Vitamin B1 and B6 in the malaria parasite: requisite or dispensable?

Vitamins are essential compounds mainly involved in acting as enzyme co-factors or in response to oxidative stress. In the last two years it became apparent that apicomplexan parasites are able to generate B vitamers such as vitamin B1 and B6 de novo. The biosynthesis pathways responsible for vitamin generation are considered as drug targets, since both provide a high degree of selectivity due to their absence in the human host. This report updates the current knowledge about vitamin B1 and B6 biosynthesis in malaria and other apicomplexan parasites. Owing to the urgent need for novel antimalarials, the significance of the biosynthesis and salvage of these vitamins is critically discussed in terms of parasite survival and their exploitation for drug development.

Synthesis and antifilarial evaluation of N1,Nn- xylofuranosylated diaminoalkanes.

A series of N(1),N(n)-xylofuranosylated diaminoalkanes (3-9 and 11-18) has been synthesized either by reductive amination of deoxy xylouloses (2a, 2b) with amines followed by one pot reduction with NaBH(4) or NaCNBH(3); or by 1,4-conjugate addition of amines to glycosyl olefinic esters (10a, 10b). The compounds were screened for their interference with filarial worms' glutathione metabolism, a potential target for chemotherapeutic attack. Interestingly, these compounds affected intracellular glutathione, gamma-glutamyl cysteine synthetase, glutathione reductase and glutathione-S-transferase(s) of bovine filarial worms to varying degrees. Some of the compounds though effected the motility and MTT reduction potential of filarial worms Brugia malayi, however, little microfilaricidal and macrofilaricidal were noted with compounds at 50mg/kg oral dose. Compounds 6, 16 and 17 were evaluated also for in vivo activity.

DBU-assisted cyclorelease elimination: combinatorial synthesis and gamma-glutamyl cysteine synthetase and glutathione-S-transeferase modulatory effect of C-nucleoside analogs.

A combinatorial library of 60C- nucleoside analogs was synthesized by sequential coupling of building blocks followed by cyclative cleavage with DBU in an efficient manner. Only DMSO soluble compounds were tested for their modulatory effect against filarial gamma-glutamyl cysteine synthetase (gamma-GCase) and glutathione-S-transeferases (GSTs). Several compounds were found to be weak inhibitors of filarial gamma-GCase, whereas, most of them stimulated filarial GSTs.

Thioredoxin and glutathione system of malaria parasite Plasmodium falciparum.

Plasmodium falciparum is the causative agent of malaria tropica. Due to the increasing resistance towards the commonly used plasmodicidal drugs there is an urgent need to identify and assess new targets for the chemotherapeutic intervention of parasite development in the human host. It is established that P. falciparum-infected erythrocytes are vulnerable to oxidative stress, and therefore efficient antioxidative systems are required to ensure parasite development within the host cell. The thioredoxin and glutathione redox systems represent two powerful means to detoxify reactive oxygen species and this article summarizes some of the recent work which has led to a better understanding of these systems in the parasite and will help to assess them as potential targets for the development of new chemotherapeutics of malaria.

The Plasmodium falciparum bifunctional ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase, enables a well balanced polyamine synthesis without domain-domain interaction.

In the human malaria parasite Plasmodium falciparum (Pf), polyamines are synthesized by a bifunctional enzyme that possesses both ornithine decarboxylase (ODC) and S-adenosyl-l-methionine decarboxylase (AdoMetDC) activities. The mature enzyme consists of the heterotetrameric N-terminal AdoMetDC and the C-terminal dimeric ODC, which results in the formation of a heterotetrameric complex. For the native bifunctional protein a half-life longer than 2 h was determined, which is in contrast to the extreme short half-life of its mammalian monofunctional counterparts. The biological advantage of the plasmodial bifunctional ODC/AdoMetDC might be that the control of polyamine synthesis is achieved by only having to regulate the abundance and activity of one protein. An interesting feature in the regulation of the bifunctional protein is that putrescine inhibits PfODC activity approximately 10-fold more efficiently than the mammalian ODC activity, and in contrast to the mammalian AdoMetDC the activity of the PfAdoMetDC domain is not stimulated by the diamine. To analyze post-translational processing, polymerization, and domain-domain interactions, several mutant proteins were generated that have single mutations in either the PfODC or PfAdoMetDC domains. The exchange of amino acids essential for the activity of one domain had no effect on the enzyme activity of the other domain. Even prevention of the post-translational cleavage of the AdoMetDC domain or ODC dimerization and thus the interference with the folding of the protein hardly affected the activity of the partner domain. In addition, inhibition of the activity of the PfODC domain had no effect on the activity of the PfAdoMetDC domain and vice versa. These results demonstrate that no domain-domain interactions occur between the two enzymes of the bifunctional PfODC/AdoMetDC and that both enzymatic activities are operating as independent catalytic sites that do not affect each other.

The malaria parasite Plasmodium falciparum possesses a functional thioredoxin system.

The thioredoxin system consists of the NADPH dependent disulphide oxidoreductase thioredoxin reductase (TrxR) which catalyses the reduction of the small protein thioredoxin. This system is involved in a variety of biological reactions including the reduction of deoxyribonucleotides, transcription factors and hydrogen peroxide. In recent years the TrxR of the malaria parasite Plasmodium falciparum was isolated and characterised using model substrates like 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) and Escherichia coli thioredoxin. Here we report on the isolation of a cDNA encoding for P. falciparum thioredoxin (PfTrx) and the expression and characterisation of the recombinant protein, the natural substrate of PfTrxR. The deduced amino acid sequence of PfTrx encodes for a polypeptide of 11715 Da and possesses the typical thioredoxin active site motif CysGlyProCys. Both cysteine residues are essential for catalytic activity of the protein, as shown by mutational analyses. Steady state kinetic analyses with PfTrxR and PfTrx in several coupled assay systems resulted in K(m)-values for PfTrx in the range of 0.8--2.1 microM which is about 250-fold lower than for the model substrate E. coli thioredoxin. Since the turnover of both substrates is similar, the catalytic efficiency of PfTrxR to reduce the isolated PfTrx is at least 250-fold higher than to reduce E. coli thioredoxin. PfTrx contains a cysteine residue in position 43 in addition to the active-site cysteine residues, which is partially responsible for dimer formation of the protein as demonstrated by changing this amino acid into an alanine residue. Using DTNB we showed that all three cysteine residues present in PfTrx are accessible to modification by this compound. Surprisingly the first cysteine residue of the active site motif (Cys30) is less accessible than the second cysteine (Cys33), which is highly prone to the modification. These results suggest a difference in the structure and reaction mechanism of PfTrx compared to other known thioredoxins.

The ornithine decarboxylase domain of the bifunctional ornithine decarboxylase/S-adenosylmethionine decarboxylase of Plasmodium falciparum: recombinant expression and catalytic properties of two different constructs.

The polyamines putrescine, spermidine and spermine play an essential role in cell differentiation and proliferation. Inhibition of the rate-limiting enzymes of polyamine biosynthesis, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), has been proposed as a therapeutic strategy against cancer and parasitic infections. In the case of Plasmodium falciparum, the causative agent of malaria tropica, this approach is especially interesting, because here both key enzymes, ODC and AdoMetDC, are combined in a bifunctional protein, ODC/AdoMetDC. This arrangement has not been found in any other organism investigated so far. We report the cloning and recombinant expression of the ODC domain of P. falciparum in Escherichia coli. First, we expressed the mere recombinant ODC domain (rPfODC). Secondly, we expressed the recombinant ODC domain in conjunction with the preceding part of the hinge region of the bifunctional ODC/AdoMetDC (rPfHinge-ODC). K(m) values for L-ornithine were 47.3 microM for the rPfHinge-ODC and 161. 5 microM for the rPfODC. Both recombinant enzymes were inhibited by putrescine, but the K(i) value for the rPfHinge-ODC was 50.4 microM (IC(50)=157 microM), whereas the IC(50) for the rPfODC was 500 microM. Spermidine was a weak inhibitor in both cases. alpha-Difluoromethylornithine inhibited the rPfHinge-ODC with a K(i) value of 87.6 microM. For two novel ODC inhibitors, CGP52622A and CGP54619A, the K(i) values of the rPfHinge-ODC were in the nanomolar range.

Intersubunit interactions in Plasmodium falciparum thioredoxin reductase.

The thioredoxin redox system is composed of the NADPH-dependent homodimeric flavoprotein thioredoxin reductase (TrxR) and the 12-kDa protein thioredoxin. It is responsible for the reduction of disulfide bridges in proteins such as ribonucleotide reductase and several transcription factors. Furthermore, thioredoxin is involved in the detoxification of hydrogen peroxide and protects the cell against oxidative damage. There exist two classes of TrxRs: the high M(r) and the low M(r) proteins. The well characterized Escherichia coli TrxR represents a member of the low M(r) class of proteins, whereas the mammalian, Caenorhabditis elegans, and Plasmodium falciparum proteins belong to the family of high M(r) proteins. The primary structure of these proteins is very similar to that of glutathione reductase and lipoamide dehydrogenase. However, the high M(r) TrxRs possess, in addition to their redox active N-terminal pair of cysteines, a pair of cysteine residues or a selenenylsulfide motif at their C terminus. These residues have been shown to be crucial for the reduction of thioredoxin. In this study we address the question whether the active site residues of P. falciparum TrxR are provided by one or both subunits. Differentially tagged wild-type and PfTrxR mutants were co-expressed in E. coli and the recombinant protein species were purified by affinity chromatography specific for the respective tags of the recombinant proteins. Co-expression of PfTrxR wild-type and mutant proteins resulted in the formation of three different protein species: homodimeric PfTrxR wild-type proteins, homodimeric mutant proteins, and heterodimers composed of one PfTrxR wild-type subunit and one PfTrxR mutant subunit. Co-expression of the double mutant PfTrxRC88AC535A with PfTrxR wild-type generated an inactive heterodimer, which indicates that PfTrxR possesses intersubunit active sites. In addition, the data presented possibly imply a coopertive interaction between both active sites of PfTrxR.

Plasmodium falciparum-infected red blood cells depend on a functional glutathione de novo synthesis attributable to an enhanced loss of glutathione.

During the erythrocytic cycle, Plasmodium falciparum is highly dependent on an adequate thiol status for its survival. Glutathione reductase as well as de novo synthesis of GSH are responsible for the maintenance of the intracellular GSH level. The first and rate-limiting step of the synthetic pathway is catalysed by gamma-glutamylcysteine synthetase (gamma-GCS). Using L-buthionine-(S, R)-sulphoximine (BSO), a specific inhibitor of the gamma-GCS, we show that the infection with P. falciparum causes drastic changes in the GSH metabolism of red blood cells (RBCs). Infected RBCs lose GSH at a rate 40-fold higher than non-infected RBCs. The de novo synthesis of the tripeptide was found to be essential for parasite survival. GSH depletion by BSO inhibits the development of P. falciparum with an IC(50) of 73 microM. The effect of the drug is abolished by supplementation with GSH or GSH monoethyl ester. Our studies demonstrate that the plasmodicidal effect of the inhibitor BSO does not depend on its specificity towards its target enzyme in the parasite, but on the changed physiological needs for the metabolite GSH in the P. falciparum-infected RBCs. Therefore the depletion of GSH is proposed as a chemotherapeutic strategy for malaria, and gamma-GCS is proposed as a potential drug target.

In the human malaria parasite Plasmodium falciparum, polyamines are synthesized by a bifunctional ornithine decarboxylase, S-adenosylmethionine decarboxylase.

The polyamines putrescine, spermidine, and spermine are crucial for cell differentiation and proliferation. Interference with polyamine biosynthesis by inhibition of the rate-limiting enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) has been discussed as a potential chemotherapy of cancer and parasitic infections. Usually both enzymes are individually transcribed and highly regulated as monofunctional proteins. We have isolated a cDNA from the malaria parasite Plasmodium falciparum that encodes both proteins on a single open reading frame, with the AdoMetDC domain in the N-terminal region connected to a C-terminal ODC domain by a hinge region. The predicted molecular mass of the entire transcript is 166 kDa. The ODC/AdoMetDC coding region was subcloned into the expression vector pASK IBA3 and transformed into the AdoMetDC- and ODC-deficient Escherichia coli cell line EWH331. The resulting recombinant protein exhibited both AdoMetDC and ODC activity and co-eluted after gel filtration on Superdex S-200 at approximately 333 kDa, which is in good agreement with the molecular mass of approximately 326 kDa determined for the native protein from isolated P. falciparum. SDS-polyacrylamide gel electrophoresis analysis of the recombinant ODC/AdoMetDC revealed a heterotetrameric structure of the active enzyme indicating processing of the AdoMetDC domain. The data presented describe the occurrence of a unique bifunctional ODC/AdoMetDC in P. falciparum, an organization which is possibly exploitable for the design of new antimalarial drugs.

The gamma-glutamylcysteine synthetase of Onchocerca volvulus.

The tripeptide glutathione (GSH) plays an important role in the maintenance of the intracellular thiol redox state and in detoxification processes. The intracellular GSH level depends on glutathione reductase as well as on GSH synthesis. The first and rate limiting step in the synthetic pathway is catalysed by gamma-glutamylcysteine synthetase (gamma-GCS). The gamma-GCS was partially purified from the filarial parasite Onchocerca volvoulus and preliminary steady state kinetics were performed. The Ki-value for L-buthionine-S,R-sulphoximine (BSO), a specific inhibitor of gamma-GCS, was determined to be 0.13 microM, which is 54-fold lower than the Ki-value for the mammalian enzyme. Filarial gamma-GCS was also inhibited by cystamine with a Ki-value of 3.9 microM compared with 22.2 microM determined for the rat enzyme. Further, the cDNA and the gene of the O. volvulus gamma-GCS were cloned and sequenced. The gene of 5762 bp is composed of 14 exons and 13 introns. Southern blot analysis indicates that the gamma-GCS gene is present as a single-copy gene. In accordance with Northern blot analysis, the entire cDNA sequence encompasses 2377 bp. At its 5' end a nematode-specific spliced leader 130 bp upstream of the first in frame methionine was identified. The cDNA encodes a polypeptide of 652 amino acids with 50 and 69% sequence identity to the human and the Caenorhabditis elegans counterparts, respectively. The filarial gamma-GCS is proposed as a potential drug target.

Ascaridia galli fatty acid-binding protein, a member of the nematode polyprotein allergens family.

A fatty acid-binding protein from the nematode Ascaridia galli was characterized. The gene was isolated and recombinantly expressed in Escherichia coli. According to the deduced amino acid sequence A. galli fatty acid-binding protein (AgFABP) belongs to the family of nematode polyprotein allergens, as shown by Western blotting and PCR analysis with genomic DNA and cDNA. Both native and recombinant proteins bind fatty acids and retinoids with high affinity. The fluorescent fatty acid analogue 11-[(5-dimethylaminonaphthalene-1-sulfonyl)amino] undecanoic acid (DAUDA) shows substantial changes in its emission spectrum when bound to AgFABP; this binding is reversed by fatty acids such as oleate. Moreover, changes of the intrinsic fluorescence of retinol and retinoic acid confirm retinoid binding activity of AgFABP. Fluorescence titration experiments with DAUDA indicate stoichiometric binding to a single binding site per monomer unit with affinities (Kd) of 1.6 and 1.8 x 10(-7) m for native and the recombinant protein, respectively. The apparent binding affinities of the nonfluorescent ligands were calculated in displacement experiments with DAUDA and values in the same range were obtained for myristic, palmitic, oleic, linoleic, arachidonic and retinoic acid. Additionally, the binding affinity of AgFABP for oleate and palmitate was determined by direct and indirect radiochemical analysis and the values obtained were similar to those from the fluorescent experiments. Both proteins show a preference for the binding of long-chain saturated and unsaturated fatty acids, but not for short chain (C3-C12) and branched fatty acids, cholesterol and tryptophan.

The putative gamma-glutamylcysteine synthetase from Plasmodium falciparum contains large insertions and a variable tandem repeat.

The tripeptide glutathione plays a pivotal role in the maintenance of the thiol redox state of the cell and for the detoxification of reactive oxygen species. Glutathione is synthesized in two consecutive reactions by y-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase, respectively. The former enzyme represents the rate limiting step of the synthetic pathway. We have cloned the cDNA and gene of a putative gamma-GCS from Plasmodium falciparum. The contiguous cDNA sequences obtained from various cDNA libraries of P. falciparum K1 and 3D7 encompass 4206 bp or 4038 bp and encode polypeptides of 1119 and 1063 amino acids, respectively. The deduced amino acid sequences show four regions of homology (identity: 31.3-43.9%) to human and Trypanosoma brucei gamma-GCS. These regions are interrupted by three large insertions between 94 and 239 amino acids. Within the first insert a variable repetitive motif was identified, which is responsible for the differing sizes of the sequences. We have analysed this phenomenon in five additional P. falciparum strains and found a high degree of variability in the number of the repeated octamer (Y/C)S(N/D)LQQ(Q/R). Therefore the predicted molecular mass of the proteins from different P. falciparum strains ranges from 124.4 to 133.2 kDa, which is almost twice that of the catalytic subunit of the human host enzyme. Isolation of three genomic clones revealed that the gene does not contain introns. P. falciparum gamma-GCS transcription peaks in trophozoites (24-30 h) suggesting that the antioxidant glutathione is predominantly produced at a time where hemoglobin degradation and the simultaneous formation of reactive oxygen species is maximal.

Molecular and biochemical characterization of S-adenosylmethionine decarboxylase from the free-living nematode Caenorhabditis elegans.

S-Adenosylmethionine decarboxylase (SAMDC) is a major regulatory enzyme in the polyamine biosynthesis and is considered a potentially important drug target for the chemotherapy of proliferative and parasitic diseases. To study regulatory mechanisms which are involved in the expression of SAMDC of the free-living nematode Caenorhabditis elegans, we have isolated the SAMDC gene and cDNA. Genomic Southern-blot analysis suggests that the C. elegans SAMDC is encoded by a single-copy gene which spans 3.9 kb and consists of six exons and five introns. The first two introns are located in the 5'-untranslated region (UTR). Analyses of the 5'-flanking region of the gene revealed several consensus sequences for the binding of different transcription factors such as CBP, AP2, cMyb, VPE2 and others. The C. elegans SAMDC mRNA possesses an open reading frame (ORF) which encodes a polypeptide of 368 amino acids, corresponding to a SAMDC proenzyme with a calculated molecular mass of 42141 Da. The active form of the C. elegans SAMDC is a heterotetramer, consisting of two subunits of 32 and 10 kDa derived from cleavage of the pro-enzyme. The SAMDC mRNA has an unusually long 5'-UTR of 477 nucleotides. This region has a small ORF which could encode a putative peptide of 17 residues. Moreover, the C. elegans SAMDC mRNA is trans-spliced with the 22 nucleotides spliced leader sequence at the 5'-end.

The role of the C-terminus for catalysis of the large thioredoxin reductase from Plasmodium falciparum.

The thioredoxin system is one of the major thiol reducing systems of the cell. Recent studies have revealed that Plasmodium falciparum and human thioredoxin reductase represent a novel class of enzymes, which are substantially different from the isofunctional prokaryotic Escherichia coli enzyme. We identified the cysteines Cys88 and Cys93 as the redox active disulfide and His509 as the active site base [Gilberger, T.-W., Walter, R.D. and Müller, S., J. Biol. Chem. 272 (1997) 29584-29589]. In addition to the active site thiols Cys88 and Cys93 the P. falciparum enzyme has another pair of cysteines at the C-terminus: Cys535 and Cys540. To assess the possible role of these peripheral cysteines in the catalytic process the single mutants PfTrxRC535A and PfTrxRC540A, the double mutant P/TrxRC535AC540A and the deletion mutant PfTrxRdelta9 (C-terminal deletion of the last nine amino acids) were constructed. All mutants are defective in their thioredoxin reduction activity, although they still show reactivity with 5,5'-dithiobis (2-nitrobenzoate). These data imply that the C-terminal cysteines are crucially involved in substrate coordination and/or electron transfer during reduction of the peptide substrate.

MGBG analogues as potent inhibitors of S-adenosylmethionine decarboxylase of Onchocerca volvulus.

Polyamines are essential for cell growth and differentiation and therefore, S-adenosylmethionine decarboxylase (SAMDC), a key regulatory enzyme of the polyamine biosynthesis, is considered as a potentially important target for chemotherapy of filarial infections. Recombinant Onchocerca volvulus SAMDC was expressed in Escherichia coli and characterised. The enzyme activity was found to be stimulated 15-fold by addition of 1 mM putrescine. The Km-value for S-adenosylmethionine was determined to be 36 microM. Furthermore, the efficiencies of SAMDC inhibitors were analysed: Berenil inhibits the enzyme activity competitively with a Ki-value of 0.1 microM. MDL 73811 acts as an irreversible inhibitor with a Ki-value of 1.4 microM. Recently synthesised aromatic methylglyoxal bis(guanylhydrazone) analogues demonstrated high efficacy as inhibitors of the SAMDCs. Some of these analogues exhibited Ki-values of 5 and 14 nM for the Onchocerca enzyme, a result which shows an up to 100-fold increase in specificity compared to the value of 0.47 microM for methylglyoxal bis(guanylhydrazone). These inhibitors might have potential as drug candidates against filarial worms.

Identification and characterization of the functional amino acids at the active site of the large thioredoxin reductase from Plasmodium falciparum.

The thioredoxin system, composed of the pyridine nucleotide-disulfide oxidoreductase thioredoxin reductase, the small peptide thioredoxin, and NADPH as a reducing cofactor, is one of the major thiol-reducing systems of the cell. Recent studies revealed that Plasmodium falciparum and human thioredoxin reductase represent a novel class of enzymes, called large thioredoxin reductases. The large thioredoxin reductases are substantially different from the isofunctional prokaryotic Escherichia coli enzyme. The putative essential amino acids at the catalytic center of large thioredoxin reductase from P. falciparum were determined by using site-directed mutagenesis techniques. To analyze the putative active site cysteines (Cys88 and Cys93) three mutant proteins were constructed substituting alanine or serine residues for cysteine residues. Further, to evaluate the function of His509 as a putative proton donor/acceptor of large thioredoxin reductase this residue was replaced by either glutamine or alanine. All mutants were expressed in the E. coli system and characterized. Steady state kinetic analysis revealed that the replacement of Cys88 by either alanine or serine and Cys93 by alanine resulted in a total loss of enzymatic activity. These results clearly identify Cys88 and Cys93 as the active site thiols of large thioredoxin reductase. The replacement of His509 by glutamine yielded in a 95% loss of thioredoxin reductase activity; replacement by alanine provoked a loss of 97% of enzymatic activity. These results identify His509 as active site base, but imply that its function can be substituted, although inefficiently, by an alternative proton donor, similar to glutathione reductase. Spectral analysis of wild-type P. falciparum thioredoxin reductase revealed a 550-nm absorption band upon reduction which resembles the EH2 form of glutathione reductase and lipoamide dehydrogenase. This spectral feature, recently also reported for the human placenta protein (Arscott, L. D., Gromer, S., Schirmer, R. H., Becker K., and Williams, C. H., Jr. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3621-3626), further illustrates the similarity between large thioredoxin reductases and glutathione reductases and stresses the profound differences to small E. coli thioredoxin reductase.

Localization and functional analysis of the cytosolic and extracellular CuZn superoxide dismutases in the human parasitic nematode Onchocerca volvulus.

This study describes the histological localization of two CuZn superoxide dismutases (SOD1 and SOD2) in the parasitic nematode Onchocerca volvulus, and a functional characterization of the 'extracellular' form of this enzyme (SOD2) which provides evidence that it is involved in the defense against environmental superoxide anion radicals. These essential enzymes are detected in larval and adult stages of the parasite, determined at the mRNA and protein levels by in situ hybridization and immunolocalization studies. These proteins are distributed throughout the worm, at various concentrations with particularly high levels produced in the hypodermis. In vitro maintenance of parasites indicated that SOD2 was secreted outside the parasite into the medium. Baculovirus constructs designed to test the ability of the SOD2 hydrophobic N-terminal region to function in processing and secretion confirmed the ability of this polypeptide sequence to direct the secretion of a marker protein, as well as of the mature SOD2 enzyme. Analyses of the native, mature SOD2 enzyme molecular mass, and the primary and quaternary structure, indicate that unlike other extracellular SODs, the SOD2 is active as a non-glycosylated dimer, rather than as a tetrameric glycoprotein. The detection of SOD2 outside of the parasite maintained in vitro, and the confirmation that the SOD2 is a secreted enzyme, indicate that this enzyme plays a role in the interactive biology of parasitic nematodes with their hosts.

Molecular characterization and expression of Onchocerca volvulus glutathione reductase.

Glutathione metabolism represents a potential target for anti-parasite drug design. The central role of glutathione reductase (GR) in maintenance of the thiol redox state and in anti-oxidative defence has to be evaluated in more detail in order to establish the essential function of this enzyme for the survival of the filarial parasite Onchocerca volvulus. The O. volvulus GR (OvGR) gene was cloned and sequenced. The gene is composed of 13 exons and 12 introns and spans 4065 bp. The first intron is located within the 5'-untranslated region of the gene, 16 nucleotides upstream of the translation initiation codon. Southern-blot analysis and structural characterization of the genomic sequence indicate that OvGR is encoded by a single-copy gene. Isolation of various cDNA clones revealed a polymorphism of polyadenylation initiation with no consensus polyadenylation sites in any of the cDNAs analysed. The entire cDNA is 1977 bp long and carries the nematode-specific spliced leader sequence SL1 at its 5' end, 236 nucleotides upstream of the first in-frame methionine. The cDNA codes for a polypeptide of 462 amino acids with 53.5% sequence identity with human GR (HsGR). A total of 18 out of 19 residues contributing to glutathione binding are identical in OvGR and HsGR. However, one of the arginine residues (Arg-224 in HsGR) involved in discrimination between NADPH and NADH in all known GRs is substituted by tryptophan (Trp-207 in OvGR). The coding region of OvGR was expressed in Escherichia coli as a histidine-fusion protein, and it was established that the parasite protein still favours the binding of NADPH (Km 10.9 microM) over NADH (Km 108 microM). The histidine-fusion protein has a subunit size of 54 kDa and is active as a homodimer of 110 kDa.