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 α2β2 tetramers, in which the β subunit is identical to protein disulfide isomerase (PDI). Two isoforms of the catalytic a 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 α subunit isoform, PRY-3. It is much shorter than the previously characterized vertebrate and C. elegans a 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.