Molecular cloning, characterization and expression analysis of a prolyl 4-hydroxylase from the marine sponge Chondrosia reniformis

Prolyl 4-hydroxylase (P4H) catalyzes the hydroxylation of proline residues in collagen. P4H has two functional subunits, \u3b1 and \u3b2. Here, we report the cDNA cloning, characterization, and expression analysis of the \u3b1 and \u3b2 subunits of the P4H derived from the marine sponge Chondrosia reniformis. The amino acid sequence of the \u3b1 subunit is 533 residues long with an Mr of 59.14 kDa, while the \u3b2 subunit counts 526 residues with an Mr of 58.75 kDa. Phylogenetic analyses showed that \u3b1P4H and \u3b2P4H are more related to the mammalian sequences than to known invertebrate P4Hs. Western blot analysis of sponge lysate protein cross-linking revealed a band of 240 kDa corresponding to an \u3b12\u3b22 tetramer structure. This result suggests that P4H from marine sponges shares the same quaternary structure with vertebrate homologous enzymes. Gene expression analyses showed that \u3b1P4H transcript is higher in the choanosome than in the ectosome, while the study of factors affecting its expression in sponge fragmorphs revealed that soluble silicates had no effect on the \u3b1P4H levels, whereas ascorbic acid strongly upregulated the \u3b1P4H mRNA. Finally, treatment with two different tumor necrosis factor (TNF)-alpha inhibitors determined a significant downregulation of \u3b1P4H gene expression in fragmorphs demonstrating, for the first time in Porifera, a positive involvement of TNF in sponge matrix biosynthesis. The molecular characterization of P4H genes involved in collagen hydroxylation, including the mechanisms that regulate their expression, is a key step for future recombinant sponge collagen production and may be pivotal to understand pathological mechanisms related to extracellular matrix deposition in higher organism