Species-specific Differences in Proteasomal Processing and Tapasin-mediated Loading Influence Peptide Presentation by HLA-B27 in Murine Cells
2003; Elsevier BV; Volume: 278; Issue: 47 Linguagem: Inglês
10.1074/jbc.m308816200
ISSN1083-351X
AutoresLaura Sesma, Iñaki Álvarez, Miguel Marcilla, Alberto Paradela, José A. Łópez de Castro,
Tópico(s)Drug-Induced Adverse Reactions
ResumoExpression of HLA-B27 in murine cells has been used to establish animal models for human spondyloarthritis and for antigen presentation studies, but the effects of xenogeneic HLA-B27 expression on peptide presentation are little known. The issue was addressed in this study. HLA-B27-bound peptide repertoires from human and murine cells overlapped by 75–85%, indicating that many endogenous HLA-B27 ligands are generated and presented in both species. Of 20 differentially presented peptides that were sequenced, only 40% arose from obvious inter-species protein polymorphism, suggesting that differences in antigen processing-loading accounted for many species-specific ligands. Digestion of synthetic substrates with human and murine 20 S proteasomes revealed cleavage differences that accounted for or correlated with differential expression of particular peptides. One HLA-B27 ligand found only in human cells was similarly generated in vitro by human and murine proteasomes. Differential presentation correlated with significantly decreased amounts of this ligand in human tapasin-deficient cells reconstituted with murine tapasin, indicating that species-specific interactions between HLA-B27, tapasin, and/or other proteins in the peptide-loading complex influenced presentation of this peptide. Our results indicate that differences in proteasomal specificity and in interactions involving tapasin determine differential processing and presentation of a significant number of HLA-B27 ligands in human and murine cells. Expression of HLA-B27 in murine cells has been used to establish animal models for human spondyloarthritis and for antigen presentation studies, but the effects of xenogeneic HLA-B27 expression on peptide presentation are little known. The issue was addressed in this study. HLA-B27-bound peptide repertoires from human and murine cells overlapped by 75–85%, indicating that many endogenous HLA-B27 ligands are generated and presented in both species. Of 20 differentially presented peptides that were sequenced, only 40% arose from obvious inter-species protein polymorphism, suggesting that differences in antigen processing-loading accounted for many species-specific ligands. Digestion of synthetic substrates with human and murine 20 S proteasomes revealed cleavage differences that accounted for or correlated with differential expression of particular peptides. One HLA-B27 ligand found only in human cells was similarly generated in vitro by human and murine proteasomes. Differential presentation correlated with significantly decreased amounts of this ligand in human tapasin-deficient cells reconstituted with murine tapasin, indicating that species-specific interactions between HLA-B27, tapasin, and/or other proteins in the peptide-loading complex influenced presentation of this peptide. Our results indicate that differences in proteasomal specificity and in interactions involving tapasin determine differential processing and presentation of a significant number of HLA-B27 ligands in human and murine cells. The xenogeneic expression of HLA-B27, an MHC 1The abbreviations used are: MHCmajor histocompatibility complexTAPtransporter associated with antigen processingmAbmonoclonal antibodyPBSphosphate-buffered salineHPLChigh-performance liquid chromatographyMSmass spectrometryMALDI-TOFmatrix-assisted laser desorption ionization time-of-flightPSDpostsource decayIPGimmobilized pH gradientCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidIEFisoelectric focusing. class I molecule, strongly associated with spondyloarthritis (1Brewerton D.A. Hart F.D. Nicholls A. Caffrey M. James D.C. Sturrock R.D. Lancet. 1973; 1: 904-907Abstract PubMed Scopus (1451) Google Scholar, 2Brewerton D.A. Caffrey M. Nicholls A. Walters D. Oates J.K. James D.C. Lancet. 1973; 2: 996-998Abstract Scopus (264) Google Scholar), has been used to establish transgenic animal disease models and to study the antigen-presenting and other properties of this molecule. HLA-B27 transgenic rats develop a spontaneous disease with many similarities to human spondyloarthropathy (3Hammer R.E. Maika S.D. Richardson J.A. Tang J.P. Taurog J.D. Cell. 1990; 63: 1099-1112Abstract Full Text PDF PubMed Scopus (828) Google Scholar). Disease manifestations are dependent on the genetic background and transgene copy number (4Taurog J.D. Maika S.D. Simmons W.A. Breban M. Hammer R.E. J. Immunol. 1993; 150: 4168-4178PubMed Google Scholar) and are modulated by alterations of the HLA-B27-bound peptide repertoire (5Zhou M. Sayad A. Simmons W.A. Jones R.C. Maika S.D. Satumtira N. Dorris M.L. Gaskell S.J. Bordoli R.S. Sartor R.B. Slaughter C.A. Richardson J.A. Hammer R.E. Taurog J.D. J. Exp. Med. 1998; 188: 877-886Crossref PubMed Scopus (48) Google Scholar). Transgenic mice have also been used as a possible model for human HLA-B27-associated disease. Development of spontaneous inflammatory arthritis in HLA-B27 transgenic mice lacking β2m (6Khare S.D. Luthra H.S. David C.S. J. Exp. Med. 1995; 182: 1153-1158Crossref PubMed Scopus (205) Google Scholar) may be related to absence of this polypeptide rather than to presence of the HLA-B27 heavy chain (7Kingsbury D.J. Mear J.P. Witte D.P. Taurog J.D. Roopenian D.C. Colbert R.A. Arthritis Rheum. 2000; 43: 2290-2296Crossref PubMed Scopus (50) Google Scholar). In contrast, HLA-B27 transgenic mice expressing β2m are being used in reactive arthritis studies (8Kuon W. Holzhutter H.G. Appel H. Grolms M. Kollnberger S. Traeder A. Henklein P. Weiss E. Thiel A. Lauster R. Bowness P. Radbruch A. Kloetzel P.M. Sieper J. J. Immunol. 2001; 167: 4738-4746Crossref PubMed Scopus (102) Google Scholar, 9Kuon W. Lauster R. Bottcher U. Koroknay A. Ulbrecht M. Hartmann M. Grolms M. Ugrinovic S. Braun J. Weiss E.H. 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Immunol. 1998; 28: 625-635Crossref PubMed Scopus (23) Google Scholar, 15Tishon A. LaFace D.M. Lewicki H. van Binnendijk R.S. Osterhaus A. Oldstone M.B. Virology. 2000; 275: 286-293Crossref PubMed Scopus (16) Google Scholar, 16Cheuk E. D'Souza C. Hu N. Liu Y. Lang H. Chamberlain J.W. J. Immunol. 2002; 169: 5571-5580Crossref PubMed Scopus (27) Google Scholar). major histocompatibility complex transporter associated with antigen processing monoclonal antibody phosphate-buffered saline high-performance liquid chromatography mass spectrometry matrix-assisted laser desorption ionization time-of-flight postsource decay immobilized pH gradient 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid isoelectric focusing. However, antigen presentation by HLA-B27 or any other HLA class I molecule expressed on murine cells implies some inherent differences, relative to human cells, that have not been sufficiently characterized at a molecular level. Thus, species-related differences in the proteome, in proteasome cleavage specificity, in the peptide specificity of the transporter associated with antigen processing (TAP), and in the interaction of the human class I molecule with human or murine tapasin or other proteins in the peptide-loading complex, all might influence the HLA-B27-bound peptide repertoire and antigen presentation upon expression on murine cells. Numerous studies have addressed the peptide-transporting preferences of human and murine TAP (reviewed in Refs. 17Schmitt L. Tampe R. Chembiochemistry. 2000; 1: 16-35Crossref PubMed Google Scholar and 18Lankat-Buttgereit B. Tampe R. Physiol. Rev. 2002; 82: 187-204Crossref PubMed Scopus (155) Google Scholar), but the actual influence of species-related differences in this and other steps of the processing-loading pathway on HLA class I-mediated antigen presentation in murine cells is little known. Knowledge of such differences is critical for assessing the validity of HLA class I transgenic models for antigen presentation and human disease. In this study, we have comparatively analyzed the HLA-B27-bound peptide repertoires expressed on human and murine cells and have characterized the origin of the differential expression of multiple HLA-B27 ligands in only one cell type. The results indicate a substantial lack of overlap between both peptide repertoires, which is only partially explained by species- or cell type-related protein differences. Both proteasome specificity differences and heterologous interactions in the peptide-loading complex contribute to differential peptide presentation by HLA-B27 on either human or murine cells. Our results have general implications for human MHC class I-mediated antigen presentation in murine systems. Cell Lines and Monoclonal Antibodies—HMy2.C1R (C1R) is a human lymphoid cell line with low expression of its endogenous class I antigens (19Storkus W.J. Howell D.N. Salter R.D. Dawson J.R. Cresswell P. J. Immunol. 1987; 138: 1657-1659PubMed Google Scholar, 20Zemmour J. Little A.M. Schendel D.J. Parham P. J. Immunol. 1992; 148: 1941-1948PubMed Google Scholar). B*2705-C1R transfectant cells were described elsewhere (21Calvo V. Rojo S. Lopez D. Galocha B. Lopez de Castro J.A. J. Immunol. 1990; 144: 4038-4045PubMed Google Scholar). P815-HTR (P815) is a murine mastocytome cell line. B*2705-P815 transfectant cells were previously described (22Rojo S. Lopez D. Calvo V. Lopez de Castro J.A. J. Immunol. 1991; 146: 634-642PubMed Google Scholar). Both the human and murine transfectants express high and similar HLA-B27 levels (23Galocha B. Lopez D. Lopez de Castro J.A. J. Immunol. 1993; 150: 1653-1662PubMed Google Scholar). These were periodically checked by flow cytometry to ensure stable expression of this molecule. The C1R and P815 cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 7.5% fetal bovine serum (both from Invitrogen, Paisley, UK). 721.220 is a human lymphoblastoid cell line (a gift from Dr. James McCluskey, University of Melbourne, Australia) in which HLA-A and HLA-B genes have been deleted and a non-functional tapasin protein is expressed (24Greenwood R. Shimizu Y. Sekhon G.S. DeMars R. J. Immunol. 1994; 153: 5525-5536PubMed Google Scholar, 25Copeman J. Bangia N. Cross J.C. Cresswell P. Eur. J. Immunol. 1998; 28: 3783-3791Crossref PubMed Scopus (45) Google Scholar). This cell line expresses low levels of endogenous HLA-Cw*0102. Transfections of HLA-B*2705 and wild type human or murine tapasin into 721.220 have been previously described (26Peh C.A. Burrows S.R. Barnden M. Khanna R. Cresswell P. Moss D.J. McCluskey J. Immunity. 1998; 8: 531-542Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). These cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. The mAb used in this study were W6/32 (IgG2a, specific for a monomorphic HLA-A, -B, and -C determinant) (27Barnstable C.J. Bodmer W.F. Brown G. Galfre G. Milstein C. Williams A.F. Ziegler A. Cell. 1978; 14: 9-20Abstract Full Text PDF PubMed Scopus (1598) Google Scholar) and ME1 (IgG1, specific for HLA-B27, -B7, and -B22) (28Ellis S.A. Taylor C. McMichael A. Hum. Immunol. 1982; 5: 49-59Crossref PubMed Scopus (201) Google Scholar). Flow Cytometry—About 6 × 104 cells were washed twice in 200 μl of PBS and resuspended in 50 μl of undiluted mAb supernatant. After incubating 30 min, cells were washed twice in 200 μl of PBS and resuspended in 50 μl of fluorescein isothiocyanate-conjugated anti-mouse IgG rabbit antiserum (Calbiochem-Novabiochem GmbH, Schwalbach, Germany), incubated for 30 min, and washed two times in 200 μl of PBS. All operations were done at 4 °C. Flow cytometry was carried out on a BD Biosciences FACSCalibur instrument using CellQuest software. Isolation of B*2705-bound Peptides—This was carried out from 1010 C1R or P815 transfectant cells lysed in 1% Nonidet P-40 in the presence of a mixture of protease inhibitors, after immunopurification of HLA-B27 with the W6/32 mAb and acid extraction, exactly as described elsewhere (29Paradela A. Garcia-Peydro M. Vazquez J. Rognan D. Lopez de Castro J.A. J. Immunol. 1998; 161: 5481-5490PubMed Google Scholar). HLA-B27-bound peptide pools were fractionated by HPLC at a flow rate of 100 μl/min as previously described (30Paradela A. Alvarez I. Garcia-Peydro M. Sesma L. Ramos M. Vazquez J. Lopez de Castro J.A. J. Immunol. 2000; 164: 329-337Crossref PubMed Scopus (37) Google Scholar), and 50-μl fractions were collected. Mass Spectrometry Analysis and Sequencing—The peptide composition of HPLC fractions was analyzed by matrix-assisted desorption ionization time-of-flight (MALDI-TOF) MS using a calibrated Kompact Probe instrument (Kratos-Shimadzu) operating in the positive linear mode, as previously described (30Paradela A. Alvarez I. Garcia-Peydro M. Sesma L. Ramos M. Vazquez J. Lopez de Castro J.A. J. Immunol. 2000; 164: 329-337Crossref PubMed Scopus (37) Google Scholar). Alternatively, a Bruker Reflex™ III MALDI-TOF mass spectrometer (Bruker-Franzen Analytic GmbH, Bremen, Germany) equipped with the SCOUT™ source in positive ion reflector mode was also used, as described elsewhere (31Alvarez I. Sesma L. Marcilla M. Ramos M. Martí M. Camafeita E. Lopez de Castro J.A. J. Biol. Chem. 2001; 276: 32729-32737Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Peptide sequencing was carried out by quadrupole ion trap nanoelectrospray MS/MS in an LCQ instrument (Finnigan ThermoQuest, San Jose, CA), as previously described (32Yague J. Vazquez J. Lopez de Castro J.A. Tissue Antigens. 1998; 52: 416-421Crossref PubMed Scopus (21) Google Scholar, 33Marina A. Garcia M.A. Albar J.P. Yague J. Lopez de Castro J.A. Vazquez J. J. Mass Spectrom. 1999; 34: 17-27Crossref PubMed Scopus (58) Google Scholar). In a few cases microelectrospray MS/MS was used, using the same procedure, except that samples were injected, through an HPLC equipped with a C18 capillary column (150 × 0.18 mm) connected online, at a flow rate of 1.5 μl/min. In some cases, peptide sequencing was also done by post-source decay (PSD)-MALDI-TOF MS, as previously described (31Alvarez I. Sesma L. Marcilla M. Ramos M. Martí M. Camafeita E. Lopez de Castro J.A. J. Biol. Chem. 2001; 276: 32729-32737Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). In all cases peptide-containing HPLC fractions were dried and resuspended in 5 μl of methanol/water (1:1) containing 0.1% formic acid. Aliquots of 0.5 or 1 μl were used for MALDI-TOF or nanoelectrospray MS analyses, respectively. For microelectrospray MS/MS-dried samples were resuspended in 0.5% acetic acid. Synthetic Peptides—Peptides were synthesized using the standard solid-phase Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry and were purified by HPLC. The correct composition and molecular mass of purified peptides were confirmed by amino acid analysis using a 6300 Amino Acid Analyzer (Beckman Coulter, Palo Alto, CA), which also allowed their quantification, and MALDI-TOF MS, respectively. Purification of 20 S Proteasome—The 20 S proteasome was purified from 3 × 109 B*2705-C1R or B*2705-P815 cell lysates by ion-exchange chromatography and centrifugation in a glycerol gradient as previously described (30Paradela A. Alvarez I. Garcia-Peydro M. Sesma L. Ramos M. Vazquez J. Lopez de Castro J.A. J. Immunol. 2000; 164: 329-337Crossref PubMed Scopus (37) Google Scholar) with the following modifications. Proteasome-containing fractions from the previous purification step were identified by 12% SDS-PAGE and further subjected to anion-exchange chromatography by fast protein liquid chromatography using a Mono-Q SR5/5 column (Amersham Biosciences, Uppsala, Sweden), at a flow rate of 1 ml/min, as follows: isocratic conditions with buffer A (50 mm Tris/HCl, 50 mm KCl, pH 8) for 1 h, followed by a linear gradient of 0–100% buffer B (50 mm Tris/HCl, 0.5 m KCl, pH 8) for 1 h. Purity of the fractions was assessed by SDS-PAGE. Aliquots of purified proteasome were stored at –80 °C. Absence of contaminant proteases in the 20 S proteasome samples was assessed by inhibition of proteolytic cleavage of a synthetic peptide substrate, histone 2A-(77–105), with the irreversible proteasome inhibitors lactacystine (50 μg/ml) and epoxomicin (1 μg/ml). Two-dimensional Gel Electrophoresis of 20 S Proteasomes—Samples of purified 20 S proteasomes were loaded by hydration of immobilized pH gradient (IPG) strips, non-linear pH 3–10, of 18-cm length (Amersham Biosciences), previously diluted to a total volume of 350 μl in 6 m urea, 2 m thiourea, 2% CHAPS, IPG non-linear pH 3–10, 1 mm Tris-(2-carboxymethyl)phosphine-HCl, and bromphenol blue. In the first dimension, IEF was performed in a IPGphor (Amersham Biosciences) under the following conditions: 30 V for 6 h, 60 V for 6 h, 500 V for 30 min, 1,000 V for 30 min, a gradient of 1,000–8,000 V for 30 min, and 8,000 V up to 32,000 Vh. After IEF, strips were equilibrated in 6 m urea, 30% glycerol, 2% SDS, and bromphenol blue, twice for 20 min. Dithiothreitol (2%) and 4% iodoacetamide were added in the first and second equilibration steps, respectively. The second dimension was performed using 12.5% SDS-PAGE. Gels were stained with silver nitrate and analyzed using the software ImageMaster (Amersham Biosciences). Spots were assigned by tryptic digestion followed by MS fingerprinting. Digestion of Synthetic Substrates—Peptide substrates (125 μg/ml) were incubated at 37 °C with purified 20 S proteasome at an enzyme/substrate ratio of 1:10 (w/w) in 20 mm Hepes buffer, pH 7.6. Digestion was stopped by adding 1/5 volume of 0.4% aqueous trifluoroacetic acid. Digestion mixtures were dried down to 100 μl in a Speed-Vac and fractionated by HPLC using the same conditions as for HLA-B27-bound peptides. Individual digestion products were identified on the basis of their molecular mass by MALDI-TOF MS and, when necessary for unambiguous assignment, by PSD-MALDI-TOF or electrospray MS/MS sequencing. HLA-B27 Presents Distinct Peptide Repertoires on Human and Murine Cells—Peptide pools were isolated by acid extraction from HLA-B*2705 immunopurified from C1R and P815 transfectant cells and fractionated by HPLC under identical conditions and consecutive runs. The peptide composition of correlative HPLC fractions from both peptide pools were systematically compared by MALDI-TOF MS, using the same strategy as previously used to compare HLA-B27 subtype-bound peptide repertoires (34Sesma L. Montserrat V. Lamas J.R. Marina A. Vazquez J. Lopez de Castro J.A. J. Biol. Chem. 2002; 277: 16744-16749Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 35Ramos M. Paradela A. Vazquez M. Marina A. Vazquez J. Lopez de Castro J.A. J. Biol. Chem. 2002; 277: 28749-28756Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). In short, the MS spectrum of any given HPLC fraction from one peptide pool was compared with the MS spectra of the correlative, previous, and following HPLC fractions from the other peptide pool. This was done to account for slight shifts in retention time that may occur between consecutive chromatographic runs. Ion peaks of the same (±1) mass-to-charge (m/z) ratio and retention time were considered to reflect shared peptides on human and murine cells. Identity of retention time and m/z does not necessarily indicate peptide identity, because in very complex mixtures unrelated peptides sharing these features might eventually co-elute. However, it is reasonable to assume that the over-whelming majority of identical peptide masses compared correspond to identical peptides. Indeed, in four of four cases in which ion peaks of the same m/z and retention time were sequenced from both the human and murine peptide pools they corresponded to identical peptides (Fig. 1). In nine additional cases a peptide sequenced from the murine peptide pool showed an identical counterpart in the human pool known from previous sequencing studies of HLA-B27-bound peptides from C1R cells to be the same peptide (Fig. 1). Ion peaks found in only one cell type in two independent experiments were considered as differentially expressed peptides. A total of 1372 and 1551 molecular species were compared from C1R and P815, respectively (Table I). Of these, 211 (15%) and 390 (25%) peptides were found only in human or in murine cells, respectively. In addition, of 351 shared peptides that showed particularly strong intensity signals in the MALDI-TOF spectra of at least one cell line, 54 (15%) and 82 (23%) showed 10-fold or higher intensity in the human or murine cells in two independent experiments, respectively. This is consistent with substantially higher expression of the shared ligand in the corresponding cell line. No significant differences in the average size of peptides expressed on either cell type were found (Table I).Table IComparison of HLA-B*2705-bound peptides from human and murine cellsC1R-B*2705P815-B*2705Total peptides compared13721551Mass range860-1667 Da860-1667 DaAverage mass1154 Da1154 DaShared peptides1161 (85%)1161 (75%)Specific peptides211 (15%)390 (25%)Average mass of shared peptides1144 Da1144 DaAverage mass of specific peptides1205 Da1193 DaMajor peaks countedaIon peaks that showed particularly strong intensity in the MALDI-TOF spectrum from one or both cell lines.351351Quantitative differencesbIon peaks that showed 10-fold or more intensity in the MALDI-TOF spectrum from one cell line.54 (15%)82 (23%)a Ion peaks that showed particularly strong intensity in the MALDI-TOF spectrum from one or both cell lines.b Ion peaks that showed 10-fold or more intensity in the MALDI-TOF spectrum from one cell line. Open table in a new tab The reproducibility of the MALDI-TOF spectra was assessed in two ways: by obtaining independent spectra from the same sample and by performing two independent comparisons with different peptide preparations. In both cases, the MS spectra of the same, or equivalent, HPLC fraction were in general very reproducible both in the nature of the ion peaks detected and in their relative intensities, although occasionally some variation was found. For this reason, assignment of both qualitative and quantitative differences was always done on the basis of reproducibility in two independent experiments. These results indicate that the HLA-B27-bound peptide repertoires on human and murine cells, although highly overlapping, contain a significant number of differentially bound ligands, as well as shared ones presented at substantially different amounts Murine TAP Does Not Impair Presentation of B*2705 Ligands with C-terminal Basic Residues—A total of 27 shared ligands, including 3 octamers, 13 nonamers, 7 decamers, 3 undecamers, and 1 dodecamer, were sequenced by MS (Fig. 1). In addition, the sequence of 20 B*2705 ligands found only in human (9 peptides) or murine cells (11 peptides) was also determined (Fig. 2). All shared peptides corresponded to conserved sequences between both species. All the peptides sequenced contained the canonic anchor motif of HLA-B27, Arg2. Shared ligands also presented the same variety of C-terminal peptide residues previously defined for HLA-B*2705 ligands from human cells, including aliphatic, aromatic, and basic residues. The number of shared ligands with C-terminal basic residues was 6 of 27. Among differential peptides (Fig. 2), 5 of 11 found only in C1R cells and 2 of 11 found only in P815 cells showed a C-terminal basic motif. Thus, of a total of 36 peptides from human cells and 38 peptides from murine cells, 11 (31%) and 8 (21%), respectively, contained C-terminal basic residues. The percentage from human cells is nearly the same as that (32%) previously reported in an independent compilation (36Lamas J.R. Paradela A. Roncal F. Lopez de Castro J.A. Arthritis Rheum. 1999; 42: 1975-1985Crossref PubMed Scopus (35) Google Scholar) and is only moderately higher than the percentage of B*2705 ligands with C-terminal basic residues found in murine cells. These results strongly suggest that the low preference of murine TAP for C-terminal basic residues reported from in vitro transport studies (37Momburg F. Roelse J. Howard J.C. Butcher G.W. Hammerling G.J. Neefjes J.J. Nature. 1994; 367: 648-651Crossref PubMed Scopus (304) Google Scholar) introduces little bias against presentation of peptides with these motifs by HLA-B27 on murine cells. Differential Presentation of HLA-B27 Ligands Is Only Partially Due to Protein Polymorphism—The 20 B*2705 ligands found only in either human or murine cells that were sequenced could be classified in three subsets (Fig. 2). Group 1, which included two peptides (10%), consisted of ligands arising from species- or cell type-specific proteins. Group 2, which included six peptides (30%), consisted of ligands arising from proteins present on both cell types but differing in one or more residues within the sequence of the peptide. Group 3, which included 12 peptides (60%) consisted of ligands arising from proteins that are either identical in both cell types (i.e. the HLA-B27 and human β2m transgene products) or identical in the region corresponding to the peptide ligand and its neighborhood. Thus, less than half of the species- or cell type-related differences in the HLA-B27-bound peptide repertoire are explained by obvious differences in the parental proteins (groups 1 and 2). These results imply that differential processing, transport, and/or loading have a significant influence on differential peptide presentation by HLA-B27 on human or murine cells. Distinct Proteasomal Cleavage Contributes to Differential Presentation of HLA-B27 Ligands—To assess the contribution of proteasomal processing to differential expression of particular HLA-B27 ligands in one cell type, we used synthetic precursors of four differentially expressed ligands from group 3 (Fig. 2), including two peptides found only in C1R cells and two others found only in P815 cells, with the sequence of the parental proteins in and around the sequence of the ligand. Each of these substrates was digested in vitro, in parallel experiments, with 20 S proteasomes purified from C1R and from P815 cells. Two-dimensional gel electrophoresis analysis indicated that both the human and murine proteasome preparations contained a mixture of proteasome and immunoproteasome. The proteasome/immunoproteasome ratio was similar in both cases, within the limits of the technique used, as judged from the relative intensities of the spots corresponding to β1/β1i and β2/β2i subunits (the murine β5 subunit did not appear in the two-dimensional gel due to its very basic pI) in the human and murine samples (Fig. 3). This technique does not allow us to rule out putative small differences in proteasome composition between cell lines, due to inaccuracies inherent to silver staining, whose intensity is variable for different proteins. However, given the similarity in proteasome/immunoproteasome composition between both cell lines, it is very unlikely that the cleavage differences observed (described below: Figs. 4,5,6,7), in particular differential cleavage of specific peptide bonds by the 20 S proteasome of only one cell line, can be attributed to cell-dependent variation in proteasome composition.Fig. 4Digestion pattern of the histone 2A-(77–105) synthetic substrate by purified 20 S proteasome from C1R (A) or P815 (B) cells. The sequence of the HLA-B27 ligand is shaded. Thick, medium, and thin lines indicate peptide products recovered with >5%, 1–5%, and 0.1% yield are indicated. The IRNDEELNK peptide is indicated by a horizontal arrow. Thick, medium, and thin vertical arrows indicate cleavage sites that generated peptides with total yields >10%, 1–10%, and <1% of the total digest, respectively. C, cleavage at individual peptide bonds of the histone 2A-(77–105) substrate by 20 S proteasomes from C1R or P815 cells; figures are cleavage yields estimated as the total yield of peptides whose N-terminal or C-terminal ends corresponded to that peptide bond. The results obtained in two independent digestion experiments using the same preparations of the human or murine proteasome are shown in each column. Peptide bonds cleaved only with the proteasome of one species, or cleaved by both proteasomes with a 10-fold or larger difference, are in boldface.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5Digestion pattern of the fatty acid synthase (1689–1718) synthetic substrate by purified 20 S proteasome from C1R (A) or P815 (B) cells. Both substrates differ by the S1712D change, corresponding to the polymorphism of the human and murine proteins at this position. C, cleavage at individual peptide bonds of the fatty acid synthase (1689–1718) substrate by 20 S proteasomes from C1R or P815 cells. Conventions are as in Fig. 4.View Large Image
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