A cryptochrome-based photosensory system in the siliceous sponge Suberites domuncula (Demospongiae)

Based on the light-reactive behavior of siliceous sponges, their intriguing quartz glass-based spicular system and the existence of a light-generating luciferase [Muller WEG et al. (2009) Cell Mol Life Sci 66, 537–552], a protein potentially involved in light reception has been identified, cloned and recombinantly expressed from the demosponge Suberites domuncula. Its sequence displays two domains characteristic of cryptochrome, the N-terminal photolyase-related region and the C-terminal FAD-binding domain. The expression level of S. domuncula cryptochrome depends on animal’s exposure to light and is highest in tissue regions rich in siliceous spicules; in the dark, no cryptochrome transcri…

Isolation and characterization of two T-box genes from sponges, the phylogenetically oldest metazoan taxon

It is now well established that all metazoan phyla derived from one common ancestor, the hypothetical Urmetazoa. Due to the basal position of Porifera (Demospongiae) in the phylogenetic tree of Metazoa, studies on the mechanisms controlling the development of these animals can provide clues on the understanding of the origin of multicellular animals and on how the first organization of the body plan evolved. In this report we describe the isolation and genomic characterization of two T-box genes from the siliceous sponge Suberites domuncula. The phylogenetic analysis classifies one into the subfamily of Brachyury, Sd-Bra, and the second into the Tbx2 subfamily, Sd-Tbx2. Analyses of the Sd-B…

Biosilicification of loricate choanoflagellate: organic composition of the nanotubular siliceous costal strips of Stephanoeca diplocostata.

SUMMARY Loricate choanoflagellates (unicellular, eukaryotic flagellates; phylum Choanozoa) synthesize a basket-like siliceous lorica reinforced by costal strips (diameter of approximately 100 nm and length of 3 μm). In the present study, the composition of these siliceous costal strips is described, using Stephanoeca diplocostata as a model. Analyses by energy-dispersive X-ray spectroscopy (EDX), coupled with transmission electron microscopy (TEM), indicate that the costal strips comprise inorganic and organic components. The organic, proteinaceous scaffold contained one major polypeptide of mass 14 kDa that reacted with wheat germ agglutinin. Polyclonal antibodies were raised that allowed …

Bio-Sintering/Bio-Fusion of Silica in Sponge Spicules

The synthesis of siliceous spicules in both demosponges and hexactinellids is enzymatically driven via silicatein. This enzyme exists both intra-spicularly and in the extra-spicular space. It catalyzes the formation of bio-silica constituting the silica lamellae that are formed during the appositional (layer-by-layer) growth of the spicules. The extent of (bio-silica forming) activity of silicatein from the demosponge Suberites domuncula measured in vitro reflects the amount of bio-silica required for the formation of spicules in vivo. It is shown that during growth and maturation of the spicules in demosponges a bio-fusion process occurs that results in an intra-spicular sintering of the s…

Phenylalanine hydroxylase from the sponge Geodia cydonium: implication for allorecognition and evolution of aromatic amino acid HYDROXYLASESfn1fn1Thesequence reported here is deposited in the EMBL\GenBank data base (Accession no. Y16353).

Abstract The prophenoloxidase activating system is a defense system, frequently reported both in protostomes and in deuterostomes. The final product of the phenoloxidase activity is melanin which is ubiquitously present throughout the metazoan kingdom. The melanin synthesis pathway starts with the amino acid [aa] phenylalanine which is converted to tyrosine by the phenylalanine hydroxylase [PAH]. We show that after allo-transplantation in the marine sponge Geodiacydonium PAH is upregulated in the grafts. Enzyme determination studies revealed that PAH activity increases by three-fold two d after transplantation and reaches its maximum after 3 d (by 3.7-fold). This finding was supported by de…

Biosilica-Based Strategies for Treatment of Osteoporosis and Other Bone Diseases

Osteoporosis is a common disease in later life, which has become a growing public health problem. This degenerative bone disease primarily affects postmenopausal women, but also men may suffer from reduced bone mineral density. The development of prophylactic treatments and medications of osteoporosis has become an urgent issue due to the increasing proportion of the elderly in the population. Apart from medical/hormonal treatments, current strategies for prophylaxis of osteoporosis are primarily based on calcium supplementation as a main constituent of bone hydroxyapatite mineral. Despite previous reports suggesting an essential role in skeletal growth and development, the significance of …

The Unique Invention of the Siliceous Sponges: Their Enzymatically Made Bio-Silica Skeleton

Sponges are sessile filter feeders that, among the metazoans, evolved first on Earth. In the two classes of the siliceous sponges (the Demospongiae and the Hexactinellida), the complex filigreed body is stabilized by an inorganic skeleton composed of amorphous silica providing them a distinct body shape and plan. It is proposed that the key innovation that allowed the earliest metazoans to form larger specimens was the enzyme silicatein. This enzyme is crucial for the formation of the siliceous skeleton. The first sponge fossils with body preservation were dated back prior to the “Precambrian-Cambrian” boundary [Vendian (610–545 Ma)/Ediacaran (542–580 Ma)]. A further molecule required for t…

Hardening of bio-silica in sponge spicules involves an aging process after its enzymatic polycondensation: evidence for an aquaporin-mediated water absorption.

Abstract Background Spicules, the siliceous skeletal elements of the siliceous sponges, are synthesized enzymatically via silicatein. The product formed, bio-silica, constitutes their inorganic matrix. It remained unexplored which reactions are involved in molding of the amorphous bio-silica and formation of a solid and rigid biomaterial. Methods Cell and molecular biological techniques have been applied to analyze processes resulting in the hardening of the enzymatically synthesized bio-silica. The demosponge Suberites domuncula has been used for the studies. Results Cell aggregates (primmorphs) from the sponge S . domuncula , grown in the presence of Mn-sulfate, form spicules that compris…

Caspase-mediated apoptosis in sponges: cloning and function of the phylogenetic oldest apoptotic proteases from Metazoa

AbstractSponges (phylum Porifera) represent the phylogenetically oldest metazoan phylum. These animals have complex cell adhesion and powerful immune systems which allow the formation of a distinct body plan. Consequently, an apoptotic machinery has to be predicted that allows sponges to eliminate unwanted cells accumulating during development. With the marine sponge Geodia cydonium, it is shown that allografts of these animals undergo apoptosis as demonstrated by apoptotic DNA fragmentation. Extracts from allografts contain an enzymic activity characteristic for caspases; as substrate to determine the cleavage activity, Ac-DEVD-AMC was applied. cDNAs encoding predicted caspase-3-related pr…

Deep metazoan phylogeny: When different genes tell different stories

11 páginas, 4 figuras, 1 tabla.

Silintaphin-1 - interaction with silicatein during structure-guiding bio-silica formation

Silicateins are unique enzymes of sponges (phylum Porifera) that template and catalyze the polymerization of nanoscale silicate to siliceous skeletal elements. These multifunctional spicules are often elaborately shaped, with complex symmetries. They carry an axial proteinaceous filament, consisting of silicatein and the scaffold protein silintaphin-1, which guides silica deposition and subsequent spicular morphogenesis. In vivo, the synthesis of the axial filament very likely proceeds in three steps: (a) assembly of silicatein monomers to form one pentamer; (b) assembly of pentamers to form fractal-like structures; and finally (c) assembly of fractal-like structures to form filaments. The …

Sponge biosilica formation involves syneresis following polycondensation in vivo.

Syneresis is a process observed during the maturation/aging of silica gels obtained by sol-gel synthesis that results in shrinkage and expulsion of water due to a rearrangement and increase in the number of bridging siloxane bonds. Here we describe how the process of biosilica deposition during spicule ("biosilica" skeleton of the siliceous sponges) formation involves a phase of syneresis that occurs after the enzyme-mediated polycondensation reaction. Primmorphs from the demosponge Suberites domuncula were used to study syneresis and the inhibition of this mechanism. We showed by scanning electron microscopy that spicules added to primmorphs that have been incubated with manganese sulfate …

Bioengineering of the silica-polymerizing enzyme silicatein-alpha for a targeted application to hydroxyapatite.

Since its discovery, numerous biotechnological approaches have aimed to explore the silica-polymerizing catalytic activity of the enzyme silicatein. In vivo, silicatein catalyzes polymerization of amorphous silica nanospheres from soluble precursors. In vitro, it directs the formation of nanostructured biosilica. This is of interest for various applications that strive to benefit from both the advantages of the biological system (i.e., silica synthesis under physiological conditions) and the cell mineralization-stimulating effect of biosilica. However, so far immobilization of silicatein has been hampered by the complex multistep procedure required. In addition, the chemical surface modific…

Front Cover Advanced Materials 3/2012

Evagination of Cells Controls Bio-Silica Formation and Maturation during Spicule Formation in Sponges

The enzymatic-silicatein mediated formation of the skeletal elements, the spicules of siliceous sponges starts intracellularly and is completed extracellularly. With Suberites domuncula we show that the axial growth of the spicules proceeds in three phases: (I) formation of an axial canal; (II) evagination of a cell process into the axial canal, and (III) assembly of the axial filament composed of silicatein. During these phases the core part of the spicule is synthesized. Silicatein and its substrate silicate are stored in silicasomes, found both inside and outside of the cellular extension within the axial canal, as well as all around the spicule. The membranes of the silicasomes are inte…

Acquisition of Structure-guiding and Structure-forming Properties during Maturation from the Pro-silicatein to the Silicatein Form

Silicateins are the key enzymes involved in the enzymatic polycondensation of the inorganic scaffold of the skeletal elements of the siliceous sponges, the spicules. The gene encoding pro-silicatein is inserted into the pCold TF vector, comprising the gene for the bacterial trigger factor. This hybrid gene is expressed in Escherichia coli and the synthesized fusion protein is purified. The fusion protein is split into the single proteins with thrombin by cleavage of the linker sequence present between the two proteins. At 23 °C, the 87 kDa trigger factor-pro-silicatein fusion protein is cleaved to the 51 kDa trigger factor and the 35 kDa pro-silicatein. The cleavage process proceeds and res…

Bioinspired self-assembly of tyrosinase-modified silicatein and fluorescent core-shell silica spheres.

Inspired by the intermolecular cross-linking of mussel foot proteins and their adhesive properties, tyrosinase has been used to modify recombinant silicatein. DOPA/DOPAquinone-mediated cross-linking and interfacial interactions enhanced both self-assembly of silicatein building blocks and templating of core–shell silica spheres, resulting in fluorescent biomimetic silicatein–silica hybrid mesofibers.

Differential Expression of the Demosponge (Suberites domuncula) Carotenoid Oxygenases in Response to Light: Protection Mechanism Against the Self-Produced Toxic Protein (Suberitine)

The demosponge Suberites domuncula has been described to contain high levels of a proteinaceous toxin, Suberitine, that displays haemolytic activityIn the present study this 7–8 kDa polypeptide has been isolated and was shown to exhibit also cytotoxic effects on cells of the same species. Addition of retinal, a recently identified metabolite of β-carotene that is abundantly present in S. domuncula was found to reduce both the haemolytic and the cell toxic activity of Suberitine at a molar ratio of 1:1. Spectroscopic analyses revealed that the interaction between β-carotene and Suberitine can be ascribed to a reversible energy transfer reaction. The enzyme that synthesises retinal in the spo…

Biosilica

Biomineralization, biosilicification in particular (i.e. the formation of biogenic silica, SiO(2)), has become an exciting source of inspiration for the development of novel bionic approaches following 'nature as model'. Siliceous sponges are unique among silica-forming organisms in their ability to catalyze silica formation using a specific enzyme termed silicatein. In this study, we review the present state of knowledge on silicatein-mediated 'biosilica' formation in marine demosponges, the involvement of further molecules in silica metabolism and their potential applications in nano-biotechnology and bio-medicine. While most forms of multicellular life have developed a calcium-based skel…

Cellular effects of bacterial N-3-Oxo-dodecanoyl-L-Homoserine lactone on the sponge Suberites domuncula (Olivi, 1792): insights into an intimate inter-kingdom dialogue.

International audience; Sponges and bacteria have lived together in complex consortia for 700 million years. As filter feeders, sponges prey on bacteria. Nevertheless, some bacteria are associated with sponges in symbiotic relationships. To enable this association, sponges and bacteria are likely to have developed molecular communication systems. These may include molecules such as N-acyl-L-homoserine lactones, produced by Gram-negative bacteria also within sponges. In this study, we examined the role of N-3-oxododecanoyl-L-homoserine lactone (3-oxo-C12-HSL) on the expression of immune and apoptotic genes of the host sponge Suberites domuncula. This molecule seemed to inhibit the sponge inn…

Cover Picture: NanoSIMS: Insights into the Organization of the Proteinaceous Scaffold within Hexactinellid Sponge Spicules (ChemBioChem 8/2010)

An evolutionary perspective on the role of mesencephalic astrocyte-derived neurotrophic factor (MANF): At the crossroads of poriferan innate immune and apoptotic pathways

The mesencephalic astrocyte-derived neurotrophic factor (MANF) belongs to a recently discovered family of neurotrophic factors. MANF can be secreted but is generally resident within the endoplasmic reticulum (ER) in neuronal and non-neuronal cells, where it is involved in the ER stress response with pro-survival effects. Here we report the discovery of the MANF homolog SDMANF in the sponge Suberites domuncula. The basal positioning of sponges (phylum Porifera) in the animal tree of life offers a unique vantage point on the early evolution of the metazoan-specific genetic toolkit and molecular pathways. Since sponges lack a conventional nervous system, SDMANF presents an enticing opportunity…

Morphology of Sponge Spicules: Silicatein a Structural Protein for Bio-Silica Formation

Most forms of multicellular life have developed a calcium-based skeleton, while only a few specialized organisms complement their body plan with silica, such as sponges (phylum Porifera). However, the way in which sponges synthesize their silica is exceptional. They use an enzyme, silicatein, for the polymerization/polycondensation of silica, and thereby form their highly resistant and stabile massive siliceous skeletal elements (spicules). During this biomineralization process (i.e., biosilicification), hydrated amorphous silica is deposited within highly specialized sponge cells, ultimately resulting in structures that range in size from micrometers to meters. This peculiar phenomenon has…

The role of biosilica in the osteoprotegerin/RANKL ratio in human osteoblast-like cells

Abstract Earlier studies have demonstrated that biosilica, synthesized by the enzyme silicatein, induces hydroxyapatite formation in osteoblast-like SaOS-2 cells. Here we study the effect of biosilica on the expressions of osteoprotegerin [OPG] and the receptor activator for NF-κB ligand [RANKL] in the SaOS-2 cell model. We show that during growth of SaOS-2 cells on biosiliceous matrices hydroxyapatite formation is induced, while syntheses of cartilaginous proteoglycans and sulfated glycosaminoglycans are down-regulated. Furthermore, quantitative real-time RT-PCR analysis revealed a strong time-depended increase in expression of OPG in biosilica exposed SaOS-2 cells while the steady-state e…

Bauplan of Urmetazoa: Basis for Genetic Complexity of Metazoa

Sponges were first grouped to the animal-plants or plant-animals then to the Zoophyta or Mesozoa and finally to the Parazoa. Only after the application of molecular biological techniques was it possible to place the Porifera monophyletically with the other metazoan phyla, justifying a unification of all multicellular animals to only one kingdom, the Metazoa. The first strong support came from the discovery that cell-cell and cell-matrix adhesion molecules that were cloned from sponges and were subsequently expressed share a high DNA sequence and protein function similarity with the corresponding molecules of other metazoans. Besides these evolutionary novelties for Metazoa, sponges also hav…

Bio-vaterite formation by glycoproteins from freshwater pearls

Abstract A 48 kDa acidic and putative calcium-binding glycoprotein was isolated from pearls of the freshwater mussel Hyriopsis cumingii . This protein was compared with a related 46 kDa polypeptide, obtained from the nacreous shell of the same species. Separation by two-dimensional gel electrophoresis revealed that the difference in molecular weight is due to the higher extent of glycosylation of the 48 kDa protein existing in pearls. Evidence is presented that the sugar moieties of the protein contribute to crystal growth, starting with the nucleation step. In in vitro precipitation experiments, the 48 kDa glycoprotein of pearls directed the formation of round-shaped vaterite crystals whil…

Towards a Molecular Systematics of the Lake Baikal/Lake Tuva Sponges

Lake Baikal is famous for its extensive biodiversity that is equaled only by few other lakes. Fascinatingly, about 80% of all the animals the lake hosts are endemic. Sponges (Porifera) that live in symbiosis with photosynthetic algae are the most abundant animal taxon found in the littoral zone of Lake Baikal and have been grouped to the family Lubomirskiidae. In recent years, several attempts to determine the phylogenetic relationship between Lubomirskiidae and cosmopolitan freshwater sponges have been undertaken. Yet the results obtained remain inconclusive. Here, we strive to determine the phylogeny of freshwater sponges with the focus on endemic Lake Baikal species, also taking into acc…

The silicatein propeptide acts as inhibitor/modulator of self-organization during spicule axial filament formation.

Silicateins are crucial enzymes that are involved in formation of the inorganic biosilica scaffold of the spicular skeleton of siliceous sponges. We show that silicatein acquires its structure-guiding and enzymatically active state by processing of silicatein from pro-silicatein to the mature enzyme. A recombinant propeptide (PROP) of silicatein from the siliceous demosponge Suberites domuncula was prepared, and antibodies were raised against the peptide. In sponge tissue, these antibodies reacted with both surface structures and the central region of the spicules. Using phage display expression, spicule-binding 12-mer peptides were identified that are rich in histidine residues. In the pre…

Sponges (Porifera) as living metazoan witnesses from the Neoproterozoic: biomineralization and the concept of their evolutionary success

Terra Nova, 22, 1–11, 2010 Abstract The emergence of the Metazoa can be dated back to the Neoproterozoic Era which comprises the Cryogenian Period during which two major glaciations occurred, the Sturtian and the Varanger-Marinoan. At that time, the phylum Porifera (sponges) evolved as the first animals and developed a hard skeleton. The two classes of siliceous sponges, the Hexactinellida and the Demospongiae, are already provided with the major genetic repertoire and gene regulatory networks that also exist in modern multicellular animals. Besides these metazoan innovations, the siliceous sponges display one autapomorphic character, silicatein, an enzyme which mediates bio-silica formatio…

Silicatein: Nanobiotechnological and Biomedical Applications

Silica-based materials are used in many high-tech products including microelectronics, optoelectronics, and catalysts. Siliceous sponges (Demospongiae and Hexactinellida) are unique in their ability to synthesize silica enzymatically. We have cloned the silica-forming enzymes, silicateins, from both demosponges (marine and freshwater sponges) and hexactinellid sponges. The recombinant enzymes allow the synthesis of silica under environmentally benign ambient conditions, while the technical (chemical) production of silica commonly requires high temperatures and pressures, and extremes of pH. Silicateins can be used for the fabrication of highly-ordered inorganic–organic composite materials w…

Silicate modulates the cross-talk between osteoblasts (SaOS-2) and osteoclasts (RAW 264.7 cells): inhibition of osteoclast growth and differentiation

It has been shown that inorganic monomeric and polymeric silica/silicate, in the presence of the biomineralization cocktail, increases the expression of osteoprotegerin (OPG) in osteogenic SaOS-2 sarcoma cells in vitro. In contrast, silicate does not affect the steady-state gene expression level of the osteoclastogenic ligand receptor activator of NF-κB ligand (RANKL). In turn it can be expected that the concentration ratio of the mediators OPG/RANKL increases in the presence of silicate. In addition, silicate enhances the growth potential of SaOS-2 cells in vitro, while it causes no effect on RAW 264.7 cells within a concentration range of 10-100 µM. Applying a co-cultivation assay system,…

Bioinspired Fabrication of Bio-Silica-Based Bone-Substitution Materials

The gold standard for bone reconstruction is the use of autogeneic grafts from various donor regions, since they possess osteoinductive as well as osteoconductive potential. Only a few synthetic materials possess/display properties that allow optimal bone reconstitution. Previously, we showed that the natural product, bio-silica, comprises osteoinductive, and probably also osteoconductive activity. Bio-silica is formed enzymatically via silicatein; this enzyme has been isolated from siliceous sponges and has also been cloned and prepared recombinantly. In the present study, silicatein was encapsulated together with its substrate, sodium metasilicate, in poly(D,L-lactide)/poly(vinyl pyrrolid…

Silicateins, the major biosilica forming enzymes present in demosponges: protein analysis and phylogenetic relationship.

Silicateins are enzymes, which are restricted to sponges (phylum Porifera), that mediate the catalytic formation of biosilica from monomeric silicon compounds. The silicatein protein is compartmented in the sponges in the axial filaments which reside in the axial canals of the siliceous spicules. In the present study silicatein has been isolated from the freshwater sponge Lubomirskia baicalensis where it occurs in isoforms with sizes of 23 kDa, 24 kDa and 26 kDa. Since the larger protein is glycosylated we posit that it is a processed form of one of the smaller size forms. The silicatein isoforms are post-translationally modified by phosphorylation; at least four isoforms exist with pI's of…

Silicateins, silicatein interactors and cellular interplay in sponge skeletogenesis: formation of glass fiber-like spicules

Biomineralization processes are characterized by controlled deposition of inorganic polymers/minerals mediated by functional groups linked to organic templates. One metazoan taxon, the siliceous sponges, has utilized these principles and even gained the ability to form these polymers/minerals by an enzymatic mechanism using silicateins. Silicateins are the dominant protein species present in the axial canal of the skeletal elements of the siliceous sponges, the spicules, where they form the axial filament. Silicateins also represent a major part of the organic components of the silica lamellae, which are cylindrically arranged around the axial canal. With the demosponge Suberites domuncula …

Aging in Sponges

The sponges (phylum Porifera) are the lowest and phylogenetically oldest, still extant phylum of Metazoa [1, 2]. These animals are considered as the evolutionary earliest living relicts of the successful transition from the (perhaps) unicellular to the multicellular state [2]. Evidence has been presented by molecular phylogenetic analysis that sponges share a common ancestor with the other metazoan phyla, the hypothetical Urmetazoa [2, 3].

Uptake of polyphosphate microparticles in vitro (SaOS-2 and HUVEC cells) followed by an increase of the intracellular ATP pool size

Recently two approaches were reported that addressed a vitally important problem in regenerative medicine, i. e. the successful treatment of wounds even under diabetic conditions. Accordingly, these studies with diabetic rabbits [Sarojini et al. PLoS One 2017, 12(4):e0174899] and diabetic mice [Müller et al. Polymers 2017, 9, 300] identified a novel (potential) target for the acceleration of wound healing in diabetes. Both studies propose a raise of the intracellular metabolic energy status via exogenous administration either of ATP, encapsulated into lipid vesicles, or of polyphosphate (polyP) micro-/nanoparticles. Recently this physiological polymer, polyP, was found to release metabolic …

Inhibitory effects of extracts from the marine alga Caulerpa taxifolia and of toxin from Caulerpa racemosa on multixenobiotic resistance in the marine sponge Geodia cydonium

The invasive growth of the introduced green alga Caulerpa taxifolia, already affecting the richness and diversity of the littoral ecosystems, has become a major ecological problem in the Mediterranean Sea. Previously, we demonstrated that the water pollutant tributyltin induces apoptosis in tissue of the marine sponge Geodia cydonium at concentrations of 3 μM and higher. Here we show that exposure of G. cydonium to low (non-toxic) concentrations of Caulerpa extract or purified caulerpin (10 μg/ml) together with low doses of tributyltin (1 μM; non-toxic), results in a strong apoptotic effect. Evidence is presented that the enhancement of toxicity of tributyltin by Caulerpa extract is at leas…

Nocturnin in the demosponge Suberites domuncula: a potential circadian clock protein controlling glycogenin synthesis in sponges

Sponges are filter feeders that consume a large amount of energy to allow a controlled filtration of water through their aquiferous canal systems. It has been shown that primmorphs, three-dimensional cell aggregates prepared from the demosponge Suberites domuncula and cultured in vitro , change their morphology depending on the light supply. Upon exposure to light, primmorphs show a faster and stronger increase in DNA, protein and glycogen content compared with primmorphs that remain in the dark. The sponge genome contains nocturnin, a light/dark-controlled clock gene, the protein of which shares a high sequence similarity with the related molecule of higher metazoans. The sponge nocturnin …

Retinoid X receptor and retinoic acid response in the marine sponge Suberites domuncula

SUMMARY To date no nuclear receptors have been identified or cloned from the phylogenetically oldest metazoan phylum, the Porifera (sponges). We show that retinoic acid causes tissue regression in intact individuals of the demosponge Suberites domuncula and in primmorphs, special three-dimensional cell aggregates. Primmorphs were cultivated on a galectin/poly-L-lysine matrix in order to induce canal formation. In the presence of 1 or 50 μmol l–1 retinoic acid these canals undergo regression, a process that is reversible. We also cloned the cDNA from S. domunculaencoding the retinoid X receptor (RXR), which displays the two motifs of nuclear hormone receptors, the ligand-binding and the DNA-…

Characterization and osteogenic activity of a silicatein/biosilica-coated chitosan-graft-polycaprolactone.

Several attempts have been made in the past to fabricate hybrid materials that display the complementary properties of the polyester polycaprolactone (PCL) and the polysaccharide chitosan (CHS) for application in the field of bone regeneration and tissue engineering. However, such composites generally have no osteogenic activity per se. Here we report the synthesis of a chitosan-graft-polycaprolactone (CHS-g-PCL) and its subsequent characterization, including crystallinity, chemical structure and thermal stability. Upon surface-functionalization of CHS-g-PCL with osteogenic biosilica via the surface-immobilized enzyme silicatein, protein adsorption, surface morphology and wettability were a…

The role of the silicatein-alpha interactor silintaphin-1 in biomimetic biomineralization.

Biosilicification in sponges is initiated by formation of proteinaceous filaments, predominantly consisting of silicateins. Silicateins enzymatically catalyze condensation of silica nanospheres, resulting in symmetric skeletal elements (spicules). In order to create tailored biosilica structures in biomimetic approaches it is mandatory to elucidate proteins that are fundamental for the assembly of filaments. Silintaphin-1 is a core component of modularized filaments and also part of a spicule-enfolding layer. It bears no resemblance to other proteins, except for the presence of an interaction domain that is fundamental for its function as scaffold/template. In the presence of silicatein sil…

Biogenic Inorganic Polysilicates (Biosilica): Formation and Biomedical Applications

The siliceous sponges, the demosponges and hexactinellid glass sponges, are unique in their ability to form biosilica structures with complex architectures through an enzyme-catalyzed mechanism. The biosilica skeleton of these sponges with its hierarchically structure and exceptional opto-mechanical properties has turned out to be an excellent model for the design of biomimetic nanomaterials with novel property combinations. In addition, biosilica shows morphogenetic activity that offers novel applications in the field of bone tissue engineering and repair. In recent years, much progress has been achieved towards the understanding of the principal enzymes, the silicateins that form the spon…

Origin of the interferon-inducible (2′-5′)oligoadenylate synthetases: cloning of the (2′-5′)oligoadenylate synthetase from the marine spongeGeodia cydonium1

In vertebrates cytokines mediate innate (natural) immunity and protect them against viral infections. The cytokine interferon causes the induction of the (2′-5′)oligoadenylate synthetase [(2-5)A synthetase], whose product, (2′-5′)oligoadenylate, activates the endoribonuclease L which in turn degrades (viral) RNA. Three isoforms of (2-5)A synthetases exist, form I (40–46 kDa), form II (69 kDa), and form III (100 kDa). Until now (2-5)A synthetases have only been cloned from birds and mammals. Here we describe the cloning of the first putative invertebrate (2-5)A synthetase from the marine sponge Geodia cydonium. The deduced amino acid sequence shows signatures characteristic for (2-5)A synthe…

17β-Estradiol-dependent regulation of chaperone expression and telomerase activity in the marine sponge Geodia cydonium

It is known that species belonging to the lowest metazoan phylum, the Porifera (sponges), do not develop tumors. Sponge cells share with tumor cells of higher animals at least one characteristic; they contain high levels of telomerase activity, suggesting that they possess a high proliferation capacity. This assumption, however, has not been substantiated experimentally. In addition, sponges show a specific bauplan, leading us to postulate that they undergo apoptosis to replace a given set of cells at a given time. In the present study, 17β-estradiol (βE2) was used as a defined agent to assess its effect on both the telomerase activity and the process of apoptosis in the marine sponge Geodi…

Arginine kinase in the demosponge Suberites domuncula:regulation of its expression and catalytic activity by silicic acid

SUMMARY In Demospongiae (phylum Porifera) the formation of the siliceous skeleton,composed of spicules, is an energetically expensive reaction. The present study demonstrates that primmorphs from the demosponge Suberites domuncula express the gene for arginine kinase after exposure to exogenous silicic acid. The deduced sponge arginine kinase sequence displays the two characteristic domains of the ATP:guanido phosphotransferases; it can be grouped to the `usual' mono-domain 40 kDa guanidino kinases (arginine kinases). Phylogenetic studies indicate that the metazoan guanidino kinases evolved from this ancestral sponge enzyme; among them are also the `unusual'two-domain 80 kDa guanidino kinas…

Sponge Bcl-2 homologous protein (BHP2-GC) confers distinct stress resistance to human HEK-293 cells

It is established that sponges, the phylogenetically oldest still extant phylum of Metazoa, possess key molecules of the apoptotic pathways, that is members from the Bcl-2 family and a pro-apoptotic molecule with death domains. Here we report on transfection studies of human cells with a sponge gene, GCBHP2. Sponge tissue was exposed to heat shock and tributyltin, which caused an upregulation of gene expression of GCBHP2. The cDNA GCBHP2 was introduced into human HEK-293 cells and mouse NIH-3T3 cells; the stable transfection was confirmed by the identification of the transcripts, by Western blotting as well as by immunofluorescence using antibodies raised against the recombinant polypeptide…

Demosponge EST sequencing reveals a complex genetic toolkit of the simplest metazoans.

Sponges (Porifera) are among the simplest living and the earliest branching metazoans. They hold a pivotal role for studying genome evolution of the entire metazoan branch, both as an outgroup to Eumetazoa and as the closest branching phylum to the common ancestor of all multicellular animals (Urmetazoa). In order to assess the transcription inventory of sponges, we sequenced expressed sequence tag libraries of two demosponge species, Suberites domuncula and Lubomirskia baicalensis, and systematically analyzed the assembled sponge transcripts against their homologs from complete proteomes of six well-characterized metazoans--Nematostella vectensis, Caenorhabditis elegans, Drosophila melanog…

Common genetic denominators for Ca++-based skeleton in Metazoa: role of osteoclast-stimulating factor and of carbonic anhydrase in a calcareous sponge.

Calcium-based matrices serve predominantly as inorganic, hard skeletal systems in Metazoa from calcareous sponges [phylum Porifera; class Calcarea] to proto- and deuterostomian multicellular animals. The calcareous sponges form their skeletal elements, the spicules, from amorphous calcium carbonate (ACC). Treatment of spicules from Sycon raphanus with sodium hypochlorite (NaOCl) results in the disintegration of the ACC in those skeletal elements. Until now a distinct protein/enzyme involved in ACC metabolism could not been identified in those animals. We applied the technique of phage display combinatorial libraries to identify oligopeptides that bind to NaOCl-treated spicules: those oligop…

Toll-like receptors are part of the innate immune defense system of sponges (demospongiae: Porifera).

During evolution and with the emergence of multicellular animals, the need arose to ward off foreign organisms that threaten the integrity of the animal body. Among many different receptors that participate in the recognition of microbial invaders, toll-like receptors (TLRs) play an essential role in mediating the innate immune response. After binding distinct microbial components, TLRs activate intracellular signaling cascades that result in an induced expression of diverse antimicrobial molecules. Because sponges (phylum Porifera) are filter feeders, they are abundantly exposed to microorganisms that represent a potential threat. Here, we describe the identification, cloning, and deduced …

Two-Armed Activation of Bone Mineral Deposition by the Flavones Baicalin and Baicalein, Encapsulated in Polyphosphate Microparticles

In this study, we investigated the effect of the two flavonoids, baicalin (baicalein 7-O-[Formula: see text]- d-glucuronic acid) and its aglycone, baicalein (5,6,7-trihydroxyflavone), after encapsulation into amorphous calcium polyphosphate (Ca-polyP) microparticles on mineralization of primary human osteoblasts (phOSB). Both flavonoids, which come from root extracts of Scutellaria baicalensis Georgi, are used in Traditional Chinese Medicine, and are nontoxic in cells up to a concentration of 3[Formula: see text][Formula: see text]g/ml. The morphogenetically active, energy-rich Ca-polyP particles with a stoichiometric P:Ca ratio of 1:2 are degraded by cellular alkaline phosphatase (ALP) to…

Novel photoreception system in sponges?

Abstract Sponges (phylum Porifera) of the classes Hexactinellida and Demospongiae possess a skeleton composed of siliceous spicules, which are synthesized enzymatically. The longest spicules are found among the Hexactinellida, with the stalk spicules (length: 30 cm; diameter: 300 μm) of Hyalonema sieboldi as prominent examples. These spicules are constructed around a central axial filament, which is formed by approximately 40 siliceous layers. The stratified spicules function as optical glass fibers with unique properties. If free-spaced coupled with a white light source (WLS), the entire fiber is illuminated. Special features of the light transmission: (i) only wavelengths between 615 and …

The 2′-5′-oligoadenylate synthetase in the lowest metazoa: isolation, cloning, expression and functional activity in the sponge Lubomirskia baicalensis

Aquatic animals, especially filter feeders such as sponges [phylum Porifera], are exposed to a higher viral load than terrestrial species. Until now, the antiviral defense system in the evolutionary oldest multicellular organisms, sponges, is not understood. One powerful protection of vertebrates against virus infection is mediated by the interferon (IFN)-inducible 2'-5'-oligoadenylate synthetase [(2-5)A synthetase] system. In the present study we cloned from the freshwater sponge Lubomirskia baicalensis a cDNA encoding a 314 aa long ORF with a calculated size of 35748Da, a putative (2-5)A synthetase, and raised antibodies against the recombinant protein. The native enzyme was identified in…

Towards an understanding of the molecular basis of immune responses in sponges: The marine demospongeGeodia cydonium as a model

The phylogenetic position of the phylum Porifera (sponges) is near the base of the kingdom Metazoa. During the last few years, not only rRNA sequences but, more importantly, cDNA/genes that code for proteins have been isolated and characterized from sponges, in particular from the marine demosponge Geodia cydonium. The analysis of the deduced amino acid sequences of these proteins allowed a molecular biological approach to the question of the monophyly of the Metazoa. Molecules of the extracellular matrix/basal lamina, with the integrin receptor, fibronectin, and galectin as prominent examples, and of cell-surface receptors (tyrosine kinase receptor), elements of sensory systems (crystallin…

Biologization of Allogeneic Bone Grafts with Polyphosphate: A Route to a Biomimetic Periosteum

Osteogenic potential of a biosilica-coated P(UDMA-co-MPS) copolymer

A P(UDMA-co-MPS) copolymer was surface-functionalized through the polycondensation activity of the enzyme silicatein. The resulting biosilica coating significantly enhanced mineralization of osteoblastic cells, thereby revealing its osteogenic potential. Consequently, the functionalized copolymer may be explored as an alternative to conventionally used acrylics in applications where stable bone-material interfaces are required.

Prolidase in the Marine Sponge Suberites domuncula: Enzyme Activity, Molecular Cloning, and Phylogenetic Relationship

: The enzyme prolidase hydrolyzes the peptide bond that involves the imino nitrogen of proline or hydroxyproline; hence, it catalyzes the final step in collagen degradation. From mammals it is known that this enzyme plays a major role in the recycling of proline for collagen synthesis and can be considered to be essential for the control of cell growth. The dominant organic exoskeleton in sponges, especially in Demospongiae, is collagen and the collagen-related spongin. Here we demonstrate that crude extracts of the demosponge Suberites domuncula contain prolidase or prolidase-like activity. The complementary DNA encoding the putative prolidase was cloned from a library of the same animal. …

Utilization of metabolic energy in treatment of ocular surface disorders: polyphosphate as an energy source for corneal epithelial cell proliferation.

Impaired regeneration of the corneal epithelium, as found in many ocular surface diseases, is a major clinical problem in ophthalmology. We hypothesized that corneal epithelial regeneration can be promoted by the physiological, energy-delivering as well as “morphogenetically active” polymer, inorganic polyphosphate (polyP). Corneal limbal explants (diameter, 4 mm) were cultivated on collagen-coated well plates in the absence or presence of polyP (chain length, ∼40 Pi units; 50 μg ml−1) or human platelet lysate (hp-lysate; 5% v/v). Cell outgrowth and differentiation were analyzed after staining with DRAQ5 (nuclei) and rhodamine phalloidin (cytoskeleton), as well as by environmental scanning …

Chemical Mimicry: Hierarchical 1D TiO2@ZrO2 Core−Shell Structures Reminiscent of Sponge Spicules by the Synergistic Effect of Silicatein-α and Silintaphin-1

In nature, mineralization of hard tissues occurs due to the synergistic effect of components present in the organic matrix of these tissues, with templating and catalytic effects. In Suberites domuncula, a well-studied example of the class of demosponges, silica formation is mediated and templated by an axial proteinaceous filament with silicatein-α, one of the main components. But so far, the effect of other organic constituents from the proteinaceous filament on the catalytic effect of silicatein-α has not been studied in detail. Here we describe the synthesis of core-shell TiO(2)@SiO(2) and TiO(2)@ZrO(2) nanofibers via grafting of silicatein-α onto a TiO(2) nanowire backbone followed by …

Dual effect of inorganic polymeric phosphate/polyphosphate on osteoblasts and osteoclasts in vitro

Inorganic polymeric phosphate/polyphosphate (polyP) is a natural polymer existing in both pro- and eukaryotic systems. In the present study the effect of polyP as well as of polyP supplied in a stoichiometric ratio of 2 m polyP:1 m CaCl2 [polyP (Ca2+ complex)] on the osteoblast-like SaOS-2 cells and the osteoclast-like RAW 264.7 cells was determined. Both polymers are non-toxic for these cells up to a concentration of 100 µm. In contrast to polyP, polyP (Ca2+ complex) significantly induced hydroxyapatite formation at a concentration > 10 µm, as documented by alizarin red S staining and scanning electron microscopic (SEM) inspection. Furthermore, polyP (Ca2+ complex) triggered in SaOS-2 cell…

Interaction of the retinoic acid signaling pathway with spicule formation in the marine sponge Suberites domuncula through activation of bone morphogenetic protein-1

Abstract Background The formation of the spicules in siliceous sponges involves the formation of cylinder-like structures in the extraspicular space, composed of the enzyme silicatein and the calcium-dependent lectin. Scope of review Molecular cloning of the cDNAs (carotene dioxygenase, retinal dehydrogenase, and BMB-1 [bone morphogenic protein-1]) from the demosponge Suberites domuncula was performed. These tools were used to understand the retinoid metabolism in the animal by qRT-PCR, immunoblotting and TEM. Major conclusions We demonstrate that silintaphin-2, a silicatein-interacting protein, is processed from a longer-sized 15-kDa precursor to a truncated, shorter-sized 13 kDa calcium-b…

NanoSIMS: insights into the organization of the proteinaceous scaffold within Hexactinellid sponge spicules.

The giant basal spicules (GBS) from Monorhaphis chuni (Porifera [sponges], Hexactinellida) represent the largest biosilica structures on Earth and can reach lengths of 300 cm (diameter of 1.1 cm). The amorphous silica of the inorganic matrix is formed enzymatically by silicatein. During this process, the enzyme remains trapped inside the lamellar-organized spicules. In order to localize the organic silicatein scaffold, the inside of a lamella has been analyzed by nano-secondary ion mass spectrometry (NanoSIMS). It is shown that the GBSs are composed of around 245 concentrically arranged individual siliceous lamellae. These surround an internal siliceous axial cylinder. The lamellae adjacent…

Axial (apical-basal) expression of pro-apoptotic and pro-survival genes in the lake baikal demosponge Lubomirskia baicalensis.

Like in all other Metazoa, also in sponges (Porifera) proliferation, differentiation, and death of cells are controlled by apoptotic processes, thus allowing the establishment of a Bauplan (body plan). The demosponge Lubomirskia baicalensis from the Lake Baikal is especially suitable to assess the role of the apoptotic molecules, since its grade of construction is highly elaborated into an encrusting base and branches composed of modules lined up along the apical-basal axis. The four cDNAs, ALG-2, BAK, MA-3, and Bcl-2, were isolated from this sponge species. The expression levels of these genes follow characteristic gradients. While the proapoptotic genes are highly expressed at the base of…

Poriferan survivin exhibits a conserved regulatory role in the interconnected pathways of cell cycle and apoptosis

Survivin orchestrates intracellular pathways during cell division and apoptosis. Its central function as mitotic regulator and inhibitor of cell death has major implications for tumor cell proliferation. Analyses in early-branching Metazoa so far propose an exclusive role of survivin as a chromosomal passenger protein, whereas only later during evolution a complementary antiapoptotic function might have arisen, concurrent with increased organismal complexity. To lift the veil on the ancestral function(s) of this key regulator, a survivin-like protein (SURVL) of one of the earliest-branching metazoan taxa was identified and functionally characterized. SURVL of the sponge Suberites domuncula …

Concept of Biogenic Ferromanganese Crust Formation: Coccoliths as Bio-seeds in Crusts from Central Atlantic Ocean (Senghor Seamount/Cape Verde)

At depths of 2,000 to 3,000 m, seamounts from the Cape Verde archipelago (Central Atlantic Ocean) are largely covered with ferromanganese crusts. Here we studied 60 to 150 mm thick crusts from the Senghor Seamount (depth: 2257.4 m). The crusts have a non lamellated texture and are covered with spherical nodules. The chemical composition shows a dominance of MnO2 (26.1%) and Fe2O3 (38.8%) with considerable amounts of Co (0.74%) and TiO2 (2.1%). Analysis by scanning electron probe microanalyzer (EPMA) revealed a well defined compositional zonation of micro-layers; the distribution pattern of Mn does not match that of Fe. Analysis by high resolution scanning electron microscopy (SEM) revealed…

Differentiation capacity of epithelial cells in the sponge Suberites domuncula.

Sponges (phylum Porifera) represent the oldest metazoans. Their characteristic metazoan adhesion molecules and transcription factors enable them to establish a complex "Bauplan" ; three major differentiated cell types (epithelial cells, skeletal cells/sclerocytes, and contractile cells) can be distinguished. Since no molecular markers are as yet available to distinguish these somatic cells or the corresponding embryonic cells from which they originate, we have selected the following three genes for their characterization: noggin (a signaling molecule in development), a caspase that encodes an apoptotic molecule, and silicatein. Silicatein is an enzyme that is involved in the synthesis of si…

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

ABSTRACT 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 ca…

Modulation of 2′-5′oligoadenylate synthetase by environmental stress in the marine spongeGeodia cydonium

Recently we established the presence of relatively high amounts of 2′-5′oligoadenylates (2′-5′A) and 2′-5′oligoadenylate synthetase (2′-5′A synthetase) in the marine sponge Geodia cydonium. Here we determined by applying radioimmunoassay and high-performance liquid chromatographical methods that the concentration of 2′-5′A and the activity of 2′-5′A synthetase change following exposure of G. cydonium tissue to environmental stress. The 2′-5′A content and the activity of 2′-5′A synthetase, present in crude sponge extract, increase by up to three-fold after treating sponge cubes for 2 h with natural stressors including heat shock (26°C), cold shock (6°C), pH shock (pH 6), and hypertonic shock…

Flashing light signaling circuit in sponges: Endogenous light generation after tissue ablation in Suberites domuncula

The skeleton of siliceous sponges (phylum Porifera: classes Demospongiae and Hexactinellida), composed of tightly interacting spicules that assemble to a genetically fixed scaffold, is formed of bio-silica. This inorganic framework with the quality of quartz glass has been shown to operate as light waveguide in vitro and very likely has a similar function in vivo. Furthermore, the molecular toolkit for endogenous light generation (luciferase) and light/photon harvesting (cryptochrome) has been identified in the demosponge Suberites domuncula. These three components of a light signaling system, spicules—luciferase—cryptochrome, are concentrated in the surface layers (cortex) of the poriferan…

Induction of heat-shock (stress) protein gene expression by selected natural and anthropogenic disturbances in the octocoral Dendronephthya klunzingeri

Previously it was found that the expression of selected heat-shock proteins is upregulated in corals after exposure to elevated temperature. We published that HSPs are suitable markers in sponges to monitor the degree of environmental stress on these animals. In the present study the heat-shock proteins (HSPs) with a molecular weight of 90 kDa have been selected to prove their potential usefulness as biomarkers under controlled laboratory conditions and in the field. The studies have been performed with the octocoral Dendronephthya klunzingeri from which the cDNA coding for HSP90 was cloned first. The expression of the HSP90 gene is upregulated by thermal stress; treatment of the animals fo…

Molecular Evolution of Apoptotic Pathways: Cloning of Key Domains from Sponges (Bcl-2 Homology Domains and Death Domains) and Their Phylogenetic Relationships

Cells from metazoan organisms are eliminated in a variety of physiological and pathophysiological processes by apoptosis. In this report, we describe the cloning and characterization of molecules from the marine sponges Geodia cydonium and Suberites domuncula, whose domains show a high similarity to those that are found in molecules of the vertebrate Bcl-2 superfamily and of the death receptors. The Bcl-2 proteins contain up to four Bcl-2 homology regions (BH). Two Bcl-2-related molecules have been identified from sponges that are provided with two of those regions, BH1 and BH2, and are termed Bcl-2 homology proteins (BHP). The G. cydonium molecule, BHP1_GC, has a putative size of 28,164, w…

Pathogen-Mimicking MnO Nanoparticles for Selective Activation of the TLR9 Pathway and Imaging of Cancer Cells

Here, design of the first pathogen-mimicking metal oxide nanoparticles with the ability to enter cancer cells and to selectively target and activate the TLR9 pathway, and with optical and MR imaging capabilities, is reported. The immobilization of ssDNA (CpG ODN 2006) on MnO nanoparticles is performed via the phosphoramidite route using a multifunctional polymer. The multifunctional polymer used for the nanoparticle surface modification not only affords a protective organic biocompatible shell but also provides an efficient and convenient means for loading immunostimulatory oligonucleotides. Since fluorescent molecules are amenable to photodetection, a chromophore (Rhodamine) is introduced …

Bio-sintering processes in hexactinellid sponges: Fusion of bio-silica in giant basal spicules from Monorhaphis chuni☆

The two sponge classes, Hexactinellida and Demospongiae, comprise a skeleton that is composed of siliceous skeletal elements (spicules). Spicule growth proceeds by appositional layering of lamellae that consist of silica nanoparticles, which are synthesized via the sponge-specific enzyme silicatein. While in demosponges during maturation the lamellae consolidate to a solid rod, the lamellar organization of hexactinellid spicules largely persists. However, the innermost lamellae, near the spicule core, can also fuse to a solid axial cylinder. Similar to the fusion of siliceous nanoparticles and lamella, in several hexactinellid species individual spicules unify during sintering-like processe…

Molecular Mechanism of Spicule Formation in the Demosponge Suberites domuncula: Silicatein-Collagen-Myotrophin

In living organisms four major groups of biominerals exist: (1) iron compounds, which are restricted primarily to Prokaryota; (2) calcium phosphates, found in Metazoa; (3) calcium carbonates, used by Prokaryota, Protozoa, Plantae, Fungi and Metazoa and (4) silica (opal) present in sponges and diatoms (reviewed in: Bengtson 1994; Baeuerlein 2000). It is surprising that the occurrence of silica as a major skeletal element is restricted to some Protozoa and to sponges (Porifera). The element silicon (Si) contributes to 28% of the earth crust and is - after oxygen - the second most abundant element on earth (Windholz 1983).

Circumferential spicule growth by pericellular silica deposition in the hexactinellid sponge Monorhaphis chuni.

SUMMARY The giant basal spicule of the hexactinellid sponge Monorhaphis chuni represents the longest natural siliceous structure on Earth. This spicule is composed of concentrically arranged lamellae that are approximately 10 μm thick. In the present study, we investigated the formation of outer lamellae on a cellular level using microscopic and spectroscopic techniques. It is shown that the formation of an outermost lamella begins with the association of cell clusters with the surface of the thickening and/or growing spicule. The cells release silica for controlled formation of a lamella. The pericellular (silica) material fuses to a delimited and textured layer of silica with depressions …

Bio-silica and bio-polyphosphate: applications in biomedicine (bone formation)

Bio-silica represents the main mineral component of the sponge skeletal elements (siliceous spicules), while bio-polyphosphate (bio-polyP), a multifunctional polymer existing in microorganisms and animals acts, among others, as reinforcement for pores in cell membranes. These natural inorganic bio-polymers, which can be readily prepared, either by recombinant enzymes (bio-silica and bio-polyP) or chemically (polyP), are promising materials/substances for the amelioration and/or treatment of human bone diseases and dysfunctions. It has been demonstrated that bio-silica causes in vitro a differential effect on the expression of the genes OPG and RANKL, encoding two mediators that control the …

Inducible ASABF-Type Antimicrobial Peptide from the Sponge Suberites domuncula: Microbicidal and Hemolytic Activity in Vitro and Toxic Effect on Molluscs in Vivo

Since sponges, as typical filter-feeders, are exposed to a high load of attacking prokaryotic and eukaryotic organisms, they are armed with a wide arsenal of antimicrobial/cytostatic low-molecular-weight, non-proteinaceous bioactive compounds. Here we present the first sponge agent belonging to the group of ASABF-type antimicrobial peptides. The ASABF gene was identified and cloned from the demospongeSuberites domuncula. The mature peptide, with a length of 64 aa residues has a predicted pI of 9.24, and comprises the characteristic CSαβ structural motif. Consequently, the S. domuncula ASABF shares high similarity with the nematode ASABFs ; it is distantly related to the defensins. The recom…

Innate Immune Defense of the Sponge Suberites domuncula against Bacteria Involves a MyD88-dependent Signaling Pathway

Sponges (phylum Porifera) are the phylogenetically oldest metazoa; as filter feeders, they are abundantly exposed to marine microorganisms. Here we present data indicating that the demosponge Suberites domuncula is provided with a recognition system for Gram-negative bacteria. The lipopolysaccharide (LPS)-interacting protein was identified as a receptor on the sponge cell surface, which recognizes the bacterial endotoxin LPS. The cDNA was isolated, and the protein (Mr 49,937) was expressed. During binding to LPS, the protein dimerizes and interacts with MyD88, which was also identified and cloned. The sponge MyD88 (Mr 28,441) is composed of two protein interaction domains, a Toll/interleuki…

Molecular Biomineralization: Toward an Understanding of the Biogenic Origin of Polymetallic Nodules, Seamount Crusts, and Hydrothermal Vents

Polymetallic nodules and crusts, hydrothermal vents from the Deep Sea are economically interesting, since they contain alloying components, e.g., manganese or cobalt, that are used in the production of special steels; in addition, they contain rare metals applied for plasma screens, for magnets in hard disks, or in hybrid car motors. While hydrothermal vents can regenerate in weeks, polymetallic nodules and seamount crusts grow slowly. Even though the geochemical basis for the growth of the nodules and crusts has been well studied, the contribution of microorganisms to the formation of these minerals remained obscure. Recent HR-SEM (high-resolution scanning electron microscopy) analyses of …

Increased expression of the potential proapoptotic molecule DD2 and increased synthesis of leukotriene B4 during allograft rejection in a marine sponge

Sponges (Porifera) are a classical model to study the events during tissue transplantation. Applying the 'insertion technique' autografts from the marine sponge Geodia cydonium fuse within 5 days. In contrast, allografts are rejected and destroyed. Here we show that during allograft rejection the cells in the grafts undergo apoptosis; 5 days after transplantation 46% of the cells show signs of apoptosis. In a previous study it was shown that during this process a tumor necrosis factor-like molecule is induced in allo- and xenografts. Molecules grouped to the superfamily of tumor necrosis factor receptors and a series of associated adapter molecules contain the characteristic death domain. T…

A synthetic biology approach for the fabrication of functional (fluorescent magnetic) bioorganic–inorganic hybrid materials in sponge primmorphs

During evolution, sponges (Porifera) have honed the genetic toolbox and biosynthetic mechanisms for the fabrication of siliceous skeletal components (spicules). Spicules carry a protein scaffold embedded within biogenic silica (biosilica) and feature an amazing range of optical, structural, and mechanical properties. Thus, it is tempting to explore the low-energy synthetic pathways of spiculogenesis for the fabrication of innovative hybrid materials. In this synthetic biology approach, the uptake of multifunctional nonbiogenic nanoparticles (fluorescent, superparamagnetic) by spicule-forming cells of bioreactor-cultivated sponge primmorphs provides access to spiculogenesis. The ingested nan…

Assessing the root of bilaterian animals with scalable phylogenomic methods.

A clear picture of animal relationships is a prerequisite to understand how the morphological and ecological diversity of animals evolved over time. Among others, the placement of the acoelomorph flatworms, Acoela and Nemertodermatida, has fundamental implications for the origin and evolution of various animal organ systems. Their position, however, has been inconsistent in phylogenetic studies using one or several genes. Furthermore, Acoela has been among the least stable taxa in recent animal phylogenomic analyses, which simultaneously examine many genes from many species, while Nemertodermatida has not been sampled in any phylogenomic study. New sequence data are presented here from org…

Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca2+ level in osteoblasts (SaOS-2 cells) in vitro

Inorganic polymeric phosphate is a physiological polymer that accumulates in bone cells. In the present study osteoblast-like SaOS-2 cells were exposed to this polymer, complexed in a 2:1 stoichiometric ratio with Ca(2+), polyP (Ca(2+) salt). At a concentration of 100 μM, polyP (Ca(2+) salt) caused a strong increase in the activity of the alkaline phosphatase and also an induction of the steady-state expression of the gene encoding this enzyme. Comparative experiments showed that polyP (Ca(2+) salt) can efficiently replace β-glycerophosphate in the in vitro hydroxyapatite (HA) biomineralization assay. In the presence of polyP (Ca(2+) salt) the cells extensively form HA crystallites, which r…

The Chemokine Networks in Sponges: Potential Roles in Morphogenesis, Immunity and Stem Cell Formation

Porifera (sponges) are now well accepted as the phylum which branched off first from the common ancestor of all metazoans, the Urmetazoa. The transition to the Metazoa became possible because during this phase, cell-cell as well as cell-matrix adhesion molecules evolved which allowed the formation of a colonial stage of animals. The next prerequisite for the evolution to the Urmetazoa was the establishment of an effective immune system which, flanked by apoptosis, allowed the formation of a first level of individuation.

Formation of a micropatterned titania photocatalyst by microcontact printed silicatein on gold surfaces

The enzyme silicatein has been bioengineered to carry a thiol-bearing Au-affinity tag (Cys-tag) for direct immobilization on gold carriers in shortest time without the need for prior surface functionalization. Through microcontact printing, defined silicatein micropatterns were created on gold surfaces, facilitating the subsequent enzymatically controlled synthesis of photocatalytically active TiO(2).

Regional and modular expression of morphogenetic factors in the demosponge Lubomirskia baicalensis

Some sponges [phylum Porifera], e.g. the demosponges Lubomirskia baicalensis or Axinella polypoides, show an arborescent growth form. In the freshwater sponge L. baicalensis this morphotype is seen mostly in depths below 4 m while in more shallow regions it grows as a crust. The different growth forms are determined in nature very likely by water current and/or light. The branches of this species are composed of modules, arranged along the apical-basal axis. The modules are delimited by a precise architecture of the spicule bundles; longitudinal bundles originate from the apex of the earlier module, while at the basis of each module these bundles are cross-linked by traverse bundles under f…

Stress Response in Marine Sponges: Genes and Molecules Involved and Their use as Biomarkers

Sponges (Porifera) are sessile filter feeders that are able to accumulate compounds from the surrounding water and thus are highly exposed to environmental stress by pollutants of both anthropogenic and natural origin. However, these animals possess a number of protective mechanisms against environmental stress, such as the expression of heat-shock proteins, the induction of the multi xenobiotic resistance mechanism, and the apoptotic elimination of cells. In the last years, a number of genes involved in the stress response of sponges have been cloned and characterized. In thischapter, the various molecular mechanisms by which cells of the lowest multicellular organisms—the marine sponges—r…

Isolation of the silicatein-α interactor silintaphin-2 by a novel solid-phase pull-down assay.

The skeleton of siliceous sponges consists of amorphous biogenous silica (biosilica). Biosilica formation is driven enzymatically by means of silicatein(s). During this unique process of enzymatic polycondensation, skeletal elements (spicules) that enfold a central proteinaceous structure (axial filament), mainly comprising silicatein, are formed. However, only the concerted action of silicatein and other proteins can explain the genetically controlled diversity of spicular morphotypes, from simple rods with pointed ends to intricate structures with up to six rays. With the scaffold protein silintaphin-1, a first silicatein interactor that facilitates the formation of the axial filament and…

A (1->3)-beta-D-glucan recognition protein from the sponge Suberites domuncula. Mediated activation of fibrinogen-like protein and epidermal growth factor gene expression

Sponges (phylum Porifera) live in a symbiotic relationship with microorganisms, primarily bacteria. Until now, molecular proof for the capacity of sponges to recognize fungi in the surrounding aqueous milieu has not been available. Here we demonstrate, for the demosponge Suberites domuncula (Porifera, Demospongiae, Hadromerida), a cell surface receptor that recognizes (1--3)-beta-D-glucans, e.g. curdlan or laminarin. This receptor, the (1--3)-beta-D-glucan-binding protein, was identified and its cDNA analysed. The gene coding for the 45 kDa protein was found to be upregulated in tissue after incubation with carbohydrate. Simultaneously with the increased expression of this gene, two further…

Mitochondrial genome of Suberites domuncula: palindromes and inverted repeats are abundant in non-coding regions.

The 26,300-nucleotide sequence of the mitochondrial DNA (mtDNA) molecule of the demosponge Suberites domuncula (Olivi, 1792), the largest in size yet found in Porifera, has been determined. We describe the second hadromerid sponge mitochondrial genome that contains the same set of 41 genes as the hadromerid sponge Tethya actinia, including trnMe(cau), trnI2(cau), trnR2(ucu), and atp9, all of which are transcribed in the same direction. Furthermore, rRNA genes for the small and large ribosomal subunit are very long, rns is indeed the longest among Metazoa (1833 bp). Intergenic regions (IGR) comprise about 25% of S. domuncula mtDNA and include numerous direct and inverted repeats, as well as …

Manganese/polymetallic nodules: Micro-structural characterization of exolithobiontic- and endolithobiontic microbial biofilms by scanning electron microscopy

Polymetallic/ferromanganese nodules (Mn-nodules) provide a rich source for manganese. It is not yet known if the nodules have a biogenic or an abiogenic origin. Here we applied the technique of high-resolution scanning electron microscopy, in combination with energy dispersive X-ray spectroscopical (EDX) analysis, to trace the existence of microbial biofilms. Two spatially separated assemblies exist, the exolithobiontic- and endolithobiontic colonizations. The exolithobiontic colonization is seen in the micro-canals, which traverse the outer surface layer of the nodules and are formed by elongated filamentous organisms, which show no signs of mineralization. In the center of the nodules thr…

Evidence for a biogenic, microorganismal origin of rock varnish from the Gangdese Belt of Tibet

In the present study we examined material from the Ashikule Basin of Tibet. Chemical analyses were performed by use of energy dispersive X-ray spectroscopy and electron probe microanalysis to clarify whether the varnish layers that had developed on the surface of the rhyolite are indeed composed of varnish bodies and silica glaze. Electron microscopic analyses revealed that the surface of the varnish is covered both by filamentous hyphae bacterial and cocci-shaped forms. Within the varnish mineral layer in those samples two forms of bacteria-like microorganisms exist; cocci as tightly packed bacterial aggregates [within varnish bodies], and bacillus-like microorganisms [within the varnish m…

Ubiquitins (polyubiquitin and ubiquitin extension protein) in marine sponges: cDNA sequence and phylogenetic analysis

The complete nucleotide sequences of twoSuberites domunculacDNAs and oneSycon raphanuscDNA, all encoding ubiquitin, have been determined. One cDNA fromS. domunculacodes for polyubiquitin with four tandemly repeated monomeric units and the second cDNA encodes ubiquitin fused to a ribosomal protein of 78 amino acids (aa).S. domunculapossesses at least one additional polyubiquitin gene, from which the last two monomers were also sequenced. All analysed genes fromS. domunculaencode identical ubiquitin proteins, with only one aa difference (Ala19) to the human/higher animals ubiquitin (Pro19). Ubiquitin inS. domunculais identical with the ubiquitin found in another Demospongia,Geodia cydonium. T…