Abstract
The collagen prolyl 4-hydroxylases (P4Hs, EC ) play a critical role in the synthesis of the extracellular matrix. The enzymes characterized from vertebrates and Drosophila are alpha(2)beta(2) tetramers, in which protein disulfide isomerase (PDI) serves as the beta subunit. Two conserved alpha subunit isoforms, PHY-1 and PHY-2, have been identified in Caenorhabditis elegans. We report here that three unique P4H forms are assembled from these polypeptides and the single beta subunit PDI-2, both in a recombinant expression system and in vivo, namely a PHY-1/PHY-2/(PDI-2)(2) mixed tetramer and PHY-1/PDI-2 and PHY-2/PDI-2 dimers. The mixed tetramer is the main P4H form in wild-type C. elegans but phy-2-/- and phy-1-/- (dpy-18) mutant nematodes can compensate for its absence by increasing the assembly of the PHY-1/PDI-2 and PHY-2/PDI-2 dimers, respectively. All three of the mixed tetramer-forming polypeptides PHY-1, PHY-2, and PDI-2 are coexpressed in the cuticle collagen-synthesizing hypodermal cells. The catalytic properties of the mixed tetramer are similar to those of other P4Hs, and analogues of 2-oxoglutarate were found to produce severe temperature-dependent effects on P4H mutant strains. Formation of the novel mixed tetramer was species-specific, and studies with hybrid recombinant PHY polypeptides showed that residues Gln(121)-Ala(271) and Asp(1)-Leu(122) in PHY-1 and PHY-2, respectively, are critical for its assembly.
Highlights
The free-living nematode Caenorhabditis elegans represents an excellent model system for studying the extracellular matrix (ECM)[1] and the enzymes involved in its biosynthesis and modification (1–3)
In order to study whether an association could be achieved between the C. elegans PHY-2 and the C. elegans protein disulfide isomerase (PDI)-1, PDI-2, or human PDI, insect cells were coinfected with recombinant viruses coding for PHY-2 and one of the PDI subunits
We describe here a detailed characterization of the unique prolyl 4-hydroxylases (P4Hs) forms that are involved in the synthesis of the cuticular ECM collagens in C. elegans
Summary
The free-living nematode Caenorhabditis elegans represents an excellent model system for studying the extracellular matrix (ECM)[1] and the enzymes involved in its biosynthesis and modification (1–3). We describe here a detailed in vitro and in vivo biochemical characterization of the second conserved C. elegans P4H ␣ subunit isoform PHY-2, demonstrating the presence of a novel mixed PHY-1/PHY-2/(PDI-2)[2] tetramer formed by the three polypeptides both in recombinant insect cell coexpression experiments and in vivo. This mixed tetramer formation is species-specific, and the regions of PHY-1 and PHY-2 required for the association were identified. A nuclear-excluded colocalization of all three polypeptides together with the newly synthesized chains of a cuticle collagen is consistent with their role in the ER modification of collagens synthesized by hypodermal cells and destined for the cuticular ECM
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