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• W2783165679 abstract "The HLA-DRB1 locus is strongly associated with rheumatoid arthritis (RA) susceptibility, whereupon citrullinated self-peptides bind to HLA-DR molecules bearing the shared epitope (SE) amino acid motif. However, the differing propensity for citrullinated/non-citrullinated self-peptides to bind given HLA-DR allomorphs remains unclear. Here, we used a fluorescence polarization assay to determine a hierarchy of binding affinities of 34 self-peptides implicated in RA against three HLA-DRB1 allomorphs (HLA-DRB1*04:01/*04:04/*04:05) each possessing the SE motif. For all three HLA-DRB1 allomorphs, we observed a strong correlation between binding affinity and citrullination at P4 of the bound peptide ligand. A differing hierarchy of peptide-binding affinities across the three HLA-DRB1 allomorphs was attributable to the β-chain polymorphisms that resided outside the SE motif and were consistent with sequences of naturally presented peptide ligands. Structural determination of eight HLA–DR4–self-epitope complexes revealed strict conformational convergence of the P4-Cit and surrounding HLA β-chain residues. Polymorphic residues that form part of the P1 and P9 pockets of the HLA-DR molecules provided a structural basis for the preferential binding of the citrullinated self-peptides to the HLA-DR4 allomorphs. Collectively, we provide a molecular basis for the interplay between citrullination of self-antigens and HLA polymorphisms that shape peptide–HLA-DR4 binding affinities in RA. The HLA-DRB1 locus is strongly associated with rheumatoid arthritis (RA) susceptibility, whereupon citrullinated self-peptides bind to HLA-DR molecules bearing the shared epitope (SE) amino acid motif. However, the differing propensity for citrullinated/non-citrullinated self-peptides to bind given HLA-DR allomorphs remains unclear. Here, we used a fluorescence polarization assay to determine a hierarchy of binding affinities of 34 self-peptides implicated in RA against three HLA-DRB1 allomorphs (HLA-DRB1*04:01/*04:04/*04:05) each possessing the SE motif. For all three HLA-DRB1 allomorphs, we observed a strong correlation between binding affinity and citrullination at P4 of the bound peptide ligand. A differing hierarchy of peptide-binding affinities across the three HLA-DRB1 allomorphs was attributable to the β-chain polymorphisms that resided outside the SE motif and were consistent with sequences of naturally presented peptide ligands. Structural determination of eight HLA–DR4–self-epitope complexes revealed strict conformational convergence of the P4-Cit and surrounding HLA β-chain residues. Polymorphic residues that form part of the P1 and P9 pockets of the HLA-DR molecules provided a structural basis for the preferential binding of the citrullinated self-peptides to the HLA-DR4 allomorphs. Collectively, we provide a molecular basis for the interplay between citrullination of self-antigens and HLA polymorphisms that shape peptide–HLA-DR4 binding affinities in RA. Rheumatoid arthritis (RA) 5The abbreviations used are: RArheumatoid arthritisSEshared epitopeACPAanti-citrullinated protein antibodyBistris propane1,3-bis[tris(hydroxymethyl)methylamino]propanePTMpost-translational modificationPADpeptidyl-arginine deiminaseCitcitrullineTCRT cell responsePBMCperipheral blood mononuclear cellFDRfalse discovery rateTAMRA6-carboxy-N,N,N′,N′-tetramethylrhodamine. is an autoimmune disease of the synovial joints. A characteristic of RA is the presence of anti-citrullinated protein antibodies (ACPA) in sera, for which ∼70% of all patients are seropositive (1El-Gabalawy H. The preclinical stages of RA: lessons from human studies and animal models.Best Pract. Res. Clin. Rheumatol. 2009; 23 (19233045): 49-58https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). ACPA target proteins that have undergone citrullination, a post-translational modification (PTM) process driven by a family of enzymes known as peptidyl-arginine deiminases (PAD), convert arginine to citrulline (2Malmström V. Catrina A.I. Klareskog L. The immunopathogenesis of seropositive rheumatoid arthritis: from triggering to targeting.Nat. Rev. Immunol. 2017; 17 (27916980): 60-75https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 3Trouw L.A. Rispens T. Toes R.E. Beyond citrullination: other post-translational protein modifications in rheumatoid arthritis.Nat. Rev. Rheumatol. 2017; 13 (28275265): 331-339https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 4Van Steendam K. Tilleman K. Deforce D. The relevance of citrullinated vimentin in the production of antibodies against citrullinated proteins and the pathogenesis of rheumatoid arthritis.Rheumatology. 2011; 50 (21278075): 830-837https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). PAD type 2 (PAD-2) and PAD-4 expression is closely associated with synovial joint inflammation in RA patients (5Foulquier C. Sebbag M. Clavel C. Chapuy-Regaud S. Al Badine R. Méchin M.C. Vincent C. Nachat R. Yamada M. Takahara H. Simon M. Guerrin M. Serre G. Peptidyl arginine deiminase type 2 (PAD-2) and PAD-4 but not PAD-1, PAD-3, and PAD-6 are expressed in rheumatoid arthritis synovium in close association with tissue inflammation.Arthritis Rheum. 2007; 56 (17968929): 3541-3553https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Citrullination creates neo-self-antigens, and given the high rate of ACPA in RA patients, these antigens are considered the prime generators of the autoimmune CD4+ T cell response in ACPA+ RA. Citrulline-specific, antigen-experienced CD4+ T cells are found in HLA-DR4 (DRA1*01:01/HLA-DRB1*04:01) RA patients as well as in HLA-DR4 (DRA1*01:01/HLA-DRB1*04:01) transgenic mice primed with citrullinated self-antigens (6Feitsma A.L. van der Voort E.I. Franken K.L. el Bannoudi H. Elferink B.G. Drijfhout J.W. Huizinga T.W. de Vries R.R. Toes R.E. Ioan-Facsinay A. Identification of citrullinated vimentin peptides as T cell epitopes in HLA-DR4-positive patients with rheumatoid arthritis.Arthritis Rheum. 2010; 62 (20039411): 117-125https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 7Hill J.A. Bell D.A. Brintnell W. Yue D. Wehrli B. Jevnikar A.M. Lee D.M. Hueber W. Robinson W.H. Cairns E. Arthritis induced by posttranslationally modified (citrullinated) fibrinogen in DR4-IE transgenic mice.J. Exp. Med. 2008; 205 (18391064): 967-979https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 8Gerstner C. Dubnovitsky A. Sandin C. Kozhukh G. Uchtenhagen H. James E.A. Ronnelid J. Ytterberg A.J. Pieper J. Reed E. Tandre C. Rieck M. Zubarev R.A. Ronnblom L. Sandalova T. et al.Functional and structural characterization of a novel HLA-DRB1*04:01-restricted α-enolase T cell epitope in rheumatoid arthritis.Front. Immunol. 2016; 7 (27895642): e494https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 9Snir O. Rieck M. Gebe J.A. Yue B.B. Rawlings C.A. Nepom G. Malmström V. Buckner J.H. Identification and functional characterization of T cells reactive to citrullinated vimentin in HLA-DRB1*0401-positive humanized mice and rheumatoid arthritis patients.Arthritis Rheum. 2011; 63 (21567378): 2873-2883https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 10von Delwig A. Locke J. Robinson J.H. Ng W.F. Response of Th17 cells to a citrullinated arthritogenic aggrecan peptide in patients with rheumatoid arthritis.Arthritis Rheum. 2010; 62 (20039419): 143-149https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 11Law S.C. Street S. Yu C.H. Capini C. Ramnoruth S. Nel H.J. van Gorp E. Hyde C. Lau K. Pahau H. Purcell A.W. Thomas R. T-cell autoreactivity to citrullinated autoantigenic peptides in rheumatoid arthritis patients carrying HLA-DRB1 shared epitope alleles.Arthritis Res. Ther. 2012; 14 (22594821): R118https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Furthermore, citrulline-specific Th1 and Th17 cells are increased in the HLA-DRB1*04:01+ RA patients, and pro-inflammatory cytokines are produced by CD4+ T cells in response to citrullinated self-antigens (8Gerstner C. Dubnovitsky A. Sandin C. Kozhukh G. Uchtenhagen H. James E.A. Ronnelid J. Ytterberg A.J. Pieper J. Reed E. Tandre C. Rieck M. Zubarev R.A. Ronnblom L. Sandalova T. et al.Functional and structural characterization of a novel HLA-DRB1*04:01-restricted α-enolase T cell epitope in rheumatoid arthritis.Front. Immunol. 2016; 7 (27895642): e494https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 9Snir O. Rieck M. Gebe J.A. Yue B.B. Rawlings C.A. Nepom G. Malmström V. Buckner J.H. Identification and functional characterization of T cells reactive to citrullinated vimentin in HLA-DRB1*0401-positive humanized mice and rheumatoid arthritis patients.Arthritis Rheum. 2011; 63 (21567378): 2873-2883https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 10von Delwig A. Locke J. Robinson J.H. Ng W.F. Response of Th17 cells to a citrullinated arthritogenic aggrecan peptide in patients with rheumatoid arthritis.Arthritis Rheum. 2010; 62 (20039419): 143-149https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 11Law S.C. Street S. Yu C.H. Capini C. Ramnoruth S. Nel H.J. van Gorp E. Hyde C. Lau K. Pahau H. Purcell A.W. Thomas R. T-cell autoreactivity to citrullinated autoantigenic peptides in rheumatoid arthritis patients carrying HLA-DRB1 shared epitope alleles.Arthritis Res. Ther. 2012; 14 (22594821): R118https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Sources for the primary RA-associated autoantigens may be from the site of disease, including articular cartilage and synovial fluids (12van Beers J.J. Schwarte C.M. Stammen-Vogelzangs J. Oosterink E. Božič B. Pruijn G.J. The rheumatoid arthritis synovial fluid citrullinome reveals novel citrullinated epitopes in apolipoprotein E, myeloid nuclear differentiation antigen, and β-actin.Arthritis Rheum. 2013; 65 (23044660): 69-80https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 13Snir O. Widhe M. Hermansson M. von Spee C. Lindberg J. Hensen S. Lundberg K. Engström A. Venables P.J. Toes R.E. Holmdahl R. Klareskog L. Malmström V. Antibodies to several citrullinated antigens are enriched in the joints of rheumatoid arthritis patients.Arthritis Rheum. 2010; 62 (20039432): 44-52https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 14Biswas S. Sharma S. Saroha A. Bhakuni D.S. Malhotra R. Zahur M. Oellerich M. Das H.R. Asif A.R. Identification of novel autoantigen in the synovial fluid of rheumatoid arthritis patients using an immunoproteomics approach.PLoS One. 2013; 8 (23418544): e56246https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar), but others may be derived from blood plasma or surrounding mucosal tissues that are susceptible to inflammation (2Malmström V. Catrina A.I. Klareskog L. The immunopathogenesis of seropositive rheumatoid arthritis: from triggering to targeting.Nat. Rev. Immunol. 2017; 17 (27916980): 60-75https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). These proteins could undergo PTMs during numerous physiologic processes, including infection, apoptosis, and cellular stress. Some of the best-characterized autoantigens that bind ACPAs are citrullinated vimentin, fibrinogen, α-enolase, and type II collagen, which are present at high levels in the joint synovium (3Trouw L.A. Rispens T. Toes R.E. Beyond citrullination: other post-translational protein modifications in rheumatoid arthritis.Nat. Rev. Rheumatol. 2017; 13 (28275265): 331-339https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 15Kinloch A. Tatzer V. Wait R. Peston D. Lundberg K. Donatien P. Moyes D. Taylor P.C. Venables P.J. Identification of citrullinated α-enolase as a candidate autoantigen in rheumatoid arthritis.Arthritis Res. Ther. 2005; 7 (16277695): R1421-R1429https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Google Scholar). rheumatoid arthritis shared epitope anti-citrullinated protein antibody 1,3-bis[tris(hydroxymethyl)methylamino]propane post-translational modification peptidyl-arginine deiminase citrulline T cell response peripheral blood mononuclear cell false discovery rate 6-carboxy-N,N,N′,N′-tetramethylrhodamine. One of the key inherited risk factors that contribute to ACPA-positive RA is the human leukocyte antigen (HLA) class II loci, namely HLA-DRB1, which encodes the HLA class II antigen-presenting molecules (16Raychaudhuri S. Sandor C. Stahl E.A. Freudenberg J. Lee H.S. Jia X. Alfredsson L. Padyukov L. Klareskog L. Worthington J. Siminovitch K.A. Bae S.C. Plenge R.M. Gregersen P.K. de Bakker P.I. Five amino acids in three HLA proteins explain most of the association between MHC and seropositive rheumatoid arthritis.Nat. Genet. 2012; 44 (22286218): 291-296https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 17Padyukov L. Seielstad M. Ong R.T. Ding B. Rönnelid J. Seddighzadeh M. Alfredsson L. Klareskog L. Epidemiological Investigation of Rheumatoid Arthritis (EIRA) study groupA genome-wide association study suggests contrasting associations in ACPA-positive versus ACPA-negative rheumatoid arthritis.Ann. Rheum. Dis. 2011; 70 (21156761): 259-265https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 18Okada Y. Wu D. Trynka G. Raj T. Terao C. Ikari K. Kochi Y. Ohmura K. Suzuki A. Yoshida S. Graham R.R. Manoharan A. Ortmann W. Bhangale T. Denny J.C. et al.Genetics of rheumatoid arthritis contributes to biology and drug discovery.Nature. 2014; 506 (24390342): 376-381https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 19van Beers J.J. Willemze A. Stammen-Vogelzangs J. Drijfhout J.W. Toes R.E. Pruijn G.J. Anti-citrullinated fibronectin antibodies in rheumatoid arthritis are associated with human leukocyte antigen-DRB1 shared epitope alleles.Arthritis Res. Ther. 2012; 14 (22339947): R35https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Google Scholar, 20Koning F. Thomas R. Rossjohn J. Toes R.E. Coeliac disease and rheumatoid arthritis: similar mechanisms, different antigens.Nat. Rev. Rheumatol. 2015; 11 (25986717): 450-461https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 21Scally S.W. Law S.C. Ting Y.T. Heemst J.V. Sokolove J. Deutsch A.J. Bridie Clemens E. Moustakas A.K. Papadopoulos G.K. van der Woude D.V. Smolik I. Hitchon C.A. Robinson D.B. Ferucci E.D. Bernstein C.N. et al.Molecular basis for increased susceptibility of indigenous North Americans to seropositive rheumatoid arthritis.Ann. Rheum. Dis. 2017; 76 (28801345): 1915-1923https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). The antigen-binding groove of the HLA class II molecule can accommodate peptide ligands that vary in length, but the main pockets that interact most strongly with the bound peptide are P1, P4, P6, P7, and P9, which can accommodate the side chains of the peptide residues 1, 4, 6, 7, and 9 (22Rossjohn J. Gras S. Miles J.J. Turner S.J. Godfrey D.I. McCluskey J. T cell antigen receptor recognition of antigen-presenting molecules.Annu. Rev. Immunol. 2015; 33 (25493333): 169-200https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 23Adams E.J. Luoma A.M. The adaptable major histocompatibility complex (MHC) fold: structure and function of nonclassical and MHC class I-like molecules.Annu. Rev. Immunol. 2013; 31 (23298204): 529-561https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). A conserved amino acid sequence QKRAA, QRRAA, or RRRAA in position 70–74 of the HLA-DRB1 chain, known as the shared epitope (SE) motif, is highly prevalent (∼90%) among ACPA seropositive patients (11Law S.C. Street S. Yu C.H. Capini C. Ramnoruth S. Nel H.J. van Gorp E. Hyde C. Lau K. Pahau H. Purcell A.W. Thomas R. T-cell autoreactivity to citrullinated autoantigenic peptides in rheumatoid arthritis patients carrying HLA-DRB1 shared epitope alleles.Arthritis Res. Ther. 2012; 14 (22594821): R118https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 16Raychaudhuri S. Sandor C. Stahl E.A. Freudenberg J. Lee H.S. Jia X. Alfredsson L. Padyukov L. Klareskog L. Worthington J. Siminovitch K.A. Bae S.C. Plenge R.M. Gregersen P.K. de Bakker P.I. Five amino acids in three HLA proteins explain most of the association between MHC and seropositive rheumatoid arthritis.Nat. Genet. 2012; 44 (22286218): 291-296https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). This SE motif defines the P4 pocket of the high-risk HLA-DRB1 RA-associated allomorphs. Subsequent genome-wide association studies have shown that two polymorphisms encoding β-chain residues at positions 11 and 13 at the base of the P4 pocket are also strongly associated with RA susceptibility (16Raychaudhuri S. Sandor C. Stahl E.A. Freudenberg J. Lee H.S. Jia X. Alfredsson L. Padyukov L. Klareskog L. Worthington J. Siminovitch K.A. Bae S.C. Plenge R.M. Gregersen P.K. de Bakker P.I. Five amino acids in three HLA proteins explain most of the association between MHC and seropositive rheumatoid arthritis.Nat. Genet. 2012; 44 (22286218): 291-296https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). We have previously reported the structural basis for the association of the SE, citrullination, and RA by showing the size and charge of the P4 pocket accommodate antigens with a citrulline residue in the P4 position but prevents the binding of peptide ligands with the natively-encoded positively-charged P4-Arg (24Scally S.W. Petersen J. Law S.C. Dudek N.L. Nel H.J. Loh K.L. Wijeyewickrema L.C. Eckle S.B. van Heemst J. Pike R.N. McCluskey J. Toes R.E. La Gruta N.L. Purcell A.W. Reid H.H. et al.A molecular basis for the association of the HLA-DRB1 locus, citrullination, and rheumatoid arthritis.J. Exp. Med. 2013; 210 (24190431): 2569-2582https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Although citrullinated peptides have generally shown enhanced affinity to several SE HLA-DRB1 allomorphs relative to native peptides (25Hill J.A. Southwood S. Sette A. Jevnikar A.M. Bell D.A. Cairns E. Cutting edge: the conversion of arginine to citrulline allows for a high-affinity peptide interaction with the rheumatoid arthritis-associated HLA-DRB1*0401 MHC class II molecule.J. Immunol. 2003; 171 (12847215): 538-541https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Google Scholar), the affinity of individual peptides has not been compared, with respect to polymorphisms outside the SE motif. Here, we show the affinity of citrullinated peptides across three different HLA-DRB1 allomorphs and reveal the key interactions between RA-associated antigens with RA-susceptible HLA-SE allomorphs. The crystals structures of eight citrullinated peptides bound to HLA-DRB1*04:01/*04:04/*04:05 demonstrate the close convergence of the binding modes of the P4-Cit, with polymorphisms outside this pocket accounting for the hierarchy of binding of citrullinated self-peptides to the three HLA-DR4 allomorphs. These specificities were also revealed in the natural repertoire of bound peptides, suggesting similar selective pressure operates on the self-peptide repertoire (immunopeptidome). Accordingly, we provide a molecular understanding of the interplay between the citrullination of self-antigens and HLA polymorphism that shape peptide-HLA binding affinity in RA. The extent to which polymorphisms outside the P4 pocket contribute to the natural selection of peptides was established using monoallelic HLA-DR4+ antigen-presenting cells. For this purpose, we generated transfectants of the T2 cell line (class II-deficient) that expressed HLA-DM and either HLA DRB1*04:01, 04:04, or 04:05. We have previously reported the immunopeptidomes of the HLA-DRB1*04:01+ and 04:04+ cells (24Scally S.W. Petersen J. Law S.C. Dudek N.L. Nel H.J. Loh K.L. Wijeyewickrema L.C. Eckle S.B. van Heemst J. Pike R.N. McCluskey J. Toes R.E. La Gruta N.L. Purcell A.W. Reid H.H. et al.A molecular basis for the association of the HLA-DRB1 locus, citrullination, and rheumatoid arthritis.J. Exp. Med. 2013; 210 (24190431): 2569-2582https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Each of these datasets contained over 1000–3000 unique and high confidence peptides, and although earlier reports have identified some HLA-DRB1*04:05 peptide ligands (26Rammensee H. Bachmann J. Emmerich N.P. Bachor O.A. Stevanovič S. SYFPEITHI: database for MHC ligands and peptide motifs.Immunogenetics. 1999; 50 (10602881): 213-219https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Google Scholar), here we report a large dataset of peptide ligands (n = 2935) from the same parental T2 cell line (Table S1). These endogenous peptide sequences determined from multiple peptide elution experiments were identified with high confidence using strict bioinformatic criteria that includes the removal of common contaminants (27Dudek N.L. Tan C.T. Gorasia D.G. Croft N.P. Illing P.T. Purcell A.W. Constitutive and inflammatory immunopeptidome of pancreatic beta-cells.Diabetes. 2012; 61 (22872234): 3018-3025https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). The motif for HLA-DRB1*04:05 generated using this approach (Fig. 1A) was in general agreement with previously determined motifs (28Friede T. Gnau V. Jung G. Keilholz W. Stevanovič S. Rammensee H.G. Natural ligand motifs of closely related HLA-DR4 molecules predict features of rheumatoid arthritis associated peptides.Biochim. Biophys. Acta. 1996; 1316 (8672555): 85-101https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 29Kinouchi R. Kobayasi H. Sato K. Kimura S. Katagiri M. Peptide motifs of HLA-DR4/DR53 (DRB1*0405/DRB4*0101) molecules.Immunogenetics. 1994; 40 (7927542): 376-378https://doi.org/10.1074/jbc.RA117.001013Abstract Full Text Full Text PDF PubMed Google Scholar) and, like the other SE+ DR4 allomorphs, disfavored Arg at P4 and showed strong preferences at P1 (Phe, Tyr, and Ile), P6 (Asp, Asn, and Thr), and P9 (Asp and Glu). To compare these preferences to the other SE+ HLA-DR4 allomorphs examined in this study, we generated a heat map of difference matrices to highlight differences in their naturally selected ligands (Fig. 1B). These heat maps show the difference in amino acid usage at each position of the core 9-mer of peptides bound to HLA-DRB1*04:05 compared with HLA-DRB1*04:01 or HLA-DRB1*04:04. Of note, HLA-DRB1*04:05 selected peptides with similar P1 usage to HLA-DR*04:01 but a quite different selection of peptides with acidic P9 residues. In contrast, P1 and P9 differences were observed when HLA-DR*04:05-bound peptides were compared with HLA-DR*04:04-bound peptides, with an increase in aromatic amino acid selection at P1 and increased acidic residue selection at P9. Given the peptide preferences for the given HLA-DR4 allomorphs identified by mass spectrometry analyses, we next aimed to determine the propensity of self-antigens implicated in RA to bind to HLA-DRB1 molecules possessing the SE motif. To establish this, we modified and undertook a fluorescence polarization assay across 34 peptides (Table 1 and Fig. 2) to measure the IC50 of each peptide bound to HLA-SE+ allomorphs, namely HLA-DRB1*04:01, HLA-DRB1*04:04, and HLA-DRB1*04:05. The HLA-DRB1*04:04 molecule varies from HLA-DRB1*04:01 at two polymorphic positions, K71R and G86V, whereas HLA-DRB1*04:05 also differs from HLA-DRB1*04:01 at two positions, namely K71R and D57S within the antigen-binding cleft (Fig. 3A). The predicted core (P1–P9) registers for the HLA–peptide interaction are underlined in Table 1, generally with a hydrophobic P1-anchor residue and a P4-Arg or P4-Cit. Arginine residues located outside the P4 pocket were also modified to citrulline and assayed for binding to the HLA-DRB1 allomorphs. The 34 peptides are predicted to place the citrulline in the P4 pocket, with the exception of the following: aggrecan-93Cit(89–103) and collagen type II-1240Cit(1237–1249) with a P2-Cit; histone-2B-73,80Cit(68–82) with a P3-Cit; and CILP-305Cit(297–309) with a P7-Cit. Four peptides possessed more than one citrullinated residue, namely aggrecan-93,95Cit(89–103), CILP-988,991,994Cit(983–995), vimentin-64,69,71Cit(59–71), and fibrinogen β-72,74Cit(69–81).Table 1IC50 values in (μm) candidate RA associated autoantigens bound to HLA-SE alleles: DRB1*04:01, DRB1*04:04 and DRB1*04:05 Open table in a new tab Figure 2Titration curve of self-peptide binding reflected by displacement of reporter fluorescent peptide in HLA-DRB1*04:01 (A), HLA-DRB1*04:04 (B), and HLA-DRB1:04:05 (C). Each data point represents normalized relative binding (in percentage) from three independent experiments. Mean values are plotted, and error bar showed SE Nat, native peptide without citrulline; Cit, citrullinated peptide. Curve fit for each set of peptides for all three allomorphs HLA-DRB1*04:01, HLA-DRB1*04:04, and HLA-DRB1:04:05 are displayed in Table S2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2Titration curve of self-peptide binding reflected by displacement of reporter fluorescent peptide in HLA-DRB1*04:01 (A), HLA-DRB1*04:04 (B), and HLA-DRB1:04:05 (C). Each data point represents normalized relative binding (in percentage) from three independent experiments. Mean values are plotted, and error bar showed SE Nat, native peptide without citrulline; Cit, citrullinated peptide. Curve fit for each set of peptides for all three allomorphs HLA-DRB1*04:01, HLA-DRB1*04:04, and HLA-DRB1:04:05 are displayed in Table S2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2Titration curve of self-peptide binding reflected by displacement of reporter fluorescent peptide in HLA-DRB1*04:01 (A), HLA-DRB1*04:04 (B), and HLA-DRB1:04:05 (C). Each data point represents normalized relative binding (in percentage) from three independent experiments. Mean values are plotted, and error bar showed SE Nat, native peptide without citrulline; Cit, citrullinated peptide. Curve fit for each set of peptides for all three allomorphs HLA-DRB1*04:01, HLA-DRB1*04:04, and HLA-DRB1:04:05 are displayed in Table S2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Polymorphic residues in the peptide-binding cleft of HLA-SE molecules. Polymorphic residues in the P1, P4, and the P9 pocket of HLA-DRB1*04:01, DRB1*04:04, DRB1*04:05, and the shared-epitope motif are shown (A). The G86V variation on the DRβ chain of HLA-DRB1*04:04 may obstruct binding of hydrophobic residues such as Tyr in the P1 pocket (B). The P4-Cit forms a conserved hydrogen bond with the Lys-71β/Arg-71β, a polymorphic residue in the P4 pocket between HLA-DRB1*04:01 and DRB1*04:04/*04:05 (C). The P9 pocket of HLA-DRB1*04:01 and DRB1*04:04 consists of a Asp-57β that shapes the P9 pocket of these two alleles by forming a salt bridge with the conserved Arg-76α (D). The D57S variation on the DRβ chain of HLA-DRB1*04:05 forms extensive hydrogen bonds with P9-Asp in the peptide (E). The P9 pocket of HLA-DRB1*04:05 with a Ser-57β that has a shorter side chain has been shown to have a preference for negatively charged residue in the P9 position of peptides.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Peptides with relative IC50 >250 μm and <5 μm were considered non-binding and moderate binders to the HLA allomorphs, respectively. Peptides with relative IC50 <1 μm (Table 1) for the HLA allomorphs were considered to have high affinity. The fluorescence polarization values were converted to percentage of binding using the values for fully bound (∼150–250 mP) and free fluorescence (∼50 mP). Our aim was to determine the following: (a) whether the enhanced capacity of HLA-SE allomorphs to present self-antigens is restricted to P4-Cit or whether additional positions influence HLA bindin" @default.
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• W2783165679 date "2018-03-01" @default.
• W2783165679 modified "2023-10-01" @default.
• W2783165679 title "The interplay between citrullination and HLA-DRB1 polymorphism in shaping peptide binding hierarchies in rheumatoid arthritis" @default.
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• W2783165679 doi "https://doi.org/10.1074/jbc.ra117.001013" @default.
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