Silica-associated proteins from hexactinellid sponges support an alternative evolutionary scenario for biomineralization in Porifera.

Metazoans use silicon traces but rarely develop extensive silica skeletons, except for the early-diverging lineage of sponges. The mechanisms underlying metazoan silicification remain incompletely understood, despite significant biotechnological and evolutionary implications. Here, the characterization of two proteins identified from hexactinellid sponge silica, hexaxilin and perisilin, supports that the three classes of siliceous sponges (Hexactinellida, Demospongiae, and Homoscleromorpha) use independent protein machineries to build their skeletons, which become non-homologous structures. Hexaxilin forms the axial filament to intracellularly pattern the main symmetry of the skeletal parts, while perisilin appears to operate in their thickening, guiding extracellular deposition of peripheral silica, as does glassin, a previously characterized hexactinellid silicifying protein. Distant hexaxilin homologs occur in some bilaterians with siliceous parts, suggesting putative conserved silicifying activity along metazoan evolution. The findings also support that ancestral Porifera were non-skeletonized, acquiring silica skeletons only after diverging into major classes, what reconciles molecular-clock dating and the fossil record.

Silica adsorption tag derived from the silica polycondensation protein glassin for the immobilization of soluble proteins.

Glassin is a water-soluble protein from the siliceous skeleton of a marine sponge that adsorbs tightly to silica at pH 7.0-9.0 and accelerates silica particle formation from silicic acid. Glassin comprises three distinct domains: a His and Asp-rich (HD) domain, a Pro-rich (P) domain, and a His and Thr-rich (HT) domain. Here, we investigated the roles of these three domains in silica adsorption by using glutathione S-transferase (GST) fused with glassin or with each domain. GST fused with the HD domain exhibited tight adsorption, equivalent to that of GST fused with the full-length glassin sequence at values above 7.0. The apparent Kd values for the binding of full-length glassin and HD to fumed silica at pH 7.0 were 20.8 and 22.7 nM, respectively, indicating that this domain greatly contributes to the silica adsorption ability of glassin. In addition, no internal cleavage was observed in the HD domain, whereas GST fused with the full-length glassin sequence exhibited internal cleavage. The HD domain adsorbed on silica did not dissociate even at pH 6.0. Given these findings, we concluded that the HD domain has potential as a tag for the immobilization of soluble proteins.

Identification of the Domains Involved in Promotion of Silica Formation in Glassin, a Protein Occluded in Hexactinellid Sponge Biosilica, for Development of a Tag for Purification and Immobilization of Recombinant Proteins.

Glassin, a protein occluded in biosilica of the hexactinellid sponge Euplectela, promotes silica formation from silicic acid at room temperature and neutral pH and is characterized by its primary structure which consists of a tandem repeat carrying three distinct domains, histidine and aspartic acid-rich (HD) domain, proline-rich (P) domain, and histidine and threonine-rich (HT) domain. The present study aims to clarify the domain responsible for the promotion of silica formation and to demonstrate usefulness of glassin and its domain as a tag for purification and immobilization of recombinant proteins. When each domain was mixed with silicic acid at neutral pH, silica was formed with HD domain as well as glassin, or a single repeat, but not with P or HT domain. Neither of amino or carboxy-terminal half of HD domain induced silica formation. The amount of silica formed with HD domain was significantly lower than that of glassin or a single repeat. HD domain fused with HT domain raised the amount of silica formed, while a HD domain fused with P domain, a mixture of HD and P domains, or a mixture of HD and HT domains has little effect on the promotion of silica formation. Collectively, a minimum sequence for promotion of silica formation is HD domain, whose activity can be enhanced by HT domain through a topological effect. In addition, practicality of glassin and HD domain was demonstrated by fusion of these sequences to green fluorescent protein which was successfully purified with Ni affinity chromatography and immobilized on silica.