Comparative analysis of p4ha1 and p4ha2 expression during Xenopus laevis development.

Collagen prolyl 4-hydroxylases (c-P4Hs) are evolutionary conserved enzymes whose activity is essential for the correct folding of stable triple helical molecules of collagen and collagen-like proteins. They play crucial roles in embryo development, connective tissue functional organization, tumor growth and metastasis. Despite the important function of these enzymes, little is known about their expression during vertebrate development. In this study, we determine and compare the previously undescribed spatio-temporal expression patterns of the p4ha1 and p4ha2 genes, which encode the main subunits containing the enzyme active site, during Xenopus development. The two genes are maternally inherited and share expression in dorsal mesoderm, branchial arches and their derivatives, as well as in the central nervous system, although with distinct spatio-temporal patterns. A major co-expression domain for p4ha1 and p4ha2 is represented by the developing notochord, where these genes are transcribed from early neurula stage to stage 42 tadpole, thus paralleling the profile of collagen II production and suggesting a coordination between collagen synthesis and its post-translational modifications.

Characterization and expression of enzymatically active recombinant filarial prolyl 4-hydroxylase.

The cuticle of parasitic nematodes consists primarily of a network of collagen molecules. The enzyme responsible for collagen maturation is prolyl 4-hydroxylase, making this enzyme a central activity in cuticle biosynthesis and a potentially important chemotherapeutic target. Adult and embryonic Brugia malayi are shown to be susceptible to inhibitors of vertebrate prolyl 4-hydroxylase, with exposed parasites exhibiting pathologies consistent with a disruption in cuticle biosynthesis. A full-length cDNA (Ov-phy-1) encoding a catalytically active alpha-subunit of Onchocerca volvulus prolyl 4-hydroxylase was isolated and characterized. The derived amino acid sequence of Ov-phy-1 encoded a peptide that was most similar to the two Caenorhabditis elegans prolyl 4-hydroxylase homologues and to the isoform II enzymes of vertebrates. Expressed sequence tag (EST) analysis and developmental polymerase chain reaction (PCR) studies demonstrated that Ov-phy-1 was expressed in L3 and adult parasites. The gene encoding the Ov-phy-1 open reading frame contained 11 introns, similar in structure to the gene encoding human prolyl 4-hydroxylase isoform I. Genomic Southern blot, EST and genomic PCR studies demonstrated that the O. volvulus genome contained between three and eight genes closely related to Ov-phy-1. Co-expression of Ov-phy-1 with the O. volvulus homologue of protein disulfide isomerase in a baculovirus system resulted in the production of enzymatically active O. volvulus prolyl 4-hydroxylase. In vitro production of enzymatically active O. volvulus prolyl 4-hydroxylase should facilitate identification of specific inhibitors of the parasite enzyme.

Prolyl 3-hydroxylase 1, enzyme characterization and identification of a novel family of enzymes.

The collagen prolyl hydroxylases are enzymes that are required for proper collagen biosynthesis, folding, and assembly. They reside within the endoplasmic reticulum and belong to the group of 2-oxoglutarate and iron-dependent dioxygenases. Although prolyl 4-hydroxylase has been characterized as an alpha2beta2 tetramer in which protein disulfide isomerase is the beta subunit with two different alpha subunit isoforms, little is known about the enzyme prolyl 3-hydroxylase (P3H). It was initially characterized and shown to have an enzymatic activity distinct from that of prolyl 4-hydroxylase, but no amino acid sequences or genes were ever reported for the mammalian enzyme. Here we report the characterization of a novel prolyl 3-hydroxylase enzyme isolated from embryonic chicks. The primary structure of the enzyme, which we now call P3H1, demonstrates that P3H1 is a member of a family of prolyl 3-hydroxylases, which share the conserved residues present in the active site of prolyl 4-hydroxylase and lysyl hydroxylase. P3H1 is the chick homologue of mammalian leprecan or growth suppressor 1. Two other P3H family members are the genes previously called MLAT4 and GRCB. In this study we demonstrate prolyl 3-hydroxylase activity of the purified enzyme P3H1 on a full-length procollagen substrate. We also show it to specifically interact with denatured collagen and to exist in a tight complex with other endoplasmic reticulum-resident proteins. Immunohistochemistry with a monoclonal antibody specific for chick P3H1 localizes P3H1 specifically to tissues that express fibrillar collagens, suggesting that other P3H family members may be responsible for modifying basement membrane collagens.