Affinage

P3H1

Prolyl 3-hydroxylase 1 · UniProt Q32P28

Length
736 aa
Mass
83.4 kDa
Annotated
2026-04-29
32 papers in source corpus 9 papers cited in narrative 8 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

P3H1 (encoded by LEPRE1) is a 2-oxoglutarate- and Fe(II)-dependent dioxygenase that functions within a trimeric endoplasmic reticulum complex with CRTAP and cyclophilin B (PPIB) to catalyze prolyl 3-hydroxylation at Pro986 of the α1 chain of type I and type II collagen (PMID:18566967, PMID:19088120). The KDEL ER-retention signal at the C-terminus of P3H1 is essential for retaining the entire ternary complex in the ER; its loss results in co-secretion of P3H1 and CRTAP and abolishes hydroxylation activity (PMID:22615817, PMID:30993352). Cryo-EM structures reveal that the P3H1 and PPIB active sites form a face-to-face bifunctional reaction center that couples prolyl 3-hydroxylation with cis-trans isomerization on collagen substrates, and the complex also serves as a collagen chaperone whose loss—independent of hydroxylation—causes collagen misfolding, ER stress, and the skeletal fragility characteristic of recessive osteogenesis imperfecta type VIII (PMID:39245686, PMID:32173581).

Mechanistic history

Synthesis pass · year-by-year structured walk · 6 steps
  1. 1999 Medium

    Establishing the identity and localization of P3H1: the gene product was identified as a novel chondroitin sulfate proteoglycan bearing a KDEL ER-retention signal, placing it in the secretory pathway and at basement membranes.

    Evidence cDNA cloning, immunostaining of tissue sections, Western blot with chondroitinase ABC, stable expression in CHO K-1 cells

    PMID:10455179

    Open questions at the time
    • Enzymatic activity was not identified
    • Physiological substrates unknown
    • No interacting partners defined
  2. 2008 High

    Resolving the enzymatic function: P3H1 was shown to form a trimeric ER complex with CRTAP and PPIB that catalyzes prolyl 3-hydroxylation specifically at Pro986 of the α1 chain of type I and II collagen, with loss-of-function mutations abolishing this modification and causing collagen overmodification indicative of delayed helix folding. The KDEL-bearing 736-aa splice isoform was identified as the enzymatically active form.

    Evidence Patient fibroblast biochemistry (SDS-urea PAGE, mass spectrometry), splice isoform analysis, loss-of-function mutation analysis across multiple patient cohorts

    PMID:18566967 PMID:19088120 PMID:19862557

    Open questions at the time
    • Structural basis of complex assembly and catalysis unknown
    • Whether the complex has functions beyond hydroxylation was untested
    • Mechanism by which KDEL retains the ternary complex not directly shown
  3. 2012 Medium

    Demonstrating the necessity of ER retention: a natural LEPRE1 mutation that selectively eliminates only the KDEL sequence was sufficient to cause OI, proving that ER retention is not just correlated but required for P3H1 function.

    Evidence Compound heterozygous mutation analysis in patient fibroblasts, real-time PCR, RNA and protein analysis

    PMID:22615817

    Open questions at the time
    • Single-family observation; broader genotype-phenotype correlation not established
    • Whether KDEL loss affects chaperone function versus only enzymatic activity was not distinguished
  4. 2019 High

    Mapping the ternary complex architecture: the KDEL of P3H1 was shown to retain all three subunits in the ER, with the P3H1 N-terminal domain binding CRTAP and the C-termini of both P3H1 and CRTAP positioned near PPIB; a disease-associated PPIB mutation on the binding interface disrupted complex formation without affecting PPIB isomerase activity.

    Evidence Co-immunoprecipitation, crosslinking-mass spectrometry, cysteine modification, secretion assay, site-directed mutagenesis

    PMID:30993352

    Open questions at the time
    • Atomic-resolution structure not yet available
    • How the complex engages collagen substrates was unknown
    • Stoichiometry of the functional complex in vivo not resolved
  5. 2020 High

    Separating hydroxylation from chaperone function: P3H1 and CRTAP knockout zebrafish, which naturally lack the equivalent Pro986 3-hydroxylation, still displayed OI-like phenotypes with collagen misfolding, ER retention, and altered fibril diameter, establishing that the complex's chaperone activity—not hydroxylation—is the primary driver of skeletal pathology.

    Evidence CRISPR/Cas9 knockout in zebrafish, transmission electron microscopy, collagen biochemistry, bone histomorphometry

    PMID:32173581

    Open questions at the time
    • Molecular mechanism of chaperone activity not defined
    • Whether the chaperone function operates independently of PPIB isomerase activity unknown
    • Applicability to mammalian systems where 3-hydroxylation does occur needs formal testing
  6. 2024 High

    Revealing the coupled catalytic mechanism: cryo-EM structures showed that P3H1 and PPIB active sites form a face-to-face bifunctional reaction center coupling prolyl 3-hydroxylation with cis-trans isomerization, identified a collagen substrate-interacting zone, and discovered a dual-ternary (dimer of trimers) assembly state modulated by active-site mutations and PPIB inhibitors.

    Evidence Cryo-EM structure determination, mutagenesis, PPIB inhibitor treatment, biochemical assembly assays

    PMID:39245686

    Open questions at the time
    • Functional significance of the dual-ternary (dimer of trimers) state in vivo unknown
    • Whether the coupled mechanism is processive along the collagen chain not tested
    • Structural basis of the chaperone function distinct from catalysis not resolved

Open questions

Synthesis pass · forward-looking unresolved questions
  • It remains unresolved how the P3H1/CRTAP/PPIB complex mechanistically prevents collagen misfolding independently of its enzymatic activity, and whether pharmacological modulation of the dual-ternary equilibrium could be therapeutically exploited in OI.
  • Molecular mechanism of chaperone function structurally undefined
  • No reconstituted in vitro chaperone assay distinguishing hydroxylation-independent folding assistance
  • Therapeutic targeting of the complex not explored

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0016491 oxidoreductase activity 3 GO:0044183 protein folding chaperone 1
Localization
GO:0005783 endoplasmic reticulum 4
Pathway
R-HSA-1643685 Disease 2 R-HSA-392499 Metabolism of proteins 2
Partners
CRTAPPPIB
Complex memberships
P3H1/CRTAP/PPIB ternary complex

Evidence

Reading pass · 8 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1999 Leprecan (P3H1) was identified as a novel chondroitin sulfate proteoglycan with a KDEL endoplasmic reticulum retention signal, and was shown to localize to basement membranes and participate in the secretory pathway; expression of the full coding sequence in CHO K-1 cells confirmed secretion as a chondroitin sulfate proteoglycan. cDNA cloning, immunostaining of tissue sections, Western blot with chondroitinase ABC digestion, stable transfection in CHO K-1 cells The Journal of biological chemistry Medium 10455179
2001 Sequence profile analysis predicted that leprecan (P3H1) belongs to the 2-oxoglutarate- and Fe(II)-dependent dioxygenase superfamily and is a novel protein hydroxylase, likely involved in generating substrates for protein glycosylation. Computational sequence profile searches (fold recognition) Genome biology Low 11276424
2008 P3H1 (encoded by LEPRE1) forms a trimeric complex with cartilage-associated protein (CRTAP) and cyclophilin B (CypB/PPIB) in the endoplasmic reticulum; this complex catalyzes prolyl 3-hydroxylation specifically at Pro986 in the α1 chain of type I and type II collagen. Loss-of-function mutations in LEPRE1 abolish Pro986 hydroxylation and cause helical overmodification of collagen chains, indicating delayed helix folding. Biochemical characterization of patient fibroblasts (SDS-urea PAGE, mass spectrometry), analysis of LEPRE1 loss-of-function mutations Human mutation High 18566967 19862557
2008 The prolyl 3-hydroxylation activity of P3H1 is restricted to the 736 amino acid splice form that contains a C-terminal KDEL ER-retention signal; a splice mutation eliminating only this isoform severely reduces α1(I)Pro986 3-hydroxylation and causes collagen overmodification. Western blot, immunocytochemistry, mass spectrometry, SDS-urea PAGE on patient fibroblasts, RNA splice form analysis Journal of medical genetics High 19088120
2012 The KDEL ER-retrieval sequence at the carboxyl terminus of P3H1 is essential for its functionality; a LEPRE1 mutation that specifically eliminates only the KDEL sequence (while leaving other functional domains intact) is sufficient to cause OI, demonstrating that ER retention is required for P3H1 to perform prolyl 3-hydroxylation. RNA analysis, real-time PCR, patient fibroblast biochemical analysis, identification of compound heterozygous mutations in LEPRE1 PloS one Medium 22615817
2019 The KDEL sequence of P3H1 is essential for retaining the entire P3H1/CRTAP/PPIB ternary complex in the ER; removal of KDEL results in co-secretion of P3H1 and CRTAP out of the cell, mediated by the binding of the P3H1 N-terminal domain with CRTAP. The C-termini of P3H1 and CRTAP are close to PPIB in the ternary complex, and PPIB surface residues involved in complex formation were identified by cysteine modification, crosslinking, and mass spectrometry. A disease-associated PPIB mutation on the binding interface disrupted ternary complex formation without affecting PPIB prolyl-isomerase activity. Co-immunoprecipitation, crosslinking, mass spectrometry, cysteine modification assay, secretion assay, mutagenesis Cellular and molecular life sciences : CMLS High 30993352
2020 P3H1 and CRTAP knockout zebrafish (generated by CRISPR/Cas9) display OI phenotype with intracellular type I collagen overmodification, partial ER retention, enlarged ER cisternae, and extracellular collagen fibers with altered diameter. Since zebrafish lack collagen type I 3-hydroxylation at the equivalent Pro986 site in wild type, these results demonstrate that the chaperone function of the P3H1/CRTAP complex—not the hydroxylation per se—is the primary cause of skeletal pathology. CRISPR/Cas9 knockout in zebrafish, transmission electron microscopy, collagen biochemistry, bone histomorphometry Matrix biology : journal of the International Society for Matrix Biology High 32173581
2024 Cryo-EM structures of the P3H1/CRTAP/PPIB ternary complex reveal that the active sites of P3H1 and PPIB form a face-to-face bifunctional reaction center, indicating a coupled prolyl 3-hydroxylation and cis-trans isomerization mechanism. Structure of the P3H1/CRTAP/PPIB/collagen peptide complex identifies multiple collagen-binding sites forming a substrate interacting zone. A dual-ternary (dimer of trimers) complex was observed; the balance between ternary and dual-ternary states is modulated by mutations in the P3H1/PPIB active site and by PPIB inhibitors. Cryo-EM structure determination, mutagenesis, PPIB inhibitor treatment, biochemical assembly assays Nature communications High 39245686

Source papers

Stage 0 corpus · 32 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2001 The DNA-repair protein AlkB, EGL-9, and leprecan define new families of 2-oxoglutarate- and iron-dependent dioxygenases. Genome biology 359 11276424
2008 CRTAP and LEPRE1 mutations in recessive osteogenesis imperfecta. Human mutation 173 18566967
2009 Null mutations in LEPRE1 and CRTAP cause severe recessive osteogenesis imperfecta. Cell and tissue research 84 19862557
2008 Recessive osteogenesis imperfecta caused by LEPRE1 mutations: clinical documentation and identification of the splice form responsible for prolyl 3-hydroxylation. Journal of medical genetics 68 19088120
2011 The identification of novel mutations in COL1A1, COL1A2, and LEPRE1 genes in Chinese patients with osteogenesis imperfecta. Journal of bone and mineral metabolism 58 21667357
1999 Molecular characterization of a novel basement membrane-associated proteoglycan, leprecan. The Journal of biological chemistry 51 10455179
2012 A founder mutation in LEPRE1 carried by 1.5% of West Africans and 0.4% of African Americans causes lethal recessive osteogenesis imperfecta. Genetics in medicine : official journal of the American College of Medical Genetics 41 22281939
2020 Crtap and p3h1 knock out zebrafish support defective collagen chaperoning as the cause of their osteogenesis imperfecta phenotype. Matrix biology : journal of the International Society for Matrix Biology 34 32173581
2009 The evolutionarily conserved leprecan gene: its regulation by Brachyury and its role in the developing Ciona notochord. Developmental biology 32 19217895
2004 LEPREL1, a novel ER and Golgi resident member of the Leprecan family. Biochemical and biophysical research communications 31 15063763
2000 Gros1, a potential growth suppressor on chromosome 1: its identity to basement membrane-associated proteoglycan, leprecan. Oncogene 30 10951563
2012 A novel mutation in LEPRE1 that eliminates only the KDEL ER- retrieval sequence causes non-lethal osteogenesis imperfecta. PloS one 27 22615817
2013 Allelic background of LEPRE1 mutations that cause recessive forms of osteogenesis imperfecta in different populations. Molecular genetics & genomic medicine 20 24498616
2015 Mutational characterization of the P3H1/CRTAP/CypB complex in recessive osteogenesis imperfecta. Genetics and molecular research : GMR 16 26634552
2010 Lethal/severe osteogenesis imperfecta in a large family: a novel homozygous LEPRE1 mutation and bone histological findings. Pediatric and developmental pathology : the official journal of the Society for Pediatric Pathology and the Paediatric Pathology Society 14 20946018
2013 Osteogenesis imperfecta due to compound heterozygosity for the LEPRE1 gene. Fetal and pediatric pathology 13 23301918
2019 Characterization of PPIB interaction in the P3H1 ternary complex and implications for its pathological mutations. Cellular and molecular life sciences : CMLS 12 30993352
2024 The structural basis for the collagen processing by human P3H1/CRTAP/PPIB ternary complex. Nature communications 11 39245686
2022 Biomarker LEPRE1 induces pelitinib-specific drug responsiveness by regulating ABCG2 expression and tumor transition states in human leukemia and lung cancer. Scientific reports 9 35190588
2007 Leprecan distribution in the developing and adult kidney. Kidney international 8 17495866
2021 A novel P3H1 mutation is associated with osteogenesis imperfecta type VIII and dental anomalies. Oral surgery, oral medicine, oral pathology and oral radiology 6 33737016
2020 Upregulated LEPRE1 correlates with poor outcome and its knockdown attenuates cells proliferation, migration and invasion in osteosarcoma. Anti-cancer drugs 6 32197005
2016 Targeted exome sequencing identifies novel compound heterozygous mutations in P3H1 in a fetus with osteogenesis imperfecta type VIII. Clinica chimica acta; international journal of clinical chemistry 6 27864101
2021 Severe cases of osteogenesis imperfecta type VIII due to a homozygous mutation in P3H1 (LEPRE1) and review of the literature. Advances in clinical and experimental medicine : official organ Wroclaw Medical University 5 34637196
2018 Expression characterization and functional implication of the collagen-modifying Leprecan proteins in mouse gonadal tissue and mature sperm. AIMS genetics 5 30417103
2024 Pan-Cancer Analysis of P3H1 and Experimental Validation in Renal Clear Cell Carcinoma. Applied biochemistry and biotechnology 4 38175417
2022 Phenotypic Variation in Vietnamese Osteogenesis Imperfecta Patients Sharing a Recessive P3H1 Pathogenic Variant. Genes 4 35327962
2023 A Founder Intronic Variant in P3H1 Likely Results in Aberrant Splicing and Protein Truncation in Patients of Karen Descent with Osteogenesis Imperfecta Type VIII. Genes 3 36833249
2023 Milder presentation of osteogenesis imperfecta type VIII due to compound heterozygosity for a predicted loss-of-function variant and novel missense variant in P3H1-further expansion of the phenotypic spectrum. Cold Spring Harbor molecular case studies 2 36963805
2025 P3H1 promotes malignant progression of esophageal squamous cell carcinoma (ESCC) and modulates the immune microenvironment. Journal of thoracic disease 1 41229743
2025 Rare Variants in the P3H1 Gene in Patients With Osteogenesis Imperfecta of Bashkir Origin From Russia. Clinical genetics 0 41499654
2024 A non-lethal presentation of osteogenesis imperfecta type VIII due to homozygous mutation in P3H1 gene. BMJ case reports 0 39455078