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Purification, Characterization, and Immunolocalization of a Thioredoxin Reductase from Adult Fasciola Hepatica

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Purification, Characterization, and Immunolocalization of a Thioredoxin Reductase from Adult Fasciola Hepatica J. Parasitol., 90(2), 2004, pp. 205±211 q American Society of Parasitologists 2004 PURIFICATION, CHARACTERIZATION, AND IMMUNOLOCALIZATION OF A THIOREDOXIN REDUCTASE FROM ADULT FASCIOLA HEPATICA Gabriela Maggioli, LucõÂa Piacenza, Beatriz Carambula, and Carlos Carmona* Unidad de BiologõÂa Parasitaria, Facultad de Ciencias, Instituto de Higiene, Av. A. Navarro 3051, CP 11600 Montevideo, Uruguay. e-mail: [email protected] ABSTRACT: Thioredoxin reductase (TrxR), an enzyme belonging to the ¯avoprotein family of pyridine nucleotide-disul®de oxi- doreductases, was isolated from the deoxycholate-soluble extract of the common liver ¯uke, Fasciola hepatica. Puri®cation to homogeneity of the 60-kDa enzyme from the adult worm was achieved by a combination of ammonium sulfate fractionation, anion exchange, and af®nity chromatography on 29,59-adenosine diphosphate±Sepharose. Using the 5,59-dithiobis(2-nitrobenzoic acid) assay, the puri®ed TrxR showed a speci®c activity of 7,117 U min21 mg21. The enzyme activity was completely inhibited by the presence of the gold compound aurothioglucose (IC50 5 120 nm), indicating that F. hepatica TrxR is a selenoenzyme. Also, the enzyme was capable of reducing disul®de bonds in insulin and was activated by the presence of the reduced form of ¯avin adenine dinucleotide, properties shared with mammalian TrxRs. Furthermore, the isolated enzyme showed very low glu- taredoxin (Grx) activity (0.47 U mg21), but no glutathione reductase activity was detected. Af®nity-puri®ed IgGs (20 mgml21) from the antisera produced against the puri®ed TrxR inhibited its activity about 80% with respect to the control. The enzyme was immunolocalized in cells located within the parenchyma and in the testes, but it was not found in the tegument of the adult ¯uke. 2x Thiol-disul®de reactions are ideally suited to control protein diffuse widely and is potentially more damaging than O2 ; function through the redox state of structural or catalytic SH therefore, production of SOD must be accompanied by a mech- groups. This mechanism, emerging as a major regulatory mech- anism to detoxify H2O2. However, no catalase or glutathione anism in cellular biology, allows the maintenance of intracel- peroxidase activity has been detected in any of the F. hepatica lular protein disul®des generally in reduced form (Gilbert, extracts (E/S products, detergent-soluble and somatic extract of 1990). Another critical function by which this mode of electron immature and adult worms) analyzed so far by spectrophoto- transportation operates is the reduction of H2O2 by peroxidoxins metric assays (Piacenza et al., 1998). (Mustacich and Powis, 2000). It is now believed that the peroxidoxin system functions as The protein protection ability of the peroxidoxins was ®rst the ``missing link'' in peroxide metabolism in helminths, as reported for yeast thioredoxin peroxidase (TrxP) (Kim et al., proposed by McGonicle et al. (1998). In this sense, a TrxP gene 1988). TrxP was originally thought to be speci®c for oxidation was cloned from an adult F. hepatica complementary DNA reactions using thiols, and the enzyme was proposed to have a (cDNA) expression library using antisera raised against the E/ thiyl radical±scavenging role. However, later on, it became ap- S antigens (McGonicle et al., 1997), and a recombinant TrxP parent that TrxP could act as a peroxidase, reducing hydrogen expressed in E. coli protected proteins against by-products of peroxide and the alkyl hydroperoxides with the use of hydrogen aerobic metabolism (Salazar-Calderon et al., 2000). Also, the provided by thioredoxin (Trx), thioredoxin reductase (TrxR), 12-kDa Trx protein was identi®ed using F. hepatica±immune and the reduced form of nicotinamide adenine dinucleotide sera and detected in the tegument of both juvenile and adult phosphate (NADPH) (Chae et al., 1994). The reduction of TrxP worms (Shoda et al., 1999). More recently, Trx was found in involves a redox cycle in which the small dithiol protein Trx the E/S products of mature ¯ukes, and functional studies reduces the oxidized TrxP. The oxidized Trx, in turn, is reduced showed that the recombinant Trx has antioxidant activity (Sa- by the action of TrxR using the reducing power of NADPH. lazar-Calderon et al., 2001). TrxR is a selenocysteine ¯avin adenine dinucleotide (FAD)± In the present study we report the puri®cation and character- containing enzyme with some structural and functional similar- ization of the third member of the peroxidoxin system, TrxR, ities to glutathione reductase (Holmgren and Bjorsnstedt, 1995). from adult liver ¯ukes. The enzyme was isolated from the deox- The enzyme from Escherichia coli shows a homodimeric struc- ycholic soluble preparation and characterized, and the antibod- ture of 34-kDa subunits, whereas mammalian TrxRs are com- ies raised against it enabled us to localize the TrxR in parasite posed of 52- to 57-kDa subunits. The TrxR enzyme mechanism tissues. involves the transfer of reducing equivalents from NADPH to a disul®de bond in the enzyme within the sequence -Cys-Ala- MATERIALS AND METHODS Thr-Cys- by way of FAD. Materials The common liver ¯uke, Fasciola hepatica, is the causative Deoxycholic acid (DOC), Freund complete and incomplete adjuvants, agent of fascioliasis, affecting principally ruminants and caus- sodium dodecyl sulfate (SDS), alkaline phosphatase±conjugated anti- ing important economic losses in agricultural countries. Previ- rabbit IgG, anti-rabbit IgG±¯uorescein isothiocyanate (FITC), Tween- ous studies showed the presence of high levels of CuZn super- 20, nitro blue tetrazolium salt (NBT), 5-bromo-4-chloro-3-indolyl phos- oxide dismutase (SOD) activity in the excretion±secretion (E/ phate (BCIP), aurothioglucose, reduced form of ¯avin adenine dinucle- otide (FADH2), ethylenediaminetetraacetic acid (EDTA), 5,59-dithiob- S) products of immature and adult worms (Piacenza et al., is(2-nitrobenzoic acid) (DTNB), NADPH, bovine serum albumin 2x 1998). The H2O2 generated by SOD dismutation of O2 can (BSA), DL-dithiothreitol (DTT), oxidized form of glutathione (GSSG), reduced form of glutathione (GSH), yeast glutathione reductase, b-hy- droxyethyl disul®de (HED), GSH±Sepharose, 29,59-adenosine diphos- Received 10 April 2003; revised 8 August 2003; accepted 8 August phate (ADP)±Sepharose, and E. coli Trx were obtained from Sigma (St. 2003. Louis, Missouri), and the BCA protein quanti®cation kit was purchased * To whom correspondence should be addressed. from Pierce (Rockford, Illinois). 205 206 THE JOURNAL OF PARASITOLOGY, VOL. 90, NO. 2, APRIL 2004 MAGGIOLI ET AL.ÐTHIOREDOXIN REDUCTASE FROM F. HEPATICA 207 Parasite homogenates eighty milliliters of DOC (324 mg total protein) was submitted to am- monium sulfate fractionation. Ammonium sulfate precipitation was car- Mature ¯ukes were removed from the bile ducts of bovine livers ried out in 3 steps of 30, 50, and 80% saturation. The pellets obtained obtained at a local abattoir. For the preparation of the detergent-soluble extract, previously washed adult ¯ukes were killed by freezing for 30 were dissolved in 10 mM Tris±HCl (pH 7.5) with 1 mM EDTA and min at 220 C, washed twice with phosphate-buffered saline (PBS), and dialyzed overnight against the same buffer. Afterward, fractions were drained. One-gram wet weight of tissue was incubated in 10 ml of 1% heated up to 60 C and rapidly cooled at 4 C in an ice bath; then, the DOC in 0.15 M glycine, pH 9, and 0.5 M NaCl for 1 hr at room precipitate was removed by centrifugation at 20,000 g for 30 min, and temperature, 30 min at 37 C, and then 30 min at 4 C. The DOC- the resulting supernatant was assayed for TrxR activity. The fraction extracted material was centrifuged at 20,000 g for 1 hr, and the super- obtained with 80% ammonium sulfate precipitation (enriched in TrxR natant was stored at 280 C until use. The somatic extract from F. activity) was further fractionated using anion exchange chromatography hepatica was prepared from live adult ¯ukes essentially as described on a Hi-Trap Mono-Q column (Amersham Biosciences, Piscataway, by Acosta et al. (1998). To obtain E/S products, mature ¯ukes were New Jersey) equilibrated in 10 mM Tris±HCl, pH 7.5, with 1 mM washed 6 times in 0.01 M PBS, pH 7.3, at 37 C and maintained for 6 EDTA, and the proteins were eluted with a discontinuous gradient of hr (1 mature ¯uke per milliliter) in Roswell Park Memorial Institute NaCl (0±200 mM). Fractions containing TrxR activity were pooled, 1640 medium, pH 7.3, containing 2% glucose, 30 mM N-2-hydroxy- concentrated, and dialyzed overnight at 4 C against 50 mM Tris±HCl, ethylpiperazine-N9-2-ethane-sulfonic acid (HEPES), and 25 mg L21 gen- pH 7.5, with 1 mM EDTA. Proteins were applied onto a 29,59-ADP± tamycin at 37 C (Dalton and Heffernan, 1989). The medium containing Sepharose column equilibrated in the same buffer and eluted with 0.2, the E/S products was then removed and centrifuged at 48,000 g for 30 0.5, and 0.8 M NaCl. One-milliliter fractions were collected, and 100- min at 4 C. The supernatant was ®ltered sequentially through a What- ml aliquots were used for determination of TrxR activity. The puri®ca- man ®lter paper No. 1 and a 0.22-mm ®lter membrane, aliquoted, and tion process was assessed by 15% SDS±polyacrylamide gel electropho- stored at 220 C until use. resis (PAGE) under nonreducing and reducing conditions (®nal concen- tration of 10 mM DTT). After electrophoresis the gels were silver Determination of TrxR activity stained. Protein concentration was determined using a microplate BCA TrxR activity in F. hepatica extracts and during the puri®cation pro- protein assay kit according to the method of Redinbaugh and Turley cedure was spectrophotometrically measured by the reduction of DTNB (1986). in the presence of NADPH (Holmgren and Bjorsnstedt, 1995).
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