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Purification and Partial Characterization of Trypanosoma cruzi Triosephosphate Isomerase SC Bourguignon/^+, MN Meirelles*, RS Pacheco**, S Giovanni De Simone/*/^++
Departamento de Biologia Celular e Molecular, Instituto de Biologia,
Universidade Federal Fluminense, Niteroi, RJ, Brasil Suported in part by CNPq, Finep and Fiocruz. Received 20 May 1997; Accepted 5 November 1997
Code Number:OC98042 The enzyme triosephosphate isomerase (TPI, EC 5.3.1.1) was purified from extracts of epimastigote forms of Trypanosoma cruzi. The purification steps included: hydrophobic interaction chromatography on phenyl-Sepharose, CM-Sepharose, and high performance liquid gel filtration chromatography. The CM-Sepharose material contained two bands (27 and 25 kDa) with similar isoelectric points (pI 9.3-9.5) which could be separated by gel filtration in high performance liquid chromatography. Polyclonal antibodies raised against the porcine TPI detected one single polypeptide on western blot with a molecular weight (27 kDa) identical to that purified from T. cruzi. These antibodies also recognized only one band of identical molecular weight in western blots of several other trypanosomatids (Blastocrithidia culicis, Crithidia desouzai, Phytomonas serpens, Herpertomonas samuelpessoai). The presence of only one enzymatic form of TPI in T. cruzi epimastigotes was confirmed by agarose gel activity assay and its localization was established by immunocytochemical analysis. The T. cruzi purified TPI (as well as other trypanosomatid' TPIs) is a dimeric protein, composed of two identical subunits with an approximate mw of 27,000 and it is resolved on two dimensional gel electrophoresis with a pI of 9.3. Sequence analysis of the N-terminal portion of the 27 kDa protein revealed a high homology to Leishmania mexicana and T. brucei proteins. Key words: Trypanosoma cruzi - triosephosphate isomerase - purification - sequence Chagas' disease is caused by the protozoan hemoflagellate Trypanosoma cruzi. This and other kinetoplastids are distinguished from diverse eucaryotic cells by the presence of glycosomes, a microbody-like organelle containing glycolytic and glycerol metabolic enzymes (Opperdoes 1987, Michels 1989, Blattner et al. 1992). Corresponding glycolytical isoenzymes were also found to be present in the cytoplasm of most protozoa studied so far. Most of these proteins are encoded by different genes (Sullivan et al. 1985, Huang et al. 1989) and the glycosomal enzymes are synthesised in the cytoplasm by free ribosomes and imported post-translationally without apparent cleavage or modification. Several of these glycosomal and cytosolic isoenzymes have been purified from T. brucei (Misset et al. 1986, 1987) and T. cruzi (Bourguignon et al. 1997) and were distinguished structurally from their mammalian counterparts (Michels & Opperdoes 1991). In view of this and based on the observation that the inhibition of some glycosomal enzymes from T. brucei (Durieux et al. 1991) may result in the death of the parasite (Opperdoes et al. 1990, Willson et al. 1993) these enzymes have suggested as promising targets for drug design. The T. brucei (Swinkels et al. 1986) and Leishmania mexicana (Kokl et al. 1994) triose-phosphate isomerase (TPI, EC 5.3.1.1) genes have been cloned and characterized. This enzyme was shown to be a dimer composed by identical subunits and it was also shown to catalyze the isomerization of the two products of aldolase, D-glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, in the absence of carbohydrates. Moreover, it presents a key position in the metabolism, generating dihydroxyacetone phosphate in anaerobic condition, through reverse action of the glycerol kinase and glyceraldehyde-phosphate dehydrogenase. The overall structures of these enzymes are strictly conserved despite substantial variation, and approximately 50% in its amino acid sequence has remained identical (Fothergill-Gilmore & Michels 1993). In this work we describe the purification and partial characterization of the TPI enzyme from T. cruzi. In addition, using an enzymatic assay by agarose gel electrophoresis we show that the T. cruzi Dm28c clone expresses only one form of this enzyme. MATERIALS AND METHODS Materials - Phenyl- and CM-Sepharose were obtained from Pharmacia Fine Chemicals (Uppsala, Sweden); ethylenediamino tetratacetic acid (EDTA), dithiotreitol (DTT), ammonium sulfate, rabbit GAPDH, agarose, trietanolamine, porcine TPI, nonidet P40 (NP40), (3-[4,5-dimethylthiazol-2yl]5-2,5 diphenyltetrazolium bromide) (MTT), phenazine metnosulfate (PMS), glutaraldehyde, paraformaldehyde, cacodilate, chromatographic weight standard proteins and goat anti-rabbit peroxidase conjugated from Sigma Chemical Co. (St. Louis, Mo, U.S.A.); ampholines, silver staining kit, SDS-molecular weight standard proteins, poly (vinylidenedifluoride) (PVDF) and nitrocellulose membrane from Bio-Rad (Richmond, CA, U.S.A.). The Shinpack Diol-150 HPLC column and the sequencing reagents were from Wako Pure Chemicals (Richmond, CA, U.S.A.) while the electrophoresis reagents were obtained from Serva (Heidelberg, Germany). Freund's adjuvants and P10 filters were from Gibco and Amicon (Beverly, MA, U.S.A.) respectively. Liver infusion tryptose (LIT) and brain heart infusion-tryptose (BHI) medium were obtained from Difco (Detroit, Michigan, U.S.A.). All other reagents and chemicals were from Merck (Darmstadt, Germany). Parasites - Epimastigote forms from Dm 28c clone were maintained in liver infusion tryptose (LIT) medium (Contreras et al. 1985) while amastigotes from cellular culture were maintained according to Giovanni De Simone et al. (1987). Blastocrithidia culicis, Crithidia desouzai, Phytomonas serpens, L. guianensis and Her-pertomonas samuelpessoai were cultivated in BHI medium (Giovanni De Simone et al. 1987). All parasites were harvested at log phase and washed three times by centrifugation (3,000 g, 10 min) in PBS. Extraction of the enzyme and binding to phenyl-Sepharose column - After washes with PBS, T. cruzi epimastigote forms were freeze-thawed three times in the same buffer and the insoluble material was pelleted by centrifugation (40,000 g, 30 min, 4 C). The supernatant was designed crude extract and ammonium sulfate was added to a final concentration of 1M. The extract was applied to a 20 ml phenyl-Sepharose CL-4B column equilibrated with 25mM Tris-HCl pH 7.8, containing 5 mM EDTA, 1mM DTT and 1M (NH4)2SO4 and fractionated using an ammonium sulfate reverse gradient. Cation-exchange chromatography - After release from phenyl-Sepharose, the fraction (peak 3) was concentrated on a Centripep P10 filter followed by replacement of the buffer (0.1M trietanolamine-HCl pH 8.0 containing 1 mM EDTA) and fractionation of the sample on a fast flow CM-Sepharose column. Fractions of 3 ml were collected and concentrated, separately, using Centripep P10 filters and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Size exclusion HPLC - The CM-Sepharose pooled peak 2 was concentrated using centricon filters and injected in a Shinpack Diol-150 HPLC column (5 mm; 50 cm x 7.9 mm I.D.), previously equilibrated in 50 mM phosphate buffer, pH 7.2. The proteins were fractioned on an automatic HPLC system (Shimadzu, 6A model) during 28 min at 25 C (Fig. 4). Fractions of 1 ml were collected and concentrated as described before. For molecular mass characterization the column was calibrated in the same buffer with the following markers: beta-galactosidase (105 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa) and carbonic anhydrase (29 kDa).
Agarose gel electrophoresis - This electrophoresis was conduced in a Multifor apparatus using a continuous buffer (414 mM Tris and 50 mM citric acid pH 8.1) under 50 V in a water-cooled apparatus for approximately 2 hr ( Harry & Hopkinson 1976). Afterwards the gels were removed, washed (0.2 M Tris-HCl, pH 8.0) and incubated in the same buffer (50 ml) containing 0.1% (w/v) of sodium pyruvate and sodium arsenate, 20% (w/v) NAD^+, 4% (w/v) MTT and 2% (w/v) PMS. The staining was allowed to proceed at 25^ C in the dark. Extract from L. guianensis was run simultaneously and the development of the TPI used as internal standard. Preparation of anti-TPI antibodies - Polyclonal antibodies were raised in rabbits by subcutaneous injection of 20 ug commercially available porcine TPI protein emulsified with complete (first booster) and incomplete (second and third booster) Freund's adjuvant. The animals were bled seven days after the last booster injection. Antibody title and specificity were evaluated by ELISA and western blotting. Immunocytochemistry - T. cruzi epimastigote and amastigote forms were processed for Lowicryl K4M at -20 C as previously described (Bendayan et al. 1987). Ultra-thin sections were cut with a RMC MT-7 ultramicrotome and sequentially labelled with polyclonal antibody anti-TPI (diluted in TBS 0.1 M, BSA 1%, Tween 1%) and (1:10 dilution) of protein A-gold complexes (15 nm). Photographs were taken using a Zeiss EM 10C electron microscope (Fig. 7).
Protein microsequencing - For sequencing experiments, the isolated protein was analyzed directly by the polybrene method or it was run a 15%-acrylamide SDS-PAGE (Laemmli 1970) and blotted onto a PVDF membrane using a semidry blotting apparatus. NH2-terminal amino acid sequence analysis was performed by automatic Edman degradation using a gas-phase protein microsequencer (Model PSQ-1; Shimadzu) coupled to an on line HPLC system (model 6A; Shimadzu) using the conditions described by Giovanni De Simone et al. (1994). The obtained amino acid sequence was compared with the GenBank and Swiss-PROT protein database. RESULTS AND DISCUSSION In this work we report the purification, in apparent homogeneity, of triose phosphate isomerase from T. cruzi epimastigote forms by combining phenyl-Sepharose, CM-Sepharose and gel filtration high performance liquid chromatography. The soluble freeze-thawed extract (40,000 g) of T. cruzi was fractionated by phenyl-Sepharose column using an ammonium sulfate reverse gradient (Fig. 1). When the fraction 3 was subsequently applied to a CM-Sepharose column at pH 8.0, four principal peaks were revealed. The TPI activity was eluted at 45-70 mM of NaCl ( Fig. 2). The SDS-PAGE analysis showed that the peak 1 contained proteins with 27 and 15 kDa (Fig. 2, lane A) while the peak 2 represented bands with approximately 27 and 25 kDa (Fig. 2 lane B, insert). These last two proteins presented a similar isoelectric point 9.3-9.5 (data not shown), however by western blot studies using the rabbit sera anti-TPI (Fig. 3), it was shown that only the 27 kDa molecular species was the immuno active protein.
Figure 2: cation exchange chromatography of the triosephosphate isomerase fraction (peak 3, Fig. 1) and SDS-PAGE (12%) of the main peak 1 (a) and 2 (b) recovered from CM-Sepharose column followed by silver staining. The molecular weight of the standard proteins are shown in the right side. The CM-Sepharose column (6.5 x 1.0 cm, I.D.) was equilibrated with 25 mM Tris-HCl, pH 8.0, buffer and the fractions (1,4 ml) collected at a flow rate of 5 ml h^-1 using a gradient of NaCl (0-200 mM) in the same buffer. Figure 3: western blotting analysis of fractions in the course of triosephosphate isomerase purification incubated with rabbit serum anti-porcine TPI. Lanes a, peak 3 from phenyl-Sepharose column (Fig. 1); b, peak 2 from CM-Sepharose column (Fig. 2); c, porcine TPI. Approximately 50 mg of protein was applied in each slot. Size markers are indicated on the left side proteins.
The partial N-terminal sequence data analysis of the first 15 amino acid of the protein revealed 93% and 100% of homology with the TPI from T. brucei and L. mexicana, respectively ( Table). This high homology in TPI of different organisms is reinforced by the results of the immunoblot tests. The use of a heterologous serum anti-TPI revealed only one identical migrating band in different parasites (B. culicis, P. serpens, C. desouzai, H. samuel- pessoai) extracts (Fig. 5), suggesting that only one form of enzyme exists in these parasites including T. cruzi (Fig. 3).
In summary, we have been able to purify homogeneous T. cruzi TPI using an alternative procedure. The enzyme was localized preferentially in the cytoplasm of both amastigote and epimastigote forms and it showed a native molecular weight of 52 kDa identical to other TPI studied thus far. Moreover, the T. cruzi enzyme differs from L. guianensis (as shown in this work), T. brucei (pI 9.8; Willson et al. 1993) and L. mexicana (pI 9.0; Kohl et al. 1994) TPIs in that its isoelectric point is slightly more acid and basic, respectively. ACKNOWLEDGEMENTS To M Ortigao for the formal revision of the manuscript and the Laboratorio de Insumos e Conjugados do Instituto de Tecnologia (Biomanguinhos) of Fiocruz for yielding the peroxidase labelled goat-anti rabbit IgG. REFERENCES
Copyright 1998 Fundacao Oswaldo Cruz - Fiocruz The following images related to this document are available:Photo images[oc98042a.jpg] [oc98042f.jpg] [oc98042c.jpg] [oc98042h.jpg] [oc98042e.jpg] [oc98042b.jpg] [oc98042g.jpg] [oc98042d.jpg] |
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