A Role for Prolyl 3-Hydroxylase 2 in Post-translational Modification of Fibril-forming Collagens

Abstract

The fibrillar collagen types I, II, and V/XI have recently been shown to have partially 3-hydroxylated proline (3Hyp) residues at sites other than the established primary Pro-986 site in the collagen triple helical domain. These sites showed tissue specificity in degree of hydroxylation and a pattern of D-periodic spacing. This suggested a contributory role in fibril supramolecular assembly. The sites in clade A fibrillar ␣1(II), ␣2(V), and ␣1(I) collagen chains share common features with known prolyl 3-hydroxylase 2 (P3H2) substrate sites in ␣1(IV) chains implying a role for this enzyme. We pursued this possibility using the Swarm rat chondrosarcoma cell line (RCS-LTC) found to express high levels of P3H2 mRNA. Mass spectrometry determined that all the additional candidate 3Hyp substrate sites in the pN type II collagen made by these cells were highly hydroxylated. In RNA interference experiments, P3H2 protein synthesis was suppressed coordinately with prolyl 3-hydroxylation at Pro-944, Pro-707, and the C-terminal GPP repeat of the pN␣1(II) chain, but Pro-986 remained fully hydroxylated. Furthermore, when P3H2 expression was turned off, as seen naturally in cultured SAOS-2 osteosarcoma cells, full 3Hyp occupancy at Pro-986 in ␣1(I) chains was unaffected, whereas 3-hydroxylation of residue Pro-944 in the ␣2(V) chain was largely lost, and 3-hydroxylation of Pro-707 in ␣2(V) and ␣2(I) chains were sharply reduced. The results imply that P3H2 has preferred substrate sequences among the classes of 3Hyp sites in clade A collagen chains.Collagen, the major structural protein of vertebrates has evolved a range of post-translational modifications that are essential for triple helix assembly and stability, intermolecular cross-linking, and strength of fibrils and tissue function. Enzymes from the family of 2-oxo-glutarate-dependent dioxygenases are responsible for most of these modifications. For example, prolyl 4-hydroxylase catalyzes proline 4-hydroxylation, a modification necessary for the secondary and tertiary structure of collagen (1, 2), and the lysyl hydroxylase isoenzymes catalyze lysine hydroxylation, a modification essential for the formation of intermolecular cross-links in collagen (2-4).Prolyl 3-hydroxlase 1 (P3H1), 2 another member of the 2-oxo-glutarate-dependent dioxygenase family, catalyzes the post-translational 3-hydroxylation of certain proline residues in fibril-forming collagens. This enzyme was only recently cloned from chicken tissues (5) after being partially purified Ͼ25 years ago (6). P3H1 was identified as the chick homologue of rat leprecan, a glycoprotein, which was first isolated from a rat parietal yolk sac tumor cell line (7). Within the past few years, novel mutations in the human P3H1 gene and genes encoding P3H1-associated proteins were shown to cause recessive forms of osteogenesis imperfecta (8 -11). By mass spectrometry, we and other investigators have shown that Pro-986 in the ␣1(I) collagen chain of bone and skin from such patients can be severely under-3-hydroxyated. The P3H1 enz...

Post-translationally Abnormal Collagens of Prolyl 3-Hydroxylase-2 Null Mice Offer a Pathobiological Mechanism for the High Myopia Linked to Human LEPREL1 Mutations

“…The hydroxylation ladder was absent in collagens isolated from P3h2 n/n mouse tissues (Fig. 5B), supporting previous cell culture knockdown assays that suggested the (GPP) n motif was a P3H2 substrate (16).…”

“…However, P3H1 is one of a family of genes that includes three isoenzymes, P3H1 (LEPRE1), P3H2 (LEPREL1), and P3H3 (LEPREL2) (15). It is believed these isoenzymes have evolved differences in their preferred collagen substrate and tissue specificities (16). Indeed, P3H2 mutations have recently been associated with autosomally recessive nonsyndromic severe myopia in humans (17,18).…”

Posttranslational Modifications in Type I Collagen from Different Tissues Extracted from Wild Type and Prolyl 3-Hydroxylase 1 Null Mice

“…This places one of the other two enzymes, P3H2 or P3H3, in the position to fulfill the function of this site 3-hydroxylation. In a recent study (30) based on cell culture experiments, the suppression of P3H2 by siRNA was associated with the sharp decrease in 3-hydroxylation of the A3 site in the ␣-2 chain of type I collagen. Our quantitative real-time PCR results, which summarize tissue-specific expression patters of the prolyl 3-hydroxylase family members, lead to the same conclusion.…”

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

“…cDNA was synthesized from 1 g of RNA using the SuperScript II Reverse Transcriptase kit (Invitrogen) with random hexamers. Subsequently, PCR was performed for 35 cycles with primer sets for human prolyl 3-hydroxylase 1 (P3H1), P3H2, P3H3, cartilage-associated protein (CRTAP), and PPIB previously described by Fernandes et al (31) and for GAPDH previously described by Shah et al (43).…”

“…Expression of Prolyl 3-Hydroxylases in 293-HEK Cells-Differential 3 hydroxylation of prolines at some, but not other sites in clade A fibrillar collagen chain COL1 domains can be due to differential expression of the enzyme prolyl 3-hydroxylase 2 (P3H2) in different cell types and tissues (31). To test whether the absence of 3-Hyp residues at some sites in human recombinant pro-␣1(V) chains that had been 3-hydroxylated in ␣1(V) chains from bovine placenta might be due to deficiency in levels of P3H2, we tested for P3H2 expression in 293-HEK cells.…”