MOLECULAR CHARACTERIZATION AND EXPRESSION OF ONCHOCERCA-VOLVULUS GLUTATHIONE-REDUCTASE

Glutathione metabolism represents a potential target for antiparasite drug design. The central role of glutathione reductase (GR) in mainten ance of the thiol redox state and in antioxidative defence has to be e valuated in more detail in order to establish the essential function o f this enzyme for the survival of the filarial parasite Onchocerca vol vulus. The O. volvulus GR (OvGR) gene was cloned and sequenced. The ge ne 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 n ucleotides upstream of the translation initiation codon. Southern-blot analysis and structural characterization of the genomic sequence indi cate that OvGR is encoded by a single-copy gene. Isolation of various cDNA clones revealed a polymorphism of poly-adenylation initiation wit h 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 fi rst 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 identica l in OvGR and HsGR. However, one of the arginine residues (Arg-224 in HsGR) involved in discrimination between NADPH and NADH in all known G Rs is substituted by tryptophan (Trp-207 in OvGR). The coding region o f OvGR was expressed in Escherichia coli as a histidine-fusion protein , and it was established that the parasite protein still favours the b inding of NADPH (K-m 10.9 mu M) over NADH (K-m 108 mu M). The histidin e-fusion protein has a subunit size of 54 kDa and is active as a homod imer of 110 kDa.