Produção Nacional
Revisado por pares
A Solanesyl-diphosphate Synthase Localizes in Glycosomes of Trypanosoma cruzi
2006; Elsevier BV; Volume: 281; Issue: 51 Linguagem: Inglês
10.1074/jbc.m607451200
ISSN1083-351X
AutoresMarcela Ferella, Andrea Montalvetti, Peter Rohloff, Kildare Miranda, Jianmin Fang, Silvia Reina, Makoto Kawamukai, Jacqueline Búa, Daniel Nilsson, Carlos Pravia, Alejandro M. Katzin, María B. Cassera, Lena Åslund, Björn Andersson, Roberto Docampo, Esteban J. Bontempi,
Tópico(s)Biochemical and Molecular Research
ResumoWe report the cloning of a Trypanosoma cruzi gene encoding a solanesyl-diphosphate synthase, TcSPPS. The amino acid sequence (molecular mass ∼ 39 kDa) is homologous to polyprenyl-diphosphate synthases from different organisms, showing the seven conserved motifs and the typical hydrophobic profile. TcSPPS preferred geranylgeranyl diphosphate as the allylic substrate. The final product, as determined by TLC, had nine isoprene units. This suggests that the parasite synthesizes mainly ubiquinone-9 (UQ-9), as described for Trypanosoma brucei and Leishmania major. In fact, that was the length of the ubiquinone extracted from epimastigotes, as determined by high-performance liquid chromatography. Expression of TcSPPS was able to complement an Escherichia coli ispB mutant. A punctuated pattern in the cytoplasm of the parasite was detected by immunofluorescence analysis with a specific polyclonal antibody against TcSPPS. An overlapping fluorescence pattern was observed using an antibody directed against the glycosomal marker pyruvate phosphate dikinase, suggesting that this step of the isoprenoid biosynthetic pathway is located in the glycosomes. Co-localization in glycosomes was confirmed by immunogold electron microscopy and subcellular fractionation. Because UQ has a central role in energy production and in reoxidation of reduction equivalents, TcSPPS is promising as a new chemotherapeutic target. We report the cloning of a Trypanosoma cruzi gene encoding a solanesyl-diphosphate synthase, TcSPPS. The amino acid sequence (molecular mass ∼ 39 kDa) is homologous to polyprenyl-diphosphate synthases from different organisms, showing the seven conserved motifs and the typical hydrophobic profile. TcSPPS preferred geranylgeranyl diphosphate as the allylic substrate. The final product, as determined by TLC, had nine isoprene units. This suggests that the parasite synthesizes mainly ubiquinone-9 (UQ-9), as described for Trypanosoma brucei and Leishmania major. In fact, that was the length of the ubiquinone extracted from epimastigotes, as determined by high-performance liquid chromatography. Expression of TcSPPS was able to complement an Escherichia coli ispB mutant. A punctuated pattern in the cytoplasm of the parasite was detected by immunofluorescence analysis with a specific polyclonal antibody against TcSPPS. An overlapping fluorescence pattern was observed using an antibody directed against the glycosomal marker pyruvate phosphate dikinase, suggesting that this step of the isoprenoid biosynthetic pathway is located in the glycosomes. Co-localization in glycosomes was confirmed by immunogold electron microscopy and subcellular fractionation. Because UQ has a central role in energy production and in reoxidation of reduction equivalents, TcSPPS is promising as a new chemotherapeutic target. Trypanosoma cruzi is the etiological agent of Chagas disease or American trypanosomiasis, which is the leading cause of cardiac death in endemic areas throughout Latin America. More than 18 million people are infected with the parasite, and some 40 million more are at risk (1Urbina J.A. Docampo R. Trends Parasitol. 2003; 19: 495-501Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar).Chemotherapy of Chagas disease is unsatisfactory because of toxicity and lack of efficacy of existing drugs, and it is important to identify enzymes and metabolic processes in T. cruzi that might be potential targets for drug development. One pathway that has been particularly useful for the identification of new targets is the isoprenoid pathway. Enzymes studied so far involved in the synthesis of sterols (2Urbina J.A. Curr. Pharm. Des. 2002; 8: 287-295Crossref PubMed Scopus (192) Google Scholar), farnesyl diphosphate (3Docampo R. Moreno S.N.J. Curr. Drug Targets Infect. Disord. 2001; 1: 51-61Crossref PubMed Scopus (75) Google Scholar), and protein prenylation (4Gelb M.H. Van Voorhis W.C. Buckner F.S. Yokoyama K. Eastman R. Carpenter E.P. Panethymitaki C. Brown K.A. Smith D.F. Mol. Biochem. Parasitol. 2003; 126: 155-163Crossref PubMed Scopus (129) Google Scholar) have been reported to be good drug targets against this parasite. The farnesyl-diphosphate synthase, for example, has been demonstrated to be the target of bisphosphonates that have activity in vitro and in vivo against T. cruzi (3Docampo R. Moreno S.N.J. Curr. Drug Targets Infect. Disord. 2001; 1: 51-61Crossref PubMed Scopus (75) Google Scholar, 5Montalvetti A. Bailey B.N. Martin M.B. Severin G.W. Oldfield E. Docampo R. J. Biol. Chem. 2001; 276: 33930-33937Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 6Urbina J.A. Moreno B. Vierkotter S. Oldfield E. Payares G. Sanoja C. Bailey B.N. Yan W. Scott D.A. Moreno S.N.J. Docampo R. J. Biol. 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Res. 2005; 96: 184-187Crossref PubMed Scopus (37) Google Scholar).Polyprenyl-diphosphate synthases are responsible for chain elongation in isoprenoid biosynthesis and catalyze the sequential condensation of isopentenyl diphosphate (IPP, 3The abbreviations used are: IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; UQ, ubiquinone; SPP, solanesyl diphosphate; SPPS, solanesyl-diphosphate synthase; TcSPPS, T. cruzi solanesyl-diphosphate synthase; PPDK, pyruvate phosphate dikinase; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; HPLC, high-performance liquid chromatography; MOPS, 4-morpholinepropanesulfonic acid; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 3The abbreviations used are: IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; UQ, ubiquinone; SPP, solanesyl diphosphate; SPPS, solanesyl-diphosphate synthase; TcSPPS, T. cruzi solanesyl-diphosphate synthase; PPDK, pyruvate phosphate dikinase; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; HPLC, high-performance liquid chromatography; MOPS, 4-morpholinepropanesulfonic acid; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. C5) with allylic prenyl diphosphates (10Ohnuma S.-I. Hirooka K. Tsuruoka N. Yano M. Ohto C. Nakane H. Nishino T. J. Biol. Chem. 1998; 273: 26705-26713Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). These condensations are catalyzed by a family of prenyltransferases, which are classified into two groups according to the stereochemistry of the E or Z double bond that is formed (10Ohnuma S.-I. Hirooka K. Tsuruoka N. Yano M. Ohto C. Nakane H. Nishino T. J. Biol. Chem. 1998; 273: 26705-26713Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Z-Polyprenyl-diphosphate synthases are used for the synthesis of dolichols for N-linked glycoprotein biosynthesis, Z-polyprenols for peptidoglycan biosynthesis in bacteria, and natural rubber, whereas E-polyprenyl-diphosphate synthases are used for the synthesis of a vast variety of important natural isoprenoids, such as steroids, cholesterol, sesquiterpenes, heme a, dolichols, farnesylated proteins, carotenoids, diterpenes, geranylgeranylated proteins, chlorophylls, and archaebacterial ether-linked lipids (10Ohnuma S.-I. Hirooka K. Tsuruoka N. Yano M. Ohto C. Nakane H. Nishino T. J. Biol. Chem. 1998; 273: 26705-26713Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Long E-polyprenyl-diphosphate synthases producing compounds with chain lengths from C30 to C50 are involved in respiratory quinone biosynthesis (10Ohnuma S.-I. Hirooka K. Tsuruoka N. Yano M. Ohto C. Nakane H. Nishino T. J. Biol. Chem. 1998; 273: 26705-26713Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar).So far, only the genes encoding farnesyl diphosphate (FPP) synthases have been studied in trypanosomatids (5Montalvetti A. Bailey B.N. Martin M.B. Severin G.W. Oldfield E. Docampo R. J. Biol. Chem. 2001; 276: 33930-33937Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 11Montalvetti A. Fernandez A. Sanders J.M. Ghosh S. Van Brussel E. Oldfield E. Docampo R. J. Biol. Chem. 2003; 278: 17075-17083Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). This is despite the presence of ubiquinone 9 (UQ-9), the product of a biosynthetic pathway beginning with the condensation of p-hydroxybenzoic acid and solanesyl diphosphate (SPP, C45), in Leishmania (12Ellis J.E. Setchell K.D.R. Kaneshiro E.S. Mol. Biochem. Parasitol. 1994; 65: 213-224Crossref PubMed Scopus (33) Google Scholar, 13Ranganathan G. Mukkada A.J. Int. J. 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Chait B.T. Menon A.K. J. Biol. Chem. 1991; 266: 19250-19257Abstract Full Text PDF PubMed Google Scholar), labeled precursors (acetate and mevalonate, and mevalonate, respectively) are incorporated into UQ. These results imply the presence of a solanesyl-diphosphate synthase (SPPS) in these parasites.The localization of the trypanosomatid enzymes involved in isoprenoid metabolism has been little studied, although some of them, like the T. cruzi FPP synthase (5Montalvetti A. Bailey B.N. Martin M.B. Severin G.W. Oldfield E. Docampo R. J. Biol. Chem. 2001; 276: 33930-33937Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), bear predicted targeting signals for the glycosomes. Glycosomes are specialized peroxisomes that, like them, contain several enzymes in pathways of ether lipid synthesis, fatty acid β-oxidation, and peroxide metabolism, and, in addition, contain the Embden-Meyerhof segment of glycolysis (19Michels P.A. Moyersoen J. Krazy H. Galland N. Herman M. Hannaert V. Mol. Membr. Biol. 2005; 22: 133-145Crossref PubMed Scopus (56) Google Scholar).In the present study, we report the cloning, sequencing, and heterologous expression of a T. cruzi gene designated TcSPPS that encodes a functional SPPS. The expressed TcSPPS gene could complement the function of the corresponding polyprenyl-diphosphate synthase of Escherichia coli, and the cells produced mainly UQ-9. The kinetic properties of the recombinant TcSPPS were analyzed, and the enzyme was shown to localize in the glycosomes, supporting the role of these organelles in isoprenoid synthesis.EXPERIMENTAL PROCEDURESMaterials—Newborn calf serum, Dulbecco's phosphate-buffered saline, protease inhibitor mixture, dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP), FPP, geranylgeranyl diphosphate (GGPP), and IPP were purchased from Sigma. [4-14C]IPP (57.5 mCi/mmol) was from PerkinElmer Life Sciences. Adsorbosil RP HPTLC plates were from Alltech (Deerfield, IL). Benzonase™ nuclease was from Novagen (Madison, WI). Nickel-nitrilotriacetic acid-agarose was obtained from Qiagen (Valencia, CA). PD-10 desalting column was from Amersham Biosciences. Plasmid and cosmid DNA was obtained using the Wizard miniprep kits (Promega, Madison, WI). PCR products were purified using the Concert kit (Life Technologies, Rockville, MD). Affinity purified T. cruzi SPPS antibodies were obtained as described previously (11Montalvetti A. Fernandez A. Sanders J.M. Ghosh S. Van Brussel E. Oldfield E. Docampo R. J. Biol. Chem. 2003; 278: 17075-17083Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Anti T. brucei pyruvate phosphate dikinase (PPDK)-producing mouse hybridoma culture supernatant was a gift from Frederique Bringaud (University of Bordeaux, France); rabbit anti-TbgGAPDH antibody was provided by Fred Opperdoes (University of Louvain, Belgium); anti-T. brucei vacuolar pyrophosphatase (TbVP1) was a gift from Norbert Bakalara (Ecole Nationale Superiéure de Chimie de Montpellier, France); MitoTracker Red CMXRos, anti-mouse Alexa 488, and anti-rabbit Alexa 546 were from Molecular Probes (Eugene, OR). Co-enzyme Q10 was purchased from Sigma. Co-enzyme Q8 was isolated from E. coli by extraction with hexane and further purification by high-performance liquid chromatography (HPLC) as described by Okamoto and co-workers (20Okamoto T. Fukui K. Nakamoto M. Kishi T. Okishio T. Yamagami T. Kanamori N. Kishi H. Hiraoka E. J. Chromatogr. 1985; 342: 35-46Crossref PubMed Scopus (56) Google Scholar). All solvents were HPLC grade.Culture Methods and Cell Extraction—T. cruzi amastigotes and trypomastigotes (Y strain) were obtained from the culture medium of L6E9 myoblasts as described previously (21Furuya T. Kashuba C. Docampo R. Moreno S.N. J. Biol. Chem. 2000; 275: 6428-6438Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). T. cruzi epimastigotes (Y strain) were grown at 28 °C in liver infusion tryptose medium (22Bone G.J. Steinert M. Nature. 1956; 178: 308-309Crossref PubMed Scopus (132) Google Scholar) supplemented with 10% newborn calf serum. T. cruzi epimastigotes (CL Brener clone) were grown as described before (23Gerez de Burgos N.M. Burgos C. Blanco A. Paulone I. Segura E.L. Acta Physiol. Lat. Am. 1976; 26: 10-19PubMed Google Scholar).DNA Sequencing and Bioinformatics—Sequencing grade DNA was obtained using a Qiagen kit. Sequencing was performed on an ABI 377 using a BigDye Terminator Cycle Sequencing Kit (PerkinElmer Life Sciences), or on a MegaBACE 1000 using the DYEnamic ET dye terminator kit (Amersham Biosciences). Vector primers and the following sequencing primers were used: Fwd (antisense), 5′-CACGTGCCACCATGGCAAAC-3′; Fwd2 (antisense), 5′-CAATGCCTTCTGCCATGTC-3′. Chromatograms were analyzed using Bio Edit software (24Hall T.A. Nucl. Acids Symp. Ser. 1999; 41: 95-98Google Scholar). Homology searches were performed at the NCBI Blast server (25Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69088) Google Scholar), and sequences were aligned using ClustalX VI 1.81. The theoretical molecular weight and isoelectric point were obtained from the ExPASy Server (cn.expasy.org). The superimposed hydrophobicity profiles were calculated using the Kyte-Doolittle hydropathy algorithm (26Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (16999) Google Scholar) at bioinformatics.weizmann.ac.il/hydroph. The presence of a signal peptide was assessed by the SignalP 3.0 software (www.cbs.dtu.dk/) (27Bendtsen J.D. Nielsen H. von Heijne G. Brunak S. J. Mol. Biol. 2004; 340: 783-795Crossref PubMed Scopus (5608) Google Scholar).Hybridization to Cosmid Filters—Cosmid filters from a CL Brener cosmid library were used (28Hanke J. Sanchez D.O. Henriksson J. Åslund L.C. Pettersson U. Frasch A.C.C. Hoheisel J. BioTechniques. 1996; 21: 686-693Crossref PubMed Scopus (29) Google Scholar). The whole coding sequence of the gene was generated by PCR, purified from agarose gels using DEAE membranes (29Dretzen G. Bellard M. Sassone-Corsi P. Chambon P. Anal. Biochem. 1981; 112: 295-298Crossref PubMed Scopus (502) Google Scholar), and 30 ng was labeled with [α-32P]dCTP by random priming (Prime a Gene, Promega). Cosmid filters were prehybridized and hybridized as described (28Hanke J. Sanchez D.O. Henriksson J. Åslund L.C. Pettersson U. Frasch A.C.C. Hoheisel J. BioTechniques. 1996; 21: 686-693Crossref PubMed Scopus (29) Google Scholar), using a Micro 4 oven (Hybaid, UK). Two of the positive clones (20i8 and 69i5) were further studied.Hybridization to Pulsed Field Gel Electrophoresis and Northern Filters—Chromosomes from the T. cruzi CL Brener clone were separated by pulsed field gel electrophoresis using different running conditions (30Búa J. Garcia G.A. Galindo M. Galanti N. Ruiz A.M. Medicina (B Aires). 1999; 59: 11-17PubMed Google Scholar) and transferred to nylon filters (kindly provided by Mario Galindo, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile). Schizosaccharomyces pombe and Saccharomyces cerevisiae chromosomes were used as markers (Bio-Rad). Total RNA from epimastigotes was isolated using an SV Prep Total RNA kit (Sigma), according to the manufacturer's instructions. For Northern blot analysis, epimastigotes total RNA was subjected to electrophoresis in 1% agarose gel containing 1× MOPS buffer and 6.29% (v/v) formaldehyde after boiling for 10 min in 50% (v/v) formamide, 1× MOPS buffer, and 5.9% (v/v) formaldehyde. The RNA was transferred to a Hybond-N filter. A T. cruzi probe encoding the 19-kDa cyclophilin, TcCyP19, was used as a positive control.RT-PCR—T. cruzi CL Brener epimastigote mRNA was isolated by using a QuickPrep Micro mRNA kit (GE Healthcare Bio-Sciences), and RT-PCR was performed with the Access RT-PCR System (Promega) using the following primers at 1 μm final concentration: Miniexon (sense), 5′-AACGCTATTATTGATACAGTTTCTGTACTATATTG-3′, Fwd2 (antisense). As an internal positive control TcCyP19 was amplified.Southern Blot Analysis and Genome Organization—T. cruzi CL Brener genomic DNA (3 μg) was digested by NcoI and AatII (Fermentas), separated on a 1% agarose gel and transferred to Hybond-N+ membrane (Amersham Biosciences). Efficient transfer was confirmed by methylene blue staining (Sigma). Probe generation and target detection was performed using the Gene Images AlkPhos Direct Labeling & Detection system (Amersham Biosciences) following the manufacturer's instructions. Blast searches of the T. cruzi genome (www.genedb.org/genedb/tcruzi/) were performed with TcSPPS nucleotide sequence. In silico restriction analysis was performed at The Sequence Manipulation Suite web site (bioinformatics.org/sms/).Expression and Purification of TcSPPS from E. coli—For expression in E. coli, the entire coding sequence of the TcSPPS gene was amplified by PCR using primers (PS5, 5′-CCGGATCCATGCTGAAAACAGGCCTTT-3′; PS3, 5′-CCAAGCTTCATACTTGTCGCGTTAAAA-3′) that introduced BamHI and HindIII restriction sites for convenient cloning into the expression vector pET-28a+ to yield pET-TcSPPS. The joining region was sequenced for confirmation. E. coli BL21(DE3) bacterial cells transformed with pET-TcSPPS were induced, and the recombinant protein was purified by nickel-nitrilotriacetic acid-agarose, following the standard Qiagen procedure. The eluted fraction was desalted with a PD-10 desalting column. Proteins were quantified by the Bradford method (31Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213377) Google Scholar) with bovine serum albumin as a standard and the absence of protein contaminants was checked by SDS-PAGE.Measurement of Activity and Product Analysis—Enzyme activity was measured by determination of the amount of [4-14C]IPP incorporated into butanol-extractable polyprenyl diphosphates. Because removal of the polyhistidine tag resulted in complete loss of activity of other prenyltransferases from trypanosomatids (5Montalvetti A. Bailey B.N. Martin M.B. Severin G.W. Oldfield E. Docampo R. J. Biol. Chem. 2001; 276: 33930-33937Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 11Montalvetti A. Fernandez A. Sanders J.M. Ghosh S. Van Brussel E. Oldfield E. Docampo R. J. Biol. Chem. 2003; 278: 17075-17083Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), this was not done. The standard assay mixture contained, in a total volume of 100 μl, 100 mm Tris-HCl buffer (at physiological pH 7.4), 1 mm MgCl2, 1% (v/v) Triton X-100, 100 μm [4-14C]IPP (1 μCi/μmol), allylic substrate (400 μm DMAPP, 400 μm GPP, 30 μm FPP, or 50 μm GGPP), and 0.5-3 μg of the purified protein. The mixture was incubated at 37 °C for 30 min, and the reaction was stopped by chilling quickly in an ice bath. The reaction products were then extracted with 1 ml of 1-butanol saturated with water. The 1-butanol layer was washed with water saturated with NaCl, and radioactivity in the butanol extract was determined with a liquid scintillation counter. One unit of enzyme activity was defined as the activity required to incorporate 1 nmol of [4-14C]IPP into extracted product in 1 min. To identify the reaction products after the enzymatic reaction, the radioactive prenyl diphosphates in the mixture were hydrolyzed to the corresponding alcohols with potato acid phosphatase as described before (32Koyama T. Fujii H. Ogura K. Methods Enzymol. 1985; 110: 153-155Crossref PubMed Scopus (41) Google Scholar). The alcohols were extracted with n-pentane and analyzed by TLC on a reversed-phase Adsorbosil HPTLC plate with a solvent system of acetone/water (12:1, v/v). The positions of authentic standards were visualized by iodine vapors. The radioactivity was visualized by autoradiography.Glycosome Enrichment—T. cruzi CL Brener epimastigotes (∼109 cells) were centrifuged for 10 min at 2,000 × g, and washed twice in TEDS buffer (25 mm Tris-HCl, pH 7.4, 1 mm EDTA, 250 mm sucrose, 1 mm dithiothreitol) containing protease inhibitors (P8340, Sigma). After freezing at -80 °C for 20 min and thawing at 37 °C, cells were centrifuged and resuspended in homogenization buffer (250 mm sucrose, 1 mm EDTA, 0.1% v/v ethanol, 5 mm MOPS, pH 7.2, and protease inhibitors). The parasites were grinded in a pre-chilled mortar with 1× wet weight silicon carbide until no intact cells were observed under the light microscope. The lysate was centrifuged at 100 × g for 10 min to remove the silicon carbide, which was washed in homogenization buffer, and both supernatants were combined (Fraction A). A centrifugation at 1,000 × g for 15 min was performed to remove the nuclei, and the supernatant (Fraction B) was centrifuged at 33,000 × g to enrich in glycosomes. The supernatant was Fraction C (cytoplasm) and the pellet (Fraction D) was the glycosomal enriched fraction. The whole procedure was performed twice. Protein concentration of each fractionation step was measured by a colorimetric assay (Protein Assay, Bio-Rad).Western Blot Analysis—To investigate for protein expression in the different stages, total trypanosome proteins (30 μg of protein/lane) were separated by SDS-polyacrylamide gel (10%) and transferred to nitrocellulose. Membranes were probed with 1:3,000 dilution of a rabbit anti-SPPS and then with horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:10,000). Immunoblots were developed using the ECL™ chemiluminescent detection kit (Amersham Biosciences).For Western blot analysis of the different subcellular fractions, the blots were sequentially probed with a rabbit anti-TbgGAPDH antibody as a marker for glycosomes at a dilution of 1:3,000, and after a stripping step, with rabbit anti-TcSPPS antibody.Complementation Analysis—The TcSPPS was tested for its capacity to complement the ispB gene of E. coli. Strain KO229, whose essential ispB gene was disrupted and complemented by the ispB expression vector pKA3 (spectinomycin-resistant), was subjected to a plasmid-swapping experiment (33Okada K. Minehira M. Zhu X. Suzuki K. Nakagawa T. Matsuda H. Kawamukai M. J. Bacteriol. 1997; 179: 3058-3060Crossref PubMed Google Scholar). Because the pET construct would not be inducible in strain KO229, the gene was subcloned into pQE30 vector (pQE-TcSPPS). After transformation with pQE-TcSPPS, the colonies were grown and passaged for several days in LB medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride, pH 7.5) supplemented with ampicillin (to select pQE-TcSPPS-carrying colonies) and isopropyl 1-thio-β-d-galactopyranoside (to induce expression of the His6-TcSPPS fusion protein). Isolated colonies were checked for ampicillin resistance and spectinomycin sensitivity.Ubiquinone Extraction and Measurement—Ubiquinone was extracted as previously described (34Okada K. Kainou T. Tanaka K. Nakagawa T. Matsuda H. Kawamukai M. Eur. J. Biochem. 1998; 255: 52-59Crossref PubMed Scopus (83) Google Scholar). The crude extract of UQ was analyzed by normal-phase TLC with authentic standard UQ-10. Normal-phase TLC was carried out on a Kieselgel 60 F254 plate (Merck) with benzene/acetone (93:7, v/v). The band containing UQ was collected from the TLC plate following UV visualization and extracted with chloroform/methanol (1:1, v/v). Samples were dried and re-dissolved in ethanol. The purified UQ was further analyzed by HPLC with ethanol as the solvent.Fluorescence Microscopy—For co-localization with a glycosomal marker, T. cruzi Y strain epimastigotes slides were prepared as previously described (35Rohloff P. Montalvetti A. Docampo R. J. Biol. Chem. 2004; 279: 52270-52281Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Antibody concentrations were as follows: affinity-purified rabbit anti-TcSPPS antibody at 1:4,000; supernatant from an anti-TbPPDK producing mouse hybridoma culture at 1:10; anti-mouse Alexa 488 at 1:1,000; anti-rabbit Alexa 546 at 1:1,000. For co-localization studies with MitoTracker and the vacuolar pyrophosphatase, epimastigotes were fixed for 30 min in 4% paraformaldehyde in 0.1 m cacodylate buffer, washed twice in Dulbecco's phosphate-buffered saline, pH 7.2, adhered to poly-l-lysine-coated coverslips, and permeabilized for 3 min with 0.3% Triton X-100. Cells were blocked for 30 min in 50 mm NH4Cl and 3% bovine serum albumin in phosphate-buffered saline, pH 8.0, and incubated for 1 h with polyclonal primary antibodies raised against T. cruzi SPPS (1:1000), and monoclonal antibodies raised against T. brucei VP1 (1:200). For mitochondrial staining, cells were previously incubated for 30 min in culture medium containing 100 nm MitoTracker before fixation. Cells were then washed in 3% bovine serum albumin, incubated with secondary antibodies anti-mouse Alexa 488 (1:1,000), anti-rabbit Alexa 488, and anti-rabbit Alexa 546 (1:1,000) and mounted in prolong Antifade. Cells were observed in a Deltavision fluorescence microscope. Images were recorded with a Photometrics CoolSnap HQ camera and deconvolved for 15 cycles using Softwarx deconvolution software.Immunogold Electron Microscopy—Immunogold electron microscopy experiments were performed as described previously (35Rohloff P. Montalvetti A. Docampo R. J. Biol. Chem. 2004; 279: 52270-52281Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) using the rabbit anti-SPPS antibody (1:100) and a monoclonal antibody against T. brucei pyruvate-phosphate dikinase (1:10). After washing, the grids were incubated with 18 nm colloidal gold-AffiniPure-conjugated anti-rabbit IgG (H + L) and 12 nm colloidal gold-conjugated goat anti-mouse IgG (H + L). Images were acquired on a Phillips CM-200 transmission electron microscope operating at 120 kV.RESULTSIdentification of T. cruzi SPPS—We determined the complete sequence of the T. cruzi cDNA clone TENU4155 (accession number AW324852) (36Porcel B.M. Tran A.N. Tammi M. Nyarady Z. Rydaker M. Urmenyi T.P. Rondinelli E. Pettersson U. Andersson B. Aslund L. Genome Res. 2000; 10: 1103-1107Crossref PubMed Scopus (37) Google Scholar), which showed similarities to polyprenyl synthases. The sequence surrounding the first ATG complied with the published rules for start codons in protozoa (37Yamauchi K. Nucleic Acids Res. 1991; 19: 2715-2720Crossref PubMed Scopus (64) Google Scholar). To obtain further upstream sequences a cDNA probe was hybridized to high density cosmid filters, and the sequence obtained from two of the positive clones (20i8 and 69i5) with the forward primer displayed a stop codon in the same reading frame as the first putative ATG, confirming the protein coding region. This sequence has been submitted to the GenBank™ under the accession number AF282771.Translation of the open reading frame of 1092 bp yielded a polypeptide of 363 amino acids with a predicted molecular mass of 39 kDa and an isoelectric point of 6.01. A small residue (Ala) is found at position -5 before the first aspartate-rich motif. This position is diagnostic, determining the final product length (for a review, see Ref. 38Liang P.-H. Ko T.-P. Wang A.H.-J. Eur. J. Biochem. 2002; 269: 3339-3354Crossref PubMed Scopus (353) Google Scholar). Bulky amino acids do not allow nascent long chains to extend further inside the hydrophobic cavity of the enzyme. A BLAST search of the protein data base showed that the amino acid sequence from T. cruzi shared up to 38% identity and up to 61% similarity with other polyprenyl synthases. Considering specifically the human homologue (accession number NP_055132), the identity reached 33%.The amino acid sequence from the T. cruzi enzyme was aligned with other representative polyprenyl synthases (Fig. 1A). All the conserved motifs involved in catalysis or binding (regions I-VII) identified in other polyprenyl synthases (39Koyama T. Biosci. Biotechnol. Biochem. 1999; 63: 1671-1676Crossref PubMed Scopus (75) Google Scholar) a
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