Plasmoredoxin, a novel redox-active protein unique for malarial parasites

Authors


  • Note: K.B. and S.M.K. contributed equally to this work.

K. Becker, Interdisciplinary Research Center, Heinrich-Buff-Ring 26–32, Justus-Liebig-University, D-35392 Gießen, Germany, Fax: + 49 641 9939129; Tel.: + 49 641 9939120. E-mail: becker.katja@gmx.de

Abstract

Thioredoxins are a group of small redox-active proteins involved in cellular redox regulatory processes as well as antioxidant defense. Thioredoxin, glutaredoxin, and tryparedoxin are members of the thioredoxin superfamily and share structural and functional characteristics. In the malarial parasite, Plasmodium falciparum, a functional thioredoxin and glutathione system have been demonstrated and are considered to be attractive targets for antimalarial drug development.

Here we describe the identification and characterization of a novel 22 kDa redox-active protein in P. falciparum. As demonstrated by in silico sequence analyses, the protein, named plasmoredoxin (Plrx), is highly conserved but found exclusively in malarial parasites. It is a member of the thioredoxin superfamily but clusters separately from other members in a phylogenetic tree. We amplified the gene from a gametocyte cDNA library and overexpressed it in E. coli. The purified gene product can be reduced by glutathione but much faster by dithiols like thioredoxin, glutaredoxin, trypanothione and tryparedoxin. Reduced Plrx is active in an insulin-reduction assay and reduces glutathione disulfide with a rate constant of 640 m−1·s−1 at pH 6.9 and 25 °C; glutathione-dependent reduction of H2O2 and hydroxyethyl disulfide by Plrx is negligible. Furthermore, plasmoredoxin provides electrons for ribonucleotide reductase, the enzyme catalyzing the first step of DNA synthesis. As demonstrated by Western blotting, the protein is present in blood-stage forms of malarial parasites.

Based on these results, plasmoredoxin offers the opportunity to improve diagnostic tools based on PCR or immunological reactions. It may also represent a specific target for antimalarial drug development and is of phylogenetic interest.

Abbreviations
BSA

bovine serum albumin

GR

glutathione reductase

Grx

glutaredoxin

GSH

glutathione, reduced

GSSG

glutathione, oxidized

GST

glutathione S-transferase

Plrx

plasmoredoxin

Trx

thioredoxin

TrxR

thioredoxin reductase

TPx

thioredoxin dependent peroxidase

The malarial parasite, Plasmodium falciparum is responsible for more than 2 million deaths per year and novel antiparasitic drugs are urgently and continuously required [1,2]. Malarial parasites are exposed to high fluxes of reactive oxygen species (ROS) and for this reason, proteins involved in antioxidant defense are promising targets for antimalarial drug development [3–6]. P. falciparum has been shown recently to possess two major functional redox systems: a thioredoxin system [7,8] comprising NADPH, thioredoxin reductase (TrxR), thioredoxin (Trx) [8,9] and thioredoxin dependent peroxidases (TPx) [10–14] and a glutathione system comprising NADPH, glutathione reductase (GR) [15], glutathione, glutathione S-transferase [16] and glutaredoxin (Grx) [17].

The thioredoxin superfamily includes the redox-active proteins thioredoxin, glutaredoxin, tryparedoxin, protein disulfide isomerase and DsbA (disulfide bond forming proteins of bacteria) [18,19]. All members of this family share the ‘thioredoxin-fold’ consisting of a central five-stranded β-sheet surrounded by four α-helices [20], and an active site with two conserved cysteine residues that specify the biological activity of the protein [18,19]. Thioredoxins are a group of small (≈ 12 kDa) proteins with the classical active site sequence, CGPC. They contribute to a range of essential cellular functions including protection from ROS, reduction of enzymes such as ribonucleotide reductase and thioredoxin peroxidase, and regulation of transcription factors [18–21]. Mammalian Trx have been shown to function as cellular growth factors, to modulate apoptosis and to be highly expressed and secreted by certain tumor cells [22].

Glutaredoxins with a similar size are part of the glutathione system and characterized by the active site sequence, CPYC. They also protect against oxidative damage, serve as hydrogen-donors for ribonucleotide reductase and are associated with transcriptional control [17,19,21,23]. As shown for yeast cells, at least one out of four Trx and Grx genes has to be present for viability [24]. The presence of both thioredoxins and glutaredoxins in different organisms, together with the conservation of their active sites through evolution, point to the importance of these antioxidative and regulatory proteins for central cellular functions. As a third family of redox-active proteins with functions comparable to Trx and Grx, tryparedoxins have been described in trypanosomes and crithidiae, unicellular parasites lacking a glutathione system [25,26].

Here we describe the identification and characterization of a novel functional redox-active protein in the malarial parasite, P. falciparum. Together with thioredoxins, glutaredoxins and tryparedoxins, this protein represents a member of the thioredoxin superfamily. The presence of the protein is restricted to malarial parasites where it is likely to be involved in ribonucleotide reduction and glutathione homeostasis.

Materials and methods

PCR

Perfect match primers (forward: 5′-ATGGCGTGCCAAGTTGATAA-3′; reverse: 5′-TGCTGTCTGTAACCACACA-3′) were designed and PCR was carried out with a P. falciparum gametocyte cDNA as a template; the forward primer introduced a BamHI restriction site, the reverse primer a PstI restriction site. The PCR conditions were chosen as follows: (a) 94 °C, 30 sec; (b) 80 °C, hold; (c) 94 °C, 30 sec; (d) 60 °C, 30 sec; (e) 72 °C, 2 min; (f) 30 × steps c–e; (g) 72 °C, 3 min; (h) 15 °C, hold. The amplified 570 bp PCR product was digested with the corresponding restriction enzymes, purified and cloned into the expression vector, pQE30, that had been cleaved previously with BamHI/PstI. The resulting plasmid-construct was sequenced and showed 100% identity to the genome sequence.

Overexpression and purification of PfPlrx

The Qiagen expression-system (pQE30 vector, that adds an N-terminal hexahistidyl-tag to the protein for affinity-purification, and M15 E. coli expression cells) was used for overexpression and purification of Plrx. The relative molecular mass of the pure protein (as judged by silver stained SDS/PAGE and gel filtration using a calibrated Sephadex G-75 column) was 21.4 kDa (calculated 21 684 Da). The calculated absorption coefficient, ε280nm, of PfPlrx was determined to be 31.4 mm−1·cm−1.

Immunoblotting

Intraerythrocytic stages of P. falciparum were cultured in vitro as described previously [17]. Rabbit antiserum raised against recombinant PfPlrx was obtained from BioScience, Göttingen, Germany. The reaction of the antibodies with authentic PfPlrx in P. falciparum trophozoite extracts, as well as with recombinant protein, was studied by Western blotting. Samples were subjected to 12% SDS/PAGE and then blotted on a polyvinylidene difluoride membrane using a semi-dry blot procedure (50 mA for 55 min). As a secondary antibody, peroxidase-conjugated porcine anti-(rabbit Ig) Igs (Dako Diagnostika, Hamburg, Germany) were used.

Enzyme assays

Ribonucleotide reductase activity was determined from the rate of conversion of [3H]GDP into [3H]dGDP essentially as described for CDP reduction [27]. The assay mixture (200 µL) contained 50 mm Hepes, 100 mm KCl, 6.4 mm MgCl2, 500 µm GDP (including 1.25 µCi [3H]GDP), 100 µm dTTP, variable concentrations of PfPlrx, E. coli thioredoxin, and Trypanosoma brucei thioredoxin, respectively. T. brucei R1 subunit (1 mU, 1.48 µm) with a 67-fold molar excess of the R2 subunit (99 µm) was used (1 U corresponds to 1 µmol dGDP formation per min−1). The mixture was incubated at 37 °C for 20 min and the reaction was stopped by boiling for 10 min. Precipitated protein was removed by centrifugation at 13 000 g, and products and educts were dephosphorylated by 45 min incubation with 10 U alkaline phosphatase. Nucleoside, deoxynucleoside and free bases were then separated isocratically by HPLC on an Aminex A9 anion exchange column (250 × 4 mm) in 100 mm sodium borate, pH 8.3 [28,29].

Glutathione reductase [15], thioredoxin reductase [9] and trypanothione reductase activities [29,30] were determined spectrophotometrically at 340 nm monitoring the consumption of NADPH as described previously. In these assays up to 100 µm PfPlrx was tested as the substrate. The detection limit in these assays is ΔA = 0.002·min−1, that corresponds to an NADPH oxidation rate of 0.3 µm·min−1 and an activity of 0.3 mU·mL−1 of an NADPH dependent disulfide reductase. The insulin-reduction assay is described in the legend to Fig. 3; P. falciparum thioredoxin used for this assay was expressed and purified as described previously [9].

Figure 3.

Insulin-reduction activity of PfPlrx in comparison with PfTrx. In this assay, the precipitation of reduced insulin B-chains is followed at 600 nm. One ml of reaction mixture contained 0.17 mm porcine insulin in 50 mm Tris/HCl, 2 mm EDTA at pH 7.4. The reaction was started at 25 °C by adding 1 mm dithiothreitol in the presence of 2 µm PfTrx (closed square), 2 µm PfPlrx (closed triangle) or 5 µm PfPlrx (cross). 1 mm dithiothreitol without protein (closed diamond) served as a negative control. Addition of 5 µm bovine serum albumin to the dithiothreitol control gave identical results. In additional assays, dithiothreitol was replaced by a reducing system consisting of either 200 µm NADPH, 1 U·mL−1 PfGR, 2 or 10 mm GSH, 5 µm Plrx or of 200 µm NADPH, 1 U·mL−1 PfTrxR, 5 µm Plrx at pH 6.9, 7.4 and 8.0. Only at pH 8.0 and 10 mm GSH was a clear insulin reducing activity observed within 30 min (open square). At pH 8.0 the reduction of 5 µm Plrx by 1 mm dithiothreitol (closed circle) was also more efficient than at pH 7.4.

Reaction of PfPlrx with different reducing agents

Reduction of PlrxS2 by trypanothione and tryparedoxin. In trypanosomes and other Kinetoplastida, a major relay system of electron transferring reactions exists that comprises of NADPH, trypanothione reductase, trypanothione, tryparedoxin and a terminal acceptor such as ribonucleotide reductase [30]. The interactions of Plrx with this system were tested as described for the GHOST assay [7].

Briefly, 1 mL assay mixture at 25 °C were used. The compounds were added in the following order: buffer (40 mm Hepes, 1 mm EDTA at pH 7.5), NADPH (200 µm final concentration), Trypanosoma cruzi trypanothione reductase (80 nm = 0.25 enzyme units), PlrxS2, trypanothione disulfide (20–50 µm) and T. brucei tryparedoxin disulfide (4.5 µm). In a series of assays, the order of additions was changed so that PlrxS2 was added at differing steps in the sequence of additions.

In the course of the reaction sequence, essentially, each disulfide is reduced completely to the corresponding dithiol, NADPH oxidation being the driving force. After each addition to the assay mixture, the absorbance decrease at 340 nm due to NADPH oxidation was registered and the rate of the respective reaction was calculated according to the equation: v = Δc × min−1= ΔA/(1 min × ε × 1 cm) [µM × min−1], where the ε−value for NADPH is 6.22 mm−1·cm−1. From a given value of v, the rate constant k was determined using the equation for a second order reaction: k = v/{[R(SH)2] × [PlrxS2]}. Assay conditions for the reduction of Plrx by other reducing agents are given in the legend to Table 1.

Table 1. Reduction of PlrxS2 by dithiols and glutathione at 25 °C.
Reductantk × 103m−1·min−1]k[m−1·s−1]Conditions
  1. a Assays were performed in 100 mm Tris, 1 mm EDTA, at different pH values adjusted at 25 °C, in the presence of 200 µm NADPH, 1 U·mL−1 PfGR, 0.5–10 mm GSH, and 50 µm PlrxS2. For the reaction of PlrxS2 with glutathione, the limited data set did not allow us to distinguish between pseudosecond and third order kinetics. b Assays were performed as above but in the presence of 1 mm GSH and 10–60 µm PfGrx. At Grx concentrations ≥20 µm no clear increase in ΔA·min−1 value was detected. The rate constant was therefore calculated on the basis of the value determined for 10 µm Grx. cThe reaction of Plrx (25–50 µm) with thioredoxin (100 µm) was determined in 100 mm potassium phosphate, 2 mm EDTA, pH 7.4 in the presence of 1 U·mL−1 PfTrxR and 200 µm NADPH. dAssay conditions for the reaction of Plrx with the trypanothione system are given in the Materials and methods section.

NADPH≤ 0.01  
Dithiothreitol1.4 23pH 8.0, 100 mm Tris
Dihydrolipoamide2.0 33pH 7.4, 50 mM phosphate, 1 mm EDTA
GSHa0.28  4.7pH 8.0, 100 mm Tris
0.10  1.6pH 7.4, 100 mm Tris
0.03  0.5pH 6.9, 100 mm Tris
P. falciparum glutaredoxinb14230pH 7.4, 100 mm Tris
P. falciparum thioredoxinc2.2 37pH 7.4, 100 mm phosphate
Trypanothioned0.67 11pH 7.5, 40 mm Hepes
T. bruceibrucei tryparedoxind30503pH 7.5, 40 mm Hepes

Reduction of GSSG by PfPlrx

PfPlrx was prereduced with 1 mm dithiothreitol. The protein was then separated rapidly from excess dithiothreitol by affinity chromatography using Ni-nitrilotriacetic acid agarose. Reduced PfPlrx (12.5 or 25 µm) was then incubated for 30 s and 15 min at 4 °C and 25 °C, respectively, with GSSG (25 or 50 µm) in 50 mm potassium phosphate, 1 mm EDTA, 200 mm KCl at pH 6.9. This incubation was followed by addition of 100 µm NADPH and 50 mU·mL−1 human glutathione reductase in order to determine the concentration of residual GSSG. PfPlrx was found to reduce GSSG in a nonenzymatic reaction. The rate constant, k, of this reaction was calculated as v/[Plrx(SH)2] ×[GSSG] × min on the basis of the following experiment. Reduction of 25 µm GSSG (25 °C for 30 s) with PfPlrx (12.5 µm) led to 19 µm residual GSSG; this corresponds to the reduction of 12 µm GSSG per min. Thus, k was calculated to be 0.0384 µm−1·min−1. In parallel experiments, we removed Plrx after the reaction with GSSG using Ni-nitrilotriacetic acid agarose. Subsequently, the thiol content, representing the formed GSH, was measured in the solution.

Results and discussion

In the genome of the malarial parasite, P. falciparum[31] a gene showing sequence similarities with thioredoxin genes was identified. The sequence consisted of an exon containing 537 bp located on chromosome 3. The gene was amplified by PCR using a gametocyte cDNA as a template, sequenced, cloned into an expression vector, and overexpressed in E. coli. The deduced amino acid sequence (PfPlrx; accession number AAF87222) comprised 179 residues (22 kDa) and contained the unique active site motif, WCKYC, when compared with other members of the thioredoxin superfamily. The novel protein was named plasmoredoxin (Plrx). Putative plasmoredoxins of comparable size were also identified by in silico analyses in the genomes of the Plasmodium species, P. vivax[32], P. berghei, P. yoelii, and P. knowlesi (this paper). The corresponding amino acid sequence alignments showed identities of 67.4, 66.9, 72.6 and 67.2% with P. falciparum plasmoredoxin (Fig. 1). The identity of PfPlrx with other members of the thioredoxin superfamily, for example PfTrx (31.4%) or PfGrx (27.5%) were significantly lower. Apart from members of this superfamily, the highest degrees of identity (31.3 and 32.6%) were with ResA (P35160), a respiration regulating protein of Bacillus subtilis, and HelX (M96013), a putative periplasmic disulfide oxidoreductase of the photosynthetic bacterium, Rhodobacter capsulatus, respectively. Homology modelling based on the swiss prot program resulted in a partial three-dimensional structure of Plrx. Residues 43–94, representing 28% of the complete amino acid sequence were modelled and indicated a characteristic thioredoxin fold including the active site sequence, WCKYC. In a reconstructed phylogenetic tree, plasmoredoxins cluster as one group separate from thioredoxins, glutaredoxins and tryparedoxins (Fig. 2). Within the plasmoredoxins, the rodent parasites P. yoelii and P. berghei Plrx share the highest degree of amino acid identity (91%), followed by the P. vivax/P. knowlesi pair with 87.6%. P. falciparum are in between these two groups. This result suggests a close relationship between P. knowlesi that infects monkeys and P. vivax that causes tertian malaria in man.

Figure 1.

Alignment of the amino acid sequence of plasmoredoxin from Plasmodium falciparum with putative homologues of different Plasmodium species. Pf, P. falciparum (GenBank AAF87222); Pv, P. vivax (GenBank AAF99466); Py, P. yoelii (GenBank EAA16465; gnl|py|TIGR_c5m141); Pk, P. knowlesi (gnl|pk|Sanger_PKN.0.004551); Pb, P. berghei (gnl|pbgss|UFL_249PbC01, gnl|pbgss|UFL_204PbH08, gnl|pbgss|UFL_225PbD05), this sequence was generated from three different genomic clones and is likely to lack a small fragment of the sequence. Identical amino acids are highlighted, the putative active site is boxed.

Figure 2.

Phylogenetic relations of plasmoredoxins, thioredoxins, glutaredoxins, and tryparedoxins. Plasmoredoxins represent a novel family of redox-active proteins belonging to the thioredoxin superfamily. The sequence comparisons were carried out using the clustal w program of the EMBL European Bioinformatics Institute (www2.ebi.ac.uk.clustalW). Pk, P. knowlesi; Pv, P. vivax; Pf, P. falciparum; Py, P. yoelii; Pb, P. berghei; Hs, Homo sapiens; Ec, E. coli; Tb, T. brucei; Cf, Crithidia fasciculata; Tc, T. cruzi; Plrx, plasmoredoxin; Trx, thioredoxin; Trp, tryparedoxin; Grx, glutaredoxin.

Interestingly, two similar sequence annotations (GenBank accesson numbers, NP_473166 and CAB38989) were available that proposed a large protein of 2417 and 2396 amino acids, respectively, with a putative structural function in the cytoskeleton of P. falciparum. These annotations suggested that plasmoredoxin might be part of this large protein as a possible second exon. To check this possibility, a PCR with ‘exon-overlapping’ primers [one primer in putative exon 1 (the big structural protein), the other primer in putative exon 2 (the plasmoredoxin)] was performed using PfcDNA as a template. Under various PCR conditions, however, no product was obtained indicating that PfPlrx is unlikely to represent a part of the protein encoded by exon 1 and indeed, very recently both former sequence predictions were updated and split into two parts resulting in a putative protein of 2226 amino acids and a second predicted protein of 179 amino acids representing plasmoredoxin.

As summarized in Table 1, PfPlrx can be reduced by different dithiols as well as by GSH. Most effective were P. falciparum glutaredoxin and T. brucei tryparedoxin. Whether the reduction of Plrx by GSH is physiologically significant might be questioned as the pseudosecond order rate constant was only 1.6 m−1·s−1 at pH 7.4 and 25 °C. Concentration-dependent redox activity of Plrx was demonstrated by its ability to cleave disulfide bonds of insulin when using dithiothreitol as a source of reducing equivalents (Fig. 3). In this assay, P. falciparum thioredoxin served as a positive control and dithiothreitol as well as bovine serum albumin as negative controls. Using the glutathione system as a primary source of reducing equivalents, the insulin-reduction by 5 µm Plrx was too slow to be detected at the physiological pH of 7.4 but a clear reaction was apparent at pH 8.0.

Interestingly, PfPlrx was found to be no substrate for thioredoxin reductase from P. falciparum, E. coli, and man; of glutathione reductase from P. falciparum and man and trypanothione reductase from T. cruzi. In each case, the specific activity was below the detection limit of 25 mU·mg−1 enzyme protein.

To test whether Plrx modulates glutathione reductase and thioredoxin reductase activity, respectively, Plrx was prereduced by incubation with 2 mm dithiothreitol. Residual dithiothreitol was removed by affinity chromatography on a Ni-nitrilotriacetic acid column. Directly after elution, 20 µm Plrx(SH)2 was added to a standard GR assay, pH 6.9 [15], and a TrxR assay, pH 7.4, containing 20 µm PfTrx [9], respectively. The addition of reduced Plrx did not influence the reaction catalysed by the disulfide reductases at 25 °C.

The ability of PfPlrx to reduce hydroxyethyl disulfide GSH-dependently was tested in an assay system typically used for characterizing glutaredoxins [17]. The assay (in 100 mm Tris, 1 mm EDTA, pH 8.0) contained 100 µm NADPH, 0.25 U·mL−1 PfGR, 1 mm GSH as well as different concentrations of PfGrx and PfPlrx, and was started with 735 µm hydroxyethyl disulfide. In a reference cuvette containing no Grx/Plrx, the spontaneous reaction between GSH and hydroxyethyl disulfide was accounted for. Grx (20 nm) produced an ΔA·min−1 value of 0.051, corresponding to a kcat of 410 min−1 (see also [17]). Plrx (25 and 75 µm) resulted in ΔA·min−1 values of 0.025 and 0.070, respectively, corresponding to a kcat of 0.15 min−1. Thus, the GSH-dependent hydroxyethyl disulfide reducing activity of PfPlrx is by a factor of almost 3000 lower than the activity of PfGrx1 [17].

Peroxidase activity of PfPlrx was tested in 100 mm Tris, 1 mm EDTA, pH 7.4 (or 8.0) in the presence of 200 µm NADPH, 1 U·mL−1 PfGR, 2 mm GSH and 50 µm PlrxS2. After 15 min preincubation, which guaranteed the reduction of PlrxS2, 200 µm H2O2 was added. The resulting ΔA·min−1 value was higher by ≤ 0.01 than the one of the controls carried out in the absence of Plrx at both pH values. This indicated an extremely slow reaction between Plrx(SH)2 and H2O2– the second order rate constant being ≤ 1.6 × 10−4µM−1·min−1– when comparing Plrx with known peroxidases of P. falciparum[10–14].

Plasmoredoxin, in its dithiothreitol-reduced form, was tested successfully as a hydrogen donor for T. brucei ribonucleotide reductase. This result points to an in vivo contribution of PfPlrx to DNA synthesis. The reduction of ribonucleotide reductase is, in most organisms, produced by Trx and Grx; in Trypanosomes, tryparedoxin was shown to have a comparable function [18,19,25,29].

Reduced PfPlrx was furthermore shown to reduce quantitatively glutathione disulfide. A 15-min incubation of 25 µm PfPlrx with 50 µm GSSG resulted in the formation of 50 µm GSH, as indicated by a decrease of the GSSG concentration from 50−25 µm. The concomitant determination of 44.2 µm GSH makes it unlikely that glutathionylated Plrx is a major reaction product. The following reaction scheme is therefore proposed:

image

According to the data obtained with different substrate concentrations and incubation times, the lower limit of the k-value for this chemical reaction can be estimated as 0.01 µm−1·min−1 at 4 °C and of 0.04 µm−1·min−1 at 25 °C. For many thioredoxins (with the notable exception of PfTrx) the corresponding rate constant is ≤ 0.01 µm−1·min−1 at 25 °C [9].

The reduction of GSSG is, in most organisms, conducted by the NADPH-dependent flavoenzyme, glutathione reductase (GR) [3]. However, we have shown recently that insects including Drosophila melanogaster and Anopheles gambiae lack a genuine GR although they contain high concentrations of glutathione [33]. In this context, a nonenzymatic reduction of GSSG by reduced thioredoxin was described for different organisms and proposed to have in vivo relevance [9,33]. Obviously, PfPlrx, as a member of the thioredoxin superfamily, is also able to fulfil this function. The stoichiometric reaction observed for PfPlrx and GSSG may contribute to antioxidant defense and specific redox regulatory processes in malarial parasites that grow and multiply in an environment of high oxygen tension [34].

P. falciparum plasmoredoxin is a member of a novel family of redox active proteins belonging to the thioredoxin superfamily. PfPlrx is larger than classical thioredoxins, glutaredoxins and tryparedoxins, it shares, however, typical structural and functional characteristics with the other three groups. The reactions of P. falciparum plasmoredoxin with rabbit IgG raised against the recombinant protein were demonstrated by Western blotting. As shown in Fig. 4, single bands of expected sizes (24 kDa, due to the His-tag and 22 kDa) appeared when probing the recombinant protein and a trophozoite extract of P. falciparum. This result, and the fact that PfPlrx was amplified from a cDNA library, indicate that the gene is transcribed and the protein is present in blood-stage forms of the parasite that cause malaria in the human host. Considering the above may allow a unique avenue for developing diagnostic tools based on PCR or immunological methods. Many organisms, including E. coli, D. melanogaster, yeast and man possess more than one Trx or Grx. As indicated by our studies, P. falciparum possesses at least one thioredoxin [9], at least two glutaredoxin-like proteins [8,17] and the newly discovered plasmoredoxin. In this context it is furthermore interesting to note that until now the gene of only one glutathione S-transferase has been detected in the genome of P. falciparum[16] and that no glutathione-dependent peroxidase has been discovered so far. The gene believed to represent a GPx, was found to code for a thioredoxin dependent peroxidase [12]. Taken together, these data might indicate that P. falciparum does not use glutathione dependent reactions to the extent described for other organisms. In other words, malarial parasites might have developed a unique additional defense line against oxidative stress, and an additional source of reducing equivalents for deoxyribonucleotide synthesis as well as for signalling processes. Indeed, the multiplication rate of P. falciparum is among the fastest in eukaryotic organisms. Potential roles of plasmoredoxin in redox metabolism of P. falciparum are delineated in Fig. 5.

Figure 4.

Western blot of P. falciparum plasmoredoxin. Lane 1: Recombinantly produced P. falciparum plasmoredoxin (200 ng); lane 2: extract of the P. falciparum strain 3D7 (18 µg total protein). The molecular masses of the standard proteins on lane 3 are given on the right hand side.

Figure 5.

The putative roles of P. falciparum plasmoredoxin(Plrx) in redox metabolism of the parasite. Only proteins/pathways that have been verified to exist in P. falciparum are shown. NADPH represents the major source of reducing equivalents in the infected erythrocyte. Both, thioredoxin reductase (TrxR) and glutathione reductase (GR) reduce their respective substrates, thioredoxin (Trx) and glutathione disulfide (GSSG) by using NADPH. Trx reduces Trx-dependent peroxidases as well as ribonucleotide reductase (RiboR). Reduced glutathione (GSH) serves as a substrate of glutathione S-transferase (GST) or reduces glutaredoxin, which in turn is able to provide RiboR with electrons. Plasmoredoxin is, like thioredoxin, able to reduce RiboR as well as GSSG and can be reduced by GSH, Trx, and glutaredoxin.

According to our data, Plrx seems to be present in Plasmodium species only, rendering the protein and/or the gene a specific diagnostic tool for clinical and epidemiological studies. Furthermore, Plrx as an antioxidant protein that is also involved in DNA synthesis may represent a potential drug target.

Acknowledgements

The technical assistance of Elisabeth Fischer, Petra Harwaldt and Beate Hecker is acknowledged. The authors wish to thank R. L. Krauth-Siegel, Heidelberg, for performing the ribonucleotide reductase assays and for kindly providing the components of the tryparedoxin-reducing system. We also thank Julia K. Ulschmid and Scott Mulrooney for helpful discussion. Sequence data for P. vivax/berghei/falciparum was obtained from the University of Florida Gene Sequence Tag Project Website at: http://parasite.vetmed.ufl.edu. Funding was provided by the National Institute of Allergy and Infectious Diseases (for P. berghei and P. vivax data) and University of Florida Division of Sponsored Research and the Burroughs Wellcome Fund (for P. falciparum data). Sequence data for P. falciparum chromosome 3 was obtained from The Sanger Centre website at http://www.sanger.ac.uk/Projects/P_falciparum/. Sequencing of P. falciparum chromosome 3 was accomplished as part of the Malaria Genome Project with support by The Wellcome Trust. Our work on redox metabolism of malarial parasites is supported by the Deutsche Forschungsgemeinschaft (grants, SFB 535 to K. B. and Schi 102/8-1 to R. H. S.).

Ancillary