Differences in collagen prolyl 4-hydroxylase assembly between two Caenorhabditis nematode species despite high amino acid sequence identity of the enzyme subunits

The collagen prolyl 4-hydroxylases (P4Hs) are essential for proper extracellular matrix formation in multicellular organisms. The vertebrate enzymes are α2β2 tetramers, in which the β subunits are identical to protein disulfide isomerase (PDI). Unique P4H forms have been shown to assemble from the <i>Caenorhabditis</i> <i>elegans</i> catalytic α subunit isoforms PHY-1 and PHY-2 and the β subunit PDI-2. A mixed PHY-1/PHY-2/(PDI-2)<sub>2</sub> tetramer is the major form, while PHY-1/PDI- 2 and PHY-2/PDI-2 dimers are also assembled but less efficiently. Cloning and characterization of the orthologous subunits from the closely related nematode <i>Caenorhabditis</i> <i>briggsae</i> revealed distinct differences in the assembly of active P4H forms in spite of the extremely high amino acid sequence identity (92-97%) between the <i>C. briggsae</i> and <i>C. elegans</i> subunits. In addition to a PHY-1/PHY-2(PDI-2)<sub>2</sub> tetramer and a PHY-1/PDI-2 dimer, an active (PHY- 2)<sub>2</sub>(PDI-2)<sub>2</sub> tetramer was formed in <i>C. briggsae</i> instead of a PHY-2/PDI-2 dimer. Site-directed mutagenesis studies and generation of inter-species hybrid polypeptides showed that the N-terminal halves of the <i>Caenorhabditis</i> PHY-2 polypeptides determine their assembly properties. Genetic disruption of <i>C. briggsae phy-1</i> (<i>Cb-dpy-18</i>) via a <i>Mos1</i> insertion resulted a small (short) phenotype that is less severe than the dumpy (short and fat) phenotype of the corresponding <i>C. elegans</i> mutants (<i>Ce-dpy-18</i>). <i>C. briggsae</i> <i>phy-2</i> RNA interference produced no visible phenotype in the wild type nematodes but produced a severe dumpy phenotype and larval arrest in <i>phy-1</i> mutants. Genetic complementation of the <i>C. briggsae</i> and <i>C. elegans</i> <i>phy-1</i> mutants was achieved by injection of a wild type <i>phy-1</i> gene from either species

Prolyl 4-hydroxylase:studies on collagen prolyl 4-hydroxylases and related enzymes using the green alga <em>Chlamydomonas reinhardtii</em> and two <em>Caenorhabditis</em> nematode species as model organisms

Abstract Collagen prolyl 4-hydroxylases (C-P4Hs) and related enzymes catalyze the hydroxylation of certain proline residues in animal collagens and plant hydroxyproline-rich proteins, respectively. Animal C-P4Hs and their isoenzymes have been characterized to date from humans, rodents, insects and nematodes. Most of the animal C-P4Hs are α2β2 tetramers in which protein disulphide isomerase (PDI) serves as the β subunit, but the nematode C-P4Hs characterized so far have unique molecular compositions. Two P4Hs have been cloned from the plant Arabidopsis thaliana and one from the Paramecium bursaria Chlorella virus-1, these being monomeric enzymes. This thesis reports on the identification of a large P4H family in the green alga Chlamydomonas reinhardtii and the cloning and characterization of one member, Cr-P4H-1. This is a soluble monomer that hydroxylates in vitro several peptides representing sequences found in C. reinhardtii cell wall proteins. Lack of its activity led to a defective cell wall structure, indicating that Cr-P4H-1 is essential for proper cell wall assembly and that the other P4Hs cannot compensate for the lack of its activity. Two C. elegans genes, Y43F8B.4 and C14E2.4, predicted to code for C-P4H α subunit-like polypeptides were analyzed. Three transcripts were generated from Y43F8B.4, one of them coding for a functional C-P4H α subunit named PHY-4.1. C14E2.4 turned out not to be a C-P4H α subunit gene, as a frame-shift led to the omission of codons for two catalytically critical residues. PHY-4.1 formed active tetramers and dimers with PDI-2 and had unique substrate requirements in that it hydroxylated certain other proline-rich sequences besides collagen-like peptides. Inactivation of the Y43F8B.4 gene led to no obvious morphological abnormalities. Spatial expression of the phy-4.1 transcript and PHY-4.1 polypeptide was localized to the pharynx and the excretory duct. Taken together, these data indicate that PHY-4.1 is not involved in the hydroxylation of cuticular collagens but is likely to have other substrates in vivo. Cloning and characterization of the PHY-1 and PHY-2 subunits from the closely related nematode Caenorhabditis briggsae revealed distinct differences in assembly properties between the C. elegans and C. briggsae PHY-2 subunits in spite of their high amino acid sequence identity. Genetic disruption of C. briggsae phy-1 resulted in a less severe phenotype than that observed in C. elegans, evidently on account of its more efficient assembly of the C. briggsae PHY-2 to an active C-P4H explaining the milder phenotype. Rescue of C. elegans and C. briggsae phy-1 mutants was achieved by injection of a wild-type phy-1 gene from either species