Silicatein-mediated incorporation of titanium into spicules from the demosponge Suberites domuncula

Primmorphs (a three-dimensional sponge primary cell culture system) have been revealed to be a cell/tissue nano-factory for the production of tailor-made hybrid nanostructures. Growth of primmorphs is stimulated by the presence of a titanium alkoxide precursor tolerating titania (TiO₂) concentrations up to 250 μM. The presence and activity of silicatein in primmorphs has been analyzed by gel electrophoresis and Western blotting. Results of studies by scanning transmission electron microscopy with energy-dispersive X-ray spectroscopy have revealed silica and titania to be co-localized on nanosized spicules. Our findings suggest that the incorporation of titania into the nanosized spicule is enzymatically mediated via active silicatein in an orchestrated mechanism.     

Histochemical and electron microscopic analysis of spiculogenesis in the demosponge Suberites domuncula.

The skeleton of demosponges is built of spicules consisting of biosilica. Using the primmorph system from Suberites domuncula, we demonstrate that silicatein, the biosilica-synthesizing enzyme, and silicase, the catabolic enzyme, are colocalized at the surface of growing spicules as well as in the axial filament located in the axial canal. It is assumed that these two enzymes are responsible for the deposition of biosilica. In search of additional potential structural molecules that might guide the mineralization process during spiculogenesis to species-specific spicules, electron microscopic studies with antibodies against galectin and silicatein were performed. These studies showed that silicatein forms a complex with galectin; the strings/bundles of this complex are intimately associated with the surface of the spicules and arranged concentrically around them. Collagen fibers are near the silactein/galectin complexes. The strings/bundles formed from silicatein/galectin display a lower degree of orientation than the collagen fibers arranged in a highly ordered pattern around the spicules. These data indicate that species-specific formation of spicules involves a network of (diffusible) regulatory factor(s) controlling enzymatic silica deposition; this mineralization process proceeds on a galectin/collagen organic matrix.     

Okadaic acid, an apoptogenic toxin for symbiotic/parasitic annelids in the demosponge Suberites domuncula

The role of okadaic acid (OA) in the defense system of the marine demosponge Suberites domuncula against symbiotic/parasitic annelids was examined. Bacteria within the mesohyl produced okadaic acid at concentrations between 32 ng/g and 58 ng/g of tissue (wet weight). By immunocytochemical methods and by use of antibodies against OA, we showed that the toxin was intracellularly stored in vesicles. Western blotting experiments demonstrated that OA also existed bound to a protein with a molecular weight of 35,000 which was tentatively identified as a galectin (by application of antigalectin antibodies). Annelids that are found in S. domuncula undergo apoptotic cell death. OA is one candidate inducer molecule of this process, since this toxin accumulated in these symbionts/parasites. Furthermore, we identified the cDNA encoding the multifunctional prosurvival molecule BAG-1 in S. domuncula; it undergoes strong expression in the presence of the annelid. Our data suggest that sponges use toxins (here, OA) produced from bacteria to eliminate metazoan symbionts/parasites by apoptosis.     

Polarity factor 'Frizzled' in the demosponge Suberites domuncula: identification, expression and localization of the receptor in the epithelium/pinacoderm(1)

Until recently, it was assumed that polarity and axis formation have evolved only in metazoan phyla higher than Cnidaria. One key molecule involved in the signal transduction causing tissue polarity is Frizzled, a seven-transmembrane receptor that is activated by the Wnt family of secreted proteins. We report the isolation and characterization of a Frizzled gene from the demosponge Suberites domuncula (Sd-Fz). The deduced polypeptide comprises all characteristic domains known from Frizzled receptors of higher metazoans. In situ hybridization studies show that Sd-Fz is expressed in cells close to the surface of the sponges and in the pinacocytes of some canals. Northern blot analysis demonstrates its upregulation during the formation of three-dimensional sponge cell aggregates in culture. These data provide for the first time experimental evidence that already in the lowest metazoan phylum (Porifera) genes are present which are very likely involved in tissue polarity.     

Apposition of silica lamellae during growth of spicules in the demosponge Suberites domuncula: biological/biochemical studies and chemical/biomimetical confirmation

Recently it has been discovered that the formation of the siliceous spicules of Demospongiae proceeds enzymatically (via silicatein) and occurs matrix guided (on galectin strings). In addition, it could be demonstrated that silicatein, if immobilized onto inorganic surfaces, provides the template for the synthesis of biosilica. In order to understand the formation of spicules in the intact organism, detailed studies with primmorphs from Suberites domuncula have been performed. The demosponge spicules are formed from several silica lamellae which are concentrically arranged around the axial canal, harboring the axial filament composed of silicatein. Now we show that the appositional growth of the spicules in radial and longitudinal direction proceeds in the extracellular space along hollow cylinders; their surfaces are formed by silicatein. The extracellularly located spicules are surrounded by sclerocytes which are filled with both electron-dense and electron-poor vesicles; energy dispersive X-ray analysis/scanning electron microscopical studies revealed that the electron-dense vesicles are filled of silicon/silica and therefore termed silicasomes. The release of the content of the silicasomes into the hollow cylinder suggests that the newly formed silica lamella originate there; in addition the data are compatible with the view that the silicatein molecules, attached at the centripetal and centrifugal surfaces, mediate biosilica formation. In a chemical/biomimetical approach silicatein is linked onto the organic material-free spicules after their functionalization with aminopropyltriethoxysilane [amino groups]-poly(acetoxime methacrylate) [reactive ester polymer]-N(epsilon)-benzyloxycarbonyl L-lysine tert-butyl ester-Ni(II); finally His-tagged silicatein is immobilized. The matrix-bound enzyme synthesized a new biosilica lamella. These bioinspired findings are considered as the basis for a technical use/application/utilization of hollow cylinders formed by matrix-guided silicatein molecules for the biocatalytic synthesis of nanostructured tubes.     

The complete set of ribosomal proteins from the marine sponge Suberites domuncula

The siliceous marine sponge Suberites domuncula is a member of the most ancient and simplest extant phylum of multicellular animals-Porifera, which have branched off first from the common ancestor of all Metazoa. We have determined primary structures of 79 ribosomal proteins (r-proteins) from S. domuncula: 32 proteins from the small ribosomal subunit and 47 proteins from the large ribosomal subunit. Only L39 and L41 polypeptides (51 and 25 residues long in rat, respectively) are missing. The sponge S. domuncula is, after nematode Caenorhabditis elegans and insect Drosophila melanogaster the third representative of invertebrates with known amino acid sequences of all r-proteins. The comparison of S. domuncula r-proteins with r-proteins from D. melanogaster, C. elegans, rat, Arabidopsis thaliana and Saccharomyces cerevisiae revealed very interesting findings. The majority of the sponge r-proteins are more similar to their homologues from rat, than to those either from invertebrates C. elegans and D. melanogaster, or yeast and plant. With few exceptions, the overall sequence conservation between sponge and rat r-proteins is 80% or higher. The phylogenetic tree of concatenated r-proteins from 6 eukaryotic species (rooted with archaeal r-proteins) has the shortest branches connecting sponge and rat. Both model invertebrate organisms experienced recently accelerated evolution and therefore sponge r-proteins very probably better reflect structures of proteins in the ancestral metazoan ribosome, which changed only little during metazoan evolution. Furthermore, r-proteins from the plant A. thaliana are significantly closer to metazoan r-proteins than are those from the yeast S. cerevisiae.     

Iron induces proliferation and morphogenesis in primmorphs from the marine sponge Suberites domuncula

Dissociated cells from marine demosponges retain their proliferation capacity if they are allowed to form special aggregates, the primmorphs. On the basis of incorporation studies and septin gene expression, we show that Fe3+ ions are required for the proliferation of cells in primmorphs from Suberites domuncula. In parallel, Fe3+ induced the expression of ferritin and strongly stimulated the synthesis of spicules. This result is supported by the finding that the enzymatic activity of silicatein, converting organosilicon to silicic acid, depends on Fe3+. Moreover, the expression of a scavenger receptor molecule, possibly involved in the morphology of spicules, depends on the presence of Fe3+. We conclude that iron is an essential factor in proliferative and morphogenetic processes in primmorphs.     

Origin of the integrin-mediated signal transduction. Functional studies with cell cultures from the sponge Suberites domuncula.

Sponges (phylum Porifera) represent the phylogenetically oldest metazoan animals. Recently, from the marine sponge Geodia cydonium a first cDNA encoding a putative integrin receptor molecule was isolated. In the present study basic functional experiments have been conducted to test the hypothesis that in sponges integrin polypeptides also function as adhesion molecules and as outside-in signaling molecules. The sponge Suberites domuncula has been used for the experiments because from this sponge only has a cell culture been established. Here we report that aggregation factor (AF)-mediated cell-cell adhesion is blocked by the RGDS peptide which is known to interact with beta integrin. Both RGDS and AF were found to stimulate DNA synthesis within 24 h. The beta subunit of the integrin receptor was cloned from S. domuncula; the estimated 91-kDa molecule comprises the characteristic signatures. Evolutionary conservation of the beta integrin was assessed by comparison with corresponding beta integrin subunits from evolutionary higher metazoan taxa. Addition of RGDS or of AF to isolated cells of S. domuncula causes a rapid (within 1-2 min) increase in the intracellular Ca2+ concentration which is further augmented in the presence of Ca2+. Furthermore, incubation of the cells with RGDS or AF causes an activation of the GTP-binding protein Ras. In addition it is shown that after a prolonged incubation of the cells with RGDS and AF the expression of the genes coding for Ras and for calmodulin is upregulated. These results suggest that the integrin receptor functions in the sponge system not only as adhesion molecule but also as a molecule involved in outside-in signaling.     

Partial characterization of suberitine,the neurotoxic protein purified from Suberites domuncula

• 1.1. The protein (mol. wt 28.000) consists of four subunits, which are not equivalent as regards the chemical composition, although all of them lack free terminal amino groups.
• 2.2. The single subunits also possess different conformations since both photosensitized oxidation studies and acrylamide-quenching experiments of the protein fluorescence emission show that the three tryptophyl residues have a different accessibility to the aqueous solvent.
• 3.3. Circular dichroism and fluorescence polarization studies suggest that suberitine has a remarkable tight three-dimensional organization: e.g., exposure of suberitine to 6 M urea for several hours is necessary to obtain a general unfolding of the protein molecule.

     

Okadaic Acid, an Apoptogenic Toxin for Symbiotic/Parasitic Annelids in the Demosponge Suberites domuncula

The role of okadaic acid (OA) in the defense system of the marine demosponge Suberites domuncula against symbiotic/parasitic annelids was examined. Bacteria within the mesohyl produced okadaic acid at concentrations between 32 ng/g and 58 ng/g of tissue (wet weight). By immunocytochemical methods and by use of antibodies against OA, we showed that the toxin was intracellularly stored in vesicles. Western blotting experiments demonstrated that OA also existed bound to a protein with a molecular weight of 35,000 which was tentatively identified as a galectin (by application of antigalectin antibodies). Annelids that are found in S. domuncula undergo apoptotic cell death. OA is one candidate inducer molecule of this process, since this toxin accumulated in these symbionts/parasites. Furthermore, we identified the cDNA encoding the multifunctional prosurvival molecule BAG-1 in S. domuncula; it undergoes strong expression in the presence of the annelid. Our data suggest that sponges use toxins (here, OA) produced from bacteria to eliminate metazoan symbionts/parasites by apoptosis.     

Origin of metazoan stem cell system in sponges: first approach to establish the model (Suberites domuncula)

It is established that Porifera (sponges) represent the earliest phylum which branched off from the common ancestor of all multicellular animals, the Urmetazoa. In the present study, the hypothesis is tested if, during this transition, pluripotent stem cells were formed which are provided-similar to the totipotent cells (archaeocytes/germ cells)-with a self-renewal capacity. As a model system, primmorphs from the sponge Suberites domuncula were used. These 3D-cell aggregates were cultivated in medium (RPMI 1640/seawater) either lacking silicate and ferric iron or in medium which was supplemented with these 'morphogenetic' factors. As molecular markers for the potential existence of stem cells in primmorphs, two genes which encode proteins found in stem cells of higher metazoan species, were cloned from S. domuncula. First, the noggin gene, which is present in the Spemann organizer of amphibians and whose translation product acts during the formation of dorsal mesoderm derivatives. The second gene encodes the mesenchymal stem cell-like protein. Both cDNAs were used to study their expression in primmorphs in dependence on the incubation conditions. It was found that noggin expression is strongly upregulated in primmorphs kept in the presence of silicate and ferric iron, while the expression of the mesenchymal stem cell-like protein was downregulated. These data are discussed with respect to the existence of stem cells in sponges.     

Genome size and chromosomes in marine sponges [Suberites domuncula,Geodia cydonium]

The genome size of the marine sponges Suberites domuncula and Geodia cydonium has been determined by flow cytofluorometric analysis using diamidino-phenylindole [DAPI]. Using human lymphocytes as reference the amount of DNA in cells from S. domuncula has been determined to be 3.7 pg and that of G. cydonium 3.3 pg. While no chromosomes could be identified in G. cydonium, the karyotype of the Suberites domuncula is 32 chromsomes in the diploid state. The size of the chromosomes was between 0.25 and 1.0 μm. No pronounced banding pattern was visible.     

Bruton tyrosine kinase-like protein, BtkSD, is present in the marine sponge Suberites domuncula

Sponges, the simplest and most ancient phylum of Metazoa, encode in their genome complex and highly sophisticated proteins that evolved together with multicellularity and are found only in metazoan animals. We report here the finding of a Bruton tyrosine kinase (BTK)-like protein in the marine sponge Suberites domuncula (Demospongiae). The nucleotide sequence of one sponge cDNA predicts a 700-aa-long protein, which contains all of the characteristic domains for the Tec family of protein tyrosine kinases (PTKs). The highest homology (38% identity, 55% overall similarity) was found with human BTK and TEC PTKs. Sponge PTK was therefore named BtkSD. Human BTK is involved in the maturation of B cells and mutations in the BTK gene cause X-linked agammaglobulinemia. Kinases from the Tec family are not present in Caenorhabditis elegans and, until now, they were found only in insects and higher animal taxa. Our finding implies that the BTK/TEC genes are of a very ancient origin.     

Synthesis of the Neurotoxin Quinolinic Acid in Apoptotic Tissue from Suberites domuncula: Cell Biological, Molecular Biological, and Chemical Analyses

Sessile marine animals, such as sponges, are prone to infection by prokaryotic as well as by eukaryotic attacking organisms. Using the sponge Suberites domuncula we document for the first time that in its apoptotic tissue a toxic compound is produced that very likely controls the elimination of the dying tissue. Apoptosis was induced by exposing the sponges to 2,2'-dipyridyl or by maintaining them under nonaeration conditions. After that treatment at least one eukaryotic epibiont (Bittium sp.) could be found grazing on apoptotic tissue. Cell proliferation assays demonstrated that aqueous extracts from unaffected sponge tissue displayed no cytotoxicity. However, addition of an extract from apoptotic tissue to neuronal cells from rat brain exerted strong toxicity. The underlying compound was identified as quinolinic acid; quantitative determination showed that quinolinic acid is present only in apoptotic tissue (4.8 mg/g dry wet weight). The complementary DNA encoding the key enzyme of the quinolinic acid pathway, 3-hydroxyanthranilate 3,4-dioxygenase, was cloned and characterized. The expression of this gene is up-regulated in apoptotic tissue. These data suggest that a complex molecular network controls apoptotic elimination of sponge tissue, which results in the synthesis of the bioactive compound quinolinic acid that controls the elimination of the tissue, perhaps via differential effects on grazing epibionts.