Differences in collagen prolyl 4-hydroxylase assembly between two Caenorhabditis nematode species despite high amino acid sequence identity of the enzyme subunits
Winter, Alan D.
Page, Antony P.
- Publisher: Elsevier BV
The collagen prolyl 4-hydroxylases (P4Hs) are\ud essential for proper extracellular matrix\ud formation in multicellular organisms. The\ud vertebrate enzymes are α2β2 tetramers, in\ud which the β subunits are identical to protein\ud disulfide isomerase (PDI). Unique P4H forms\ud have been shown to assemble from the\ud <i>Caenorhabditis</i> <i>elegans</i> catalytic α subunit\ud isoforms PHY-1 and PHY-2 and the β subunit\ud PDI-2. A mixed PHY-1/PHY-2/(PDI-2)<sub>2</sub>\ud tetramer is the major form, while PHY-1/PDI-\ud 2 and PHY-2/PDI-2 dimers are also assembled\ud but less efficiently. Cloning and\ud characterization of the orthologous subunits\ud from the closely related nematode\ud <i>Caenorhabditis</i> <i>briggsae</i> revealed distinct\ud differences in the assembly of active P4H\ud forms in spite of the extremely high amino\ud acid sequence identity (92-97%) between the\ud <i>C. briggsae</i> and <i>C. elegans</i> subunits. In\ud addition to a PHY-1/PHY-2(PDI-2)<sub>2</sub> tetramer\ud and a PHY-1/PDI-2 dimer, an active (PHY-\ud 2)<sub>2</sub>(PDI-2)<sub>2</sub> tetramer was formed in <i>C.\ud briggsae</i> instead of a PHY-2/PDI-2 dimer.\ud Site-directed mutagenesis studies and\ud generation of inter-species hybrid polypeptides\ud showed that the N-terminal halves of the\ud <i>Caenorhabditis</i> PHY-2 polypeptides\ud determine their assembly properties. Genetic\ud disruption of <i>C. briggsae phy-1</i> (<i>Cb-dpy-18</i>)\ud via a <i>Mos1</i> insertion resulted a small (short)\ud phenotype that is less severe than the dumpy\ud (short and fat) phenotype of the corresponding\ud <i>C. elegans</i> mutants (<i>Ce-dpy-18</i>). <i>C. briggsae</i>\ud <i>phy-2</i> RNA interference produced no visible\ud phenotype in the wild type nematodes but\ud produced a severe dumpy phenotype and larval\ud arrest in <i>phy-1</i> mutants. Genetic\ud complementation of the <i>C. briggsae</i> and <i>C.\ud elegans</i> <i>phy-1</i> mutants was achieved by\ud injection of a wild type <i>phy-1</i> gene from either\ud species.