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

Winter, A. D., Keskiaho, K., Kukkalo, L., McCormack, G., Felix, M.-A., Myllyharju, J. and Page, A. P. (2007) Differences in collagen prolyl 4-hydroxylase assembly between two Caenorhabditis nematode species despite high amino acid sequence identity of the enzyme subunits. Matrix Biology, 26(5), pp. 382-395. (doi: 10.1016/j.matbio.2007.01.008)

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Abstract

The collagen prolyl 4-hydroxylases (P4Hs) are essential for proper extracellular matrix formation in multicellular organisms. The vertebrate enzymes are α2β2 tetramers, in which the β subunits are identical to protein disulfide isomerase (PDI). Unique P4H forms have been shown to assemble from the <i>Caenorhabditis</i> <i>elegans</i> catalytic α subunit isoforms PHY-1 and PHY-2 and the β subunit PDI-2. A mixed PHY-1/PHY-2/(PDI-2)<sub>2</sub> 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 <i>Caenorhabditis</i> <i>briggsae</i> revealed distinct differences in the assembly of active P4H forms in spite of the extremely high amino acid sequence identity (92-97%) between the <i>C. briggsae</i> and <i>C. elegans</i> subunits. In addition to a PHY-1/PHY-2(PDI-2)<sub>2</sub> tetramer and a PHY-1/PDI-2 dimer, an active (PHY- 2)<sub>2</sub>(PDI-2)<sub>2</sub> tetramer was formed in <i>C. briggsae</i> 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 <i>Caenorhabditis</i> PHY-2 polypeptides determine their assembly properties. Genetic disruption of <i>C. briggsae phy-1</i> (<i>Cb-dpy-18</i>) via a <i>Mos1</i> insertion resulted a small (short) phenotype that is less severe than the dumpy (short and fat) phenotype of the corresponding <i>C. elegans</i> mutants (<i>Ce-dpy-18</i>). <i>C. briggsae</i> <i>phy-2</i> RNA interference produced no visible phenotype in the wild type nematodes but produced a severe dumpy phenotype and larval arrest in <i>phy-1</i> mutants. Genetic complementation of the <i>C. briggsae</i> and <i>C. elegans</i> <i>phy-1</i> mutants was achieved by injection of a wild type <i>phy-1</i> gene from either species.

Item Type:Articles
Keywords:Prolyl 4-hydroxylase, Collagen biosynthesis, C. elegans.
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Page, Professor Tony
Authors: Winter, A. D., Keskiaho, K., Kukkalo, L., McCormack, G., Felix, M.-A., Myllyharju, J., and Page, A. P.
Subjects:Q Science > QH Natural history > QH345 Biochemistry
College/School:College of Medical Veterinary and Life Sciences
College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Research Group:Page Group
Journal Name:Matrix Biology
Publisher:Elsevier BV
ISSN:0945-053X
ISSN (Online):1569-1802
Copyright Holders:Copyright © 2007 Elsevier
First Published:First published in Matrix Biology 26(5):382-395
Publisher Policy:Reproduced in accordance with the copyright policy of the publisher
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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
229993The Molecular Enzymology of Collagen Assembly and Post-Translational Modification: a Nematode Model SystemAntony PageMedical Research Council (MRC)G117/476Infection Immunity and Inflammation Life Sciences