Silicatein alpha: cathepsin L-like protein in sponge biosilica

Abstract

Earth's biota produces vast quantities of polymerized silica at ambient temperatures and pressures by mechanisms that are not understood. Silica spicules constitute 75% of the dry weight of the sponge Tethya aurantia, making this organism uniquely tractable for analyses of the proteins intimately associated with the biosilica. Each spicule contains a central protein filament, shown by x-ray diffraction to exhibit a highly regular, repeating structure. The protein filaments can be dissociated to yield three similar subunits, named silicatein alpha, beta, and gamma. The molecular weights and amino acid compositions of the three silicateins are similar, suggesting that they are members of a single protein family. The cDNA sequence of silicatein alpha, the most abundant of these subunits, reveals that this protein is highly similar to members of the cathepsin L and papain family of proteases. The cysteine at the active site in the proteases is replaced by serine in silicatein alpha, although the six cysteines that form disulfide bridges in the proteases are conserved. Silicatein alpha also contains unique tandem arrays of multiple hydroxyls. These structural features may help explain the mechanism of biosilicification and the recently discovered activity of the silicateins in promoting the condensation of silica and organically modified siloxane polymers (silicones) from the corresponding silicon alkoxides. They suggest the possibility of a dynamic role of the silicateins in silicification of the sponge spicule and offer the prospect of a new synthetic route to silica and siloxane polymers at low temperature and pressure and neutral pH.

Figure 1

Scanning electron micrographs of isolated silica spicules (×130) (A) and axial filaments (×1,000) (B) from Tethya aurantia.

Figure 2

Electrophoretic profile of proteins from axial filaments. Filaments (10 μg of protein) were subjected to SDS/PAGE on a 4–20% gradient polyacrylamide gel followed by staining with Coomassie brilliant blue R250. Silicatein α, β, and γ are indicated by arrows. Numbers indicate molecular masses of standard proteins.

Figure 3

X-ray diffraction of axial protein filaments. The diffraction pattern was obtained from filaments oriented parallel to a glass substrate by using a Scintag x-ray diffractometer. The peak at 2θ = 0.51° represents a periodicity of 17.245 nm.

Figure 4

(A) Amino acid sequence of silicatein α deduced from cDNA. The putative signal peptidase cleavage site is marked with an arrow; the observed boundary between the propeptide and mature peptide is shown by an arrowhead. Amino acid sequences obtained from peptide sequencing are underlined as follows: solid line, N-terminal sequence; long-dashed, endoproteinase Asp-N fragments; short dashed, endoproteinase Glu-C fragment; and dotted, endoproteinase Lys-C fragment. (B) Northern hybridization analysis of silicatein α transcript. Total RNA prepared from regenerating sponge tissue was subjected to Northern blot analysis with digoxigenin-labeled RNA probe as described in the text.

Figure 5

Alignment and comparison of silicatein α with human cathepsin L. (A) Propeptide regions. (B) Mature proteins. Identical amino acids are indicated by vertical bars; similar residues indicated by colons. Cysteine residues involved in disulfide bonds in cathepsin L are shaded. Catalytic triad amino acids of the active site of cathepsin L and corresponding amino acids in silicatein α are highlighted. Hydroxy amino acid residues in silicatein α and cathepsin L are overlined and underlined, respectively. Silicatein α-specific hydroxy amino acid cluster is boxed.