Silicateins , silicase and spicule-associated proteins : synthesis of demosponge silica skeleton and nanobiotechnological applications

The major skeletal elements of the Demospongiae and Hexactinellida are spicules formed from amorphous silica (biosilica). Demosponges are unique in their ability to synthesize their silica skeleton enzymatically. In the past few years, we have cloned several isoforms of the silica-forming enzyme, silicatein, from both marine sponges (example: Suberites domuncula) and freshwater sponges (example: Lubomirskia baikalensis). The silicateins are very similar to cathepsins, a family of cysteine proteases which do not precipitate silica. In the silicatein sequence, the cysteine residue of the catalytic triad of these proteases is replaced by a serine residue which is essential for the catalytic mechanism of the enzyme. In addition, a hydroxy amino acid (serine) cluster is present in the molecule. Silicatein undergoes posttranslational modifications of the protein, e.g. phosphorylation, as revealed by 2-dimensional gel electrophoresis. Using primmorphs (a special form of 3-dimensional cell aggregates) we established that spicule formation begins intracellularly and is completed extracellularly. Immunoblotting and immunogold labeling experiments revealed that silicatein is not only present in the axial filament but also at the surface of the spicules. Applying the technique of differential display of transcripts, we identified a further enzyme involved in silica metabolism, the silicase, which is able to depolymerize amorphous silica. The silicase shares highest similarity to the carbonic anhydrases, a family of zinc metal enzymes. The recombinant sponge enzymes, silicatein and silicase allow the synthesis/degradation of silica under ambient conditions that do not damage biomolecules. They are key enzymes for a variety of potential applications in (nano)biotechnology and medicine, including (i) surface modification of biomaterials, (ii) encapsulation of biomolecules and (iii) biofabrication of nanostructure materials for optoand micro-electronics.