Characterization of Recombinant Human Prolyl 3-Hydroxylase Isoenzyme 2, an Enzyme Modifying the Basement Membrane Collagen IV

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 K m and K i 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.Collagen synthesis involves many unusual co-translational and post-translational modifications, including the formation of 4-hydroxyproline (4Hyp), 2 3-hydroxyproline, and hydroxylysine in -X-Pro-Gly-, -Pro-4Hyp-Gly-, and -X-Lys-Glysequences, respectively. These reactions take place within the lumen of the endoplasmic reticulum and are catalyzed by collagen prolyl 4-hydroxylases (C-P4Hs), prolyl 3-hydroxylases

Location of 3-Hydroxyproline Residues in Collagen Types I, II, III, and V/XI Implies a Role in Fibril Supramolecular Assembly

“…It is possible therefore that the non-A1 sites in A-clade and B-clade collagen chains are not hydroxylated by P3H1. However, P3H1 does seem to be the main isoform expressed by cells that make fibrillar collagens, whereas P3H2 is most prominently expressed in basement membrane-rich tissues (5). Alternatively the non-A1 3Hyp sites may not require the same enzyme complex as site A1.…”

“…Three different genes encoding P3H1, P3H2, and P3H3 are present in the human genome, which show tissue specificity in their expression (4,5). Substrate proline residues occur in a prerequisite sequence -Pro-4Hyp-Gly.…”

Comprehensive Mass Spectrometric Mapping of the Hydroxylated Amino Acid residues of the α1(V) Collagen Chain

“…Thus, the 106 Y-position Hyp residues reported here are likely 4-Hyp. Gly-Pro-Hyp triplets are thought to constitute the substrate sequence for P3Hs, of which there are three (55,56). We conclude the 22 Hyp residues in the X-position of Gly-Hyp-Hyp triplet repeats in the COL1 domain of bovine placental ␣1(V) chain are likely to be 3-Hyp residues.…”

Biological role of prolyl 3-hydroxylation in type IV collagen

“…Limited information is available about the substrate specificity of the P3H2 and P3H3 members of the family. Although cell culture studies have indicated that P3H2 exhibits some activity toward fibril-forming collagens (7), type IV collagen was suggested as the main substrate of this enzyme (8).…”