Final Report Summary - BIOMINTEC (Biomineralization: Understanding of basic mechanisms for the design of novel strategies in nanobiotechnology)
Biomineralization is the formation of minerals by living cells and organisms. To understand the processes involved in biomineralization, at the cutting edge between inorganic and organic world, the cooperation between molecular and cell biologists, inorganic chemists, and physical chemists, but also computational scientists is required. The ability of organisms to form nanostructured biominerals with high precision and in large copy number under biological, environmentally benign conditions makes the mechanisms underlying biomineral formation extremely interesting for nanotechnology, a key technology of the 21st century. In this ITN, we focussed on the formation of biominerals consisting of calcium carbonate or biosilica-glass due to the enormous interest and importance that these biominerals have achieved in the last few years. There is an urgent need for training of young researchers in Europe in this pioneering area of research. The goal of this network was to understand basic principles of biomineralization (biosilicification and biocalcification) in order to develop novel strategies to apply the biological mechanisms in the field of nanotechnology.
In this training network, we have studied biosilicification using siliceous sponges and a loricate choanoflagellate as a model. First proteins involved in biosilica formation in the hexactinellid sponge Euplectella aspergillum have been identified and characterized. The optical-mechanical properties of the sponge spicules have been studied. The structural properties of the siliceous strips of loricate choanoflagellates have been analyzed. Several matrix proteins have been isolated and characterized. To determine the X-ray structure of the principle silica-forming enzyme in sponges, silicatein, refolding experiments with the recombinant protein have been performed to obtain single crystals for X-ray crystallography. In addition, basic proteins and other factors which interact with silicatein and are involved in regulation of the biosilicification process have been investigated. Binding studies of proteins involved in biosilicification have been performed using advanced calorimetric techniques. Particular efforts have been taken to characterise the protein applying circular dichroism, dynamic light scattering, and fluorescence immunoassays. Moreover, we have investigated the role of silica in hydroxyapatite deposition (bone formation). A further topic of research has been the investigation of organic-inorganic hybrid nanomaterials with complex architectures and properties. The biomimetic formation of synthetic nanocomposite materials based on isolated organic components of sponge spicules has been studied. Among others, we have been able to synthesize for the first time nanostructured crystalline tin dioxide using silicatein, under mild physiological conditions. This material showed interesting structural and catalytic properties. In addition, we focused on new strategies for surface functionalization with silicatein. For that we have studied recombinant silicateins which possess additional engineered groups (tags) that allow specific binding to surfaces. Using these different tags, patterning of silicatein on gold and metal oxide surfaces using a soft lithography method has been achieved. Using the ability of the protein to synthesize silica and other metal oxides it was been possible to obtained nano and micro patterned surfaces. Moreover, a growth model of spiculogenesis has been developed. In order to obtain novel hierarchical nanomaterials, the catalytic activity of silicatein has been exploited to synthesize nanocomposite materials of various metal oxides that mimic the layered structure of spicules. In order to produce superhydrophobic surfaces by silicatein-mediated silica deposition on self-assembled S-layers, recombinant silicatein and S-layer proteins have been expressed and purified. Silicatein has been immobilized to S-layer proteins recrystalized on different surfaces. Furthermore, we have developed new methodologies for the use of mass spectrometry for the speciation of the early stages of silica formation enabling the interaction of small, biologically relevant amine containing molecules and small polysilicic acid species to be determined for the first time. We have shown that proteins and peptides, relevant in biomineralisation processes can be combined in very different ways in the laboratory to yield novel materials and composites with unusual composition and form. We have identified trace mineral components in biosilicas that ultimately affect how the material changes under thermal treatment. In addition, the calcium carbonate biomineralization, in particular in molluscs and corals has been studied. New proteins associated to the outer calcitic shell layer of the fan mussel Pinna nobilis have been characterized. By combining biochemical as well as molecular biology screening methods, we have been able to identify new clones encoding putative shell proteins. One of them, provisionally named CSP3, has been characterized further. This intracrystalline protein exhibits an extremely acidic C-terminal end. One antibody against an immunogenic peptide of CSP3 has been developed and used for immuno-localization. A further topic, realized in cooperation with the Institut de Biologie et Chimie des Protéines (IBCP, Lyon) concerns the construction of a database (CaBioMin db), comprising all the proteins associated to calcium carbonate biomineralization in metazoans, including different tools for protein sequence analysis. In addition, a small protein, Upsalin, associated to the nacre of the freshwater bivalve Unio pictorum, has been characterized, at transcript and protein levels. The full transcript sequence has been retrieved from degenerated oligonucleotides, and in parallel, the protein has been purified from nacre extracts, and its sequence verified by MS-MS. An antibody has been produced for localizing upsalin in the shell. Furthermore, an identification, via a proteomic approach associated to genomic and transcriptomic data, of the full set of proteins associated to the aragonite skeleton of the scleractinian coral Acropora millepora, have been performed; so far, 37 proteins have been identified, and sorted in families according to their primary structures. Deepened in silico analyses have been were performed in order to compare the Acropora’s ‘protein toolbox’ required for calcifying to that of non-calcifying corals and to other calcifying metazoans. In addition, ultrastructural investigations on the prisms of Pinna nobilis, and, in parallel, an exploration of the shell matrix composition and properties of the crossed-lamellar bivalve Glycimeris glycimeris has been performed. Moreover, the recombinant proteins for Pl-galectin-8 and Pl-carbonic anhydrase have been synthesized in vitro and tested for their functional activity by biochemical and cellular assays. Mouse/rabbit polyclonal antibodies have been produced against the two recombinant proteins. Moreover, the expression of genes involved in formation of the magnesium calcite skeleton of the sea urchin Paracentrotus lividus has been studied using embryos and adults as a model system. Several cDNAs for spicule matrix proteins have been isolated and characterized in P. lividus for the first time, including carbonic anhydrase, tetraspanin, p16, p19 as well as the lectins advillin and galectin. Their temporal and spatial expression profiles were monitored by comparative qPCR and whole mount in situ hybridization during development. Furthermore, orthologues of sm30, sm50 and msp130RI expressed in P. lividus have been characterized. In order to study the functional activities of Pl-galectin-8 and Pl-carbonic anhydrase, the recombinant proteins expressed in E. coli have been prepared and used in biochemical and cellular assays. In addition to the transcriptomic studies, biochemical analyses on tests from P. lividus adults have been performed. In preliminary experiments, acetic acid-soluble and -insoluble matrix have been extracted and analysed by SDS-PAGE, ELISA and Western blots. Furthermore, the effects of SM on CaCO3 crystallization were tested by an in vitro assay.
The aim of this ITN was - through training of young researchers in the most advanced techniques to study biomineralization - to strengthen the competitiveness of the European Union in this important field of present-day research. Understanding of the principles of biomineralization is of high importance for the development of novel strategies in nanotechnology, a key technology of this century with enormous economic impact. Therefore, the BIOMINTEC Initial Training Network was of benefit to the future career of the young researchers. This network enhanced their skills and knowledge in this fascinating field of research. The new knowledge generated in this network allowed the students to be trained in applied, industry-relevant research topics.
Address of project public website:
http://www.biomintec.de(opens in new window)
In this training network, we have studied biosilicification using siliceous sponges and a loricate choanoflagellate as a model. First proteins involved in biosilica formation in the hexactinellid sponge Euplectella aspergillum have been identified and characterized. The optical-mechanical properties of the sponge spicules have been studied. The structural properties of the siliceous strips of loricate choanoflagellates have been analyzed. Several matrix proteins have been isolated and characterized. To determine the X-ray structure of the principle silica-forming enzyme in sponges, silicatein, refolding experiments with the recombinant protein have been performed to obtain single crystals for X-ray crystallography. In addition, basic proteins and other factors which interact with silicatein and are involved in regulation of the biosilicification process have been investigated. Binding studies of proteins involved in biosilicification have been performed using advanced calorimetric techniques. Particular efforts have been taken to characterise the protein applying circular dichroism, dynamic light scattering, and fluorescence immunoassays. Moreover, we have investigated the role of silica in hydroxyapatite deposition (bone formation). A further topic of research has been the investigation of organic-inorganic hybrid nanomaterials with complex architectures and properties. The biomimetic formation of synthetic nanocomposite materials based on isolated organic components of sponge spicules has been studied. Among others, we have been able to synthesize for the first time nanostructured crystalline tin dioxide using silicatein, under mild physiological conditions. This material showed interesting structural and catalytic properties. In addition, we focused on new strategies for surface functionalization with silicatein. For that we have studied recombinant silicateins which possess additional engineered groups (tags) that allow specific binding to surfaces. Using these different tags, patterning of silicatein on gold and metal oxide surfaces using a soft lithography method has been achieved. Using the ability of the protein to synthesize silica and other metal oxides it was been possible to obtained nano and micro patterned surfaces. Moreover, a growth model of spiculogenesis has been developed. In order to obtain novel hierarchical nanomaterials, the catalytic activity of silicatein has been exploited to synthesize nanocomposite materials of various metal oxides that mimic the layered structure of spicules. In order to produce superhydrophobic surfaces by silicatein-mediated silica deposition on self-assembled S-layers, recombinant silicatein and S-layer proteins have been expressed and purified. Silicatein has been immobilized to S-layer proteins recrystalized on different surfaces. Furthermore, we have developed new methodologies for the use of mass spectrometry for the speciation of the early stages of silica formation enabling the interaction of small, biologically relevant amine containing molecules and small polysilicic acid species to be determined for the first time. We have shown that proteins and peptides, relevant in biomineralisation processes can be combined in very different ways in the laboratory to yield novel materials and composites with unusual composition and form. We have identified trace mineral components in biosilicas that ultimately affect how the material changes under thermal treatment. In addition, the calcium carbonate biomineralization, in particular in molluscs and corals has been studied. New proteins associated to the outer calcitic shell layer of the fan mussel Pinna nobilis have been characterized. By combining biochemical as well as molecular biology screening methods, we have been able to identify new clones encoding putative shell proteins. One of them, provisionally named CSP3, has been characterized further. This intracrystalline protein exhibits an extremely acidic C-terminal end. One antibody against an immunogenic peptide of CSP3 has been developed and used for immuno-localization. A further topic, realized in cooperation with the Institut de Biologie et Chimie des Protéines (IBCP, Lyon) concerns the construction of a database (CaBioMin db), comprising all the proteins associated to calcium carbonate biomineralization in metazoans, including different tools for protein sequence analysis. In addition, a small protein, Upsalin, associated to the nacre of the freshwater bivalve Unio pictorum, has been characterized, at transcript and protein levels. The full transcript sequence has been retrieved from degenerated oligonucleotides, and in parallel, the protein has been purified from nacre extracts, and its sequence verified by MS-MS. An antibody has been produced for localizing upsalin in the shell. Furthermore, an identification, via a proteomic approach associated to genomic and transcriptomic data, of the full set of proteins associated to the aragonite skeleton of the scleractinian coral Acropora millepora, have been performed; so far, 37 proteins have been identified, and sorted in families according to their primary structures. Deepened in silico analyses have been were performed in order to compare the Acropora’s ‘protein toolbox’ required for calcifying to that of non-calcifying corals and to other calcifying metazoans. In addition, ultrastructural investigations on the prisms of Pinna nobilis, and, in parallel, an exploration of the shell matrix composition and properties of the crossed-lamellar bivalve Glycimeris glycimeris has been performed. Moreover, the recombinant proteins for Pl-galectin-8 and Pl-carbonic anhydrase have been synthesized in vitro and tested for their functional activity by biochemical and cellular assays. Mouse/rabbit polyclonal antibodies have been produced against the two recombinant proteins. Moreover, the expression of genes involved in formation of the magnesium calcite skeleton of the sea urchin Paracentrotus lividus has been studied using embryos and adults as a model system. Several cDNAs for spicule matrix proteins have been isolated and characterized in P. lividus for the first time, including carbonic anhydrase, tetraspanin, p16, p19 as well as the lectins advillin and galectin. Their temporal and spatial expression profiles were monitored by comparative qPCR and whole mount in situ hybridization during development. Furthermore, orthologues of sm30, sm50 and msp130RI expressed in P. lividus have been characterized. In order to study the functional activities of Pl-galectin-8 and Pl-carbonic anhydrase, the recombinant proteins expressed in E. coli have been prepared and used in biochemical and cellular assays. In addition to the transcriptomic studies, biochemical analyses on tests from P. lividus adults have been performed. In preliminary experiments, acetic acid-soluble and -insoluble matrix have been extracted and analysed by SDS-PAGE, ELISA and Western blots. Furthermore, the effects of SM on CaCO3 crystallization were tested by an in vitro assay.
The aim of this ITN was - through training of young researchers in the most advanced techniques to study biomineralization - to strengthen the competitiveness of the European Union in this important field of present-day research. Understanding of the principles of biomineralization is of high importance for the development of novel strategies in nanotechnology, a key technology of this century with enormous economic impact. Therefore, the BIOMINTEC Initial Training Network was of benefit to the future career of the young researchers. This network enhanced their skills and knowledge in this fascinating field of research. The new knowledge generated in this network allowed the students to be trained in applied, industry-relevant research topics.
Address of project public website:
http://www.biomintec.de(opens in new window)