Abstract
The single 3-hydroxyproline residue in the collagen I polypeptides is essential for proper fibril formation and bone development as its deficiency leads to recessive osteogenesis imperfecta. The vertebrate prolyl 3-hydroxylase (P3H) family consists of three members, P3H1 being responsible for the hydroxylation of collagen I. We expressed human P3H2 as an active recombinant protein in insect cells. Most of the recombinant polypeptide was insoluble, but small amounts were also present in the soluble fraction. P3H1 forms a complex with the cartilage-associated protein (CRTAP) that is required for prolyl 3-hydroxylation of fibrillar collagens. However, coexpression with CRTAP did not enhance the solubility or activity of the recombinant P3H2. A novel assay for P3H activity was developed based on that used for collagen prolyl 4-hydroxylases (C-P4H) and lysyl hydroxylases (LH). A large amount of P3H activity was found in the P3H2 samples with (Gly-Pro-4Hyp)5 as a substrate. The Km and Ki values of P3H2 for 2-oxoglutarate and its certain analogues resembled those of the LHs rather than the C-P4Hs. Unlike P3H1, P3H2 was strongly expressed in tissues rich in basement membranes, such as the kidney. 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. These findings suggest that P3H2 is responsible for the hydroxylation of collagen IV, which has the highest 3-hydroxyproline content of all collagens. It is thus possible that P3H2 mutations may lead to a disease with changes in basement membranes.
Highlights
These reactions take place within the lumen of the endoplasmic reticulum and are catalyzed by collagen prolyl 4-hydroxylases (C-P4Hs), prolyl 3-hydroxylases (P3Hs), and lysyl hydroxylases (LHs), which belong to the 2-oxoglutarate-dependent dioxygenases [1,2,3,4]. 4-Hydroxyproline residues have an essential role in the folding of the newly synthesized collagen polypeptide chains into thermally stable triple-helical molecules and their amounts in different collagen types are relatively similar, around 100 residues per 1000 amino acids
Chick P3H1 copurifies with cyclophilin B and the cartilage-associated protein (CRTAP), indicating that it forms a tight complex with these endoplasmic reticulum-resident proteins [11]
These results indicate that P3H1 can hydroxylate collagen chains in vitro without the presence of CRTAP [11], the latter is required for its efficient function in vivo
Summary
PCR Analysis of P3H mRNA Expression in Various Mouse and Human Tissues—Expression of mouse and human P3H1–3 mRNAs in various tissues was studied by PCR analysis of the mouse and human multiple tissue cDNA (MTCTM) panel I and the human fetal MTC panel (Clontech) according to the manufacturer’s protocol, using the Phusion polymerase (Finnzymes). The reaction was performed in a final volume of 0.5 ml, which contained 50 –75 l of the Triton X-100 extracts of the insect cells as a source of the enzyme, differing amounts of a peptide substrate (Gly-Pro-4Hyp) (Innovagen), 0.025 mol of FeSO4, 0.05 mol of 2-oxo-[114C]glutarate, 1 mol of ascorbate, 30 g of catalase (Sigma), 0.05 mol of dithiothreitol, 1 mg of bovine serum albumin, and 25 mol of Tris-HCl buffer, adjusted to pH 7.8 at 25 °C. The adult mouse specimens for use in the immunofluorescence studies were immediately frozen in liquid nitrogen and stored at Ϫ70 °C Samples from these were cut into 5-m cryosections on SuperFrost plus glass (Menzel-Glaser) slides and the sections were fixed in pre-cooled acetone for 10 min at 4 °C. Images were captured by CCD camera equipped with a TCL-EM-Menu, version 3, from Tietz Video and Image Processing Systems GmbH (Gaunting)
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