Abstract
Sponges (phylum Porifera) of the class of Demospongiae are stabilized by a siliceous skeleton. It is composed of silica needles (spicules), which provide the morphogenetic scaffold of these metazoans. In the center of the spicules there is an axial filament that consists predominantly of silicatein, an enzyme that catalyzes the synthesis of biosilica. By differential display of transcripts we identified additional proteins involved in silica formation. Two genes were isolated from the marine demosponge Suberites domuncula; one codes for a galectin and the other for a fibrillar collagen. The galectin forms aggregates to which silicatein molecules bind. The extent of the silicatein-mediated silica formation strongly increased if associated with the galectin. By applying a new and mild extraction procedure that avoids hydrogen fluoride treatment, native axial filaments were extracted from spicules of S. domuncula. These filaments contained, in addition to silicatein, the galectin and a few other proteins. Immunogold electron microscopic studies underscored the role of these additional proteins, in particular that of galectin, in spiculogenesis. Galectin, in addition to silicatein, presumably forms in the axial canal as well as on the surface of the spicules an organized net-like matrix. In the extraspicular space most of these complexes are arranged concentrically around the spicules. Taken together, these additional proteins, working together with silicatein, may also be relevant for potential (nano)-biotechnological applications of silicatein in the formation of surface coatings. Finally, we propose a scheme that outlines the matrix (galectin/silicatein)-guided appositional growth of spicules through centripetal and centrifugal synthesis and deposition of biosilica.
Highlights
Sponges of the class of Demospongiae are stabilized by a siliceous skeleton
In S. domuncula the differential display of transcripts revealed one dominant galectin, termed galectin-2
This lectin comprises, in contrast to galectin-1 from G. cydonium [35], two galactose-binding sites and one hydrophobic region at its N terminus
Summary
Materials, and Enzymes—Restriction enzymes, a Total RNA isolation kit, and reagents for the RACE procedure were purchased from Invitrogen; TriplEx2 vector from BD Biosciences; pET41a(ϩ) vector and BugBuster reagent from Novagen (Darmstadt, Germany); TRIzol reagent from Invitrogen; Hybond-Nϩ nylon membrane from Amersham Biosciences; Cy3-conjugated F(abЈ) goat antirabbit IgG from Jackson ImmunoResearch; PCR-DIG probe-synthesis kit, a BM chemiluminescence blotting substrate kit, proteinase inhibitor mixture, and CDP-Star from Roche Applied Science; sodium metasilicate from Aldrich; a silicon test colorimetric assay kit and silicon standards from Merck (Darmstadt, Germany); natural seawater (containing 5 M silicate) and tetraethoxysilane from Sigma; protein A/G-agarose beads from Santa Cruz Biotechnology (Santa Cruz, CA); 1.4-nm nanogold anti-rabbit IgG from Nanoprobes (Yapbank, NY); and rhodamineconjugated goat anti-rabbit immunoglobulin from Dako (Carpinteria, CA). Polyclonal antibodies (pAb) were raised against the purified, recombinant galectin-2 (rGALEC2_SUBDO) in female rabbits (New Zealand White) as described [28]. They were shock-frozen in liquid nitrogen, subjected to twice the volume of lysis buffer (1ϫ Tris-buffered saline, pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.1% Nonidet-P40, 10 mM NaF, protease inhibitor mixture (1 tablet/10 ml), and 1 mM sodium orthovanadate), homogenized, incubated for 60 min at 37 °C, and brought to 4 °C. In control experiments 100 l of the respective antibodies pAb-aGALEC2 and pAb-aSILIC were adsorbed with 20 g of the recombinant proteins (rGALEC2_SUBDO or rSILIC_SUBDO) during an incubation period of 30 min (4 °C) prior to their use. Electron Microscopy—For transmission microscopic analysis, sponge primmorphs or tissue samples were cut into pieces (1 mm3), incubated in phosphate buffer, and washed in phosphate-buffered saline, pH 7.4, at room temperature as described [5, 8]. After size separation by SDS-gel electrophoresis the N terminus of galectin-2 was determined by direct protein sequencing
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