Isolation and Characterization of Adhesive Secretion from Cuvierian Tubules of Sea Cucumber Holothuria forskåli (Echinodermata: Holothuroidea)

The sea cucumber Holothuria forskåli possesses a specialized system called Cuvierian tubules. During mechanical stimulation white filaments (tubules) are expelled and become sticky upon contact with any object. We isolated a protein with adhesive properties from protein extracts of Cuvierian tubules from H. forskåli. This protein was identified by antibodies against recombinant precollagen D which is located in the byssal threads of the mussel Mytilus galloprovincialis. To find out the optimal procedure for extraction and purification, the identified protein was isolated by several methods, including electroelution, binding to glass beads, immunoprecipitation, and gel filtration. Antibodies raised against the isolated protein were used for localization of the adhesive protein in Cuvierian tubules. Immunostaining and immunogold electron microscopical studies revealed the strongest immunoreactivity in the mesothelium; this tissue layer is involved in adhesion. Adhesion of Cuvierian tubule extracts was measured on the surface of various materials. The extracted protein showed the strongest adhesion to Teflon surface. Increased adhesion was observed in the presence of potassium and EDTA, while cadmium caused a decrease in adhesion. Addition of antibodies and trypsin abolished the adhesive properties of the extract

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 interspersed by pores of ≈2 nm that are likely associated with aquaporin channels which are implicated in the hardening of the initial bio-silica products formed by silicatein. We can summarize the sequence of events that govern spicule formation as follows: differential genetic readout (of silicatein) → fractal association of the silicateins → evagination of cells by hydro-mechanical forces into the axial canal → and finally processive bio-silica polycondensation around the axial canal. We termed this process, occurring sequentially or in parallel, bio-inorganic self-organization

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 oligopeptides allowed us to detect proteins that bind to those spicules. Two molecules have been identified, the (putative) enzyme carbonic anhydrase and the (putative) osteoclast-stimulating factor (OSTF), that are involved in the catabolism of ACC. The complete cDNAs were isolated and the recombinant proteins were prepared to raise antibodies. In turn, immunofluorescence staining of tissue slices and qPCR analyses have been performed. The data show that sponges, cultivated under standard condition (10 mM CaCl2) show low levels of transcripts/proteins for carbonic anhydrase or OSTF, compared to those animals that had been cultivated under Ca2+-depletion condition (1 mM CaCl2). Our data identify with the carbonic anhydrase and the OSTF the first two molecules which remain conserved in cells, potentially involved in Ca-based skeletal dissolution, from sponges (sclerocytes) to human (osteoclast)

Sponge spicules as blueprints for the biofabrication of inorganic–organic composites and biomaterials

While most forms of multicellular life have developed a calcium-based skeleton, a few specialized organisms complement their body plan with silica. However, of all recent animals, only sponges (phylum Porifera) are able to polymerize silica enzymatically mediated in order to generate massive siliceous skeletal elements (spicules) during a unique reaction, at ambient temperature and pressure. 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. Spicules lend structural stability to the sponge body, deter predators, and transmit light similar to optic fibers. This peculiar phenomenon has been comprehensively studied in recent years and in several approaches, the molecular background was explored to create tools that might be employed for novel bioinspired biotechnological and biomedical applications. Thus, it was discovered that spiculogenesis is mediated by the enzyme silicatein and starts intracellularly. The resulting silica nanoparticles fuse and subsequently form concentric lamellar layers around a central protein filament, consisting of silicatein and the scaffold protein silintaphin-1. Once the growing spicule is extruded into the extracellular space, it obtains final size and shape. Again, this process is mediated by silicatein and silintaphin-1, in combination with other molecules such as galectin and collagen. The molecular toolbox generated so far allows the fabrication of novel micro- and nanostructured composites, contributing to the economical and sustainable synthesis of biomaterials with unique characteristics. In this context, first bioinspired approaches implement recombinant silicatein and silintaphin-1 for applications in the field of biomedicine (biosilica-mediated regeneration of tooth and bone defects) or micro-optics (in vitro synthesis of light waveguides) with promising results

The relevance of the silica metabolizing enzyme silicatein-alpha to biomineralization and the formation of biogenic silica in siliceous sponges

Die technische Silikatproduktion erfordert in der Regel hohe Temperaturen und extreme pH-Werte. In der Natur hingegen haben insbesondere Kieselschwämme die außergewöhnliche Fähigkeit, ihr Silikatskelett, das aus einzelnen sogenannten Spiculae besteht, enzymatisch mittels des Proteins Silicatein zu synthetisieren. rnIm Inneren der Spiculae, im zentralen Kanal, befindet sich das Axialfilament, welches hauptsächlich aus Silicatein-α aufgebaut ist. Mittels Antikörperfärbungen und Elektronenmikroskopischen Analysen konnte festgestellt werden, dass Silicatein in mit Kieselsäure-gefüllten Zellorganellen (silicasomes) nachzuweisen ist. Mittels dieser Vakuolen kann das Enzym und die Kieselsäure aus der Zelle zu den Spiculae im extrazellulären Raum befördert werden, wo diese ihre endgültige Länge und Dicke erreichen. Zum ersten Mal konnte nachgewiesen werden, dass rekombinant hergestelltes Silicatein-α sowohl als Siliciumdioxid-Polymerase als auch Siliciumdioxid-Esterase wirkt. Mittels Massenspektroskopie konnte die enzymatische Polymerisation von Kieselsäure nachverfolgt werden. Durch Spaltung der Esterbindung des künstlichen Substrates Bis(p-aminophenoxy)-dimethylsilan war es möglich kinetische Parameter der Siliciumdioxid-Esterase-Aktivität des rekombinanten Silicateins zu ermitteln.rnZu den größten biogenen Silikatstukuren auf der Erde gehören die Kieselnadeln der Schwammklasse Hexactinellida. Nadelextrakte aus den Schwammklassen Demospongien (S. domuncula) und Hexactinellida (M. chuni) wurden miteinander verglichen um die potentielle Existenz von Silicatein oder Silicatein-ähnliche Molekülen und die dazu gehörige proteolytischen Aktivität nachzuweisen. Biochemische Analysen zeigten, dass das 27 kDA große isolierte Polypeptid in Monoraphis mehrere gemeinsame Merkmale mit den Silicateinen der Demospongien teilt. Dazu gehören die Größe und die Proteinase-Aktivität. rnUm die Frage zu klären, ob das axiale Filament selbst zur Formbildung der Skelettelemente beiträgt, wurde ein neues mildes Extraktionsverfahren eingeführt. Dieses Verfahren ermöglichte die Solubilisierung des nativen Silicateins aus den Spiculae. Die isolierten Silicateine lagen als Monomere (24 kDa) vor, die Dimere durch nicht-kovalente Bindungen ausbildeten. Darüber hinaus konnten durch PAGE-Gelelektrophorese Tetramere (95 kDa) und Hexamere (135 kDa) nachgewiesen werden. Die Monomere zeigten eine beträchtliche proteolytische Aktivität, die sich während der Polymerisationsphase des Proteins weiter erhöhte. Mit Hilfe der Lichtmikroskopie und Elektronenmikroskopie (TEM) konnte die Assemblierung der Proteine zu filamentartigen Strukturen gezeigt werden. Die Selbstorganisation der Silicatein-α-Monomeren scheint eine Basis für Form- und Musterbildung der wachsenden Nadeln zu bilden.rn Um die Rolle des kürzlich entdeckten Proteins Silintaphin-1, ein starker Interaktionspartner des Silicatein-α, während der Biosilifizierung zu klären, wurden Assemblierungs-Experimente mit den rekombinanten Proteinen in vitro durchgeführt. Zusätzlich wurde deren Effekt auf die Biosilikatsynthese untersucht. Elektronenmikroskopische Analysen ergaben, dass rekombinantes Silicatein-α zufällig verteilte Aggregate bildet, während die Koinkubation beider Proteine (molekulares Verhältnis 4:1) über fraktal artige Strukturen zu Filamenten führt. Auch die enzymatische Aktivität der Silicatein-α-vermittelte Biosilikatsynthese erhöhte sich in Gegenwart von Silintaphin-1 um das 5,3-fache. rnIndustrial production of silica requires usually high temperature conditions and extreme pH. Siliceous sponges have the exceptional ability to synthesize their siliceous skeleton enzymatically via the protein silicatein under physiological conditions. In the central canal of spicules the axial filament is situated, which is composed mainly of silicatein.rnIt is found that silicatein exists in silica-filled cell organelles (silicasomes) that transport the enzyme to the spicules. For the first time it is shown that recombinant silicatein-α acts as a silica polymerase and also as a silica esterase. The enzymatic polymerization of silicic acid follows a distinct course, which was visualized by mass spectroscopy. Following the cleavage of the ester-like bond in the artificial substrate bis(p-aminophenoxy)-dimethylsilane by the recombinant silicatein, the kinetic parameters for silica esterase activity are evaluated. rnThe sponge class Hexactinellida also forms siliceous spicules which are the largest biogenic silica structures on earth. Spicule extracts from S. domuncula and M. chuni are compared and the potential existence of silicatein, or silicatein-like molecules and the potential proteolytic activity of the silicateins in hexactinellids are approached. The results indicate that the isolated 27•kDa polypeptide in Monorhaphis has several characteristics in common with the silicateins found in demosponges like the size, the post-translational modifications and the proteinase activity. rnIn order to approach the question whether it is the axial filament itself, which contributes to the shape formation of the spicules a new mild extraction procedure is introduced. This procedure allows the solubilization of native silicateins from the silica shell and immediately yields monomeric (24kDa) protein, which readily forms dimers through non-covalent linkages. Furthermore polymerization to tetramers (95 kDa) and hexamers (135 kDa) can be demonstrated. The monomers show a considerable proteolytic activity that increases during the polymerization phase of the protein. It is proposed that the basic self-assembly of the silicatein-α monomers provides the general platform for the shape and pattern formation of growing spicules. rnTo clarify the role of the newly identified protein silintaphin-1, a strong interactor of silicatein- α, during the aggregation process the re-assembly experiments are performed with recombinant silicatein-α and recombinant silintaphin-1 at different stoichiometric ratios in vitro. Additionally the effect on the biosilica synthesis is evaluated. Whereas recombinant silicatein-α reaggregates to randomly organized structures, co-incubation of both proteins (molecular ratio 4 : 1) resulted in synthetic filaments via fractal-like patterned self-assemblies, as observed by electron microscopy. Owing to the concerted action of both proteins, the enzymatic activity of silicatein-α strongly increased by 5.3-fold. r

Model for the roles of the OSTF and the carbonic anhydrase in sclerocytes of the sponge <i>S. raphanus</i>.

<p>Based on the finding that the expression of the two molecules is upregulated during Ca⁺⁺ depletion condition, it is proposed that these proteins are involved in the development of the precursor sclerocytes to the functionally active catabolic sclerocytes. OSTF forms with Cbl and Src a triple complex that stimulates the membrane-associated PI-3K and lipid metabolism. This metabolic chain is initiated by clustering integrins. These processes finally result in an increased mobility/migration of the catabolic sclerocytes and in an increased expression of the <i>carbonic anhydrase</i> gene. The latter enzyme generates protons that dissolve spicular CaCl₂ (ACC), and causes spicule resorption. Presumably occurring pH shifts within the cells are counterbalanced by a vacuolar H⁺-transporting adenosine triphosphatase and the release of Cl⁻ via the chloride/bicarbonate exchanger (see: <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034617#pone.0034617-Pfaffl1" target="_blank">[58]</a>). In analogy to the differentiation pathway of mammalian osteoclasts it is proposed that also in sponges the differentiation of sclerocytes is under control of, hitherto unknown, differentiation factors and their receptors acting similar like OPG, RANKL and RANK in mammals.</p

Ultrastructure and immunoelectron microscopy prove the specificity of the antibodies.

<p>Sections through spicules were prepared and inspected by TEM analysis. Parallel specimens were reacted either with antibodies or with the preimmune serum kept from this immunization. (<b>A</b> and <b>B</b>) Silicatein: (A) reaction with PAb-aSILIC_SUBDO; (B) incubation with the corresponding pre-immune serum. (<b>C</b> and <b>D</b>) Aquaporin: (C) reaction with PoAb-aAQP_SUBDO; (D) corresponding pre-immune serum. (<b>E</b> and <b>F</b>) Arginine kinase: (E) reaction with PoAb-aAK_SUBDO; (F) corresponding pre-immune serum. The axial canal (ac), the axial filament (af) and the silica shell (si) as well as the intra-cellular space (ics) and the extra-cellular space (ecs) are marked.</p

The calcareous sponge <i>Sycon raphanus</i> (Schmidt, 1862).

<p>(<b>A</b>) The species <i>S. raphanus</i> had been grouped by Haeckel <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034617#pone.0034617-Haeckel1" target="_blank">[28]</a> to the taxon <i>Sycandra</i>. Here a scheme of the morphology and the skeletal structure of <i>Sycandra hystrix</i> is given <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034617#pone.0034617-Haeckel1" target="_blank">[28]</a>. (<b>B</b>) <i>S. raphanus</i> specimens, growing on the mussel (m) <i>Mytilus galloprovincialis</i>. The oscule of the specimens is surrounded by a pronounced corona (co), formed of spicules. On the basis of the specimens stolons/buds (st) are seen. They develop after release from the parent sponge asexually to a descendent. (<b>C</b> and <b>D</b>) Cross section through <i>S. raphanus</i> specimens, displaying the external and internal surface layer. In the center the atrium (a) is shown into which the water canals flow in. Radial aquiferous canals traverse the body that originate at the surface of the animal, via the inhalant openings (io), and end at the internal surface via exhalant pores (eo). Between the canals the mesenchyme (m) compartments is radially arranged. The slices were stained with ASTRIN. (<b>E</b>) Non-stained section through the outer part of the sponge showing the location of the two major types of spicules; (i) the diactines spicules (ds), protruding from the distal cones of the outer surface of the specimens, and (ii) the triactines (ts) that are localized within the mesohyl. The mesohyl compartment is filled with eggs/embryos (e).</p

Immunogold labeling electron microscopy [TEM] of the axial canal of spicules.

<p>Antibodies against silicatein (<b>A</b> to <b>C</b>), against aquaporin (<b>D</b> to <b>F</b>) and against arginine kinase (<b>G</b> to <b>I</b>) were used. (A to C) The silicatein antibodies reacted with the axial filament (af), which was surrounded by the silica mantel (si), while (D to F) the aquaporin antibodies recognized their antigens primarily at the rim of the axial canal (ac) towards the silica mantel (si). (G to I) The anti-arginine kinase antibodies reacted in a more scattered pattern with the antigen in the axial canal, primarily recognizing membranous structures. The size of all bars represents 1 µm.</p