The Exoskeleton Collagens in Caenorhabditis elegans Are Modified by Prolyl 4-Hydroxylases with Unique Combinations of Subunits

Abstract

The collagen prolyl 4-hydroxylases (P4Hs, EC 1.14.11.2) play a critical role in the synthesis of the extracellular matrix. The enzymes characterized from vertebrates and Drosophila are ␣ 2 ␤ 2 tetramers, in which protein disulfide isomerase (PDI) serves as the ␤ subunit. Two conserved ␣ 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 ␤ 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.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). The major ECM formed in C. elegans is the collagenous cuticle or exoskeleton, a protective structure that is synthesized repeatedly during development. Over 150 small collagen genes are involved in the formation of this structure (2), producing stage-specific cuticles that are both structurally and chemically distinct (3).Collagen prolyl 4-hydroxylases (P4Hs) are enzymes resident in the endoplasmic reticulum (ER) (4, 5) that play a critical role in the synthesis and processing of all collagens (6). Recently, an additional family of cytoplasmic P4Hs has been shown to play an essential role in O 2 sensing and the hypoxia response (7,8).The collagen P4Hs present in vertebrates (4, 5) and in Drosophila (9) are ␣ 2 ␤ 2 tetramers in which the multifunctional chaperone protein disulfide isomerase (PDI) serves as the ␤ subunit. The ␣ subunit binds Fe 2ϩ , 2-oxoglutarate, and ascorbate (4, 5) and possesses the peptide substrate-binding site (10). The main role of PDI is to retain the ␣ subunits in a catalytically active, nonaggregated conformation within the ER (11-13). Two conserved P4H ␣ subunit isoforms have been described in human and mouse tissues, which form [␣(I)] 2 ␤ 2 or [␣(II)] 2 ␤ 2 tetramers (14 -16). Initial characterization of a recombinant C. elegans ␣ subunit isoform (PHY-1) revealed a unique association in that it formed an active ␣␤ dimer with both human PDI and its C. elegans orthologue PDI-2 but no...

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

“…A collagen P4H tetramer containing two different catalytically active subunits has not been found in any other organism, and insect cell co-expression studies have shown that the human (I) and (II) subunits do not assemble into mixed tetramers of this type (10). Formation of the C. elegans mixed tetramer is species-specific as the C. elegans PHY-1, PHY-2 or PDI-2 could not be replaced in insect cell coexpression experiments by a recombinant collagen P4H or subunit from human or Drosophila (20). Detailed studies with hybrid recombinant PHY-1/PHY-2 polypeptides revealed that PHY-1 residues Gln121-Ala271 and PHY-2 residues Asp1-Leu122 are essential for the assembly of the mixed tetramer (20).…”

“…The nematode Caenorhabditis elegans has two conserved genes encoding collagen P4H subunits, named phy-1 (also known as dpy -18) and phy-2 that are involved in the synthesis of cuticle collagens (17)(18)(19)(20). Based on in vivo and in vitro studies these subunits assemble into unique collagen P4H forms with a subunit encoded by the pdi-2 gene (17,(21)(22)(23).…”

“…Based on in vivo and in vitro studies these subunits assemble into unique collagen P4H forms with a subunit encoded by the pdi-2 gene (17,(21)(22)(23). The main collagen P4H form in C. elegans is a PHY-1/PHY-2/(PDI-2) 2 tetramer, but a PHY-1/PDI-2 dimer and a PHY-2/PDI-2 dimer can also be assembled albeit at a much lower efficiency (20)(21)(22). A collagen P4H tetramer containing two different catalytically active subunits has not been found in any other organism, and insect cell co-expression studies have shown that the human (I) and (II) subunits do not assemble into mixed tetramers of this type (10).…”

“…Formation of the C. elegans mixed tetramer is species-specific as the C. elegans PHY-1, PHY-2 or PDI-2 could not be replaced in insect cell coexpression experiments by a recombinant collagen P4H or subunit from human or Drosophila (20). Detailed studies with hybrid recombinant PHY-1/PHY-2 polypeptides revealed that PHY-1 residues Gln121-Ala271 and PHY-2 residues Asp1-Leu122 are essential for the assembly of the mixed tetramer (20).…”

“…The C. elegans phy-1, phy-2 and pdi-2 genes are expressed in the hypodermis (17,20), a tissue whose main role is to secrete the collagenous exoskeleton known as the cuticle (23). All three genes are expressed temporally in a cyclical pattern that matches the expression of the exoskeleton collagens, namely the molting cycle (17).…”