A 52-kDa Leucyl Aminopeptidase from Treponema denticola Is a Cysteinylglycinase That Mediates the Second Step of Glutathione Metabolism
2008; Elsevier BV; Volume: 283; Issue: 28 Linguagem: Inglês
10.1074/jbc.m801034200
ISSN1083-351X
AutoresLianrui Chu, Yanlai Lai, Xiaoping Xu, Scott Eddy, Shuang Yang, Li Song, David Kolodrubetz,
Tópico(s)Folate and B Vitamins Research
ResumoThe metabolism of glutathione by the periodontal pathogen Treponema denticola produces hydrogen sulfide, which may play a role in the host tissue destruction seen in periodontitis. H2S production in this organism has been proposed to occur via a three enzyme pathway, γ-glutamyltransferase, cysteinylglycinase (CGase), and cystalysin. In this study, we describe the purification and characterization of T. denticola CGase. Standard approaches were used to purify a 52-kDa CGase activity from T. denticola, and high pressure liquid chromatography electrospray ionization tandem mass spectrometry analysis of this molecule showed that it matches the amino acid sequence of a predicted 52-kDa protein in the T. denticola genome data base. A recombinant version of this protein was overexpressed in and purified from Escherichia coli and shown to catalyze the hydrolysis of cysteinylglycine (Cys-Gly) with the same kinetics as the native protein. Surprisingly, because sequence homology indicates that this protein is a member of a family of metalloproteases called M17 leucine aminopeptidases, the preferred substrate for the T. denticola protein is Cys-Gly (kcat/Km of 8.2 μm–1 min–1) not l-Leu-p-NA (kcat/Km of 1.1 μm–1 min–1). The activity of CGase for Cys-Gly is optimum at pH 7.3 and is enhanced by Mn2+, Co2+, or Mg2+ but not by Zn2+ or Ca2+. Importantly, in combination with the two other previously purified T. denticola enzymes, γ-glutamyltransferase and cystalysin, CGase mediates the in vitro degradation of glutathione into the expected end products, including H2S. These results prove that T. denticola contains the entire three-step pathway to produce H2S from glutathione, which may be important for pathogenesis. The metabolism of glutathione by the periodontal pathogen Treponema denticola produces hydrogen sulfide, which may play a role in the host tissue destruction seen in periodontitis. H2S production in this organism has been proposed to occur via a three enzyme pathway, γ-glutamyltransferase, cysteinylglycinase (CGase), and cystalysin. In this study, we describe the purification and characterization of T. denticola CGase. Standard approaches were used to purify a 52-kDa CGase activity from T. denticola, and high pressure liquid chromatography electrospray ionization tandem mass spectrometry analysis of this molecule showed that it matches the amino acid sequence of a predicted 52-kDa protein in the T. denticola genome data base. A recombinant version of this protein was overexpressed in and purified from Escherichia coli and shown to catalyze the hydrolysis of cysteinylglycine (Cys-Gly) with the same kinetics as the native protein. Surprisingly, because sequence homology indicates that this protein is a member of a family of metalloproteases called M17 leucine aminopeptidases, the preferred substrate for the T. denticola protein is Cys-Gly (kcat/Km of 8.2 μm–1 min–1) not l-Leu-p-NA (kcat/Km of 1.1 μm–1 min–1). The activity of CGase for Cys-Gly is optimum at pH 7.3 and is enhanced by Mn2+, Co2+, or Mg2+ but not by Zn2+ or Ca2+. Importantly, in combination with the two other previously purified T. denticola enzymes, γ-glutamyltransferase and cystalysin, CGase mediates the in vitro degradation of glutathione into the expected end products, including H2S. These results prove that T. denticola contains the entire three-step pathway to produce H2S from glutathione, which may be important for pathogenesis. The volatile sulfur compound H2S can be produced by the metabolic activity of numerous oral bacteria, including several periodontal pathogens (1Persson S. Claesson R. Carlsson J. Oral Microbiol. Immunol. 1989; 4: 169-172Crossref PubMed Scopus (106) Google Scholar, 2Claesson R. Edlund M.B. Persson S. Carlsson J. Oral Microbiol. Immunol. 1990; 5: 137-142Crossref PubMed Scopus (104) Google Scholar, 3Persson S. Edlund M.B. Claesson R. Oral Microbiol. Immunol. 1990; 5: 195-201Crossref PubMed Scopus (387) Google Scholar). This gas, which is malodorous and highly toxic (4United States National Research CouncilHydrogen Sulfide in Medical and Biologic Effects of Environmental Pollutants. University Park Press, Baltimore, MD1979: 1-50Google Scholar, 5Beauchamp R.O. Bus Jr., J.S. Popp J.A. Boreiko C.J. Andjelkovich D.A. Crit. Rev. Toxicol. 1984; 13: 25-97Crossref PubMed Scopus (840) Google Scholar, 6Reiffenstein R.J. Hulbert W.C. Roth S.H. Annu. Rev. Pharmacol. Toxicol. 1992; 32: 109-134Crossref PubMed Scopus (800) Google Scholar), is found in high concentrations in periodontal pockets (7Morhart R.E. Mata L.J. Sinskey A.J. Harris R.S. J. Periodontol. 1970; 41: 644-649Crossref PubMed Scopus (31) Google Scholar, 8Horowitz A. Folke L.E. J. Periodontal. 1973; 44: 390-395Crossref PubMed Scopus (39) Google Scholar, 9Persson S. Oral Microbiol. Immunol. 1992; 7: 378-379Crossref PubMed Scopus (92) Google Scholar) and may play a role in some of the tissue destruction seen in periodontal diseases (7Morhart R.E. Mata L.J. Sinskey A.J. Harris R.S. J. Periodontol. 1970; 41: 644-649Crossref PubMed Scopus (31) Google Scholar, 8Horowitz A. Folke L.E. J. Periodontal. 1973; 44: 390-395Crossref PubMed Scopus (39) Google Scholar, 10Rizzo A.A. Periodontics. 1967; 5: 233-236PubMed Google Scholar). H2S can be produced from the metabolism of several molecules, but glutathione (l-γ-glutamyl-l-cysteinylglycine) is believed to be the major source for H2S production in the oral cavity; human cells, especially polymorphonuclear leucocytes, have high concentrations (up to 4 mm) of glutathione that can be released when host cells are damaged in the periodontal pocket. Although a number of oral bacteria have been tested, only a few of them are able to catabolize glutathione into H2S (3Persson S. Edlund M.B. Claesson R. Oral Microbiol. Immunol. 1990; 5: 195-201Crossref PubMed Scopus (387) Google Scholar, 11Carlsson J. Larsen J.T. Edlund M.B. Oral Microbiol. Immunol. 1993; 8: 42-45Crossref PubMed Scopus (58) Google Scholar, 12Chu L. Dong Z. Xu X. Cappelli D. Ebersole J. Infect. Immun. 2002; 70: 1113-11207Crossref PubMed Scopus (48) Google Scholar). Treponema denticola, which appears to play a significant role in the development of acute and chronic periodontal diseases in humans (13Simonson L.G. Goodman C.H. Bial J.J. Morton H.E. Infect. Immun. 1988; 56: 726-728Crossref PubMed Google Scholar, 14Moore W.E.C. J. Periodont. Ris. 1987; 22: 335-341Crossref PubMed Scopus (281) Google Scholar, 15Aimetti M. Romano F. Nessi F. J. Periodontol. 2007; 78: 1718-1723Crossref PubMed Scopus (86) Google Scholar, 16Sela M.N. Crit. Rev. Oral Biol. Med. 2001; 12: 399-413Crossref PubMed Scopus (144) Google Scholar, 17Holt S.C. Ebersole J.L. Periodontol. 2005; 38: 72-122Crossref Scopus (698) Google Scholar), is the only oral pathogen in which the proteins involved in this catabolic pathway have begun to be identified and characterized (18Chu L. Ebersole J.L. Kurzben G.P. Holt S.C. Infect. Immun. 1997; 65: 3231-3238Crossref PubMed Google Scholar, 19Chu L. Xu X. Dong Z. Cappelli D. Ebersole J.D. Infect. Immun. 2003; 71: 335-342Crossref PubMed Scopus (18) Google Scholar, 20Chu L. Holt S.C. Microb. Pathog. 1994; 16: 197-212Crossref PubMed Scopus (47) Google Scholar, 21Chu L. Burgum A. Kolodrubetz D. Holt S.C. Infect. Immun. 1995; 63: 4448-4455Crossref PubMed Google Scholar, 22Makinen P.L. Makinen K.K. Infect. Immun. 1997; 65: 685-691Crossref PubMed Google Scholar). Glutathione catabolism to H2S (and glutamate, glycine, ammonia, and pyruvate) has been proposed to occur via a three-step enzyme pathway in this spirochete (12Chu L. Dong Z. Xu X. Cappelli D. Ebersole J. Infect. Immun. 2002; 70: 1113-11207Crossref PubMed Scopus (48) Google Scholar). In the first step, glutathione is split into glutamate and Cys-Gly. This dipeptide is then hydrolyzed into glycine and l-cysteine followed by the breakdown of l-cysteine into pyruvate, ammonia, and H2S. The enzymes involved in the first and third steps have been purified from T. denticola and characterized (19Chu L. Xu X. Dong Z. Cappelli D. Ebersole J.D. Infect. Immun. 2003; 71: 335-342Crossref PubMed Scopus (18) Google Scholar, 20Chu L. Holt S.C. Microb. Pathog. 1994; 16: 197-212Crossref PubMed Scopus (47) Google Scholar, 21Chu L. Burgum A. Kolodrubetz D. Holt S.C. Infect. Immun. 1995; 63: 4448-4455Crossref PubMed Google Scholar, 22Makinen P.L. Makinen K.K. Infect. Immun. 1997; 65: 685-691Crossref PubMed Google Scholar, 23Chu L. Ebersole J.L. Kurzben G.P. Holt S.C. Clin. Infect. Dis. 1999; 28: 442-450Crossref PubMed Scopus (25) Google Scholar, 24Chu L. Ebersole J.L. Holt S.C. Oral Microbiol. Immunol. 1999; 14: 293-303Crossref PubMed Scopus (22) Google Scholar, 25Kurzben G.P. Chu L. Ebersole J.L. Holt S.C. Oral Microbiol. Immunol. 1999; 14: 153-164Crossref PubMed Scopus (43) Google Scholar, 26Krupka H.I. Huber R. Holt S.C. Clausen T. EMBO J. 2000; 19: 3168-3178Crossref PubMed Scopus (67) Google Scholar, 27Bertoldi M. Cellini B. Clausen T. Voltattorni C.B. Biochemistry. 2002; 41: 9153-9164Crossref PubMed Scopus (43) Google Scholar, 28Cellini B. Bertoldi M. Paiardini A. D'Aguanno S. Voltattorni C.B. J. Biol. Chem. 2004; 279: 36898-36905Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 29Cellini B. Bertoldi M. Montioli R. Laurents D.V. Paiardini A. Voltattorni C.B. Biochemistry. 2006; 45: 14140-14154Crossref PubMed Scopus (20) Google Scholar, 30Cellini B. Montioli R. Bossi A. Bertoldi M. Laurents D.V. Voltattorni C.B. Arch. Biochem. Biophys. 2006; 455: 31-39Crossref PubMed Scopus (4) Google Scholar). γ-Glutamyltransferase (GGT) 2The abbreviations used are: GGT, γ-glutamyltransferase; HPLC, high pressure liquid chromatography; ESI-MS/MS, electrospray ionization tandem mass spectrometry; CGase, cysteinylglycinase; l-Leu-p-NA, l-leucine-p-nitroanilide; PBS, phosphate-buffered saline; LAP, leucine aminopeptidase. is a 27-kDa protein that catalyzes the cleavage of glutathione into glutamate and Cys-Gly. Cystalysin (l-cysteine desulfhydrase) is a 46-kDa protein that converts l-cysteine into H2S, ammonia, and pyruvate. However, neither of these enzymes can utilize Cys-Gly as a substrate (18Chu L. Ebersole J.L. Kurzben G.P. Holt S.C. Infect. Immun. 1997; 65: 3231-3238Crossref PubMed Google Scholar, 19Chu L. Xu X. Dong Z. Cappelli D. Ebersole J.D. Infect. Immun. 2003; 71: 335-342Crossref PubMed Scopus (18) Google Scholar, 25Kurzben G.P. Chu L. Ebersole J.L. Holt S.C. Oral Microbiol. Immunol. 1999; 14: 153-164Crossref PubMed Scopus (43) Google Scholar), indicating that T. denticola has an additional enzyme, presumably a cysteinylglycinase (CGase), to cleave the peptide bond of Cys-Gly. In the present study, the purification and characterization of a CGase from T. denticola is described. The T. denticola CGase is a 52-kDa protein that should be a leucyl aminopeptidase based upon its sequence homology to members of that protein family (31Seshadri R. Myers G.S. Tettelin H. Eisen J.A. Heidelberg J.F. Dodson R.J. Davidsen T.M. DeBoy R.T. Fouts D.E. Haft D.H. Selengut J. Ren Q. Brinkac L.M. Madupu R. Kolonay J. Durkin S.A. Daugherty S.C. Shetty J. Shvartsbeyn A. Gebregeorgis E. Geer K. Tsegaye G. Malek J. Ayodeji B. Shatsman S. McLeod M.P. Smajs D. Howell J.K. Pal S. Amin A. Vashisth P. McNeill T.Z. Xiang Q. Sodergren E. Baca E. Weinstock G.M. Norris S.J. Fraser C.M. Paulsen I.T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5646-5651Crossref PubMed Scopus (215) Google Scholar). Characterization of a recombinant version of the T. denticola 52-kDa protein indicates that its preferred substrate is Cys-Gly, not leucine aminopeptides. The T. denticola CGase can, in combination with GGT and cystalysin from T. denticola, catalyze the production of H2S from glutathione in vitro, thus proving that the proposed three-step pathway for glutathione catabolism to H2Sin T. denticola is correct. Unless otherwise indicated, all of the chemicals and reagents were obtained from Sigma. T. denticola 35404 and other treponema ATCC strains were from the American Type Culture Collection (Manassas, VA), and the clinic isolates were from Dr. Stanley Holt (32Weinberg A. Holt S.C. Infect. Immun. 1990; 58: 1720-1729Crossref PubMed Google Scholar). All of the bacteria used in this study were cultured anaerobically in a Coy anaerobic chamber (5% CO2, 10% H2, and 85%N2) at 37 °C in GM-1 broth (33Blakemore R.P. Canale-Parola E. J. Bacteriol. 1976; 128: 616-622Crossref PubMed Google Scholar) supplemented with 3.4% rabbit serum. The standard reaction buffer for CGase assays was 50 mm Tris-HCl (pH 7.3) with 0.2 mm MnCl2. Protein samples were incubated in the reaction buffer with 2 mm Cys-Gly, unless another concentration is indicated, for 20 min at 37 °C, and the reactions were stopped by the addition of 5% trichloroacetic acid. The amount of the reaction product, l-cysteine, produced was measured as described by Gaitonde (34Gaitonde M.K. Biochem. J. 1967; 104: 627-633Crossref PubMed Scopus (1037) Google Scholar). l-Cysteine concentrations, as the optical density at 560 nm, were calculated from a standard curve with known amounts of l-cysteine, after subtracting a blank. Leucine aminopeptidase activity was measured using l-Leu-p-NA as substrate (35Morty R.E. Morehead J. J. Biol. Chem. 2002; 277: 26057-26065Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The reaction was done in 50 mm Tris-HCl (pH 8.0) with 0.2 mm MnCl2 and 2 mm l-Leu-p-NA, unless noted otherwise. Enzyme was added, and the reaction was incubated at 37 °C for 20 min before being stopped by cooling on ice. The concentration of the reaction product was measured by the absorbance at 405 nm. All of the enzyme assays were carried out in triplicate unless otherwise indicated. The purification of the CGase activity from T. denticola 35404 was carried out in three steps. Step 1: Initial Enrichment of CGase—Briefly, whole cell pellets were diluted with PBS buffer containing 2 mm phenylmethylsulfonyl fluoride to a protein concentration of 5 mg/ml. The cells were sonicated (Branson Cell Disrupter 200; Sonic Power Co.) for 3 min at maximum output at 4 °C. The soluble material was separated from cell and membrane debris by two consecutive centrifugations at 20,000 × g, 45 min. Stepwise ammonium sulfate precipitation (2.0, 2.8, and 3.6 m ammonium sulfate) of the soluble cell fraction was then used to further enrich the CGase activity. The precipitate formed at 3.6 m ammonium sulfate was collected by centrifugation, dissolved in 15 ml of 10 mm phosphate buffer (pH 7.5), and dialyzed at 4 °C against the same buffer with 2 mm β-mercaptoethanol and 0.02 mm Mn2+ (final concentrations) for 48 h with three changes of the same buffer. Step 2: Ultrafiltration by Microcon—Approximately 15 ml of the 3.6 m ammonium sulfate fraction (3.5 mg protein/ml) was applied to a Microcon 100 filter device and centrifuged at 8,000 × g at 4 °C for 10 min. The flow-through (<100-kDa proteins) was then put onto a Microcon 50 filter device and centrifuged to remove proteins smaller than 50 kDa and to concentrate the CGase activity. This material was suspended in 50 mm Tris-HCl and dialyzed overnight at 4 °C against a large volume of the same buffer containing 2 mm β-mercaptoethanol and 0.2 mm Mn2+. Step 3: Purification by HiTrap Sepharose Q FF Ion Exchange Columns—After centrifugation, 14,000 rpm in a microcentrifuge for 15 min, of the dialysate from the previous step to remove precipitated protein, about 5 ml of sample was loaded into a 1-ml HiTrap Sepharose Q FF cation exchange column (GE Healthcare). Chromatography was carried out on a Beckman Coulter System Gold HPLC system at a flow rate of 1 ml/min (Beckman Coulter, Inc., Fullerton, CA). The column was washed with 50 mm Tris-HCl (pH 8.0) for 5 min to remove unbound protein, and then the bound protein, including CGase, was eluted by a 0–0.5 m gradient of NaCl in 50 mm Tris-HCl, pH 8.0. The eluted proteins were monitored by a UV detector at 280 nm. The CGase activity eluted at about 0.18 m NaCl and was usually distributed across one to three fractions. The three Coomassie-stained bands seen after the final purification step were excised from an SDS gel and identified, after digestion with trypsin, by capillary HPLC electrospray ionization tandem mass spectrometry (36Thomas J.A. Hardies S.C. Rolando M. Hayes S. Lieman K. Carroll C.A. Weintraub S.T. Serwer P. Virology. 2007; 368: 405-421Crossref PubMed Scopus (52) Google Scholar). The resulting CID spectra were searched against the NCBI nr data base by means of Mascot (Matrix Science). Cross-correlation with X! Tandem and determination of probabilities of correct protein identification were done with Scaffold (Proteome Software). Cloning and Sequencing of the cga Gene in E. coli—Based upon the genomic DNA sequence of T. denticola strain 35405 (31Seshadri R. Myers G.S. Tettelin H. Eisen J.A. Heidelberg J.F. Dodson R.J. Davidsen T.M. DeBoy R.T. Fouts D.E. Haft D.H. Selengut J. Ren Q. Brinkac L.M. Madupu R. Kolonay J. Durkin S.A. Daugherty S.C. Shetty J. Shvartsbeyn A. Gebregeorgis E. Geer K. Tsegaye G. Malek J. Ayodeji B. Shatsman S. McLeod M.P. Smajs D. Howell J.K. Pal S. Amin A. Vashisth P. McNeill T.Z. Xiang Q. Sodergren E. Baca E. Weinstock G.M. Norris S.J. Fraser C.M. Paulsen I.T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5646-5651Crossref PubMed Scopus (215) Google Scholar), two primers, (forward, 5′-CCGCTCGAGATGAAATTTAATATTGCAAAAAAAG-3′; reverse: 5′-GGGGTACCATATTTGCTTCCCTGCGGC-3′) were designed to amplify the entire TDE0300 open reading frame, which we will refer to as cga, and to put an XhoI site (forward) and KpnI site (reverse) at the ends of the PCR product. With T. denticola 35404 genomic DNA as template, a 1.6-kb fragment was amplified by PCR, using standard protocols, and ligated into the XhoI/KpnI sites of the expression vector pRsetA (Invitrogen). The insert from a plasmid containing the cga gene of strain 35404 was sequenced, independently from both strands, in the Center for Advanced DNA Technology at the University of Texas Health Science Center at San Antonio. Expression and Purification of Recombinant CGase—To express recombinant CGase, isopropyl β-d-thiogalactopyranoside (final concentration, 0.5 mm) was added to a culture of E. coli cells containing the expression plasmid. After 4 h, the bacterial culture was centrifuged at 6,000 × g for 5 min, and the cells were washed once using 20 mm PBS (pH 7.4). The cells were resuspended in 60 ml of PBS and sonicated (Branson Sonifier 450, VWR Scientific) for 5 min on ice. The soluble fraction of the cell sonicate was recovered after centrifugation at 16,500 × g for 30 min. Because the recombinant CGase has a His6 tag, it was purified on a 2.5-ml nickel-nitrilotriacetic acid gel column (Qiagen). After loading 15 ml of the soluble cell material onto the column, it was washed with 20× volume of 50 mm Tris-HCl (pH 8.0), 300 mm NaCl, and 20 mm imidazole. A step gradient elution with imidazole (40–250 mm in the wash buffer) was used to elute the CGase. The identity of the purified recombinant CGase was confirmed by HPLC ESI-MS/MS as described above for the native protein. Determination of Optimal pH—The effect of pH on CGase activity was determined in the standard reaction buffer, except the pH was varied from pH 5.0 to 9.5 in ∼0.3 to 0.5 pH units. CGase (final concentration, 5 μg/ml) and either 2 mm Cys-Gly or l-Leu-p-NA were added, and the reaction was done at 37 °C for 10 min. Substrate Specificity—To assess the substrate specificity profile of the T. denticola 52-kDa CGase, its activity with a number of different substrates was measured in the standard reaction buffer at 37 °C for 15 min. At the end of the reaction, the concentrations of the appropriate products, l-cysteine, glycine, H2S, pyruvate, or ammonia, were determined (12Chu L. Dong Z. Xu X. Cappelli D. Ebersole J. Infect. Immun. 2002; 70: 1113-11207Crossref PubMed Scopus (48) Google Scholar, 37Siegel L.M. Anal. Biochem. 1965; 11: 126-132Crossref PubMed Scopus (380) Google Scholar, 38Zheng L. White R.H. Cash V.L. Jack R.F. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2754-2758Crossref PubMed Scopus (512) Google Scholar, 39Bauer J.D. Ackermann P.G. Toro P. Clinical Laboratory Methods. The C. V. Mosby Company, St. Louis, MO1974: 399-401Google Scholar). Because the pH optima could vary for different substrates, each substrate was tested at two different pH values. Initially, each substrate was used at a concentration of 2 mm; if there was enzyme activity at that concentration, then enzyme activity was measured over a range of substrate concentrations. The Km, Vmax, and kcat values were calculated as previously described (25Kurzben G.P. Chu L. Ebersole J.L. Holt S.C. Oral Microbiol. Immunol. 1999; 14: 153-164Crossref PubMed Scopus (43) Google Scholar). Effect of Cations on CGase Activity—For this set of reactions, the control reaction contained 5 μg/ml (final concentration) of purified recombinant CGase with 2 mm Cys-Gly in 50 mm Tris-HCl (pH 7.3), 0.2 mm MnCl2 and was incubated at 37 °C for 20 min. To see whether metal ions were required for enzyme activity, 0.2 mm EDTA was added to one set of tubes. To determine which cations were best at enhancing activity, 2 mm of different cations, MgCl2, MnCl2, CaCl2, CoCl2, or ZnCl2, were preincubated separately with the enzyme in 50 mm Tris-HCl (pH 7.3) for 20 min at 37 °C, and then 2 mm Cys-Gly was added, and the reaction was allowed to proceed for an additional 20 min. Antibody against the purified recombinant CGase was produced in New Zealand White rabbits (100 μg of purified protein mixed with Freund's incomplete adjuvant/each injection) as described previously (18Chu L. Ebersole J.L. Kurzben G.P. Holt S.C. Infect. Immun. 1997; 65: 3231-3238Crossref PubMed Google Scholar). Rabbit anti-CGase IgG (20 mg) was purified, coupled to activated Sepharose 4B (40Chu L. Kong X. Shao J. Lee M. Xie J. Shanghai J. Immunol. 1986; 6: 243-248Google Scholar), and resuspended in 6 ml of distilled H2O. For immunodepletion, 1 × 1010 T. denticola cells were lysed by 1% Triton X-100 in PBS containing 2 mm β-ME and 0.2 mm MnCl2. The lysate was clarified by centrifugation at 12,000 × g for 5 min. One ml of the soluble material was combined with 0.3 ml of anti-CGase-IgG-Sepharose 4B and mixed gently by shaking at 25 °C for 30 min. The supernatant was collected for analysis of CGase activity. Bacterial lysate that was not put over the Sepharose 4B column and cell supernatant that was depleted on a Sepharose 4B column that had been coupled to preimmune serum IgG were used as controls. After 2 days of growth in an anaerobic chamber, cells of various Treponema strains were harvested by centrifugation, washed once with PBS, and then assayed, at 1 mg protein/ml, for CGase activity. To see whether the cga gene was present in other strains of T. denticola or in other bacteria, genomic DNA was isolated from isolated from several T. denticola, Treponema vincentii, and E. coli strains. The DNAs were used as templates for PCR. Two primers, the forward primer (5′-ATGAAATTTAATATTGCAAAAAAAG-3′), which starts exactly at the first codon of the cga open reading frame, and the reverse primer (5′-ATATTTGCTTCCCTGCGGC-3′), which is 18 bases after the end of the cga open reading frame, were used for DNA amplification using standard reaction conditions. Recombinant T. denticola GGT and cystalysin were purified from E. coli cells with the appropriate expression clones, as previously described (18Chu L. Ebersole J.L. Kurzben G.P. Holt S.C. Infect. Immun. 1997; 65: 3231-3238Crossref PubMed Google Scholar, 19Chu L. Xu X. Dong Z. Cappelli D. Ebersole J.D. Infect. Immun. 2003; 71: 335-342Crossref PubMed Scopus (18) Google Scholar). Purified CGase, GGT, and cystalysin, either alone or in various combinations, were added to the standard reaction buffer so that the final concentration of each enzyme was 2 μg/ml. Glutathione was added (final concentration, 2 mm), and the reaction was allowed to proceed for 30 min at 37 °C. The reaction was quenched on ice, and then the production of H2S, ammonia, pyruvate, glutamic acid/glutamine and glycine was determined (12Chu L. Dong Z. Xu X. Cappelli D. Ebersole J. Infect. Immun. 2002; 70: 1113-11207Crossref PubMed Scopus (48) Google Scholar, 37Siegel L.M. Anal. Biochem. 1965; 11: 126-132Crossref PubMed Scopus (380) Google Scholar, 38Zheng L. White R.H. Cash V.L. Jack R.F. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2754-2758Crossref PubMed Scopus (512) Google Scholar, 39Bauer J.D. Ackermann P.G. Toro P. Clinical Laboratory Methods. The C. V. Mosby Company, St. Louis, MO1974: 399-401Google Scholar). As a control, T. denticola 35404 cells, at a final concentration of 0.5 mg protein/ml, were also incubated with 2 mm glutathione under the same conditions as the purified proteins. Purification of Cysteinylglycinase from T. denticola—We have proposed that glutathione is catabolized by a three-step pathway in T. denticola and have previously identified and characterized the enzymes involved in steps 1 and 3. However, the T. denticola enzyme (CGase) involved in the proposed second step, cleavage of Cys-Gly into glycine and l-cysteine, has not been identified. To rectify this, we first showed that sonicated extracts of T. denticola strain ATCC 35404 had CGase activity (Table 1). The protein responsible for this activity was then purified more than 120-fold with a 7.5% yield of the initial total enzyme activity. Almost all of the enzyme activity was found in one fraction from the final HPLC ion exchange purification (Fig. 1a). This fraction contained one major protein band, with an approximate molecular mass of 52 kDa, on an SDS-PAGE gel (Fig. 1b) and two minor protein bands with molecular masses of 35 and 19 kDa.TABLE 1Purification of CGase from T. denticola 35404FractionationsTotal proteinEnzymatic activityYield of proteinRetained activitymgmmol/mg/min%%Whole cell sonicated425.51.3100.0100Soluble cell fraction250.21.958.8852.8-3.6 m ASP precipitation81.44.519.166Microcon cut-off (50-100 kDa)50.538.2511.949Second HiTrap Q FF column0.25165.40.057.5 Open table in a new tab To determine which of the three proteins was most likely to be the CGase, the three protein species were digested in situ with trypsin, and the resulting peptides were subjected to HPLC ESI-MS/MS analysis. The tandem mass spectra obtained from the 52-kDa major protein band had a significant match to the amino acid sequence of a predicted 52-kDa protein (Gene ID TDE0300) in the genomic data base of T. denticola strain 35405 (The Institute for Genomic Research, Rockville, MD) (31Seshadri R. Myers G.S. Tettelin H. Eisen J.A. Heidelberg J.F. Dodson R.J. Davidsen T.M. DeBoy R.T. Fouts D.E. Haft D.H. Selengut J. Ren Q. Brinkac L.M. Madupu R. Kolonay J. Durkin S.A. Daugherty S.C. Shetty J. Shvartsbeyn A. Gebregeorgis E. Geer K. Tsegaye G. Malek J. Ayodeji B. Shatsman S. McLeod M.P. Smajs D. Howell J.K. Pal S. Amin A. Vashisth P. McNeill T.Z. Xiang Q. Sodergren E. Baca E. Weinstock G.M. Norris S.J. Fraser C.M. Paulsen I.T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5646-5651Crossref PubMed Scopus (215) Google Scholar). The two minor protein bands (19 and 35 kDa) also matched up with a part of the amino acid sequence of the predicted TDE0300 protein, suggesting that they are proteolytic fragments of the full-length protein. We concluded that the 52-kDa protein encoded by TDE0300 has CGase activity. Cloning, Sequencing, and Analysis of the 52-kDa CGase Gene of T. denticola—To prove that the correct protein had been identified as a cysteinylglycinase, it is easiest to clone its gene, which we will refer to as cga, in E. coli so that the recombinant gene product can be overexpressed and shown to have CGase activity. To accomplish this, the gene encoding the 52-kDa protein first had to be cloned and sequenced, because we identified the protein in a strain (strain 35404) different from the one whose genome has been sequenced (strain 35405). Thus, PCR with primers designed from the known sequence of strain 35405 (31Seshadri R. Myers G.S. Tettelin H. Eisen J.A. Heidelberg J.F. Dodson R.J. Davidsen T.M. DeBoy R.T. Fouts D.E. Haft D.H. Selengut J. Ren Q. Brinkac L.M. Madupu R. Kolonay J. Durkin S.A. Daugherty S.C. Shetty J. Shvartsbeyn A. Gebregeorgis E. Geer K. Tsegaye G. Malek J. Ayodeji B. Shatsman S. McLeod M.P. Smajs D. Howell J.K. Pal S. Amin A. Vashisth P. McNeill T.Z. Xiang Q. Sodergren E. Baca E. Weinstock G.M. Norris S.J. Fraser C.M. Paulsen I.T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5646-5651Crossref PubMed Scopus (215) Google Scholar) was used to amplify a 1.6-kb DNA fragment from T. denticola strain 35404. This PCR fragment, which contains the entire cga gene, was cloned and sequenced. The open reading frame of the T. denticola 35404 cga gene consists of 1428 bp encoding 476 amino acids (predicted molecular mass of 52.2 kDa). The cga nucleotide sequence from strain 35404 differs from the published 35405 sequence at 12 positions; nine of the differences result in amino acid changes in the predicted protein sequence (Fig. 2). Somewhat surprisingly, only five of the nine changes are conservative amino acid substitutions. By sequence homology, the CGase protein from T. denticola is a member of the M17 family of metal-dependent leucyl aminopeptidases. This family is defined by a cluster of seven amino acids, five of which are involved in metal binding and two of which have a direct role in catalysis (41Colloms S.D. Barrett A.J. Rawlings N.D. Woesner J.F. Handbook of Proteolytic Enzymes. 2nd Ed. Elsevier/Academic Press, San Diego, CA2004: 905-908Crossref Scopus (13) Google Scholar, 42Strater N. Lipscomb W.N. Barrett A.J. Rawlings N.D. Woesner J.F. Handbook of Proteolytic Enzymes. 2nd Ed. Elsevier/Academic Press, San Diego, CA2004: 896-899Crossref
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