BIOCHEMICAL-ANALYSIS, GENE STRUCTURE AND LOCALIZATION OF THE 24 KDA GLUTATHIONE-S-TRANSFERASE FROM ONCHOCERCA-VOLVULUS

Survival of Onchocerca volvulus, a pathogenic human filarial parasite, is likely to depend upon the detoxification activities of the glutath ione S-transferases (GSTs). The 24 kDa O. volvulus GST, OvGST2, was ex pressed in a bacterial system and the recombinant protein was purified to homogeneity by affinity chromatography. Specific activities of the recombinant OvGST2 (rOvGST2) with a variety of substrates: and in the presence of inhibitors, were determined. With the universal substrate 1-chloro-2,4-dinitrobenzene, the specific activity of rOvGST2 was 213 0 nmol min(-1) mg(-1). The rOvGST2 showed relatively limited selenium- independent glutathione peroxidase activity, but secondary products of lipid peroxidation, namely members of the trans,trans-alka-2,4-dienal , trans-alk-2-enal and 4-hydroxyalk-2-enal series, were conjugated to glutathione via OvGST2 dependent activity. The gene encoding the OvGST 2 was isolated and the nucleotide sequence determined. The ovgst2 gene was found to possess seven exons with six intervening sequences, with all except one having consensus splice-site junctions. This intron/ex on organisation of the ovgst2 gene is almost identical with those desc ribed for the mammalian Pi class GST genes, consistent with the protei n structural evidence that the OvGST2 is related to the Pi class GSTs. Southern blot analysis with total parasite genomic DNA indicated a si ngle copy gene, with a restriction pattern consistent with that of the isolated gene. The tissue distribution of the OvGST2 was examined in O. volvulus by immunohistochemistry;and was shown to be distinct from that of the OvGST1. The OvGST2 was located throughout the syncytial hy podermis of male and female adult worms, as well as in the uterine epi thelium. Microfilariae, and infective third stage larvae of O. volvulu s; isolated from Simulium neavei, were immunopositive for OvGST2.