From the Division of Biological Chemistry and Molecular Microbiology, The Wellcome Trust Biocentre, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom
Received for publication, March 10, 2003 , and in revised form, April 2, 2003.
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ABSTRACT |
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INTRODUCTION |
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AEP was first identified as a minor component in acid hydrolysates of lipid and glycolipid extracts of the intracellular protozoan parasite Trypanosoma cruzi (5), the causative agent of Chagas' disease. Although dismissed as a minor component of lipopeptidophosphoglycan (now known as glycosylinositolphospholipid (GIPL)), subsequent studies indicate that AEP may be a universal component of the dense coat of glycoconjugates (mucins and GIPLs) that cover the surface of different stages of the parasite (68). Mucins are highly glycosylated proteins that are anchored in the plasma membrane via a glycosylphosphatidylinositol (GPI) moiety. Although the lipid moiety of GPI anchors varies throughout the life cycle, most GPIs and GIPLs share a common Man4(AEP)GlcN-Ins-PO4 core (6) (Fig. 1B). AEP can also substitute for phosphoethanolamine in the linkage between the GPI anchor and the polypeptide chain of mucins, although this is not obligatory. In contrast, an additional AEP substituent on the O-6 of glucosamine of T. cruzi GPI anchors appears to be universal (6) and has also been identified in glycolipids from other members of the Kinetoplastida, including the dixenic bat trypanosome Trypanosoma dionisii (9) and the monoxenic insect parasites Leptomonas samueli (10) and Herpetomonas samuelpessoai (11). However, the most abundant surface glycoconjugate of Leishmania (lipophosphoglycan, a GPI-anchored polymer of the repeating disaccharide phosphate units) does not contain AEP (12). Likewise, AEP is absent from the African trypanosome, Trypanosoma brucei (5), where ethanolamine is an integral component of the variant surface glycoprotein (13).
Apart from the demonstration that 32P can be incorporated into AEP (5), the biosynthesis of this intermediate has not been studied. The identification of pyruvate phosphate dikinase in T. cruzi (14), which catalyzes formation of phosphoenolpyruvate from pyruvate, prompted us to search for other genes in the biosynthetic pathway to AEP. Here we report the isolation and functional expression of PEP mutase, some of its kinetic properties, and its subcellular localization in T. cruzi.
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EXPERIMENTAL PROCEDURES |
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Cloning of PEP Mutase Gene from T. cruziA BLAST search of conserved sequence regions of PEP mutase from M. edulis and Tetrahymena pyriformis against the T. cruzi genome data base yielded a partial sequence (accession number AQ445225 [GenBank] .2) with significant homology to other PEP mutases. This partial PEP mutase gene fragment was amplified using oligonucleotides 5'-GGGGTTCCGCGACACAACGAAGCG and 3'-CTGCATGGCGGCAATGCAGGCCC under the following conditions: denaturation at 95 °C for 10 min followed by 30 cycles of denaturation at 95 °C for 1 min, annealing at 50 °C for 1 min, and extension at 72 °C for 1 min followed by a final extension at 72 °C for 10 min. The PCR product was cloned into the Novagen vector ZEROBLUNT TOPO. The fluorescein-labeled PEP mutase PCR fragment was used as a probe in the initial isolation and characterization of this gene. A size-selected library (24 kilobases) was constructed in pUC18 with genomic DNA digested with HindIII and SacI. Positive clones were identified from a colony screen using the labeled probe and found to contain the same size insert. One of these was sequenced using primers designed to the known partial PEP mutase gene sequence, and the full-length gene was obtained.
For expression studies the open reading frame of PEP mutase was amplified by PCR as described above using the primers PEPM-pET-15bF, GGCTCGAGATGCGTCACTGCTGTGGTCTG, and PEPM-pET-15bR, GGCTCGAGTTATTTCTTCGGCAAGTACATTTCC, engineered with XhoI sites in the 5' and 3' primers respectively, for cloning into the Invitrogen E. coli expression vector pET15b.
Southern Blot AnalysisT. cruzi genomic DNA (5 µg) was digested with selected restriction endonucleases (BamHI, SacI, HindIII, and NdeI), separated by gel electrophoresis using a 0.8% agarose gel, and transferred to a positively charged nylon membrane (Hybond N+, Amersham Biosciences). Hybridization and signal detection were performed using the Gene Images labeling and detection kit (Amersham Biosciences) following the manufacturer's recommendations.
Western Blot AnalysisTotal extracts of T. cruzi and other trypanosomatids (1 x 106 parasites per lane) and M. edulis (5 µg) were fractionated by electrophoresis on 412% gradient SDS-PAGE gels (18). Immunoblot analysis was performed essentially as described (19) using polyclonal antisera to PEP mutase and GAPDH at a dilution of 1:50. Blots were developed by chemiluminescence following the manufacturer's instructions (ECL kit, Amersham Biosciences).
Soluble Expression of Recombinant PEP MutaseA 6-liter culture of BL21(DE3)pLysS/pET15b-PepM, derived from a single colony, was grown at 37 °C with vigorous agitation in Terrific Broth containing 50 µg ml1 carbenicillin and 12.5 µg ml1 chloramphenicol. When the culture reached an A600 of 0.8, the culture was cooled to 25 °C, agitation was reduced to 100 rpm, and the culture was induced with a final concentration of 0.5 mM isopropyl-
-D-thiogalactopyranoside. Cultures were grown for 16 h and harvested by centrifugation. Cells were washed with 20 mM Tris, pH 8.0, and lysed in 50 ml of breaking buffer (20 mM Tris, pH 8.0, 0.5 M NaCl, 5 mM MgCl2, 100 µg ml1 DNase I and protease inhibitor mixture, Roche Applied Science) by flash-freezing in an ethanol/dry ice bath followed by rapid thawing and bead beating. Cell debris was separated and discarded after centrifugation at 30,000 x g for 30 min at 4 °C.
Purification and Properties of Recombinant PEP Mutase Protein The supernatant containing soluble protein was diluted 2-fold with 20 mM Bis-Tris propane, 20 mM Tris, pH 7.4, 0.5 M NaCl, passed through a 0.2-µm Steriflip filter, and loaded onto a nickel-chelating Sepharose high performance column (Amersham Biosciences) pre-equilibrated with the same buffer. Protein was eluted using a linear gradient from 0 to 1 M imidazole, and active fractions were pooled, dialyzed against phosphate-buffered saline (10 mM phosphate buffer, 2.7 mM KCl, 137 mM NaCl, pH 7.4), and digested with human thrombin (50 µg ml1) for 2 h at 25 °C to remove the hexahistidine (His6) tag. The sample was then dialyzed for 2 h in 20 mM Bis-Tris propane, 20 mM Tris, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol, and loaded onto a 6-ml ResourceQ column (Amersham Biosciences). Protein was eluted with a linear gradient from 0 to 0.5 M NaCl, and the active fractions were pooled and dialyzed against 50 mM (K+) Hepes, pH 8.0, containing 1 mM dithiothreitol and 0.01% sodium azide. Aliquots of 50 µl were dispensed, rapidly frozen, and stored at 80 °C. Under these conditions the enzyme lost less than 10% of its activity over 4 months.
Native Mr was determined by Superdex 200 column chromatography (Amersham Biosciences) against gel filtration standards (Bio-Rad). Molecular mass was determined by matrix-assisted laser desorption ionization time-of-flight spectroscopy in linear mode using sinapinic acid as a matrix on a Voyager-DE STR mass spectrometer (PerSeptive Biosystems). Cross-linking experiments were conducted using the BS3 cross-linker (bis[sulfosuccinimidyl]suberate, Pierce) following the manufacturer's instructions.
Kinetic Analysis of PEP MutaseThe pH optimum of the enzyme was determined over the pH range 510 using a mixed buffer system of 0.1 M CHES, 0.1 M MES, 0.1 M HEPPS. Enzyme activity was determined using a continuous spectrophotometric assay at 340 nm and 25 °C in which ATP synthesis was coupled through pyruvate kinase and lactate dehydrogenase to the oxidation of NADH. Each 1-ml assay contained 50 mM (K+) HEPES, pH 8.0, 0.5 mM dithiothreitol, 0.2 mM NADH, 1 mM ADP, 5 mM MgSO4, 16 units of pyruvate kinase, and 27 units of lactate dehydrogenase (both enzymes as solutions in 50% glycerol from Roche Applied Science), 88 nM T. cruzi PEP mutase, and 1 mM phosphonopyruvate. The Km for phosphonopyruvate was determined using this method by varying phosphonopyruvate concentration in the presence of 5mM MgSO4 or5mM magnesium acetate. Concentrations of phosphonopyruvate were standardized by spectrophotometric assay in the presence of excess PEP mutase. Protein concentration was determined based on the calculated extinction coefficient at 280 nm (34850 M1 cm1). This method yields a 1.5-fold higher protein concentration than that determined by the bicinchoninic acid method (Pierce). One unit of enzyme activity is defined as 1µmol of NADH oxidized/min. Because Mg2+ is required for the coupled assay, the requirement for divalent metal ions was determined using a direct assay after the formation of phosphoenolpyruvate. Each 1-ml assay contained 50 mM (K+) HEPES, pH 8.0, 0.5 mM dithiothreitol, 450 nM T. cruzi recombinant PEP mutase, 1 mM phosphonopyruvate, and varying amounts of MgSO4. The production of phosphoenolpyruvate was assayed by the increase in absorbance at 233 nm using a = 1.5 mM1cm1 (20). Data were fitted by nonlinear regression analysis to either the Michaelis-Menten equation (for phosphonopyruvate) or the Hill equation (for Mg2+) using the computer program GraFit.
Sub-cellular FractionationThe following procedures were performed at 4 °C. T. cruzi epimastigotes were centrifuged and washed twice in STE buffer (0.32 M sucrose, 25 mM Tris-HCl, and 1 mM EDTA, pH 7.8). Cells were mixed with silicon carbide to form a paste and disrupted by grinding with a pestle and mortar (21). Grinding was continued until 90% of the cells were lysed as viewed by phase-contrast microscopy. The suspension was diluted 510-fold in STE buffer and briefly centrifuged for 3 min at 100 x g. The pellet was washed once in STE buffer and centrifuged. The combined supernatants were centrifuged for 10 min at 1000 x g to remove nuclei and unbroken cells. The resultant supernatant was then centrifuged at 14,500 x g for 10 min. The pellet obtained contained the large granule fraction. The supernatant was spun for 1 h at 139,000 x g; the resulting supernatant contained the cytosolic fraction. The pellet was dissolved in STE buffer and contained the small granule or microsomal fraction.
Production of T. cruzi PEP Mutase AntibodyAntiserum was raised in mice against recombinant T. cruzi PEP mutase (100 µg). The initial injection was emulsified in complete Freund adjuvant and the second in incomplete Freund adjuvant.
Immunolocalization StudiesMid-log phase epimastigotes were airdried onto microscope slides and fixed in 4% paraformaldehyde in PBS (0.15 M NaCl, 5 mM potassium-phosphate buffer, pH 7.4) for 10 min at room temperature followed by methanol at 20 °C for 2 min. Slides were then incubated in PBS, 1% saponin, and 1 mg ml1 heat-treated RNase for 30 min followed by blocking in 5% fetal calf serum, PBS for 5 min. The slides were then incubated in anti-T. cruzi PEP mutase diluted 1:50 in PBS for 1 h at room temperature in a dark humid chamber. After washing in PBS, slides were incubated for 1 h in fluorescein isothiocyanate-conjugated goat anti-mouse secondary antibody diluted 1:50 in PBS. Slides were washed again, incubated in 4,6-diamidino-2-phenylindole (DAPI) (1 µg ml1) for 2 min followed by a further wash in PBS, and mounted in Mowiol containing phenylenediamine (1 µg ml1).
For double-labeling experiments using the anti-GAPDH (rabbit) and anti-PEP mutase (mouse), slides were treated as above except the primary antibody was a mixture of 1:100 and 1:50 dilution, respectively, of each antibody in PBS, and the secondary was a mixture of anti-mouse fluorescein isothiocyanate and anti-rabbit TRITC.
Double-labeling experiments of monoclonal antibody to vacuolar type protein pyrophosphatase (V-H+-PPiase) and anti-PEP mutase used ZenonTM One mouse IgG1 labeling kit (Molecular Probes) to label anti-V-H+-PPiase, as instructed by the manufacturers. After staining with the anti-V-H+-PPiase, slides were stained with anti-PEP mutase as above.
Mitochondrial labeling of T. cruzi was conducted using MitoTracker Red 580 (Molecular Probes) as instructed by the manufacturers. Slides were double-labeled with anti-PEP mutase as described above.
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RESULTS |
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Expression and Purification of Recombinant T. cruzi PEP MutaseT. cruzi PEPM was sub-cloned into the expression vector pET15b containing an N-terminal His6 tag and expressed in E. coli. The recombinant protein was purified on a nickel-chelating Sepharose high performance column, digested with thrombin to remove the His6 tag, and further purified by anionic exchange chromatography (Fig. 3A). The final yield of recombinant protein was 3 mg liter1 of culture. Matrix-assisted laser desorption ionization time-of-flight analysis of recombinant PEP mutase revealed a nominal molecular mass of 33,832 Da that correlates well with the predicted molecular mass of 33,482 Da after cleavage with thrombin. Migration on SDS-PAGE shows an Mr of
36,800 (Fig. 3A), similar to that reported for T. pyriformis (33,000) (4) and M. edulis (34, 000) (20). On gel filtration chromatography T. cruzi PEP mutase migrates as a single symmetrical peak corresponding to an Mr of 86,000 (n = 2.6, mean of 2 experiments), suggestive of either a homodimer (as reported for the T. pyriformis enzyme (23)) or a novel trimeric species. To test this possibility the native enzyme was cross-linked with BS3 and analyzed by SDS-PAGE (Fig 3C). The resulting gel indicates that the majority of the PEP mutase is recovered as a homotetramer with only trace amounts of trimer and dimer. Identical results were obtained when PEP mutase was held constant (1.5 mg ml1) and cross-linker was varied (0.252.0 mM) over 0.54 h (data not shown). Crystallographic studies on the T. cruzi enzyme also reveal a tetrameric arrangement2 as reported for the enzyme from M. edulis (22). The reason for the anomalous behavior on gel filtration is not known.
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Kinetic Characterization of Recombinant PEP Mutase ProteinThe pH profile for PEP mutase enzyme activity follows a bell-shaped curve with a pH optimum of 8 and apparent pKa values of 7.05 ± 0.09 and 9.12 ± 0.09 (data not shown). Under optimal conditions (pH 8.0, 1 mM phosphonopyruvate, 5 mM MgSO4) the specific activity of the purified recombinant T. cruzi enzyme (54 units mg1) was similar to the enzymes from M. edulis (90 units mg1 (20)) and T. pyriformis (22 units mg1 (23) or 113 units mg1, (24)). Using the coupled assay, the enzyme followed Michaelis-Menten kinetics with phosphonopyruvate (Fig. 4A), yielding an apparent Km value of 7.5 ± 0.6 µM, intermediate between 10 and 3.3 µM reported at pH 7.5 for T. pyriformis and M. edulis, respectively (20, 25). A similar Km value (8.4 ± 1.1 µM) was obtained with 5 mM magnesium acetate replacing 5 mM MgSO4 (not shown). The kcat values for the T. cruzi enzyme (12.4 ± 0.2 s1 with MgSO4; 11 ± 0.3 s1 with magnesium acetate) was somewhat lower than those for T. pyriformis (150 s1) and M. edulis (34 s1).
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The requirement for divalent metal ions (1 mM, all as chloride salts) was determined by measuring the thermodynamically favored direction of the reaction, i.e. formation of phosphoenolpyruvate at 233 nm. The enzyme displayed a pronounced requirement for divalent metal ions with <1% activity in the absence of Mg2+. The extent of activation was identical to that reported for the M. edulis enzyme (20), with the order being Mg2+ > Co2+ > Mn2+ > Zn2+ > Ni2+, with Ca2+ showing no activation (not shown). Sulfate and other oxyanions have been reported to be inhibitory with the T. pyriformis enzyme (25). However, with the T. cruzi PEP mutase is not inhibitory when compared with Cl anion. In kinetic studies where MgSO4 was the variable substrate, double-reciprocal plots were nonlinear (not shown). However, the data fitted well to the Hill equation yielding an S0.5 of 0.40 ± 0.04 µM, n = 0.46 ± 0.03, kcat 19 ± 0.4 s1, consistent with a negative co-operative effect between subunits (Fig. 4B). Co-operative behavior has not been reported with Mg2+ for PEP mutase from M. edulis (Km 4 µM) (20) or T. pyriformis (Km 6 µM) (23).
Species Distribution and Intracellular LocationWestern analysis using antibodies raised against the T. cruzi recombinant protein indicated that PEP mutase is constitutively expressed in the insect epimastigote, trypomastigote, and mammalian amastigote stages of the life cycle (data not shown). The antiserum also detects a 34-kDa band in M. edulis extracts but not in more closely related trypanosomatid species, suggesting that PEP mutase is absent in T. brucei, Crithidia fasciculata, Leishmania major, and Leishmania donovani (Fig. 5A). Two other trypanosomatids, T. dionisii and Herpetomonas muscarum, which contain AEP glycolipids, also show a band corresponding to T. cruzi PEP mutase (Fig. 5A).
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Subcellular fractionation studies and immunolabeling experiments indicate that PEP mutase has a dual location in the cell. Western blots of subcellular fractions show that the enzyme is present in the cytosol and large granule fraction, which is enriched in mitochondria, glycosomes, and other large vesicular organelles (Fig. 5B). This dual location is unlikely to be a preparative artifact since antiserum to T. cruzi trypanothione synthetase (26) showed a largely cytosolic location.
Immunofluorescence staining shows a diffuse cytosolic staining together with a punctate pattern in the cells (Fig. 6). This distribution is reminiscent of glycosomal staining even though the amino acid sequence does not show a type I or type II glycosomal targeting signal (27). Staining with anti-serum to glycosomal GAPDH is also punctate, but examination of the merged image indicates that anti-PEP mutase does not co-localize exactly with the glycosome (Fig. 6A). Double-labeling with anti-PEP mutase antibody and Mitotraker indicates that PEP mutase is not located to the mitochondrion either (Fig. 6B). The presence of glycoconjugates in acidocalcisomes has been reported recently (30). However, double labeling of slides with anti-PEP mutase and anti-H+-PPiase, an acidocalcisome marker, clearly do not co-localize (Fig. 6C). These results suggest that PEP mutase is not specifically localized to the glycosome, mitochondria, or acidocalcisome, but to some other unidentified organelle.
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DISCUSSION |
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Phosphonolipids constitute 23% of total phospholipids in T. pyriformis, and it has been proposed that surface phosphonolipids are important for protection against phospholipases secreted by itself or by other organisms (29). However, this is unlikely to be the case for T. cruzi since phosphonolipids constitute a minor fraction (0.34%) of total phospholipids (5). However, the occurrence of PEP mutase in trypanosomatids correlates exactly with those parasites that have been reported to possess AEP glycolipids, and thus, one function of the pathway could be to supply AEP for glycolipid synthesis. Although AEP and ethanolamine phosphate can be used interchangeably to link the GPI anchor to the polypeptide chain of mucins, the glucosamine moiety is exclusively substituted by AEP (6). Thus, the universality of AEP modification of the 6-O position of glucosamine in GIPLs and mucins suggests an essential role for this moiety in these surface glycoconjugates. Because they are thought to play a role in attachment, invasion, and intracellular survival in the parasite, the AEP moiety may be an important determinant in one or more of these functions. Likewise, GPI anchors from T. cruzi, which show pronounced proinflammatory activity, play an important role in activation of innate immunity during infection (7, 8). Conceivably, the AEP substituent could protect these molecules from degradation in the gut of the insect vector or the cytoplasm of vertebrate host cells. Gene knockout studies are planned to examine these possibilities and to evaluate whether this pathway could represent a therapeutic target since it is absent in the mammalian host.
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FOOTNOTES |
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* This work was supported by a program grant from the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Current address: Centre for Carbohydrate Chemistry, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, UK.
A Wellcome Trust Principal Research Fellow. To whom correspondence should be addressed. Tel.: 44-1382-345155; Fax: 44-1382-345542; E-mail: a.h.fairlamb{at}dundee.ac.uk.
1 The abbreviations used are: PEP, phosphoenolpyruvate; PEPM, PEP mutase; GIPL, glycosylinositolphospholipid; GPI, glycosylphosphatidylinositol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CHES, 2-(cyclohexylamino)ethanesulfonic acid; MES, 4-morpholineethanesulfonic acid; HEPPS, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid; PBS, phosphate-buffered saline; AEP, 2-aminoethylphosphonate; His6, hexahistidine; V-H+-PPiase, vacuolar type proton pyrophosphatase; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; TRITC, tetramethylrhodamine isothiocyanate.
2 D. J. McNulty, M. Sarkar, A. H. Fairlamb, and D. M. F. van Aalten, unpublished results.
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ACKNOWLEDGMENTS |
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REFERENCES |
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