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

Abstract

The collagen prolyl 4-hydroxylases (P4Hs) are essential for proper extracellular matrix formation in multicellular organisms. The vertebrate enzymes are α 2β 2 tetramers, in which the β subunits are identical to protein disulfide isomerase (PDI). Unique P4H forms have been shown to assemble from the Caenorhabditis elegans catalytic α subunit isoforms PHY-1 and PHY-2 and the β 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.