title: A hypodermally expressed prolyl 4-hydroxylase from the filarial nematode Brugia malayi is soluble and active in the absence of protein disulfide isomerase.
abstract: The collagen prolyl 4-hydroxylase (P4H) class of enzymes catalyze the hydroxylation of prolines in the X-Pro-Gly repeats of collagen chains. This modification is central to the synthesis of all collagens. Most P4Hs are alpha(2)beta(2) tetramers with the catalytic activity residing in the alpha subunits. The beta subunits are identical to the enzyme protein disulfide isomerase. The nematode cuticle is a collagenous extracellular matrix required for maintenance of the worm body shape. Examination of the model nematode Caenorhabditis elegans has demonstrated that its unique P4Hs are essential for viability and body morphology. The filarial parasite Brugia malayi is a causative agent of lymphatic filariasis in humans. We report here on the cloning and characterization of a B. malayi P4H with unusual properties. The recombinant B. malayi alpha subunit, PHY-1, is a soluble and active P4H by itself, and it does not become associated with protein disulfide isomerase. The active enzyme form is a homotetramer with catalytic and inhibition properties similar to those of the C. elegans P4Hs. High levels of B. malayi phy-1 transcript expression were observed in all developmental stages examined, and its expression was localized to the cuticle-synthesizing hypodermal tissue in the heterologous host C. elegans. Although active by itself, the B. malayi PHY-1 was not able to replace enzyme function in a C. elegans P4H mutant.
authors.affiliation: Wellcome Centre for Molecular Parasitology, Anderson College, University of Glasgow, Scotland, United Kingdom.
authors.forename: Alan D
authors.initials: AD
authors.lastname: Winter
authors.forename: Johanna
authors.initials: J
authors.lastname: Myllyharju
authors.forename: Antony P
authors.initials: AP
authors.lastname: Page
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D017178:Brugia malayi / Q000201:enzymology*; D017173:Caenorhabditis elegans; D002460:Cell Line; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D006801:Humans; D007313:Insecta; D008957:Models, Genetic; D008969:Molecular Sequence Data; D011393:Procollagen-Proline Dioxygenase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D011401:Promoter Regions, Genetic; D019704:Protein Disulfide-Isomerases / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
chemical_list: D018076:DNA, Complementary; C471558:Phy-1 protein, Brugia malayi; D011994:Recombinant Proteins; D011393:Procollagen-Proline Dioxygenase; D019704:Protein Disulfide-Isomerases