Characterization of a Thioredoxin-1 Gene from Taenia solium and Its Encoding Product

Taenia solium thioredoxin-1 gene (TsTrx-1) has a length of 771 bp with three exons and two introns. The core promoter gene presents two putative stress transcription factor binding sites, one putative TATA box, and a transcription start site (TSS). TsTrx-1 mRNA is expressed higher in larvae than in adult. This gene encodes a protein of 107 amino acids that presents the Trx active site (CGPC), the classical secondary structure of the thioredoxin fold, and the highest degree of identity with the Echinococcus granulosus Trx. A recombinant TsTrx-1 (rTsTrx-1) was produced in Escherichia coli with redox activity. Optimal activity for rTsTrx-1 was at pH 6.5 in the range of 15 to 25°C. The enzyme conserved activity for 3 h and lost it in 24 h at 37°C. rTsTrx-1 lost 50% activity after 1 h and lost activity completely in 24 h at temperatures higher than 55°C. Best storage temperature for rTsTrx-1 was at −70°C. It was inhibited by high concentrations of H2O2 and methylglyoxal (MG), but it was inhibited neither by NaCl nor by anti-rTsTrx-1 rabbit antibodies that strongly recognized a ~12 kDa band in extracts from several parasites. These TsTrx-1 properties open the opportunity to study its role in relationship T. solium-hosts.


Introduction
Thioredoxin (Trx) is a small (∼12 kDa) enzyme that belongs to the reductase family. Trx reduces disulfides in several proteins using its conserved dithiol active site. It is ubiquitous and multifunctional; it is involved in processes such as maintenance of cellular homeostasis, cell proliferation, detoxification of peroxides (H 2 O 2 , hydroperoxides), DNA synthesis, signaling, and inhibition of apoptosis. Likewise it reduces diverse molecules of low molecular weights, such as glutathione disulfide, as well antioxidants dehydroascorbate, lipoic acid, and lipoamine [1][2][3][4]. All these events oxidize Trx, and it is reduced by thioredoxin glutathione reductase (TGR) and NADPH + H + ; these components form the thioredoxin system in platyhelminths [5].
Trx has been classified into cytosolic (Trx-1) and mitochondrial (Trx-2); the latter is synthesized with an additional N-terminal extension that targets the mitochondrial protein, where it is cleaved to yield the ∼12 kDa form [3]. All Trx enzymes have a similar structure, the Trx fold that is formed by a central domain with five-stranded -sheet, surrounded by four -helices, and the active site (CGPC), located between strand 2 and -helix 2 [3]. In Cestoda, Trx and TGR have been reported in Echinococcus granulosus and Taenia crassiceps. On the other hand, these organisms and Taenia solium possess a typical 2-Cys peroxiredoxin, which reduces H 2 O 2 and hydroperoxides to water and its corresponding alcohol using the thioredoxin system. This shows that these organisms are able to regulate hydroperoxides levels and repair enzymes inactivated by oxidative stress [5][6][7][8][9].
Neurocysticercosis is the most common parasitic brain disease worldwide; moreover the high relationship between epilepsy and neurocysticercosis is considered now as a "biological marker" of the social and economic development of a community [10]. No commercial vaccine exists to prevent this parasitic disease and the treatment relies on two drugs, albendazole and praziquantel, to which T. solium has started to develop resistance [11,12]. Therefore, the identification and biochemical characterization of new targets are important tools for development of vaccines or therapeutic drugs.
In this study, we describe the cloning and characterization of a gene that encodes a thioredoxin-1 from Taenia solium (TsTrx-1) and present a partial biochemical characterization of its encoding product.

Taenia solium Trx Gene and cDNA Isolation.
A Trx probe was generated by RT-PCR using the SuperScript One Step RT-PCR Kit (Invitrogen, Carlsbad, CA) with 1 g of T. solium larval total RNA prepared by TRIzol (Invitrogen, Carlsbad, CA) and two degenerated primers called TRX-1 and TRX-2 designed from the well conserved regions (TWCGPCK and MPTLFVFK) in Trx enzymes. The RT-PCR program for cDNA synthesis was 1 cycle at 50 ∘ C for 30 min, 30 cycles at 94 ∘ C for 1 min, 54 ∘ C for 30 sec, and 72 ∘ C for 1 min, and a final extension cycle at 72 ∘ C for 15 min. The fragment (probe) obtained was cloned into pCRII vector (Invitrogen), sequenced on an automated DNA sequencer ABI Prism model 373 (Perkin-Elmer, Applied Biosystem, Foster City, CA), and the nucleotide translation to amino acids sequence was analyzed with the PCGENE program. Screenings for T. solium cysticerci cDNA and genomic DNA libraries were carried out using 45,000 and 120,000 ZAPII phages, respectively. Both libraries were hybridized with the aforementioned probe, as previously described [8,9]. Phage positive clones obtained after three screening rounds of each library were converted to Bluescript plasmids using ExAssist helper phage (Stratagene, La Jolla, CA). Plasmids were sequenced and analyzed as before. Intron detection was carried out with the PCGENE and analyses of amino acid sequences were performed through BLAST (National Center for Biotechnology Information NCBI (http://www.ncbi.nlm.nih.gov/BLAST/)). Alignment of the multiple amino acid sequences was performed by Clustal X (http://www.clustal.org/). The proximal promoter analysis for detecting putative transcription binding sites was carried out with the TRANSFAC program (http://www.gene-regulation.com/pub/databases.html).

Transcription Start Site
Determination. Taenia solium larval total RNA (200 ng) was used as template for the transcription start site (TSS) determination using the Smart RACE cDNA Amplification Kit (Clontech Mountain View, CA). RACE fragments were amplified by PCR using reverse primer TRXRE-1 designed from the region DEMAKENAN (5 -GTTAGCATTCTCCTTTGCCATTTCGTC-3 ) and forward primer SMARTII from kit (5 -AAGCAGTGGTAT-CAACGCAGAGTACGCGGG-3 ) following manufacturer's directions. The resulting bands were cloned into pCRII (Invitrogen), sequenced, and compared with the results obtained with the neural network analysis tool (http://www .fruitfly.org/) to confirm transcription start site (TSS) found by the 5 -RACE method.

Transcripts Relative Expression.
For the real time-PCR, 3 g of total RNA from T. solium larval and adult stages was reverse-transcribed to cDNA using SMARTScribe Reverse Transcriptase and 5 -CDS primer A (Clontech) according to manufacturer's instructions. cDNA 200 ng was used for each reaction in a volume of 10 L using the primers TRX-X1 and TRX-X2 designed from the regions (MSVEAVV) and (IQANV-) of TsTrx-1. Primers SOZ-2 and SOZ-6 were designed on the regions (KHGFHVH) and (GNAGGR-) of T. solium Cu/Zn superoxide dismutase (TsCu/ZnSOD) [13]. The reactions were performed with LightCycler 480 SYBR Green I Master in the LightCycler 480 System (Roche, Germany). The real time-PCR program used was 95 ∘ C for 10 min and then 40 cycles at 95 ∘ C for 15 sec and 52 ∘ C for 1 min and 72 ∘ C for 30 sec. The mRNA levels of TsTrx-1 were normalized using the TsCu/ZnSOD as a housekeeping gene, and relative amounts of mRNA were calculated using the comparative CT method.

Purification of Recombinant TsTrx (rTsTrx-1).
Plasmid pRSET containing the cDNA coding region from TsTrx-1 was expressed on BL21(DE3) bacteria with 1 mM IPTG during 4 h. Bacteria were centrifuged at 10,000 ×g and the pellet was disrupted by sonication in a TrisED buffer (10 mM Tris, 1 mM EDTA, and 1 mM DTT, pH 7.5) plus 4 M urea. The supernatant was applied onto a Ni+ sepharose column (His Trap HP GE Healthcare) and eluted with TrisED plus urea using a linear gradient of imidazole (0, 50, 100, 200, 300, and 400 mM). Fractions containing high Trx activity were dialyzed in TrisED buffer and reloaded in the Ni+ sepharose column for a second purification process without urea. The Trx obtained was concentrated and proteins concentration was determined by the Lowry method. Purification process of rTsTrx-1 was visualized by 15% SDS-PAGE staining with Coomassie Brilliant Blue.

Production of Antibodies and Western Blot.
A 10-weekold New Zealand rabbit was immunized subcutaneously with 100 g of the purified recombinant enzyme plus 10 g of saponin as adjuvant. Immunizations were conducted on days 1, 15, and 30. Antisera were obtained one week after the third immunization.
For western blot analysis, 1 g/mm of rTsTrx-1, rTrx-E. coli, and human T-cell recombinant Trx (rTrx-human, Sigma-Aldrich, St. Louis. MO) and likewise 5 g/mm of parasites crude extract and T. solium cysticerci excretionsecretion antigens (E/S Ag), prepared as described in [14], were separated by 15% SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham Biosciences, Sweden). The membrane was blocked with 1% BSA in PBS containing 0.05% Tween 20 buffer and incubated for 2 h at room temperature with the anti-rTsTrx-1 serum (dilution 1 : 100). After 3 washes of 5 min with PBS containing 0.05% Tween, the membrane was incubated for 1 h with horseradish peroxidase-(HRP-) conjugated goat anti-rabbit antibody (1 : 2000). Bands recognized by the anti-rTsTrx-1 serum were visualized with 3,3 -diaminobenzidine (DAB) and H 2 O 2 as substrate; normal rabbit serum was used as negative control at the same dilution.  dithiothreitol (DTT)/insulin reduction method described by Holmgren [15]. Briefly thioredoxin reactions are coupled to DTT using insulin as the protein substrate. rTsTrx-1 or rTrx-E. coli at 1, 10, and 20 g were added to 160 M of insulin in PE buffer (100 mM potassium phosphate, pH 6.5, containing 2 mM EDTA and 1 mM DTT). Insulin reduction was monitored by measuring turbidity at 650 nm for 30 min in a spectrophotometer Ultrospec 3100 Pro (Amersham Biosciences). A sample without rTsTrx-1 was used as reference control.
Assays to determine the pH enzymatic stability were carried out with rTsTrx-1 dialyzed for 8 h in citrate buffer at pH of 3, 5, and 6, in PE buffer at pH of 6.5, 7.5, and in Tris buffer at pH of 8, 9, and 10. The thermal stability of the enzyme was assayed incubating rTsTrx-1 at temperatures between 15 and 100 ∘ C during 1, 3, and 24 h. In both assays the Trx residual activity was measured. In addition, the optimal storage temperature was analyzed by incubating the enzyme at temperatures ranging from 25 ∘ to −70 ∘ C during 1 to 28 days.
To determine whether concentrations of 0 to 8 mM of MG, 0 to 2 M of NaCl, 0 to 200 mM H 2 O 2 , and 1, 10, and 20 g of IgG fraction, coming from sera of rabbit immunized with rTsTrx-1 and normal rabbit serum (control), affected the TsTrx-1 activity, the enzyme was incubated in each one for 30 min at 37 ∘ C and the activity was measured as before. For all these assays 20 g of enzyme was used.

Isolation of cDNA and Gene Encoding TsTrx-1 and Its
Characterization. Through RT-PCR, using total RNA from larval T. solium and primers designed on two conserved regions from several Trxs, we obtained a ∼153 bp DNA fragment that evidenced high homology with Trx genes. This was used as probe to isolate the transcript and the coding TsTrx-1 gene by screening a genomic DNA and cDNA ZAPII libraries, respectively. Figure 1 shows the isolate genomic DNA sequence; it spans 771 bp and codes for the same Trx-1 as the cDNA. The 5 -RACE experiments on the proximal promoter of the TsTrx-1 gene showed that the transcription start site (TSS) corresponds to an adenine (A) located within the initiator (Inr, ACAATGC) sequence and mapped at 81 bp upstream of the translation start codon (ATG). Moreover, we identified a putative TATA box located at −19 pb and a GGCTGT motif (downstream promoter element, DPE) at +22 pb, both from the TSS. Additionally, putative binding sites for Nrf2 and XBP1 transcription factors were found at −32 bp and +14 bp, respectively (GenBank accession for TsTrx-1 gene is KM401604). The structural coding region for the TsTrx-1 gene spans over 770 pb. It has three exons split by two introns (intron I: 218bp length; intron II: 65bp length) that possess the donor-acceptor sites (NGT-AGN). Moreover, the putative binding sites for the splicing machinery U1 (intron I: 121 GTACGT 126 ; intron II: 495 GTATGG 500 ) and U2AF (intron I: 326 TTCGTCTCTTCAG 338 ; intron II: 547 CCTTCAATTATAG 459 ) were identified. Additionally, the putative branching point in each intron (intron I: 281 TGTCGAC 287 ; intron II: 522 TTTCTAT 528 ) was identified too. Figure 1 also shows the isolated cDNA with 427 bp with an open reading frame (ORF) from 31 (ATG) to 354 (TGA) bp that encodes for a protein with 107 amino acids with a theoretical molecular weight of 11,579 Da and pI of 4.39. It presents the motif (CGPC) that corresponds to the catalytic active site of Trx enzymes. Furthermore, a putative classic polyadenylation (AATAAA) site was located between 380 and 385 bp downstream of the stop codon (GenBank accession for TsTrx-1 cDNA is KM401605). Table 1 depicts a comparison of the coding structural region of the TsTrx-1 gene with Trx-1 genes from E. granulosus, Schistosoma mansoni, human, and mouse [16][17][18][19]. It shows that the Trx-1 genes of T. solium, E. granulosus, and S. mansoni have a similar size, in contrast to mammalian Trx-1 genes that are bigger. It also depicts that T. solium and E. granulosus present an identical structural coding region to that of the Trx-1 gene composed by three exons and two introns. Similarly, S. mansoni Trx-1 gene also presents three exons and two introns, which are slightly larger than T. solium and E. granulosus Trx-1 genes. In contrast, human and mouse genes present 5 exons and 4 introns with similar sizes between them, but introns are larger than those of cestodes. It is noteworthy that the second introns of TsTrx-1 and E. granulosus genes coincide with the second intron of S. mansoni and third intron of human and mouse Trx-1 genes. Figure 2 shows that TsTrx-1 mRNA was expressed higher in larvae than in adult, as determined by real time-PCR assays.
The comparison of the deduced amino acid sequence of TsTrx-1 with E. granulosus Trx revealed (Figure 3(a)) an 87.85% identity, followed by a 46.72% with S. mansoni. In contrast, a low identity, between 41.12 and 43.92%, was found with pig and human Trx-1, respectively. TsTrx-1 has the   two conserved cysteines (Cys34 and Cys37) in its active site and cysteine 64, which is conserved in cestodes and mammalian, but it is not presented in S. mansoni Trx-1. Cysteine 27 is shared only by T. solium and E. granulosus Trx-1; unfortunately, its function is still unknown. T. solium and E. granulosus Trx-1 as well as S. mansoni lack tyrosine 49 and cysteines 69 and 73, present in mammalians, which are involved in nitration, glutathionylation, and Snitrosylation and dimer formation [20]. Figure 3(b) shows a model constituted by a central domain with five-strandedsheet, surrounded by four -helices, showing the classical Trx fold and the active site (CGPC) located between strand 2 and -helix 2 [3].

Production and Characterization of rTsTrx-1. Escherichia
coli containing the expression vector pRSETB with the coding region for TsTrx-1 were induced with IPTG for 4 h; bacteria were centrifuged and the pellet was disrupted with 4 M urea. Because rTsTrx-1 was produced with six histidines in the amino terminal, the supernatant was through to a nickel affinity chromatography. Figure 4 Figure 5(a) shows the insulin reduction activity performed with concentrations of 1 to 20 g of rTsTrx-1 at room temperature; all showed detectable insulin precipitation, but velocity and quantity of insulin reduction were dependent on rTsTrx-1 concentration. The maximal rate of precipitation was obtained with 20 g (1.5 M); however, there are no significant differences between 10 and 20 g; even 5 g reduced by half the amount of insulin precipitated. Therefore, the 20 g concentration was used for the characterization assays. Similar results were obtained with rTrx-E. coli (control assay) which showed a better activity at 20 g. A control reaction without rTsTrx-1 enzyme was carried out in parallel. Figure 5(b) depicts the enzyme exhibiting a triangle-shaped curve showing high activity (more than 84%) over a broad range of pH, between 6 and 7.5, but the optimal pH was 6.5 in PE buffer. The same figure shows that the activity was maintained around 58% at pH of 3, 5, 8, and 9 and was lost completely at pH 10. Figure 6(a) shows the residual activity after exposing the rTsTrx-1 to different temperatures (15 to 100 ∘ C) at times (1, 3, and 24 h). The activity/temperature plots show a descending pattern. The enzyme maintained 100% activity for 3 h and lost it in 24 h at 37 ∘ C; at 55 ∘ C, 100% activity was maintained for 1 h; ∼50% activity was lost at 3 h and lost activity completetly in 24 h. Finally at 70 and 100 ∘ C, the enzyme lost ∼50%, ∼75%, and 100% of activity at 1, 3, and 24 h, respectively. As negatives controls, we used a similar reaction without the Trx enzyme at all times; as expected, there was no activity. Noteworthy is that the loss of activity was not reversible in any condition. To determine the best temperature to store the rTsTrx-1, assays such as those showed in Figure 6(b) were done. It shows that the enzyme stored at 25 ∘ C gradually lost 10%, 30%, and 60% of activity at 3, 7, and 14 days, respectively; at this same temperature, the enzyme lost the activity completely at 28 days. Decrease in activity at 15 ∘ C was of 10,33,42, and 70% at the tested times. Storage at 4 ∘ C induced a ∼10% activity loss at 14 days and activity loss of 50% at day 28, whereas storage at 20 ∘ C induced a gradual activity loss of 33%, 50%, 85%, and almost 100% between days 3 and 28; at −70 ∘ C, the enzyme presented the best stability; it lost only 9% activity at 3 days and ∼25% of activity between days 7 and 28.

Discussion
The analysis of the 5 -flanking region of the TsTrx-1 gene reveals putative sites for Nrf2 and XBP1 transcription factors; these sites are presented in promoters for typical 2-Cysperoxiredoxin of T. solium and Trx-1 from human genes and both factors are positive regulators of antioxidant genes under stress condition [9,16,[21][22][23]. This suggests Trx-1 gene in cestodes could be regulated in this way. On the other hand, a putative TATA box was found at −19 bp and a DPE putative site at +22 bp, both related to TSS. It is known that the TATA box usually appears at −30 bp and DPE at +28 to +32 bp, even in Taeniidae family genes [9,24,25], and, for these reasons, neither TATA box nor DPE in the TsTrx-1 gene is at a classical distance to be functional [26]. However, these assertions must be corroborated with functional studies. The comparison analysis of Inr sequences from the TsTrx-1 gene with other Inr sequences from genes of the Taeniidae family suggests conservation in this motif with similarities to the mammalian consensus Inr sequence YYANWYY [9,13,[24][25][26].
Comparison analyses of the coding region for the Trx-1 gene show that T. solium (0.605 kb) and E. granulosus (0.609 kb) were identical. Even S. mansoni Trx-1 gene was showed to have similar structure with three exons and two small introns; the composition of nucleotides and amino acids sequences is different. In contrast, they are different from the structure of human (∼17 kb) and mouse (13 kb) genes, which have five exons and four big introns. These differences between number and sequence of introns could be used to design specific primers for the diagnosis of cysticercosis/taeniasis caused by T. solium using PCR with cerebrospinal fluid (CSF) and human feces [27]. This point is important for epidemiological studies, because it would allow identifying active infection of carriers of this parasite, which will be easy to treat with antihelminthic drugs. Moreover, the difference in introns and the presence of an intron in the same position in different Trx-1 genes of different organisms suggest that it was present in the ancestor; however more detailed studies should be done to use intron position as marker of evolution [28].
The confrontation of cysticerci with the host immune response (inflammation) and oxidative stress in tissues with high oxygen, such as brain and muscle, in contrast to the adult stage that lives in the small intestine, where it is exposed less to these factors, could be the reason why RNA expression of the TsTrx-1 gene is higher in cysticerci than in adults.
The primary sequence shows a typical catalytic site (CGPC), which executes the oxidoreductase activity, and the typical thioredoxin fold; even more, it shows higher identity with Trx from E. granulosus, a cestode, less identity with S. mansoni, a trematode, and poor identity with pigs and humans (intermediate and accidental hosts of T. solium). Polyclonal antibodies produced against rTsTrx-1 strongly recognized a ∼12 kDa band in various Taenia species and F. hepatica and weakly in E. histolytica but did not recognize E. coli and human Trxs. These points suggest that specific regions of TsTrx-1 could be used in vaccination assays against T. solium. Noteworthy, TsTrx-1, E. granulosus, and S. mansoni Trx-1 have a cysteine at position 27 close to the cysteines of the active site; moreover they lack cysteines 69 and 73, which regulate the activity and biological functions of the mammalian Trx-1. These findings give rise to the following questions. Does cysteine 27 have a role in the catalytical activity? Are the helminths Trx-1 not regulated by posttranscriptional modifications, and which is the biological consequence of this? [20].
The rTsTrx-1 enzyme exerted its optimal reductase activity in a range of pH and temperature of 6.5 to 7.5 and 4 to 37 ∘ C, respectively. Likewise, it presented 100% activity in a buffer with 100 mM NaCl and lost it gradually in a buffer with concentrations higher than 250 mM NaCl, losing up to 50% activity at 2 mM NaCl. On the other hand, the best way to store this enzyme to not lose its activity was at −70 ∘ C for 28 days and at 4 ∘ C for 14 days. The known biochemical properties of the enzyme, such as pH, temperature, buffers, cofactors, salt concentration, additives as glycerol, and oxidants and inhibitors, let us preserve its activity, which will help to determine enzymatic mechanisms with inhibitors or observe its effect on cells or organisms in vitro and in vivo.
The presence of MG and ROS especially H 2 O 2 triggers oxidative stress, DNA damage, and apoptosis in cells. MG is a reactive carbonyl compound that causes glycation of proteins and is formed principally by glucose metabolism; it induces oxidative stress by inactivating antioxidant enzymes, such as Cu/Zn superoxide dismutase, glutathione peroxidase, and Trx reductase and decreases Trx protein level [29]. Activity of rTsTrx-1 was not affected by 2 mM of MG. On the other hand, ROS are continuously produced by the host's inflammation caused by the immune response. It is known that helminths lack catalase and present low activity of glutathione peroxidase; cysticerci of T. crassiceps (cestode) resist concentration of 2.5 mM of H 2 O 2 in vitro for 2.5 h. We observed that rTsTrx-1 enzyme resists 1 mM H 2 O 2 . Both findings indicate that rTsTrx-1 is highly resistant to oxidant molecules and, together with the 2-Cys-peroxiredoxins of T. solium, could constitute the hydroperoxides-regulating system in this parasite [8,9].
Trx is an essential component of the thioredoxin system, where it performs functions such as antioxidative, proteinreducing, and signal-transducing ones. In mammalian and helminths, the antioxidative activity is the most studied. However, there is evidence that Trx participates in signaling pathways, interacting with different proteins, to control processes such as development, proliferation, migration, apoptosis, inflammation, and metabolism [30]. No signal sequence was found on the TsTrx-1 gene; however Trx-1 was found in the cysticerci E/S Ag and several reports have observed that mammalian cells stimulated with lipopolysaccharide and viral infections are able to secrete Trx-1 [31,32]. In addition, now it is known that helminths constitutively secrete Trx-1 and molecules that are able to modify the immune response by altering the normal signaling of host immune cells, letting them drive Th2 immune response, which allows for their long term establishment, such as the cases of 2-Cysperoxiredoxins from S. mansoni and F. hepatica [33,34].
In conclusion, the Trx-1 tools presented here could help to perform studies to know the role that Trx-1 plays in the hostparasite relationship; likewise its antioxidant and biochemical properties could be used to inactivate TsTrx-1 by drug or by vaccine. Furthermore, its importance in immune signaling pathways could let us think of it as a therapeutical molecule to other diseases.