Abstract
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.
Highlights
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]
Amino Acid Sequence of PHY-3 and Its Comparison with Those of Other P4H ␣ Subunits—A sequence homology search indicated that the C. elegans genome contains an open reading frame T20B3.7 consisting of four exons that encodes a 239amino acid polypeptide showing sequence similarity to the conserved C-terminal region of the vertebrate P4H ␣(I) and ␣(II) subunits [7, 8, 30] and C. elegans PHY-1 and PHY-2 [12, 14, 15] (Figs. 1 and 2B)
Expression of phy-3 was detected only in embryos and in the late larval and adult nematodes, and expression in the latter was restricted to the spermatheca, indicating that PHY-3 is not involved in the synthesis of cuticle collagens
Summary
The prolyl 4-hydroxylases (P4Hs), 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]. Two P4H ␣ subunits, PHY-1 ( known as DPY-18) and PHY-2, have previously been characterized from Caenorhabditis elegans [12,13,14,15,16]. 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]. The phy-1 and phy-2 genes are expressed in collagen-synthesizing hypodermal cells at times of maximal collagen synthesis, suggesting an important role in cuticle formation at all developmental stages [14]. The phy-1;phy-2 double mutant was embryonic lethal, suggesting that phy-2 is required for phy-1 mutant viability [14, 15]
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