Egg Shell Collagen Formation in Caenorhabditis elegans Involves a Novel Prolyl 4-Hydroxylase Expressed in Spermatheca and Embryos and Possessing Many Unique Properties

Abstract

The collagen prolyl 4-hydroxylases (EC 1.14.11.2) play a critical role in the synthesis of all collagens. The enzymes from all vertebrate species studied are ␣ 2 ␤ 2 tetramers, in which the ␤ subunit is identical to protein disulfide isomerase (PDI). Two isoforms of the catalytic ␣ 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 ␣ subunit isoform, PHY-3. It is much shorter than the previously characterized vertebrate and C. elegans ␣ 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.The prolyl 4-hydroxylases (P4Hs), 1 enzymes residing within the lumen of the endoplasmic reticulum, catalyze the formation of 4-hydroxyproline in collagens and more than 15 other proteins (1) by the hydroxylation of proline in X-Pro-Gly repeats (2, 3). P4Hs have a central role in the synthesis of all collagens, as 4-hydroxyproline residues are essential for the folding of the collagen triple helix. In addition, a family of three cytoplasmic P4Hs plays a critical role in the regulation of the hypoxiainducible transcription factor HIF␣ (4, 5).The P4Hs require Fe 2ϩ , 2-oxoglutarate, O 2 , and ascorbate (2, 3). The vertebrate enzymes, and also a P4H from Drosophila melanogaster (6), are ␣ 2 ␤ 2 tetramers in which the ␤ subunits are identical to protein disulfide isomerase (PDI) (2, 3). At least two isoforms of the catalytic ␣ subunit are found in human (7) and mouse (8) tissues, the [␣(I)] 2 ␤ 2 tetramer being the main form in many cell types, whereas [␣(II)] 2 ␤ 2 is found especially in chondrocytes and vascular endothelial cells (9, 10). The properties of the two isoenzymes are very similar, but differences are found between them in the binding of peptide substrates and peptide inhibitors (7, 11).Two P4H ␣ subunits, PHY-1 (also known as DPY-18) and PHY-2, have previously been characterized from Caenorhabditis elegans (12-16). Both are 43-46% identical to the human ␣(I) and ␣(II) subunits, the highest degree of identity being found within the catalytically important C-terminal regions (12,14,16). Unlike the vertebrate ␣ subunits, the C. elegans PHY-1 formed an ␣␤ dimer in insect cell coexpression experiments with human PDI or its corresponding C. ...

Characterization of a Novel Caenorhabditis elegans Prolyl 4-Hydroxylase with a Unique Substrate Specificity and Restricted Expression in the Pharynx and Excretory Duct

“…The recombinant C. elegans PHY-3 differs from PHY-1 and PHY-2 in that C-P4H activity is only generated when it is coexpressed with another C. elegans PDI isoform, PDI-1, but not with PDI-2 (37,38). Whether PHY-3 assembles into a complex with PDI-1 or whether PDI-1 just assists in its folding is as yet unknown, however (37).…”

“…Although enhanced assembly of the PHY-2/PDI-2 dimer and (PHY-2) 2 /(PDI-2) 2 tetramer occurs in phy-1 null C. elegans and C. briggsae, respectively, these C-P4H forms cannot fully compensate for the lack of the mixed tetramer and the PHY-1/PDI-2 dimer (36,40). The recombinant C. elegans PHY-3 differs from PHY-1 and PHY-2 in that C-P4H activity is only generated when it is coexpressed with another C. elegans PDI isoform, PDI-1, but not with PDI-2 (37,38). Whether PHY-3 assembles into a complex with PDI-1 or whether PDI-1 just assists in its folding is as yet unknown, however (37).…”

“…Analyses of genetically mutant nematodes or nematodes subjected to RNA interference (RNAi) have shown that PHY-1 and PHY-2 are involved in the synthesis of cuticle collagens in C. elegans and its close homologue Caenorhabditis briggsae (33)(34)(35)(36)38). C. elegans PHY-3 is expressed in the embryos, late larval stages, and adult nematodes, its expression in the larval and adult stages being restricted to the spermatheca, a specialized region of the gonad where fertilization of the oocytes occurs (37). Nematodes lacking PHY-3 have no obvious phenotypic abnormalities, but the 4-hydroxyproline content of their embryos is markedly reduced, suggesting that PHY-3 is involved in the synthesis of collagens in early embryos, most likely the collagens present in the egg shell (37).…”

“…C. elegans PHY-3 is expressed in the embryos, late larval stages, and adult nematodes, its expression in the larval and adult stages being restricted to the spermatheca, a specialized region of the gonad where fertilization of the oocytes occurs (37). Nematodes lacking PHY-3 have no obvious phenotypic abnormalities, but the 4-hydroxyproline content of their embryos is markedly reduced, suggesting that PHY-3 is involved in the synthesis of collagens in early embryos, most likely the collagens present in the egg shell (37). C. elegans has three PDI isoforms, PDI-1, PDI-2, and PDI-3, of which PDI-2 functions as the C-P4H ␤ subunit (38,39).…”

“…Three C-P4H ␣ subunits, PHY-1, PHY-2, and PHY-3, have been characterized in C. elegans (32)(33)(34)(35)(36)(37), two of which, PHY-1 and PHY-2, are expressed in the cuticle collagen-synthesizing hypodermal cells in a cyclical fashion that corresponds the moulting cycle and the times of maximal cuticle collagen synthesis (34,35). Analyses of genetically mutant nematodes or nematodes subjected to RNA interference (RNAi) have shown that PHY-1 and PHY-2 are involved in the synthesis of cuticle collagens in C. elegans and its close homologue Caenorhabditis briggsae (33)(34)(35)(36)38).…”