P4HA3 (prolyl 4-hydroxylase subunit alpha-3) is the third catalytic alpha-subunit isoform of collagen prolyl 4-hydroxylase (C-P4H). The active enzyme is an alpha2-beta2 heterotetramer in which the two catalytic alpha subunits combine with two beta subunits that are identical to protein disulfide isomerase (PDI/P4HB). P4HA3 is an endoplasmic reticulum lumenal, Fe(II)- and 2-oxoglutarate-dependent dioxygenase that, using L-ascorbate as a cofactor and molecular oxygen as co-substrate, hydroxylates proline residues in -X-Pro-Gly- triplets of procollagen and related proteins to form trans-4-hydroxyproline (consuming 2-oxoglutarate and producing succinate and CO2). 4-hydroxyproline formation is essential for folding and thermal stability of the collagen triple helix. The alpha-3 isoenzyme has catalytic properties similar to the type I and type II C-P4Hs but with intermediate peptide-substrate binding properties, and its mRNA is expressed broadly but at much lower levels than the alpha(I)/alpha(II) isoforms, being most abundant in placenta, liver, and fetal skin and detectable in vascular smooth muscle and the fibrous cap of atherosclerotic lesions.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004656 procollagen-proline 4-dioxygenase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic (IBA) assignment of the core collagen prolyl 4-hydroxylase activity, consistent with the direct experimental EXP evidence for P4HA3 and with conserved catalytic residues across the P4HA family. Reason: This is the defining core molecular function of P4HA3; the IBA inference agrees with the experimental characterization of the recombinant alpha(III) enzyme. Supporting Evidence: PMID:14500733 led to the formation of an active enzyme that hydroxylated collagen chains and a collagen-like peptide and appeared to be an [alpha(III)]2 beta 2 tetramer |
| GO:0016222 procollagen-proline 4-dioxygenase complex | IBA GO_REF:0000033 | ACCEPT | Summary: P4HA3 is the catalytic alpha subunit of the alpha2-beta2 collagen prolyl 4-hydroxylase tetramer, in which the beta subunit is PDI. Membership in the prolyl 4-hydroxylase complex is the correct cellular-component assignment. Reason: Directly supported; the recombinant alpha(III) assembles with PDI into an [alpha(III)]2-beta2 tetramer, i.e. the procollagen-proline 4-dioxygenase complex. Supporting Evidence: PMID:14500733 The vertebrate enzymes are alpha 2 beta 2 tetramers, the beta-subunit being identical to protein-disulfide isomerase (PDI) |
| GO:0004656 procollagen-proline 4-dioxygenase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic assignment of the core dioxygenase activity from the EC/Rhea mapping (EC 1.14.11.2; RHEA:18945), redundant with the experimental and IBA evidence. Reason: Correct core molecular function; the EC/Rhea reaction matches the curated catalytic activity of P4HA3. Supporting Evidence: file:human/P4HA3/P4HA3-uniprot.txt EC=1.14.11.2 {ECO:0000269|PubMed:14500733} |
| GO:0005506 iron ion binding | IEA GO_REF:0000002 | ACCEPT | Summary: P4HA3 binds one Fe(2+) ion per subunit as the catalytic metal of its Fe(II)/2-oxoglutarate-dependent dioxygenase reaction; the InterPro-based electronic assignment is accurate. Reason: Iron coordination (residues 440, 442, 510) is essential for catalysis; correct cofactor molecular function supporting the core activity. Supporting Evidence: file:human/P4HA3/P4HA3-uniprot.txt Binds 1 Fe(2+) ion per subunit |
| GO:0005783 endoplasmic reticulum | IEA GO_REF:0000120 | ACCEPT | Summary: Collagen prolyl 4-hydroxylation occurs in the ER; the electronic ER localization is consistent with the UniProt subcellular location and the secretory-pathway signal peptide. Reason: Correct compartment; the more specific ER lumen term is also annotated. Supporting Evidence: file:human/P4HA3/P4HA3-uniprot.txt SUBCELLULAR LOCATION: Endoplasmic reticulum lumen |
| GO:0005788 endoplasmic reticulum lumen | IEA GO_REF:0000044 | ACCEPT | Summary: The C-P4H tetramer resides and acts in the ER lumen, the site of procollagen folding; this is the precise correct compartment for P4HA3. Reason: Correct and specific subcellular location, supported by the UniProt subcellular location and Reactome curation. Supporting Evidence: file:human/P4HA3/P4HA3-uniprot.txt SUBCELLULAR LOCATION: Endoplasmic reticulum lumen |
| GO:0016705 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen | IEA GO_REF:0000002 | ACCEPT | Summary: P4HA3 is a 2-oxoglutarate/Fe(II)-dependent dioxygenase that incorporates oxygen using paired donors (prolyl substrate and 2-oxoglutarate); this is a correct parent molecular function of the specific procollagen-proline 4-dioxygenase activity. Reason: Accurate general oxidoreductase chemistry; the specific GO:0004656 captures the core function more informatively, but this parent term is not wrong. Supporting Evidence: file:human/P4HA3/P4HA3-uniprot.txt Reaction=L-prolyl-[collagen] + 2-oxoglutarate + O2 = trans-4-hydroxy-L- prolyl-[collagen] + succinate + CO2 |
| GO:0031418 L-ascorbic acid binding | IEA GO_REF:0000002 | ACCEPT | Summary: L-ascorbate (vitamin C) is a required cofactor of collagen prolyl 4-hydroxylases, maintaining the iron in the reduced state; the electronic assignment is accurate (KM 370 uM for L-ascorbate). Reason: Correct cofactor-binding molecular function supporting the core dioxygenase activity. Supporting Evidence: file:human/P4HA3/P4HA3-uniprot.txt Name=L-ascorbate |
| GO:0005515 protein binding | IPI PMID:25416956 A proteome-scale map of the human interactome network. | KEEP AS NON CORE | Summary: Bare protein binding derived from a large-scale binary (Y2H) interactome reference map. The captured partners are systematic-screen hits unrelated to the physiological PDI/collagen interactions, and the term is uninformative. Reason: Real IntAct interactome captures but uninformative bare protein binding; not the functional alpha2-beta2/PDI interaction, so not elevated to core (per curation guidance). Supporting Evidence: PMID:25416956 a systematic map of ?14,000 high-quality human binary protein-protein interactions |
| GO:0005515 protein binding | IPI PMID:26871637 Widespread Expansion of Protein Interaction Capabilities by ... | KEEP AS NON CORE | Summary: Bare protein binding from a high-throughput interactome screen examining alternative splicing isoform interactions; uninformative for P4HA3's catalytic function. Reason: Valid high-throughput interaction capture but uninformative bare protein binding; does not reflect the core collagen-hydroxylase function. Supporting Evidence: file:human/P4HA3/P4HA3-uniprot.txt Q7Z4N8; O43379: WDR62 |
| GO:0005515 protein binding | IPI PMID:31515488 Extensive disruption of protein interactions by genetic vari... | KEEP AS NON CORE | Summary: Bare protein binding from a high-throughput interactome screen assaying disruption of protein interactions by genetic variants; partners are screen captures unrelated to the core enzymatic function. Reason: Real interactome capture but uninformative bare protein binding; not a core function. Supporting Evidence: file:human/P4HA3/P4HA3-uniprot.txt Q7Z4N8; P43356: MAGEA2B |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | KEEP AS NON CORE | Summary: Bare protein binding from the HuRI binary interactome reference map; the large partner list comprises systematic Y2H captures (keratin-associated proteins, MAGE proteins, transcription factors, etc.) not relevant to collagen prolyl 4-hydroxylation. Reason: Real interactome captures but uninformative bare protein binding; per guidelines, kept non-core rather than elevated or removed. Supporting Evidence: file:human/P4HA3/P4HA3-uniprot.txt Q7Z4N8; O14964: HGS |
| GO:0004656 procollagen-proline 4-dioxygenase activity | EXP PMID:14500733 Identification and characterization of a third human, rat, a... | ACCEPT | Summary: Direct experimental demonstration that recombinant human alpha(III), coexpressed with PDI, forms an active enzyme that hydroxylates collagen chains and a collagen-like peptide. This is the founding biochemical characterization establishing P4HA3's core function. Reason: Core molecular function with direct experimental (EXP) support; the recombinant enzyme hydroxylated collagen substrates with catalytic properties similar to the type I/II C-P4Hs. Supporting Evidence: PMID:14500733 led to the formation of an active enzyme that hydroxylated collagen chains and a collagen-like peptide and appeared to be an [alpha(III)]2 beta 2 tetramer |
| GO:0005788 endoplasmic reticulum lumen | TAS Reactome:R-HSA-1650808 | ACCEPT | Summary: Reactome curation of P4HA3 in the ER lumen, where the collagen prolyl 4-hydroxylase reaction occurs; consistent with the UniProt subcellular location. Reason: Correct and specific compartment; redundant with the IEA ER lumen annotation. Supporting Evidence: file:human/P4HA3/P4HA3-uniprot.txt SUBCELLULAR LOCATION: Endoplasmic reticulum lumen |
| GO:0005788 endoplasmic reticulum lumen | TAS Reactome:R-HSA-9918779 | ACCEPT | Summary: Reactome curation of ER-lumen localization in the context of proline hydroxylation of a (viral) polyprotein substrate; the compartment assignment for P4HA3 is correct. Reason: Correct compartment; redundant with other ER lumen annotations. Supporting Evidence: file:human/P4HA3/P4HA3-uniprot.txt SUBCELLULAR LOCATION: Endoplasmic reticulum lumen |
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Download this section (compressed HTML)Q: Does P4HA3-containing C-P4H have a distinct substrate preference (e.g. for particular collagen types or non-collagenous Pro-Gly-containing substrates) that distinguishes it from the alpha(I)- and alpha(II)-containing isoenzymes, given its intermediate peptide-binding properties?
Q: Is the elevated P4HA3 expression in vascular smooth muscle, atherosclerotic fibrous cap, and certain tumors associated with a direct collagen-hydroxylation role in ECM remodeling, or with non-catalytic/contextual functions?
Experiment: Reconstitute recombinant [alpha(III)]2-beta2 (P4HA3/PDI) C-P4H and measure steady-state kinetics and substrate specificity against a panel of collagen-type peptides versus the type I and type II C-P4Hs to define isoenzyme-specific substrate preferences.
Experiment: CRISPR knockout or knockdown of P4HA3 in cells with high endogenous expression (e.g. placental or vascular smooth muscle cells) followed by mass spectrometry quantification of collagen 4-hydroxyproline content to assess its non-redundant contribution to collagen hydroxylation in vivo.
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