Characterization of a Novel Caenorhabditis elegans Prolyl 4-Hydroxylase with a Unique Substrate Specificity and Restricted Expression in the Pharynx and Excretory Duct

Alan D Winter,Katriina Keskiaho,Päivi Riihimaa,Antony P Page,Jussi Vuoristo,Ritva Nissi,Liisa Kukkola,Johanna Myllyharju,Raija Sormunen

doi:10.1074/jbc.m800972200

Alan D Winter, Katriina Keskiaho + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m800972200

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Abstract

Collagen prolyl 4-hydroxylases (C-P4Hs) have a critical role in collagen synthesis, since 4-hydroxyproline residues are necessary for folding of the triple-helical molecules. Vertebrate C-P4Hs are alpha(2)beta(2) tetramers in which the beta subunit is identical to protein-disulfide isomerase (PDI). Three isoforms of the catalytic alpha subunit, PHY-1, PHY-2, and PHY-3, have been characterized from Caenorhabditis elegans, PHY-1 and PHY-2 being responsible for the hydroxylation of cuticle collagens, whereas PHY-3 is predicted to be involved in collagen synthesis in early embryos. We have characterized transcripts of two additional C. elegans alpha subunit-like genes, Y43F8B.4 and C14E2.4. Three transcripts were generated from Y43F8B.4, and a polypeptide encoded by one of them, named PHY-4.1, assembled into active (PHY-4.1)(2)/(PDI-2)(2) tetramers and PHY-4.1/PDI-2 dimers when coexpressed with C. elegans PDI-2 in insect cells. The C14E2.4 transcript was found to have a frameshift leading to the absence of codons for two residues critical for P4H catalytic activity. Thus, C. elegans has altogether four functional C-P4H alpha subunits, PHY-1, PHY-2, PHY-3, and PHY-4.1. The tetramers and dimers containing recombinant PHY-4.1 had a distinct substrate specificity from the other C-P4Hs in that they hydroxylated poly(l-proline) and certain other proline-rich peptides, including ones that are expressed in the pharynx, in addition to collagen-like peptides. These data and the observed restricted expression of the phy-4.1 transcript and PHY-4.1 polypeptide in the pharyngeal gland cells and the excretory duct suggest that in addition to collagens, PHY-4.1 may hydroxylate additional proline-rich proteins in vivo.

Highlights

Two animal prolyl 4-hydroxylase (P4H)2 families are currently known that are functionally distinct, they both belong to the 2-oxoglutarate-dependent dioxygenases and require Fe2ϩ, 2-oxoglutarate, and molecular oxygen for catalysis [1,2,3,4,5,6]
collagen P4Hs (C-P4Hs)-I is the major form in most tissues, and lack of its function leads to early embryonic death in mice, 2 The abbreviations used are: P4H, prolyl 4-hydroxylase; C-P4H, collagen prolyl 4-hydroxylase; HIF, hypoxia-inducible transcription factor; HIF-P4H, HIF prolyl 4-hydroxylase; PHY, ␣ subunit of C. elegans P4H; protein-disulfide isomerase (PDI), proteindisulfide isomerase; RNAi, RNA interference; PBS, phosphate-buffered saline; PBS containing 0.1% Tween 20 (PBST), PBS-Tween; PBCV-1, Paramecium bursaria Chlorella virus-1; E3, ubiquitin-protein isopeptide ligase; RACE, rapid amplification of cDNA ends; HSP, heat shock protein
C. elegans P4H Tetramers and Dimers Containing PHY-4.1 as the Catalytic Subunit Have a Unique Substrate Specificity— Since the Triton X-100-soluble extracts from insect cells expressing the PHY-4.1/PDI-2 dimers and (PHY-4.1)2/(PDI2)2 tetramers seemed to hydroxylate the collagen-like (ProPro-Gly)10 peptide relatively poorly, we studied whether they would hydroxylate other proline-rich sequences (Table 1)

Summary

The abbreviations used are

P4H, prolyl 4-hydroxylase; C-P4H, collagen prolyl 4-hydroxylase; HIF, hypoxia-inducible transcription factor; HIF-P4H, HIF prolyl 4-hydroxylase; PHY, ␣ subunit of C. elegans P4H; PDI, proteindisulfide isomerase; RNAi, RNA interference; PBS, phosphate-buffered saline; PBST, PBS-Tween; PBCV-1, Paramecium bursaria Chlorella virus-1; E3, ubiquitin-protein isopeptide ligase; RACE, rapid amplification of cDNA ends; HSP, heat shock protein. The D. melanogaster genome contains a large gene family of 20 members that encode C-P4H ␣-subunit-like polypeptides [29] One of these has been characterized as a recombinant protein and shown to assemble into an ␣2␤2 tetramer with PDI and to hydroxylate collagen-like peptides [30]. The recombinant (PHY-4.1)2/(PDI-2) tetramer and PHY4.1/PDI-2 dimer had a distinct substrate specificity from other C-P4Hs, in that they hydroxylated poly(L-proline) and certain other proline-rich peptides in addition to collagen-like peptides These data together with the unique expression pattern suggest that in addition to collagens, PHY-4.1 may be involved in the hydroxylation of some other proline-rich proteins in vivo

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