P3H2 (prolyl 3-hydroxylase 2, also LEPREL1/MLAT4) is an endoplasmic reticulum-lumenal, 2-oxoglutarate/Fe(II)-dependent dioxygenase (EC 1.14.11.7) of the leprecan/prolyl 3-hydroxylase family (P3H1/P3H2/P3H3). It catalyzes post-translational formation of (trans-)3-hydroxyproline on procollagen, hydroxylating the first proline in Gly-Pro-4Hyp triplets and requiring a pre-existing 4-hydroxyproline in the third position. P3H2 shows high activity toward the basement-membrane collagen type IV (COL4A1), which has the highest 3-hydroxyproline content of all collagens, and lower activity toward collagen I; it is thus considered the principal modifier of basement-membrane collagens, complementing the fibrillar-collagen isoenzyme P3H1. The enzyme uses Fe(II) and L-ascorbate (vitamin C) as cofactors and 2-oxoglutarate as a co-substrate. The 708-residue precursor has an N-terminal signal sequence, tetratricopeptide (TPR) repeats, an Fe2OG dioxygenase (prolyl 4-hydroxylase alpha-type) catalytic domain, and a C-terminal KDEL-type ER-retention motif that retains it in the ER/sarcoplasmic reticulum; it is also detected in the Golgi. P3H2 is broadly expressed with enrichment in basement-membrane-rich tissues such as kidney and in muscle. Loss-of-function variants cause autosomal-recessive high myopia with cataract and vitreoretinal degeneration (MCVD), consistent with a role in ocular/lens-capsule basement-membrane collagen biogenesis.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005783 endoplasmic reticulum | IBA GO_REF:0000033 | ACCEPT | Summary: P3H2 acts in the endoplasmic reticulum, where collagen prolyl hydroxylation occurs prior to secretion. The phylogenetic (IBA) ER localization is consistent with direct experimental evidence (IDA) and the C-terminal ER-retention motif. Reason: ER is the correct catalytic compartment; supported by IDA (PMID:15063763), the KDEL-type ER-retention motif, and the UniProt subcellular location. Supporting Evidence: file:human/P3H2/P3H2-uniprot.txt SUBCELLULAR LOCATION: Endoplasmic reticulum |
| GO:0019797 procollagen-proline 3-dioxygenase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Procollagen-proline 3-dioxygenase activity is the core molecular function of P3H2, conserved across the P3H/leprecan family and directly demonstrated for human P3H2 on collagen IV-derived peptides. Reason: Core molecular function; corroborated by IDA (PMID:18487197), EC 1.14.11.7, and family-level conservation. Supporting Evidence: file:human/P3H2/P3H2-uniprot.txt Prolyl 3-hydroxylase that catalyzes the post-translational formation of 3-hydroxyproline on collagens |
| GO:0032963 collagen metabolic process | IBA GO_REF:0000033 | ACCEPT | Summary: By 3-hydroxylating procollagen prolyl residues, P3H2 participates in collagen biosynthesis/metabolism; the phylogenetic annotation is consistent with the experimental IDA evidence. Reason: Correct biological process; redundant with IDA (PMID:18487197) collagen metabolic process annotation. Supporting Evidence: file:human/P3H2/P3H2-uniprot.txt post-translational formation of 3-hydroxyproline on collagens |
| GO:0005506 iron ion binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: As a 2-oxoglutarate/Fe(II)-dependent dioxygenase, P3H2 coordinates a catalytic Fe(II) ion (binding residues 580/582/652) required for hydroxylation. This cofactor binding supports, but is subsidiary to, the informative procollagen-proline 3-dioxygenase activity. Reason: Accurate catalytic cofactor attribute (Fe cation cofactor; Fe-binding residues in the Fe2OG domain) but a supporting feature rather than the standalone core function. Supporting Evidence: file:human/P3H2/P3H2-uniprot.txt Name=Fe cation |
| GO:0005783 endoplasmic reticulum | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic transfer of ER localization from the UniProt subcellular location, consistent with the stronger IDA/IBA evidence. Reason: Correct compartment; redundant with experimental ER localization. Supporting Evidence: file:human/P3H2/P3H2-uniprot.txt SUBCELLULAR LOCATION: Endoplasmic reticulum |
| GO:0005794 Golgi apparatus | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Electronic transfer of the Golgi localization from the UniProt subcellular location. Golgi residence is a reported secondary localization; the catalytic site of action is the ER lumen. Reason: Accurate reported localization (UniProt/IDA) but secondary to the ER, where collagen prolyl hydroxylation occurs; not the core site of action. Supporting Evidence: file:human/P3H2/P3H2-uniprot.txt Golgi apparatus |
| GO:0016529 sarcoplasmic reticulum | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: The sarcoplasmic reticulum is the specialized ER of muscle; this localization is consistent with the strong muscle expression and muscle-specific transcript of P3H2 reported by Jaernum et al. Reason: Accurate UniProt-derived localization reflecting muscle-tissue ER, but a tissue-specific variant of the ER compartment rather than a distinct core function. Supporting Evidence: file:human/P3H2/P3H2-uniprot.txt Sarcoplasmic reticulum |
| GO:0016705 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: This is the parent oxidoreductase class for the dioxygenase reaction P3H2 catalyzes (it incorporates one atom of O2 into proline while reducing 2-oxoglutarate). Correct but less informative than the specific procollagen-proline 3-dioxygenase activity. Reason: Correct but generic parent term; the specific GO:0019797 captures the core catalytic activity. Supporting Evidence: file:human/P3H2/P3H2-uniprot.txt EC=1.14.11.7 |
| GO:0019797 procollagen-proline 3-dioxygenase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic (multi-method, EC/Rhea-based) assignment of the core procollagen-proline 3-dioxygenase activity, consistent with the experimental IDA evidence and EC 1.14.11.7. Reason: Correct core molecular function; redundant with IDA/IBA evidence. Supporting Evidence: file:human/P3H2/P3H2-uniprot.txt EC=1.14.11.7 |
| GO:0031418 L-ascorbic acid binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: P3H2 uses L-ascorbate (vitamin C) as a cofactor to maintain the active-site iron in the reduced state, a hallmark of collagen prolyl hydroxylases. This cofactor binding supports the catalytic activity. Reason: Accurate catalytic cofactor attribute (L-ascorbate cofactor; KM=110 uM for ascorbate) but subsidiary to the informative procollagen-proline 3-dioxygenase activity. Supporting Evidence: file:human/P3H2/P3H2-uniprot.txt Name=L-ascorbate |
| GO:0032963 collagen metabolic process | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic assignment of the collagen metabolic process, consistent with the experimental IDA evidence. Reason: Correct biological process; redundant with IDA (PMID:18487197). Supporting Evidence: file:human/P3H2/P3H2-uniprot.txt 3-hydroxyproline on collagens |
| GO:0005604 basement membrane | IEA GO_REF:0000107 | MARK AS OVER ANNOTATED | Summary: Basement membrane is the location of P3H2's collagen IV substrate/product, not where the enzyme itself acts. P3H2 is an ER-resident enzyme (KDEL-type retention motif) that modifies procollagen in the ER lumen before secretion; it is not a structural component of the assembled basement membrane. This annotation is transferred electronically from the mouse ortholog. Reason: Ortholog-transferred (Ensembl/ISS from Q8CG71) localization to basement membrane conflates the enzyme's ER site of action with the destination of its collagen IV substrate; P3H2 acts in the ER and is retained there by a KDEL-type motif. Supporting Evidence: file:human/P3H2/P3H2-uniprot.txt Prevents secretion from ER |
| GO:0005794 Golgi apparatus | IDA GO_REF:0000052 | KEEP AS NON CORE | Summary: Immunofluorescence (HPA) evidence for Golgi localization, consistent with the reported ER-and-Golgi distribution of P3H2. The catalytic site of action remains the ER lumen. Reason: Direct (IDA) reported localization but secondary to the ER catalytic compartment; not the core site of action. Supporting Evidence: file:human/P3H2/P3H2-uniprot.txt Golgi apparatus |
| GO:0005788 endoplasmic reticulum lumen | TAS Reactome:R-HSA-1980233 | ACCEPT | Summary: Reactome curation places P3H2 in the ER lumen, the precise compartment where collagen prolyl 3-hydroxylation occurs. This is the most informative cellular-component term for the enzyme. Reason: Correct and precise catalytic compartment; consistent with the ER localization and ER-retention motif. Supporting Evidence: file:human/P3H2/P3H2-uniprot.txt SUBCELLULAR LOCATION: Endoplasmic reticulum |
| GO:0005788 endoplasmic reticulum lumen | TAS Reactome:R-HSA-8948226 | ACCEPT | Summary: Reactome curation of P3H2 in the ER lumen during the collagen 3-hydroxylation reaction cycle. Reason: Correct precise catalytic compartment; redundant with the other ER lumen TAS annotation. Supporting Evidence: file:human/P3H2/P3H2-uniprot.txt SUBCELLULAR LOCATION: Endoplasmic reticulum |
| GO:0005788 endoplasmic reticulum lumen | TAS Reactome:R-HSA-8948230 | ACCEPT | Summary: Reactome curation of P3H2 in the ER lumen, where it binds 4-Hyp collagen propeptides. Reason: Correct precise catalytic compartment; redundant with the other ER lumen TAS annotations. Supporting Evidence: file:human/P3H2/P3H2-uniprot.txt SUBCELLULAR LOCATION: Endoplasmic reticulum |
| GO:0005604 basement membrane | ISS GO_REF:0000024 | MARK AS OVER ANNOTATED | Summary: Sequence-similarity (ISS, from mouse ortholog Q8CG71) transfer of basement membrane localization. As with the IEA version, this reflects the location of the collagen IV substrate/product rather than the ER-resident enzyme's site of action. Reason: ISS transfer from the mouse ortholog conflates enzyme site of action (ER) with the destination of its collagen IV substrate; P3H2 is ER-retained via a KDEL-type motif. Supporting Evidence: file:human/P3H2/P3H2-uniprot.txt Prevents secretion from ER |
| GO:0019511 peptidyl-proline hydroxylation | IDA PMID:18487197 Characterization of recombinant human prolyl 3-hydroxylase i... | ACCEPT | Summary: P3H2 directly hydroxylates peptidyl-proline residues; the recombinant enzyme assay demonstrated robust activity on (Gly-Pro-4Hyp)5 and collagen IV-derived peptides. This is the biological-process view of the core catalytic function. Reason: Directly demonstrated peptidyl-proline hydroxylation activity (IDA); the central biological process of P3H2. Supporting Evidence: PMID:18487197 A large amount of P3H activity was found in the P3H2 samples with (Gly-Pro-4Hyp)5 as a substrate |
| GO:0019797 procollagen-proline 3-dioxygenase activity | IDA PMID:18487197 Characterization of recombinant human prolyl 3-hydroxylase i... | ACCEPT | Summary: Direct experimental demonstration that recombinant human P3H2 catalyzes prolyl 3-hydroxylation, with high activity toward collagen IV sequences. This is the core molecular function of the enzyme. Reason: Core molecular function with direct experimental (IDA) support; defines P3H2 as the collagen IV-preferring prolyl 3-hydroxylase isoenzyme (EC 1.14.11.7). Supporting Evidence: PMID:18487197 P3H2 hydroxylated more effectively two synthetic peptides corresponding to sequences that are hydroxylated in collagen IV than a peptide corresponding to the 3-hydroxylation site in collagen I |
| GO:0032963 collagen metabolic process | IDA PMID:18487197 Characterization of recombinant human prolyl 3-hydroxylase i... | ACCEPT | Summary: P3H2 contributes to collagen metabolism by modifying procollagen prolyl residues, with a preference for the basement-membrane collagen IV. Reason: Directly supported (IDA) collagen-modifying process; central to P3H2's biological role. Supporting Evidence: PMID:18487197 P3H2 is responsible for the hydroxylation of collagen IV, which has the highest 3-hydroxyproline content of all collagens |
| GO:0005783 endoplasmic reticulum | IDA PMID:15063763 LEPREL1, a novel ER and Golgi resident member of the Lepreca... | ACCEPT | Summary: Direct immunofluorescence evidence that LEPREL1/P3H2 localizes to the ER, consistent with its role as an ER-lumenal collagen-modifying enzyme and its KDEL-type retention motif. Reason: IDA-supported ER localization agrees with the catalytic site of action and the ER-retention motif. Supporting Evidence: PMID:15063763 LEPREL1 is localized to the ER and Golgi network |
| GO:0005794 Golgi apparatus | IDA PMID:15063763 LEPREL1, a novel ER and Golgi resident member of the Lepreca... | KEEP AS NON CORE | Summary: Direct immunofluorescence evidence that LEPREL1/P3H2 also localizes to the Golgi. This is a reported secondary localization; the ER lumen is the catalytic compartment. Reason: Direct (IDA) reported localization but secondary to the ER site of action for collagen prolyl hydroxylation. Supporting Evidence: PMID:15063763 LEPREL1 is localized to the ER and Golgi network |
| GO:0008285 negative regulation of cell population proliferation | IDA PMID:19436308 The prolyl 3-hydroxylases P3H2 and P3H3 are novel targets fo... | KEEP AS NON CORE | Summary: P3H2 is epigenetically silenced by promoter CpG methylation in breast cancer, and ectopic re-expression suppresses colony formation, indicating a tumor-suppressor-like anti-proliferative effect. The colony-suppression assay was performed with P3H2 cDNA in P3H2-silenced breast cancer cell lines (MCF7, T47D), so the experimental annotation is gene-specific and should be retained. However, this is a context-dependent, likely indirect phenotype rather than the enzyme's core ER collagen-modifying function. Reason: Real gene-specific experimental (IDA) phenotype, but an indirect/context-dependent tumor-suppressor effect of overexpression in cancer cell lines, not the core catalytic function; retained as non-core per guidelines. Supporting Evidence: PMID:19436308 Ectopic expression of P3H2 and P3H3 in cell lines with silencing of the endogenous gene results in suppression of colony growth |
Loading supporting content…
Download this section (compressed HTML)Q: Does P3H2 act as a monomer or within a multiprotein complex in the ER lumen, and does it (like P3H1/CRTAP/PPIB) have additional chaperone/foldase or non-catalytic roles in basement-membrane collagen biogenesis?
Q: Is the anti-proliferative/tumor-suppressor effect of P3H2 in breast cancer a consequence of its collagen-modifying enzymatic activity (e.g. altered ECM signaling) or an activity-independent moonlighting function?
Experiment: Quantitative mass spectrometry of 3-hydroxyproline sites on collagen IV (and other basement-membrane collagens) from P3H2-knockout versus wild-type cells/tissues to define the in vivo substrate site repertoire and confirm collagen IV specificity.
Experiment: Structure-function analysis of the MCVD G508V variant (and active-site mutants) measuring prolyl 3-hydroxylase activity, folding/solubility, and ER retention to establish the molecular basis of the loss of function underlying high myopia.
Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)