Parasitol Res (2015) 114:2119–2127 DOI 10.1007/s00436-015-4400-8

ORIGINAL PAPER

Molecular cloning and characterization of Fasciola gigantica thioredoxin-glutathione reductase Narin Changklungmoa & Pornanan Kueakhai & Kant Sangpairoj & Pannigan Chaichanasak & Wipaphorn Jaikua & Suda Riengrojpitak & Prasert Sobhon & Kulathida Chaithirayanon

Received: 15 January 2015 / Accepted: 27 February 2015 / Published online: 19 March 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract The Fasciola gigantica thioredoxin-glutathione reductase (FgTGR) gene is a fusion between thioredoxin reductase (TR) and a glutaredoxin (Grx) gene. FgTGR was cloned by polymerase chain reaction (PCR) from adult complementary DNA (cDNA), and its sequences showed two isoforms, i.e., the cytosolic and mitochondrial FgTGR. Cytosolic FgTGR (cytFgTGR) was composed of 2370 bp, and its peptide had no signal sequence and hence was not a secreted protein. Mitochondrial FgTGR (mitFgTGR) was composed of 2506 bp with a signal peptide of 43 amino acids; therefore, it was a secreted protein. The putative cytFgTGR and mitFgTGR peptides comprised of 598 and 641 amino acids, respectively, with a molecular weight of 65.8 kDa for cytFgTGR and mitFgTGR, with a conserved sequence (CPYC) of TR, and ACUG and CVNVGC of Grx domains. The recombinant FgTGR (rFgTGR) was expressed in Escherichia coli BL21 (DE3) and used for production for a polyclonal antibody in rabbits (antirFgTGR). The FgTGR protein expression, estimated by indirect ELISA using the rabbit anti-rFgTGR as probe, showed high N. Changklungmoa : W. Jaikua : S. Riengrojpitak Department of Pathobiology, Faculty of Science, Mahidol University, Rama VI Rd, Bangkok 10400, Thailand N. Changklungmoa e-mail: [email protected] N. Changklungmoa : P. Kueakhai Faculty of Allied Health Sciences, Burapha University, Long-Hard Bangsaen Rd, Mueang District, Chonburi 20131, Thailand P. Chaichanasak Faculty of Veterinary Medicine, Mahanakorn University of Technology, Cheum-Sampan Rd, Nong Chok, Bangkok 10530, Thailand K. Sangpairoj : P. Sobhon : K. Chaithirayanon (*) Department of Anatomy, Faculty of Science, Mahidol University, Rama VI Rd, Bangkok 10400, Thailand e-mail: [email protected]

levels of expression in eggs, and 2- and 4-week-old juveniles and adults. The rFgTGR exhibited specific activities in the 5,5′dithiobis (2-nitro-benzoic acid) (DTNB) reductase assay for TR activity and in β-hydroxyethul disulfide (HED) for Grx activity. When analyzed by immunoblotting and immunohistochemistry, rabbit anti-rFgTGR reacted with natural FgTGR at a molecular weight of 66 kDa from eggs, whole body fraction (WB) of metacercariae, NEJ, 2- and 4-week-old juveniles and adults, and the tegumental antigen (TA) of adult. The FgTGR protein was expressed at high levels in the tegument of 2- and 4-weekold juveniles. The FgTGR may be one of the major factors acting against oxidative stresses that can damage the parasite; hence, it could be considered as a novel vaccine or a drug target.

Keywords Fasciola gigantica . Thioredoxin-glutathione reductase . Cloning . Molecular characteristics . Tissue expression

Introduction Fasciola gigantica is a prevalent trematode parasite causing fasciolosis in tropical regions, which results in serious losses of domestic animals, especially cattle, sheep, and goats. The parasite can also infect humans (Fairweather et al. 1999; Torgerson and Claxton 1999). Metacercariae are the stages that infect the hosts who ingest metacercaria-contaminated grass or vegetables. In the small intestine of the host, metacercariae excyst, to become newly eaxcysted juveniles (NEJ) by the actions of gastric enzymes, bile salts, and proteases released from the metacercariae themselves. The NEJ immediately penetrate through the intestinal wall and enter the abdominal cavity, from where they migrate over a period of approximately 7 days to the liver. As the young parasites develop and migrate through the liver, they feed mainly on blood

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and parenchymal cells for about 6 weeks, which result in hemorrhage and liver damage. After this period, the parasites enter the bile duct and mature into adults at about 3 months after the infection. During their sojourn in the liver, the parasites are exposed to reactive oxygen species (ROS) generated by the host’s immune cells, including neutrophils, eosinophils, and macrophages (Badwey and Karnovsky 1980; Tagaya et al. 1989; Bertini et al. 1999; Nakamura et al. 2001). However, the parasites can evade the damage resulting from the ROS through their possession of a series of antioxidant proteins and enzymes that played essential roles in cellular redox response, including superoxide dismutase (SOD; Assady et al. 2011), peroxiredoxin (Prx; Sangpairoj et al. 2014; Chaithirayanon and Sobhon 2010), thioredoxin glutathione reductase (TGR; Maggioli et al. 2011), and thioredoxin (Trx; Changklungmoa et al. 2014; Alger et al. 2002). The antioxidant system is the first defense line against the damaging effects from the ROS. SOD is the first among the series of antioxidant enzymes that accelerates the dismutation of superoxide to hydrogen peroxide (H2O2). The Trx and glutathione (GSH) systems are the next major thiol-dependent redox pathways. Both systems have overlapping yet distinct properties but function in a similar manner in maintaining redox homeostasis and providing defense against oxidative stress and also convert H2O2 to water. In Fasciola spp., TGR is a combination molecule containing both Trx and GSH domains (Maggioli et al. 2011); thus, TGR showed both Trx and GSH activities (Angelucci et al. 2008; Salinas et al. 2004). Because of the crucial role of TGR in the antioxidation system, it is regarded as a potential target for drug and possibly as a novel vaccine against fasciolosis gigantica. In this study, we report on the characteristics and expression of TGR in F. gigantica.

Materials and methods Parasites specimens F. gigantica metacercariae were obtained from experimentally infected snails, Lymnea ollula. NEJs were produced by activating the excystment of metacercariae according to the method described by Sethadavit et al. (2009). The 2- and 4-week-old juvenile parasites were collected from the livers of the Golden Syrian hamsters experimentally infected with metacercariae (Changklungmoa et al. 2012, 2014). Eggs as well as adult parasites were collected from the bile ducts and gallbladders of the naturally infected cattle killed at a local abattoir in Pathum Thani Province, Thailand. They were then washed several times with 0.85 % NaCl and kept for further experiments. Cloning and SEQUENCE analyses of FgTGR gene The adult F. gigantica complementary DNA (cDNA) was used for amplification of the DNA fragment of TGR gene

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using specific primers designed from the F. hepatica TGR sequence (GenBank accession no. AM709787), which comprise of a forward primer (5′ TGT GTT AAC GTT GGC TGC 3′) and a reverse primer (5′ GCA GCC AAC GTT AAC ACA 3′). The fragment of FgTGR was cloned by polymerase chain reaction (PCR) using λTriplEx2 sequencing primers and specific primers of TGR gene and then inserted into the pGEM-T easy vector (Promega, Madison, USA). Finally, the purified plasmid-DNA isolated from a single colony was sequenced by Macrogen Inc. (South Korea). The nucleotide and deduced amino acid sequences were analyzed by BLAST (The National Center for Biotechnology Information, NCBI, http://ww.ncbi.nlm.nih. gov/BLAST/), ExPaSy (http://au.expasy.org/tools/) and SignalP 3.0 (Bendtsen et al. 2004; http://www.cbs.dtu.dk/ services/SignalP/). Alignment of multiple homologous sequences from closely related parasites and host species was carried out by ClustalW. Expression and purification of the recombinant FgTGR protein The full-length FgTGR cDNA was mutated from Selenocysteine insertion sequence (SECIS) element to Sec incorporation (Maggioli et al. 2011), by using specific FgTGR forward primer (5′ CAT ATG CTG CGC GCC 3′) and reverse primer (5′ CTC GAG ACC TGA GCA AGC 3′), and subcloned into the pET-30b vector and transformed into Escherichia coli BL21(DE). The rFgTGR was expressed at 37 °C for 3 h by inducing the bacteria with 1 mM isopropyl-β-D-thiogalactoside (IPTG). The bacterial cells were collected and resuspended in a lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) and sonicated for 10 s, with a 10-s burst cycle (using 5 cycles) on ice. The lysate was purified using nickel-nitrilotriacetic acid (Ni-NTA) at 4 °C, washed twice with a washing buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 20 mM imidazole, pH 8.0), and then eluted by an elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). The rFgTGR protein was dialyzed in phosphate buffer saline (PBS) (137 mM NaCl, 2.7 mM KCl, 1.4 mM KH2PO4, 10 mM Na2HPO4) pH 7.4, and concentrated using Amicon Ultra Centrifugal Devices, 10000 NMWL (Millipore Corporation, Massachusettes, USA). Dectection of rFgTGR activity TR was assayed by the 5,5′-dithiobis (2-nitro-benzoic acid) (DTNB) reductase assay (Luthman et al. 1979). The purified rFgTGR was added to a reaction mixture containing PBS (pH 7.6), 5 mM EDTA, 1 mM DTNB, and 0.1 mM NADP H. The reaction was carried out at 25 °C and monitored by the increase in absorbance at 412 nm (A412). One enzyme unit

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was defined as the NADPH-dependent production of 2 μmol of DTNB/min as described in Maggioli et al (2011). Glutaredoxin (Grx) activity was assayed using βhydroxyethyl disulfide (HED) as the substrate (Holmgren and Aslund 1995; Carlberg and Mannervik 1985). The activity was monitored by measuring the decrease in absorbance at 340 nm (A340) at 25 °C. The purified rFgTGR was added to the reaction mixture containing 1 mM GSH, 1 mM HED, 0.5 mM NADPH, and 5 mM EDTA in PBS, pH 7.0. One unit of Grx activity was defined as the oxidation of 1 μmol of NADPH/min at 25 °C. Both thioredoxin reductase and glutaredoxin activity assays were done in triplicate.

separated on a 12.5 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. The membranes were blocked with 5 % skim milk (Merck KGaA, Darmstadt, Germany) in PBS containing 0.1 % Tween-20 (PBST) for 1 h and then incubated with rabbit anti-rFgTGR serum (diluted at 1:1000) for 1 h. Positive bands were visualized using alkaline phosphatase (AP)-conjugated goat anti-rabbit IgG (Invitrogen-Life Technologies, Carlsbad, CA, USA) diluted at 1:2000 in PBS, and the color developed with nitro-blue tetrazolium chloride/5-bromo-4-chloro-3-indodyl phosphate (NBT/ BCIP) substrates (Roche, Mannheim, Germany).

Production of a polyclonal antibody against rFgTGR

Estimation of the expression levels of natural FgTGR in developmental stages of F. gigantica by ELISA

A polyclonal anti-rFgTGR was produced by immunizing subcutaneously a New Zealand white rabbit three times at 2-week intervals, with 200 μg of rFgTGR protein mixed with Freund’s complete (first immunization) or incomplete adjuvant (boosting immunization). Blood was taken at 2 weeks post-third boost, and the serum was kept at −20 °C for further experiment. Rabbits were kept in suspended cages, wire bottom in an air-conditioned room at approximately 22–25 °C with the dark-light period of 12:12 h, 50–60 % humidity, and provided with food and water ad libitum. All experiments involving animals were approved and performed following to the standard protocols set by Mahidol University Animal Care and Use Committee (SCMU-ACUC), Faculty of Science, Mahidol University, Thailand (MUSC55-003-249). Preparation of parasite antigens Whole body (WB) extracts of eggs, metacercariae, NEJ, and 2- and 4-week-old juveniles and adults were obtained by homogenizing the samples in a buffer containing 10 mM Tris– HCl, 150 mM NaCl, 0.5 % Triton X-100, and 10 mM ethylene diamine tetra acetic acid (EDTA), pH 7.4, and sonicated for 5 min in an ice bath with 15 pulses (Kueakhai et al. 2011). Excretory–secretory antigens (ES) of adult F. gigantica were obtained by incubating the adult parasites in PBS for 3 h at 37 °C (Changklungmoa et al. 2013, 2014). Tegumental antigens (TA) of adult F. gigantica were obtained by extracting the adult parasites with a nonionic detergent (1 % Triton X-100, 0.05 M Tris buffer, 0.01 M EDTA, 0.15 M NaCl, pH 8.0) at 37 °C for 20 min (Chaithirayanon et al. 2002). After centrifugation at 10,000g for 30 min at 4 °C, each antigen-containing supernatant was collected and stored at −80 °C until used. Immunoblot analysis of FgTGR A 1.0 μg per well of WB fractions of developmental stages of F. gigantica and adult TA, ES (5 μg each) and rFgTGR were

A 96-well plate was coated with 100 μl of 10 μg/ml of WBs of eggs, metacercariae, NEJ, 2- and 4-week-old juveniles, adults, adult ES and TA, and a serial dilution of rFgTGR from 10−15 to 10−7 g/ml in the coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6), incubated at RT for 1 h. The coated plates were washed three times with PBST (0.05 % Tween 20), and nonspecific bindings were blocked with 1 % bovine serum albumin (BSA) at RT for 1 h. Then, the coated plates were washed three times with PBST. Rabbit polyclonal antirFgTGR, diluted at 1:2000 with PBS, was added at 100 μl per well and incubated at RT for 1 h. The plates were washed three times with PBST and incubated with 100 μl per well of HRP-conjugated goat anti-rabbit IgG (SouthernBiotech, Birmingham, USA), diluted at 1:5000 with PBS, at RT for 1 h. Then, the plates were washed three times with PBST and incubated with 100 μl per well of 3,3′,5,5′tetramethylbenzidine (TMB) (KPL, Gaithersburg, USA) at RT for 5 min. Finally, enzymatic reaction was stopped by adding 1 N HCl at 100 μl per well. The optical density (OD450) was measured at 450 nm in an automatic Titertek Multiscan spectrophotometer (Flow Laboratories, VA, USA). The experiments were performed in triplicate. Localization of FgTGR protein by immunolocolization The 5 μm thick paraffin sections of 2- and 4-week-old juveniles and adult were treated with fresh xylene twice for 10 min each and then rehydrated in ethanol 100, 95, 80, and 70 % and in ddH 2 O twice for 5 min each. The sections were microwaved at 700 W in citrate buffer (10 mM citric acid, pH 6.0) for 5 min three times. The sections were washed thoroughly with tap water for 5 min followed by PBST, incubated in PBS containing 4 % BSA for 30 min, and in the rabbit polyclonal anti-rFgTGR at dilution 1:1000, for 1 h at RT. Finally, the sections were incubated in PBS containing HRPconjugated goat anti-rabbit IgG (Southern Biotech, Birmingham, USA) at 1:2000 dilution for 30 min at RT and

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ƒFig. 1

Alignment of TGR amino acid sequence from F. gigantica with TGRs from other closely related trematode parasites retrieved from GenBank and SwissProt: F. hepatica CAM96615, S. mansoni AAK85233, S. japonicum ACH86016, cytisolic E. granulosus AAN63052, and mitochondrial E. granulosus AAN63051. Asterisk indicates identical residues, colon indicates highly conserved residues, and full stop indicates moderately conserved residues

washed with PBST before incubating in 3,3′-diaminobenzidine (DAB) substrates (Invitrogen-Life Technologies, Carlsbad, CA, USA) in the dark. The optimal color development was stopped by soaking the sections in ddH2O. The sections were counterstained with hematoxylin, mounted in 90 % glycerol, and photographed in a light microscope (Nikon, ECLIPSE E600).

Results Molecular cloning and characterization of cDNA encoding FgTGR gene

Fig. 3 The immunoblot analysis of the native FgTGR protein by probing with a rabbit polyclonal antibody against rFgTGR that reacted with whole body fraction (WB) of eggs (lane 1), metacercariae (Meta; lane 2), newly excyst juveniles (NEJ; lane 3), 2-week-old juveniles (2 wk; lane 4), 4week-old juveniles (4 wk; lane 5), adults (AD; lane 6), adult tegumental antigens (TA; lane 7), and adult excretory-secretory antigens (ES; lane 8). The arrow head indicates molecular weight of the native FgTGR which is at 66 kDa. The standard MW markers are shown in the left side

The PCR products of cytosolic FgTGR (cytFgTGR) with an estimated size of 2370 base pairs (bp) and mitochondrial FgTGR (mitFgTGR) with 2506 bp were separated in a 1 % agarose gel. The putative cytFgTGR ORF comprised of 598 amino acids, with a predicted molecular mass of 65.8 kDa. The putative mitFgTGR ORF comprised of 641 amino acids, with molecular mass of 65.8 kDa without signal peptide. The cytFgTGR peptide had no signal sequence; hence, it was not a

secreted protein, but mitFgTGR peptide had signal sequence. Three conserved motifs “CPYC” of the TR domain, “CVNVGC” and “ACUG” of the Grx domain, were also detected (Fig. 1). Comparison of the deduced FgTGR amino acid sequence with those of F. hepatica, and other closely related parasites, i.e., Echinococcus granulosus, Schistosoma japonicum, and Schistosoma mansoni as well as two host species Mus musculus, Homo sapiens, showed that FgTGR protein shared a high identity with TGR proteins of F. hepatica (98 %), S. mansoni (62 %), S. japonicum (62 %), E. granulosus (58 %), M. musculus (48 %), and H. sapiens (49 %).

Fig. 2 Coomassie blue-stained 15 % SDS-PAGE of rFgTGR expression purified by the Ni-NTA affinity chromatography. Bacterial proteins from noninduced condition (lane 1), whole bacterial lysate after induction (lane 2), and purified rFgTGR (lane 3). The position of purified FgTGR is indicated by the arrow head. MW markers are shown in the left side

Fig. 4 Semiquantitation of the expression levels of the native FgTGR in eggs, metacercariae, NEJ, and 2- and 4-week-old juveniles and adult F. gigantica as estimated by its reactivity with rabbit anti-rFgTGR using indirect ELISA. The high expression levels of FgTGR were shown in eggs, 4-week-old juveniles, and adult F. gigantica. The estimations were performed in triplicate

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Expression of rFgTGR and detection of TGR protein during the life cycle by immunoblotting

Estimation of the expression levels of native FgTGR in developmental stages of F. gigantica by ELISA

The FgTGR cDNA was subcloned into pET-30b vector, and rFgTGR protein coupled with six His-tag at the Nterminus was expressed and purified from the culture supernatant by Ni-NTA chromatography. The rFgTGR protein was resolved as a single band with an apparent molecular weight (MW) of 66 kDa on SDS-PAGE (Fig. 2). By immunoblot analysis, WB of eggs, metacercariae, 2- and 4-week-old juveniles, adults, and adult TA showed one positive band with rabbit antirFgTGR at MW 66 kDa, whereas adult ES did not (Fig. 3).

The lowest concentration of rFgTGR protein that was detected by rabbit polyclonal anti-rFgTGR was at 10 fg/ml. Using the same antibody, the native FgTGR detected was expressed in all stages of the F. gigantica life cycle, with continuously increasing levels from metacercariae, NEJ, and 2–4-weekold juveniles and adults. The eggs also contained a very high level of FgTGR (Fig. 4a).

Dectection of rFgTGR activity The rFgTGR activity was assayed by the 5,5′-dithiobis (2nitro-benzoic acid) (DTNB) reductase assay and HED assay. For the TR activity, a concentration-dependent increase in DTNB reduction was observed (Fig. 5a). For the Grx activity, a concentration-dependent decrease in HED reduction was observed (Fig. 5b).

Distribution of FgTGR protein in 2- and 4-week-old juvenile tissues The immunodetection for TGR protein was shown in the tegument and tegumental cell of 2- and 4-week-old juveniles, but the caecum, muscle, and parenchyma were not stained (Fig. 6h–k). In the adult parasite, a positive signal for FgTGR was detected in the tegument, parenchyma, eggs, testes, ovary, and vitelline cells (Fig. 6c–g). The spine and muscle were not stained (Fig. 6c). The negative control, using preimmune serum, did not show a positive signal (Fig. 6a).

Fig. 5 The rFgTGR was tested for TR activity using the 5,5′-dithiobis (2nitro-benzoic acid) (DTNB) reduction assay. DTNB reduction reactions with 8 mM rFgTGR (circle), 4 mM rFgTGR (square), 2 mM rFgTGR (triangle), and in the absence of rFgTGR (diamond), a concentrationdependent increase in DTNB reduction was observed (Fig. 5a). The rFgTGR was tested for Grx activity using β-hydroxyethul disulfide (HED) assay. HED reactions with 8 mM rFgTGR (circle), 4 mM rFgTGR (square), 2 mM rFgTGR (triangle), and in the absence of rFgTGR (diamond), a concentration-dependent decrease in HED reduction was observed (Fig. 5b)

Fig. 6 The detection of FgTGR protein in the tissues of 2- and 4-week-„ old juveniles and adult F. gigantica by immunohistochemistry, using rabbit anti-rFgTGR as probe and observed by a light microscope. a Negative control of adult parasite section probed with pre-immune sera. b A low magnification of adult parasite section showing positive signal in the tegument (Tg) and parenchyma (Pc), but not in caecum (Ca). c A medium magnification of adult parasite section showing positive signal in the tegument (Tg) and parenchyma (Pc), but not in muscle (Mu) and spine (Sp). d A high magnification of adult F. gigantica section showing positive signal in the testis (Ti). e A high magnification of adult F. gigantica section showing positive signal in the vetilline gland (Vi). f A high magnification of adult F. gigantica section showing positive signal in the ovary (Ov). g A high magnification of adult F. gigantica section showing positive signal in the egg (Eg). h A medium magnification of 2week-old juvenile section showing strong signal in the tegument (Tg), but not in ceacum (Ca), ventral sucker (Vs), and parenchyma (Pc). i A high magnification of 2-week-old juvenile section showing strong signal in the tegument (Tg) and tegumental cells (arrows), but not in ceacum (Ca), and parenchyma (Pc). j A medium magnification of 4-week-old juvenile section showing strong signal in the tegument (Tg), but not in caecum (Ca), and parenchyma (Pc). k A high magnification of 4-week-old juvenile section showing strong signal in the tegument (Tg) and tegumental cells (arrows), but not in muscle (Mu), and parenchyma (Pc)

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Discussion We found two isoforms of FgTGR, i.e., cytosolic and mitochondrial FgTGR. The cytFgTGR had no signal peptide,

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while mitFgTGR contained a signal peptide, which is similar to the E. granulosus TGR (EgTGR) (Agorio et al. 2003). The FgTGR sequence showed high conservation with two cysteines at the active site, CXXC, indicating that FgTGR gene is a

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member of thioredoxin family (Powis et al. 1997; Alger et al. 2002). Furthermore, the FgTGR sequence exhibited a conserved sequence of Grx as CVNVGC and the selenocysteine residue at the C-terminal. The presence of senocysteine at the C-terminal redox center of TGR has also been described in other trematodes, including S. mansoni (Angelucci et al. 2008; Alger and Williams 2002), S. japonicum (Han et al. 2012), F. hepatica (Maggioli et al. 2011), the cestode E. granunulisus (Agorio et al. 2003), and in mammals (Sun et al. 2005). The full sequence of rFgTGR was composed of 1797 and 1926 bp of cytosolic and mitochondrial FgTGR, respectively. The putative FgTGR peptides for both isoforms were composed of the same 598 amino acids, with an MW of 65.8 kDa, with an extra 43 amino acid signal peptide at the N-terminal in mitFgTGR. Multiple alignments of cyt- and mitFgTGR putative amino acid sequences exhibited the same conserved sequences at CPYC of TR and CVNVGC and ACUG of Grx domains which are identical to the other related cytEgTGR and mitEgTGR of parasites, including F. hepatica, S. japonicum, S. mansoni, and E. granulosus (Agorio et al. 2003; Angelucci et al. 2008; Han et al. 2012; Maggioli et al. 2011). The FgTGR protein shares a high identity with F. hepatica (98 %), S. mansoni (62 %), S. japonicum (62 %), E. granulosus (58 %), M. musculus (48 %), and H. sapiens (49 %). The rFgTGR activities were tested for both TR and Grx domians. For TR activity, the substrate DNTB and various concentrations of rFgTGR reacted together and showed that the NADPH production was directly correlated with rFgTGR concentrations similar to previously reported for FhTGR (Guevara-Flores et al. 2011) and EgTGR (Agorio et al. 2003). For Grx activity, the HED and various concentrations of rFgTGR reacted together and also showed the rFgTGR concentration-dependent NADPH reduction that is also similar to that reported for F. hepatica TGR (Guevara-Flores et al. 2011) and EgTGR (Agorio et al. 2003). The verified rFgTGR activities indicate that both TR and Grx domains were encoded in the rFgTGR and that both thioredoxin and glutathione systems were functionally active. All stages of F. gigantica were exposed to ROS generated from the host’s immune cells, as well as from their own metabolism and cell proliferation, especially in the reproductive organs (vitelline gland, testis, and ovary). By immunohistochemistry, the expression of FgTGR protein was detected at a fairly high level in the tegument of 2- and 4-week-old juveniles similar to the FgTrx expression (Changklungmoa et al. 2014). The tegument is an organ that is directly exposed to the ROS, especially H2O2 from the host’s immune cells, while the juvenile stages of parasite encountered during their migration. Hence, they protect themselves by expressing a high level of antioxidant enzymes, especially Trx (Changklungmoa et al. 2014) and TGR in the tegument for ROS neutralization. However, the level of TGR expression in different host’

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species (cattle, hamster, mice, and buffalo) may differ, and the washing process may reduce the level of protein in parasite tissues. In the adult parasite, FgTGR was detected in the reproductive organs, especially vitelline gland, testis, egg, and ovary. These indicated that tissue expression of FgTGR depends on the stages of the parasites and their exposure to ROS. As the 2- and 4-week-old juveniles resided in liver parenchyma, the high level of ROS in this tissue induced a high expression level of FgTGR in the tegument, while in the adult parasites, the FgTGR expression was higher in reproductive organs as a result of cell proliferation as opposed to the tegument which was less exposed to the ROS as the parasites was in the bile ducts (Changklungmoa et al. 2014). Quantitatively, by using ELISA, it was shown that FgTGR expression levels were high in eggs and 2- and 4-week-old juveniles and adult parasites but low in metacercariae and NEJ, perhaps because these stages were exposed to relatively a low level of free radicals. High expression level of TGR in the eggs and the adult reproductive organs was similar to the Trx expression that previously described in F. gigantica (Changklungmoa et al. 2014) and S. mansoni (Alger et al. 2002). This is expected since the two proteins complement each other in the Trx antioxidation system. FgTGR may be considered as a good vaccine candidate to prevent F. gigantica infection, by blocking or killing NEJ and juvenile stages, as FgTGR was expressed at a high level in the tegument of these stages, thus protecting them during the early phase of infection. Furthermore, a vaccine using FgTGR combined with other F. gigantica proteins that are essential for the migration processes, such as cathepsin B2 and B3 (Chantree et al. 2012, 2013), and cathepsin L1H (Sansri et al. 2013) could even be more effective in stopping the invasion and migration of the juvenile parasites. FgTGR may also be a drug target against fasciolosis gigantica as previously reported for S. japonicum TGR (SjTGR), which is an essential enzyme for maintaining the thiol-disulfide redox homeostasis of S. japonicum (Song et al. 2012) and also a multifunctional target for antischistosomal drugs for S. mansoni (Sharma et al. 2009). We are presently investigating the vaccine potential of FgTGR. Acknowledgments This research was supported by grants from Faculty of Science, Mahidol University, to Prasert Sobhon and the Research Grant of Mahidol University through National Research Council of Thailand to Kulathida Chaithirayanon.

References Agorio A, Chalar C, Cardozo S, Salinas G (2003) Alternative mRNAs arising from Trans-splicing code for mitochondrial and cytosolic variants of Echinococcus glanulosus thioredoxin glutathione reductase. J Biol Chem 278(15):12920–12928

Parasitol Res (2015) 114:2119–2127 Alger HM, Williams DL (2002) The disulfide redox of Schistosoma mansoni and the importance of a multifunctional enzyme, thioredoxin glutayhione reductase. Mol Biochem Parasitol 121: 129–139 Alger HM, Sayed AA, Stadecker MJ, Williams DL (2002) Molecular and enzymatic characterization of Schistosoma mansoni thioredoxin. Int J Parasitol 32:1285–1295 Angelucci F, Miele AE, Boumis G, Dimastrogiovanni D, Brunori M, Bellelli A (2008) Glutathione reductase and thioredoxin reductase at the crossroad: the structure of Schistosoma mansoni thioredoxin glutathione reductase. Proteins 72:936–945 Assady M, Farahnak A, Golestani A, Esharghian MR (2011) Superoxide dismutase (SOD) enzyme activity assay in Fasciola spp. parasites and liver tissue extract. Iranian J Parasitol 6(4):17–22 Badwey JA, Karnovsky ML (1980) Active oxygen species and the functions of phagocytic leucocytes. Annu Rev 49:695–726 Bendtsen JD, Nielsen H, Heijne GV, Brunak S (2004) Improved of signal peptide: SignalP 3.0. J Mol Biol 340:783–795 Bertini R, Howard OMZ, Dong HF, Oppenheim JJ, Bizzarri C, Sergi R, Caselli G, Pagliei S, Romines B, Wilshire JA, Mengozzi M, Nakamura H, Yodoi J, Pekkari K, Gurunath R, Holmgren A, Herzenberg LA, Ghezzi P (1999) Thioredoxin, a redox enzyme released in infection and inflammation, is a unique chemoattractant for neutrophils, monocytes, and T cells. J Exp Med 189:1783–1789 Carlberg L, Mannervik B (1985) Glutathione reductase. Methods Enzymol 113:484–490 Chaithirayanon K, Sobhon P (2010) Molecular cloning and characterization of 2-Cys Peroxiredoxin from Fasciola gigantica. Exp Parasitol 125:106–113 Chaithirayanon K, Wanichanon C, Vichasri-Grams S, Ardseungneon P, Grams R, Viyanant V, Upatham ES, Sobhon P (2002) Production and characterization of a monoclonal antibody against 28.5 kDa tegument antigen of Fasciola gigantica. Acta Trop 84:1–8 Changklungmoa N, Chaithirayanon K, Kueakhai P, Meemon K, Sobhon P, Riengrojpitak S (2012) Molecular cloning and characterization of leucine aminopeptidase from Fasciola gigantica. Exp Parasitol 131: 283–291 Changklungmoa N, Kueakhai P, Riengrojpitak S, Chaithirayanon K, Chaichanasak P, Preyavichyapugdee N, Chantree P, Sansri V, Itagaki T, Sobhon P (2013) Immunization with recombinant leucine aminopeptidase showed protection against Fasciola gigantica in mice. Parasitol Res 112:3653–3659 Changklungmoa N, Kueakhai P, Apisawetakan S, Riengrojpitak S, Sobhon P, Chaithirayanon K (2014) Identification and expression of Fasciola gigantica thioredoxin. Parasitol Res 113(6):2335–2343 Chantree P, Wanichanon C, Phatsara M, Meemon K, Sobhon P (2012) Characterization and expression of cathepsin B2 in Fasciola gigantica. Exp Parasitol 132:249–256 Chantree P, Phatsara M, Meemon K, Chaichanasak P, Changklungmoa N, Kueakhai P, Lorsuwannarat N, Sangpairoj K, Songkoomkrong S, Wanichanon C, Itagaki T, Sobhon P (2013) Vaccine potential of recombinant cathepsin B against Fasciola gigantica. Exp Parasitol 135:102–109 Fairweather I, Threadgold LT, Hanna REB (1999) Development of Fasciola hepatica in the mammalian host. In: Dalton JP (ed) Fasciolosis. CABI publishing, Oxon, pp 47–111

2127 Guevara-Flores A, Pardo JP, Rendon JL (2011) Hysteresis in thioredoxinglutathione reductase (TGR) from the adult stage of the liver fluke Fasciola hepatica. Rarasitol Int 60:156–160 Han Y, Zhang M, Hong Y, Zhu Z, Li D, Li X, Fu Z, Lin J (2012) Characterization of thioredoxin glutathione reductase in Schistosoma japonicum. Parasitol Int 61:475–480 Holmgren A, Aslund F (1995) Glutaredoxin. Methods Enzymol 252: 283–292 Kueakhai P, Meemon K, Changklungmoa N, Chaithirayanon K, Reingrojpitak S, Sobhon P (2011) Characterization and localization of saposin-like protein-2 (SAP-2) in Fasciola gigantica. Parasitol Res 108:1493–1500 Luthman M, Eriksson S, Holmgren A, Thelander L (1979) Glutathionedependent hydrogen donor system for calf thymus ribonucleosidediphosphate reductase. Proc Natl Acad Sci U S A 76:2158–2162 Maggioli G, Silveira F, Martin-Alonso JM, Salinas G, Carmona C, Parra F (2011) A recombinant thioredoxin-glutathione reductase from Fasciola hepatica induces a protective response in rabbits. Exp Parasitol 129:323–330 Nakamura H, Herzenberg LA, Bai J, Araya S, Kondo N, Nishinaka Y, Yo d o i J ( 2 0 0 1 ) C i r c u l a t i n g t h i o r e d o x i n s u p p r e s s e s lipopolysaccharide-induced neutrophil chemotaxis. Proc Natl Acad Sci U S A 98:15143–15148 Powis G, Gasdaska JR, Baker A (1997) Redox signaling and the control of cell growth and death. Adv Pharmacol 38:329–359 Salinas G, Selkirk ME, Chalar C, Maizels RM, Fernandez C (2004) Linked thioredoxin-glutathione systems in platyhelminths. Trends Parasitol 20(7):340–346 Sangpairoj K, Changklungmoa N, Vanichviriyakit R, Sobhon P, Chaithirayanon K (2014) Analysis of the expression and antioxidant activity of 2-Cys peroxiredoxin protein in Fasciola gigantica. Exp Parasitol 140:24–32 Sansri V, Changklungmoa N, Chaichanasak P, Sobhon P, Meemon K (2013) Molecular cloning, characterization and functional analysis of a novel juvenile-specific cathepsin L of Fasciola gigantica. Acta Trop 128(1):76–84 Sethadavit M, Meemon K, Jardim A, Spithill TW, Sobhon P (2009) Identification, expression and immunolocalization of cathepsin B3, a stage-specific antigen expressed by juvenile Fasciola gigantica. Acta Trop 122:164–173 Sharma M, Khanna S, Bulusu G, Mitra A (2009) Comparative modeling of thioredoxin glutathione reductase from Schistosoma mansoni: a multifunctional target for antischistosomal theraphy. J Mol Graph Mod 27:665–675 Song L, Li J, Xie S, Qian C, Wang J, Zhang W, Yin X, Hua Z, Yu C (2012) Thioredoxin glutathione reductase as a novel drug target: evidence from Schistosoma japonicum. PloS ONE 7(2):e31456 Sun QA, Su D, Novoselov SV, Carlson BA, Hatfield DL, Gladyshev VN (2005) Reaction mechanism and regulation of mammalian thioredoxin/glutathione reductase. Biochemistry 44:14528–14537 Tagaya Y, Maeda Y, Mitsui A, Kondo N, Matsui H, Hamuro J, Brown N, Arai K, Yokota T, Wakasugi H, Yodoi J (1989) ATL-derived factor (ADF), an IL-2 receptor/Tac inducer homologous to thioredoxin; possible involvement of dithiol-reduction in the IL-2 receptor induction. EMBO J 8:757–764 Torgerson P, Claxton J (1999) Epidemiology and control. In: Dalton JP (ed) Fasciolosis. CABI Publishing, Oxon, pp 113–149