Dataset Information

Reduced Bone Mass in Collagen Prolyl 4-Hydroxylase P4ha1 ^+/-; P4ha2 ^-/- Compound Mutant Mice.

ABSTRACT: Proper deposition of the extracellular matrix and its major components, the collagens, is essential for endochondral ossification and bone mass accrual. Collagen prolyl 4-hydroxylases (C-P4Hs) hydroxylate proline residues in the -X-Pro-Gly- repeats of all known collagen types. Their product, 4-hydroxyproline, is essential for correct folding and thermal stability of the triple-helical collagen molecules in physiological body temperatures. We have previously shown that inactivation of the mouse P4ha1 gene, which codes for the catalytic α subunit of the major C-P4H isoform, is embryonic lethal, whereas inactivation of the P4ha2 gene produced only a minor phenotype. Instead, mice with a haploinsufficiency of the P4ha1 gene combined with a homozygous deletion of the P4ha2 gene present with a moderate chondrodysplasia due to transient cell death of the growth plate chondrocytes. Here, to further characterize the bone phenotype of the P4ha1 ^+/-; P4ha2 ^-/- mice, we have carried out gene expression analyses at whole-tissue and single-cell levels, biochemical analyses, microcomputed tomography, histomorphometric analyses, and second harmonic generation microscopy to show that C-P4H α subunit expression peaks early and that the C-P4H deficiency leads to reduced collagen amount, a reduced rate of bone formation, and a loss of trabecular and cortical bone volume in the long bones. The total osteoblast number in the proximal P4ha1 ^+/-; P4ha2 ^-/- tibia and the C-P4H activity in primary P4ha1 ^+/-; P4ha2 ^-/- osteoblasts were reduced, whereas the population of osteoprogenitor colony-forming unit fibroblasts was increased in the P4ha1 ^+/-; P4ha2 ^-/- marrow. Thus, the P4ha1 ^+/-; P4ha2 ^-/- mouse model recapitulates key aspects of a recently recognized congenital connective tissue disorder with short stature and bone dysplasia caused by biallelic variants of the human P4HA1 gene. Altogether, the data demonstrate the allele dose-dependent importance of the C-P4Hs to the developing organism and a threshold effect of C-P4H activity in the proper production of bone matrix. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

SUBMITTER: Tolonen JP

PROVIDER: S-EPMC9189910 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Reduced Bone Mass in Collagen Prolyl 4-Hydroxylase P4ha1 +/-; P4ha2 -/- Compound Mutant Mice.

Tolonen Jussi-Pekka JP Salo Antti M AM Finnilä Mikko M Aro Ellinoora E Karjalainen Emma E Ronkainen Veli-Pekka VP Drushinin Kati K Merceron Christophe C Izzi Valerio V Schipani Ernestina E Myllyharju Johanna J

JBMR plus 20220509 6

Proper deposition of the extracellular matrix and its major components, the collagens, is essential for endochondral ossification and bone mass accrual. Collagen prolyl 4-hydroxylases (C-P4Hs) hydroxylate proline residues in the -X-Pro-Gly- repeats of all known collagen types. Their product, 4-hydroxyproline, is essential for correct folding and thermal stability of the triple-helical collagen molecules in physiological body temperatures. We have previously shown that inactivation of the mouse < ...[more]

PMID: 35720665

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Project description:BackgroundWe describe a method for specific, quantitative and quick detection of human collagen prolyl 4-hydroxylase (C-P4H), the key enzyme for collagen prolyl-4 hydroxylation, in crude samples based on a sandwich ELISA principle. The method is relevant to active C-P4H level monitoring during recombinant C-P4H and collagen production in different expression systems. The assay proves to be specific for the active C-P4H alpha2beta2 tetramer due to the use of antibodies against its both subunits. Thus in keeping with the method C-P4H is captured by coupled to an anti-alpha subunit antibody magnetic beads and an anti-beta subunit antibody binds to the PDI/beta subunit of the protein. Then the following holoenzyme detection is accomplished by a goat anti-rabbit IgG labeled with alkaline phosphatase which AP catalyzes the reaction of a substrate transformation with fluorescent signal generation.ResultsWe applied an experimental design approach for the optimization of the antibody concentrations used in the sandwich ELISA. The assay sensitivity was 0.1 ng of C-P4H. The method was utilized for the analysis of C-P4H accumulation in crude cell extracts of E. coli overexpressing C-P4H. The sandwich ELISA signals obtained demonstrated a very good correlation with the detected protein activity levels measured with the standard radioactive assay. The developed assay was applied to optimize C-P4H production in E. coli Origami in a system where the C-P4H subunits expression acted under control by different promoters. The experiments performed in a shake flask fed-batch system (EnBase) verified earlier observations that cell density and oxygen supply are critical factors for the use of the inducer anhydrotetracycline and thus for the soluble C-P4H yield.ConclusionsHere we show an example of sandwich ELISA usage for quantifying multimeric proteins. The method was developed for monitoring the amount of recombinant C-P4H tetramer in crude E. coli extracts. Due to the specificity of the antibodies used in the assay against the different C-P4H subunits, the method detects the entire holoenzyme, and the signal is not disturbed by background expression of the separate subunits.

Dataset Information

Reduced Bone Mass in Collagen Prolyl 4-Hydroxylase P4ha1 ^+/-; P4ha2 ^-/- Compound Mutant Mice.

Publications

Reduced Bone Mass in Collagen Prolyl 4-Hydroxylase <i>P4ha1</i> <sup>+/-</sup>; <i>P4ha2</i> <sup>-/-</sup> Compound Mutant Mice.

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