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

Abstract

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.

Figure 1

Genomic (gDNA) and complementary DNA (cDNA) nucleotides sequences and deduced protein from Taenia solium thioredoxin-1. Putative transcription factors sites (Nrf2, TBP, and XBP-1) are placed inside a box; DPE is in a white letter inside the grey box; TSS inside the Inr is underlined and signalized by an arrow. Start (ATG) and stop (TGA) codons are inside the box; donor (GT) and acceptor (AG) introns sequences are underlined. Putative branch point is underlined by a black bar; putative U1 and U2AF splicing binding sites are in grey inside a box; polyadenylation sites are indicated by asterisks (∗). Thioredoxin residues from the active site (CGPC) are highlighted in white on a black background.

Figure 2

Relative transcription of T. solium thioredoxin-1 (TsTrx-1) gene from larvae and adult stages of T. solium was done by real time-PCR using TRX-X1 and TRX-X2 primers.

Figure 3

(a) Alignment of Trx-1 from Taenia solium (Ts. GenBank accession KM401605) with other thioredoxins from Echinococcus granulosus (Eg. GenBank: AF034637.1), Schistosoma mansoni (Sm. GenBank: AAL79841.1), Sus scrofa (Ss. GenBank NM_214313.2), and Homo sapiens (Hs. GenBank AF085844.1). Identical residues are highlighted in white on a black background. The symbols in the residues indicate (-) absence and (:) homology. Tyrosine 49 where nitration occurs (■) in mammalian. Cysteines: (▲) from active site, (○) conserved cysteine in mammalian and helminths, and (⧫) only present in mammalians where S-nitrosylation occurs; likewise (⧫) it is involved in glutathionylation. In a box is the active site and underlined are the residues used for primer design to produce the Trx-1 probe. Secondary structure elements are shown above the alignment. (b) Structure model of TsTrx-1. It shows the Trx fold formed by a central domain with five-stranded β-sheet, surrounded by four α-helices. The model was drawn with the Swiss Model program (http://swissmodel.expasy.org/).

Figure 4

Purification process of the recombinant T. solium thioredoxin-1 (rTsTrx-1) and specificity of rabbit anti-TsTrx-1 serum. (a) 15% SDS-PAGE showing the crude extract of Escherichia coli produced with 4 M urea induced with IPTG, 1: after and 2: before. Crude extract was run through the nickel chelator column; 3: wash column. Eluted fractions with imidazole at 4: 50 mM, 5: 100 mM, 6: 200 mM, and 7: 400 mM. Eluted fractions from 100 and 200 mM were mixed and dialyzed and run through the same column to obtain 8: a pure rTsTrx-1. (b) Western blot showing the reaction from anti-TsTrx-1 serum with 1: pure rTsTrx-1 and crude extracts from 2: Taenia solium larvae, 3: T. solium adult, 4: T. saginata adult, 5: T. taeniaeformis adult, 6: T. crassiceps larvae, 7: Hymenolepis diminuta adult, 8: Fasciola hepatica adult, 9: Entamoeba histolytica, 10: E. coli, and 11: Homo sapiens recombinant Trxs. 12: a preimmune serum was incubated with a crude extract of T. solium larvae as a negative control. 13: T. solium cysticerci E/S Ag. Strips 14 and 15 show the rTrx-E. coli and Trx-Homo sapiens stained with Ponceau red. Molecular mass standards are indicated in the middle of both figures.

Figure 5

(a) Thioredoxin-catalyzed reduction of insulin. The increase in turbidity at 650 nm is plotted against the 1, 10, and 20 μg of E. coli rTrx (dark bars) and 5, 10, and 20 μg of Taenia solium thioredoxin-1 (rTsTrx-1, grey bars). (C−) Control lacking rTsTrx-1. (b) The pH enzymatic stability was determined incubating 20 μg of rTsTrx-1 at different pH between 3 and 10. The residual activity was measured as before.

Figure 6

Effect of temperature on T. solium thioredoxin-1 (rTsTrx-1) activity. (a) rTsTrx-1 was incubated for 1, 3, and 24 hours at 15°C, 25°C, 37°C, 55°C, 70°C, and 100°C. (C−) Control lacking rTsTrx-1. (b) rTsTrx-1 was incubated during 3 to 28 days at 25°C, 15°C, 4°C, −20°C, and −70°C. (C+) Control of enzymatic activity was performed with a freshly made rTsTrx-1. Residual activity was determined by reduction of insulin assay.

Figure 7

Effect on T. solium thioredoxin-1 (TsTrx-1) after incubation with different concentrations of (a) NaCl (25–2 M), (b) methylglyoxal (MG, 0.05–8 mM), (c) H₂O₂ (10 μM–200 mM), and (d) IgG rabbit anti-T. solium Trx-1 and IgG from preimmune serum (1, 10, and 20 μg). (C−) Control lacking rTsTrx-1 and (C+) control of enzymatic activity was performed with a freshly made rTsTrx-1.