Purification and kinetic analysis of cytosolic and mitochondrial thioredoxin glutathione reductase extracted from Taenia solium cysticerci

ARTICLE IN PRESS Experimental Parasitology ■■ (2014) ■■–■■

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Purification and kinetic analysis of cytosolic and mitochondrial thioredoxin glutathione reductase extracted from Taenia solium cysticerci Q1 A. Plancarte *, G. Nava Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México, UNAM., México, D.F. 04510, México

H I G H L I G H T S

• • • • •

G R A P H I C A L

A B S T R A C T

Thioredoxin glutathione reductases (TGRs) from Taenia solium tissues were purified. T. solium TGRs are multifunctional enzymes as classic TGRs described. Hysteretic kinetic properties showed both cytoplasm and mitochondrial T. solium TGRs. T. solium TGRs as selenoproteins were inhibited by nanomolar amounts of auranofin. Thioredoxin and glutathione systems are now demonstrated simultaneously in T. solium.

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A R T I C L E

I N F O

Article history: Received 12 August 2014 Received in revised form 10 December 2014 Accepted 15 December 2014 Available online Keywords: Thioredoxin Glutathione Selenoprotein Hysteresis Cestodes Taenia solium

A B S T R A C T

Thioredoxin glutathione reductases (TGRs) (EC 1.8.1.9) were purified to homogeneity from the cytosolic (cTsTGR) and mitochondrial (mTsTGR) fractions of Taenia solium, the agent responsible for neurocysticercosis, one of the major central nervous system parasitic diseases in humans. TsTGRs had a relative molecular weight of 132,000, while the corresponding value per subunit obtained under denaturing conditions, was of 62,000. Specific activities for thioredoxin reductase and glutathione reductase substrates for both TGRs explored were in the range or lower than values obtained for other platyhelminths and mammalian TGRs. cTsTGR and mTsTGR also showed hydroperoxide reductase activity using hydroperoxide as substrate. Km(DTNB) and Kcat(DTNB) values for cTsTGR and mTsTGR (88 μM and 1.9 s−1; 45 μM and 12.6 s−1, respectively) and Km(GSSG) and Kcat(GSSG) values for cTsTGR and mTsTGR (6.3 μM and 0.96 s−1; 4 μM and 1.62 s−1, respectively) were similar to or lower than those reported for mammalian TGRs. Mass spectrometry analysis showed that 12 peptides from cTsTGR and seven from mTsTGR were a match for gi|29825896 thioredoxin glutathione reductase [Echinococcus granulosus], confirming that both enzymes are TGRs. Both T. solium TGRs were inhibited by the gold compound auranofin, a selective inhibitor of thiol-dependent flavoreductases (I50 = 3.25, 2.29 nM for DTNB and GSSG substrates, respectively for cTsTGR; I50 = 5.6, 25.4 nM for mTsTGR

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* Corresponding author. Tel. +52 55 56232384; fax: +52 55 56232384. E-mail address: [email protected] (A. Plancarte). http://dx.doi.org/10.1016/j.exppara.2014.12.009 0014-4894/© 2014 Published by Elsevier Inc.

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toward the same substrates in the described order). Glutathione reductase activity of cTsTGR and mTsTGR exhibited hysteretic behavior with moderate to high concentrations of GSSG; this result was not observed either with thioredoxin, DTNB or NADPH. However, the observed hysteretic kinetics was suppressed with increasing amounts of both parasitic TGRs. These data suggest the existence of an effective substitute which may account for the lack of the detoxification enzymes glutathione reductase and thioredoxin reductase in T. solium, as has been described for very few other platyhelminths. © 2014 Published by Elsevier Inc.

1. Introduction Neurocysticercosis (NCC), a condition arising from the larval stage of Taenia solium in the central nervous system (CNS) of humans, is a neurological complex parasitic disease which may or may not yield symptoms in infected human hosts (Carpio, 2002). Symptoms include a severe immunological reaction developed by polymorfonuclear cells surrounding the metacestodes and with the ability to destroy them. When symptoms are absent, a few inflammatory cells may be observed in the vicinity of the unaffected parasite tissues. In NCC the production of toxic lipid peroxidation metabolites appears as a consequence of inflammatory cell activation by T. solium cysticerci antigens (Comporti, 1993; Sotelo and Del Bruto, 2002). Patients with NCC exhibit high lipid peroxidation products in their cerebrospinal fluid (CSF) related to neurological symptoms (Rodriguez et al., 2008), a condition which is absent in asymptomatic patients lodging cysticerci in their CNS. The existence of the latter condition despite long-term infection (Dixon and Lipscomb, 1961), suggests that the parasite not only behaves as a passive onlooker during infection but that it also contributes to disrupt the expression of host resistance carried out by lipid peroxidation metabolites. Consequently, it appears that T. solium must possess adequate detoxification mechanisms, as may be concluded from the existence of asymptomatic carriers. In mammalian tissue, antioxidant defenses against lipid peroxidation metabolites rely on two major independent pathways: the glutathione (GSH) and the thioredoxin (Trx) systems (Fernandes and Holmgren, 2004; Winyard et al., 2005). The flavoenzyme thioredoxin reductase (TrxR) constitutes the enzyme center in the Trx system given its ability to accept reducing equivalents from NADPH and to transfer them to Trx, which in turn, may reduce the detoxification enzymes peroxiredoxins (Pxs) that reduce H2O2 producing H2O. TrxR is characterized by the GCUG motif at the C terminus, where U indicates selenocysteine (Sec) which is essential for its activity and function. Glutathione reductase (GR) is the corresponding enzyme center of the GSH system, given its ability to transfer electrons from NADPH to oxidized glutathione (GSSG), which results in the formation of two GSH molecules. Glutathione, in turn, may transfer electrons to oxidized dithiol and glutaredoxin (Grx), a small thiol-disulfide oxidoreductase capable of reducing several different targets. Peroxiredoxin and glutathione transferases, also detoxification enzymes, accept electrons proceeding respectively from the Trx and GSH pathways, and thus reduce H2O2 and other organic peroxides which constitute the source of lipid peroxidation. At present, specialized TrxR and GR in T. solium have not been detected, and it may be useful to bear in mind that TrxR and GR enzymes in Schistosoma mansoni and other platyhelminths have not been isolated as independent entities (Alger and Williams, 2002; Bonilla et al., 2008). Instead of those proteins, some platyhelminths have a GR and TrxR molecular link exhibiting the fusion of glutaredoxin (Grx) and thioredoxin reductase (TrxR) domains into a single protein, a selenocysteine-containing enzyme which acts as thioredoxin glutathione reductase (TGR) (Salinas et al., 2004; Sun et al., 2001). TGR thus plays a central role in thiol-disulfide redox reactions by providing electrons to essential detoxification enzymes such as GR and Prx. GR reduces the tripeptide GSSG to GSH, which

acts as main reducing agent in the catalytic functions displayed by GSTs (Mannervik and Danielson, 1988). It has also been shown that the most abundant GST in T. solium (Ts26GST) catalyzes lipid peroxidation (Plancarte et al., 2004) and that a T. solium peroxiredoxin (Ts2-CysPrx) was able to inactivate H2O2 and cummene hydroperoxide (Molina-López et al., 2006), most likely due to the presence of a Trx system present in T. solium tissues. In order to arrive at a better understanding of the detoxification mechanism exhibited by T. solium, we decided to investigate the presence of TGRs in this parasite’s tissues. 2. Materials and methods 2.1. Materials Chromatography resins were from the following source: DEAEsepharose and 2′5′ADP-sepharose 4B from GE Healthcare Co. (Fairfield, CT, USA). NADPH, GSH, GSSG, 2-hydroxyethyl disulfide (HED), Escherichia coli and thioredoxin were from Aldrich Chemical Company (Milwaukee, WI, USA); Amicon PM 10 membrane from Diaflo, (Danvers, MA, USA); polyvinylidene difluoride (PVDF) from Millipore Corporation, (Billerica, MA, USA). Auranofin was purchased from ICN Biomedicals Inc. (Santa Ana, CA, USA). All other chemicals were reagent grade and obtained from common commercial sources. 2.2. Parasites Taenia solium cysticerci were obtained from naturally infected cysticercotic pigs from the states of Morelos and Guerrero in Mexico. Parasites were dissected from their skeletal muscle and washed thoroughly with 150 mM NaCl, partially dried in filter paper, and immediately frozen at −70 °C until used, taking special care to avoid host tissues. 2.3. Isolation of the cytosolic protein fraction (CPF) Taenia solium TGRs were purified by using a modified procedure previously developed by Sun et al. (2001). Briefly, T. solium cysticerci (15 g) were homogenized in one and a half volume of working buffer made of 50 mM imidazole-HCl (pH 6.5), containing 1 mM EDTA and 0.1 mM phenylmethyl-sulfonyl-fluoride (PMSF), using a Polytron homogenizer (Brinkmann Instruments). The homogenate containing 35 mg·ml−1 was spun 20 minutes at 10,000 gav, the supernatant thus obtained was spun again for 1 h at 105,000 gav to obtain the CPF. 2.4. Isolation of the soluble mitochondrial matrix proteins (SMMP) Purification of T. solium mitochondrial fraction (MF) was done at 4 °C by differential centrifugation procedures as described by Ernest et al. (1962) with some modifications. Briefly, 15 g of parasites were homogenized in 15 ml of working buffer with 1 mM EGTA and 0.25 M sucrose (WBES), using a Polytron homogenizer (Brinkmann Instruments, Inc. Westbury, NY, USA). The homogenate with 9 mg·ml−1 was spun 20 minutes at 180 gav and the supernatant 1 (Sn1) was kept aside. Pellet 1 (P1) (detritus

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fraction) was suspended in 15 ml of WBES and spun as previously described to obtain supernatant 1’ (Sn1’). Sn1 plus Sn1’ were pooled and spun 20 minutes at 900 gav (nuclear fraction) to obtain supernatant 2 (Sn2). Sn 2 was spun a further 20 min at 14,600 gav to obtain pellet 3 containing mitochondria. Pellet 3 was suspended immediately in 15 ml of WBES and was washed twice. Washed pellet 3 was suspended in 2.5 ml of working buffer without sucrose, frozen overnight at −70 °C, and labeled mitochondria fraction (MF). An aliquot of MF (41.6 mg/2.5 ml) was sonicated for 15 s at 3 amp 5X in ice cold water with an Ultrasonic Processor (Daigger Co., Vernon Hills, IL, USA). The sonicated MF fraction was then centrifuged at 105,000 gav for 1 h. The supernatant contained the soluble mitochondrial matrix proteins (SMMP). Mitochondrial purity was determined by two marker enzymes, lactate dehydrogenase (cytosolic) and succinate dehydrogenase (mitochondrial). The cytosolic protein fraction (CPF) employed as control solution for the cytosol marker enzyme evaluation was obtained from supernatant 3. 2.5. Ionic exchange chromatography CPF and SMMP were applied onto DEAE Sepharose columns (2.6 cm × 15 cm for the former and 2.6 cm × 5 cm for the latter), equilibrated in working buffer with either 1 mM EDTA for CPF or 1 mM EGTA for SMMF. After washing the non-adsorbed protein, a gradient of 0.5 M NaCl in working buffer was applied for enzyme elution, (three times the column volume). TGRs were detected in column fractions by recognition of thioredoxin reductase activity using DTNB as substrate. Fractions containing TGR activity (CPFTGR off DEAE) were pooled and concentrated to 2 ml by ultrafiltration (YM-5 membrane; Amicon-Millipore, Bedford, MA, USA) under nitrogen. CPFTGR off DEAE were dialyzed overnight against 0.1 M Tris– HCl (pH 7.8), with 1 mM EDTA. A similar procedure was performed for the fractions containing SMMF TGR activity (SMMFTGR off DEAE), except that 1 mM EGTA was used in the dialysis buffer. 2.6. Affinity chromatography Either of CPFTGR off DEAE or SMMFTGR off DEAE was applied to a 2′,5′-ADP Sepharose 4B column (AS) (GE Healthcare Co., Fairfield, CT, USA) (1.6 cm × 7 cm) previously equilibrated in the sample buffer. After thoroughly washing the column, the parasite TGRs were eluted by using a linear NADPH concentration gradient (0–120 μM) prepared in the same buffer (three times the column volume). Fractions of 1 ml showing TGR activity (detected as described previously), either cytosolic TGR (cTsTGR) from CPF or mitochondrial TGR (mTsTGR) from SMMP, were pooled and then concentrated, dialyzed overnight and stored at 4 °C until use. 2.7. Molecular weight determination A total of 70 mg/0.5 ml of CPF was applied to a Sephacryl S-200 chromatography column (0.9 cm × 60 cm) equilibrated in a 10 mM sodium phosphate, 150 mM sodium chloride, pH 7.2 solution. Myoglobin, carbonic anhydrase, ovoalbumin, bovine serum albumin, alcoholic dehydrogenase and β-galactosidase were used for molecular weight calibration (Hagel, 1998). Under denatured conditions, the molecular weight of the cTsTGR and mTsTGR monomers was determined by SDS-polyacrylamide gel electrophoresis according to established procedures (Andrews, 1986). 2.8. Protein determination Protein concentration of cTsTGR or mTsTGR was determined by absorbance measurements at 280 nm; an extinction coefficient of 0.920 mM−1·cm−1 at this wavelength was employed. An extinction

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coefficient of 1.0 mM−1·cm−1 was used for non-purified protein fractions (Gill and von Hippel, 1989).

66 67 68 2.9. Polyacrylamide gel electrophoresis (PAGE) 69 70 Aliquots of cTsTGR and mTsTGR, and fractions obtained in the 71 TGRs’ purification procedures were subjected to PAGE in the pres72 ence of SDS at room temperature in slab gels (15% acrylamide; 73 Laemmli, 1970). The gels were stained for protein with Commassie 74 or silver nitrate. 75 76 2.10. Tandem mass spectrometry (LC/ESI-MS/MS) 77 78 Following electrophoresis, cTsTGR and mTsTGR were excised from 79 the stained SDS gel, destained, reduced, carbamidomethylated, and 80 digested with modified porcine trypsin. Peptide mass spectromet81 ric analysis was carried out using a 3200 Q TRAP hybrid tandem mass 82 spectrometer following the procedure of Xolapa et al. (2007). Ob- Q2 83 tained spectrometric data were subjected to a NCBI-BLAST search 84 employing the ProteinProspector and Mascot programs. 85 86 2.11. Mitochondrial marker enzymes’ controls 87 88 Lactate dehydrogenase (LDH) activity was measured by study89 ing the oxidation of NADH in the presence of pyruvate as substrate 90 at 340 nm (Vassault, 1983). Succinate dehydrogenase (SDH) was 91 assayed measuring the reduction of 2,6-dichlorophenolindophenol 92 (DCIP) at 600 nm after the addition of succinate, in the presence 93 of phenazine methosulfate (Arrigoni and Singer, 1962); all assays 94 were performed in triplicate. 95 96 2.12. Enzyme assays 97 98 Five different enzyme activities, independent of each other, were 99 assayed for cytosolic and mitochondrial TsTGRs; all assays were per100 formed at room temperature using an Ultrospec 3100 Pro 101 spectrophotometer (Pharmacia-Biochem, Uppsala, Sweden). One unit 102 of enzyme activity is defined as the amount of enzyme which 103 reduces one μmol of substrate formed per minute under de104 scribed conditions. Specific activities are expressed as units of 105 enzyme per milligram of protein. For each assay, measured absor106 bance values were corrected by subtraction of the absorbance values 107 obtained for the reaction control mixture without enzyme. Kinetic 108 data analyses were performed using ORIGIN software (Origin Lab). 109 Individual points represent the obtained means of multiple assays 110 (in triplicate or higher) performed independently on different days 111 and with different enzyme preparations. 112 113 2.12.1. Thioredoxin reductase (TrxR) assay 114 Both the 5-5′-dithiobis (2-nitrobenzoic acid) (DTNB) reduction 115 assay and the insulin reduction assay were performed to evaluate 116 TrxR activity. For the DTNB assay (Arnér et al., 1999), either of cTsTGR 117 (0.5–1.2 μM) or mTsTGR (3–40 nM) was added to a mixture of 50 mM 118 Tris–HCl (pH 7.8), 1 mM EDTA (assay buffer), 0.1 mM NADPH and 119 0.35 mM DTNB. The increase in absorbance at 412 nm was mea120 sured (ε412 = 13.6 mM−1·cm−1). For the insulin assay, the decrease in 121 absorbance at 340 nm (ε340 = 6.2 mM−1·cm−1) due to the oxidation 122 of NADPH by thioredoxin was measured after addition of any of 123 cTsTGR (0.5–1.2 μM) or mTsTGR (3–40 nM) to a reaction mixture 124 composed of assay buffer, 0.1 mM NADPH, 8.6 μM insulin and 120 μM 125 thioredoxin from E. coli. 126 127 2.12.2. Glutathione reductase (GR) assay 128 The method employed was based on Worthington and Rosemeyer 129 (1976). Either of cTsTGR (0.5–1.2 μM) or mTsTGR (3–40 nM) was 130 added to a mixture of assay buffer, containing 0.1 mM NADPH and 131

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0.05 mM GSSG. The decrease in absorbance at 340 nm was measured (ε340 = 6.2 mM−1·cm−1). 2.12.3. Glutharedoxin (Grx) assay The method employed was that of Holmgren and Aslund (1995). Briefly, a β-hydroxyethyl disulfide (HED) is reduced in the presence of GSH yielding glutathione disulfide (GSSG), which is then reduced by GR with depletion of NADPH. A reaction mixture containing 1 mM GSH and 0.7 mM HED in assay buffer was preincubated for 2 min. Either of cTsTGR (0.5–1.2 μM) or mTsTGR (3–40 nM), plus 0.05 units of yeast glutathione reductase and 0.1 mM NADPH were added; the reduction of the mixed glutathione-hydroxyethyl disulfide was followed by the oxidation of NADPH at 340 nm (ε340 = 6.2 mM−1·cm−1). 2.12.4. Hydroperoxide reductase activity The assay was performed as described by Zhong and Holmgren (2000). The reduction of H2O2 by the transference of NADPH reducing equivalents was followed by NADPH depletion. A 0.5 ml sample containing the assay buffer with 0.1 mM NADPH and either of cTsTGR (0.5–1.2 μM) or mTsTGR (3–40 nM) was preincubated for 2 min. NADPH oxidation was followed at 340 nm (ε340 = 6.2 mM−1·cm−1) for 2 min after addition of H2O2 (1–60 mM). 2.12.5. Thioredoxin peroxidase activity The ability of cTsTGR and mTsTGR to catalyze the Trx-dependent reduction of H2O2 was evaluated by mixing 400 μM H2O2, 20 μM E. coli Trx, and 100 μM NADPH in 0.5 ml of 50 mM Tris/HCl buffer (pH 7.8) containing 1 mM EDTA. The reaction was initiated by addition of cTsTGR (10–18 μm) or mTsTGR (70–110 nM) and NADPH depletion was followed at 340 nm. 2.13. Kinetic studies The cTsTGR and mTsTGR kinetic constants for five of the six substrates employed (Km and Vmax) were determined through initial velocity studies. In these studies one substrate was varied at fixed co-factor concentrations (NADPH; 0.1 mM). The assays were performed as described above (enzyme assays) with concentration variations in the ranges of 10–900 μm for DTNB; 2–300 μm for Trx E. coli; 2–240 μm for GSSG; 20–1000 μm for HED and 10–250 mM for H2O2. Fixed concentrations of either of cTsTGR (0.2–0.4 μm) or mTsTGR (1.8–10 μM) were preincubated for 2 minutes in the assay buffer in the presence of one of the substrates at varied concentrations, and the reaction was initiated by NADPH addition. Data for each reaction were adjusted to Michaelis–Menten equations using the Origin Pro program. Lineweaver–Burk plots were used in order to linearize the results of the initial velocity plots. Thioredoxin peroxidase activity was determined only as the specific activity of each enzyme, as described above. 2.14. Effect of pH on activity and stability pH dependence on either cTsTGR or mTsTGR enzyme function was carried out by measuring its TrxR and GR activity, in the 4.0– 12.0 pH range, as described above. 2.15. Effect of temperature Temperature effect on the catalytic TrxR and GR activities of cTsTGR and mTsTGR was examined under standard assay conditions. The reactions were carried out at temperatures ranging from 5 to 80 °C, and the residual activities of the enzymes were measured; assays for enzymes maintained at 37 °C (T. solium host body temperature) during 20 minutes were also performed.

2.16. Inhibition studies The effect of auranofin on TrxR and GR activities of cTsTGR and mTsTGR was determined as described by Gromer et al. (1998). Briefly, an enzyme sample was incubated during 1 minute in working buffer in the presence of 100 μM NADPH and auranofin from a fresh 100 μM stock solution in dimethylsulfoxide (dilutions of inhibitor 0–100 nM). Either of DTNB or GSSG was added to start the reaction; the oxidation of NADPH was followed spectrophotometrically at 340 nm.

66 67 68 69 70 71 72 73 74 75 76

2.17. Hysteretic behavior Kinetic full-time courses of GR activity with cTsTGR and mTsTGR were obtained to evaluate the GSSG and enzyme concentrations on initial velocity reactions. For the effect of GSSG, cytosolic and mitochondrial parasite enzymes (0.6 and 3.2 μM, respectively) were incubated in the presence of different concentrations (0.05– 0.4 mM) of GSSG at constant NADPH concentration (100 μM). Variations in absorbance at 340 nm were registered during 1–3 h after assays were initialized. Effect of protein was studied by assays performed at varying concentrations: 0.6–1.2 μM for cTsTGR or 3.2–6.4 μM for mTsTGR; NADPH concentration was kept constant (100 μM), as was GSSG concentration (0.3 mM), a value at which hysteretic behavior is maintained. Variations in absorbance measured at 340 nm were registered as previously described.

77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93

3. Results 3.1. Enzyme purification cTsTGR and mTsTGR were purified separately through the same purification scheme: a DEAE-Sepharose anion exchange chromatography followed by 2′5′ADP-Sepharose affinity column was employed. TGR activities with DTNB and GSSG as substrates were measured in order to monitor the enzymes during their chromatogaphy purification. Both CPFTGR off DEAE and SMMFTGR off DEAE fractions were eluted from DEAE-Sepharose at conductivities of 12–22, 10–16 mS·cm−1 for the cytosolic and mitochondrial fractions respectively. cTsTGR and mTsTGR were eluted as homogeneous proteins from the affinity column and characterized by SDSPAGE electrophoresis (Fig 1). NADPH concentration at specific elution of any enzyme in the affinity chromatography could not be estimated due to overlapping absorbance from NADPH with proteins at 280 nm. It is interesting to note that both reductase activities were coeluted in a single peak from the anionic exchange and affinity chromatography. This result was further supported by the constant ratio maintained between the two reductase activities throughout the purification procedure for both T. solium reductases; in other words, no TrxR or GR independent activity could be detected during the purification schemes. This behavior has been observed for other helminthes’ TGRs (Guevara-Flores et al., 2010; Rendón et al., 2004), indicating that both TrxR and GR activities are located within the same protein. No TGR activity was detected in the excluded 2′5′ADPSepharose fraction showing that the two enzymes were firmly attached to the affinity resin. Both purification steps were repeated 12-fold, yielding the same results. The purification of cTsTGR and mTsTGR, respectively from cytosolic and mitochondrial soluble extracts, is summarized in Table 1. TGR activity was observed to increase with increasing purity. Specific activities of cTsTGR and mTsTGR toward DTNB were 2140 and 10,100 mU·mg–1 protein, respectively; and 980 and 1300 mU·mg–1 protein toward GSSG. cTsTGR was purified 17-fold and mTsTGR 416-fold.

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3.3. Physical properties

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Fig. 1. Electrophoretic analysis of purified TsTGRs. The samples were run in a 15% polyacrylamide gel by established procedures and stained and unstained by conventional methods. (A) Molecular weight markers, (B and C) Enzymes obtained from the last purification step, cTsTGR and mTsTGR respectively.

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3.2. Mitochondrial marker enzymes’ controls In order to ensure purification of parasitic MF, the procedure employed was partially characterized by two enzyme markers acting upon subcellular fractionations of T. solium cysticerci. Succinate dehydrogenase (SDH) was used as a marker for MF and lactate dehydrogenase (LDH) as a marker for CPF. The average of the total activity percent distribution of SDH was 0.09 ± 0.007, 1.63 ± 0.03, 97.4 ± 2.5, 0.9 ± 0.02, respectively for CPF, nuclear fraction, MF and microsomal fraction. Average values for LDH activity were 91.1 ± 3, 5.9 ± 0.05, 1.13 ± 0.09, and 1.8 ± 0.07, respectively for CPF, nuclear fraction, MF and microsomal fraction. Additionally, all the supernatants obtained from MF washes, showed neither TGR nor marker enzyme activity. These markers provide a useful indication of the distribution of these cell structures. The results obtained are in agreement with those obtained for vertebrate organisms (De Duve et al., 1955).

43 44 45 3.3.1. Biochemical analysis (SDS-PAGE) 46 Figure 1 shows a single stained protein band for cTsTGR (2) and 47 for mTsTGR (3). Both proteins were eluted in the last purification 48 step with an apparent molecular weight of ~62 ± 3 kDa as ana49 lyzed by SDS-PAGE on 15% gels under reducing conditions and 50 compared with the molecular weight markers (1), (average of five 51 separate determinations). Both enzymes clearly showed TGR ac52 tivity (as DTNB reduction and GR activity). 53 54 3.3.1.1. . Determination of molecular weights and subunit composition. The native molecular weight of cTsTGR was 132 ± 3 kDa Q3 55 56 (data not shown). Together with SDS-PAGE electrophoresis results, 57 these data suggest a unique dimeric structure for cTsTGR. Due to 58 minimal amounts of native mTsTGR, gel permeation chromatog59 raphy was not performed. 60 61 3.3.2. Tandem mass spectrometry (LC/ESI-MS/MS) 62 In order to investigate the molecular nature of both cTsTGR and 63 mTsTGR, peptide mass spectrometric analyses were carried out with 64 the purified enzymes. Fig. 2 shows the peptide mass spectromet65 ric results obtained for cTsTGR and mTsTGR. These results are a match 66 for the TGR amino acid sequences of T. crassiceps, E. granulosus and 67 S. mansoni; the latter two reported in the GenPept database (NCBI68 BLAST). A better match to TGRs from cestodes (94%) over trematodes 69 (58%) was also observed. 70 71 3.3.3. Effect of pH and temperature 72 The optimum pH for cTsTGR was 8.0 when DTNB and GSSG sub73 strates were employed; however, optimum pH for mTsTGR was 8.5 74 with the former and 9.5 with the latter substrate. In both enzymes 75 with both substrates a rapid decrease on either side of these values 76 was observed as reported for T. crassiceps (Rendón et al., 2004). 77 Enzyme activity for both enzymes toward both substrates was un78 affected in the temperature range of 15–40 °C. Activity decreased 79 beyond 40 °C, until none was detected at 65 °C. In addition, the 80 enzymes proved to be stable for 30 min at 20 and 37 °C. 81 82 3.4. Kinetic properties 83 84 cTsTGR and mTsTGR were characterized in accordance with the 85 five catalytic activities well known for TGRs (Table 2). Except for hy86 droperoxide reductase activity, the mitochondrial enzyme showed 87 a higher reductase activity than the cytosolic enzyme. Both enzymes 88 exhibited a greater activity toward HED than that observed for TrxR 89 (in both assays), followed by GR.

22 23 24

Table 1 Purification of TGRs from cytosolic and mitochondrial cysticerci of T. soliuma.

25 26 27 28 29

Step

30 31 32 33 34 35 36 37 38

Crude extract cyt mt Anion exchange (DEAE-sepharose) cyt mt Affinity chromatography (2′5′ADP-sepharose) cyt mt

39 40 41 42

Volume (ml)

Protein (mg)

TrxR

27 2.5

241.6 41.6

30.5 22.2

73.2 24.9

2.25 1.35

0.9 0.0045

Activity ratio TrxR/GR

Total activityb (U) DTNB

Yieldb (%) DTNB

43 2.2

2.9 11

30.2 1.01

100 100

129 3.2

2.6 10.3

24.16 0.82

Specific activity (mU/mg)b,c

125 24.3 330 33 2140 10100

GR

980 1300

2.2 7.7

1.926 0.045

80 81.2 6.4 4.46

Purificationb (folds) DTNB

1 1 2.64 1.36 17.12 416

a

Starting material for both enzymes was 15 g. Data obtained with DTNB and NADPH as substrates. Data obtained with GSSG and NADPH as substrates. cyt, cytosolic; mt, mitochondrial. b

c

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Fig. 2. Amino acid sequences of cTsTGR and mTsTGR and homologous proteins from helminthes, showing alignment of the deduced amino acid sequences of cytosolic and mytocondrial TGR from T. solium (cTsTGR and mTsTGR respectively) with sequences of TcTGR thioredoxin glutathione reductase from T. crassiceps (Rendón et al., 2004), EgTGR thioredoxin glutathione reductase from E. granulosus (GenBank™ accession number AY147416) and SmTGR thioredoxin glutathione reductase from S. mansoni (GenBank™ accession number AF395822). Peptide mass spectrometric analysis was carried out as described in Section 2, Materials and methods.

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Except for HED, mTsTGR had the lowest Km values for DTNB, GSSG and H2O2 when compared with cTsTGR. A similar trend was observed for both enzymes regarding specific activities toward the aforementioned substrates, including thioredoxine peroxidase. The catalytic efficiency (Kcat/Km) values between both enzymes differed by five orders of magnitude. cTsTGR and mTsTGR also showed a hydroperoxide reductase specific activity of 210 and 23 mU·mg– 1, respectively toward hydroperoxide as substrate. Both T. solium enzymes also exhibited thioredoxine hydroperoxidase activity and were able to reduce H 2 O 2 with a specific activity of 25 and

1270 mU·mg–1, respectively for cytosolic and mitochondrial TGRs from parasite samples. As observed for all reported TGRs, cTsTGR and mTsTGR showed specificity toward NADPH as electron donor. cTsTGR and mTsTGR also exhibited glutaredoxin activity with specific activities of 3050 and 131,000 mU·mg–1, respectively, toward HED. Auranofin is a gold compound known specifically to inhibit seleno-dependent enzymes (Saccoccia et al., 2012). The enzymes investigated were also inhibited with this compound. The half maximal inhibitory concentration (IC50) values obtained with auranofin for cTsTGR and mTsTGR were very similar for both reductase activities investigated (Fig. 3). Total enzyme activity inhibition was observed at nanomolar concentrations of inhibitor. 3.5. Hysteretic behavior Hysteretic behavior (i.e. the existence of a time lag before catalysis occurs) was observed for cTsTGR and mTsTGR only when GSSG was used as substrate. This behavior has been reported for the cytosolic TGR of the cestodes T. crassiceps (Rendón et al., 2004) and E. granulosus (Bonilla et al., 2008), and in the trematode Fasciola hepatica (Guevara-Flores et al., 2011); however, for mitochondrial TGR, this behavior has only been reported for T. crassiceps (Guevara-Flores

20 21 22

Table 2 Kinetic parameters of cTsTGR and mTsTGR.

23 24 25

Activity

Substrate

TGR

26 27 28 29 30 31 32 33 34 35 36 37 38 39

Trx reductase

DTNB

Trx reductasa

NADPH

Trx reductase

Trx (E. coli)

GR

GSSG

Glutaredoxine

HED

Hydroperoxide reductasa

H2O2

cyt mt cyt mt cyt mt cyt mt cyt mt cyt

Trx peroxidase

H2O2

40

cyt mt

Km (mM) 0.088 0.045 0.048 0.0016 0.067 0.0063 0.004 0.184 0.41 19 2.3 ND

Specific activity (mU/mg) 2,140 10,100 2,140 10,100 0.223 1020 980 1,300 3,050 131,000 210 23 25 1270

Kcat (s−1)

Kcat/Km (M−1 s−1)

1.855 12.6 1.637 12.6 0.35

2.1 × 104 2.8 × 105 3.4 × 104 7.9 × 106 5.2 × 103

0.96 1.62 5.13 163.5 0.306 0.028 ND

1.5 × 105 4.2 × 105 2.8 × 104 3.9 × 105 1.6 × 101 1.2 × 101 ND

cyt, cytosolic; mt, mitochondrial; ND, not determined.

41

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Fig. 3. The auranofin inhibitory effect on TsTGRs reductase activities. IC50 plots were obtained at concentrations of 0–100 nM of auranofin in the presence of cTsTGR (A) and mTsTGR (B). Diamonds represent 0.5 mM of GSSG and open circles 0.35 mM of DTNB. Enzyme and constant concentrations necessary for the development of initial velocity assays are described in Section 2, Materials and methods.

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Fig. 4. Hysteretic behavior of GR activity for cTsTGR and mTsTGR. Full-time courses of GR activity were obtained using different assay conditions. Plots A and B show assays employing GSSG as substrate; 0.6 μM of cTsTGR (A); 3.2 μM of mTsTGR (B). In both assays NADPH concentration was 1 mM and the reactions were started by the addition of GSSG at the indicated concentrations. Plots C and D show enzyme concentration effect, respectively for cTsTGR and mTsTGR. Assays were performed at the abovementioned NADPH concentration; reactions were started by the addition of a constant 0.3 mM concentration of GSSG shown to contribute to hysteresis.

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et al., 2010). Fig. 4 shows that hysteretic behavior was observed for cTsTGR and mTsTGR regardless of the different assay conditions employed. Similar assay conditions with thioredoxin or NADPH as substrates failed to produce a time lag (data not shown). Figures 4A and B show a typical full time course kinetics indicating that at GSSG concentrations greater than 50 μM both cTsTGR and mTsTGR responded more slowly as GSSG concentration increased. A more marked hysteresis in mTsTGR compared with cTsTGR may also be observed. Figures 4C and D, respectively for cTsTGR and mTsTGR, show that hysteretic behavior disappears with increasing enzyme concentration. Hysteretic kinetics for both enzymes practically disappeared upon duplication of enzyme concentration. 4. Discussion It has been suggested that antioxidants play an important role in protecting some helminthes from the host’s immune response (Li et al., 2004; LoVerde, 1998; Salazar-Calderón et al., 2000). In T. solium, no direct evidence supports this mechanism, except for the existence of asymptomatic patients with a healthy immune system known to lodge up to dozens of cysticerci in their central nervous system for long periods of time (Flisser et al., 1993). At present, several antioxidant enzymes have been characterized for T. solium. These include Cu-Zn superoxide dismutase (SOD) (González et al.,

2002), three isoforms of glutathione transferase (GSTs) (Neguyen et al., 2010; Plancarte et al., 2004; Vibanco et al., 2002) and one peroxiredoxine (Prx) (Molina-López et al., 2006). SOD in T. solium tissues is a source of hydrogen peroxide (H2O2) and this oxidant allows for lipid peroxidation when the Fenton reaction produces the hydroxide radical. This process results in the formation of lipid and phospholipid hydroperoxydes, which decompose further into secondary products, such as extremely toxic carbonyl species (Comporti, 1985). The presence of glutathione transferases and Prx in T. solium tissues requires other enzymes capable of supplying reducing equivalents to both GSH/GSSG and Trx systems, shuttling electrons to GSSG and oxidized thioredoxin (Salinas et al., 2004). Thus, it is likely that T. solium achieves detoxification through the linked thioredoxin– glutathione systems present in cTsTGR and mTsTGR, allowing for efficient reduction of GSH and Prx by transferases, a necessary condition for adequate detoxification. In addition to the essential detoxification function of TGRs in flatworm parasites described above, it is necessary to include the TGR activities associated with the Grx domain, such as deglutathionylase activity of GSH-protein mixed disulfides (protein-S-SG). Protein glutathionylation is the mechanism in which protein-SH groups form mixed disulphides with glutathione to avoid protein-SH groups’ oxidation (Popov, 2014). Also, TGRs play essential participations in the redox cell signaling and sensing. Cell signaling transduction is the way in which outside cell

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stimulus are transferred to its inner compartments resulting in the activation or inhibition of genes and cell sensing is the oxidative modifications on protein cysteines with consequent events such as changes in its activities and interactions with other biomacromolecules (Go and Jones, 2013). In the present work, we purified and partially characterized cTsTGR and mTsTGR from T. solium cysticerci. The purification procedure followed was similar for both enzymes, as has been described by other authors (Guevara-Flores et al., 2010; Sun et al., 2001). This procedure included an ion exchange and an affinity coenzyme chromatography. The former technique showed that both TGRs were anionic due to their interaction with the resin; the latter technique showed the enzyme’s NADPH-dependence. Comparison of the peptide sequences of both homogeneous parasite proteins (Fig. 2) by LC/MS/MS proved to be a match to the TGR Echinococcus Q4 granulosus amino acid sequence (http://blast.nvbi.nlm.nih .gov/BLAST.cgi). Studies performed by gel filtration (data not shown) and by SDS-PAGE showed the dimeric nature of cTsTGR as composed of two equivalent (or very similar) subunits with identical native molecular mass (Fig. 1). The observed molecular weight of cTsTGR falls within the range for TGRs (Sun et al., 2001). The small amount purified of mTsTGR made gel filtration analysis impossible; however, its monomer molecular mass of 62 ± 3 kDa was similar to that obtained for cTsTGR. In addition, it has been shown that in parasitic flatworms, mitochondrial and cytosolic TGR variants are derived from a single gene, and that the encoded mature polypeptides are identical in sequence (Otero et al., 2010). The aforementioned results suggest mTsTGR also to be a dimer. Specificity toward the Trx and GSH systems was exhibited by both parasite TGRs. cTsTGR was less active toward both systems than mTsTGR, but similar to the activities of the corresponding enzymes from E. granulosus and mouse testes (Agorio et al., 2003; Sun et al., 2001); activities were also five to seven times lower than those reported for T. cracciceps (Rendón et al., 2004) and trematodes (Alger and Williams, 2002; Song et al., 2012). mTsTGR exhibited a greater TR and GR activity than T. crassiceps’ mitochondrial TGR (Guevara-Flores et al., 2010), and very similar to that of trematodes’ cytosolic TGRs (Han et al., 2012; Song et al., 2012). T. solium, however, exhibited a lower GR activity than that observed for S. mansoni (Kuntz et al., 2007). Remarkably, mTsTGR Grx activity was the highest when compared with other catalytic activities displayed by this enzyme and by cTsTGR. This Grx activity value is 6–65 times higher than that observed for mammalian and other helminthes different to T. solium (Agorio et al., 2003; Alger and Williams, 2002; Sun et al., 2001). Grx activity is associated with electron donation in the reduction of an intramolecular disulfide bond in ribonucleotide reductase (Holmgren and Aslund, 1995). Grx activity also partakes of the defense of cells against reactive oxygen species (ROS) by means of its deglutathionylation activity (Gravina and Mieyal, 1993). Recently, a new role for Grxs as ancillary proteins has been proposed, whereby reducing equivalents are shunted from main catabolic pathways to recycle GSSG via a lypoil group, thus performing biochemical functions which involve GSH but without NAD(P)H depletion (Porras et al., 2002). Perhaps Grx activity in mTsTGR is not an ancillary but the main activity, a possibility which needs to be explored in future. mTsTGR Km values obtained with Trx and GSH substrates related indirectly to catalytic activities: the higher the activities, the lower the Km constants. These data suggest improved catalytic functions for mTsTGR over cTsTGR, further confirmed by the time required by mTsTGR to “turn over” one substrate molecule; by its Kcat values, and also by the enzyme efficiency Kcat/Km values, where a large value of kcat (rapid turnover) or a small value of Km (high affinity for substrate) contribute to increase the kcat/Km ratio. When cTsTGR and mTsTGR were incubated in presence of nanomolar concentrations of the gold compound auranofin (AF), both

T. solium enzymes’ TR and GR activities were inhibited. These results are in agreement with the ability of AF to inhibit the family members of the selenocysteine enzymes that have an essential selenocysteine amino acid in their active site (Gly-Cys-SeCys-Gly) (Kuntz et al., 2007). Additionally, AF inhibitory values on cTsTGR and mTsTGR were close to those obtained with T. crassiceps, (Rendón et al., 2004) S. mansoni (Alger and Williams, 2002) and mammalian selenocysteines (Gromer et al., 1998; Rigobello et al., 2005). All enzymes mentioned have (or are thought to have) a selenocysteine residue that plays an essential role in their catalytic activity. These results are important in that cTsTGR and mTsTGR may be explored as possible drug targets. This idea has been suggested by Angelucci et al. (2009) regarding S. manssoni; Bonilla et al. (2008) regarding E. granulosus; and del Arenal regarding T. crassiceps (Martínez-González et al., 2010), due the potential role of these enzymes in the detoxification pathway of reactive oxygen species of T. solium. Rendon et al. suggest that a hysteretic kinetics (the appearance of a lag period in the enzymatic assays when GSSG is used as disulfide substrate) is a common feature of TGR from any parasitic flatworm (Guevara-Flores et al., 2011). Our results support this idea because cTsTGR and mTsTGR exhibited this behavior. It implies that GSSG exerts a strong substrate inhibition: as greater GSSG concentrations were employed in the enzymatic assays larger lag periods were observed for both T. solium reductases (Fig 4). Additionally, reversible hysteretic kinetics was observed in assays with high GSSG concentrations upon increasing cTsTGR and mTsTGR concentrations. Similar results have been observed for T. crassiceps and Echinococcus granulosus TGRs (Bonilla et al., 2008; Rendón et al., 2004); lag times were similar to those observed for T. crassiceps but higher than those for E. granulosus TGRs under similar experimental conditions. Thus, hysteretic behavior for cTsTGR and mTsTGR is qualitatively identical to that reported for enzymes from the parasitic flatworm T. crassiceps, F. hepatica and E. granulosus. Enzymes showing hysteretic behavior can be classified as regulatory ones if TGRs are involved in the control of cellular redox homeostasis (Frieden, 1979). cTsTGR and mTsTGR, as has clearly been shown in this work, may participate in maintaining a reduced intracellular environment, thus protecting macromolecules from oxidative damage in the T. solium tissues, particularly when they are exposed to reactive oxidants produced by the host’s immune response (Bartsch and Nair, 2005; Rodriguez et al., 2008).

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Acknowledgements The present study was funded by a grant from the Universidad Nacional Autónoma de México, DGPA (PAPIIT IN211313-3). All experiments complied with the currents laws of the Universidad Nacional Autónoma de México. The authors wish to thank to Dr. Gerardo Medina for valuable English corrections.

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