Characterization of an omega-class glutathione S -transferase from Schistosoma mansoni with glutaredoxin-like dehydroascorbate reductase and thiol transferase activities Javier Girardini 1, *, Alejandro Amirante 2,† , Khalid Zemzoumi 1 and Esteban Serra 1 1 Instituto de Biologı ´ a Molecular y Celular de Rosario, IBR-CONICET, Facultad de Ciencias Bioquı ´ micas y Farmace ´ uticas, UNR; and 2 Facultad de Odontologı ´ a, UNR, Rorario, Argentina Glutathione S-transferases (EC 2.5.1.18) (GSTs), are a family of multifunctional enzymes present in all living organisms whose main function is the detoxification of electrophilic compounds. GSTs are considered the most prominent detoxifying class II enzymes in helminths. We describe here the characterization of novel dehydroascorbate reductase and thiol transferase activities that reside in the human parasite Schistosoma mansoni GSTx. Protein sequence analysis of this parasite product showed lower identity to known GSTs. However, phylogenic analysis placed SmGSTx among the recently described omega class GSTs (GSTO1-1). We report here that SmGSTO protein is a 28-kDa polypeptide, detected in all life stages of the parasite, being highly expressed in adult worms. Like other omega class GSTs, SmGSTO showed very low activity toward classical GSTs substrates as 1-chloro-2,4-dinitrobenzene, and no binding affinity to glutathione–agarose matrix but showed some biochemical characteristics related with thio- redoxins/glutaredoxins. Interestingly, SmGSTO was able to bind S-hexyl glutathione matrix and displayed significant glutathione-dependent dehydroascorbate reductase and thiol transferase enzymatic activities. Keywords: glutathione S-transferase; dehydroascorbate reductase; thiol transferase; Schistosoma. Glutathione S-transferases (GSTs, EC 2.5.1.18) constitute a family of multifunctional enzymes that mainly catalyze the nucleophilic attack of reduced glutathione (GSH) to a wide variety of electrophilic endogenous and exogenous com- pounds. GSTs are found in all living organisms tested to date, present as unique enzymes in lower organisms and as a large number of tissue-specific isoforms in more complex species like mammals [1,2]. The expression level of GSTs is modulated by many compounds including carcinogens, drugs and oxidative- stress metabolites [1]. Several additional functions were attributed to GSTs including the transport of hydrophobic ligands, binding to bilirubin and carcinogens [3,4], the isomerization of maleylacetoacetate and the regulation of stress kinases and apoptosis [5,6]. Based on their sequence structure, catalytic activitiy, immunogenicity, substrate specificity and sensitivity to inhibitors, the mammalian GSTs form six evolutionary distinct classes termed alpha, kappa, mu, pi, sigma, and zeta [1,7,8]. A new class of the GST superfamily, designated GST omega (GSTO) in accordance with the established human GST nomenclature convention [9], has been recently characterized in humans on the basis of structural data [10]. This new enzyme (GSTO1-1) has similar functional characteristics with previously described proteins in rats [11] and mouse [12]. Although the mammalian GSTO has low sequence similarity to other known GSTs, its crystallo- graphic structure showed a GST fold composed of an N-terminal glutathione-binding domain and a C-terminal domain composed entirely of a-helices. In contrast, unlike other GSTs, GSTO has an active site cysteine that is able to form a disulfide bond with GSH and exhibits glutathione- dependent thiol transferase (TTase) and dehydroascorbate reductase (DHA) activities, reminiscent of thioredoxin (Trx) and glutaredoxin (Grx) enzymes [10]. Recent studies have shown new additional functions for human GSTO including monomethylarsonic acid (MMA) reductase activity and the modulation of ryanodine calcium channels [13,14]. A particular member of the GST superfamily, designated Tc52, exhibiting GSH-dependent TTase activity, has been characterized in the human causative agent of Chagas’ disease, Trypanosoma cruzi [15,16]. Further studies have shown that Tc52 is essential for the parasite development and is involved in the immunomodulatory processes asso- ciated with Chagas’ disease [17–20]. Correspondence to E. Serra, Instituto de Biologı ´ a Molecular y Celular de Rosario, IBR-CONICET, Facultad de Ciencias Bioquı ´ micas y Farmace ´ uticas, UNR, Suipacha 531 CP 2000, Rorario, Argentina. Fax: 54 3414390465, Tel.: 54 3414370008, E-mail: eserra@arnet.com.ar Abbreviations: CDNB, 1-chloro-2,4-dinitrobenzene; DHA, dehydro- ascorbate reductase; DCNB, 1,2-dichloro-4-nitrobenzene; Grx, glutaredoxin; GSH, glutathione; GST, glutathione S-transferase; GSTO, GST omega; HEDS, hydroxyethyl disulfide; MMA, mono- methylarsonic acid; Trx, thioredoxin; TTase, thiol transferase. Enzyme: glutathione S-transferase (EC 2.5.1.18). Note: nucleotide sequence data are available in the GeneBank database under the accession number AF484940. *Note: These authors contributed equally to this work. Present address:Hoˆ pital N-Dame, Allergy, M4211-K, 1560 rue sherbrooke Est, Montre ´ al, QC, H2L 4M1, Canada (Received 8 July 2002, revised 3 September 2002, accepted 11 September 2002) Eur. J. Biochem. 269, 5512–5521 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03254.x At least five GST activities have been described in the human parasite Schistosoma mansoni (SmGST-1 to SmGST-5). SmGST-5 has been characterized as an unstable enzyme that may be involved in the conjugation of epoxide substrates and dichlorovos, the active form of the anti-schistosomal drug metrifonate, but no corres- ponding parasite gene has been cloned to date [21–23]. Two members of SmGSTs, 28-kDa Sm28GST and 26-kDa Sm26GST, have been cloned and were found to correspond to the previously reported SmGST-1, -2, -3 and -4 isoenzymes, respectively [22,24]. Although DHA activity was first described in plants several years ago, and then in mammals, insects and protozoans, little is known about nonvertebrate GSH-dependent DHA proteins at the molecular level. We report here the characterization of a new member of the GST superfamily from S. mansoni. When compared with other GSTs, S. mansoni protein showed a limited sequence identity with omega-class GSTs. Additional phylogenic analysis, including known GSTs classes and S. mansoni GSTs, allowed us to place the new parasite product among the newly identified GSTO class, and the previously characterized Sm28GST and Sm26GST as mu- and sigma-class, respectively. Additional evidence placed the S. mansoni protein among the omega class of GSTs, as the recombinant parasite protein (a) did not have significant affinity for glutathione, but bound strongly to S-hexyl glutathione matrix; (b) exhibited low activity towards the classical GST substrate 1-chloro-2,4- dinitrobenzene (CDNB); and (c) showed significant GSH- dependent TTase and DHA activities. The data presented here provide the first evidence for a potential new ascorbic pathway within S. mansoni. EXPERIMENTAL PROCEDURES Materials Reduced glutathione, oxidized glutathione, S-hexyl gluta- thione, 1-chloro-2,4-dinitrobenzene, 1,2 dichloro-4-nitro- benzene, cibacron blue 3GA, and t-butyl hydroquinone, were obtained from Sigma; 7-chloro-4-nitrobenzo-2-oxa- 1,3-diazole, ethacrynic acid, p-nitrobenzyl chloride, dehydroascorbate, bromosulfophthalein, Evan’s blue, hem- atine, and p-chloranil were obtained from ICN; hydroxy- ethyl disulfide (HEDS) and vinylene trithiocarbonate was obtained from Aldrich. EST identification and cDNA isolation The S. mansoni EST data base search was performed using the t BLAST n version of blast program [25] with the T. cruzi Tc52 amino acid sequence as query. An EST encoding an unknown protein was obtained (GeneBank accession num- ber AI975843). A 549-bp fragment was amplified by PCR using oligonucleotides GSTX1: 5¢- GTTGTCGACAAA CATCTCAACTAG-3 and GSTX3: 5¢-GTAAGTGTGG GAATAAGATCAAATC from adult S. mansoni reverse- transcribed RNA. The product of the PCR was sequenced and used as probe to screen an adult worm S. mansoni lambda gt10 cDNA library by conventional methods [26]. Southern blot analysis was performed using S. mansoni cercariae purified DNA as described [26]. Alignments and phylogenetic analysis of S. mansoni GSTs Sm26GST, Sm28GST and SmGSTO amino acid sequences were aligned manually on the alignment provided by L. Jermiin (see [10]). From the original alignment used by Board et al. [10] eight not clearly class-defined sequences were avoided. A phylogenetic tree was obtained by maximum likelihood analysis of all the sites in the above- mentioned alignment. The data was analyzed using the JTT- F substitution model [27], and local bootstrap probabilities were estimated for the internal branches using the PROTML program [28]. More than one analysis was performed by using different input order of the sequences. Each analysis involved two steps: stepwise addition and nearest neighbor interchanges. The most likely tree was obtained by using the test of Kishino and Hasegawa [29]. All calculations were performed using the MOLPHY 2.3 molecular phylogenetics programs package [28]. Expression and purification of recombinant SmGSTO Recombinant SmGSTO was expressed in Escherichia coli and purified by two separate methods using either the pQE30 vector (Qiagen) and nickel agarose affinity chroma- tography, or the pT7-7 vector [30] and S-hexyl glutathione affinity chromatography. To produce N-terminal 6 · His tag fused SmGST, the SmGSTO cDNA was amplified by polymerase chain reaction (sense primer GSTX2: 5¢-AAGGATCCATGCACCTTAAACGAAATGACC-3¢; antisense primer odTSalI: 5¢-AAGTCGACTTTTTTTTTT TTTTTTTTTS-3¢) and was inserted between the BamHI and SalI sites of the bacterial expression vector pQE30 (Qiagen), the cloned vector was transformed into BL21 [DE3] cells (Novagen, Milwaukee, WI, USA). Briefly, a seed culture of the transformed cells was grown to D 600 of 0.4–0.6, scaled up, grown again to the same density, induced with IPTG (0.5 l M ), and grown for a further 3 h at 30 °C. His-tagged SmGSTO product was purified on nickel agarose as described by manufacturers (Qiagen). The enzyme was eluted with 250 m M imidazole, 50 m M potas- sium phosphate, pH 7.6, and exhaustively dialyzed against the same buffer to remove imidazole before storage at 80 °C in 50% glycerol. Recombinant SmGSTO was also expressed from it’s own methionine initiation codon. Briefly, SmGSTO cDNA was amplified by polymerase chain reaction (sense primer GSTX4: 5¢-AAACATATGAT GCACCTTAAACGAAATGACC-3¢; antisense primer odTSalI) and cloned between the NdeIandSalIsitesof the expression vector pT7-7. Protein was purified from soluble extracts on S-hexyl glutathione agarose (Sigma) as previously described [31]. The enzyme was eluted with 5 m M S-hexyl glutathione, 50 m M Hepes, pH 8, and dialyzed against 50 m M Hepes, pH 8.0, before storage. Purification yield approximately 500 lg of protein per milliliter of S-hexyl glutathione agarose. In all cases, protein purity was determined by SDS/PAGE and protein concentration was measured by bicinchoninic acid method following manu- facturers indications (Sigma). Antiserum against the puri- fied protein was raised in rabbits using standard immunization protocols. Glutathione and S-hexyl glutathione affinity assays were performed in batch. Briefly, 20 lL of 50% resuspended Ó FEBS 2002 Omega-class GST from Schistosoma mansoni (Eur. J. Biochem. 269) 5513 resin was centrifuged and the supernatant was eliminated. Ten microliters of 1 mgÆmL )1 purified enzyme was added to the same tube and incubated in ice with gentle agitation. After 30 min the supernatant was recovered by centrifuga- tion. The resin was washed four times with 250 lLof 50 m M Hepes, pH 8.0, and eluted by adding 10 lLofthe same buffer containing 5 m M GSH, GSSG or S-hexyl GSH. Ten microliters of the original protein, binding assay supernatant and eluate were analyzed by SDS/PAGE. Expression of SmGSTO along the parasite life cycle Total proteins were prepared as follows: S. mansoni schist- osomule or sporocysts were resuspended in extraction buffer (100 m M Tris, 3 m M EDTA, 1 m M phenylmethylsulfonyl fluoride, 2.5 lgÆmL )1 leupeptin, 4 lgÆmL )1 pepstatin A); adults or cercariae were first homogenized with liquid nitrogen and then resuspended in extraction buffer. The extracts were homogenized by pulses of 1 min at 25% amplitude and centrifuged at 10 000 g for 20 min at 4 °C. The soluble fraction was ultracentrifuged at 105 000 g for 30 min at 4 °C and the supernatants were used for assays. One hundred micrograms protein from each extract were separated by SDS/PAGE, electroblotted to a nitrocellulose membrane (Amersham Pharmacia). Western blot experi- ments were carried out according to standard techniques. Ten micrograms of DNAse I-treated total RNA from S. mansoni miracidia, sporocyst, cercariae and adult worm were reverse transcribed by using 100 U of SuperScript TM reverse transcriptase (Life Technologies) in 50 lLof supplied reaction buffer. PCRs were performed on 1 lL of each reverse transcription reaction and resolved by agarose gel electrophoresis. Primers used were: GSTX1/ GSTX3 for SmGSTO and TUB3 (5¢-GAAGTGGAT ACGAGGATAAGGTACCAG-3¢)/TUB4 (TGGAACTT ATCGTCAACTTTTCCATCC-3¢)forS. mansoni a-tubu- lin. SmGSTO amplification bands were quantified by using GelPro and normalized by comparing to a-tubulin ampli- fication products. Enzyme assays Enzymatic activity towards a range of substrates and inhibitors was determined as described [32]. Thiol trans- ferase activity was measured according to Axelsson et al. [33] using HEDS as substrate. The reaction mixture contained 0.2 m M NADPH, 0.5 m M GSH, 50 m M phos- phate buffer, 0.5 units of glutathione reductase and an aliquot of the protein solution. The reaction was initiated by the addition of 2 m M of HEDS at 30 °C and followed by 340 nm absorption decrease. Absorption coefficient used for NADPH oxidation at 340 nm was 6.22 m M )1 Æcm )1 . Glutathione-dependent dehydroascorbate reductase activity was determined by following the dehydroascorbate (DHA) reduction spectrophotometrically at 265 nm. The standard reaction mixture contained 50 m M phosphate buffer, pH 8, 1m M GSH, and was started with the addition of 0.25 m M DHA after a 1 min preincubation [34]. Construction of a homology model of SmGSTO SmGSTO structure was built using the human GSTO 1–1 structure as template in the SWISS-MODEL modeling environment and structure accuracy was assayed by WHAT CHECK tool in the same environment [35]. Briefly, structurally driven alignments were performed using SmGSTO sequence. Best alignment, obtained based on GSTO1-1, was refined and used to obtain an optimized model. The WhatCheck tool (from the WHAT IF package program) was used to estimate accuracy of the structure obtained. Parameters taken into account were: Ramachandran plot appearance Z-score, chi- 1/chi-2/Z-score, packing quality Z-score and RMS Z-scores (http://www.cmbi.kun.nl/gv/pdbreport/checkhelp/). RESULTS SmGSTO DNA and protein sequence BLAST search of the S. mansoni EST database with the complete sequence of Tc52 revealed a clone (EST AI975843) with around 25% sequence identity with the GST-like domain of the T. cruzi protein. A fragment of 549 bp was amplified by PCR using specific primers, designed based on the EST sequence, and cDNA from S. mansoni adult worms. The amplified fragment was sequenced and used as probe to screen an adult worm cDNA library. After three rounds of hybridization, two independent clones were purified and sequenced. The sequences obtained were identical in both clones and corresponded to a cDNA of 934 bp including an open reading frame encoding for a 241 amino acid polypeptide, with a predicted molecular mass of 27.6 kDa (Fig. 1). Sequence identity ranging from 18 to 25% was obtained with human GST-theta, mouse p28, rat DHA, human GSTO 1-1, as well as with several plant DHAs and non characterized GST-like proteins. In all cases, the most conserved amino acids were localized in the N-terminal domain of SmGSTO. The best hit obtained (E ¼ 8.1e )11 ) when searched at HMM motifs databases was the pfam glutathione S-transferase N-terminal domain (GST-Nter, PF02996). In order to determine whether our sequence belongs to the GST omega class, a phylogenetic analysis using the maximum-likelihood approach was carried out. To achieve this, our sequence, as well as Sm28GST and Sm26GST sequences, were included in a multiple-sequence alignment similar to that previously used to characterize human omega class GST [10]. The tree in Fig. 2 is the most likely tree obtained by neighbor-interchange analysis of 2000 likeli- hood trees, and shows the eight families previously described by Board et al. [10] The tree grouped the new SmGST with human, rat, mouse and Caenorhabditis elegans omega GSTs as well as two plant GST-like sequences already proposed to belong to the omega class by Board et al. [10] leading us to name it SmGSTO. Concerning the two other S. mansoni GSTs, Sm26GST grouped within mu-class GSTs with a high local bootstrap value and Sm28GST remains in a nondefined position between sigma and pi classes. A Southern blot analysis was performed in order to ascertain that the cloned sequence belonged to the parasite and was not due to an artifact, and also to examine the copy number of SmGSTO genes in the parasite genome. Cerca- riae genomic DNA was digested with several restriction enzymes and hybridized with radiolabeled SmGSTO probe (Fig. 3). The pattern of hybridization obtained suggested that one copy of SmGSTO exists per haploid genome in S. mansoni. 5514 J. Girardini et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Fig. 1. Nucleotide and deduced amino acid sequences of SmGSTO 1. The codon corres- ponding to the initial methionine is underlined. Fig. 2. Unrooted phylogeny showing the most likely relationship between class representative GSTs and S. mansoni GST amino acid sequences. Branch lengths are proportional to estimates of evolutionary change. The number associated with each internal branch is the local bootstrap probability that is an indicator of confidence. The sequences are (species name; GenBank TM accession number): Schistosoma omega (Schistosoma mansoni, AF484940), nematode omega (Caenorhabditis elegans, L23651), mouse omega (Mus musculus, U80819), rat omega (Rattus rattus, AB008807), human omega (Homo sapiens, AF212303), soybean heat-shock protein (HsPr) (Glycine max, M20363), potato GST (Solanum tuberosum, J03679), nematode zeta (Caenorhabditis elegans, Z66560), human zeta (Homo sapiens, NM_001513), carnation zeta (Dianthus caryophyllus, M64268), mouse theta (Mus musculus, U48419), human theta (Homo sapiens, NM_000854), blowfly delta (L. cuprina, L23126), house fly delta (Musca domestica, X61302), fruit fly Delta (Drosophila melanogaster, X14233), Arabidopsis phi (Arabidopsis thaliana, D17672), Petunia phi (Petunia hybrida, Y07721), mouse mu (Mus musculus, J03952), human mu (Homo sapiens, NM_000848), chicken mu (Gallus gallus, X58248), rat Pi (Rattus norvegicus, L29427), human pi (Homo sapiens, NM_000852), rat sigma (Rattus norvegicus, D82071), human sigma (Homo sapiens, D82073), squid2 sigma (Ommastrephens sloanei, M36938), squid1 sigma (O. sloanei, M36937), Schistosoma 28 kDa (Schistosoma mansoni, S71584), human alpha (Homo sapiens, NM_000846), mouse alpha (Mus musculus, M73483), and chicken alpha (Gallus gallus, L15386), Schistosoma 26 kDa (Schistosoma mansoni, M31106). Ó FEBS 2002 Omega-class GST from Schistosoma mansoni (Eur. J. Biochem. 269) 5515 GSH binding affinity of recombinant SmGSTO Recombinant SmGSTO was first produced as a fusion to a histidine-tag and purified in one step using an Ni 2+ nitrilotriacetic acid resin (Fig. 4A). There are some discrep- ancies in the literature about the ability of omega class GSTs to bind different glutathione-coupled matrixes. To deter- mine whether SmGSTO was able to bind glutathione agarose, we have used the purified enzyme in batch assays. As showed in Fig. 4B,C, His-tagged SmGSTO was not able to bind to agarose-coupled glutathione, but was able to bind to S-hexyl glutathione agarose and was recovered after elution with S-hexyl glutathione, as was already described for Tc52 [16]. In order to ascertain our observation and to rule out the (possibility that the lack of binding of SmGSTO to glutathione was neither due to the presence of the His-tag sequence nor to a deletion in the parasite protein sequence, we constructed a second plasmid which directed the production of SmGSTO from its own methionine initiation codon, lacking the His-tag. In this way, the recombinant SmGSTO could be purified using an S-hexyl glutathione– agarose matrix (Fig. 4D). Furthermore, batch experiments performed with this protein determined that S-hexyl glutathione agarose-bound SmGSTO was not eluted by reduced nor by oxidized glutathione (Fig. 4E). These results suggested that SmGSTO has a low affinity to glutathione, which coincides with the K m value obtained for GSH in kinetic experiments (see below). Expression of SmGSTO The recombinant SmGSTO was used to produce polyclonal antibodies in immunized rabbits. Expression of SmGSTO along the parasite life cycle was analyzed by Western blot. In cercariae, sporocysts, schistosomule and adult worms, a immunoreactive band was observed with a calculated molecular weight of nearly 28 kDa (Fig. 5A). However, higher SmGSTO levels were observed in sporocysts (para- sitic stage of the intermediate host Biomphalaria glabrata) and adult worms (parasitic stage of human) than in others stages. Expression of SmGSTO was also studied at the transcriptional level by RT-PCR using cDNA prepared from miracidia, sporocysts, cercariae and adult worms (Fig. 5B). A unique amplification product of the 549 bp expected size was observed in all reactions. The intensity of the amplified products was compared after normalization using a-tubulin cDNA as internal control. Relative values showed as a bar graphic determine that SmGSTO tran- scription is higher in sporocysts and adult worms in comparison with cercariae and miracidia. This results are in agreement with those obtained by Western blot. Taken together, these results suggested that SmGSTO is expressed more in parasitic stages than in free living stages during the S. mansoni life cycle. Characterization of recombinant SmGSTO enzymatic activity Recombinant GSTO was used to assay its enzymatic activity. Results of substrate specificity are shown in Table 1. SmGSTO showed a negligible activity against CDNB and ethacrynic acid and no measurable activity against other GST substrates like 1,2-dichloro-4-nitroben- zene (DCNB), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole; p-nitrobenzyl chloride, vinylene thiocarbonate, t-butyl hydroquinone and p-chloranil. In contrast, SmGSTO showed DHA and HEDS-measured TTase activities. Kinetic parameters were obtained for DHA and TTase activities. SmGSTO showed a K m ¼ 0.23 m M for HEDS and a K m ¼ 0.32 m M for GSH in the TTase reaction. These values were similar to those obtained for the same reaction for glutaredoxins from several origins [36]. When kinetic parameters were calculated for DHA activity, a K m ¼ 2.3 m M for DHA was obtained. Graphical analysis of the data obtained for GSH concentrations between 300 l M and 4 m M in a Lineweaver–Burk plot resulted reproducibly in a K m value of 6.5 m M . As GSH concentra- tions >4 m M could not be tested, we prefer to give a K m of >4 m M for GSH in this reaction. Even though differences in K m values for GSH in the two assayed reactions were obtained, it was clear that a K m lower than 0.32 m M could not be reached. Finally, SmGSTO showed differential sensitivity to several GST inhibitors tested (Table 2). Among them, it is interesting to note the inhibitory activity of CDNB which is considered as a classical GST substrate. Specific activity was first measured for both reactions in standard conditions using a phosphate buffer pH 7.6 [33]. However, when the pH profile for SmGSTO activity was carried out, optimal activities were recorded at pH 8.0 and pH 8.6 when phosphate buffer or Tris/HCl buffer were used, respectively (Fig. 6). A similar optimal pH profile was recently reported for Plasmodium falciparum glutaredoxin 1 activity [37]. Construction of a homology model of SmGSTO SmGSTO structure was built using the human GSTO 1–1 structure as template in the SWISS-MODEL modeling Fig. 3. Southern blot analysis of SmGST. DNA from S. mansoni cercaria (10 lg) digested with different restriction enzymes was hybridized with the coding region of SmGST-O cDNA radiolabeled by a random primer. 5516 J. Girardini et al. (Eur. J. Biochem. 269) Ó FEBS 2002 environment [35]. Structure accuracy was assayed by WhatCheck tool in the same environment. As can be observed in Fig. 7(A), only N-terminal SmGSTO domain structure could be predicted with accuracy. The program was unable to model the C-terminal domain of SmGSTO due to the low rate of identity between the two proteins in this region. However, as the active sites of the GSTO1-1 are located at the N-terminal domain, several shared structural features with SmGSTO could be pointed out. Residues S(86), E(85), V(72), K(59) and C(32) which contact GSH in GSTO 1–1 correspond to S(79), E(78), V(65), K(52) and C(25) in SmGSTO, and were predicted to be placed in the same spatial orientation (Fig. 7B). DISCUSSION We report here the characterization of a new member of the GST superfamily from the human blood fluke S. mansoni. BLAST searches for sequence similarities showed that the cloned parasite gene has sequence homology to members of the recently discovered omega class of GSTs [10], and was Fig. 4. Expression and purification of His-tagged SmGSTO and native SmGSTO. (A) SDS/PAGE analysis of His-tagged SmGST purified on nickel-agarose. Lane 1, 50 lg soluble extract of BL21 (ED3) expressing His-tagged SmGSTO. Lane 2, 5 lg purified His-tagged SmGSTO. (B) Batch analysis of His-tagged SmGSTO to GSH-agarose. Lane 1, protein used in the binding step. Lane 2, supernatant of the binding-step. Lane 3, GSH eluted fraction. (C) Batch analysis of His-tagged SmGSTO to S-hexyl glutathione-agarose. Lane 1, protein used in the binding step. Lane 2, supernatant of the binding-step. Lane 3, S-hexyl glutathione eluted fraction. (D) SDS/PAGE analysis of recombinant native GST purified on S-hexyl glutathione-agarose. Lane 1, 50 lg soluble extract of BL21 (ED3) expressing native SmGSTO. Lane 2, 5 lg purified SmGSTO. (E) Elution of S-hexyl glutathione agarose-bound SmGSTO by GSH, GSSG or S-hexyl glutathione. In all cases: Lane 1, protein used in the binding step. Lane 2, supernatant of the binding-step. Lane 3, eluted fraction. Compound used to elute in each case are indicated in the figure. Fig. 5. Expression of SmGSTO in S. mansoni. (A) Immunoblotting analysis of parasite extracts. Each lane contains total protein (50 lg) from cercariae (lane 1), schistosomula (lane 2), sporocysts (lane 3), and adult worms (lane 4) electroblotted and immunodetected by a-SmG- STO serum. (B) RT-PCR analysis of different S. mansoni stages. Reverse-transcribed RNA from miracidia (lane 1), sporocysts (lane 2), cercariae (lane 3) and adult worms (lane 4) were amplified using SmGSTO and a-tubulin specific primers as indicated in experimental section. Ó FEBS 2002 Omega-class GST from Schistosoma mansoni (Eur. J. Biochem. 269) 5517 referred here as SmGST omega (SmGSTO). In addition, sequence alignment of SmGSTO with representative sequences from the recently reported GSTO class or from the EST data base highlights similarities, but also significant differences. In all cases, the most conserved amino acids were localized in the N-terminal domain of the SmGSTO protein. However, the remaining regions of SmGSTO presented significant divergences from the known GSTO, at the primary structure level. Indeed, the GSTO class represents a particular class of the GST superfamily which possesses specific structural features, such as an active site cysteine that is able to form a disulfide bond with GSH, a novel domain formed by the proximity of a specific N-terminal extension to the C-terminus and a large H-site, as well as the ability to catalyze the GSH-dependent reduction of dehydroascorbate [10]. We further undertook a phylogenetic protein sequence analysis to ascertain whether the SmGSTO was a divergent member of the GSTO class. The deduced SmGSTO protein sequence was used in a phylogenic analysis using the maximum-likelihood approach. Two other previously char- acterized S. mansoni GSTs, Sm26GST and Sm28GST, were amongst the GST proteins included in this study. The most likely tree obtained showed the eight proposed GST families [7,8]. The tree grouped the new cloned schistosome protein as a divergent member of the GSTO class. We could also place the Sm26GST among the Mu-class. Even if position of Sm28GST was not well defined in the tree, the fact that Sm28GST has a prostaglandin D2 synthetase activity, typical of the sigma class of GSTs, strongly suggest that it belongs to this group (J. F. Trottein, Institut Pasteur de Lille, France, personal communication). All S. mansoni GSTs showed particularly long branches when compared with those obtained for free living invertebrates GSTs. This high rate of evolution for Schistosoma GSTs was already reported [38]. The same abnormality was also observed when the phylogenetic analysis of S. mansoni nuclear receptors was performed [39]. The proposed explanation for this observation is that the human parasites of Schisto- soma genus were subjected during evolution to a biased selection pressure due to host biochemical characteristics, including the immune and endocrine systems. The host pressure resulted in abnormally divergent parasite sequences as shown for GSTs and nuclear receptors of the parasite, or by abnormally host-parasite converged sequences as was reported for the tropomyosins of S. mansoni and its intermediate host Biomphalaria glabrata [40]. SmGSTO expression in the different life forms of S. mansoni was performed by RT-PCR and Western blot. The results obtained support the higher expression of SmGSTO observed in S. mansoni parasitic life stages rather than in free-living life stages, suggesting that this protein may play a role in the survival of the parasite within the host. An increased expression during cercariae transforma- tion to mature adults in mammalian host was already described as a general feature for detoxifying enzymes in Schistosoma [41–43]. Immunohistochemical analysis of human tissues confirmed a widespread expression of GSTO1-1, suggesting that it has important biological functions. Specific expression of GSTO1-1 was localized in the nuclei and in nuclear membranes of many cell types [44]. However, no putative nuclear localization signals could be found within GSTO1-1 or SmGSTO. Nuclear localization Table 1. Substrate specificities of recombinant SmGSTO. Activity for each substrate was determined in standard conditions. ND, not detected. Substrate Specific activity (lmolÆmin )1 Æmg )1 ) 1,2-Dichloro-4-nitrobenzene ND 1-Chloro-2,4-dinitrobenzene 0.02 7-Chloro-4-nitrobenzo-2-oxa- 1,3-diazole ND Ethacrynic acid 0.02 p-Nitrobenzyl chloride ND Vinylene trithiocarbonate ND t-Butyl hydroquinone ND p-Chloranil ND Dehydroascorbate 0.20 Hydroxyethyl disulfide 0.11 Table 2. Inhibitor sensitivities of recombinant SmGSTO. Inhibitor sensitivities are presented as I 50 and were calculated in standard reac- tion conditions. Values in the table represent the mean of three determinations. Dehydroascorbate activity inhibitor Hydroxyethyl disulfide activity I 50 (l M )I 50 (l M ) Evan’s blue 12.5 4.8 Bromosulfophthalein 0.3 8.3 Cibacron blue 3GA < 0.001 < 0.001 S-Hexyl glutathione < 0.001 < 0.001 Ethacrinic acid 0.1 238 Hematine 6.4 4.5 CDNB 5.3 < 0.001 Fig. 6. pH optimum of SmGSTO. The SmGSTO pH profile was car- ried out using the glutathione:dehydroascorbate reductase assay. Grafic indicates relative DHA activity measured in 100 m M phosphate buffer and 100 m M Tris/HCl, using standard substrate concentrations. 5518 J. Girardini et al. (Eur. J. Biochem. 269) Ó FEBS 2002 of S. mansoni 28 kDa GST, which has no detectable nuclear localization signal, was already described [45]. A cytolocali- zation study of SmGSTO is being undertaken in our laboratory. Some contrasting findings were reported concerning the ability of omega GSTs to bind matrix-linked glutathione. Mouse p28 was reported to bind glutathione-agarose, but human GSTO1-1 was unable to bind S-linked glutathione- sepharose. Here, we report that SmGSTO binds S-hexyl glutathione-agarose but not glutathione-agarose. Moreover, S-hexyl glutathione-agarose-bound SmGSTO was not dis- placed neither by reduced nor oxidized glutathione. These results are in line with the high GSH K m value obtained for SmGSTO and the strong inhibitory effect of S-hexyl glutathione on the enzyme activity. The preference of SmGSTO for more hydrophobic alkyl-bound S-hexyl glutathione rather than glutathione could reflect some particular characteristics of the active site of this enzyme. As other GSTs from the omega class the parasite protein was unable to use known GST family substrates like CDNB and other GST substrates assayed (Table 1). The most significant enzymatic activities observed for SmGSTO were the ability to act as GSH-dependent DHA and TTase. SmGSTO activity was inhibited to different extents by classical GSTs inhibitors. In addition, GSTs’ substrate, CDNB, which inhibits thioredoxin activity, also inhibited GSTO activity. Our phylogenic analysis showed that omega, zeta and theta GST classes, which demonstrated low activities towards CDNB substrate, branch together in the left side of the tree whereas the remaining classes, which use CDNB as substrate, branch in the right side of the tree. This observation may reflect an evolutionary relationship among this group of proteins. To confirm this assumption, the inhibitory activity of CDNB over zeta- and theta-class GSTs should be tested. Finally, the SmGSTO structure was built based on homology modeling. Even if sequence divergence makes it impossible to produce a model of the whole protein, a prediction of the N-terminal domain was obtained. This GSTO domain shares structural features with the recently described glutaredoxin-2 from E. coli [46]. Glutaredoxins can catalyze the reduction of mixed disulfides between GSH and proteins or low molecular mass disulfides, in a reaction that only requires N-terminal active-site cysteine residue, and the reverse reaction called glutathionylation [47]. Xia et al. recently proposed a three subfamilies classification for glutaredoxins. The first group, which includes E. coli Gsx1 and human Grx1 amongst others, corresponds to small Ôtwo-cysteineÕ (dithiol) classical glutaredoxins which contain the consensus sequence C-P-Y-C and have a high activity with HEDS. The second group corresponds to variable molecular mass Ôone-cysteineÕ (monothiol) glutaredoxins with a conserved sequence C-G-F-S, such as yeast gluta- redoxins Grx3–5. E. coli Grx2 and GSTO 1–1 were proposed to be grouped into a third subfamily, having an N-terminal Grx-like domain and the helical C-terminal domain and the general structure reminiscent to the GST superfamily of proteins. Sequence similarity and predicted structure show Fig. 7. Model showing SmGSTO structure. Structure was built based on homology modeling using human GSTO1-1 as template in the SWISS-MODEL modeling environ- ment. (A) General chain fold view of human GSTO 1–1 and SmGSTO. (B) Scheme illus- trating position of GSH contacting residues determined for human GST 1–1 and modeled for SmGSTO. Ó FEBS 2002 Omega-class GST from Schistosoma mansoni (Eur. J. Biochem. 269) 5519 that SmGSTO belongs to this last group. At this point, it should be noted that the questions as to whether E. coli Grx- 2 is a GST or if GST-O are glutaredoxins is not completely solved. When active cysteine-containing tetramers were sought in these proteins, a striking sequence divergence was observed. E. coli Grx2 contains a Trx1-like two-cysteine sequence, C-P-Y-C; GSTO 1–1 has a one-cysteine C-P-F-A sequence; and SmGSTO contains C-P-Y-V, similar to the Trx1-like sequence but with only one cysteine. Sequence comparison at the active site level and HEDS-measured SmGSTO TTase activity strongly suggest that SmGSTO could participate in glutathionylation and reduction of mixed disulfides, a typical glutaredoxin activity. Sensitivity to CDNB and alkaline optimal pH, which stabilize the GSH thiolate at the active cysteine, are considered as GSTO biochemical characteristics that support the idea of a functional relationship between omega-class GSTs and glutaredoxins/thioredoxins superfamily. In this way, Caccuri et al. [48] recently proposed that Proteus mirabilis low molecular weight GST could be an intermediary enzyme somewhere between thioldisulfide oxidoreductases and the GST superfamily. P. mirabilis GST differs from SmGST-O because of it shows both TTase and CDNB-measured transferase activities. Keeping in mind that GSTO contains an additional domain, new activities can not be ruled out for GSTO. To resume, SmGSTO can be considered as a multifunc- tional enzyme displaying thioredoxin/glutaredoxin features. The additional C-terminal domain could allow this enzyme to react with a large substrate spectrum in comparison with low molecular weight thioredoxins and glutaredoxins. To date, very little is known about thioredoxin or glutaredoxin metabolism in S. mansoni. Recently a thioredoxin/glutathi- one reductase containing a thioredoxin/glutaredoxin-like motif at the N-terminal was described in this parasite [49]. Structural and functional relationships between these two multifuctional enzymes should be explored in the future. Finally, this work is the first evidence that S. mansoni may take advantage of host ascorbic acid. The physiological significance of all these findings will need much investigation in order to be understood. ACKNOWLEDGMENTS The authors wish to thank Dr Lars Jermiin for GST sequences alignments and for His help with Molphy 2.3 utilization, Dr Luis Esteban for His help with Linux operative system installation and utilization and Dr Eleonora Garcı ´ aVe ´ scovi for her critical reading of the manuscript. This research was supported by Fundacion Antorchas, Third World Academy of Sciences and the Research Program of the UNR. ECS is member of the National Research Council (CONICET, Argentina) and JEG is Fellow of the same institution. REFERENCES 1. 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. CRC Crit.Rev.Biochem.Mol.Biol.30, 445–600. 2. Vuilleumier, S. (1997) Bacterial glutathione S-transferases: What are they good for? J. Bacteriol. 179, 1431–1441. 3. Tipping, E. & Ketterer, B. 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(2002) The disulfide redox system of Schistosoma mansoni and the importance of a multifunctional enzyme, thioredoxin glutathione reductase. Mol. Biochem. Para- sitol. 121, 129–139. Ó FEBS 2002 Omega-class GST from Schistosoma mansoni (Eur. J. Biochem. 269) 5521 . Characterization of an omega-class glutathione S -transferase from Schistosoma mansoni with glutaredoxin-like dehydroascorbate reductase and thiol transferase. transferase enzymatic activities. Keywords: glutathione S -transferase; dehydroascorbate reductase; thiol transferase; Schistosoma. Glutathione S-transferases