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

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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.

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Acknowledgments

This work was supported by grant from EU (FP7 grant agreement no. 266033 SPonge Enzyme and Cell for Innovative AppLication—SPECIAL) to Marco Giovine and by the University of Genova Funding (PRA 2012) to Sonia Scarfì. The authors thank Carlo Cerrano for the supplying of fresh marine sponges.

Conflict of Interest

The authors declare no conflict of interests.

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Authors and Affiliations

  1. Department of Territory Environment and Life Sciences, University of Genova, Via Pastore 3, 16132, Genova, Italy

    Marina Pozzolini, Sonia Scarfì, Sara Ferrando, Lorenzo Gallus & Marco Giovine

  2. Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV, 1, 16132, Genova, Italy

    Francesca Mussino

Authors
  1. Marina Pozzolini
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  2. Sonia Scarfì
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  3. Francesca Mussino
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  4. Sara Ferrando
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  5. Lorenzo Gallus
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  6. Marco Giovine
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Corresponding author

Correspondence to Marina Pozzolini.

Electronic supplementary material

Below is the link to the electronic supplementary material.

3D reconstruction of P4H immunoreactivity and nuclei in choanocyte chambers in C. reniformis. (MP4 649 kb)

Movie 2

3D reconstruction of P4H immunoreactivity and nuclei close to sand grains in C. reniformis. (MP4 446 kb)

Movie 3

3D reconstruction of P4H immunoreactivity and nuclei close to sand grains in C. reniformis. (MP4 776 kb)

Movie 4

3D reconstruction of P4H immunoreactivity and nuclei close to sand grains in C. reniformis. (MP4 850 kb)

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Pozzolini, M., Scarfì, S., Mussino, F. et al. Molecular Cloning, Characterization, and Expression Analysis of a Prolyl 4-Hydroxylase from the Marine Sponge Chondrosia reniformis . Mar Biotechnol 17, 393–407 (2015). https://doi.org/10.1007/s10126-015-9630-3

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