Characterization and expression of enzymatically active recombinant filarial prolyl 4-hydroxylase.
The cuticle of parasitic nematodes consists primarily of a network of collagen molecules. The enzyme responsible for collagen maturation is prolyl 4-hydroxylase, making this enzyme a central activity in cuticle biosynthesis and a potentially important chemotherapeutic target. Adult and embryonic Brugia 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) encoding a catalytically active alpha-subunit of Onchocerca volvulus prolyl 4-hydroxylase was isolated and characterized. The derived amino acid sequence of Ov-phy-1 encoded a peptide that was most similar to the two Caenorhabditis elegans prolyl 4-hydroxylase homologues and to the isoform II enzymes of vertebrates. Expressed sequence tag (EST) analysis and developmental polymerase chain reaction (PCR) studies demonstrated that Ov-phy-1 was expressed in L3 and adult parasites. The gene encoding the Ov-phy-1 open reading frame contained 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. volvulus homologue of protein disulfide isomerase in a baculovirus system resulted in the production of enzymatically active O. volvulus prolyl 4-hydroxylase. In vitro production of enzymatically active O. volvulus prolyl 4-hydroxylase should facilitate identification of specific inhibitors of the parasite enzyme.
Merriweather
A
A
Division of Geographic Medicine, University of Alabama at Birmingham, BBRB 203, 1530 3rd Avenue South, Birmingham, AL 35294-2170, USA.
Guenzler
V
V
Brenner
M
M
Unnasch
T R
TR
D000595:Amino Acid Sequence; D000818:Animals; D015139:Blotting, Southern; D017178:Brugia malayi / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D017238:Genes, Helminth; D006801:Humans; D007527:Isoenzymes / Q000235:genetics; D008969:Molecular Sequence Data; D017181:Onchocerca volvulus / Q000196:embryology / Q000201:enzymology / Q000235:genetics*; D016366:Open Reading Frames; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011347:Procollagen / Q000378:metabolism; D011393:Procollagen-Proline Dioxygenase / Q000037:antagonists & inhibitors / Q000145:classification / Q000235:genetics*; D011753:Pyrones / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011347:Procollagen; D011753:Pyrones; D011994:Recombinant Proteins; D011393:Procollagen-Proline Dioxygenase; C052808:coumalic acid