P4HA1 is the catalytic alpha-1 subunit of collagen prolyl 4-hydroxylase (C-P4H; EC 1.14.11.2), an endoplasmic reticulum-lumenal, Fe(II)- and 2-oxoglutarate-dependent dioxygenase. The active enzyme is an alpha2-beta2 heterotetramer in which the beta subunit is P4HB (protein disulfide-isomerase, PDI); the two catalytic alpha subunits provide the peptide-substrate-binding and Fe2OG dioxygenase domains, while P4HB acts as a structural subunit and retains the tetramer in the ER lumen through its C-terminal KDEL sequence (the alpha subunit lacks a retention signal). C-P4H catalyzes the post-translational formation of trans-4-hydroxy-L-proline from proline residues in -Xaa-Pro-Gly- sequences of procollagen and other proteins, consuming O2 and 2-oxoglutarate (which is decarboxylated to succinate and CO2) and requiring ascorbate (vitamin C) to maintain the active-site iron in the reduced state. 4-Hydroxyproline is essential for the thermal stability of the collagen triple helix, making this the key rate-limiting modification in collagen maturation and a central enzyme of collagen biosynthesis. Two enzyme isotypes exist (type I, with P4HA1, and type II, with the paralog P4HA2); the alpha-1 and alpha-2 subunits do not form mixed tetramers. P4HA1 is broadly expressed, with highest levels in collagen-producing tissues.
Definition: The hydroxylation of peptidyl-proline to peptidyl-4-hydroxy-L-proline, the direct biological process catalyzed by P4HA1 (corresponds to the existing term GO:0018401).
Justification: The existing BP annotations (collagen fibril organization) are downstream/indirect. The direct biological process performed by P4HA1 is peptidyl-proline hydroxylation to 4-hydroxy-L-proline (GO:0018401), which would more precisely capture its core process role; consider adding an involved_in annotation to GO:0018401 or a collagen biosynthetic process term.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004656 procollagen-proline 4-dioxygenase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic (IBA) assignment of the core catalytic molecular function of P4HA1 as the alpha subunit of collagen prolyl 4-hydroxylase. Reason: This is the defining, experimentally demonstrated molecular function of P4HA1; the IBA call is consistent with the direct IDA evidence (PMID:9211872) and EC 1.14.11.2. Supporting Evidence: file:human/P4HA1/P4HA1-uniprot.txt Catalyzes the post-translational formation of 4- |
| GO:0016222 procollagen-proline 4-dioxygenase complex | IBA GO_REF:0000033 | ACCEPT | Summary: P4HA1 is part of the alpha2-beta2 collagen prolyl 4-hydroxylase complex (alpha = P4HA1, beta = P4HB/PDI); phylogenetic assignment of complex membership. Reason: The active enzyme is the alpha2-beta2 tetramer; P4HA1 is a constitutive catalytic subunit of this complex. Core cellular component. Supporting Evidence: file:human/P4HA1/P4HA1-uniprot.txt Heterotetramer of two alpha-1 chains and two beta chains |
| GO:0030199 collagen fibril organization | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: P4HA1-catalyzed 4-hydroxyproline formation stabilizes the collagen triple helix and is a prerequisite for proper collagen fibril formation; this is a downstream process role. Reason: P4HA1 contributes indirectly to fibril organization by modifying procollagen, but fibril assembly itself is carried out by other machinery. The direct function is peptidyl-proline hydroxylation; this BP is a valid non-core involvement. Supporting Evidence: file:human/P4HA1/P4HA1-uniprot.txt this tetramer catalyzes the formation of 4-hydroxyproline in collagen |
| GO:0004656 procollagen-proline 4-dioxygenase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic assignment of the core procollagen-proline 4-dioxygenase activity, redundant with the IDA/TAS/IBA evidence. Reason: Correct core molecular function; consistent with EC 1.14.11.2 and the experimental IDA annotation. Supporting Evidence: file:human/P4HA1/P4HA1-uniprot.txt EC=1.14.11.2 |
| GO:0005506 iron ion binding | IEA GO_REF:0000002 | ACCEPT | Summary: P4HA1 binds one catalytic Fe(2+) per subunit in its Fe2OG dioxygenase domain; this cofactor binding is required for the dioxygenase mechanism. Reason: Supported by the UniProt COFACTOR (Fe(2+), one per subunit) and the Fe-binding residues in the Fe2OG domain; a genuine, mechanistically essential molecular function. Supporting Evidence: file:human/P4HA1/P4HA1-uniprot.txt Binds 1 Fe(2+) ion per subunit. |
| GO:0005783 endoplasmic reticulum | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic assignment of endoplasmic reticulum localization, the parent compartment of the documented ER lumen location. Reason: Correct compartment; consistent with the UniProt ER lumen subcellular location and the IDA (HPA) ER annotation. Supporting Evidence: file:human/P4HA1/P4HA1-uniprot.txt SUBCELLULAR LOCATION: Endoplasmic reticulum lumen. |
| GO:0005788 endoplasmic reticulum lumen | IEA GO_REF:0000044 | ACCEPT | Summary: ER lumen localization derived electronically from the UniProt subcellular location vocabulary; this is the precise documented compartment for C-P4H. Reason: Matches the curated UniProt subcellular location; the soluble tetramer acts in the ER lumen, retained there via the P4HB KDEL. Supporting Evidence: file:human/P4HA1/P4HA1-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: General oxidoreductase/dioxygenase activity term, a parent of the precise procollagen-proline 4-dioxygenase activity, reflecting incorporation of molecular oxygen. Reason: Correct and consistent with the 2-oxoglutarate/O2-dependent dioxygenase mechanism; the more specific GO:0004656 captures the precise core function. Supporting Evidence: file:human/P4HA1/P4HA1-uniprot.txt L-prolyl-[collagen] + 2-oxoglutarate + O2 = trans-4-hydroxy-L- |
| GO:0031418 L-ascorbic acid binding | IEA GO_REF:0000002 | ACCEPT | Summary: C-P4H requires ascorbate (vitamin C) as a cofactor to keep the active-site iron reduced; L-ascorbic acid binding is a genuine cofactor-binding function. Reason: Supported by the UniProt COFACTOR (L-ascorbate) and the Vitamin C keyword; mechanistically required and biologically central (collagen hydroxylation depends on vitamin C). Supporting Evidence: file:human/P4HA1/P4HA1-uniprot.txt Name=L-ascorbate |
| GO:0005515 protein binding | IPI PMID:17500595 Huntingtin interacting proteins are genetic modifiers of neu... | KEEP AS NON CORE | Summary: High-throughput huntingtin-interactome screen reporting a P4HA1-HTT interaction (IntAct P13674-P42858). Bare protein binding is uninformative and unrelated to the catalytic core function. Reason: Records a real IntAct interaction also listed in UniProt, but the bare protein binding term gives no functional information and the HTT partner does not reflect P4HA1's collagen-hydroxylase role; per guidelines not elevated to core. Supporting Evidence: file:human/P4HA1/P4HA1-uniprot.txt P13674; P42858: HTT |
| GO:0005515 protein binding | IPI PMID:30021884 Histone Interaction Landscapes Visualized by Crosslinking Ma... | KEEP AS NON CORE | Summary: Crosslinking mass spectrometry of intact nuclei capturing a P4HA1 interaction (IntAct partner P4HA2/O15460). Bare protein binding from a high-throughput screen. Reason: Uninformative bare protein binding term from a high-throughput XL-MS dataset; the partner is the paralog P4HA2 and the interaction does not inform the core catalytic function. Supporting Evidence: PMID:30021884 we use crosslinking mass spectrometry (XL-MS) to chart the protein-protein interactions in intact human nuclei |
| GO:0005515 protein binding | IPI PMID:40205054 Multimodal cell maps as a foundation for structural and func... | KEEP AS NON CORE | Summary: Multimodal cell map (AP-MS/immunofluorescence in U2OS) reporting a P4HA1 interaction (IntAct partner P4HA2/O15460). High-throughput, uninformative bare term. Reason: Bare protein binding from a proteome-scale interaction/imaging map; the paralog partner does not establish a specific function beyond the already-captured complex membership. Supporting Evidence: PMID:40205054 protein biophysical interactions and protein IF images for a matched set of more than 5,100 proteins in U2OS cells |
| GO:0042802 identical protein binding | IPI PMID:24207127 The structural motifs for substrate binding and dimerization... | KEEP AS NON CORE | Summary: P4HA1 alpha subunits homodimerize via an antiparallel coiled-coil four-helix bundle in the N domain, an assembly step of the alpha2-beta2 tetramer; this is the structural basis of the identical protein binding (self-interaction) annotation. Reason: A genuine, structurally demonstrated alpha-alpha homodimerization within the tetramer, but it is subsidiary to the informative catalytic core function and complex membership; retained as a non-core supporting attribute. Supporting Evidence: PMID:24207127 forming an extended four-helix bundle that includes an antiparallel coiled-coil dimerization motif between the two Ξ± subunits |
| GO:0016222 procollagen-proline 4-dioxygenase complex | IEA GO_REF:0000107 | ACCEPT | Summary: Ortholog-based electronic assignment of membership in the procollagen-proline 4-dioxygenase (C-P4H) complex. Reason: Correct; redundant with the IBA complex annotation. P4HA1 is a catalytic subunit of the alpha2-beta2 tetramer with P4HB. Supporting Evidence: file:human/P4HA1/P4HA1-uniprot.txt Heterotetramer of two alpha-1 chains and two beta chains |
| GO:0005783 endoplasmic reticulum | IDA GO_REF:0000052 | ACCEPT | Summary: Direct immunofluorescence (HPA) evidence for endoplasmic reticulum localization of P4HA1, consistent with its ER-lumenal site of action. Reason: IDA-supported ER localization agrees with the curated ER lumen subcellular location. Supporting Evidence: file:human/P4HA1/P4HA1-uniprot.txt SUBCELLULAR LOCATION: Endoplasmic reticulum lumen. |
| GO:0004656 procollagen-proline 4-dioxygenase activity | IDA PMID:9211872 Cloning of the human prolyl 4-hydroxylase alpha subunit isof... | ACCEPT | Summary: Direct experimental characterization of the purified human prolyl 4-hydroxylase tetramer demonstrating procollagen-proline 4-dioxygenase activity (catalytic activity, kinetics, cofactor, inhibition by poly(L-proline)). Reason: Core molecular function with direct (IDA) experimental support; this is the rate-limiting collagen-modifying activity of P4HA1. Supporting Evidence: PMID:9211872 Prolyl 4-hydroxylase (proline hydroxylase, EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens. |
| GO:0016020 membrane | HDA PMID:19946888 Defining the membrane proteome of NK cells. | MARK AS OVER ANNOTATED | Summary: P4HA1 was detected in a membrane-fraction proteomics screen of NK-like cells. P4HA1 is a soluble ER-lumenal protein, so a generic membrane localization most likely reflects co-fractionation of secretory/ER proteins with membrane preparations. Reason: The documented, mechanistically meaningful localization is the soluble ER lumen; a bulk membrane-proteome HDA hit does not establish a credible membrane localization for this lumenal enzyme. The study itself notes many identified proteins are only transiently membrane-associated. Not removed (experimental data), but over-annotated and non-core. Supporting Evidence: PMID:19946888 The remaining species were largely involved in cellular processes and molecular functions that could be predicted to be transiently associated with membranes. |
| GO:0005788 endoplasmic reticulum lumen | TAS Reactome:R-HSA-1650808 | ACCEPT | Summary: Reactome curation places P4HA1 in the ER lumen, where prolyl 4-hydroxylase converts collagen prolines to 4-hydroxyprolines. Reason: Correct compartment; consistent with the curated UniProt ER lumen location and the enzyme's site of action. Supporting Evidence: file:human/P4HA1/P4HA1-uniprot.txt SUBCELLULAR LOCATION: Endoplasmic reticulum lumen. |
| GO:0005788 endoplasmic reticulum lumen | TAS Reactome:R-HSA-9918779 | ACCEPT | Summary: Reactome curation of P4HA1 ER lumen localization (proline hydroxylation context). Reason: Correct compartment; redundant with the other ER lumen annotations and the UniProt subcellular location. Supporting Evidence: file:human/P4HA1/P4HA1-uniprot.txt SUBCELLULAR LOCATION: Endoplasmic reticulum lumen. |
| GO:0004656 procollagen-proline 4-dioxygenase activity | TAS PMID:2543975 Molecular cloning of the alpha-subunit of human prolyl 4-hyd... | ACCEPT | Summary: Traceable assertion (original cloning paper) of P4HA1 as the alpha subunit of the alpha2-beta2 prolyl 4-hydroxylase that catalyzes 4-hydroxyproline formation in collagens. Reason: Core molecular function; the foundational cloning/characterization study supports the catalytic activity, consistent with the IDA evidence. Supporting Evidence: PMID:2543975 an alpha 2 beta 2 tetramer, catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages |
| GO:0005783 endoplasmic reticulum | TAS PMID:2543975 Molecular cloning of the alpha-subunit of human prolyl 4-hyd... | ACCEPT | Summary: Traceable assertion of ER localization, inferred from the demonstration that the beta/PDI subunit retains the tetramer within the ER (the alpha subunit lacks a KDEL signal). Reason: Correct compartment; the cloning paper established ER retention of the enzyme via P4HB, consistent with the curated ER lumen location. Supporting Evidence: PMID:2543975 necessary for the retention of a polypeptide within the lumen of the endoplasmic reticulum |
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Download this section (compressed HTML)Q: To what extent are the three P4HA1 splice isoforms functionally distinct in substrate preference or tissue-specific collagen hydroxylation, given that the alternatively spliced exon lies within the catalytic region?
Q: Do the high-throughput protein binding interactions (HTT; paralog P4HA2 cross-links) reflect any genuine biology, or are they incidental captures unrelated to the C-P4H catalytic function?
Experiment: Reconstitute the alpha2-beta2 C-P4H tetramer from purified recombinant P4HA1 and P4HB and measure 4-hydroxyproline formation on defined procollagen peptides as a function of Fe(2+), 2-oxoglutarate, O2 and ascorbate concentrations to quantify cofactor dependence.
Experiment: Isoform-resolved hydroxyproline mapping (mass spectrometry) of collagen produced by cells expressing individual P4HA1 splice isoforms to test for isoform-specific differences in hydroxylation site usage or efficiency.
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