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• W2046005361 abstract "Cys-67 of HLA-B27 is located in the B pocket, which determines peptide-binding specificity. We analyzed effects of the Cys-67 → Ser mutation on cell surface expression, peptide specificity, and T-cell recognition of HLA-B*2705. Surface expression was assessed with antibodies recognizing either native or unfolded HLA proteins. Whereas native B*2705 molecules predominated over unfolded ones, this ratio was reversed in the mutant, suggesting lower stability. Comparison of B*2705- and Cys-67 → Ser-bound peptides revealed that the mutant failed to bind ∼15% of the B*2705 ligands, while binding as many novel ones. Two peptides with Gln-2 found in both B*2705 and Cys-67 → Ser are the first demonstration of natural B*2705 ligands lacking Arg-2. Other effects of the mutation on peptide specificity were: 1) average molecular mass of natural ligands higher than for B*2705, 2) bias against small residues at peptide position (P) 1, and 3) increased P2 permissiveness. The results suggest that the Cys-67 → Ser mutation weakens B pocket interactions, leading to decreased stability of the mutant-peptide complexes. This may be partially compensated by interactions involving bulky P1 residues. The effect of the mutation on allorecognition was consistent with that on peptide specificity. Our results may aid understanding of the pathogenetic role of HLA-B27 in spondyloarthropathy. Cys-67 of HLA-B27 is located in the B pocket, which determines peptide-binding specificity. We analyzed effects of the Cys-67 → Ser mutation on cell surface expression, peptide specificity, and T-cell recognition of HLA-B*2705. Surface expression was assessed with antibodies recognizing either native or unfolded HLA proteins. Whereas native B*2705 molecules predominated over unfolded ones, this ratio was reversed in the mutant, suggesting lower stability. Comparison of B*2705- and Cys-67 → Ser-bound peptides revealed that the mutant failed to bind ∼15% of the B*2705 ligands, while binding as many novel ones. Two peptides with Gln-2 found in both B*2705 and Cys-67 → Ser are the first demonstration of natural B*2705 ligands lacking Arg-2. Other effects of the mutation on peptide specificity were: 1) average molecular mass of natural ligands higher than for B*2705, 2) bias against small residues at peptide position (P) 1, and 3) increased P2 permissiveness. The results suggest that the Cys-67 → Ser mutation weakens B pocket interactions, leading to decreased stability of the mutant-peptide complexes. This may be partially compensated by interactions involving bulky P1 residues. The effect of the mutation on allorecognition was consistent with that on peptide specificity. Our results may aid understanding of the pathogenetic role of HLA-B27 in spondyloarthropathy. ankylosing spondylitis cytotoxic T lymphocyte HMy2.C1R monoclonal antibody post-source decay matrix-assisted laser desorption/ionization time of flight mass spectrometry peptide position β2-microglobulin phosphate-buffered saline fetal bovine serum high performance liquid chromatography The molecular basis for the very strong association of HLA-B27 with ankylosing spondylitis (AS)1 (1Brewerton D.A. Hart F.D. Nicholls A. Caffrey M. James D.C. Sturrock R.D. Lancet. 1973; 1: 904-907Abstract PubMed Scopus (1447) Google Scholar), reactive arthritis (2Brewerton D.A. Caffrey M. Nicholls A. Walters D. Oates J.K. James D.C. Lancet. 1973; 2: 996-998Abstract Scopus (264) Google Scholar), and other spondyloarthropathies remains unknown. Among the pathogenetic hypotheses proposed, a classical one assumes that cytotoxic T lymphocytes (CTL) activated in response to external antigen would eventually cross-react with a self-peptide constitutively presented by HLA-B27 (3Benjamin R. Parham P. Immunol. Today. 1990; 11: 137-142Abstract Full Text PDF PubMed Scopus (350) Google Scholar). This hypothesis is supported by much indirect evidence (4Zhou 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 (47) Google Scholar, 5D'Amato M. Fiorillo M.T. Carcassi C. Mathieu A. Zuccarelli A. Bitti P.P. Tosi R. Sorrentino R. Eur. J. Immunol. 1995; 25: 3199-3201Crossref PubMed Scopus (185) Google Scholar, 6Lopez-Larrea C. Sujirachato K. Mehra N.K. Chiewsilp P. Isarangkura D. Kanga U. Dominguez O. Coto E. Peña M. Setien F. Gonzalez-Roces S. Tissue Antigens. 1995; 45: 169-176Crossref PubMed Scopus (241) Google Scholar, 7Hermann E. Yu D.T. Meyer zum Buschenfelde K.H. Fleischer B. Lancet. 1993; 342: 646-650Abstract PubMed Scopus (298) Google Scholar, 8Fiorillo M.T. Maragno M. Butler R. Dupuis M.L. Sorrentino R. J. Clin. Invest. 2000; 106: 47-53Crossref PubMed Scopus (159) Google Scholar). Alternatively, HLA-B27 heavy chain homodimers, whose formation in vitro is dependent on an unpaired Cys-67 residue (9Allen R.L. O'Callaghan C.A. McMichael A.J. Bowness P. J. Immunol. 1999; 162: 5045-5048PubMed Google Scholar), were suggested to act as noncanonical peptide presenting molecules, perhaps stimulating abnormal T-cell responses (10Allen R.L. Bowness P. McMichael A. Immunogenetics. 1999; 50: 220-227Crossref PubMed Scopus (69) Google Scholar, 11Edwards J.C. Bowness P. Archer J.R. Immunol. Today. 2000; 21: 256-260Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). It has also been proposed that HLA-B27 association to AS might be unrelated to peptide presentation, but rather to B27 heavy chain misfolding and triggering of endoplasmic reticulum stress responses (12Mear J.P. Schreiber K.L. Münz C. Zhu X. Stevanovic S. Rammensee H.G. Rowland-Jones S.L. Colbert R.A. J. Immunol. 1999; 163: 6665-6670PubMed Google Scholar, 13Colbert R.A. Mol. Med. Today. 2000; 6: 224-230Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 14Colbert R.A. J. Rheumatol. 2000; 27: 1107-1109PubMed Google Scholar). The Cys-67 residue of HLA-B27 is an integral part of its B pocket, which determines the specificity of the molecule for peptides with Arg-2 (15Madden D.R. Gorga J.C. Strominger J.L. Wiley D.C. Cell. 1992; 70: 1035-1048Abstract Full Text PDF PubMed Scopus (612) Google Scholar). This residue plays a critical role in controlling the thermodynamic stability of soluble HLA-B27-peptide complexes (16Reinelt S. Dedier S. Asuni G. Folkers G. Rognan D. J. Biol. Chem. 2001; 276: 18472-18477Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). This same study claimed that mutation of Cys-67 did not significantly affect the peptide binding properties of HLA-B27, but this conclusion was based on the affinity of a single HLA-B27 ligand and a few peptide analogs. The pathogenetic role of Cys-67 was tested on HLA-B27 transgenic rats. In this system, HLA-B27 triggers a spontaneous inflammatory disease with many features of human spondyloarthropathies (17Hammer R.E. Maika S.D. Richardson J.A. Tang J.P. Taurog J.D. Cell. 1990; 63: 1099-1112Abstract Full Text PDF PubMed Scopus (826) Google Scholar). Rats transgenic for the Cys-67 → Ser (C67S) mutant showed a disease phenotype similar to wild type B*2705 transgenic rats of the same strain, suggesting that Cys-67 has little influence on disease (18Taurog J.D. Maika S.D. Satumtira N. Dorris M.L. McLean I.L. Yanagisawa H. Sayad A. Stagg A.J. Fox G.M. Le O'Brien A. Rehman M. Zhou M. Weiner A.L. Splawski J.B. Richardson J.A. Hammer R.E. Immunol. Rev. 1999; 169: 209-223Crossref PubMed Scopus (192) Google Scholar). In this study we have addressed the role of Cys-67 on cell surface expression, peptide specificity, and T-cell recognition of HLA-B27. We demonstrate that the C67S mutation decreases the stability of HLA-B27-peptide complexes at the cell surface, induces distinct alterations in the endogenous HLA-B27-bound peptide repertoire, and influences T-cell allorecognition. 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 have been described elsewhere (21Calvo V. Rojo S. Lopez D. Galocha B. Lopez de Castro J.A. J. Immunol. 1990; 144: 4038-4045PubMed Google Scholar). A genomic construct containing the C67S mutant of the B*2705 gene cloned into pUC19 (22Taurog J.D. El-Zaatari F.A. J. Clin. Invest. 1988; 82: 987-992Crossref PubMed Scopus (30) Google Scholar, 23El-Zaatari F.A. Sams K.C. Taurog J.D. J. Immunol. 1990; 144: 1512-1517PubMed Google Scholar) was a kind gift of Dr. Joel D. Taurog (University of Texas Southwestern Medical Center, Dallas, TX). This construct was designated as JHG1.1b. Exons 1 and 2 of the mutant were resequenced in our laboratory to confirm its correct structure. To obtain a C67S-C1R transfectant cell line, approximately 107C1R cells were washed twice, and resuspended in 0.8 ml of PBS. Ten μg of linearized JHG1.1b plasmid and 1 μg of linearized pSV2.neo were co-transfected by electroporation at 960 V, 300 microfarads in a Bio-Rad Gene Pulser. After 10 min at room temperature, cells were seeded in a culture flask in Dulbecco's modified Eagle's medium supplemented with 10% FBS. Twenty-four hours after electroporation, 1 mg/ml G418 (Life Technologies, Paisley, United Kingdom) was added to the cell culture. Surviving cells were tested for expression of the C67S mutant by flow cytometry as described below. C1R cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 7.5% FBS (both from Life Technologies). RMA-S is a TAP-deficient murine cell line (24Ljunggren H.G. Karre K. J. Exp. Med. 1985; 162: 1745-1759Crossref PubMed Scopus (646) Google Scholar, 25Townsend A. Ohlen C. Bastin J. Ljunggren H.G. Foster L. Karre K. Nature. 1989; 340: 443-448Crossref PubMed Scopus (882) Google Scholar). RMA-S transfectant cells expressing B*2705 and human β2m have been described previously (26Villadangos J.A. Galocha B. Lopez de Castro J.A. J. Immunol. 1994; 152: 2317-2323PubMed Google Scholar). These cells were cultured in RPMI 1640 medium supplemented with 10% FBS. The following mAbs were used: W6/32 (IgG2a, specific for a monomorphic HLA-A,B,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 (1594) Google Scholar), ME1 (IgG1, specific for HLA-B27, B7, B22) (28Ellis S.A. Taylor C. McMichael A. Hum. Immunol. 1982; 5: 49-59Crossref PubMed Scopus (201) Google Scholar), BBM.1 (IgG2b, specific for human β2m) (29Brodsky F.M. Bodmer W.F. Parham P. Eur. J. Immunol. 1979; 9: 536-545Crossref PubMed Scopus (249) Google Scholar, 30Parham P. Androlewicz M.J. Holmes N.J. Rothenberg B.E. J. Biol. Chem. 1983; 258: 6179-6186Abstract Full Text PDF PubMed Google Scholar), and HC10 (IgG2a, specific for denatured and other forms of HLA class I heavy chain not associated to β2m) (31Stam N.J. Spits H. Ploegh H.L. J. Immunol. 1986; 137: 2299-2306PubMed Google Scholar). For flow cytometry analysis, approximately 2 × 105C1R or RMA-S transfectant cells were washed twice in 200 μl of PBS, and resuspended in 50 μl of undiluted mAb supernatant. After incubating for 20 min, cells were washed three times 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 20 min, and washed three times in 200 μl of PBS. All operations were done at 4 °C. Flow cytometry was carried out in an Epics Profile XL instrument (Coulter Electronics Inc., Hialeah, FL). This was carried from 1010 C1R 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 (32Paradela 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 described previously (33Paradela 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. 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 described previously (33Paradela 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 previously (34Alvarez I. Sesma L. Marcilla M. Ramos M. Martı́ 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), exactly as detailed elsewhere (35Yague J. Vazquez J. Lopez de Castro J.A. Tissue Antigens. 1998; 52: 416-421Crossref PubMed Scopus (21) Google Scholar, 36Marina 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 some cases, peptide sequencing was also done by post-source decay (PSD) MALDI-TOF MS, as described previously (34Alvarez I. Sesma L. Marcilla M. Ramos M. Martı́ 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. 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 model 6300 amino acid analyzer (Beckman Coulter, Palo Alto, CA), which also allowed their quantification. The epitope stabilization assay used to measure peptide binding was performed as described (37Galocha B. Lamas J.R. Villadangos J.A. Albar J.P. Lopez de Castro J.A. Tissue Antigens. 1996; 48: 509-518Crossref PubMed Scopus (37) Google Scholar). Briefly, B*2705 RMA-S transfectants were incubated at 26 °C for 22 h in RPMI 1640 medium supplemented with 10% heat-inactivated FBS. They were then washed three times in serum-free medium, incubated for 1 h at 26 °C with various peptide concentrations without FBS, transferred to 37 °C, and collected for flow cytometry after 4 h. B*2705 expression was measured using 50 μl of hybridoma culture supernatant containing the mAb ME1. Binding of the RRYQKSTEL peptide, used as positive control, was expressed as C50, which is the molar concentration of the peptide at 50% of the maximum fluorescence obtained at the concentration range used (10−4 to 10−9m). Binding of other peptides was assessed as the concentration of peptide required to obtain the fluorescence value at the C50 of the control peptide. This was designated as EC50. A set of allospecific CTL clones raised against B*2705 were used. The generation, culture conditions, and fine specificity of these CTL clones has been described previously (38Lopez D. Garcia Hoyo R. Lopez de Castro J.A. J. Immunol. 1994; 152: 5557-5571PubMed Google Scholar). Recognition of B*2705 and C67S by these CTL was analyzed using a standard 51Cr release cytotoxicity assay, essentially as described elsewhere (39Aparicio P. Jaraquemada D. Lopez de Castro J.A. J. Exp. Med. 1987; 165: 428-443Crossref PubMed Scopus (44) Google Scholar). Cell surface expression of B*2705 on C1R transfectants was measured by flow cytometry with the ME1, W6/32, BBM.1, and HC10 mAbs (Fig. 1). W6/32 and ME1 recognize distinct epitopes on the heavy chain of the native HLA-B27 molecule (3Benjamin R. Parham P. Immunol. Today. 1990; 11: 137-142Abstract Full Text PDF PubMed Scopus (350) Google Scholar,22Taurog J.D. El-Zaatari F.A. J. Clin. Invest. 1988; 82: 987-992Crossref PubMed Scopus (30) Google Scholar, 40Maziarz R.T. Fraser J. Strominger J.L. Burakoff S.J. Immunogenetics. 1986; 24: 206-208PubMed Google Scholar). BBM.1 recognizes an epitope on human β2m (30Parham P. Androlewicz M.J. Holmes N.J. Rothenberg B.E. J. Biol. Chem. 1983; 258: 6179-6186Abstract Full Text PDF PubMed Google Scholar), and HC10 recognizes an epitope on unfolded, but not native, HLA class I heavy chains (31Stam N.J. Spits H. Ploegh H.L. J. Immunol. 1986; 137: 2299-2306PubMed Google Scholar). Whereas surface expression of C67S in our transfectants was lower than expression of B*2705 when measured with W6/32, ME1, or BBM.1, it was higher when measured with HC10. This result indicates that the ratio between unfolded and native HLA-B27 molecules on the cell surface was higher for C67S than for B*2705, suggesting lower stability of the mutant molecules. A systematic comparison of B*2705- and C67S-bound peptide repertoires was carried out to determine the effect of the mutation on peptide specificity. The corresponding peptide pools were isolated by acid extraction, after immunoprecipitation of the B27 molecules with the W6/32 mAb, and fractionated by HPLC under identical conditions. The peptide composition of individual HPLC fractions was analyzed by MALDI-TOF MS. The MS spectrum of each HPLC fraction from one of the molecules was compared with the MS spectra of the correlative, previous, and following HPLC fractions from the other molecule. This was done to account for slight shifts in retention times between consecutive chromatographic runs. Ion peaks with the same (±1) mass/charge (m/z) among the HPLC fractions compared were considered to reflect identical peptides shared by both molecules. Ion peaks in one HPLC fraction not found in the counterparts from the other molecule were considered to be peptides differentially bound to one molecule. One example of such comparison is shown in Fig.2. HPLC fraction 161 from B*2705 was compared with HPLC fractions 160–162 from C67S. Of 14 ion peaks compared from the B*2705 fraction, 12 had counterparts in one or more of the HPLC fractions from C67S, and 2 were not detected in the mutant. A total of 1193 peptides from B*2705 and 1175 peptides from C67S were compared in this analysis (Table I). Of the B*2705 ligands, 1005 (84%) were shared by the C67S mutant, and 188 (16%) were not found in the mutant. Conversely, 171 peptides from C67S (15%) were not found in B*2705. These results indicate that the C67S mutation is compatible with binding of a large majority, but not all, of the natural B*2705 ligands, and influences peptide specificity also by allowing binding of new ligands absent from the B*2705-bound pool.Table IComparison of B*2705- and C67S-B27-bound peptide repertoiresB*2705 ligandsC67S ligandsTotalCommonSpecificTotalCommonSpecific11931005 (84%)188 (16%)11751005 (85%)171 (15%)Quantitative differences between B*2705- and C67S-bound peptidesTotal peptides comparedPredominant in B*2705Predominant in C67S25225 (10%)31 (12%) Open table in a new tab Although MALDI-TOF MS is not a quantitative technique because ion peak signal intensity is affected by multiple factors, an attempt was made to identify the extent in which peptide amount could be affected by the C67S mutation. Thus, to identify peptides common to both molecules but much more abundant in one of them, we selected ion peaks in each HPLC fraction whose intensity was higher than 50% of the maximum signal intensity in that fraction. Their amount was measured as the total number of millivolts (mV) corresponding to each ion peak in all the HPLC fractions in which it was detected. When the total intensity of a given ion peak was more than 10 times higher in one molecule than in the other, the corresponding peptide was assigned as a quantitative difference. One example (Fig. 2), is the ion peak atm/z 1088.1, in HPLC fraction 161 from B*2705 (SRTPYHVNL). Of 252 peptides compared by these criteria (Table I), 25 (10%) were much more prominent in B*2705, and 31 (12%) predominated in C67S. These results suggest that, in addition to determining differential binding of some peptides, the C67S mutation also influences the amount of bound peptide in at least an additional 22% of the shared ligands. The size distribution of natural ligands bound to B*2705 or C67S showed a gaussian pattern in both cases, with mean peptide sizes ([M + H]+) of 1154 and 1185 Da, respectively (Fig.3A). This subtle size difference, which was reproducible in a second independent comparison (data not shown), became more obvious when the size distribution of peptides differentially bound to either B*2705 or C67S was compared (Fig. 3B). Whereas the mean size of B*2705 peptide differences was 1089 Da, that of C67S differences was 1295 Da. Thus, peptides differentially bound to the mutant had an mean size 206 Da bigger than B*2705 differences. This is compatible with a slightly bigger length (1–2 residues), a bias toward bigger residues at some positions, or both. A total of 36 natural ligands bound to B*2705 or to the C67S mutant were sequenced by quadrupole ion trap nanoelectrospray MS/MS (Fig. 4). Of these, 29 peptides, including 20 nonamers, 8 decamers, and 1 undecamer, were shared by both molecules. Among these shared ligands, nine showed quantitative differences, six of them predominant in B*2705, and three of them predominant in C67S. In addition, six peptides, including five nonamers and one decamer, were differentially bound to B*2705, and one nonamer was differentially bound to C67S. In general there were no obvious differences between peptides common to B*2705 and C67S, and peptides found only or predominantly in B*2705, except at position (P) 1. Of the 12 peptides B*2705-specific or predominant in B*2705 (Fig. 4), 11 (92%) had a small P1 residue: Gly, Ala, or Ser. Together, these three amino acids accounted only for 25% of the other shared ligands. In contrast, whereas basic P1 residues (Arg, Lys, and His) were found in 12 (60%) of the shared ligands, and in 2 of 3 peptides predominant in the mutant, these residues were absent from peptides differentially or predominantly bound to B*2705. These results strongly suggest that the C67S mutation disfavors binding of peptides with small P1 residues, while allowing basic residues at this position. There were more peptides with Pro-4 among those differentially or predominantly bound to B*2705 (58%) than among shared ones (15%). However, the significance of this is dubious because there was Pro at positions 4, 5, or 6 in 40% of the shared ligands (Fig. 4). Two peptides with Gln-2 were sequenced from the C67S mutant, both of which had counterparts in the B*2705-bound peptide pool (Fig. 4). The MALDI-TOF MS spectra of HPLC fraction 143 from C67S and B*2705 showed an ion peak atm/z 1056.4. The ion peak from the mutant was fragmented by quadrupole ion trap nanoelectrospray MS/MS, which allowed us to determine the sequence of the corresponding peptide as RQTGIVLNR (M = 1055.6 Da) (Fig.5A). Assignment of this sequence and, in particular, distinction of Gln-2 from the isobaric Lys residue was based on: (a) match of this sequence, but not of that with Lys-2, with a human protein in the data base (Fig. 4), (b) detection of the y8* fragment ion (m/z 883.3), which is consistent with neutral loss of ammonia caused by cyclation of the N-terminal Gln residue in the y8“ ion, and (c) the MS/MS spectrum of the synthetic RQTGIVLNR peptide was identical to the peptide from the mutant (data not shown). The corresponding ion peak from B*2705 was sequenced by PSD-MALDI-TOF MS. Its spectrum (Fig.5B) was essentially identical to that of the synthetic RQTGIVLNR peptide (Fig. 5C). This is the first demonstration that B*2705 binds peptides with Gln-2 in vivo. Similarly, the MALDI-TOF MS spectra of HPLC fractions 173 from C67S and B*2705 revealed an ion peak with m/z 1023.4. The sequence of this peptide obtained from the C67S mutant was RQVIPIIGK (Fig. 4). In this case, PSD-MALDI-TOF or electrospray MS/MS spectra of the B*2705 counterpart could not be obtained, so that assignment of this peptide as a natural B*2705 ligand relies only on identity of molecular mass and retention time with the peptide from C67S. A third peptide lacking Arg-2 was found in HPLC fraction 155 from the mutant. In the MALDI-TOF MS spectrum of this fraction, there was an ion peak atm/z 1105.4, which was not detected in the corresponding HPLC fractions from B*2705 (data not shown), so that this peak was assigned as a peptide difference from the mutant. The nanoelectrospray MS/MS spectrum of this peptide was consistent with the sequence YKFSGFTQK (Fig. 6A). Distinction between the isobaric Gln/Lys residues was based on: 1) unambiguous matching with a human sequence in the data base (Fig. 4), 2) lack of a prominent fragment ion corresponding to neutral loss of ammonia from the y8“+ ory8”2+ fragment ions, which would be expected if the peptide would have Gln-2, because of cyclation of this residue when it becomes N-terminal in the y8“ ions, and 3) identity of the MS/MS spectrum with that of synthetic YKFSGFTQK (Fig. 6B), but not with the synthetic analog with Gln-2 (Fig. 6C). In this latter spectrum, prominenty8* fragment ions corresponding to neutral loss of ammonia from y8”+and y8“2+ fragment ions were a clear differential feature distinguishing this spectrum from those of the C67S ligand and the synthetic YKFSGFTQK peptide. This result demonstrates that the C67S mutation relaxes the specificity of the B pocket, allowing for residues other than Arg-2 or Gln-2. The three peptide ligands lacking Arg-2 found in the previous analyses were tested for cell surface binding to B*2705 in an epitope stabilization assay (Fig. 7). The two peptides with Gln-2 bound B*2705 with high efficiency (EC50 1 μm), and only slightly worse than an Arg-2-containing B*2705 ligand, RRYQKSTEL (EC50 0.5 μm), used as a control. In contrast, the Lys-2-containing ligand found only in the mutant bound B*2705 with moderate efficiency (EC50 36 μm), and over 30-fold less efficiently than the Gln-2-containing ligands. Because most allospecific CTL are peptide-dependent, another way to look at the effect of C67S on peptide specificity is to examine the cross-reactivity of allospecific CTL raised against B*2705 with the C67S mutant. Thus, 11 anti-B*2705 CTL clones were tested for recognition of C67S-C1R transfectant target cells in a classical cytotoxicity assay (TableII). Seven CTL clones (64%) showed significant cross-reaction (>50% relative lysis), whereas four CTL clones (36%) cross-reacted more weakly (<40% relative lysis). These results indicate that many allospecific T-cell epitopes are conserved in the mutant. However, epitope sharing was lower than the peptide sharing determined by comparison of the B*2705- and C67S-bound peptide repertoires (Table I). This result suggests that some shared ligands may bind B*2705 and the mutant in different conformations, so that the corresponding T-cell epitopes are altered.Table IIReactivity of B*2705-specific alloreactive CTL with the C67S mutantCTLE:TSpecific lysisRelative lysis2-aPercentage of lysis of C67-C1R relative to the specific lysis of B*2705-C1R cells. (C67S-C1R)C1RB*2705-C1RC67S-C1R%%102DRF1:156371112.8DM51.5:11035823139DRD1:145613234S151:1150183611SRY1:1027145233S151:1645245333S690.5:1354407458GRK1:1067416127S691:1371486820.8GRK1:15273412637GRK1:10575291Data are means of two to nine independent experiments.2-a Percentage of lysis of C67-C1R relative to the specific lysis of B*2705-C1R cells. Open table in a new tab Data are means of two to nine independent experiments. The Cys-67 residue is a structural feature of HLA-B27 shared by few other allotypes such as subtypes of HLA-B14, B15, B38, B39, and B73 (41Parham P. Adams E.J. Arnett K.L. Immunol. Rev. 1995; 143: 141-180Crossref PubMed Scopus (261) Google Scholar). However, only HLA-B27 and the rare allotype HLA-B73 (42Parham P. Arnett K.L. Adams E.J. Barber L.D. Domena J.D. Stewart D. Hildebrand W.H. Little A.M. Tissue Antigens. 1994; 43: 302-313Crossref PubMed Scopus (53) Google Scholar, 43Vilches C. de Pablo R. Herrero M.J. Moreno M.E. Kreisler M. Immunogenetics. 1994; 40 (166): 166Crossref PubMed Scopus (28) Google Scholar) also have Lys-70, which confers to Cys-67 enhanced chemical reactivity (44Whelan M.A. Archer J.R. Eur. J. Immunol. 1993; 23: 3278-3285Crossref PubMed Scopus (33) Google Scholar). Although this residue might be modified by homocysteine in vivo upon bacterial invasion (45Gao X.M. Wordsworth P. McMichael A.J. Kyaw M.M. Seifert M. Rees D. Dougan G. Eur. J. Immunol. 1996; 26: 1443-1450Crossref PubMed Scopus (36) Google Scholar), it seems that the overwhelming majority of HLA-B27 molecules in normal cells have a free Cys-67. Because this residue is an integral part of the B pocket (15Madden D.R. Gorga J.C. Strominger J.L. Wiley D.C. Cell. 1992; 70: 1035-1048Abstract Full Text PDF PubMed Scopus (612) Google Scholar), it was expected to influence peptide binding and antigen presentation. Thus, in this study we investigated the role of Cys-67 in: 1) cell surface expression and stability, 2) peptide specificity, and 3) T-cell allorecognition. Early studie" @default.
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• W2046005361 date "2001-12-01" @default.
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• W2046005361 title "The Cys-67 Residue of HLA-B27 Influences Cell Surface Stability, Peptide Specificity, and T-cell Antigen Presentation" @default.
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• W2046005361 doi "https://doi.org/10.1074/jbc.m108882200" @default.
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