title: Characterization of a novel Caenorhabditis elegans prolyl 4-hydroxylase with a unique substrate specificity and restricted expression in the pharynx and excretory duct.
abstract: Collagen prolyl 4-hydroxylases (C-P4Hs) have a critical role in collagen synthesis, since 4-hydroxyproline residues are necessary for folding of the triple-helical molecules. Vertebrate C-P4Hs are alpha(2)beta(2) tetramers in which the beta subunit is identical to protein-disulfide isomerase (PDI). Three isoforms of the catalytic alpha subunit, PHY-1, PHY-2, and PHY-3, have been characterized from Caenorhabditis elegans, PHY-1 and PHY-2 being responsible for the hydroxylation of cuticle collagens, whereas PHY-3 is predicted to be involved in collagen synthesis in early embryos. We have characterized transcripts of two additional C. elegans alpha subunit-like genes, Y43F8B.4 and C14E2.4. Three transcripts were generated from Y43F8B.4, and a polypeptide encoded by one of them, named PHY-4.1, assembled into active (PHY-4.1)(2)/(PDI-2)(2) tetramers and PHY-4.1/PDI-2 dimers when coexpressed with C. elegans PDI-2 in insect cells. The C14E2.4 transcript was found to have a frameshift leading to the absence of codons for two residues critical for P4H catalytic activity. Thus, C. elegans has altogether four functional C-P4H alpha subunits, PHY-1, PHY-2, PHY-3, and PHY-4.1. The tetramers and dimers containing recombinant PHY-4.1 had a distinct substrate specificity from the other C-P4Hs in that they hydroxylated poly(l-proline) and certain other proline-rich peptides, including ones that are expressed in the pharynx, in addition to collagen-like peptides. These data and the observed restricted expression of the phy-4.1 transcript and PHY-4.1 polypeptide in the pharyngeal gland cells and the excretory duct suggest that in addition to collagens, PHY-4.1 may hydroxylate additional proline-rich proteins in vivo.
authors.affiliation: Collagen Research Unit, Department of Medical Biochemistry and Molecular Biology, University of Oulu, FIN-90014 Oulu, Finland.
authors.forename: Katriina
authors.initials: K
authors.lastname: Keskiaho
authors.forename: Liisa
authors.initials: L
authors.lastname: Kukkola
authors.forename: Antony P
authors.initials: AP
authors.lastname: Page
authors.forename: Alan D
authors.initials: AD
authors.lastname: Winter
authors.forename: Jussi
authors.initials: J
authors.lastname: Vuoristo
authors.forename: Raija
authors.initials: R
authors.lastname: Sormunen
authors.forename: Ritva
authors.initials: R
authors.lastname: Nissi
authors.forename: Päivi
authors.initials: P
authors.lastname: Riihimaa
authors.forename: Johanna
authors.initials: J
authors.lastname: Myllyharju
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000378:metabolism*; D002384:Catalysis; D003094:Collagen / Q000737:chemistry; D015971:Gene Expression Regulation, Enzymologic; D008954:Models, Biological; D008969:Molecular Sequence Data; D010614:Pharynx / Q000201:enzymology*; D011393:Procollagen-Proline Dioxygenase / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D011392:Proline / Q000737:chemistry; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D003094:Collagen; D011392:Proline; D011393:Procollagen-Proline Dioxygenase