Identification of Endogenously Presented Peptides from Chlamydia trachomatis with High Homology to Human Proteins and to a Natural Self-ligand of HLA-B27
2007; Elsevier BV; Volume: 7; Issue: 1 Linguagem: Inglês
10.1074/mcp.m700386-mcp200
ISSN1535-9484
AutoresJuan J. Cragnolini, José A. Łópez de Castro,
Tópico(s)Immune Response and Inflammation
ResumoA strategy for the stable expression of proteins, or large protein fragments, from Chlamydia trachomatis into human cells was designed to identify bacterial epitopes endogenously processed and presented by HLA-B27. Fusion protein constructs in which the green fluorescent protein gene was placed at the 5′-end of the bacterial DNA primase gene or some of its fragments were transfected into B*2705-C1R cells. One of these constructs, including residues 90–450 of the bacterial protein, was stably and efficiently expressed. Mass spectrometry-based comparative analysis of HLA-B27-bound peptide pools led to identification of three HLA-B27 ligands differentially presented in the transfectant cells. Sequencing of these peptides confirmed that they were derived from the bacterial DNA primase. One of them, spanning residues 211–221, showed 55% sequence identity with a known self-ligand of HLA-B27 derived from its own molecule. The other two bacterial ligands, P-(112–121) and P-(112–122), were derived from the same region and differed in length by one residue at the C terminus. Both peptides showed >50% identity with multiple human protein sequences that possessed the optimal peptide motifs for HLA-B27 binding. Thus, expression of proteins from arthritogenic bacteria in HLA-B27-positive human cells allows identifying bacterial peptides that are endogenously processed and presented by HLA-B27 and show molecular mimicry with known self-ligands of this molecule and human proteins. A strategy for the stable expression of proteins, or large protein fragments, from Chlamydia trachomatis into human cells was designed to identify bacterial epitopes endogenously processed and presented by HLA-B27. Fusion protein constructs in which the green fluorescent protein gene was placed at the 5′-end of the bacterial DNA primase gene or some of its fragments were transfected into B*2705-C1R cells. One of these constructs, including residues 90–450 of the bacterial protein, was stably and efficiently expressed. Mass spectrometry-based comparative analysis of HLA-B27-bound peptide pools led to identification of three HLA-B27 ligands differentially presented in the transfectant cells. Sequencing of these peptides confirmed that they were derived from the bacterial DNA primase. One of them, spanning residues 211–221, showed 55% sequence identity with a known self-ligand of HLA-B27 derived from its own molecule. The other two bacterial ligands, P-(112–121) and P-(112–122), were derived from the same region and differed in length by one residue at the C terminus. Both peptides showed >50% identity with multiple human protein sequences that possessed the optimal peptide motifs for HLA-B27 binding. Thus, expression of proteins from arthritogenic bacteria in HLA-B27-positive human cells allows identifying bacterial peptides that are endogenously processed and presented by HLA-B27 and show molecular mimicry with known self-ligands of this molecule and human proteins. Chlamydia trachomatis, an obligate intracellular parasite that primarily infects the urogenital epithelium, is one of the most common infectious agents in humans. Chlamydia-induced arthritis is the most frequent form of reactive arthritis (ReA) 1The abbreviations used are: ReA, reactive arthritis; CTL, cytotoxic T lymphocyte; GFP, green fluorescent protein; C1R, Hmy2.C1R; mAb, monoclonal antibody; MHC, major histocompatibility complex; Ab, antibody; P, primase. in Western countries (1Zeidler H. Kuipers J. Kohler L. Chlamydia-induced arthritis.Curr. Opin. Rheumatol. 2004; 16: 380-392Crossref PubMed Scopus (63) Google Scholar). Persistent forms of the bacteria, rather than actively growing forms, are found in chronically infected tissues and, presumably because they sustain chronic infection, are critical in the development of ReA. Persistent forms of Chlamydia are produced by differentiation of the metabolically active reticulate bodies in response to tryptophan starvation induced by interferon-γ (2Brunham R.C. Rey-Ladino J. Immunology of Chlamydia infection: implications for a Chlamydia trachomatis vaccine.Nat. Rev. Immunol. 2005; 5: 149-161Crossref PubMed Scopus (470) Google Scholar). Several studies (3Gerard H.C. Krausse-Opatz B. Wang Z. Rudy D. Rao J.P. Zeidler H. Schumacher H.R. Whittum-Hudson J.A. Kohler L. Hudson A.P. Expression of Chlamydia trachomatis genes encoding products required for DNA synthesis and cell division during active versus persistent infection.Mol. Microbiol. 2001; 41: 731-741Crossref PubMed Scopus (85) Google Scholar, 4Belland R.J. Nelson D.E. Virok D. Crane D.D. Hogan D. Sturdevant D. Beatty W.L. Caldwell H.D. Transcriptome analysis of chlamydial growth during IFN-γ-mediated persistence and reactivation.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15971-15976Crossref PubMed Scopus (200) Google Scholar) have established that proteins involved in DNA replication are expressed during persistence and that the bacterial chromosome is continuously replicated. Despite the multiple mechanisms used for C. trachomatis to evade the immune system, which include down-regulation of the major histocompatibility complex (MHC) class I and class II molecules (5Zhong G. Fan T. Liu L. Chlamydia inhibits interferon γ-inducible major histocompatibility complex class II expression by degradation of upstream stimulatory factor 1.J. Exp. Med. 1999; 189: 1931-1938Crossref PubMed Scopus (175) Google Scholar, 6Zhong G. Liu L. Fan T. Fan P. Ji H. Degradation of transcription factor RFX5 during the inhibition of both constitutive and interferon γ-inducible major histocompatibility complex class I expression in chlamydia-infected cells.J. Exp. Med. 2000; 191: 1525-1534Crossref PubMed Scopus (171) Google Scholar, 7Zhong G. Fan P. Ji H. Dong F. Huang Y. Identification of a chlamydial protease-like activity factor responsible for the degradation of host transcription factors.J. Exp. Med. 2001; 193: 935-942Crossref PubMed Scopus (324) Google Scholar) and persistence, the occurrence of CD4+ and CD8+ T cell responses is well established (8Loomis W.P. Starnbach M.N. T cell responses to Chlamydia trachomatis.Curr. Opin. Microbiol. 2002; 5: 87-91Crossref PubMed Scopus (106) Google Scholar), and HLA-B27-restricted cytotoxic T lymphocytes (CTLs) are found in patients with Chlamydia-induced ReA (9Kuon 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. Identification of HLA-B27-restricted peptides from the Chlamydia trachomatis proteome with possible relevance to HLA-B27-associated diseases.J. Immunol. 2001; 167: 4738-4746Crossref PubMed Scopus (102) Google Scholar, 10Appel H. Kuon W. Kuhne M. Wu P. Kuhlmann S. Kollnberger S. Thiel A. Bowness P. Sieper J. Use of HLA-B27 tetramers to identify low-frequency antigen-specific T cells in Chlamydia-triggered reactive arthritis.Arthritis Res. Ther. 2004; 6: R521-R534Crossref PubMed Google Scholar). The joint role of HLA-B27 and Chlamydia infection in determining susceptibility to ReA, especially in its chronic form, suggests that molecular mimicry between bacterial and self-antigens presented by HLA-B27 may provide an autoimmune pathogenetic mechanism for this disease (11Ramos M. Alvarez I. Sesma L. Logean A. Rognan D. Lopez de Castro J.A. Molecular mimicry of an HLA-B27-derived ligand of arthritis-linked subtypes with chlamydial proteins.J. Biol. Chem. 2002; 277: 37573-37581Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 12Bachmaier K. Penninger J.M. Chlamydia and antigenic mimicry.Curr. Top. Microbiol. Immunol. 2005; 296: 153-163Crossref PubMed Scopus (43) Google Scholar). This is supported by studies in rats showing that immunization with HLA-B27 rendered the animals capable to mount a Chlamydia-specific CD8+ T cell response after in vitro stimulation with this bacteria (13Popov I. Dela Cruz C.S. Barber B.H. Chiu B. Inman R.D. The effect of an anti-HLA-B27 immune response on CTL recognition of Chlamydia.J. Immunol. 2001; 167: 3375-3382Crossref PubMed Scopus (20) Google Scholar). Moreover HLA-B27 transgenic rats activated an autoreactive B27-directed CTL response upon exposure to C. trachomatis (14Popov I. Dela Cruz C.S. Barber B.H. Chiu B. Inman R.D. Breakdown of CTL tolerance to self HLA-B*2705 induced by exposure to Chlamydia trachomatis.J. Immunol. 2002; 169: 4033-4038Crossref PubMed Scopus (35) Google Scholar). Although these studies show an immunological interplay between HLA-B27 and Chlamydia, it must be noted that molecular mimicry is only one of the possible mechanisms of autoimmunity. Actually the elusive nature of the concept of antigenic mimicry at a molecular level, because the T cell receptor may cross-react against structurally disparate epitopes (15Evavold B.D. Sloan-Lancaster J. Wilson K.J. Rothbard J.B. Allen P.M. Specific T cell recognition of minimally homologous peptides: evidence for multiple endogenous ligands.Immunity. 1995; 2: 655-663Abstract Full Text PDF PubMed Scopus (202) Google Scholar, 16Wucherpfennig K.W. Strominger J.L. Molecular mimicry in T cell-mediated autoimmunity: viral peptides activate human T cell clones specific for myelin basic protein.Cell. 1995; 80: 695-705Abstract Full Text PDF PubMed Scopus (1292) Google Scholar), and the difficulty of actually establishing a causative link between molecular mimicry and autoimmunity cast doubts on the actual relevance of this mechanism (17Fourneau J.M. Bach J.M. Van Endert P.M. Bach J.F. The elusive case for a role of mimicry in autoimmune diseases.Mol. Immunol. 2004; 40: 1095-1102Crossref PubMed Scopus (43) Google Scholar). Nevertheless cross-reactivity between chlamydial peptides and homologous peptides from the heart muscle-specific protein α-myosin was shown to be involved in the pathogenesis of autoimmune myocarditis in mice (18Bachmaier K. Neu N. de la Maza L.M. Pal S. Hessel A. Penninger J.M. Chlamydia infections and heart disease linked through antigenic mimicry.Science. 1999; 283: 1335-1339Crossref PubMed Scopus (365) Google Scholar). To investigate the role of molecular mimicry in Chlamydia-induced HLA-B27-associated ReA, two experimental strategies have been undertaken. In the first one, chlamydial peptides recognized by HLA-B27-restricted CTLs from both transgenic mice and patients were identified by screening a panel of synthetic peptides with binding motifs and proteasome cleavage features compatible with presentation by HLA-B27 (9Kuon 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. Identification of HLA-B27-restricted peptides from the Chlamydia trachomatis proteome with possible relevance to HLA-B27-associated diseases.J. Immunol. 2001; 167: 4738-4746Crossref PubMed Scopus (102) Google Scholar, 10Appel H. Kuon W. Kuhne M. Wu P. Kuhlmann S. Kollnberger S. Thiel A. Bowness P. Sieper J. Use of HLA-B27 tetramers to identify low-frequency antigen-specific T cells in Chlamydia-triggered reactive arthritis.Arthritis Res. Ther. 2004; 6: R521-R534Crossref PubMed Google Scholar). However, given the large cross-reactive potential of CTLs, the relationship of these peptides to naturally processed chlamydial epitopes is unknown. In a second approach a peptide derived from the cytoplasmic tail of HLA-B27 and other class I molecules was shown to be presented as an endogenously processed natural ligand of three HLA-B27 subtypes, B*2702, B*2704, and B*2705, associated with spondyloarthritis and not by two subtypes, B*2706 and B*2709, weakly or not disease-associated. This peptide showed high homology with a sequence encompassing residues 211–222 of the DNA primase of C. trachomatis, and the corresponding chlamydial peptide was directly generated in vitro from a synthetic precursor by the 20 S proteasome (11Ramos M. Alvarez I. Sesma L. Logean A. Rognan D. Lopez de Castro J.A. Molecular mimicry of an HLA-B27-derived ligand of arthritis-linked subtypes with chlamydial proteins.J. Biol. Chem. 2002; 277: 37573-37581Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). This study was the first to directly address the molecular mimicry between natural ligands of HLA-B27 arising from human self-proteins and chlamydial protein sequences. However, a critical test for the relevance of the DNA primase-derived peptide was to determine whether this peptide, or a closely related one, was actually processed and presented in vivo by HLA-B27. A direct molecular approach aiming at directly mapping chlamydial peptides presented by HLA-B27 in infected cells by biochemical methods is hardly feasible due to the exceedingly low expression of bacterial antigens on these cells as reported, for instance, with Salmonella (19Ramos M. Alvarez I. Garcia-del-Portillo F. Lopez de Castro J.A. Minimal alterations in the HLA-B27-bound peptide repertoire induced upon infection of lymphoid cells with Salmonella typhimurium.Arthritis Rheum. 2001; 44: 1677-1688Crossref PubMed Scopus (17) Google Scholar, 20Ringrose J.H. Meiring H.D. Speijer D. Feltkamp T.E. van Els C.A. de Jong A.P. Dankert J. Major histocompatibility complex class I peptide presentation after Salmonella enterica serovar typhimurium infection assessed via stable isotope tagging of the B27-presented peptide repertoire.Infect. Immun. 2004; 72: 5097-5105Crossref PubMed Scopus (12) Google Scholar). In the case of Chlamydia, this approach is further complicated by the down-regulation of MHC class I expression induced by the bacteria shortly after infection (6Zhong G. Liu L. Fan T. Fan P. Ji H. Degradation of transcription factor RFX5 during the inhibition of both constitutive and interferon γ-inducible major histocompatibility complex class I expression in chlamydia-infected cells.J. Exp. Med. 2000; 191: 1525-1534Crossref PubMed Scopus (171) Google Scholar, 7Zhong G. Fan P. Ji H. Dong F. Huang Y. Identification of a chlamydial protease-like activity factor responsible for the degradation of host transcription factors.J. Exp. Med. 2001; 193: 935-942Crossref PubMed Scopus (324) Google Scholar). Thus, we adopted an alternative strategy to ask the specific question of whether the DNA primase-(211–222) peptide, or closely related ones, could be generated and presented by HLA-B27 when the bacterial protein was endogenously expressed in the cell. To this end, we devised a method to endogenously and stably express a viable construct derived from the DNA primase of C. trachomatis and demonstrated the endogenous presentation of three peptides from this protein by HLA-B27 in vivo, including one spanning residues 211–221. Green fluorescent protein (GFP)-DNA primase fusion proteins were generated by fusing the cDNA of the DNA primase gene of C. trachomatis serovar L2 (CT794) (Advanced Biotechnologies, Columbia, MD), or truncated forms of this gene, in frame to the 3′-end of the GFP gene. All the DNA primase gene constructs were cloned into the pEGFP-C1 vector (BD Biosciences Clontech) in 5′ EcoRI and 3′ BamHI sites. Three gene constructs were made (see Fig. 1A): the complete DNA primase sequence, P-(1–595), a truncated form lacking the first 89 codons, P-(90–595), and a second truncated form lacking codons 1–89 and 451–595, P-(90–450). The chlamydial DNA primase cDNA was amplified by PCR using the following primers: for P-(1–595), 5′-TCTCTCTCGAATTCTATGTATTACACAGAAGAGAGC and 3′-TCTCTCTCGGATCCTTAAGAAACTAAAGAAACCTTAAC; for P-(90–595), 5′-TCTCTCTCGAATTCTATGTTTCATGTTGATCTTGTTGTCAG and 3′-TCTCTCTCGGATCCTTAAGAAACTAAAGAAACCTTAAC; and for P-(90–450), 5′-TCTCTCTCGAATTCTATGTTTCATGTTGATCTTGTTGTCAG and 3′-TCTCTCTCGGATCCTTATCCTTCTGTAGAAGTTTGCTTA. Hmy2.C1R (C1R) is a human lymphoid cell line with low expression of its endogenous HLA class I molecules (21Storkus W.J. Howell D.N. Salter R.D. Dawson J.R. Cresswell P. NK susceptibility varies inversely with target cell class I HLA antigen expression.J. Immunol. 1987; 138: 1657-1659Crossref PubMed Google Scholar, 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). The GFP-DNA primase constructs were cotransfected with the RSV5 vector (23Long E.O. Rosen-Bronson S. Karp D.R. Malnati M. Sekaly R.P. Jaraquemada D. Efficient cDNA expression vectors for stable and transient expression of HLA-DR in transfected fibroblast and lymphoid cells.Hum. Immunol. 1991; 31: 229-235Crossref PubMed Scopus (123) Google Scholar) carrying the hygromycin resistance gene (a kind gift of Dr. D. Jaraquemada, Autonomous University, Barcelona, Spain) at a 20:1 ratio in B*2705-C1R transfectant cells (24Calvo V. Rojo S. Lopez D. Galocha B. Lopez de Castro J.A. Structure and diversity of HLA-B27-specific T cell epitopes. Analysis with site-directed mutants mimicking HLA-B27 subtype polymorphism.J. Immunol. 1990; 144: 4038-4045PubMed Google Scholar) by electroporation at 300 mV and 960 microfarads. Cells were cultured in RPMI 1640 medium supplemented with 10% FCS (both from Invitrogen). Stable GFP or GFP-DNA primase transfectants were selected with 250 μg/ml hygromycin (Invitrogen). The monoclonal antibody (mAb) W6/32 (IgG2a, specific for a monomorphic HLA-A, -B, -C determinant) (25Barnstable C.J. Bodmer W.F. Brown G. Galfre G. Milstein C. Williams A.F. Ziegler A. Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens. New tools for genetic analysis.Cell. 1978; 14: 9-20Abstract Full Text PDF PubMed Scopus (1600) Google Scholar) was used. Approximately 106 cells were washed twice in 200 μl of PBS. The GFP-associated fluorescence was directly measured in a FACSCalibur instrument, and data were analyzed using CellQuest software (both from BD Biosciences). About 2 × 106 cells were lysed in Igepal CA-630 (Sigma) lysis buffer (0.5% Igepal, 50 mm Tris-HCl, pH 7.4, 5 mm MgCl2) containing a mixture of protease inhibitors (Complete Mini, Roche Applied Science). After centrifugation to remove insoluble material, lysates were precleared with anti-rabbit IgG beads (TrueBlot, eBioscience, San Diego, CA) for 2 h. Immunoprecipitation was done by overnight incubation with the GFP-specific polyclonal Ab A6455 (Invitrogen) followed by incubation with anti-rabbit IgG beads for 1 h. All procedures were carried out at 4 °C with continuous shaking. Immunoprecipitates were washed three times with lysis buffer, boiled for 5 min in SDS sample buffer, subjected to SDS-PAGE, and transferred onto nitrocellulose membranes (Bio-Rad). Immunoblotting was done with the A6455 Ab and horseradish peroxidase-conjugated anti-rabbit IgG (TrueBlot, eBioscience) at 1:10,000 and 1:5000 dilution, respectively. Antibodies were diluted in PBS-milk buffer (PBS, 5% milk, 0.1% Tween 20). The immunoblots were developed with the ECL immunodetection system (Amersham Biosciences). B*2705-bound peptides were isolated from about 1010 or, in one case, 2 × 1010 C1R-B*2705 transfectant cells as described previously (26Paradela 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 (Sigma), 20 mm Tris/HCl, 150 mm NaCl, pH 7.5, in the presence of a mixture of protease inhibitors. After ultracentrifugation, the soluble fraction was subjected to affinity chromatography using the W6/32 mAb. HLA-B27-bound peptides were eluted with 0.1% aqueous TFA at room temperature, filtered through Centricon 3 devices (Amicon, Beverly, MA), concentrated, and subjected to reverse phase 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 previously described (27Paradela 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. HPLC fractions were analyzed by MALDI-TOF MS using a Bruker Reflex™ mass spectrometer (Bruker Daltoniks, Bremen, Germany) equipped with the SCOUT™ source operating in positive ion reflector mode as described previously (28Merino E. Montserrat V. Paradela A. Lopez de Castro J.A. Two HLA-B14 subtypes (B*1402 and B*1403) differentially associated with ankylosing spondylitis differ substantially in peptide specificity, but have limited peptide and T-cell epitope sharing with HLA-B27.J. Biol. Chem. 2005; 280: 35868-35880Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Briefly dried HPLC fractions were resuspended in 0.5 μl of TA (33% aqueous acetonitrile, 0.1% TFA), deposited onto the MALDI plate, and allowed to dry at room temperature. Then 0.5 μl of matrix solution (α-cyano-4-hydroxycinnamic acid in TA) at 2 mg/ml was added and allowed to dry again. MS spectra were processed using the MoverZ software (version 2001.02.03) (Genomic Solutions). Alternatively a MALDI-TOF/TOF Instrument (4800 Proteomics Analyzer, Applied Biosystems, Foster City, CA) was used. In this case, fractions were reconstituted with 0.6 μl of TA, loaded onto an Opti-TOF™ 384-well MALDI insert (Applied Biosystems), and allowed to dry at room temperature. Then 0.6 μl of the matrix solution (3 mg/ml) was added. These mass spectra were acquired in reflector positive mode and processed using the 4000 Series Explorer software version 3.5. Peptide sequencing was carried out by quadrupole ion trap nano-ESI MS/MS in an LCQ Classic or LCQ DECA-XP instrument (Finnigan Thermoquest, San Jose, CA) using the Xcalibur 2.0 software after on-line chromatographic separation of samples as described previously (28Merino E. Montserrat V. Paradela A. Lopez de Castro J.A. Two HLA-B14 subtypes (B*1402 and B*1403) differentially associated with ankylosing spondylitis differ substantially in peptide specificity, but have limited peptide and T-cell epitope sharing with HLA-B27.J. Biol. Chem. 2005; 280: 35868-35880Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Alternatively sequencing was performed by MALDI-TOF/TOF. The acquisition method was MS/MS at 1 kV with collision-induced dissociation where the collision gas was atmospheric air and the precursor mass window was set as ±10 Daltons. The parameter used for processing data was a signal-to-noise ratio of 10. Interpretation of mass spectra was done manually but assisted by various software tools as follows. Manual inspection of the spectrum allowed us to determine a tentative sequence. This was used to screen the chlamydial DNA primase protein sequence (UniProtKB/Swiss-Prot accession number O84799). When a match was obtained, a list of theoretical fragment ions of the corresponding peptide sequence was generated using the MS-product tool available at the Protein Prospector tools web site: prospector.ucsf.edu/prospector/4.0.8/html/msprod.htm (University of California, San Francisco, CA) as an assistance to match the putative candidate sequences to our experimental MS/MS spectrum. In addition, the corresponding synthetic peptide was made, and its MS/MS spectrum was used to confirm the manually assigned sequence of the chlamydial B27 ligand. The search for homologies between chlamydial peptides and human proteins was carried out in the UniProtKB database (release 54.0; July 24, 2007) at www.expasy.org/sprot using the Fasta 3 software (release 12.0; July 24, 2007) at www.ebi.ac.uk/fasta. This was done essentially as described previously (29Marcilla M. Cragnolini J.J. Lopez de Castro J.A. Proteasome-independent HLA-B27 ligands arise mainly from small basic proteins.Mol. Cell. Proteomics. 2007; 6: 923-938Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) with minor modifications. Briefly three batches of 6 × 108 B*2705-C1R transfectant cells expressing the chlamydial P-(90–450) fusion protein were separately cultured for 4 h in Dulbecco's modified Eagle's medium without Arg supplemented with 10% FCS. Then one flask was supplemented with standard [14N]Arg (100 μg/ml), the second flask was supplemented with 100 μg/ml l-[guanido-15N2]Arg·HCl (Cambridge Isotope Laboratories, Andover, MA) in which two nitrogen atoms of the guanidinium group have been replaced with 15N, and the third flask was treated with a 20 μm concentration of the irreversible proteasome inhibitor MG132 (Calbiochem) for 30 min prior to the addition of 100 μg/ml 15N-tagged Arg to ensure that the proteasome was inhibited from the start of the labeling; the inhibitor was left for the complete labeling period. After 5 h, cells were washed twice in 20 mm Tris/HCl, 150 mm NaCl, pH 7.5, and stored at −70 °C for further processing. This labeling time was used because the much longer times required for quantitative protein labeling are not feasible due to the limited viability of cells in the presence of proteasome inhibitors. These were obtained using standard N-(9-fluorenyl)methoxycarbonyl chemistry and purified by HPLC. The correct molecular mass of purified peptides was verified by MALDI-TOF MS. Stable transfectants expressing DNA primase protein sequences from C. trachomatis in HLA-B27-positive cells were required to analyze bacterial peptide presentation. Three fusion proteins were constructed in which GFP was fused to the N terminus of the complete DNA primase protein, P-(1–595), to a fragment lacking the N-terminal 89 residues, P-(90–595), and to a fragment lacking both the N-terminal 89 residues and the C-terminal 144 residues, P-(90–450). The deleted N-terminal and C-terminal regions included the DNA binding and helicase interaction domains of the DNA primase, respectively (Fig. 1A). The three constructs included the P-(211–222) sequence that was previously reported as homologous to a natural ligand of HLA-B27 derived from its own molecule (11Ramos M. Alvarez I. Sesma L. Logean A. Rognan D. Lopez de Castro J.A. Molecular mimicry of an HLA-B27-derived ligand of arthritis-linked subtypes with chlamydial proteins.J. Biol. Chem. 2002; 277: 37573-37581Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). In preliminary experiments the three fusion proteins were efficiently expressed in transient transfectants of COS and HeLa cells (data not shown). In contrast, stable transfectants in B*2705-C1R cells were obtained with P-(90–595) and P-(90–450) but not with the complete bacterial protein P-(1–595). The expression levels of the fusion proteins as assessed by flow cytometry were higher for P-(90–450) than for P-(90–595), although in both cases the fluorescence was much lower than in the transfectant expressing only GFP used as a control (Fig. 1B). The expression of the fusion proteins with the correct size in the corresponding transfectants was determined by immunoprecipitation with anti-GFP Ab and Western blot (Fig. 1C). The search for HLA-B27 ligands derived from the bacterial DNA primase was carried out by comparative analysis of the HLA-B27-bound peptide pools isolated from B*2705-C1R cells or transfectants of these cells expressing either GFP alone or P-(90–450). This transfectant was used, instead of the one expressing P-(90–595), due to its higher expression level of the fusion protein (Fig. 1). The strategy used was the same as that used previously for comparing HLA-B27 subtype-bound peptide repertoires (30Ramos M. Paradela A. Vazquez M. Marina A. Vazquez J. Lopez de Castro J.A. Differential association of HLA-B*2705 and B*2709 to ankylosing spondylitis correlates with limited peptide subsets but not with altered cell surface stability.J. Biol. Chem. 2002; 277: 28749-28756Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 31Sesma L. Montserrat V. Lamas J.R. Marina A. Vazquez J. Lopez de Castro J.A. The peptide repertoires of HLA-B27 subtypes differentially associated to spondyloarthropathy (B*2704 and B*2706) differ by specific changes at three anchor positions.J. Biol. Chem. 2002; 277: 16744-16749Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). HLA-B27-bound peptide pools were isolated by immunopurification of HLA-B27 with the W6/32 mAb followed by acid extraction. The peptide pools were fractionated by HPLC under identical conditions and consecutive runs, and the peptide composition of individual fractions was analyzed by MALDI-TOF MS. To look for peptides specifically presented by HLA-B27 in cells expressing the chlamydial construct, the MS spectrum of each HPLC fraction of the B27-bound peptide pool from these cells was compared with the MS spectra of the correlative HPLC fraction of the control cell lines (untransfected and GFP-transfected B*2705-C1R) as well as with the spectra of the previous and following fractions. This was done to account for small shifts in the retention times of individual peptides that might occur among distinct chromatographic runs. In a survey of 1570 ion peaks from 150 HPLC fractions from each chromatography three ion peaks were detected in the P-(90–450) transfectant but not in the controls of untransfected (Fig. 2) or GFP-transfected (not shown) B*2705-C1R: in HPLC fraction number 105 an ion peak with m/z 1247.3, in fraction number 134 an ion peak with m/z 1346.3, and in fraction number 162 an ion peak with m/z 1493.8. The intensity of the 1346.3 ion peak was about 10 times higher than the intensity of each of the other two ion peaks. These intensities were calculated by adding the intensities of the corresponding ion peak from the tw
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