Manfred Sumper

Silica formation in diatoms: the function of long-chain polyamines and silaffins

The stunning silica structures formed by diatoms are among the most remarkable examples of biological nanofabrication. In recent years, insight into the molecules and mechanism that allow diatoms to perform silica morphogenesis under ambient conditions has been gained.

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

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.

A new calcium binding glycoprotein family constitutes a major diatom cell wall component

Diatoms possess silica‐based cell walls with species‐specific structures and ornamentations. Silica deposition in diatoms offers a model to study the processes involved in biomineralization. A new wall is produced in a specialized vesicle (silica deposition vesicle, SDV) and secreted. Thus proteins involved in wall biogenesis may remain associated with the mature cell wall. Here it is demonstrated that EDTA treatment removes most of the proteins present in mature cell walls of the marine diatom Cylindrotheca fusiformis. A main fraction consists of four related glycoproteins with a molecular mass of approximately 75 kDa. These glycoproteins were purified to homogeneity. They consist of repeats of Ca2+ binding domains separated by polypeptide stretches containing hydroxyproline. The proteins in the EDTA extract aggregate and precipitate in the presence of Ca2+. Immunological studies detected related proteins in the cell wall of the freshwater diatom Navicula pelliculosa, indicating that these proteins represent a new family of proteins that are involved in the biogenesis of diatom cell walls.

Putative spermine synthases from Thalassiosira pseudonana and Arabidopsis thaliana synthesize thermospermine rather than spermine

Polyamines are involved in many fundamental cellular processes. Common polyamines are putrescine, spermidine and spermine. Spermine is synthesized by transfer of an aminopropyl residue derived from decarboxylated S‐adenosylmethionine to spermidine. Thermospermine is an isomer of spermine and assumed to be synthesized by an analogous mechanism. However, none of the recently described spermine synthases was investigated for their possible activity as thermospermine synthases. In this work, putative spermine synthases from the diatom Thalassiosira pseudonana and from Arabidopsis thaliana could be identified as thermospermine synthases. These findings may explain the previous result that two putative spermine synthase genes in Arabidopsis produce completely different phenotypes in knock‐out experiments. Likely, part of putative spermine synthases identifiable by sequence comparisons represents in fact thermospermine synthases.

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

Silica Biomineralisation in Diatoms: The Model Organism Thalassiosira pseudonana

After complete genome sequencing, the diatom Thalassiosira pseudonana has become an attractive model organism for silica biomineralisation studies. Recent progress, especially with respect to intracellular silicic acid processing, as well as to the natures of the biomolecules involved in diatom cell wall formation, is described. On the one hand, considerable progress has been made with respect to silicon uptake by special proteins (SITs) from the surrounding water, as well as to the storage and processing of silicon before cell division. On the other hand, the discovery and characterisation of remarkable biomolecules such as silaffins, polyamines and—quite recently—of silacidins in the siliceous cell walls of diatoms strongly impacts the growing field of biomimetic materials synthesis.

Algal-CAMs: isoforms of a cell adhesion molecule in embryos of the alga Volvox with homology to Drosophila fasciclin I.

Proof that plants possess homologs of animal adhesion proteins is lacking. In this paper we describe the generation of monoclonal antibodies that interfere with cell‐cell contacts in the 4‐cell embryo of the multicellular alga Volvox carteri, resulting in a hole between the cells. The number of following cell divisions is reduced and the cell division pattern is altered drastically. Antibodies given at a later stage of embryogenesis specifically inhibit inversion of the embryo, a morphogenetic movement that turns the embryo inside out. Immunofluorescence microscopy localizes the antigen (Algal‐CAM) at cell contact sites of the developing embryo. Algal‐CAM is a protein with a three‐domain structure: an N‐terminal extensin‐like domain characteristic for plant cell walls and two repeats with homology to fasciclin I, a cell adhesion molecule involved in the neuronal development of Drosophila. Alternatively spliced variants of Algal‐CAM mRNA were detected that are produced under developmental control. Thus, Algal‐CAM is the first plant homolog of animal adhesion proteins.

Biomimetic synthesis of silica nanospheres depends on the aggregation and phase separation of polyamines in aqueous solution

Long-chain polyamines extracted from the highly siliceous cell walls of diatoms are known to precipitate silica nanospheres from aqueous, silicic-acid containing solutions at near-neutral pH in vitro. The same is true for synthetic polyamines such as polyallylamine. In the present contribution we show that the microscopic phase separation of polyallylamine in aqueous solution is strictly correlated with the silica precipitation activity of polyallylamine/silicic acid solutions. Multivalent anions such as phosphate or sulfate efficiently induce this microscopic phase separation. At higher anion concentrations, macroscopic phase separation occurs. In contrast to the multivalent phosphate and sulfate ions, the monovalent chloride ions are much less efficient in polyallylamine aggregate formation.

TARGETING AND COVALENT MODIFICATION OF CELL WALL AND MEMBRANE PROTEINS HETEROLOGOUSLY EXPRESSED IN THE DIATOM CYLINDROTHECA FUSIFORMIS (BACILLARIOPHYCEAE)

Diatoms are unicellular organisms encased by silica‐based cell walls that display species‐specific structures. Morphogenesis of diatom cell walls is believed to be controlled by a polysaccharide/protein‐matrix that remains associated with mature cell walls. Recently, a family of calcium‐binding glycoproteins, the frustulins, has been identified as major diatom cell wall component. Here we describe a transformation‐based approach to investigate intracellular targeting and function of frustulins. When ε‐frustulin from the diatom Navicula pelliculosa is expressed in Cylindrotheca fusiformis, it is correctly targeted into the cell wall. Furthermore, the unique N‐terminus of ε‐frustulin was properly modified, indicating that C. fusiformis and N. pelliculosacontain homologous frustulin‐processing proteases. In a different transformation experiment, a modified version of theChlorella kessleri hexose/H+ symporter bearing a bacterial biotinyl‐acceptor domain was expressed in C. fusiformis. The transporter became biotinylated in vivo and was functionally incorporated into the plasma membrane, allowing C. fusiformis to take up 14C‐glucose and 14C‐glucosamine. Stage‐specific radioactive labeling with this transformant revealed that secretion of frustulins is strongly enhanced during cell wall development. The data presented in this study demonstrate for the first time functional expression of a membrane protein and correct targeting of a cell wall protein heterologously expressed in a diatom cell.

Halobacterial glycoprotein biosynthesis

III. Structure of the saccharides linked to the cell surface giycoprotein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 A. Sulfated high molecular weight saccliaride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 B. Sulfated low molecular weight saccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 C. Nonsulfated glycopeptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71