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Silicon is the second most common element in the Earth's crust [1]; it possesses semi-metallic as well as metalloid properties. Silicon exists in nature in combination with oxygen as silicate ions or as silica; silica has no negative charge, while silicate anions carry a negative net electrical charge, which is counterbalanced by cations. Free silica ⁄ silicate is found both in the crystalline state (such as quartz) and in the amorphous state (such as opal). Silica ⁄ silicate is widely used in industry and medicine for the fabrication of poly(silicate), e.g. in amorphous glasses, ceramics, paints, adhesives, catalysts and photonic materials [2,3]. Furthermore, poly(silicate) is an important new material in nano(bio)technology [4,5]. This multidisciplinary research field is concerned with bio-and electronic engineering at nanometer, molecular and cellular levels [4]. Currently, production of silica require high temperature conditions and extremes of pH [6]. Hydrated amorphous silica, e.g. in the form of opal, has superb properties such as low density and high porosity. In nature, amorphous silica can be produced by diatoms by passive deposition onto an organic matrix. Siliceous sponges (Demospongiae) have the exceptional ability to synthesize silica enzymatically via silicatein [7,8]. Based on its protein sequence, silicatein is related to the proteinases of the cathepsin class [9].Silicatein has been isolated from a number of siliceous sponges, e.g. Tethya aurantium or Suberites domuncula [9,10]. If the enzyme is isolated from the skeletal elements of these animals, the spicules, it can be used in vitro to catalyze polycondensation of a wide variety of alkoxides, as well as ionic and organometallic Siliceous sponges can synthesize poly(silicate) for their spicules enzymatically using silicatein. We found that silicatein exists in silica-filled cell organelles (silicasomes) that transport the enzyme to the spicules. We show for the first time that recombinant silicatein acts as a silica polymerase and also as a silica esterase. The enzymatic polymerization ⁄ polycondensation of silicic acid follows a distinct course. In addition, we show that silicatein cleaves the ester-like bond in bis(p-aminophenoxy)-dimethylsilane. Enzymatic parameters for silica esterase activity are given. The reaction is completely blocked by sodium hexafluorosilicate and E-64. We consider that the dual function of silicatein (silica polymerase and silica esterase) will be useful for the rational synthesis of structured new silica biomaterials.

The siliceous sponge Monorhaphis chuni (Hexactinellida) synthesizes the largest biosilica structures on earth (3 m). Scanning electron microscopy has shown that these spicules are regularly composed of concentrically arranged lamellae (width: 3-10 μm). Between 400 and 600 lamellae have been counted in one giant basal spicule. An axial canal (diameter:~2 μm) is located in the center of the spicules; it harbors the axial filament and is surrounded by an axial cylinder (100-150 μm) of electron-dense homogeneous silica. During dissolution of the spicules with hydrofluoric acid, the axial filament is first released followed by the release of a proteinaceous tubule. Two major proteins (150 kDa and 35 kDa) have been visualized, together with a 24-kDa protein that cross-reacts with antibodies against silicatein. The spicules are surrounded by a collagen net, and the existence of a hexactinellidan collagen gene has been demonstrated by cloning it from Aphrocallistes vastus. During the axial growth of the spicules, silicatein or the silicatein-related protein is proposed to become associated with the surface of the spicules and to be finally internalized through the apical opening to associate with the axial filament. Based on the data gathered here, we suggest that, in the Hexactinellida, the growth of the spicules is mediated by silicatein or by a silicatein-related protein, with the orientation of biosilica deposition being controlled by lectin and collagen.

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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 present study was aimed at exploring the effect of selfassembled complexes of silicatein and silintaphin-1 on biosilica synthesis in vitro. Hence, in a comparative approach, recombinant silicatein and recombinant silintaphin-1 were used at different stoichiometric ratios to form axial filaments and to synthesize biosilica. Whereas recombinant silicatein-a reaggregates to randomly organized structures, coincubation of silicatein-a and silintaphin-1 (molecular ratio 4 : 1) resulted in synthetic filaments via fractal-like patterned self-assemblies, as observed by electron microscopy. Concurrently, owing to the concerted action of both proteins, the enzymatic activity of silicatein-a strongly increased by 5.3-fold (with the substrate tetraethyl orthosilicate), leading to significantly enhanced synthesis of biosilica. These results indicate that silicatein-a-mediated biosilicification depends on the concomitant presence of silicatein-a and silintaphin-1. Accordingly, silintaphin-1 might not only enhance the enzymatic activity of silicatein-a, but also accelerate the nonenzymatic polycondensation of the silica product before releasing the fully synthesized biosiliceous polymer. Structured digital abstractl silicatein-a binds to silicatein-a by electron microscopy (View interaction) l silicatein-a and silintaphin-1 bind by electron microscopy (View interaction) Abbreviations HF, hydrofluoric acid; pAb, polyclonal antibody; SEM, scanning electron microscopy; TEM, transmission electron microscopy; TEOS, tetraethyl orthosilicate.

Biosilica is a natural polymer, synthesized by the poriferan enzyme silicatein from monomeric silicate substrates. Biosilica stimulates mineralizing activity and gene expression of SaOS-2 cells. To study its effect on the formation of hydroxyapatite (HA), SaOS-2 cells were grown on different silicatein/biosilica-modified substrates (bone slices, Ca-P-coated coverslips, glass coverslips). Growth on these substrates induced the formation of HA nodules, organized in longitudinal arrays or spherical spots. Nodules of sizes above 1 μm were composed of irregularly arranged HA prism-like nanorods, formed by aggregates of three to eight SaOS-2 cells. Moreover, growth on silicatein/biosilica-modified substrates elicited increased [(3)H]dT incorporation into DNA, indicative of enhanced cell proliferation. Consequently, an in vitro-based bioassay was established to determine the ratio between [(3)H]dT incorporation and HA formation. This ratio was significantly higher for cells that grew on silicatein/biosilica-modified substrates than for cells on Ca-P-coated coverslips or plain glass slips. Hence, we propose that this ratio of in vitro-determined parameters reflects the osteogenic effect of different substrates on bone-forming cells. Finally, qRT-PCR analyses demonstrated that growth of SaOS-2 cells on a silicatein/biosilica matrix upregulated BMP2 (bone morphogenetic protein 2, inducer of bone formation) expression. In contrast, TRAP (tartrate-resistant acid phosphatase, modulator of bone resorption) expression remained unaffected. We conclude that biosilica shows pronounced osteogenicity in vitro, qualifying this material for studies of bone replacement also in vivo.

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.

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