The exoskeleton collagens in Caenorhabditis elegans are modified by prolyl 4-hydroxylases with unique combinations of subunits.
The collagen prolyl 4-hydroxylases (P4Hs, EC ) play a critical role in the synthesis of the extracellular matrix. The enzymes characterized from vertebrates and Drosophila are alpha(2)beta(2) tetramers, in which protein disulfide isomerase (PDI) serves as the beta subunit. Two conserved alpha subunit isoforms, PHY-1 and PHY-2, have been identified in Caenorhabditis elegans. We report here that three unique P4H forms are assembled from these polypeptides and the single beta subunit PDI-2, both in a recombinant expression system and in vivo, namely a PHY-1/PHY-2/(PDI-2)(2) mixed tetramer and PHY-1/PDI-2 and PHY-2/PDI-2 dimers. The mixed tetramer is the main P4H form in wild-type C. elegans but phy-2-/- and phy-1-/- (dpy-18) mutant nematodes can compensate for its absence by increasing the assembly of the PHY-1/PDI-2 and PHY-2/PDI-2 dimers, respectively. All three of the mixed tetramer-forming polypeptides PHY-1, PHY-2, and PDI-2 are coexpressed in the cuticle collagen-synthesizing hypodermal cells. The catalytic properties of the mixed tetramer are similar to those of other P4Hs, and analogues of 2-oxoglutarate were found to produce severe temperature-dependent effects on P4H mutant strains. Formation of the novel mixed tetramer was species-specific, and studies with hybrid recombinant PHY polypeptides showed that residues Gln(121)-Ala(271) and Asp(1)-Leu(122) in PHY-1 and PHY-2, respectively, are critical for its assembly.
Myllyharju
Johanna
J
Collagen Research Unit, Biocenter Oulu and the Department of Medical Biochemistry and Molecular Biology, University of Oulu, FIN-90014 Oulu, Finland.
Kukkola
Liisa
L
Winter
Alan D
AD
Page
Antony P
AP
D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D017173:Caenorhabditis elegans / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry*; D002384:Catalysis; D002460:Cell Line; D003094:Collagen / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D007313:Insecta; D007700:Kinetics; D008856:Microscopy, Fluorescence; D017830:Octoxynol / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D011393:Procollagen-Proline Dioxygenase / Q000378:metabolism*; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
D029742:Caenorhabditis elegans Proteins; D003902:Detergents; D010455:Peptides; D011994:Recombinant Proteins; D012313:RNA; D017830:Octoxynol; D003094:Collagen; D011393:Procollagen-Proline Dioxygenase