Biochemical analysis of a recombinant glutathione transferase from the cestode Echinococcus granulosus

Acta Tropica 114 (2010) 31–36

Contents lists available at ScienceDirect

Acta Tropica journal homepage: www.elsevier.com/locate/actatropica

Biochemical analysis of a recombinant glutathione transferase from the cestode Echinococcus granulosus Laura Harispe a , Gabriela García b , Paula Arbildi b , Leticia Pascovich b , Cora Chalar a , Arnaldo Zaha c , Cecilia Fernandez b , Veronica Fernandez b,∗ a

Sección Bioquímica, Facultad de Ciencias, UdelaR, Igua 4225, Montevideo, CP 11400, Uruguay Cátedra de Inmunología, Facultad de Química, UdelaR, Av. Alfredo Navarro 3051, piso 2, Montevideo, CP 11600, Uruguay Laboratorio de Biología Molecular de Helmintos del Departamento de Biologia Molecular e Biotecnologia, Instituto de Biociências e Centro de Biotecnología, UFRGS, Avenida Bento Gonc¸alves 9500, Prédio 43421, Porto Alegre, RS, Brazil b c

a r t i c l e

i n f o

Article history: Received 13 August 2009 Received in revised form 4 December 2009 Accepted 13 December 2009 Available online 23 December 2009 Keywords: Mu-class GSTs Glutathione peroxidase Substrate specificity

a b s t r a c t Glutathione transferases (GSTs) are believed to be a major detoxification system in helminths. We describe the expression and functional analysis of EgGST, a cytosolic GST from Echinococcus granulosus, related to the Mu-class of mammalian enzymes. EgGST was produced as an enzymatically active dimeric protein (rEgGST), with highest specific activity towards the standard substrate 1-chloro2,4-dinitrobenzene (CDNB; 2.5 ␮mol min−1 mg−1 ), followed by ethacrynic acid. Interestingly, rEgGST displayed glutathione peroxidase activity (towards cumene hydroperoxide), and conjugated reactive carbonyls (trans-2-nonenal and trans,trans-2,4-decadienal), indicating that it may intercept damaging products of lipid peroxidation. In addition, classical GST inhibitors (cybacron blue, triphenylthin chloride and ellagic acid) and a number of anthelmintic drugs (mainly, hexachlorophene and rafoxanide) were found to interfere with glutathione-conjugation to CDNB; suggesting that they may bind to EgGST. Considered globally, the functional properties of rEgGST are similar to those of putative orthologs from Echinococcus multilcularis and Taenia solium, the other medically important cestodes. Interestingly, our results also indicate that differences exist between these closely related cestode GSTs, which probably reflect specific biological functions of the molecules in each parasitic organism. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Cystic hydatid disease is a zoonosis affecting humans and a wide range of livestock species caused by infection with the metacestode of the dog tapeworm Echinococcus granulosus. In intermediate hosts, eggs develop into slowly growing metacestodes (hydatid cysts) at visceral sites, mainly liver and lungs. Hydatid cysts are fluid-filled bladders, typically unilocular, and contain larval worms (protoscoleces) (Thompson and Lymbery, 1995). The disease has a worldwide distribution and constitutes an important health and economic problem (Craig and Larrieu, 2006). Glutathione transferases (GSTs; EC 2.5.1.18) are a diverse superfamily of widely distributed multifunctional proteins that catalyze the covalent addition of the tripeptide glutathione (␥glutamylcysteinylglycine, GSH) to a structurally diverse set of electrophiles (Mannervik and Danielson, 1988; Hayes and Pulford, 1995; Sheehan et al., 2001). Besides detoxifying xenobiotics – either actively, through conjugation to GSH, or passively, through binding – some of these transferases are able to inactivate endogenous

∗ Corresponding author. Tel.: +598 2 4801196; fax: +598 2 4874320. E-mail address: [email protected] (V. Fernandez). 0001-706X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2009.12.003

secondary metabolites formed during oxidative stress, such as ␣,␤unsaturated aldehydes. Furthermore, some GST isoenzymes have GSH peroxidase activity and are capable of reducing lipid hydroperoxides (Hayes et al., 2005). GSTs are generally grouped into three families according to their sub-cellular location, namely, the cytosolic family, the mitochondrial (kappa) family, and the microsomal (also known as MAPEG) family (Hayes et al., 2005). Cytosolic GSTs form the major group, with members in all aerobic organisms. Based on several criteria, including amino acid sequence similarities, physical structure of the genes, as well as substrate and inhibitor specificities, cytosolic GSTs are usually subdivided into classes (Mannervik et al., 1985; Hayes et al., 2005; Frova, 2006). Seven such classes are currently recognized for the mammalian enzymes, namely Alpha, Mu, Pi, Sigma, Theta, Omega and Zeta. These are soluble dimeric proteins of either identical or different subunits; with heterodimerization being restricted to subunits of the same class (Frova, 2006). Most organisms contain a panel of these enzymes bearing overlapping substrate specificities that reflect their capacity to metabolize structurally diverse chemicals (Hayes et al., 2005). The GSTs from several helminth parasites – especially those from nematodes and trematodes – have been extensively characterized at the molecular and biochemical level (Brophy and

32

L. Harispe et al. / Acta Tropica 114 (2010) 31–36

Pitchard, 1994; Wu et al., 2006), based on the assumption that they are good targets for chemotherapeutic and/or immunological intervention against helminth infections. On the one hand, they are believed to be major detoxification enzymes in these organisms, which usually possess low levels of “phase I” enzymes, but present several GSTs (Precious and Barrett, 1989). In addition, due to their capacity to reduce lipid hydroperoxides, some isoenzymes may intercept the damaging compounds generated by free radicals. This is particularly relevant in the case of parasites that have to deal with oxidants produced by their own metabolism and also with those derived from host defenses (Callahan et al., 1988). On the other hand, several studies have been conducted using GSTs as antigens to confer immune protection against helminth infections, mainly against schistosomiasis (Capron et al., 1992, 2001) and fasciolasis (Sexton et al., 1990). Some cestode GSTs have also been molecularly and biochemically characterized (Brophy et al., 1989; Liebau et al., 1996; Vibanco-Perez et al., 1999, 2002). In the particular case of E. granulosus, GST activity was initially described in the cytosol of protoscoleces and found to increase upon treatment of the worms with GST inducers (Morello et al., 1982). A 25 kDa protein (EgGST), whose amino-terminal sequence resembled the Mu-class of mammalian enzymes, was subsequently isolated from a protoscolex lysate by glutathione affinity and partially characterized (Fernandez and Hormaeche, 1994). More recently, we have cloned the full-coding EgGST cDNA on the basis of the sequence from the orthologous enzyme from E. multilocularis (EMGST1, Liebau et al., 1996) and have shown that the corresponding mRNA increases after phenobarbital treatment of the worms (Fernandez et al., 2000). EgGST and EMGST1 were found to be strikingly similar (99% pair-wise identity, 217/219 amino acids); a phylogenetic analysis highlighted their relatedness with mammalian Mu-GSTs. Indeed, the Echinococcus enzymes display the so-called Mu-loop (Ji et al., 1992), a structural feature distinguishing them from another family of flatworm GSTs, also related to the Mu-class, the 26-kDa family described in trematodes. A putative ortholog of EgGST and EMGST1 has also been described in Taenia solium (SGSTM1; Vibanco-Perez et al., 2002) bearing, respectively, 85% and 86% pair-wise identities with the Echinococcus enzymes. Interestingly, the amino acid differences between the two Echinococcus enzymes, and between them and SGSTM1, are concentrated in two regions of the molecule known to be critical for the enzyme interaction with its substrates: the C-terminal portion, where the residues involved in the binding of hydrophobic ligands are located in all GSTs (Mannervik and Danielson, 1988), and the Mu-loop, which is also an important determinant of affinity towards electrophiles in transferases of this class (Hearne and Colman, 2006). In this context, as a follow-up of our studies with EgGST, we report the biochemical characterization of the recombinant protein (rEgGST) and analyze its behavior taking into account the data available from the putative E. multilocularis and T. solium orthologs, and from mammalian Mu-GSTs. In order to complement the existing information on helminth GSTs, we also report the results of an analysis of the effect of different anthelmintic drugs on enzyme activity; a study of this sort has not been described for EMGST1 or SGSTM1.

2. Materials and methods 2.1. Reagents 1-Chloro-2,4-dinitrobenzene (CDNB), ethacrynic acid (EA), cumene hydroperoxide (CHP), glutathione reductase, NADPH, bithionol and cybacron blue were obtained from Sigma (St. Louis, MO). 1,2-Dichloro-4-nitrobenzene (DCNB), trans-non-2-enal (t-

non), trans,trans-deca-2,4-dienal (tt-dec), triphenyltin chloride and 4-nitropyridine-N-oxide were from Aldrich (Milkwaukee, Wis.). The sodium salt of bromosulfophthalein (BSP), ellagic acid, alizarin, hexachlorophene, bilirubin and chenodeoxycholic acid were from ICN Biochemicals Inc. (Belgium); reduced glutathione (GSH) and isopropylthiogalactoside (IPTG) from BioBasic Inc. (Canada). Albendazole, febendazole, mebendazole, closantel and rafoxanide of the highest purity were purchased from pharmaceutical laboratories in Uruguay. 1,2-Epoxy-3-(p-nitrophenoxy)propane (EPNP) was from Sigma, and was gently donated by Dr. Peter Brophy (Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Wales, UK). 2.2. Preparation of EgGST expression vector E. granulosus cysts from the lungs of naturally infected cows were obtained from a local abattoir. The hydatid fluid was aseptically aspirated and protoscoleces were recovered after decantation of the fluid, washed in phosphate-buffered saline (PBS) containing gentamicin (30 ␮g/mL), and their viability estimated by 5% eosin exclusion. Only batches showing >95% viability were used. Protoscoleces were stored in TRIZOL® reagent (Invitrogen) at −70 ◦ C until RNA extraction Total RNA was isolated following the manufacturer’s instructions. Single-stranded cDNA was synthesized from 2 ␮g of total RNA using the SuperScriptTM III RT kit (Invitrogen) with an oligo(dT)20 primer. The full-coding sequence of EgGST was PCR-amplified from the cDNA using sense (5 -aaacatatggctcccactctggcttac-3 ) and antisense (5 -aaagaattcacctaacagtcaccacgcc-3 ) primers, containing NdeI and EcoRI restriction sites (underlined), respectively. The amplified product was recovered from a 1% agarose gel (QIAquick® Gel Extraction Kit, QIAgen) and cloned into a pGEM vector (Promega). After checking the sequence of the cloned product, the EgGST coding region was isolated from the pGEM-EgGST plasmid by digestion with NdeI and EcoRI (Gibco), and subcloned into the pET-5a expression vector (Promega) to produce a non-tagged recombinant EgGST (rEgGST). 2.3. Expression and purification of rEgGST Escherichia coli BL21[DE3] were transformed with the expression construct, and rEgGST was produced from single bacterial colonies grown at 37 ◦ C in Luria–Bertani medium containing 200 ␮g/mL ampicillin following standard protocols. Briefly, expression was induced with 0.4 mM IPTG at A600 of 0.5 for 3 h. After harvesting the cells, the pellet was resuspended in PBS containing 1% Triton X-100, and the suspension was lysed by three cycles of freeze-thawing followed by sonication on ice. rEgGST was affinitypurified from the soluble fraction on GSH-Agarose (Pharmacia). The isolated recombinant protein was analyzed by SDS-PAGE (Laemmli, 1970) and its enzymatic activity checked with the standard assay (see below). Protein concentration was determined using the BCA Protein Assay kit (Novagen® ) and bovine serum albumin as standard. 2.4. Determination of rEgGST molecular weight by gel filtration chromatography and multiangle light scattering This analysis was carried out at the Laboratory of Carbohydrates and Glycoconjugates (Universidad de la República), following the procedure described by Wyatt (1993). Gel filtration was with a Superdex 75 HR 10/30 column (Amersham Pharmacia Biotech), equilibrated with 50 mM Tris–HCl pH 8.0, at a flow rate of 0.5 mL min−1 . The column was followed in-line by a Mini-DAWN light scattering detector (Wyatt Technologies). Molecular mass calculations were carried out with the ASTRA software (Wyatt

L. Harispe et al. / Acta Tropica 114 (2010) 31–36

33

Technologies), using bovine serum albumin (67 kDa) as a standard. GST activity was analyzed in the eluted fractions with the standard assay (see below). 2.5. Characterization of rEgGST activity 2.5.1. Standard assay and steady-state kinetics Activity was determined at 25 ◦ C in a reaction volume of 1 mL, in 100 mM phosphate buffer (pH 6.5), containing 7 ␮g of rEgGST, 1 mM CDNB and 4 mM GSH (Morello et al., 1982). The reaction was monitored at 340 nm (ε = 9.6 cm−1 mM−1 ) during 1 min; the non-enzymatic formation of product was concomitantly monitored and subtracted from the overall reaction rate (Habig et al., 1974; Morello et al., 1982). Steady-state kinetic studies were carried out at various concentrations of CDNB (0.2–3.0 mM) and GSH (0.25–5.0 mM) to determine apparent kinetic parameters, kcat , Km and kcat /Km . The data were fit to the Michaelis–Menten equation by non-linear regression analysis using Origin® version 6.1 (http://OriginLab.com). 2.5.2. Temperature and pH effects on activity and stability To analyze the effects of temperature (25–55 ◦ C) and pH (5–8) on rEgGST activity, rEgGST (7 ␮g) was incubated during 1 min, either in 0.1 M phosphate buffer (pH 6.5) at the corresponding temperature; or at 25 ◦ C in 0.1 M phosphate buffer of the corresponding pH. Then, GST activity was measured with 1 mM CDNB and 4 mM GSH as substrates, using a thermostatically controlled spectrophotometer (Cary-Peltier; Variant Inc., USA). The effects of temperature and pH were estimated as percentages with respect to activities under standard conditions (25 ◦ C and pH 6.5, respectively). The thermal (10–100 ◦ C) and pH (4–8) stabilities of rEgGST were similarly analyzed. rEgGST (7 ␮g) was incubated for 10 min at various temperatures and pH. Then, the remaining activity was subsequently determined under standard conditions. The effects of temperature and pH on enzyme stability were calculated as percentages of the remaining activity with respect to activity of the untreated enzyme. 2.5.3. Substrate specificity The specific activity of rEgGST with different substrates was analyzed at 25 ◦ C using 7 ␮g of the recombinant enzyme. GSHconjugation with DCNB, BSP, EA and EPNP was determined as described by Habig et al. (1974). Thus, the enzyme was incubated with: 1 mM DCNB and 5 mM GSH at 345 nm (ε = 8.5 mM−1 cm−1 ); 0.03 mM BSP and 5 mM GSH at 330 nm (ε = 4.5 mM−1 cm−1 ); 0.2 mM EA and 0.2 mM GSH at 270 nm (ε = 5.0 mM−1 cm−1 ); 5 mM EPNP and 5 mM GSH at 360 nm (ε = 0.5 mM−1 cm−1 ). In turn, GSHconjugation with t-non and tt-dec was assayed following Brophy et al. (1989), using 0.025 mM t-non and 1 mM GSH or 0.05 mM tt-dec and 2.5 mM GSH; and monitored at 225 nm (ε = −19.2 mM−1 cm−1 ) or at 280 nm (ε = −29.7 mM−1 cm−1 ), respectively. The GSH peroxidase activity was assessed with 1.2 mM CHP in the presence of 1 mM GSH at 340 nm (ε = 6.22 mM−1 cm−1 ), according to Jaffe and Lambert (1986). One activity unit was defined as the amount of enzyme catalyzing the turnover of 1 ␮mol of the corresponding substrate per minute under the specified conditions. 2.5.4. Inhibitor profile Table 2 includes a list of the molecules that were analyzed for their capacity to inhibit rEgGST activity: model inhibitors of GST subclasses, GST ligands, and several anthelmintic drugs. Activity was measured using the standard assay in the presence of five inhibitor concentrations over the studied range. In each case, the inhibitor was incubated for 10 min at 25 ◦ C with 7 ␮g of rEgGST in 0.1 M phosphate buffer pH 6.5 containing 4 mM GSH. The reaction was initiated by adding 1 mM CDNB. Some anthelmintic drugs

Fig. 1. (A) SDS-PAGE analysis of the production and purification of rEgGST. The Coomassie-stained 15% gel contains samples of: lane 1, soluble fraction of the lysate from E. coli transformed with the pET-EgGST construct and induced with IPTG; lanes 2 and 3, GSH-affinity purified rEgGST before and after dialysis against 10 mM phosphate buffer (pH 6.5), respectively. (B) Gel filtration chromatography profile of purified rEgGST. Note that a single protein peak was obtained which included all the eluted GST activity (++++). Light scattering indicated that rEgGST has a molecular weight of 50 ± 0.7 kDa. ND, indicates that no activity was detected in the fractions.

(closantel, febendazole and mebendazole) were only sparingly soluble in the assay buffer; in those cases, enzyme inhibition was determined at the highest concentration tested (50 ␮M). The sensitivity of rEgGST to each inhibitor was characterized by means of the IC50, the concentration of inhibitor decreasing enzyme activity by 50%. This parameter was graphically estimated according to Mannervik and Danielson (1988). 3. Results and discussion 3.1. Production and purification of rEgGST EgGST was produced as a non-tagged recombinant protein, rEgGST. Enzyme activity was detected in the soluble fraction of the transformed-bacterial extract (data not shown), indicating that the recombinant enzyme would be correctly folded and could be purified by GSH affinity (Smith and Johnson, 1988). Induced E. coli BL21 overexpressed a protein with an apparent molecular mass of about 25 kDa (Fig. 1A), in agreement with the molecular weight predicted for EgGST from the translated cDNA sequence (25.6 kDa; Fernandez et al., 2000). The purified recombinant migrated as a single band in SDS-PAGE (Fig. 1A), and retained GST activity (see the next section). We next carried out a gel filtration chromatography with light scattering detection to determine the actual molecular mass of rEgGST. We also analyzed GST activity in the eluted fractions. A single protein peak that overlapped with the activity peak (Fig. 1B) was thus obtained; a mass of 50.0 ± 0.7 kDa was subsequently determined for the protein, in agreement with the expected value if rEgGST were dimeric (about 51 kDa). Furthermore, dimerization appeared to be very favored because no monomers or other

34

L. Harispe et al. / Acta Tropica 114 (2010) 31–36

Table 1 Substrate specificities of rEgGST. Activity (␮M min−1 mg−1 ) rEgGST

EMGST1a

rSGSTM1b

Mu-classc

CDNB

2.5 ± 0.4

2.9

2.2

1.4–220

Model substrates CHP (Alpha-class) DCNB (Mu-class) BSP (Mu-class) EA (Pi-class) EPNP (Theta-class)

0.30 ± 0.02 ND 0.015 ± 0.007 0.87 ± 0.08 ND

0.06 ND ND 0.75 ND

1.22 0.493 0.088 0.61 ND

<0.05–0.7 <0.006–2.1 0.002–0.900 0.06–0.60 ND–0.065

Reactive carbonyls t-non tt-dec

0.15 ± 0.02 0.16 ± 0.02

0.16 0.199

0.19 –

– –

All values are the mean ± SD of at least triplicate determinations. ND indicates that no activity was detected with the particular substrate. CDNB: 1-chloro-2,4-dinitrobenzene; CHP: cumene hydroperoxide; DCNB: 1,2-dichloro-4nitrobenzene; BSP: bromosulfophthalein; EA: ethacrynic acid; EPNP: 1,2-epoxy3-(p-nitrophenoxy)propane; t-non: trans-non-2-enal; tt-dec: trans,trans-deca-2,4dienal. a Liebau et al. (1996). b Vibanco-Perez et al. (2002). c Mannervik et al. (1985) and Comstock et al. (1994).

of Mu-GSTs (Ji et al., 1992), any amino acid change in these regions may modify the catalytic properties and, consequently, the kinetic parameters. The Km for CDNB did also differ from the one of SGSTM1 (3.3 mM; Vibanco-Perez et al., 2002); a discrepancy that most likely reflects the peculiarities of the electrophilic substrate binding site from each protein, because several residues differ between them over the Mu-loop and the C-terminal portion. In any case, as mentioned before, the specific activities towards CDNB of the three cestode GSTs compared very well (Table 1). We also analyzed the effects of temperature and pH on enzyme activity and stability. rEgGST activity increased with temperature over the assayed range (25–50 ◦ C), whereas its stability remained unchanged up to 40 ◦ C (10 min incubation at 10–40 ◦ C). Beyond this temperature, the stability decreased and rEgGST was virtually inactivated if treated at more than 60 ◦ C. Regarding the influence of pH on GSH-conjugation to CDNB, rEgGST activity was maximal at pH 6.5 and slightly decreased on either side of this value (to about 80% at pH 5.5 and pH 8.0). Finally, the influence of pH in enzyme stability was also analyzed. rEgGST was stable between pH 6.0 and pH 7.0; activity decreased to about 80% at pH 5.0 and pH 8.0 and drastically fell below pH 5.0. 3.3. Characterization of rEgGST substrate specificities

oligomers were detected in the eluted fractions. Similar results have been obtained with a whole range of recombinant GSTs produced as non-tagged proteins and, in particular, with the putative EgGST orthologs from E. multilocularis (EMGST1; Liebau et al., 1996) and T. solium (SGSTM1; Vibanco-Perez et al., 1999). This is the expected behavior for these enzymes taking into account that GSTs are dimeric proteins and structural studies have highlighted the importance of dimerization for activity (Wilce and Parker, 1994). 3.2. Analysis of rEgGST enzyme activity The specific activity of rEgGST towards 1-chloro-2,4dinitrobenzene (CDNB) was 2.5 ␮mol min−1 mg−1 (Table 1), which is comparable to those described for EMGST1 (Liebau et al., 1996) and SGSTM1 (Vibanco-Perez et al., 2002) (Table 1). However, a considerably lower activity (0.4 ␮mol min−1 mg−1 ) has been reported for the GSH-affinity purified fraction from E. granulosus protoscoleces (Fernandez and Hormaeche, 1994). Although the presence of a mixture of different GSTs in the native fraction cannot be ruled out, the simplest explanation for the discrepancy between the two values is that a portion of the native enzyme was not active, due either to denaturation or to the presence of a natural inhibitor co-purified with the enzyme(s) from the parasite extract. rEgGST kinetic parameters were subsequently determined using the standard assay. Michaelis–Menten constants (Km ) of 0.5 ± 0.1 mM and 0.8 ± 0.1 mM were respectively calculated for GSH and CDNB. Interestingly, these values are similar to those reported for the cytosolic fraction of E. granulosus protoscoleces (correspondingly, 0.25 mM and 1.1 mM; Morello et al., 1982). The Km for CDNB was higher than described for EMGST1 (0.3 mM; Liebau et al., 1996), and the calculated kcat 2.0 ± 0.1 s−1 lower (3.2 ± 0.1 s−1 ; Liebau et al., 1996); the kcat /Km ratio was thus significantly smaller (2.5 ± 0.4 × 103 M−1 s−1 for rEgGST vs 10.7 × 103 M−1 s−1 for EMGST1). These differences are surprising considering the high identity of the Echinococcus enzymes; still, the two amino acids differing between EgGST and EMGST1 represent non-conservative changes in critical regions of the molecule: one of them is at the centre of the 10-residue Mu-loop (Thr38 is Ala in EMGST1); and the other one at the C-terminal end of the molecule (Arg206 is Cys in EMGST1). Given that the Mu-loop and the C-terminal tail are two structural elements forming the hydrophobic binding pocket

The specific activities of rEgGST with various substrates are shown in Table 1; for comparison, the values reported for EMGST1 (Liebau et al., 1996) and SGSTM1 (Vibanco-Perez et al., 2002) with the same substrates as well as the ranges of specific activity characteristic of the mammalian Mu-class of enzymes are also included (Mannervik et al., 1985; Comstock et al., 1994). Following CDNB, rEgGST was highly active with EA and CHP. Lower activity values were observed with t-non, tt-dec and BSP; whereas no reactivity was detected against DCNB, and EPNP. Globally, the activity profile of rEgGST was similar to those of the closely related EMGST1 and SGSTM1 and also to the mammalian Mu-class of enzymes. Going into more detail, as it could be anticipated from their protein sequences, the behavior of rEgGST almost reproduced the one of EMGST1 and differed from SGSTM1 with particular substrates. In fact, rEgGST, and EMGST1, had very low or undetectable activities towards DCNB and BSP, whereas SGSTM1 was more efficient with both substrates. The specific activities for the three enzymes are, nevertheless, within the range of the mammalian Mu-class, because the performance of Mu GSTs with these substrates is highly variable, especially with DCNB (see Table 1 and Hansson et al., 1999). The cestode enzymes are also similar to the mammalian counterparts in terms of conjugating EA with GSH and reacting with CHP. Interestingly, quantitative differences appear to exist between them regarding the latter reactivity, i.e. GSH peroxidase activity (Table 1). This may be physiologically relevant because it provides an indication of the capacity of each enzyme to react with lipid hydroperoxides, released from membrane lipids by oxidants and free radicals. Quite strikingly, rEgGST showed higher peroxidase activity than EMGST1. In turn, the performance of SGSTM1 was reported to be significantly higher. Finally, the reactivity of the three cestode enzymes towards the carbonyls tt-dec and t-non, which are models of the products generated by the break down of lipid hydroperoxides, appeared to be virtually identical. 3.4. Inhibitor studies of rEgGST The effect of potential inhibitors on GSH-conjugation to CDNB by rEgGST is shown in Table 2. Different products were tested, including three class-distinguishing inhibitors of the mammalian enzymes (Mannervik et al., 1985; Tahir et al., 1985), several anthelmintic drugs and various GST ligands. For comparison, available data from SGSTM1 (Vibanco-Perez et al., 2002) and the IC50

L. Harispe et al. / Acta Tropica 114 (2010) 31–36

35

Table 2 Sensitivity to inhibitors of rEgGST. Tested range (␮M)

IC50 (␮M) rEgGST

SGSTM1a

Mu-classb

Conventional inhibitors Cybacron blue Triphenyltin chloride Bromosulfophthalein

0–500 0–500 0–500

0.12 2.3 188

0.71 0.32 0.68

Anthelmintic drugs Benzimidazoles Albendazole Febendazole Mebendazole

0–200 0–50 0–50

160 >50 (26%) >50 (18%)

– – –

– – –

0–100 0–50 0–100 0–200

39 >50 (42%) 4.07 126

– – – –

– – – –

0–150 0–300 0–250 0–300 0–50

7.3 65 165 NI NI

– – – – –

0.13–0.30 – – – –

Halogenated phenols Rafoxanide Closantel Hexachlorophene Bithionol Other inhibitors Ellagic acid Alizarin Bilirubin Chenodeoxycholic acid 4-Nitropyridine-N-oxide

0.08–4.20 0.04–20.00 0.5–10.0

The IC50 values are concentrations giving 50% inhibition of enzyme activity in the standard assay with 1 mM CDNB. The figures with % indicate percentage inhibition at the specified concentration. NI, indicates that there was no inhibition over the assayed range. Each assay was carried out at least three times. a Vibanco-Perez et al. (2002). b Comstock et al. (1994), Mannervik et al. (1985) and Hayeshi et al. (2007).

ranges characterizing the mammalian Mu-GSTs (Mannervik et al., 1985; Comstock et al., 1994; Hayeshi et al., 2007) are also included in the table. No data are available on inhibitor studies for EMGST1. rEgGST activity was considerably reduced by the Mu-class inhibitors cybacron blue and triphenylthin chloride. These molecules also affect SGSTM1 (Table 2). rEgGST showed very low susceptibility to BSP, in contrast with the T. solium homologue and mammalian Mu-GSTs. In fact, although BSP is an inhibitor of Alpha-GSTs, it is known to interfere with the capacity of the Mu enzymes to conjugate GSH to CDNB (Table 2). Interestingly, the BSPconjugating activity of SGSTM1 is also considerably higher than the one of rEgGST (see Table 1). The sensitivity to BSP of the CDNBconjugating activity of the two enzymes may, therefore, reflect their different capacity to conjugate GSH to BSP. In addition, rEgGST appeared to interact with a number of anthelmintic drugs (Table 2). Globally, the sensitivity of enzyme activity correlated with the electrophilic character of the assayed molecules. Thus, halogenated phenolic products acted as stronger inhibitors than benzimidazole derivatives. Hexachlorophene was the most powerful, followed by rafoxanide. In this context, rEgGST sensitivity to bithionol, which was comparable to the one with albendazole, was unexpectedly low. Except for the latter result, the behavior of rEgGST reproduced those of other helminth enzymes (Torres-Rivera and Landa, 2008). Several studies have been carried out in an attempt to gain insight about the putative role of the parasite GSTs in drug metabolism. The enzymes were found to bind to the drugs but they do not appear to conjugate them with GSH. Activity was also analyzed in the presence of various molecules known to interfere with GSH-conjugation to CDNB, namely non-substrate physiological ligands of the mammalian enzymes (bilirubin and chenodeoxycolic acid), the polyphenol ellagic acid and the anthraquinone alizarin. rEgGST activity was sensitive to ellagic acid, an inhibitor of Alpha-, Mu- and Pi-GSTs; the human Mu enzymes were, though, reported to be more sensitive (Hayeshi et al., 2007; see Table 2). Finally, we also assayed the effect of 4-nitropyridine-N-oxide (4NPO) because it had been reported to act as a drastic inhibitor of GSH-conjugation to CDNB by in vitro

cultured E. granulosus protoscoleces (Repetto et al., 1986). rEgGST activity was not affected by this product (Table 2), indicating that EgGST would not be directly involved in the activity measured by Repetto et al. (1986). The results presented in this work show that, as has been described for other helminth GSTs (Torres-Rivera and Landa, 2008), this E. granulosus enzyme is a multifunctional protein with a range of potentially interesting catalytic and binding activities. In particular, it could play a role in protection against host-derived lipid peroxidation and in the passive detoxification of anthelmintic drugs. These properties may be especially relevant taking into account that EgGST has been found to be inducible (Fernandez et al., 2000). In addition, the comparative analysis of our data and similar studies carried out with the presumed E. multilocularis and T. solium orthologs highlight putative functional differences between the three proteins that may reflect their specific roles in each organism.

Acknowledgements We are grateful to Dr. Beatriz Alvarez for invaluable comments and suggestions, to Maria Ines Bessio and Dr. Fernando Ferreira for help and advice with light scattering; and to Dr. Ricardo Ehrlich for helpful discussions. This work was supported by grants from IFS 3113 (Sweden), CSIC-251 (Uruguay) and CNPq/PADCT (Brazil).

References Brophy, P.M., Pitchard, D.I., 1994. Parasitic helminth glutathione S-transferases: an update on their potential as targets for immuno- and chemotherapy. Exp. Parasitol. 79, 89–96. Brophy, P.M., Southan, C., Barret, J., 1989. Glutathione transferases in tapeworm Moniezia expansa. Biochem. J. 262, 939–946. Callahan, H.L., Crouch, R.K., James, J.E., 1988. Helminth antioxidant enzymes: a protective mechanism against host oxidants. Parasitol. Today 4, 218–225. Capron, A., Capron, M., Dombrowicz, D., Riveau, G., 2001. Vaccine strategies against schistosomiasis: from concepts to clinical trials. Int. Arch. Allergy Immunol. 124, 9–15.

36

L. Harispe et al. / Acta Tropica 114 (2010) 31–36

Capron, A., Dessaint, J.P., Capron, M., Pierce, R.J., 1992. Vaccine strategies against schistosomiasis. Immunobiology 184, 282–294. Comstock, K.E., Widersten, M., Hao, X.Y., Henner, W.D., Mannervik, B., 1994. A comparison of the enzymatic and physicochemical properties of human glutathione transferase M4-4 and three other human Mu class enzymes. Arch. Biochem. Biophys. 311, 487–495. Craig, P.S., Larrieu, E., 2006. Control of cystic echinococcosis/hydatidosis: 1863–2002. Adv. Parasitol. 61, 443–508. Fernandez, C., Hormaeche, C.E., 1994. Isolation and biochemical characterization of a glutathione S-transferase from Echinococcus granulosus protoscolices. Int. J. Parasitol. 24, 1063–1066. Fernandez, V., Chalar, C., Martinez, C., Musto, H., Zaha, A., Fernandez, C., 2000. Echinococcus granulosus: molecular cloning and phylogenetic analysis of an inducible glutathione S-transferase. Exp. Parasitol. 96, 190–194. Frova, C., 2006. Glutathione transferases in the genomics era: new insights and perspectives. Biomol. Eng. 23, 149–169. Habig, W.H., Pabst, M.J., Jacoby, W.B., 1974. Glutathione S-transferase: first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130–7139. Hansson, L.O., Bolton-Grob, R., Widersten, M., Mannervik, B., 1999. Structural determinants in domain II of human glutathione transferase M2-2 govern the characteristic activities with aminochrome, 2-cyano-1,3-dimethyl-1nitrosoguanidine, and 1,2-dichloro-4-nitrobenzene. Protein Sci. 8, 2742–2750. Hayes, J.D., Flanagan, J.U., Jowsey, I.R., 2005. Glutathione transferases. Annu. Rev. Pharmacol. Toxicol. 45, 51–88. Hayes, J.D., Pulford, D.J., 1995. The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit. Rev. Biochem. Mol. Biol. 30, 445–600. Hayeshi, R., Mutingwende, I., Mavengere, W., Masiyanise, V., Mukanganyama, S., 2007. The inhibition of human glutathione S-transferases activity by plant polyphenolic compounds ellagic acid and curcumin. Food Chem. Toxicol. 45, 286–295. Hearne, J.L., Colman, R.F., 2006. Contribution of the mu loop to the structure and function of rat glutathione transferase M1-1. Protein Sci. 15, 1277–1289. Jaffe, J.J., Lambert, R.A., 1986. Glutathione S-transferase in adult Dirofilaria immitis and Brugia pahangi. Mol. Biochem. Parasitol. 20, 199–206. Ji, X., Zhang, P., Armstrong, R.N., Gilliland, G.L., 1992. The three-dimensional structure of a glutathione S-transferase from the mu gene class. Structural analysis of the binary complex of isoenzyme 3-3 and glutathione at 2.2-A resolution. Biochemistry 31, 10169–10184. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Liebau, E., Muller, V., Lucius, R., Walter, R.D., Henkle-Duhrsen, K., 1996. Molecular cloning, expression and characterization of a recombinant glutathione

S-transferase from Echinococcus multilocularis. Mol. Biochem. Parasitol. 77, 49–56. Mannervik, B., Alin, P., Guthenberg, C., Jenson, H., Tahir, M.K., Warholm, M., Jörnvall, H., 1985. Identification of 3 classes of cytosolic glutathione transferases common to several mammalian species: correlation between structural data and enzymatic properties. Proc. Natl. Acad. Sci. U.S.A. 82, 7202–7206. Tahir, M.K., Guthenberg, C., Mannervik, B., 1985. Inhibitors for distinction of the three types of human glutathione transferase. FEBS 181, 249–252. Mannervik, B., Danielson, V.H., 1988. Glutathione transferases-structure and catalytic activity. CRC Crit. Rev. Biochem. 23, 283–337. Morello, A., Repetto, Y., Atias, A., 1982. Characterization of glutathione S-transferase activity in Echinococcus granulosus. Comp. Biochem. Physiol. 72B, 449–452. Precious, W.Y., Barrett, J., 1989. Xenobiotic metabolism in helminths. Parasitol. Today 5, 156–160. Repetto, Y., Aldunate, J., Letelier, M.E., Morello, A., 1986. Glutathione S-transferase activity in intact protoscolices of Echinococcus granulosus. Biochem. Soc. Trans. 14, 132–133. Sexton, J.L., Milner, A.R., Panaccio, M., Waddington, J., Wijffels, G., Chandler, D., Thompson, C., Wilson, L., Spithill, T.W., Mitchell, G.F., Campbell, N.J., 1990. Glutathione S-transferase. Novel vaccine against Fasciola hepatica infection in sheep. J. Immunol. 145, 3905–3910. Sheehan, D., Meade, G., Foley, V.M., Dowd, C.A., 2001. Structure, function and evolution of glutathione transferases: implications for classification of nonmammalian members of an ancient enzyme superfamily. Biochem. J. 360, 1–16. Smith, D.B., Johnson, K.S., 1988. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67, 31–40. Thompson, R.C.A., Lymbery, A.J., 1995. Echinococcus and Hydatid Disease. CAB International, Wallingford, Oxon, UK, pp. 1–50. Torres-Rivera, A., Landa, A., 2008. Glutathione transferases from parasites: a biochemical view. Acta Trop. 105, 99–112. Vibanco-Perez, N., Jimenez, L., Mendoza-Hernandez, G., Landa, A., 2002. Characterization of a recombinant mu-class glutathione S-transferase from Taenia solium. Parasitol. Res. 88, 398–404. Vibanco-Perez, N., Jimenez, L., Merchant, M.T., Landa, A., 1999. Characterization of glutathione S-transferase of Taenia solium. J. Parasitol. 85, 448–453. Wilce, M.C.J., Parker, M.W., 1994. Structure and function of glutathione Stransferases. Biochim. Biophys. Acta 1205, 1–18. Wu, Z., Wu, D., Hu, X., Xu, J., Chen, S., Wu, Z., Yu, X., 2006. Molecular cloning and characterization of cDNA encoding a novel cytosolic glutathione transferase from Clonorchis sinensis. Parasitol. Res. 98, 534–538. Wyatt, P.J., 1993. Light scattering and the absolute characterization of macromolecules. Anal. Chim. Acta 272, 1–40.