Parasitology International 64 (2015) 194–201

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Purification and characterization of Taenia crassiceps cysticerci thioredoxin: insight into thioredoxin-glutathione-reductase (TGR) substrate recognition J.J. Martínez-González, A. Guevara-Flores, J.L. Rendón, A. Sosa-Peinado, I.P. del Arenal Mena ⁎ Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), Apartado Postal 70-159, 04510 México, D.F., México

a r t i c l e

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Article history: Received 23 April 2014 Received in revised form 5 December 2014 Accepted 6 December 2014 Available online 15 December 2014 Keywords: Taenia crassiceps Taenia solium thioredoxin thioredoxin-glutathione reductase platyhelminths

a b s t r a c t Thioredoxin (Trx) is an oxidoreductase central to redox homeostasis in cells and is involved in the regulation of protein activity through thiol/disulfide exchanges. Based on these facts, our goal was to purify and characterize cytosolic thioredoxin from Taenia crassiceps cysticerci, as well as to study its behavior as a substrate of thioredoxin-glutathione reductase (TGR). The enzyme was purified N 133-fold with a total yield of 9.7%. A molecular mass of 11.7 kDa and a pI of 4.84 were measured. Native electrophoresis was used to identify the oxidized and reduced forms of the monomer as well as the presence of a homodimer. In addition to the catalytic site cysteines, cysticerci thioredoxin contains Cys28 and Cys65 residues conserved in previously sequenced cestode thioredoxins. The following kinetic parameters were obtained for the substrate of TGR: a Km of 3.1 μM, a kcat of 10 s−1 and a catalytic efficiency of 3.2 × 106 M−1 s−1. The negative patch around the α3-helix of Trx is involved in the interaction with TGR and suggests variable specificity and catalytic efficiency of the reductase toward thioredoxins of different origins. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Thioredoxin (Trx) is a small protein central to redox homeostasis in cells. Trx is widely distributed in a variety of species ranging from bacteria to vertebrates [1,2]. In the parasitic lineage of flatworms, Trx has been reported in the adult stages of the trematodes Schistosoma mansoni [3] and Fasciola hepatica [4], as well as in the larval stage of the tapeworm Echinococcus granulosus [5]. Originally described as a hydrogen donor for ribonucleotide reductase [6], Trx is now known to be involved in multiple cellular functions such as the activation of transcription factors [7–9], regulation of protein activity through thiol/disulfide exchange reactions [10], as well as in the scavenging of oxygen radicals [7,11,12]. Also important to mention are the methionine sulfoxide reductases which are involved in the regeneration of methionine sulfoxides (formed during oxidative stress) using thioredoxin as a reductant and recently reported in platyhelminth genomes [13–15]. The presence of an active redox motif containing two conserved cysteine residues (WCGPC) is critical for Trx’s function. When Trx participates in the processes noted above, the redox active dithiol is oxidized into a disulfide bond; as a result, the regeneration of the

Abbreviations: Eg, Echinococcus granulosus; Fh, Fasciola hepatica; GSH, reduced glutathione; HsTrx, human thioredoxin; Pf, Plasmodium falciparum; Sm, Schistosoma mansoni; Tc, Taenia crassiceps; Ts, Taenia solium; TE, 50 mM Tris plus 1 mM EDTA buffer; Trx, thioredoxin; TGR, thioredoxin glutathione-reductase; TrxR, thioredoxin reductase ⁎ Corresponding author. Tel.: +55 56232169; fax: +55 56162419.

http://dx.doi.org/10.1016/j.parint.2014.12.004 1383-5769/© 2014 Elsevier Ireland Ltd. All rights reserved.

reduced state of the protein is essential. This function is carried out by the NADPH-dependent thioredoxin reductase [16]. The threedimensional structure of the protein, common to all Trx described to date, is characterized by the presence of a central five-strand β-sheet surrounded by four α-helices [17,18]. Albeit similar, an interesting difference among Trx homologs from different organisms is that they vary in the number of additional non-catalytic cysteine residues, of which up to four have been reported [11,18,19]. In human Trx1, a second intramolecular disulfide bond between cysteines 62 and 69 has been reported to be involved in the regulation of the reduction of the active site [20]. When a cysteine residue at position 73 is present, the protein has the ability to dimerize through the formation of an intermolecular disulfide bond [21–23]. Different isoforms of thioredoxin have been reported, such as Trx1 and Trx2 from Escherichia coli [24], cytosolic and mitochondrial Trx in eukaryotic organisms [18] and in plants (e.g., Arabidopsis thaliana), 20 genes coding for Trx are known [25]. In most organisms, two intracellular antioxidant pathways for scavenging reactive oxygen species are present: glutathione and thioredoxin systems. After donating their electrons, the resulting oxidized glutathione and thioredoxin are reduced by specific NADPH-dependent disulfide reductases, namely, glutathione (GR) and thioredoxin (TR) reductase, respectively. However, in parasitic platyhelminths, thiol/disulfide redox homeostasis relies on the presence of multifunctional NADPHdependent thioredoxin glutathione reductase (TGR), which is involved in the regeneration of the reduced state of both thioredoxin and

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glutathione [26–29]. Both substrates share the defense against oxidative stress through the intermediation of peroxiredoxins [30,31]. As we have previously reported, Taenia crassiceps metacestode (cysticerci) mitochondria are able to produce H2O2 in significant amounts [32]. In these parasites, where the activity of the catalase and glutathione peroxidase is absent or scarce, the detoxification of H2O2 is dependent only on the Trx/thioredoxin peroxiredoxin system [33,34]. In a previous study, we described the purification and catalytic properties of TGR from T. crassiceps cysticerci (TcTGR) [29], and showed that this enzyme efficiently reduced Plasmodium falciparum Trx (PfTrx) and human Trx, but was unable to reduce prokaryote thioredoxins. To elucidate the properties of the T. crassiceps Trx system (NADPH, TGR and Trx), critical for parasite survival, in the present work, we report the purification and characterization of cytosolic Trx. To gain insight into the substrate specificity of TGR, we analyzed the crystallographic structures of the previously tested thioredoxins and modelled the tertiary structures of the partial sequence of TcTrx and the full sequence T. solium thioredoxin (TsTrx). The electrostatic complementarity between the surfaces involved in the interactions of Trx with T. solium TGR reveals the importance of the negative patch around the α3-helix of Trx in substrate-enzyme recognition and that likely explains the different activities of the reductase toward Trx from various species.

2. Materials and methods 2.1. Biological and chemicals Female Balb/c mice were inoculated intraperitoneally with 15 cysticerci of the T. crassiceps HYG strain, kindly donated by Dr. Larralde (Instituto de Investigaciones Biomédicas, UNAM). Six to eight months later, cysticerci were recovered from the peritoneal cavity and washed thoroughly with PBS (phosphate-buffered saline solution; pH 7.4) [35]. This material was used as the source of Trx. Dithiothreitol (DTT),

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Table 1 T. crassiceps thioredoxin purificationa. Total Purification Yield Fractionb Volume Protein Specific activityc (%) (mL) (mg) (nmole min−1 mg−1) activity (fold) Crude DEAE UG Phenyl-S a b c

340.0 23.0 43.0 2.1

1,632.0 0.438 95.2 4.89 30.1 6.72 1.2 145.0

714.8 474.1 202.2 172.5

1.0 9.3 15.3 331.0

100.0 66.40 28.3 24.2

Activity was measured at 340 nm. Starting from 160 g cysticerci (wet weight). nmoles NADP min−1 protein mg−1

phenylmethylsulfonyl fluoride (PMSF), DEAE-Trisacryl M, HA-Ultrogel, Phenyl-Sepharose CL-4B, insulin and E. coli Trx were obtained from Sigma-Aldrich (St. Louis, MO, USA). Protein markers for isoelectric focusing and Precision Plus Protein Standards were purchased from Bio-Rad Labs (Hercules CA, USA). Cytosolic TcTGR was purified as previously described [29]. 2.2. Thioredoxin purification Unless otherwise stated, all manipulations were carried out at 4 °C. Cysticerci were suspended in an equal volume of 100 mM Tris/HCl buffer (pH 8.5) containing 2 mM EDTA plus 172 μM PMSF and mechanically disrupted with a motor-driven Teflon pestle. The resulting homogenate was centrifuged at 800g for 20 min, the pellet discarded and the supernatant submitted to ultracentrifugation at 106,000g for 45 min. The clear solution was recovered and dialyzed overnight against 100 mM Tris/HCl buffer (pH 8.5) containing 1 mM EDTA (TE buffer). Before all of the chromatographic steps, samples were incubated with 2 mM DTT for 30 min. Similarly, all the chromatographic matrices were equilibrated in the corresponding buffer containing 0.1 mM DTT. The dialyzed high-speed supernatant was adsorbed onto a DEAETrisacryl column (2.6 × 20 cm) that had been equilibrated in TE buffer previously. After washing out the non-adsorbed protein, Trx elution was carried out through application of a 0 to 0.3 M NaCl linear gradient prepared in 10 column volumes of the same TE buffer. Fractions containing Trx activity were pooled and dialyzed overnight against 5 mM sodium phosphate buffer pH 7 (P-buffer). The dialyzed sample obtained from the DEAE-Trisacryl chromatography was applied onto a HA-Ultrogel column (1.8 × 7 cm) that had previously been equilibrated in 5 mM P-buffer. Under these conditions, Trx activity was retained in the non-adsorbed fractions, which were pooled and dialyzed against 50 mM P-buffer containing 1.7 M ammonium sulfate. The dialyzed pool obtained from the Ultrogel chromatography was adsorbed at room temperature on a Phenyl-Sepharose CL-4B column (1 × 18 cm) that had previously been equilibrated in the 50 mM Pbuffer containing 1.7 M ammonium sulfate. After washing the nonadsorbed protein with the same buffer solution, the protein was eluted with three column volumes of 1.4 M ammonium sulfate followed by five column volumes of a decreasing 1.4 M to 0.5 M ammonium sulfate

Table 2 N-Terminal sequence and LC/MS/MS of thioredoxin purified from T. crassiceps cysticerci. Coverage of 63% of the amino acid sequence and a total score of 255 was obtained by LC/MS/MS according of E. granulosus [22]. N-terminal sequence by Edman

Peptide sequenced obtained by LC/MS/MS

Matched peptide sequence to E. granulosus Trx

QVDGDALEAAIKGDK GDKLLVCDFFATWCGPCK LDAMAK LDVDECQDVAEK RVTAMPTLIVFK

1–20 9–23 21–38 44–49 59–70 72–83

MSAEVVVKQVDGDALEAAIK Fig. 1. Electrophoretic analysis of the various steps used in the purification of TcTrx. Protein samples from each corresponding step were analyzed by 16% SDS-PAGE: a) Crude extract (22 μg protein); b) DEAE chromatography (10 μg protein); c) Ultrogel chromatography (5 μg protein); Phenyl-Sepharose chromatography (8 μg protein). The gel was stained and destained using conventional methods.

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Fig. 2. Amino acid sequence alignment of TcTrx with those of related flatworms. Data were obtained from the GeneDB Project (GeneDB), with the exception of F. hepatica Trx, which was taken from ref. [55]. GeneDB access codes are as follows: E. granulosus (EgrG_000602100.1), T. solium (TsM_000941400), S. mansoni (Smp_008070) and S. japonicum (Sjp_0039090). Cysteine residues are shaded in black, while residues enclosed in the blue box correspond to the active site motif and the residues enclosed in a red box represent the negatively charged patch surrounding α helix 3. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)

linear gradient. All solutions were prepared in 50 mM P-buffer. Fractions with Trx activity were thoroughly dialyzed against distilled water, pooled and concentrated in Amicon Ultra-5 k tubes. Purified Trx was stored at −20 °C until further use. 2.3. Thioredoxin activity assay The quantitative assay for Trx activity was based on its ability as a disulfide oxidoreductase to catalyze the reduction of insulin using dithiothreitol as an electron donor [36]. The reaction mixture contained, in a final volume of 500 μL, a Trx aliquot and 100 μM human insulin in 0.1 M Tris-HCl buffer (pH 7.8) plus 2 mM EDTA. The reaction was started by the addition of 0.33 mM DTT and the turbidity produced by precipitation of the insulin β chain was monitored at 650 nm. The reduction of insulin by DTT in the absence of Trx was recorded as a control. Alternatively, Trx activity was also assayed as reported by Arner et al. [37], measuring NADPH oxidation at 340 nm. The reaction mixture contained 45 μM NADPH, a Trx aliquot and 100 μM human insulin in 500 μL of 0.1 M Tris/HCl buffer (pH 7.8) containing 1 mM EDTA. The reaction was started by adding TcTGR at a final concentration of 11 nM. 2.4. Electrophoresis and protein sequencing Samples of all purification steps were analyzed by both native [38] and denaturing PAGE (polyacrylamide gel electrophoresis) [39]. To determine the amino acid sequence of the putative Trx from T. crassiceps, the protein was separated by denaturing PAGE, blotted onto an Immobilon-P PVDF membrane (Millipore Corporation, Bedford, Mass, USA) and stained with 1% Ponceau-S red. Then, the band was excised and subjected to Edman degradation [40] in a gas-phase automated

protein sequencer (LF 3000, Beckman Instruments) equipped with an on-line Beckman Gold HPLC system using a model 126 pump and a model 168 diode array detector setting at 268 and 293 nm for signal and reference, respectively. For tandem mass spectrometry (LC/ESIMS/MS), the protein band obtained following electrophoresis was excised from the Coomassie-stained SDS gel, destained, reduced, carbamidomethylated and digested with modified porcine trypsin. Peptide mass spectrometric analysis was performed using a 3200 Q TRAP hybrid tandem mass spectrometer as previously reported [41]. Database searches (NCBI-nr) and protein identification were performed using the MS/MS spectra and Mascot Software (http://www.matrixscience.com). The criteria to accept the protein hit as a valid identification were two or more peptides that matched the same protein sequence with at least 95% confidence level (p b 0.05) and, in this case, if the individual ion scores were greater than 25. The sequence of five peptides from TcTrx obtained by mass spectroscopy were used to assess the cytosolic EgTrx sequence EgrG_000602100.1 (GeneDB) and originally deposited in NCBI http://www.ncbi.nlm.nih.gov/protein/. Analysis of Trx by two-dimensional electrophoresis was performed by running a protein aliquot under native conditions in a 12% gel slab. For the second dimension, the bands of interest were cut off from the gel and included into a minimal volume of 0.05 M Tris/HCl buffer (pH 6.8) containing 3% SDS, 3 mM 2-mercaptoethanol, 10% glycerol and 0.1% bromophenol blue. Next, the sample was loaded onto a 16% SDS-denaturing gel. 2.5. Isoelectric point determination The isoelectric point of TcTrx was determined in a 3–10 pH immobilized gradient strip (Amersham Pharmacia Biotech). Immobiline

Fig. 3. Electrophoretic analysis of purified TcTrx. A) A Trx aliquot was stored at −20 °C for 15 days and then analyzed using 12% PAGE under native conditions; four bands are clearly visible. B) The three faster migrating protein bands obtained under native conditions were cut off from the gel and analyzed in a second dimension 16% SDS-PAGE. C) Effect of oxidizing or reducing conditions on the electrophoretic pattern of TcTrx. A protein aliquot was incubated with 5 mM DTT and another with 500 μM H2O2, for 30 min, and then both were analyzed in a discontinuous native gel. An untreated sample was used as a control.

J.J. Martínez-González et al. / Parasitology International 64 (2015) 194–201 Table 3 Kinetic parameters of TGR from T. crassiceps with different thioredoxin sources and its relation with the interaction sequence enzyme substrate. TRX origin

Sequence

Km (μM)

kcat (s−1)

kcat/Km (mM s−1 × 106)

T. crassiceps H. sapiens P. falciparum S. subsalsa E. coli

DECQDVAEK-R DDCQDVASECE DEVSEVTEKEN DENPNVASQYG DQNPGTAPKYG

3.1 5.3 17 NR NR

10 11.8 19.2 NR NR

3.22 2.2 1.13 NR⁎ NR

NR, not recognized Negative residues denoted in bold. ⁎ Measured with Spirulina sp. (SIGMA), genome not available

dry strips were hydrated and focusing was performed according to standard protocols [42]. A mixture of nine protein markers with isoelectric points ranging from 4.45 to 9.6 were used.

2.6. Protein determination The method of Markwell et al. [43] was used to determine protein concentration, employing bovine serum albumin as a standard. The concentration of purified TcTrx was determined by measuring its absorbance at 280 nm using the molar extinction coefficient reported for E. granulosus cytosolic Trx (7.24 mM−1 cm−1) [25].

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2.7. Preparation of reduced and oxidized thioredoxin Samples (100 μl) of pure Trx (210 μM) were incubated for 30 min at room temperature with either 5.0 mM DTT or 500 μM H2O2. The peroxide-treated sample was diluted 40 times with water and then concentrated on an Amicon Ultra 3 K centrifugal filter (Millipore) at a centrifugal force of 4,000g to the original 100 μl volume. Reduced, oxidized and untreated samples were then analyzed by native electrophoresis using a 5% stacking gel and 12%–16% resolving gels. 2.8. Modelling of TcTrx From the amino acid sequence of TcTrx obtained as described above, a partial structural model was generated using the server I-Tasser [44]. From the best five models obtained, a model was selected with a C value of 1.14, a TM value of 0.87 ± 0.07 and a RMSD of 1.4 ± 1.3 Å. The best structural model obtained with the server I-Tasser was checked for completeness by the What-If suite [45]. This model was then energy-minimized using the AMBER force field by the steepest descent algorithm for 50 steps with the program YASARA structure version 11.6.16. 2.9. Sequence, molecular modelling and Trx–TGR complex formation. To obtain platyhelminth TGR and Trx sequences, S. mansoni, S. japonicum, E. granulosus and T. solium genomic databases (GeneDB,

Fig. 4. A) Electrostatic potential mapped on the solvent accessible surface of thioredoxin from T. solium (TsTrx), Homo sapiens (HsTrx), P. falciparum (PfTrx), S. subsalsa (SsTrx), and E. coli (EcTrx). The magnitude of the electric charge is indicated in red for negative charges and blue for positive charges. In all cases, a circle encloses the region surrounding α helix 3. B) Electrostatic potential mapped on the solvent accessible surface of T. solium TGR. The color code is as in A. C) Details of the TGR–Trx complex stressing the electrostatic complementarity, which is critical in the reduction of the disulfide bond (in yellow) of Trx by the selenocysteine residue (magenta) of the enzyme. Models were generated by PyMol. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)

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generated using PyMol (http://www.pymol.org). The superimposed models and intermolecular interactions of TsTrx-TsTGR were obtained using the crystallographic structure of the homologous interaction HsTrx-HsTrxR (PDB:3QFA) [58].

3. Results 3.1. Thioredoxin purification

Fig. 5. Ribbon representation showing the interaction between TGR (grey) and Trx (blue) from T. solium. The predicted three-dimensional structures of both TGR and Trx are superimposed on the corresponding structure of human TrxR (green) and Trx (red). A circle encloses the regions on both TGR and Trx suggesting that these two areas are to be involved in the formation of the enzyme–substrate complex. Models were generated by PyMol. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)

www.genedb.org) [46] were searched with Blastp, using sequences of human Trx1 (HsTrx, PDB 3TRX) and TrxR (HsTrxR, PDB 2CFY) as queries. The flatworm sequences obtained were aligned with Clustal W [47] and colored using JalView software [48]. The structural models for the thioredoxins and TsTGR were obtained using the automatic server from Swiss-Model [49–51] by analyzing the flatworm sequences described above and the Trx homolog from Spirulina subsalsa (NCBI, ID: WP_017305605.1). Global and local quality models were estimated according to the Swiss-Model server with Qmean [52], Analea [53] and Gromos [54]. The electrostatic potentials mapped onto the protein’s surface were obtained with ABPS [55–57] software. In addition, we evaluated electrostatic properties of P. falciparum (PDB 1YSR), Human (PDB 3TRX) and E. coli (PDB 2TRX) crystallographic-resolved Trx. The molecular representations were

Fig. 6. Superposition of the predicted tertiary structure of Trx from E. granulosus (orange) and T. crassiceps (blue). Cysteine residues of the redox motif are shown in yellow. Models were generated with PyMol. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)

To obtain cytosolic TcTrx from the cysticerci, the soluble fraction of the cysticerci (supernatant of 106,000g) was used. The purification of this protein was achieved by the use of three chromatographic matrices to which the extract was consecutively exposed as outlined in detail in the Materials and methods. Trx was eluted between 80 and 100 mM NaCl in DEAE-Trisacryl. Trx was not adsorbed in HA-Ultrogel, but most of the contaminants were bound, and the sample harboring activity was eluted at 0.9 M ammonium sulfate using Phenyl-Sepharose chromatography. The three-step procedure used in the present work for the purification of Trx from T. crassiceps cysticerci resulted in an essentially homogeneous preparation, as revealed by electrophoretic analysis (Fig. 1). As shown in Table I, TcTrx was purified 331-fold with a total yield of 24%. Clearly, the chromatographic step on Phenyl-Sepharose was the most important step in the purification procedure.

3.2. Sequence and characterization Trx was found at the expected size in the electrophoretic pattern of the purest fraction (Fig. 1). To confirm the molecular nature of cysticerci TcTrx, the N-terminal amino acid sequence was determined (20 residues). When compared with database sequences, a 97% match with cytosolic Trx (EgrG_000602100.1) from the related E. granulosus was obtained [25]. Peptide mass spectrometric analysis was also performed to obtain supplementary amino acid sequence information; five different peptide sequences that match with the EgTrx amino acid sequence were identified (Table 2). (See Table 1.) The amino acid sequences obtained from the five peptidic fragments of TcTrx represents 63% of the total amino acid content (assuming a total number of amino acid residues of 107 as in EgTrx) of the homologous protein from both cestodes and trematodes (Fig. 2). In all cases, the conserved Trp-Cys-Gly-Pro-Cys redox motif of the active site is present; however, in cestode Trx, two additional cysteine residues, Cys28 and Cys65 (cestode numbering) are present, which were not found in Trx proteins from different trematodes (SjTrx, SmTrx, FhTrx) [23,24]. An additional Cys86 is present in EgTrx. An isoelectric point of 4.84 was obtained for TcTrx, similar to its counterpart in E. granulosus [25]. The molecular mass estimated by SDS-PAGE was 11.7 employing Precision Plus Protein as the STD. The 12% native PAGE analysis of Trx stored at − 20 °C for 15 days showed four bands, which were marked as 0, 1, 2 and 3 (Fig. 3A). To clarify its identity, protein bands were excised and analyzed in the second dimension using SDS-PAGE (Figs. 3B and S1). Furthermore, N-terminal amino acid sequence was also analyzed. The results of both analyses revealed that samples marked 0, 2 and 3 correspond to thioredoxin, and 0 represents the Trx dimer, as revealed by its molecular mass. The relative abundance of the Trx dimer was increased during storage due to auto-oxidation. To analyze the possibility that Trx 2 and 3 represent alternative oxidation states, duplicate samples were incubated in either 5.0 mM DTT (a reducing reagent) or 500 μM H2O2 and then analyzed by PAGE under non-denaturing conditions. As shown in Fig. 3C, in the sample incubated with DTT (a reducing reagent), band 2 was clearly visible, indicating that it is the reduced form of Trx, while band 3 disappeared. In contrast, band 2 disappeared and band 3 was retained in the presence of H2O2 suggesting that band 3 is an oxidized state of Trx. It was not possible to clarify the identity of protein band 1.

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3.3. Kinetic studies and substrate recognition When TcTrx was assayed as the substrate of TcTGR by measuring NADPH disappearance at 340 nm, the kinetic parameters obtained were: a Km of 3.1 μM ± 0.98, a kcat of 10 s−1 and a catalytic efficiency of 3.2 × 106 M−1 s−1. These data were comparable with those reported for TGR from E. granulosus [26] and S. mansoni [28] using endogenous Trx (Table S2). In a previous study, thioredoxin from prokaryotic (Spirulina sp. and E. coli) and eukaryotic (P. falciparum and human) organisms were assayed as substrates of cysticerci TGR [29]. Substantially different activities were found among these proteins (Table 3). It was clear that the enzyme was unable to reduce prokaryotic Trx. To gain insight into the structural basis for such selectivity, a comparison of the amino acid sequences of the tested Trx proteins was performed. The results revealed that the amino acid sequence DECQDVAEKYR, which was recognized by TcTGR, is present in Trx of eukaryotic origin but not in prokaryotic Trx. Interestingly, this sequence is characterized by a predominance of negatively charged residues. As shown in Fig. 4A, differences in the electrostatic potential mapped onto the protein surface constitute a negative patch in the α3 helix of the proteins. This region is critical in the recognition of Trx by the reductase because it interacts electrostatically with the opposite positive regions on the enzyme (Fig. 4B) and is present only in the organisms recognized by TcTGR. A close-up of the TGR-Trx complex shows how the complementary charges facilitate the reduction of the disulfide of Trx (Fig. 4C, yellow) by TGR selenocysteine (Fig. 4C, magenta). No homology is present in this region in E. coli and in S. subsalsa Trx (NCBI WP_D17305605.1). We used the amino acid sequence of S. subsalsa because it is the only published available Spirulina genome. A stereo plot of superimposed structures of TsTrx (blue) and HsTrx (red) illustrate the importance of the complementary charges required for contact with the HsTGR (Fig. 5). Previous reports demonstrated the importance of this region in Trx-TrxR interaction [58,59]. Consistent with the absence of reduction, this interaction does not occur between TGR and Trx from E. coli (Fig. S2) or Spirulina. 4. Discussion As reported in 2012 by the World Health Organization (www.who. int/neglected_diseases/diseases/en/), the larval stages of some parasitic cestodes, such as E. granulosus and T. solium, can cause diseases that affect humans and animals and result in severe health and economic problems, particularly in developing countries. An example is neurocysticercosis, a serious neural disease caused by the metacestode of T. solium [60,61]. At present, drug resistance has been documented in several trematode species, such as S. mansoni [62–64] and F. hepatica [65–67]. In contrast, resistance to anthelminthic drugs has not been recognized in cestodes, even through a variety of different responses to treatments are documented [68]. For treatment against T. saginata, low efficacy of albendazole or its sulfate derivative was found [69]. To combat cestode infections, different combined treatments provide good results [70]. As has been frequently documented, the substitution of the GR and the TR systems in flatworms by a single multifunctional enzyme (TGR) that reduces both glutathione and thioredoxin makes the Trx system a potential drug target to control parasitic diseases [71–73]. In fact, silencing SmTGR by RNA interference leads to worm death [73]. Alternatively, the gold-containing compound Auranofin, an irreversible inhibitor of selenocysteine-containing enzymes such as TGR, has the ability to kill the larval stage (metacestode) of either E. granulosus or T. crassiceps [26,74]. Thus, TGR is a key enzyme required to maintain the redox balance of flatworm parasites. Since 1983 [75], Auranofin has been used as a therapy for rheumatoid arthritis with no secondary effects reported after one year of treatment [76]. Using the purification procedure protocol we developed, cytosolic Trx was obtained from T. crassiceps cysticerci in a highly purified form,

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as shown in Table I. The best results were obtained following the oxidation of NADPH at 340 nm in the presence of TGR (331-fold and 24.2% yield) compared with those using the turbidimetric method involving only Trx (Table S1 Supporting information). The results reported in the present work revealed that a functional Trx is expressed in T. crassiceps cysticerci. General physicochemical properties were similar to its counterpart isolated from related parasites [4,5]. From the sequence comparison of the proteins, the presence of two cysteine residues, Cys28 and Cys65, in addition to those of the redox motif Cys35 and Cys38 (cestodes numbering) were revealed appearing to be a general feature of cestode Trx. Such cysteines are absent in trematode Trx. Based on the modelling of the threedimensional structure of both E. granulosus and T. crassiceps Trx (Fig. 6), it was found that the above noted cysteines are far away (10.597 Å), excluding any possibility for the formation of an intramolecular disulfide bond. By contrast, Cys62 and Cys69 of the mammalian Trx were found to be involved in such intramolecular linkage, resulting in impaired reduction of the redox active disulfide bond by TrxR [20]. The possible functions of the conserved Cys28 and Cys65 residues in tapeworm Trx remain to be elucidated. Although Cys73 is absent in TcTrx, the results obtained in the present work revealed that this protein is able to aggregate. After storage at −20 °C, the electrophoretic analysis revealed that a significant fraction of Trx forms a dimer (Fig. 3A and B). This fact strengthens the reports that indicate that the presence of Cys73 this amino acid is not essential for Trx dimerization and in accord with the results obtained with mutants of the human protein when Cys73 was replaced with serine without any effect on dimerization [23]. When the capacity of TcTGR to recognize Trx from different sources was tested, the kinetic parameters revealed substrate recognition of T. crassiceps, human and P. falciparum Trx, although E. coli and Spirulina sp. thioredoxins were not recognized (Table 3). Despite the conserved tertiary structure in all of the known Trx homologs, TrxR shows substrate preference. Similar substrate specificity have been reported in D. melanogaster, S. mansoni and E. coli for their respective Trx homologs [3,73,77]. We postulate that the high activity is related to the electrostatic recognition supported by several opposite charges involved in the formation of the Trx–TGR complex. Additionally, the TsTGR and TsTrx models overlapped on the crystallographic structure of human thioredoxin reductase complexed with thioredoxin [58] (PDB code 3QFA). The crystallographic and modelled interactions of the two structures show the same complementary charges on the interacting face between Trx and TrxR. These results are in accord with previous studies in which the negative patch around the Trx α3 helix is used to make contact with the target molecules and TrxR [18,78–81]. Moreover, in P. falciparum, it has been demonstrated that residues Asp58-Ser71 of Trx are bound to helical Pf-TrxR residues Ser136–Ser151 [59]. As mentioned above, E. coli Trx is not used by TcTGR while in the Trx–TGR interaction, the number of opposite charges are low (Fig. S1). In summary, this study reports the purification method for T. crassiceps cytosolic thioredoxin and its biochemical characterization and we provide evidence that can explain the differences in recognition between the TrxR and Trx of different origin. Hence, a better understanding of the Trx-dependent redox system of cestodes will contribute to the design of effective control strategies against these kinds of parasites. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.parint.2014.12.004. Acknowledgments We would like to thank Dr. Gustavo Salinas and Dr. Rachel Duffié for critical reading of this manuscript and Abigail González Valdez for technical assistance. This work was supported by PAPIIT research grants IN220710-3 and IN219414-25 from the Dirección General de Asuntos del Personal Académico, UNAM and a Doctoral Scholarship from Consejo

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Nacional de Ciencia y Tecnología (CONACyT) to J.J. Martínez-González, student of the Programa de Doctorado en Ciencias Biológicas, UNAM. All experiments reported in this work were performed according to the bioethical regulations of the Universidad Nacional Autónoma de México. We also thank Dr. José Luis Perez Garcia for English language editing in the manuscript. This work is dedicated to Dr. Guillermo Mendoza who participated in this work, but unfortunately passed away.

[30] [31]

[32]

[33]

References [1] Holmgren A. Thioredoxin. Annu Rev Biochem 1985;54:237–71. [2] Lu J, Holmgren A. The thioredoxin antioxidant system. Free Radic Biol Med 2014;66: 7587. [3] Alger HM, Sayed AA, Stadecker MJ, Williams DL. Molecular and enzymatic characterisation of Schistosoma mansoni thioredoxin. Int J Parasitol 2002;32:1285–92. [4] Salazar-Calderón M, Martín-Alonso JM, Ruiz de Enguino AD, Parra E. Heterologous expression and functional characterization of thioredoxin from Fasciola hepática. Parasitol Res 2001;87:390–5. [5] Chalar C, Martínez C, Agorio A, Salinas G, Soto J, Ehrlich R. Molecular cloning and characterization of a thioredoxin gene from Echinococcus granulosus. Biochem Biophys Res Commun 1999;262:302–7. [6] Laurent TC, Moore EC, Reichard P. Enzymatic synthesis of deoxyribonucleotides. IV. Isolation and characterization of thioredoxin, the hydrogen donor from Escherichia coli. J Biol Chem 1964;239:3436–44. [7] Nordberg J, Arnér ES. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic Biol Med 2001;31:1287–312. [8] Sen CK, Packer L. Antioxidant and redox regulation of gene transcription. FASEB J 1996;10:709–20. [9] Sturm N, Jortzik E, Mailu BM, Komcarevic S, Deponte M, Forchhammer K, et al. Identification of proteins targeted by the thioredoxin superfamily in Plasmodium falciparum. PLoS Pathog 2009;5:1–12. [10] Powis G, Montfort WR. Properties and biological activities of thioredoxins. Annu Rev Biophys Biomol Struct 2001;30:421–55. [11] Hall G, Emsley J. Structure of human thioredoxin exhibits a large conformational change. Protein Sci 2010;19:1807–11. [12] Holmgren A. Antioxidant function of thioredoxin and glutaredoxin systems. Antioxid Redox Signal 2000;2:811–20. [13] Luo S, Levine RL. Methionine in proteins defends against oxidative stress. FASEB J 2009;23:464–72. [14] Tarrago L, Gladyshev VN. Recharging oxidative protein repair: catalysis by methionine sulfoxide reductases towards their amino acid, protein and model substrates. Biochemistry (Mosc) 2012;77:1097–107. [15] Tsai IJ, et al. The genomes of four tapeworm species reveal adaptations to parasitism. Nature 2013;496:57–63. [16] Arnér ESJ, Holmgren A. Physiological functions of thioredoxin and thioredoxin reductase. Eur J Biochem 2000;267:6102–9. [17] Eklund H, Gleason FK, Holmgren A. Structural and functional relations among thioredoxins of different species. Proteins 1991;11:3–28. [18] Wahl MC, Irmler A, Hecker B, Schirmer RS, Becker K. Comparative structural analysis of oxidized and reduced thioredoxin from Drosophila melanogaster. J Mol Biol 2005; 345:1119–30. [19] Haendeler J. Thioredoxin-1 and posttranslational modifications. Antioxid Redox Signal 2006;8:1723–8. [20] Watson WH, Pohl J, Montfort WR, Stuchlik O, Reed MS, Powis G, et al. Redox potential of human thioredoxin 1 and identification of a second dithiol/disulfide motif. J Biol Chem 2003;278:33408–15. [21] Gasdaska JR, Kirkpatrick DL, Montfort W, Kuperus M, Hill SR, Berggren M, et al. Oxidative inactivation of thioredoxin as a cellular growth factor and protection by Cys73 → Ser mutation. Biochem Pharmacol 1996;52:1741–7. [22] Ren G, Stephan D, Xu Z, Zheng Y, Tang D, Harrison RS, et al. Properties of the thioredoxin fold superfamily are modulated by a single amino acid residue. J Biol Chem J Biol Chem 2009;284(15):10150–9. [23] Weichsel A, Gasdaska GR, Powis G, Montfort WR. Crystal structures of reduced, oxidized, and mutated human thioredoxins: evidence for a regulatory homodimer. Structure 1996;4:735–51. [24] Miranda-Vizuete A, Damdimopoulos AE, Gustafsson J, Spyrou G. Cloning, expression, and characterization of a novel Escherichia coli thioredoxin. J Biol Chem 1997;272: 30841–7. [25] Meyer Y, Vignols F, Reichheld JP. Classification of plant thioredoxins by sequence similarity and intron position. Methods Enzymol 2002;347:394–402. [26] Bonilla M, Denicola A, Novoselov SV, Turanov AA, Protasio A, Izmendi D, et al. Platyhelminth mitochondrial and cytosolic redox homeostasis is controlled by a single thioredoxin glutathione reductase and dependent on selenium and glutathione. J Biol Chem 2008;283:17898–907. [27] Guevara-Flores A, Pardo JP, Rendón JL. Hysteresis in thioredoxin-glutathione reductase (TGR) from the adult stage of the liver fluke Fasciola hepatica. Parasit Int 2011;60: 156–60. [28] Williams DL. Thioredoxin glutathione reductase from Schistosoma mansoni: an essential parasite enzyme and a key drug target. PLoS Med 2007;4(6):1071–85. [29] Rendón JL, del Arenal IP, Guevara-Flores A, Uribe A, Plancarte A, Mendoza-Hernández G. Purification, characterization and kinetic properties of the multifunctional thioredoxin-

[34] [35]

[36] [37] [38] [39] [40] [41]

[42] [43]

[44] [45] [46] [47] [48]

[49]

[50] [51] [52] [53] [54] [55]

[56]

[57]

[58] [59]

[60] [61] [62] [63] [64]

glutathione reductase from Taenia crassiceps metacestode (cysticerci). Mol Biochem Parasitol 2004;133:61–9. Docampo R. Antioxidant Mechanisms. Biochemistry and Molecular Biology of Parasites. London, UK: Academic Press Limited; 1995 147–60. Vaca-Paniagua F, Parra-Unda R, Landa A. Characterization of one typical 2-Cys peroxiredoxin gene of Taenia solium and Taenia crassiceps. Parasitol Res 2009;105: 781–7. del Arenal IP, Rubio ME, Ramírez J, Rendón JL, Escamilla JE. Cyanide-resistant respiration in Taenia crassiceps metacestode (cysticerci) is explained by the H2O2producing side-reaction of respiratory complex I with O2. Parasitol Int 2005;54: 185–93. Callahan HL, Crouch RK, James ER. Helminth anti-oxidant enzymes: a protective mechanism against host oxidants? Parasitol Today 1988;4:218–25. Mei H, LoVerde PT. Schistosoma mansoni: the developmental regulation and immunolocalization of antioxidant enzymes. Exp Parasitol 1997;86:69–78. Larralde C, Sciutto E, Grun J, Diaz ML, Govezensky T, Montoya RM. Biological determinants of host–parasite relationship in mouse cysticercosis caused by Taenia crassiceps: influence of sex, major histocompatibility complex and vaccination. In: Cañedo LE, Todd LE, Packer L, Jaz J, editors. Cell Function and Disease. Plenum Publishing Corporation; 1989. p. 325–32. Holmgren A. Thioredoxin catalyzes the reduction of insulin disulfides by dithiothreitol and dihydrolipoamide. J Biol Chem 1979;254:9627–32. Arnér ESJ, Zhong L, Holmgren A. Preparation and assay of mammalian thioredoxin and thioredoxin reductase. Methods Enzymol 1999;300:226–39. Davis BJ. Method and application to human serum proteins. Ann N Y Acad Sci 1964; 121:404–7. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond) 1970;227:680–5. Edman P, Begg GA. Protein sequenator. Eur J Biochem 1967;1:80–91. Xolalpa W, Vallecillo AJ, Lara M, Mendoza-Hernández G, Comini M, Spallek R, et al. Identification of novel bacterial plasminogen-binding proteins in the human pathogen Mycobacterium tuberculosis. Proteomics 2007;7:3332–41. Berkelmen T. Stenstedt T. 2-D electrophoresis using immobilized pH gradients. Principles and Methods. UK: Amersham Pharmacia Biotech; 1998. Markwell MAK, Haa SM, Bieber LL, Tolbert NE. A modification to the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 1978;87:206–10. Roy A, Kucukural A, Zhang Y. I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc 2010;5:725–38. Vriend G. WHAT IF: a molecular modeling and drug design program. J Mol Graph 1990;8(52-56):29. Logan-Klumper FJ, et al. GeneDB—an annotation database for pathogens. Nucleic Acids Res 2012;40:D98–D108. Larkin MA, Blackshields G, Brown NP, Chenna R, Mcgettigan PA, Mcwilliam H, et al. Clustal X version 2.0. Bioinformatics 2007;23:2947–8. Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ. Jalview Version 2 a multiple sequence alignment editor and analysis workbench. Bioinformatics 2009; 25:1189–91. Arnold K, Bordoli I, Kopp J, Schwede T. The SWISS-MODEL Workspace: a web-based environment for protein structure homology modelling. Bioinformatics 2006;22: 195–201. Schwede T, Kopp J, Guex N, Peitsch MC. SWISS-MODEL: an automated protein homology-modeling server. Nucl Acid Res 2003;31:3381–5. Bordoli l, Kiefer F, Arnold K, Benkert P, Battey J, Schwede T. Protein structure homology modeling using SWISS-MODEL workspace. Nat Protoc 2009;4(1):1–13. Benkert P, Tosatto SC, Schomburg D. QMEAN: a comprehensive scoring function for model quality assessment. Proteins 2008;71:261–77. Melo F, Feytmans E. Predicting reliable regions in protein alignments from sequences profiles. J Mol Biol 1998;330:705–18. Van Gunsteren WF, et al. Biomolecular Simulations: the GROMOS96 Manual and User Guide. Zürich: VdF Hochschulverlag ETHZ; 1996. Baker NA, Sept D, Joseph S, Holst MJ, McCammon JA. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc Natl Acad Sci U S A 2001;98: 10037–41. Dolinsky TJ, Czodrowski P, Li H, Nielsen JE, Jensen JH, Klebe G, et al. PDB2PQR: expanding and upgrading automated preparation of biomolecular structures for molecular simulations. Nucl Acid Res 2007;35:W522–5. Dolinsky TJ, Nielsen JE, McCammon JA, Baker NA. PDB2PQR: an automated pipeline for the setup, execution, and analysis of Poisson–Boltzmann electrostatics calculations. Nucleic Acids Res 2004;32:W665–7. Fritz-Wolf K, Kehr S, Stumpf M, Rahlfs S, Becker K. Crystal structure of the human thioredoxin reductase-thioredoxin complex. Nat Commun 2011;2:383. Fritz-Wolf K, Jortzik E, Stumpf M, Preuss J, Lozef R, Rahlfas S, et al. Crystal structure of the Plasmodium falciparum thioredoxin reductase-thioredoxin complex. J Mol Biol 2013;425:3446–60. Carpio A, Fleury A, Allen Hauser W. Neurocysticercosis. Nutr Clin Pract 2013;3: 118–25. Moreno GJ, Valdez AP, de Sayve DM, Becerril RP, Larralde C, Sciutto E. Neurocysticercosis a persistent health problem in México. PLoS Negl Trop Dis 2010;4:e805. Pica-Mattocia L, Cioli D. Sex- and stage-related sensitivity of Schistosoma mansoni to in vivo and in vitro praziquantel treatment. Int J Parasitol 2004;34:527–33. Doenhoff MJ, Cioli D, Utzinger J. Praziquantel: mechanisms of action, resistance and new derivatives for shistosomiasis. Curr Opin Infect Dis 2008;21:659–67. Valentim CLL, Cioli D, Chevalier FD, et al. Genetic and molecular basis of drug resistance and species-specific drug action on Schistosoma parasites. Sci 2013;342: 1385–9.

J.J. Martínez-González et al. / Parasitology International 64 (2015) 194–201 [65] Coles GC, Stafford KA. Activity of oxyclozanide, nitroxynil, clorsulon and albendazole against adult triclabendazole-resistant Fasciola hepatica. Vet Rec 2001;148:723–4. [66] Mottler L, Alvarez L, Fairweather I, Lanusse C. Resistance-induced changes in triclabendazole transport in Fasciola hepatica: ivermectin reversal effect. J Parasitol 2006;92:1355–60. [67] Martínez-Valladares M, Famularo N, Férnandez-Pato N, Castañon-Ordóñez I, Cordero-Pérez C, Rojo-Vázquez FA. Efficacy of nitroxynil against Fasciola hepatica resistant to triclabendazole in a naturally infected sheep flock. Parasitol Res 2010;107: 1205–11. [68] Chai JY. Praziquantel treatment in trematode and cestode infections: An update. J Infect Chemother 2013;45:32–45. [69] Zanetti WD, et al. Historic of therapeutic efficacy of albendazol sulphoxide administered in different routes, dosages and treatment schemes, against Taenia saginata cysticercus in cattle experimentally infected. Exp Parasitol 2014;137:14–20. [70] González AE, Bustos JA, Jiménez JA, Rodríguez MI, Ramírez MG, Gilman RH, et al. Efficacy of diverse antiparasitic treatments for cysticercosis in the pig model. Am J Trop Med Hyg 2012;87:292–6. [71] Huang H, Day L, Cass C, Ballou DP, Williams DL. Investigation of the catalytic mechanism of thioredoxin glutathione reductase from Schistosoma mansoni. Biochemistry 2011;50:5870–82. [72] Ross F, Hernandez P, Porcal W, López GV, Cerecetto H, González M, et al. Identification of thioredoxin glutathione reductase inhibitors that kill cestode and trematode parasites. PLoS One 2012;7:1–7.

201

[73] Kuntz AN, Davioud-Charvet E, Sayed AA, Califf LL, Dessolin J, Arnér ES, et al. Thioredoxin glutathione reductase from Schistosoma mansoni: an essential parasite enzyme and a key drug target. Plos Med 2007;4:e264. [74] Martínez-González JJ, Guevara-Flores A, Álvarez G, Rendón GJL, del Arenal IP. vitro killing action of auranofin on Taenia crassiceps metacestode (cysticerci) and inactivation of thioredoxin-glutathione reductase (TGR). Parasitol Res 2010:227–31. [75] Sigler JW. Parenteral gold in the treatment of rheumatoid arthritis. Am J Med 1983; 75:59–62. [76] Blodgett RC. Auranofin: experience to date. Am J Med 1983;75:86–9. [77] Kanzok SM, Fechner A, Bauer H, Ulschmid JK, Müller H, Botella-Muñoz J, et al. Substitution of the thioredoxin system for glutathione reductase in Drosophila melanogaster. Science 2001;291:643–6. [78] Weichsel A, Kem M, Montfort WR. Crystal structure of human thioredoxin revealing an unraveled helix and exposed S-nitrosation site. Protein Sci 2010;19:1801–6. [79] Wang X, Ling S, Zhao D, Sun Q, Li Q, Wu F, et al. Redox regulation of actin by thioredoxin-1 is mediated by the interaction of the proteins via cysteines 62. Antioxid Redox Signal 2010;13:565–73. [80] Katti SK, LeMaster DM, Exlund H. Crystal structure of thioredoxin from Escherichia coli at 1.68 A resolution. J Mol Biol 1990;212:167–84. [81] Sandalova T, Zhong L, Lindqvist Y, Holmgren A, Schneider G. Three-dimensional structure of a mammalian thioredoxin reductase: implications for mechanism and evolution of a selenocysteine-dependent enzyme. Proc Natl Acad Sci 2001;98: 9533–8.