Identification and Functional Characterization of Thioredoxin from Trypanosoma brucei

Trypanosomes and Leishmania, the causative agents of several tropical diseases, lack the glutathione/gluta-thione reductase system but have trypanothione/ trypanothione reductase instead. The uniqueness of this thiol metabolism and the failure to detect thioredoxin reductases in these parasites have led to the suggestion that these protozoa lack a thioredoxin system. As pre-sented here, this is not the case. A gene encoding thioredoxin has been cloned from Trypanosoma brucei , the causative agent of African sleeping sickness. The single copy gene, which encodes a protein of 107 amino acid residues, is expressed in all developmental stages of the parasite. The deduced protein sequence is 56% identical with a putative thioredoxin revealed by the genome project of Leishmania major . The relationship to other thioredoxins is low. T. brucei thioredoxin is unusual in having a calculated pI value of 8.5. The gene has been overexpressed in Escherichia coli . The recombinant protein is a substrate of human thioredoxin reductase with a K m value of 6 m M but is not reduced by trypanothione reductase. T. brucei thioredoxin catalyzes the reduction of insulin by dithioerythritol, and functions as an electron donor for T. brucei ribonucleotide reductase. The parasite protein is the first classical thioredoxin G)CAIGGICC(A/G)C) was derived from the active site consensus se- quence of thioredoxins. PCR 2 amplification with this sequence-specific primer and the spliced leader primer (5 9 -TAGAACCAGTTTCTGTAC- TATATTG) yielded the 5 9 -end of the gene ( Taq -polymerase; 94 °C, 2 min; 94 °C, 30 s; 50 °C, 30 s; 72 °C, 2 min; 30 cycles; 72 °C, 5 min). The 3 9 -end was amplified by PCR using a perfect match primer (5 9 -GGATC-CGGGGAAAATGTCGGTAGTGG) and an oligo(T) primer (94 °C, 2 min; 94 °C, 30 s; 50 °C, 30 s; 72 °C, 3 min; 30 cycles; 72 °C, 5 min). The coding region was again amplified from the cDNA with Pfu polymerase (94 °C, 2 min; 94 °C, 30 s; 50 °C, 30 s; 72 °C, 3 min; 30 cycles; 72 °C, 5 min) using the 5 9 -perfect match primer described above together with a

Trypanosomes and Leishmania, the causative agents of several tropical diseases, lack the glutathione/glutathione reductase system but have trypanothione/ trypanothione reductase instead. The uniqueness of this thiol metabolism and the failure to detect thioredoxin reductases in these parasites have led to the suggestion that these protozoa lack a thioredoxin system. As presented here, this is not the case. A gene encoding thioredoxin has been cloned from Trypanosoma brucei, the causative agent of African sleeping sickness. The single copy gene, which encodes a protein of 107 amino acid residues, is expressed in all developmental stages of the parasite. The deduced protein sequence is 56% identical with a putative thioredoxin revealed by the genome project of Leishmania major. The relationship to other thioredoxins is low. T. brucei thioredoxin is unusual in having a calculated pI value of 8.5. The gene has been overexpressed in Escherichia coli. The recombinant protein is a substrate of human thioredoxin reductase with a K m value of 6 M but is not reduced by trypanothione reductase. T. brucei thioredoxin catalyzes the reduction of insulin by dithioerythritol, and functions as an electron donor for T. brucei ribonucleotide reductase. The parasite protein is the first classical thioredoxin of the order Kinetoplastida characterized so far.
Thioredoxins are small ubiquitous proteins with a molecular mass of about 12000 and a conserved redox active Cys-Gly-Pro-Cys motif. The proteins function in a wide variety of cellular processes (1). The first elucidated role was as donor of reducing equivalents for ribonucleotide reductase (2). In higher organisms, the thioredoxin system, composed of thioredoxin, NADPH, and thioredoxin reductase, seems to be a general dithiol-disulfide oxidoreductase. Thioredoxin also provides reducing equivalents for thioredoxin peroxidase. This system, which catalyzes the reduction of hydrogen peroxide and organic peroxides, is widely distributed in nature (3).
Trypanosomes and Leishmania are the causative agents of severe tropical diseases, examples being African sleeping sickness (Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense), Nagana cattle disease (Trypanosoma congolense and Trypanosoma brucei brucei), Chagas' disease (Trypanosoma cruzi), and the three manifestations of leishmaniasis (Leishmania donovani, Leishmania major, Leishmania mexicana). All these parasitic protozoa have a thiol metabolism that completely differs from that of other eukaryotes and prokaryotes. They lack the glutathione/glutathione reductase system as well as glutathione peroxidase and catalase. Trypanothione (N 1 ,N 8 -bis(glutathionyl)spermidine) and monoglutathionylspermidine are the main low molecular mass thiols (4,5). These glutathionylspermidine conjugates are kept in the reduced state by trypanothione reductase and NADPH. The dithiol trypanothione has been shown to be involved in the detoxification of hydroperoxides (6), homeostasis of ascorbate (7), as well as the synthesis of deoxyribonucleotides catalyzed by ribonucleotide reductase (8). 1 Enzymes of the trypanothione metabolism are attractive target molecules for the rational development of new antiparasitic drugs (for a recent review see Ref. 5). The uniqueness of the trypanothione metabolism and the failure to detect thioredoxin reductases in trypanosomatids have led to the suggestion that these protozoa lack a thioredoxin system (4). Recently the genome sequencing project of L. major revealed a sequence that probably codes for a thioredoxin (9). Based on this observation we have cloned and overexpressed a gene encoding thioredoxin from Trypanosoma brucei. As shown here, the protein is a classical thioredoxin with several unusual properties.

EXPERIMENTAL PROCEDURES
Materials-Escherichia coli thioredoxin was purchased from Calbiochem, and bovine pancreas insulin was from Sigma. A sample of human thioredoxin reductase was a kind gift of Drs. Katja Becker and R. Heiner Schirmer, Heidelberg. The plasmids of the two genes of T. brucei ribonucleotide reductase were kindly provided by Drs. Anders Hofer and Lars Thelander, Umeå, Sweden. Recombinant T. brucei tryparedoxin (8), T. cruzi trypanothione reductase (10,11), and T. brucei ribonucleotide reductase (12)(13)(14) were prepared as described. Polyclonal rabbit antibodies against the recombinant T. brucei thioredoxin were produced by Eurogentec.
Expression of the Gene and Purification of the Recombinant Protein-Competent E. coli SG 13009 cells were transformed with the pQE 32/trx plasmid. A 5-ml overnight culture was diluted 1:100 in 2ϫ YT medium (Life Technologies, Inc.) containing 100 g/ml carbenicillin and 100 g/ml kanamycin, and the cells were grown at 30°C to an A 600 of 0.5. Expression was induced by 1 mM isopropyl-␤-D-thiogalactopyranoside overnight at 30°C. After centrifugation the cells were suspended in 40 ml of buffer A (50 mM sodium phosphate, pH 7.0, 10 mM 2-mercaptoethanol) containing 5 mM imidazole, 150 nM pepstatin, 4 nM cystatin, and 20 M phenylmethylsulfonyl fluoride and disintegrated by sonication. After centrifugation the supernatant was applied onto a 13-ml nickel-nitrilotriacetic acid Superflow-Sepharose column (Qiagen) and washed with 60 ml of buffer A containing 20 mM imidazole followed by 300 ml of 40 mM imidazole in buffer A. The His-tagged T. brucei thioredoxin was eluted with 500 mM imidazole in buffer A.
Insulin Reduction Assay-A fresh solution of 1 mg/ml insulin was prepared in 100 mM potassium phosphate, 1 mM EDTA, pH 7.0 (16,17). The assay mixture contained in a total volume of 800 l of 100 mM potassium phosphate, 1 mM EDTA, pH 7.0, 130 M insulin, 500 M DTE, and varying amounts of T. brucei and E. coli thioredoxin, respectively. The increase in turbidity because of formation of the insoluble insulin B chain was followed at 650 nm and 30°C. The control assay contained insulin and DTE but lacked thioredoxin.
Ribonucleotide Reductase Assay-Ribonucleotide reductase activity was determined from the rate of reduction of [ 3 H]GDP to dGDP essentially as described for CDP reduction (18). The reaction mixture contained in a total volume of 200 l of 50 mM Hepes, pH 7.6, 500 M [ 3 H]GDP, 100 M dTTP, 100 mM KCl, 6.4 mM MgCl 2 , 1 mM DTE, and varying amounts of T. brucei thioredoxin, T. brucei tryparedoxin, and E. coli thioredoxin, respectively. The reaction was started by adding T. brucei ribonucleotide reductase, and the assay mixture was incubated 20 min at 37°C. After dephosphorylation educts and products were separated by high performance liquid chromatography (19).
Cultivation of T. brucei in Mice-NMRI mice were infected intraperitoneally with the pleomorphic T. brucei clone AnTat 1.1 (22). After 3 (long slender) and 5 days (short stumpy) trypanosomes were isolated from the blood by chromatography on DEAE-cellulose (24).
Isolation of Genomic DNA and Southern Blot-Genomic DNA from a culture of bloodstream TC221 T. brucei cells as well as from T. cruzi were prepared as described (25,26). 10 g of DNA was digested with StyI, and the fragments were separated on a 0.7% agarose gel. The DNA was blotted onto a Hybond TM -Nϩ membrane (Amersham Pharmacia Biotech) by capillary transfer and hybridized with the digoxigeninlabeled trx gene. Southern blot analysis was performed as described (27) using the DIG High Prime DNA labeling and detection kit (Roche Molecular Biochemicals).
Isolation of T. brucei Total RNA and Reverse Transcriptase-PCR-Total RNA of procyclic, long slender, and short stumpy T. brucei was isolated by guanidinium isothiocyanate and phenol extraction as described (28). 1.5 g of RNA was reverse transcribed and amplified using the ready to go reverse transcriptase-PCR beads (Amersham Pharmacia Biotech). The spliced leader primer 5Ј -TAGAACCAGTTTCTGTAC-TATATTG and the gene-specific primer 5Ј-TGTGCCCCTTTTCTCCG-GAACTC were used for first strand cDNA synthesis and the following amplification. 1/25 of the PCR reaction mixture was separated on a 1.5% agarose gel and hybridized in a Southern blot as described above.

RESULTS
Cloning and Sequencing of a Thioredoxin Gene from T. brucei-The gene encoding thioredoxin has been cloned from cDNA of long slender bloodstream forms of T. brucei brucei (Fig. 1). The 5Ј-region was amplified by PCR using a sequencespecific reverse primer deduced from the highly conserved active site WCGPC motif and a spliced leader primer, which is the very 5Ј-end of all trypanosomal mRNAs added by transsplicing. The spliced leader addition site is found 195 nucleotides from the start codon. The complete coding region of the gene together with the 3Ј-untranslated region was amplified by PCR using a primer starting with the initial ATG together with a poly(T) primer. Two products of about 530 and 600 base pair length were obtained. The cDNA clones were identical in the coding sequence but differed in the length of the 3Ј-untranslated region (Fig. 1), the larger fragment containing an additional 77 base pairs preceding the poly(A) stretch. Diversity in the 3Ј-noncoding region is common in trypanosomes as no specific polyadenylation signal is known (for a review see Ref. 29).
FIG. 1. Nucleotide sequence and deduced protein sequence of T. brucei thioredoxin. Two clones, which covered the coding region but differed in the length of the 3Ј-untranslated region, were amplified by PCR from cDNA of bloodstream parasites. The last nucleotide of the shorter clone is indicated by a bold G.
T. brucei trx is most probably a single copy gene. Digestion of genomic DNA with StyI, which does not cut within the gene but digests DNA into relatively small fragments, yielded a single fragment of about 1200 base pairs (Fig. 2). Hybridization of StyI-digested genomic DNA from T. cruzi did not allow detection of a trx-encoding fragment in the related trypanosomatid.
Structural Comparison of T. brucei Thioredoxin with Other Thioredoxins-The gene of T. brucei thioredoxin encodes a protein of 107 amino acid residues and a M r of 12,000 with a calculated pI value of 8.5. The protein displays 56% identity with a putative thioredoxin of L. major (9), which reflects the close phylogenetic relationship of the two organisms. The overall similarities to other thioredoxins range from 21% with the E. coli to 33% identities with thioredoxin I from yeast (Fig. 3). Classical thioredoxins are characterized by a WCGPC sequence in the active site. This motif is also present in the T. brucei thioredoxin sequence and clearly distinguishes the protein from tryparedoxin, another small T. brucei dithiol protein with a WCPPC sequence (8). Besides the active site motif, a few, mostly single, positions are conserved throughout thioredoxin sequences (36). They also occur in the trypanosomatid proteins (Fig. 3). One of these residues is Pro-73 (76 in E. coli thioredoxin), which forms a cis peptide bond stabilizing the protein (37,38). Despite the generally low sequence similarities within this protein family the folding of thioredoxins is highly conserved, which allows sequence comparisons in light of the known three-dimensional structures of the E. coli and human proteins. In human thioredoxin, the dimer interface is mainly formed by Trp-30, Val-58, Ala-65, and Met-73 of each monomer (39). Another hydrophobic area composed of Gly-33, Pro-34, Ile-75, Pro-76, plus Val-91, Gly-92, and Ala-93 has been suggested to be involved in binding E. coli thioredoxin to other proteins (37). Strictly hydrophobic residues at the respective positions are also found in the parasite proteins (Fig. 3).
Functional Characterization of T. brucei Thioredoxin-From a 1-liter culture of recombinant E. coli cells about 15 mg of T. brucei thioredoxin have been purified as a fusion protein carrying a 19-residue-long N-terminal extension with 6 His residues at the very end. The protein is Ͼ95% pure (Fig. 4). Prolonged storage can lead to the formation of covalent dimers. SDS-polyacrylamide gel electrophoresis of the stored protein sample without thiols in the sample buffer shows an additional band with a molecular mass of about 28,000, whereas under reducing conditions a single protein band of 13,000 is obtained (Fig. 4). Immunoblot analysis using the first rabbit immune serum detected both bands (not shown) in accordance with the higher molecular weight species representing a dimer. Because in T. brucei thioredoxin Cys-67 is the only cysteine in addition to the active site couple, one may speculate that it is involved in dimer formation. Mammalian thioredoxins possess two additional conserved cysteine residues corresponding to Cys-68 and Cys-72 in the human protein (Fig. 3). Oxidized and reduced human thioredoxin form inactive dimers, which contain an intermolecular disulfide bridge between Cys-72 from each monomer (39,40). Cys-67 in T. brucei and L. major thioredoxins corresponds to Cys-68 in the human protein (40), which has been shown to be rather buried in the protein structure (40,41). Yeast thioredoxin III also contains a cysteine residue at this position but dimer formation has not been observed (42). Future work should reveal the molecular structure of the dimeric protein species.
T. brucei Thioredoxin Is a Substrate of Human Thioredoxin Reductase-Thioredoxin reductases catalyze the NADPH-dependent reduction of thioredoxin disulfide to the dithiol. In the spectrophotometric assay reduction of thioredoxin is followed in the presence of excess insulin ensuring the constant reoxidation of thioredoxin-(SH) 2 formed. In contrast to the bacterial enzymes, mammalian thioredoxin reductases show broad specificities for their disulfide substrates and accept thioredoxins from other species. T. brucei thioredoxin is readily reduced by human thioredoxin reductase. The K m value of 6 M derived from a Lineweaver-Burk plot (not shown) compares well with those for human and E. coli thioredoxin, which are 4.3 and 20 M, respectively (43). To test if trypanothione reductase is able to reduce thioredoxin, we replaced human thioredoxin reductase by T. cruzi trypanothione reductase in the NADPH/thioredoxin/insulin assay (data not shown). No activity could be detected in agreement with thioredoxin not being a substrate for trypanothione reductase.
T. brucei Thioredoxin Catalyzes Reduction of Insulin-The two interchain disulfides of insulin are substrates of thioredoxins. Reduction of the disulfide bonds generates the free A and B chains of insulin, and precipitation of the insoluble B chain is measured by the increase in turbidity (16). The reduction of insulin by DTE was followed at pH 7.0 in the absence and presence of T. brucei and E. coli thioredoxin (Fig. 5). The maximal rates of precipitation measured as ⌬A 650 /min were very similar for both thioredoxins yielding a relative specific activity of 3.6 ⌬A 650 ϫ min Ϫ1 mg Ϫ1 of protein. The activities measured here are twice as high as those for E. coli and human thioredoxin reported previously (17), which may be because of slightly different assay conditions. Taken together, the data show that the three thioredoxins behave very similar in their insulin reduction capacity.
T. brucei Thioredoxin Is an Electron Donor for Ribonucleotide Reductase-Ribonucleotide reductase catalyzes the reduction of ribonucleotides to the respective deoxyribonucleotides. The reaction is dependent on the presence of thiols, physiological electron donors being the thioredoxin or glutaredoxin systems (44). In vitro DTE can function as the reductant (18), and an increase of the reaction rate is a measure for thioredoxin or glutaredoxin activities. Formation of [ 3 H]dGDP by T. brucei ribonucleotide reductase was followed in the presence of 1 mM DTE and different concentrations of dithiol proteins. Under these conditions, the increase of ribonucleotide reductase activity caused by thioredoxin is not very pronounced because at high concentrations DTE can act as a direct hydrogen donor for the enzyme (Fig. 6). Nevertheless, T. brucei thioredoxin clearly stimulates the reaction, and the activation is comparable to that caused by E. coli thioredoxin and T. brucei tryparedoxin (8).
The trx Gene Is Expressed in All Developmental Stages of the Parasite-Total RNA was isolated from long slender and short stumpy bloodstream T. brucei grown in mice. RNA of the procyclic insect form was obtained from cell culture. Northern blot analyses using the digoxigenin-labeled coding region of the trx gene as probe did not allow visualization of the thioredoxin mRNA. In comparison, the mRNA of T. brucei tryparedoxin was easily detected. 3 Therefore the trx mRNA was amplified by reverse transcriptase-PCR and hybridized with the trx gene. A fragment of the expected 560 base pairs was found in all three life stages of the parasite but the transcript obviously occurs in low abundancy (Fig. 7). DISCUSSION African trypanosomes possess a classical thioredoxin. The T. brucei protein is the first thioredoxin of an organism belonging to the order Kinetoplastida that has been characterized to date. Phylogenetically the thioredoxins of T. brucei and L. major form a new branch distinct from all other eukaryotic lineages, whereby the parasite proteins are more closely related to mammalian thioredoxins than those of yeasts and plants (not shown).
One of the best studied functions of thioredoxin is the delivery of reducing equivalents for the synthesis of deoxyribonucleotides catalyzed by ribonucleotide reductase. In many organisms the physiological electron donors are the thioredoxin and glutaredoxin systems. The disulfide form of thioredoxin generated in the reaction is then reduced by NADPH and thioredoxin reductase. In the case of glutaredoxin the dithiol is regenerated spontaneously by glutathione, and the glutathione disulfide formed is subsequently reduced by glutathione reductase at the expense of NADPH (1). The replacement of glutathione reductase by trypanothione reductase in trypanosomatids raised the question as to the donors of reducing equivalents for the parasite ribonucleotide reductase. As shown here, both T. brucei thioredoxin and tryparedoxin (8) catalyze the reduction of T. brucei ribonucleotide reductase by DTE as efficiently as E. coli thioredoxin (Fig. 6). 1 Thus, most probably trypanosomes have developed two systems that provide electrons for the synthesis of DNA precursors as it is the case in other organisms (1).
Interestingly, T. brucei and L. major thioredoxins lack the highly conserved Asp-26, which in E. coli thioredoxin has been shown to play a crucial role for catalytic activity. It is the only acidic residue not localized on the surface of the protein (41), and mutation to an Ala increased the K m value for thioredoxin reductase by a factor of 10. In addition, the mutant E. coli protein had a drastically lowered ability to serve as a hydrogen donor for ribonucleotide reductase (38). T. brucei thioredoxin is an excellent substrate of human thioredoxin reductase, and like E. coli, thioredoxin is able to deliver the electrons for T. brucei ribonucleotide reductase. These findings indicate that in the parasite thioredoxins an acidic residue at this position is not essential for catalysis.
African trypanosomes change between three main life stages. In the blood of the mammalian host the parasites occur as dividing long slender and nondividing short stumpy forms. Upon a blood meal on an infected animal the tsetse fly takes up parasites and the short stumpy cells differentiate to procyclics, which multiply in the insect vector. The thioredoxin gene is expressed in all three developmental stages of T. brucei. The occurrence of the mRNA in the nondividing short stumpy parasites may indicate that the protein is not only involved in deoxyribonucleotide synthesis but serves additional purposes. Of course, it cannot be excluded that the mRNA synthesized in long slender parasites is highly stable and thus is still present in the short stumpy stage.
Another important function of thioredoxins is to provide reducing equivalents for the detoxification of hydroperoxides by thioredoxin peroxidases. The thioredoxin peroxidases of yeast (3) and mammals (45) and the alkyl hydroperoxide reductases of bacteria (46) form the large family of peroxiredoxins found in all phyla. In trypanosomatids a unique cascade composed of trypanothione/trypanothione reductase/tryparedoxin/tryparedoxin peroxidase has been shown to detoxify hy- droperoxides (6,(47)(48)(49). The parasite peroxidase is a member of the peroxiredoxin family of proteins. Thus future work will show if tryparedoxin peroxidase also accepts electrons from the parasite thioredoxin and how the dithiol form of thioredoxin is subsequently regenerated.
T. brucei thioredoxin is rather unique in having a calculated pI value of 8.5. The protein contains several arginine residues resulting in an overall positive charge. Nearly all thioredoxins studied so far are acidic proteins with pI values between 4.5 and 5.0. Very recently another highly basic thioredoxin III has been described in mitochondria of Saccharomyces cerevisiae (42). The sequence of T. brucei thioredoxin is slightly more similar to this thioredoxin than to the cytosolic thioredoxins I and II of yeast. So far it is not known if the pronounced charge differences correlate with distinct functions or the localization of the proteins. The T. brucei sequence does not show an Nterminal extension that could serve as a mitochondrial import signal. In the putative L. major thioredoxin, most of the basic residues found in the T. brucei protein are conserved, but the protein has a theoretical pI value of 5.4 (9).
T. brucei thioredoxin is a substrate of human thioredoxin reductase but is not reduced by trypanothione reductase, which strongly suggests the presence of a thioredoxin reductase. From a phylogenetic point of view it will be highly interesting which kind of thioredoxin reductase Kinetoplastida have because two completely different types of enzymes have been realized in nature. Procaryotes, yeast, and the protozoan parasite Giardia lamblia (50) possess small homodimeric proteins with a subunit molecular mass of about 35,000 (51). Mammalian thioredoxin reductases (52) are homodimeric selenoproteins consisting of two subunits of about 55,000. These enzymes carry a selenocysteine in their C-terminal dipeptide, which is involved in the enzymes' catalytic activity (53). The malarial parasite Plasmodium falciparum also possesses a large homodimeric enzyme but with two redox active cysteine residues in the C-terminal region (54), and as shown recently, the nematode Caenorhabditis elegans obviously possesses both types of large enzymes (55). The accessibility of recombinant T. brucei thioredoxin should now allow the characterization of the first trypanosomatid thioredoxin reductase. Disruption of the trx 1.5 g of total RNA was reverse transcribed and amplified by reverse transcriptase-PCR using a spliced leader primer and a genespecific primer of the trx 3Ј-end. The product was applied onto a 1.5% agarose gel, blotted, and visualized by hybridization with the digoxigenin-labeled trx gene. Lane 1, digoxigenin-labeled DNA size marker; lane 2, procyclic culture form; lane 3, long slender bloodstream parasites isolated from mice; lane 4, short stumpy bloodstream parasites isolated from mice. bp, base pairs. gene in T. brucei is in progress to reveal if thioredoxin is essential for the viability and virulence of the parasite.