic activity (19Nguyen T.T. Chang S.C. Evnouchidou I. York I.A. Zikos C. Rock K.L. Goldberg A.L. Stratikos E. Stern L.J. Structural basis for antigenic peptide precursor processing by the endoplasmic reticulum aminopeptidase ERAP1.Nat. Struct. Mol. Biol. 2011; 18: 604-613Crossref PubMed Scopus (149) Google Scholar, 20Kochan G. Krojer T. Harvey D. Fischer R. Chen L. Vollmar M. von Delft F. Kavanagh K.L. Brown M.A. Bowness P. Wordsworth P. Kessler B.M. Oppermann U. Crystal structures of the endoplasmic reticulum aminopeptidase-1 (ERAP1) reveal the molecular basis for N-terminal peptide trimming.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 7745-7750Crossref PubMed Scopus (183) Google Scholar). The polymorphic residues found among natural ERAP1 variants (21Harvey D. Pointon J.J. Evans D.M. Karaderi T. Farrar C. Appleton L.H. Sturrock R.D. Stone M.A. Oppermann U. Brown M.A. Wordsworth B.P. Investigating the genetic association between ERAP1 and ankylosing spondylitis.Hum. Mol. Genet. 2009; 18: 4204-4212Crossref PubMed Scopus (111) Google Scholar), and often co-occurring in complex allotypes, are located in various topological regions, including some in close proximity to the catalytic site, the substrate binding cavity, or domain junctions. Therefore, they might alter ERAP1 activity by directly affecting catalysis, altering substrate binding, or modulating domain rearrangements. The association of ERAP1 with AS does not by itself reveal the specific feature(s) determining the pathogenetic role of HLA-B27. Indeed, ERAP1 might influence the generation of specific pathogenetic epitopes; have a general effect on the HLA-B27 peptidome, altering the stability or other features of the molecule; or both. This study investigated general effects of ERAP1 polymorphism on the HLA-B27 peptidome by comparing the size distribution, molecular features, and N-terminal flanking sequences of peptides from human cells expressing the AS-associated B*27:04 subtype and different natural variants of ERAP1. The following B*27:04-positive cell lines were used: JSL (HLA-A*11; B*27:04, *48; C*02), WEWAK I (WE-I: HLA-A*11, *24; B*27:04, *62; C*02, *04), and KNE (HLA-A*01, *02:04; B*27:04, *08) are lymphoblastoid cell lines (LCLs). C1R04 is a transfectant of the lymphoid HLA class I-defective Hmy2.C1R cell line (22Zemmour J. Little A.M. Schendel D.J. Parham P. The HLA-A,B "negative" mutant cell line C1R expresses a novel HLA-B35 allele, which also has a point mutation in the translation initiation codon.J. Immunol. 1992; 148: 1941-1948Crossref PubMed Google Scholar) expressing B*27:04 (23Garcia F. Marina A. Lopez de Castro J.A. Lack of carboxyl-terminal tyrosine distinguishes the B*2706-bound peptide repertoire from those of B*2704 and other HLA-B27 subtypes associated to ankylosing spondylitis.Tissue Antigens. 1997; 49: 215-221Crossref PubMed Scopus (62) Google Scholar). The cells were cultured in RPMI 1640 medium supplemented with 2 mm l-glutamine and 10% FBS (Invitrogen, Paisley, UK). ME1 (IgG1), an anti-HLA-B7/B27/B22 monoclonal antibody (mAb) that recognizes HC/β2m/peptide complexes (24Ellis S.A. Taylor C. McMichael A. Recognition of HLA-B27 and related antigens by a monoclonal antibody.Hum. Immunol. 1982; 5: 49-59Crossref PubMed Scopus (201) Google Scholar), was used for immunopurification of HLA-B27. DNA purification was performed using the High Pure PCR Template Preparation system (Roche Diagnostics, Barcelona, Spain) following the instructions of the manufacturer. Aliquots of 10 ng were added onto 384-well plates in duplicate, dried, and amplified using specific oligonucleotides for eight non-synonymous SNPs located in the coding sequence of the ERAP1 gene: rs26653, rs26618, rs27895, rs27044, rs30187, rs10050860, rs17482078, and rs2287987 (Table I). Samples were run in an HT7900 Fast Real-Time PCR System and genotyped using SDS2.2 software (both from Applied Biosystems, Invitrogen, Carlsbad, CA) for allelic discrimination. The nonsynonymous SNP rs2549782 (G/T), encoding for the K392N change in ERAP2, was typed by same procedure.Table IERAP1 polymorphism in B*27:04 positive cell linesaOnly nonsynonymous changes in the coding strands of ERAP1 are shown. Nucleotide and amino acid residue numbering and consensus sequence are from Human ERAP1 Isoform 2 (Accession No.: Q9NZ08-2). Deviations from the consensus sequence are in boldface. Polymorphisms associated with increased risk for AS are underlined. All polymorphic positions were determined via SNP typing and confirmed by genomic sequencing.SNPNucl./AAOdds ratio (Ref.)ConsensusJSLC1RKNEbKNE was heterozygous for rs26653, rs26618, and rs27895, and this was confirmed by genomic sequencing.WE-IPosition N.rs26653380/1271.3 (21Harvey D. Pointon J.J. Evans D.M. Karaderi T. Farrar C. Appleton L.H. Sturrock R.D. Stone M.A. Oppermann U. Brown M.A. Wordsworth B.P. Investigating the genetic association between ERAP1 and ankylosing spondylitis.Hum. Mol. Genet. 2009; 18: 4204-4212Crossref PubMed Scopus (111) Google Scholar)g/Rg/Rc/Pg, c/R, Pc/Prs26618828/2760.99 (21Harvey D. Pointon J.J. Evans D.M. Karaderi T. Farrar C. Appleton L.H. Sturrock R.D. Stone M.A. Oppermann U. Brown M.A. Wordsworth B.P. Investigating the genetic association between ERAP1 and ankylosing spondylitis.Hum. Mol. Genet. 2009; 18: 4204-4212Crossref PubMed Scopus (111) Google Scholar)a/Ia/Ig/Ma, g/I, Ma/Irs278951037/3461.07 (21Harvey D. Pointon J.J. Evans D.M. Karaderi T. Farrar C. Appleton L.H. Sturrock R.D. Stone M.A. Oppermann U. Brown M.A. Wordsworth B.P. Investigating the genetic association between ERAP1 and ankylosing spondylitis.Hum. Mol. Genet. 2009; 18: 4204-4212Crossref PubMed Scopus (111) Google Scholar)g/Gg/Gg/Gg, a/G, Dg/Grs22879871045/3490.71 (56Brown M.A. Breakthroughs in genetic studies of ankylosing spondylitis.Rheumatology (Oxford). 2008; 47: 132-137Crossref PubMed Scopus (105) Google Scholar)a/Ma/Ma/Ma/Mg/Vrs301871583/5281.4 (56Brown M.A. Breakthroughs in genetic studies of ankylosing spondylitis.Rheumatology (Oxford). 2008; 47: 132-137Crossref PubMed Scopus (105) Google Scholar)a/Ka/Kg/Rg/Rg/Rrs100508601723/5750.71 (56Brown M.A. Breakthroughs in genetic studies of ankylosing spondylitis.Rheumatology (Oxford). 2008; 47: 132-137Crossref PubMed Scopus (105) Google Scholar)g/Dg/Dg/Dg/Da/Nrs174820782174/7250.7 (56Brown M.A. Breakthroughs in genetic studies of ankylosing spondylitis.Rheumatology (Oxford). 2008; 47: 132-137Crossref PubMed Scopus (105) Google Scholar)g/Rg/Rg/Rg/Ra/Qrs270442188/7301.4 (56Brown M.A. Breakthroughs in genetic studies of ankylosing spondylitis.Rheumatology (Oxford). 2008; 47: 132-137Crossref PubMed Scopus (105) Google Scholar)c/Qc/Qg/Eg/Eg/Ea Only nonsynonymous changes in the coding strands of ERAP1 are shown. Nucleotide and amino acid residue numbering and consensus sequence are from Human ERAP1 Isoform 2 (Accession No.: Q9NZ08-2). Deviations from the consensus sequence are in boldface. Polymorphisms associated with increased risk for AS are underlined. All polymorphic positions were determined via SNP typing and confirmed by genomic sequencing.b KNE was heterozygous for rs26653, rs26618, and rs27895, and this was confirmed by genomic sequencing. Open table in a new tab Exons 2–20, encompassing the coding region of ERAP1, were separately amplified via PCR and cloned into M13 for sequencing. PCR products were generated using AmpliTaq Gold PCR Master Mix (Applied Biosystems) following standard procedures, purified using ExoSap (USB Corp., Cleveland, OH), and sequenced in a 3730XL instrument (Applied Biosystems). Sequencing primers were either M13-complementary oligonucleotides, for amplicons that included this extension along the ERAP1 sequence, or the specific primers themselves when the amplicons lacked M13-derived sequences. Both strands of amplicons were routinely sequenced. Total cellular RNA was extracted using the RNeasy Mini Kit (Qiagen, Madrid, Spain), and this was followed by digestion with DNase I (Invitrogen, Karlsruhe, Germany). Complementary DNA was synthesized from 250 ng of total RNA using the High Capacity cDNA reverse transcription kit (Invitrogen) according to the manufacturer's instructions. The primers used for the amplification were purchased from Applied Biosystems. Comparative quantification of gene expression was performed via quantitative RT-PCR with an AB7900HT instrument (Applied Biosystems) using TaqMan probes and Gene Expression Master Mix (Applied Biosystems). Amplifications were carried out with an initial hold at 50 °C for 2 min followed by denaturation at 95 °C for 10 min, 40 cycles of denaturation at 95 °C for 15 s, and annealing and extension at 60 °C for 60 s. The results were expressed as relative mRNA expression quantified with the RQ Manager software and normalized to glyceraldehyde-3-phospahate dehydrogenase transcript levels. About 2 × 105 cells were lysed in 0.5% Igepal CA-630 (Sigma-Aldrich, St Louis, MO), 50 mm Tris HCl, 5 mm MgCl2, pH 7.4, containing protease inhibitors (Complete Mini) (Roche, Mannheim, Germany). After SDS-PAGE (10% slab gels) of whole lysates under reducing conditions, the separated components were electroblotted onto a nitrocellulose membrane (Amersham Biosciences Hybond-ECL) (GE Healthcare, Buckinghamshire, UK) at 20 V overnight using 30% methanol in 50 mm Tris/Gly buffer, pH 8.8, 0.04% SDS. ERAP1, ERAP2, and γ-tubulin, used as an internal standard, were revealed with the 6H9 mAb (a kind gift from Peter van Endert, INSERM, Paris, France), 3F5 (R&D Systems, Minneapolis, MN), or GTU88 (Sigma-Aldrich), respectively, using peroxidase-conjugated goat anti-mouse Ig polyclonal antibody (DakoCytomation, Glostrup, Denmark). The scanned autoradiograms were quantified using TINA 2.09e image analyzer software (Raystest Isotopenmessgeräte, Straubenhardt, Germany). This was carried out as described elsewhere (25Paradela A. Garcia-Peydro M. Vazquez J. Rognan D. Lopez de Castro J.A. The same natural ligand is involved in allorecognition of multiple HLA-B27 subtypes by a single T cell clone: role of peptide and the MHC molecule in alloreactivity.J. Immunol. 1998; 161: 5481-5490Crossref PubMed Google Scholar). Briefly, cells were lysed in 1% Igepal CA-630 with a mixture of protease inhibitors (Roche). The soluble fraction was subjected to affinity chromatography using the ME1 mAb. HLA-B27-bound peptides were eluted with 0.1% aqueous TFA at room temperature, filtered through Centricon 3 (Amicon, Beverly, MA) or Vivaspin 2 Hydrosart (VS02H11, Sartorious Stedim Biotech, Gottingen, Germany), concentrated, and subjected to HPLC fractionation in a Waters Alliance system (Waters, Milford, MA) using a Vydac 218TP52-C18 column (Vydac, Hesperia, CA) at a flow rate of 100 μl/min, as described elsewhere (26Paradela A. Alvarez I. Garcia-Peydro M. Sesma L. Ramos M. Vazquez J. Lopez de Castro J.A. Limited diversity of peptides related to an alloreactive T cell epitope in the HLA-B27-bound peptide repertoire results from restrictions at multiple steps along the processing-loading pathway.J. Immunol. 2000; 164: 329-337Crossref PubMed Scopus (37) Google Scholar). Fractions of 50 μl were collected and stored at −20 °C. Individual HPLC fractions were analyzed via MALDI-TOF MS using a 4800 Proteomics Analyzer (Applied Biosystems), as described elsewhere (27Garcia-Medel N. Sanz-Bravo A. Barnea E. Admon A. Lopez de Castro J.A. The origin of proteasome-inhibitor resistant HLA Class I peptidomes: a study with HLA-A*68:01.Mol. Cell. Proteomics. 2012; 11 (M111.011486)Abstract Full Text Full Text PDF Scopus (14) Google Scholar). The mass spectra were acquired in reflector positive mode at 25 kV in the m/z range of 800–2000, using a signal-to-noise ratio (s/n) cutoff of 3, and processed using Data Explorer software, version 4.9 (Applied Biosystems). Sample handling and acquisition parameters were tightly controlled to minimize differences in the experimental conditions among samples to be compared in each experiment. Peptide sequencing was carried out with MALDI-TOF/TOF MS/MS, as described elsewhere (27Garcia-Medel N. Sanz-Bravo A. Barnea E. Admon A. Lopez de Castro J.A. The origin of proteasome-inhibitor resistant HLA Class I peptidomes: a study with HLA-A*68:01.Mol. Cell. Proteomics. 2012; 11 (M111.011486)Abstract Full Text Full Text PDF Scopus (14) Google Scholar). Interpretation of the MS/MS spectra was assisted by various tools. Manual inspection of the spectrum usually allowed us to derive a tentative sequence. This was used to screen the human proteome in the human protein entries of the Uniprot/Swiss-prot database (Release 57.6, June 28, 2009, with 20,331 entries), using a window of 0.5 m/z units for both precursor and fragment ions, for a possible match using the Mascot server 2.2 software. For those sequences showing the highest scores in this preliminary search, the MS-product tool (version 5.9.4) (University of California, San Francisco, CA) was used to match the candidate sequences to our MS/MS spectra. The susceptibility of flanking (P-2, P-1) and P1 residues to ERAP1 trimming was estimated by assigning a score to each amino acid ranging from 0 to 100, based on a previously reported assay that measured the presentation of the SIINFEKL peptide from ER-targeted precursors (28Hearn A. York I.A. Rock K.L. The specificity of trimming of MHC class I-presented peptides in the endoplasmic reticulum.J. Immunol. 2009; 183: 5526-5536Crossref PubMed Scopus (77) Google Scholar). The score assigned to each residue (supplemental Table S1) corresponded to the mean percent of SIINFEKL expressed at the cell surface from XX- SIINFEKL precursors, relative to the most susceptible residue in this assay (Y: score 100). Proline and any residue immediately preceding it were assigned a score of 0. This was estimated based on a previous study (29Evnouchidou I. Momburg F. Papakyriakou A. Chroni A. Leondiadis L. Chang S.C. Goldberg A.L. Stratikos E. The internal sequence of the peptide-substrate determines its N-terminus trimming by ERAP1.PLoS One. 2008; 3: e3658Crossref PubMed Scopus (76) Google Scholar) in which a 9-mer library was used to assess the influence of different residues at each peptide position on the trimming of P1 by ERAP1 in vitro. Each residue at a given position, from P3 to P9, was assigned a score corresponding to the percentage of depleted substrate in that experiment (supplemental Table S1). Residues not directly tested in that study were not scored. This was performed as described elsewhere (30Merino E. Galocha B. Vazquez M.N. Lopez de Castro J.A. Disparate folding and stability of the ankylosing spondylitis-associated HLA-B*1403 and B*2705 proteins.Arthritis Rheum. 2008; 58: 3693-3704Crossref PubMed Scopus (21) Google Scholar). Briefly, the cell lines were pulse-labeled for 15 min and chased at various times. At each time point, the cell lysates were incubated for 1 h at various temperatures, immunoprecipitated with ME1, and analyzed via SDS-PAGE. The amount of heterodimer precipitated at each temperature at any given time was expressed as a percentage of the amount precipitated at 4 °C and plotted as a function of the temperature. Four B*27:04-positive cell lines, including 3 LCL and C1R transfectants, were selected on the basis of their expression of eight SNPs encoding nonsynonymous substitutions in ERAP1, including 6 AS-associated ones (9WTCC Consortium Association scan of 14,500 nonsynonymous SNPs in four diseases identifies autoimmunity variants.Nat. Genet. 2007; 39: 1329-1337Crossref PubMed Scopus (1148) Google Scholar, 21Harvey D. Pointon J.J. Evans D.M. Karaderi T. Farrar C. Appleton L.H. Sturrock R.D. Stone M.A. Oppermann U. Brown M.A. Wordsworth B.P. Investigating the genetic association between ERAP1 and ankylosing spondylitis.Hum. Mol. Genet. 2009; 18: 4204-4212Crossref PubMed Scopus (111) Google Scholar). Sequencing of all the exons of this gene in the four cell lines confirmed these polymorphisms (Table I). Whereas the coding sequence of ERAP1 from JSL was identical to the consensus and carried all six polymorphisms associated with increased susceptibility to AS, that from WE-I showed six nonsynonymous changes, including all those associated with protection from AS. ERAP1 from C1R04 showed four nonsynonymous changes relative to the consensus. KNE was heterozygous for rs26653, rs26618, and rs27895. This variability allowed us to examine the effect of various combinations of amino acid changes in natural ERAP1 variants on the HLA-B27-bound peptidome. Genotyping of the four cell lines for the nonsynonymous rs2549782 SNP of ERAP2, coding for the K392N change, revealed that WE1, C1R04, and KNE were heterozygous, whereas JSL was homozygous (T/T), expressing only the N392 allotype. Western blot analyses of WE-I, C1R04, and KNE (Figs. 1A and 1B) revealed that these cell lines expressed fairly similar ERAP1 protein levels: the WE-I/C1R04 ratio was 1:1.2, and the C1R04/KNE ratio was 1:1.4 (Fig. 1B), in agreement with previous studies on LCL (31Fruci D. Ferracuti S. Limongi M.Z. Cunsolo V. Giorda E. Fraioli R. Sibilio L. Carroll O. Hattori A. Van Endert P.M. Giacomini P. Expression of endoplasmic reticulum aminopeptidases in EBV-B cell lines from healthy donors and in leukemia/lymphoma, carcinoma, and melanoma cell lines.J. Immunol. 2006; 176: 4869-4879Crossref PubMed Scopus (82) Google Scholar). Although protein levels could not be assessed for JSL because of the loss of this cell line in the course of this study, the relative mRNA expression of ERAP1 was determined for all four cell lines (Fig. 1C). This reflected closely ERAP1 protein levels in C1R04 and WE-I (WE-I/C1R04 ratio, 1:1.3). mRNA expression for KNE was similar to that for WE-I and somewhat lower than that for C1R (C1R:KNE ratio, 1:0.75). JSL showed the lowest mRNa levels (about 0.6:1 and 0.8:1 relative to C1R04 and to WE1 or KNE, respectively). ERAP2 protein expression was similar in C1R04 and KNE and about 2-fold higher in WE-I (supplemental Fig. S1). B*27:04-bound peptide pools were fractionated via HPLC, and each fraction was analyzed using MALDI-TOF MS. Three pairwise comparisons—WE-I/JSL, WE-I/C1R04, and C1R04/KNE—were carried out, following a strategy previously used for HLA-B27 subtype-bound peptidomes (32Ramos M. Paradela A. Vazquez M. Marina A. Vazquez J. Lopez
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