title: Prolyl 4-hydroxlase activity is essential for development and cuticle formation in the human infective parasitic nematode Brugia malayi.
abstract: Collagen prolyl 4-hydroxylases (C-P4H) are required for formation of extracellular matrices in higher eukaryotes. These enzymes convert proline residues within the repeat regions of collagen polypeptides to 4-hydroxyproline, a modification essential for the stability of the final triple helix. C-P4H are most often oligomeric complexes, with enzymatic activity contributed by the α subunits, and the β subunits formed by protein disulfide isomerase (PDI). Here, we characterize this enzyme class in the important human parasitic nematode Brugia malayi. All potential C-P4H subunits were identified by detailed bioinformatic analysis of sequence databases, function was investigated both by RNAi in the parasite and heterologous expression in Caenorhabditis elegans, whereas biochemical activity and complex formation were examined via co-expression in insect cells. Simultaneous RNAi of two B. malayi C-P4H α subunit-like genes resulted in a striking, highly penetrant body morphology phenotype in parasite larvae. This was replicated by single RNAi of a B. malayi C-P4H β subunit-like PDI. Surprisingly, however, the B. malayi proteins were not capable of rescuing a C. elegans α subunit mutant, whereas the human enzymes could. In contrast, the B. malayi PDI did functionally complement the lethal phenotype of a C. elegans β subunit mutant. Comparison of recombinant and parasite derived material indicates that enzymatic activity may be dependent on a non-reducible covalent link, present only in the parasite. We therefore demonstrate that C-P4H activity is essential for development of B. malayi and uncover a novel parasite-specific feature of these collagen biosynthetic enzymes that may be exploited in future parasite control.
authors.affiliation: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Garscube Estate, Bearsden Road, Glasgow G61 1QH, Scotland, United Kingdom. Alan.Winter@glasgow.ac.uk
authors.forename: Alan D
authors.initials: AD
authors.lastname: Winter
authors.forename: Gillian
authors.initials: G
authors.lastname: McCormack
authors.forename: Johanna
authors.initials: J
authors.lastname: Myllyharju
authors.forename: Antony P
authors.initials: AP
authors.lastname: Page
mesh_terms: D000818:Animals; D017178:Brugia malayi / Q000201:enzymology* / Q000235:genetics; D017173:Caenorhabditis elegans / Q000201:enzymology / Q000235:genetics; D003094:Collagen / Q000378:metabolism*; D019295:Computational Biology; D015870:Gene Expression; D005786:Gene Expression Regulation; D005816:Genetic Complementation Test; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D009154:Mutation; D011393:Procollagen-Proline Dioxygenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics
chemical_list: D015801:Helminth Proteins; D021122:Protein Subunits; D034741:RNA, Small Interfering; D003094:Collagen; D011393:Procollagen-Proline Dioxygenase