Characterization and expression of enzymatically active recombinant filarial prolyl 4-hydroxylase

The cuticle of parasitic nematodes consists primarily of a network of colla gen molecules. The enzyme responsible for collagen maturation is prolyl 4-h ydroxylase, making this enzyme a central activity in cuticle biosynthesis a nd a potentially important chemotherapeutic target. Adult and embryonic Bru gia malayi are shown to be susceptible to inhibitors of vertebrate prolyl 4 -hydroxylase, with exposed parasites exhibiting pathologies consistent with a disruption in cuticle biosynthesis. A full-length cDNA (Ov-phy-1) encodi ng a catalytically active a-subunit of Onchocerca volvulus prolyl 4-hydroxy lase was isolated ana characterized. The derived am ino acid sequence of Ov -phy-1 encoded a peptide that was most similar to the two Caenorhabditis el egans prolyl 4-hydroxylase homologues and to the isoform II enzymes of vert ebrates. Expressed sequence tag (EST) analysis and developmental polymerase chain reaction (PCR) studies demonstrated that Ov-phy-1 was expressed in L 3 and adult parasites. The gene encoding the Ov-phy-1 open reading frame co ntained 11 introns, similar in structure to the gene encoding human prolyl 4-hydroxylase isoform I. Genomic Southern blot, EST and genomic PCR studies demonstrated that the O. volvulus genome contained between three and eight genes closely related to Ov-phy-1. Co-expression of Ov-phy-1 with the O. v olvulus homologue of protein disulfide isomerase in a baculovirus system re sulted in the production of enzymatically active O. volvulus prolyl 4-hydro xylase. In vitro production of enzymatically active O. volvulus prolyl 4-hy droxylase should facilitate identification of specific inhibitors of the pa rasite enzyme. (C) 2001 Elsevier Science B.V. All rights reserved.