Biosilica formation in diatoms: characterization of native silaffin-2 and its role in silica morphogenesis

Abstract

The biological formation of inorganic materials with complex form (biominerals) is a widespread phenomenon in nature, yet the molecular mechanisms underlying biomineral morphogenesis are not well understood. Among the most fascinating examples of biomineral structures are the intricately patterned, silicified cell walls of diatoms, which contain tightly associated organic macromolecules. From diatom biosilica a highly polyanionic phosphoprotein, termed native silaffin-2 (natSil-2), was isolated that carries unconventional amino acid modifications. natSil-2 lacked intrinsic silica formation activity but was able to regulate the activities of the previously characterized silica-forming biomolecules natSil-1A and long-chain polyamines. Combining natSil-2 and natSil-1A (or long-chain polyamines) generated an organic matrix that mediated precipitation of porous silica within minutes after the addition of silicic acid. Remarkably, the precipitate displayed pore sizes in the range 100-1000 nm, which is characteristic for diatom biosilica nanopatterns.

Fig. 1.

Tricine/SDS/PAGE analysis (15) of ammonium fluoride extracts from C. fusiformis biosilica. Preparation of biosilica and extraction with ammonium fluoride were performed as described (12). Lane 1, ammonium fluoride extract from ³³P-labeled biosilica, fluorography; lane 2, ammonium fluoride extract, Stained-All stained; lane 3, ammonium fluoride extract, Coomassie bluestained; lane 4, HF-treated ammonium fluoride extract, Coomassie bluestained.

Fig. 2.

Influence of natSil-2 on the silica precipitation activities of natSil-1A and LCPA. Silica precipitation was performed under standard assay conditions in the presence of a constant concentration of LCPA (0.6 μg/μl, ▴) or natSil-1A (0.3 mM, ▪).

Fig. 3.

Chemical composition of natSil-2. (A) Analysis of natSil-2 labeled with ³⁵S and ³³P, respectively, before (-) and after (+) treatment with trifluoromethanesulfonic acid (Tricine/SDS/PAGE, fluorography). Deglycosylated (deglyc.) natSil-2 is composed of phosphorylation isoforms that appear as three discrete bands on SDS/PAGE. Residual ³⁵S label in deglycosylated [³⁵S]natSil-2 is due to its methionine content. (B) Upper trace, analysis of Fmoc-derivatized phosphoamino acids of natSil-2 by anion-exchange chromatography (photometric detection at 256 nm). Fraction numbers are indicated. Lower trace, synthetic Fmoc-phosphohydroxyproline (Hyp-P) was added before chromatography. Ser-P, Fmoc-phosphoserine; Thr-P, Fmoc-phosphothreonine. (C) Amino acid composition of natSil-2 after complete acid hydrolysis (24 h, 110°C, 6 M HCl). Relative amounts were determined from the peak areas in the reverse-phase HPLC chromatogram of the phenyl isothiocyanate (PITC) derivatives (PITC-hydroxyproline peak area defined as 10). Polyamine-modified lysines could not be quantified, because the corresponding PITC derivatives do not show up in the chromatogram. n.a., Not applicable. (D) ESI-MS spectrum of natSil-2 after complete acid hydrolysis (24 h, 110°C, 6 M HCl). Each peak represents a doubly charged positive ion. Note the constant difference of m/z = 35.5 between neighboring peaks, which is indicative of a repeated unit element of 71 Da. The absolute molecular masses, in Da, of the corresponding uncharged molecules (501.4, 272.6, 643.8, 715.0, 786.0, and 856.8) reveal 5–10 repeats (see numbers in parentheses) of N-methylpropylenimine (71 Da) attached to a lysine residue (10).

Fig. 4.

SEM analysis of silica precipitates formed by mixtures of natSil-1A and natSil-2. (A) natSil-2 at 0.5 units/μl, natSil-1A at 0.3 mM. (B) natSil-2 at 5.0 units/μl, natSil-1A at 0.3 mM. (C) natSil-2 at 2.0 units/μl, natSil-1A at 0.3 mM. (D) natSil-2 at 1.6 units/μl, natSil-1A at 0.2 mM. (Bars = 2 μm.)

Fig. 5.

Formation of a silaffin phase by ionic interactions between natSil-1A and natSil-2. Samples 1a, 1b, 2, and 3 were incubated for 2 h at 18°C and subsequently centrifuged (15 min, 20,000 × g, 18°C). Aliquots of the pellets (P) and supernatants (S) were analyzed by Tricine/SDS/PAGE for natSil-1A (Coomassie blue staining) and natSil-2 (Stains-All staining). Composition of the samples: 1a, 2.0 units/μl natSil-2; 1b, 0.3 mM natSil-1A; 2, 2.0 units/μl natSil-2 plus 0.3 mM natSil-1A; 3, same as sample 2 plus 500 mM NaCl. Each sample contained 50 mM sodium acetate/acetic acid, pH 5.5.