Molecular Cloning, Characterization, and Expression Analysis of a Prolyl 4-Hydroxylase from the Marine Sponge Chondrosia reniformis

Abstract

Prolyl 4-hydroxylase (P4H) catalyzes the hydroxylation of proline residues in collagen. P4H has two functional subunits, α and β. Here, we report the cDNA cloning, characterization, and expression analysis of the α and β subunits of the P4H derived from the marine sponge Chondrosia reniformis. The amino acid sequence of the α subunit is 533 residues long with an M r of 59.14 kDa, while the β subunit counts 526 residues with an M r of 58.75 kDa. Phylogenetic analyses showed that αP4H and βP4H 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 α2β2 tetramer structure. This result suggests that P4H from marine sponges shares the same quaternary structure with vertebrate homologous enzymes. Gene expression analyses showed that αP4H 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 αP4H levels, whereas ascorbic acid strongly upregulated the αP4H mRNA. Finally, treatment with two different tumor necrosis factor (TNF)-alpha inhibitors determined a significant downregulation of αP4H 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 organisms.

Silica-induced fibrosis: an ancient response from the early metazoans

“…Given the fibrotic effect of quartz dust in mammalian models, it has been speculated that crystalline silica sand incorporation and erosion by C. reniformis could be causal to sponge collagen biosynthesis . The identification by immunostaining of the enzyme involved in collagen maturation, prolyl-4-hydroxylase (P4H), around the numerous sand grains usually incorporated by this sponge strongly supports this hypothesis (Pozzolini et al, 2015). A functional relationship between collagen and silica has already been described in glass sponges (Hexatinellida).…”

Bioinspiring Chondrosia reniformis (Nardo, 1847) Collagen-Based Hydrogel: A New Extraction Method to Obtain a Sticky and Self-Healing Collagenous Material

“…The range was comprised between 16.8 and 6.92 mg/mL. Higher concentrations were obtained from Ec samples given the fact that Ec is characterized by a higher abundance of both non fibrillar [ 44 ] and fibrillar [ 45 , 46 ] collagen than choanosome. In this view the higher yield of extracted material from Ec (126–109 mg/g of fresh sponge tissue) compared to Ch (77–50 mg/g of fresh sponge tissue) was not surprising.…”

“…The extraction was performed separately for the two different sponge regions: the ectosome (Ec; the outer cortical layer), and the choanosome (Ch) that constitutes the main bulk of sponge. Independent collagen extractions from the two regions were run in parallel in order to evaluate potential differences in the characteristics of hydrogels given that type IV collagen is more abundant in the ectosome than in the choanosome [ 44 ].…”

The meteorology of cytokine storms, and the clinical usefulness of this knowledge

“…Cytokines can be good examples. For instance, although TNF is large in the mammalian literature it is also found in the marine sponge, Chondrosia reniformis [16], and the reef-building corals, Acropora spp. [17].…”