We report here the complete cDNA sequence of a nonfibrillar collagen (COLch) isolated from the marine sponge Chondrosia reniformis, Nardo 1847 using a PCR approach. COLch cDNA consists of 2,563 nucleotides and includes a 5' untranslated region (UTR) of 136 nucleotides, a 3' UTR of 198 nucleotides, and an open reading frame encoding for a protein of 743 amino acids with an estimated M (r) of 72.12 kDa. The phylogenetic analysis on the deduced amino acid sequence of C-terminal end shows that the isolated sequence belongs to the short-chain spongin-like collagen subfamily, a nonfibrillar group of invertebrate collagens similar to type IV collagen. In situ hybridization analysis shows higher expression of COLch mRNA in the cortical part than in the inner part of the sponge. Therefore, COLch seems to be involved in the formation of C. reniformis ectosome, where it could play a key role in the attachment to the rocky substrata and in the selective sediment incorporation typical of these organisms. qPCR analysis of COLch mRNA level, performed on C. reniformis tissue culture models (fragmorphs), also demonstrates that this matrix protein is directly involved in sponge healing processes and that soluble silicates positively regulate its expression. These findings confirm the essential role of silicon in the fibrogenesis process also in lower invertebrates, and they should give a tool for a sustainable production of marine collagen in sponge mariculture.

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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Here we report the molecular cloning and characterization of the first Tumor Necrosis Factor homologous and of its putative receptor in the marine sponge Chondrosia reniformis: chTNF and chTNFR, respectively. The deduced chTNF amino acid sequence is a type II transmembrane protein containing the typical TNFSF domain. Phylogenetic analysis reveals that chTNF is more related to Chordata TNFs rather than to other invertebrates. chTNF and chTNFR are constitutively expressed both in the ectosome and in the choanosome of the sponge, with higher levels in the ectosome. chTNF and chTNFR mRNAs were monitored in sponge fragmorphs treated with Gram(+) or Gram(-) bacteria. chTNF was significantly upregulated in Gram(+)-treated fragmorphs as compared to controls, while chTNFR was upregulated by both treatments. Finally, the possible chTNF fibrogenic role in sponge fragmorphs was studied by TNF inhibitor treatment measuring fibrillar and non fibrillar collagen gene expression; results indicate that the cytokine is involved in sponge collagen deposition and homeostasis.

The possibility to cryopreserve cells allows for wide opportunities of flexible handling of cell cultures from different sponge species. Primmorphs model, a multicellular 3D aggregate formed by dissociated sponge cells, is considered one of the best approaches to establish sponge cell culture but, in spite of the available protocols for freezing sponge cells, there is no information regarding the ability of the latter to form primmorphs after thawing. In the present work, we demonstrate that, after a freezing and thawing cycle using dissociated Petrosia ficiformis cells as a model, cells viability was high but it was not possible to obtain primmorphs. The same protocol for cryopreservation was then used to directly freeze primmorphs. In this second case, after thawing, viability and the cellular proliferative level were similar to unfrozen standard primmorphs. Spiculogenesis in thawed primmorphs was evaluated by quantifying the silicatein gene expression level and by assaying the silica amount in the newly formed spicules, then compared with the correspondent values obtained in standard unfrozen primmorphs. Results indicate that the freezing cycle does not affect the spiculogenesis rate. Finally, the expression level of heat shock protein 70, a well-known stress marker, was assayed and the results showed no differences between frozen and unfrozen samples. These findings are likely to promote relevant improvements in sponge cell culture technique, allowing for a worldwide exchange of living biological material, paving the way for cell banking of Porifera.

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Biosilica is an amazing example of natural order and complexity. Siliceous sponge spicules, in particular, are characterized by a large variety of dimensions and shapes, with an ultrastructure based on silica nanoparticles strictly packaged around an axial filament constituted by a family of proteins called silicateins. These peculiar proteins have a high sequence homology with cathepsins and they play a double role of enzyme and template in the control of biosilica precipitation. However, their natural structural organization inside the spicules is far from being understood in details. In this work, axial filaments extracted from spicules of Petrosia ficiformis have been extensively analyzed by mass spectrometry, exploiting MALDI and ESI analysis of both the intact protein and the peptides coming from digestion of the axial filament with different proteases. Results demonstrate that P. ficiformis spicules contain almost only silicatein beta. Several post-translational modifications, like methylations at the N-terminal region, three phosphorylation sites, and the oxidation of a histidine and of a cysteine to cysteic acid, are described.