Silica Morphogenesis by Alternative Processing of Silaffins in the Diatom Thalassiosira pseudonana

Abstract

For almost 200 years scientists have been fascinated by the ornate cell walls of the diatoms. These structures are made of amorphous silica, exhibiting species-specific, mostly porous patterns in the nano-to micrometer range. Recently, from the diatom Cylindrotheca fusiformis unusual phosphoproteins (termed silaffins) and long chain polyamines have been identified and implicated in biosilica formation. However, analysis of the role of silaffins in morphogenesis of species-specific silica structures has so far been hampered by the difficulty of obtaining structural data from these extremely complex proteins. In the present study, the five major silaffins from the diatom Thalassiosira pseudonana (tpSil1H, -1L, -2H, -2L, and -3) have been isolated, functionally analyzed, and structurally characterized, making use of the recently available genome data from this organism. Surprisingly, the silaffins of T. pseudonana and C. fusiformis share no sequence homology but are similar regarding amino acid composition and posttranslational modifications. Silaffins tpSil1H and -2H are higher molecular mass isoforms of tpSil1L and -2L, respectively, generated in vivo by alternative processing of the same precursor polypeptides. Interestingly, only tpSil1H and -2H but not tpSil1L and -2L induce the formation of porous silica patterns in vitro, suggesting that the alternative processing event is an important step in morphogenesis of T. pseudonana biosilica.During evolution many organisms (e.g. diatoms, sponges, radiolaria) have acquired the ability to use the ubiquitous monosilicic acid Si(OH) 4 for the formation of species specifically structured, silica-based exo-or endoskeletons (1). This interesting biomineralization phenomenon is mediated by cellular organic (macro-) molecules that accelerate silicic acid polycondensation and control morphogenesis of the forming silica (2). Diatoms are an extremely large group (Ͼ10,000 species) of unicellular eukaryotic algae that play a major role in biological silica cycling. Within the last few years diatom biosilica-associated proteins (termed silaffins) and long chain polyamines (LCPA) 1 have been identified and hypothesized to represent key components of the diatom biosilica-forming machinery. Silaffins and LCPA exhibit the remarkable ability to induce rapid silica deposition in vitro and to control the nanostructure of the forming silica (3). Therefore, unraveling the correlations between chemical structures, physical properties, and silica-forming activities of silaffins and LCPA will be important for understanding the molecular mechanism of species-specific biosilica nanopatterning. So far, silaffins have only been characterized from the diatom Cylindrotheca fusiformis. They are highly modified proteins/peptides rich in hydroxyamino acids (serine, threonine, hydroxyproline) and lysine residues. Silaffins natSil1A and -1B are O-phosphorylated at numerous sites and contain polyamine-modified lysine residues, features that enable these peptides to rapidly form silica nanospheres in vitr...

Targeted drug delivery using genetically engineered diatom biosilica

“…18) into the diatom's genome. Both S (17 amino acids) and T8 (37 amino acids) are derived from silaffin-3 (231 amino acids), which is a natural component of T. pseudonana biosilica 22 . The combination of S and T8 acts as intracellular address tag for permanently anchoring proteins into the biosilica during its formation 18,23 .…”

“…T. pseudonana (Hustedt) Hasle et Heimdal clone CCMP1335 was grown in an enriched artificial seawater medium according to the North East Pacific Culture Collection protocol (Canadian Centre for the Culture of Microorganisms, ESAWRecipe) at 18°C under constant light at 5,000-10,000 lux 22 was PCR amplified from pUC57/pgb1 (synthesized by Genescript) using the oligonucleotides 5 0 -GAA TTC AAG ACT GAC ACT TAC AAA TTA AT-3 0 and 5 0 -GCG GCC GCT TAT TCT GGT TTT TCA GTA ACT GTA A-3 0 (EcoRI site in bold and NotI site in italics) and then ligated into the EcoRI and NotI sites of vector pTpNR/T8-egfpEcoRI-NotI, resulting in the final plasmid pTpNR/ T8-egfp-pgb1. All PCR products were verified by DNA sequencing.…”

Application of AFM in understanding biomineral formation in diatoms

“…The statistically significant difference in silica particle size between species, but not between girdle bands and valves in the same species, suggested species-specificity in nanoscale silica formation. Silaffins and LCPAs from different diatom species can form silica precipitates of different size classes and morphologies [25,27,35], which could explain the underlying mechanism for nanoscale size differences identified in P. viridis and H. amphioxys [8].…”

“…From these studies, it is clear that shaping and molding of the SDV (which involves the cytoskeleton) dictate overall microscale morphology. Mesoscale structure has been proposed to occur either by templating of an organic material (protein or carbohydrate) internal to the SDV [9,33,37], by a diffusion-based chemical process [17], by self-association of silaffins and LCPAs [24,35,46], or by phase separation involving LCPAs [47]. Close examination of detailed structure formation in diatoms that make complicated threedimensional structures [41,42] indicate a level of control over all aspects that is not consistent with solely diffusionbased, self-association, or phase separation process.…”

“…Characterization of organic components tightly associated with diatom silica suggests that major determinants of nanoscale structure are long chain polyamines (LCPAs), which catalyze silica polymerization [25], and silaffins, which are (poly)peptides proposed to play a regulatory role in silica polymerization by organizing LCPAs via electrostatic interactions [26,27,35]. In vitro, LCPAs and silaffins isolated from diatoms precipitate silica to form a variety of morphologies [25][26][27]35], but these lack the higher order structure seen in a diatom frustule. In diatoms, higher order is seen on the mesoscale, in which substructures consisting of organized patterns of nanoscale polymerization products are formed within the confines of the microscale of the SDV.…”