Silacidins: highly acidic phosphopeptides from diatom shells assist in silica precipitation in vitro.

Biomineralization is the formation of inorganic materials under the control of a living cell. Silica biomineralization occurs, for example, in unicellular diatoms with cell walls composed of silica. Although amorphous, diatom silica displays intricate structures of amazing beauty even on the nanometer scale. Therefore, it is commonly assumed that the cellular processes that govern the biogenesis of this mineral may include structure-directing templates. Single molecules, however, are much too small to act as templates for the observed shapes and patterns. Thus, only supramolecular assemblies are candidates as templates. Indeed, diatom silica is a composite material that contains organic substances with the potential to promote assembly. Highly zwitterionic proteins known as silaffins and long-chain polyamines have been identified as constituents of diatom biosilica, and have been shown to promote silica formation frommonosilicic acid in vitro. To date, silaffins from Cylindrotheca fusiformis and Thalassiosira pseudonana have been characterized in detail. Silaffin-1 from C. fusiformis contains modified lysine and serine residues. Certain lysine residues are linked through their e-amino groups to long-chain polyamines (four to eight propyleneimine repeated units), and all serine residues are phosphorylated. Similar modification strategies are associated with silaffins extracted from the cell walls of T. pseudonana ; 9] however, selected lysine residues are linked with only two propyleneimine units, which are further modified by methylation. Permanent positive charges are introduced by quaternary ammonium groups. Again, negative charges are introduced by the phosphorylation of serine residues. As a result of this zwitterionic nature, individual silaffins or silaffin mixtures are able to form supramolecular aggregates. In vitro, silaffins guide silica formation only in this aggregated state. Biosilica from all diatom species investigated so far also contains long-chain polyamines that are not attached covalently to a polypeptide backbone. Their chemical structures display a remarkable degree of species specificity. Interestingly, long-chain polyamines were also detected recently as constituents of biosilica produced by sponges. In vitro, polyamines display silica-precipitation activity if polyanions or silaffins with an acidic domain are present to allow their assembly by electrostatic interactions. Evidence for the presence of phosphate and/or phosphorylated compounds in the shells of Coscinodiscus diatoms was obtained by P NMR spectroscopy. However, until now, no purely polyanionic substances that may serve as crosslinking agents for the assembly of long-chain polyamines could be identified in diatom biosilica. Herein, we describe a new class of aspartate/glutamate-rich and serine phosphate rich peptides as constituents of biosilica produced by the diatom Thalassiosira pseudonana. Owing to their presence in silica and their acidic nature, we refer to these peptides as silacidins. It has been shown previously that organic constituents can be extracted from cell walls in a native state if an aqueous solution of ammonium fluoride is used to dissolve the silica. By applying this procedure to the diatom T. pseudonana, several silaffins and polyamines could be extracted and separated by size-exclusion chromatography. The silaffins were denoted sil1/2L, sil1/2H, and sil3, respectively. If silaffin-1/2L is treated after purification by sizeexclusion chromatography (Figure 1a) with a concentrated salt solution (2m NaCl) and again subjected to size-exclusion chromatography under high-salt conditions, a previously undetected low-molecular-weight component dissociates and can be separated readily from silaffin-1/2L (Figure 1b). Amino acid sequencing of this material was only possible after treatment with anhydrous hydrogen fluoride, which indicates that this material is a peptide that contains many HF-labile posttranslational modifications. After treatment with HF and purification by reversed-phase chromatography, this material can be separated further into three related substances (Figure 2). Edman sequencing of each of these substances identified in the N-terminal amino acid sequences given in Figure 2. These very unusual sequences consist mainly of serine residues and the acidic amino acids aspartic and glutamic acid. We named these related but slightly different peptides silacidins A, B, and C. These peptides were absent in fractions containing the more anionic silaffins sil1/ 2H and sil3. The recently completed genome sequence of T. pseudonana has opened the door for genomic and proteomic approaches to compounds synthesized by this diatom. A search in the corresponding data base uncovered a gene model that indeed encoded all three silacidin sequences. The terminal part of the corresponding open reading frame [*] Dr. S. Wenzl, R. Hett, P. Richthammer, Prof. Dr. M. Sumper Lehrstuhl Biochemie I Universit+t Regensburg 93040 Regensburg (Germany) Fax: (+49)941-943-2936 E-mail: manfred.sumper@vkl.uni-regensburg.de