Characterization of a novel Caenorhabditis elegans prolyl 4-hydroxylase with a unique substrate specificity and restricted expression in the pharynx and excretory duct.

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.

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 alpha(2)beta(2) tetramers, in which the beta subunits are identical to protein disulfide isomerase (PDI). Unique P4H forms have been shown to assemble from the Caenorhabditis elegans catalytic alpha subunit isoforms PHY-1 and PHY-2 and the beta subunit PDI-2. A mixed PHY-1/PHY-2/(PDI-2)(2) 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 Caenorhabditis briggsae revealed distinct differences in the assembly of active P4H forms in spite of the extremely high amino acid sequence identity (92-97%) between the C. briggsae and C. elegans subunits. In addition to a PHY-1/PHY-2(PDI-2)(2) tetramer and a PHY-1/PDI-2 dimer, an active (PHY-2)(2)(PDI-2)(2) tetramer was formed in C. briggsae 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 Caenorhabditis PHY-2 polypeptides determine their assembly properties. Genetic disruption of C. briggsae phy-1 (Cb-dpy-18) via a Mos1 insertion resulted in a small (short) phenotype that is less severe than the dumpy (short and fat) phenotype of the corresponding C. elegans mutants (Ce-dpy-18). C. briggsae phy-2 RNA interference produced no visible phenotype in the wild type nematodes but produced a severe dumpy phenotype and larval arrest in phy-1 mutants. Genetic complementation of the C. briggsae and C. elegans phy-1 mutants was achieved by injection of a wild type phy-1 gene from either species.

Chlamydomonas reinhardtii has multiple prolyl 4-hydroxylases, one of which is essential for proper cell wall assembly.

Prolyl 4-hydroxylases (P4Hs) catalyze formation of 4-hydroxyproline (4Hyp), which is found in many plant glycoproteins. We cloned and characterized Cr-P4H-1, one of 10 P4H-like Chlamydomonas reinhardtii polypeptides. Recombinant Cr-P4H-1 is a soluble 29-kD monomer that effectively hydroxylated in vitro both poly(l-Pro) and synthetic peptides representing Pro-rich motifs found in the Chlamydomonas cell wall Hyp-rich glycoprotein (HRGP) GP1. Similar Pro-rich repeats that are likely to be Cr-P4H-1 substrates are also present in the cell wall HRGP GP2 and probably GP3. Suppression of the gene encoding Cr-P4H-1 by RNA interference led to a defective cell wall consisting of a loose network of fibrils resembling the inner and outer W1 and W7 layers of the wild-type wall, while the layers forming the dense central triplet were absent. The lack of Cr-P4H-1 most probably affected 4Hyp content of the major HRPGs of the central triplet, GP1, GP2, and GP3. The reduced 4Hyp levels in these HRGPs can also be expected to affect their glycosylation and, thus, the interactive properties and stabilities of their fibrous shafts. Interestingly, our RNA interference data indicate that the nine other Chlamydomonas P4H-like polypeptides could not fully compensate for the lack of Cr-P4H-1 activity and are therefore likely to have different substrate specificities and functions.

Egg shell collagen formation in Caenorhabditis elegans involves a novel prolyl 4-hydroxylase expressed in spermatheca and embryos and possessing many unique properties.

The collagen prolyl 4-hydroxylases (EC ) play a critical role in the synthesis of all collagens. The enzymes from all vertebrate species studied are alpha(2)beta(2) tetramers, in which the beta subunit is identical to protein disulfide isomerase (PDI). Two isoforms of the catalytic alpha subunit, PHY-1 and PHY-2, have previously been characterized from Caenorhabditis elegans. We report here on the cloning and characterization of a third C. elegans alpha subunit isoform, PHY-3. It is much shorter than the previously characterized vertebrate and C. elegans alpha subunits and shows 23-30% amino acid sequence identity to PHY-1 and PHY-2 within the catalytic C-terminal region. Recombinant PHY-3 coexpressed in insect cells with a C. elegans PDI isoform that does not associate with PHY-1 was found to be an active prolyl 4-hydroxylase. The phy-3 gene consists of five exons, and its expression pattern differs distinctly from the hypodermally expressed phy-1 and phy-2 in that it is expressed in embryos, late larval stages, and adult nematodes, expression in the latter being restricted to the spermatheca. Nematodes homozygous for a phy-3 deletion are phenotypically of the wild type and fertile, but the 4-hydroxyproline content of phy-3(-/-) early embryos was reduced by about 90%. PHY-3 is thus likely to be involved in the synthesis of collagens in early embryos, probably of those in the egg shell.