Soluble Organic Matrix Research Articles

Assembly of the Intraskeletal Coral Organic Matrix during Calcium Carbonate Formation.

Scleractinia coral skeleton formation occurs by a heterogeneous process of nucleation and growth of aragonite in which intraskeletal soluble organic matrix molecules, usually referred to as SOM, play a key role. Several studies have demonstrated that they influence the shape and polymorphic precipitation of calcium carbonate. However, the structural aspects that occur during the growth of aragonite have received less attention. In this research, we study the deposition of calcium carbonate on a model substrate, silicon, in the presence of SOM extracted from the skeleton of two coral species representative of different living habitats and colonization strategies, which we previously characterized. The study is performed mainly by grazing incidence X-ray diffraction with the support of Raman spectroscopy and electron and optical microscopies. The results show that SOM macromolecules once adsorbed on the substrate self-assembled in a layered structure and induced the oriented growth of calcite, inhibiting the formation of vaterite. Differently, when SOM macromolecules were dispersed in solution, they induced the deposition of amorphous calcium carbonate (ACC), still preserving a layered structure. The entity of these effects was species-dependent, in agreement with previous studies. In conclusion, we observed that in the setup required by the experimental procedure, the SOM from corals appears to present a 2D lamellar structure. This structure is preserved when the SOM interacts with ACC but is lost when the interaction occurs with calcite. This knowledge not only is completely new for coral biomineralization but also has strong relevance in the study of biomineralization on other organisms.

Exploring Coral Calcification by Calcium Carbonate Overgrowth Experiments

The Scleractinia coral biomineralization process is a representative example of a heterogeneous process of nucleation and growth of biogenic CaCO3 over a mineral phase. Indeed, even if the biomineralization process starts before settlement, the bulk formation of the skeleton takes place only when the larvae attach to a solid substrate, which can be Mg-calcite from coralline algae, and the following growth proceeds on the Mg-calcite surface of the formed baseplate of the planula. Despite this peculiarity and central role of the Mg-calcite substrate, the in vitro overgrowth of CaCO3 on single crystals of Mg-calcite, or calcite, in the presence of magnesium ions and the soluble organic matrix (SOM) extracted from coral skeletons has not been performed until now. In this study, the SOMs from Stylophora pistillata and Oculina patagonica skeletons were used in a set of overgrowth experiments. The overgrown CaCO3 was characterized by microscopic, diffractometric, and spectroscopic techniques. Our results showed that CaCO3 overgrowth in the presence of S. pistillata or O. patagonica SOM produces different effects. However, there appears to be a minor distinction between samples when magnesium ions are present in solution. Moreover, the Mg-calcite substrate appears to be a favorable substrate for the overgrowth of aragonite, differently from calcite. These observations fit with the observed settling of coral larvae on Mg-calcite-based substrates and with the in vivo observation that in the planula aragonite forms on first-formed Mg-calcite crystals. The overall results of this study highlight the importance of magnesium ions, either in the solution or in the substrate, in defining the shape, morphology, and polymorphism of biodeposited CaCO3. They also suggest a magnesium-dependent biological control on the deposition of coral skeletons.

The skeleton of Balanophyllia coral species suggests adaptive traits linked to the onset of mixotrophy

The diversity in the skeletal features of coral species is an outcome of their evolution, distribution and habitat. Here, we explored, from macro- to nano-scale, the skeletal structural and compositional characteristics of three coral species belonging to the genus Balanophyllia having different trophic strategies. The goal is to address whether the onset of mixotrophy influenced the skeletal features of B. elegans, B. regia, and B. europaea. The macroscale data suggest that the presence of symbiotic algae in B. europaea can lead to a surplus of energy input that increases its growth rate and skeletal bulk density, leading to larger and denser corals compared to the azooxanthellate ones, B. regia and B. elegans. The symbiosis would also explain the higher intra-skeletal organic matrix (OM) content, which is constituted by macromolecules promoting the calcification, in B. europaea compared to the azooxanthellate species. The characterization of the soluble OM also revealed differences between B. europaea and the azooxanthellate species, which may be linked to diverse macromolecular machineries responsible for skeletal biosynthesis and final morphology. Differently, the crystallographic features were homogenous among species, suggesting that the basic building blocks of skeletons remained a conserved trait in these related species, regardless of the trophic strategy. These results show changes in skeletal phenotype that could be triggered by the onset of mixotrophy, as a consequence of the symbiotic association, displaying remarkable plasticity of coral skeletons which repeatedly allowed this coral group to adapt to a range of changing environments throughout its geological history.

In Vitro Coral Biomineralization under Relevant Aragonite Supersaturation Conditions.

The biomineralization of corals occurs under conditions of high and low supersaturation with respect to aragonite, which corresponds to day- or night-time periods of their growth, respectively. Here, in vitro precipitation of aragonite in artificial seawater was investigated at a high supersaturation, allowing spontaneous nucleation and growth, as well as at low supersaturation conditions, which allowed only the crystal growth on the deliberately introduced aragonite seeds. In either chemical systems, soluble organic matrix (SOM) extracted from Balanophyllia europaea (light sensitive) or Leptopsammia pruvoti (light insensitive) was added. The analyses of the kinetic and thermodynamic data of aragonite precipitation and microscopic observations showed that, at high supersaturation, the SOMs increased the induction time, did not affect the growth rate and were incorporated within aggregates of nanoparticles. At low supersaturation, the SOMs affected the aggregation of overgrowing crystalline units and did not substantially change the growth rate. On the basis of the obtained results we can infer that at high supersaturation conditions the formation of nanoparticles, which is typically observed in the skeleton's early mineralization zone may occur, whereas at low supersaturation the overgrowth on prismatic seeds observed in the skeleton fiber zone is a predominant process. In conclusion, this research brings insight on coral skeletogenesis bridging physicochemical (supersaturation) and biological (role of SOM) models of coral biomineralization and provides a source of inspiration for the precipitation of composite materials under different conditions of supersaturation.

Formation and morphogenesis of a cuttlebone's aragonite biomineral structures for the common cuttlefish (Sepia officinalis) on the nanoscale: Revisited

This study describes and examines the structural and morphological properties of the hierarchically organized, aragonite cuttlebone forms for the common cuttlefish (Sepia officinalis, L.), including its main structural parts, the dorsal shield, and the chambers. Specifically, it complements the mechanism for the self-organized formation of aragonite, identifies the presence, and determines the role of soluble organic matrix (SOM) proteins in the morphogenesis of the cuttlebone’s biomineral structures on the nanoscale. The structure and morphology of the cuttlebone were examined using X-ray diffraction (XRD) and field-emission scanning electron microscopy (FESEM), and their thermal properties by thermo-gravimetric analysis (TGA) and differential thermal analysis (DTA). Proteins from the SOM were investigated using two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS), nano liquid chromatography tandem mass spectrometry (nano-LC ESI-MS) and Edman degradation. The results showed that the cuttlebone exhibited several diverse biomineral structures characterized by complex morphologies. Their formation is governed by the organic matrix, particularly proteins, which at the earliest stage of development provide templates for the initial extracellular nucleation of the aragonite nanocrystals. This is followed by a bottom-up morphogenesis, based on the nanoscale oriented aggregation and coalescence of primarily formed aragonite nanograins, which results in the hierarchically organized, nanostructured, aragonite forms. The molecular masses of the most pronounced SOM proteins from the dorsal shield were about 10, 15, 40 and 60kDa, while from the chambers they were 10, 20, 25, 30 and 45kDa. Peptide fragments corresponding to Sep7, Sep8, chitin synthase 1, ficoline-2, polyubiquitin and the ubiquitin carboxyl-terminal hydrolase 32-like protein were detected in the SOM, with these proteins having functional properties related to the biomineralization processes. In general, there are mostly acidic proteins present in alternatively glycosylated forms, which are common attributes of biomineralization-related proteins.

Calcium carbonate bio-precipitation in counter-diffusion systems using the soluble organic matrix from nacre and sea-urchin spine

The biomineralization process of nacre and sea-urchin spine occurs under the biological control of specific macromolecules in gelling environments through diverse mechanisms. In both cases, the formation of the crystalline phases – aragonite in nacre and magnesian calcite in sea-urchin spine – takes place through amorphous calcium carbonate precursor phases. Here, these biomineralization processes were investigated by means of counter-diffusion crystallization experiments using agarose highly viscous sols entrapping the soluble organic matrix (SOM) extracted from the two mentioned biominerals. The presence of these SOMs did not increase the supersaturation needed for precipitation, but narrowed the permitted supersaturations with respect to calcium and carbonate ions, when compared to those observed in experiments using synthetic polypeptides or SOM from corals. In the presence of SOMs and diffusing magnesium ions, the analyses of the precipitates suggested that crystallization proceeded through transient amorphous calcium carbonate phases. These favoured the crystallization of aragonite or calcite, according to the biomineral from which SOM was extracted, when a specific concentration was used. This study showed a control of SOMs on the mineralization process, which was more specific for nacre and sea-urchin spine than that for coral skeletons. It also validates the counter-diffusion system as a tool to investigate biomineralization processes in vitro .

Molecular Cloning and Characterization of First Organic Matrix Protein from Sclerites of Red Coral, Corallium rubrum

We report here for the first time the isolation and characterization of a protein from the organic matrix (OM) of the sclerites of the alcyonarian, Corallium rubrum. This protein named scleritin is one of the predominant proteins extracted from the EDTA-soluble fraction of the OM. The entire open reading frame (ORF) was obtained by comparing amino acid sequences from de novo mass spectrometry and Edman degradation with an expressed sequence tag library dataset of C. rubrum. Scleritin is a secreted basic phosphorylated protein which exhibits a short amino acid sequence of 135 amino acids and a signal peptide of 20 amino acids. From specific antibodies raised against peptide sequences of scleritin, we obtained immunolabeling of scleroblasts and OM of the sclerites which provides information on the biomineralization pathway in C. rubrum.

Fits and Misfits in Organic Matrix Analyses: Case of the Soluble Matrices of the Nacreous Layer of Pinctada margaritifera (Mollusca)

Mollusk shells, especially the nacre, are of commercial interest as well as palaeoenvironmental proxies. They are also investigated as biomaterials for medical purposes and biomimetics. Although the mineralogy is well-known and unique (aragonite tablets), the organic components are various. However, determination of the precise composition of the soluble organic matrix (SOM) of the nacreous layer is difficult. Among the range of possible techniques, 1D electrophoresis and High-performance liquid chromatography (HPLC) have previously been applied separately to differentiate pI and molecular weights. To date, no clear correlation has been established between the two parameters obtained in such conditions. Here, we report the use of preparative electrophoresis, coupled with HPLC, to determine the molecular weights of the pI fractions. The results are compared with 2D gel electrophoresis. It is shown that both methods have drawbacks and advantages, and are not redundant. The complexity of the composition of the nacreous tablet shown by scanning electron microscope (SEM) and Atomic Force Microscope (AFM) observations is also evidenced by electrophoresis and HPLC.

Specific organic matrix characteristics in skeletons of Corallium species

Corallium species have ecological, cultural and commercial importance and thus require tools to assist with their identification for both management and trade. The organic matrix (OM) of the skeletons of four Corallium species (C. rubrum, C. konojoi, C. secundum and C. elatius) was examined to provide insight into the biomineralization process and to develop a new tool of identification. The pattern of OM and the set of soluble organic matrix proteins (SOM) in the skeletons were examined by gel electrophoresis. Staining of cross-sections of skeletons showed a common cyclic, concentric pattern of OM during growth. Differences in molecular weight and isoelectric point were observed for proteins in the SOM in different Corallium species but not among different populations of Corallium rubrum. Immunolabeling with antibodies against the SOM of C. rubrum showed labeling of the OM of the three other Corallium species suggesting the presence of common epitopes.

The Skeletal Organic Matrix from Mediterranean Coral Balanophyllia europaea Influences Calcium Carbonate Precipitation

Scleractinian coral skeletons are made mainly of calcium carbonate in the form of aragonite. The mineral deposition occurs in a biological confined environment, but it is still a theme of discussion to what extent the calcification occurs under biological or environmental control. Hence, the shape, size and organization of skeletal crystals from the cellular level through the colony architecture, were attributed to factors as diverse as mineral supersaturation levels and organic mediation of crystal growth. The skeleton contains an intra-skeletal organic matrix (OM) of which only the water soluble component was chemically and physically characterized. In this work that OM from the skeleton of the Balanophyllia europaea, a solitary scleractinian coral endemic to the Mediterranean Sea, is studied in vitro with the aim of understanding its role in the mineralization of calcium carbonate. Mineralization of calcium carbonate was conducted by overgrowth experiments on coral skeleton and in calcium chloride solutions containing different ratios of water soluble and/or insoluble OM and of magnesium ions. The precipitates were characterized by diffractometric, spectroscopic and microscopic techniques. The results showed that both soluble and insoluble OM components influence calcium carbonate precipitation and that the effect is enhanced by their co-presence. The role of magnesium ions is also affected by the presence of the OM components. Thus, in vitro, OM influences calcium carbonate crystal morphology, aggregation and polymorphism as a function of its composition and of the content of magnesium ions in the precipitation media. This research, although does not resolve the controversy between environmental or biological control on the deposition of calcium carbonate in corals, sheds a light on the role of OM, which appears mediated by the presence of magnesium ions.

Electrochemical characterization of a bioceramic material: The shell of the Eastern oyster Crassostrea virginica

The shell of the Eastern oyster ( Crassostrea virginica) is composed of multiple incongruent mineralized layers. This bioceramic composite material was investigated to determine the effects of shell thickness, orientation and layer composition on its electrochemical behavior using electrochemical impedance spectroscopy, potentiodynamic polarization and scanning electron microscopy-energy dispersive spectroscopy. SEM-EDS analysis of the oyster shell revealed that the multilayered biocomposite material is composed of calcium carbonate (CaCO 3). EIS measurements in 3.5 wt.% NaCl indicated that the impedance of the whole oyster shell in the low frequency region exhibited high impedance values which exhibited a decreasing trend with increasing immersion time. In terms of overall shell thickness, limiting currents measured by potentiodynamic techniques through the shell were observed to increase when the outer layers of the shell were sequentially removed by grinding, thus decreasing the shell thickness. These limiting current values remained relatively constant when the inner layers of the shell were removed. The impedance values of the oyster shell material as measured by EIS were shown to decrease with decreasing shell thickness. These findings suggest that the prismatic (outermost) shell layer in combination with the soluble organic matrix between all shell layers may influence the ionic conductivity through the oyster shell.

Comparative analysis of the soluble organic matrix of axial skeleton and sclerites of Corallium rubrum: Insights for biomineralization

We analysed the soluble organic matrix (SOM) of two biominerals formed by the same organism but differing by their morphological characteristics: the axial skeleton and the sclerites of Corallium rubrum. The results of 1D SDS-PAGE electrophoresis show for the two biominerals that SOM proteins bands have similar apparent molecular weight but differ in quantity. Further analysis by 2D electrophoresis reveals each protein band as a line of spots with different isoelectric points. Our results suggest that each SOM protein band consists of a mix of proteins and/or one unique protein with post-translational modifications. By immunohistochemistry, we show that antibodies raised against the SOM of axial skeleton and sclerites label the SOM of the two biominerals but also label the insoluble organic matrix suggesting the presence of common epitopes between the two biominerals and the two organic fractions.

Relative influences of solution composition and presence of intracrystalline proteins on magnesium incorporation in calcium carbonate minerals: Insight into vital effects

[1] Biogenic calcites may contain considerable magnesium concentrations, significantly higher than those observed in inorganic calcites. Control of ion concentrations in the calcifying space by transport systems and properties of the organic matrix of mineralization are probably involved in the incorporation of high magnesium quantities in biogenic calcites, but their relative effects have never been quantified. In vitro precipitation experiments performed at different Mg/Ca ratios in the solution and in the presence of soluble organic matrix macromolecules (SOM) extracted from sea urchin tests and spines showed that, at a constant temperature, magnesium incorporation in the precipitated minerals was mainly dependent on the Mg/Ca ratio of the solution. However, a significant increase in magnesium incorporation was observed in the presence of SOM compared with control experiments. Furthermore, this effect was more pronounced with SOM extracted from the test, which was richer in magnesium than the spines. According to SEM observations, amorphous calcium carbonate was precipitated at high Mg/Casolution. The observed predominant effect of Mg/Casolution, probably mediated in vivo by ion transport to and from the calcifying space, was suggested to induce and stabilize a transient magnesium-rich amorphous phase essential to the formation of high magnesium calcites. Aspartic acid rich proteins, shown to be more abundant in the test than in the spine matrix, further stabilize this amorphous phase. The involvement of the organic matrix in this process can explain the observation that sympatric organisms or even different skeletal elements of the same individual present different skeletal magnesium concentrations.

Toward a Structure Determination Method for Biomineral-Associated Protein Using Combined Solid- State NMR and Computational Structure Prediction

Protein-biomineral interactions are paramount to materials production in biology, including the mineral phase of hard tissue. Unfortunately, the structure of biomineral-associated proteins cannot be determined by X-ray crystallography or solution nuclear magnetic resonance (NMR). Here we report a method for determining the structure of biomineral-associated proteins. The method combines solid-state NMR (ssNMR) and ssNMR-biased computational structure prediction. In addition, the algorithm is able to identify lattice geometries most compatible with ssNMR constraints, representing a quantitative, novel method for investigating crystal-face binding specificity. We use this method to determine most of the structure of human salivary statherin interacting with the mineral phase of tooth enamel. Computation and experiment converge on an ensemble of related structures and identify preferential binding at three crystal surfaces. The work represents a significant advance toward determining structure of biomineral-adsorbed protein using experimentally biased structure prediction. This method is generally applicable to proteins that can be chemically synthesized.

Colloid-chemical processes in the growth and design of the bio-inorganic aragonite structure in the scleractinian coral Cladocora caespitosa

This study describes the morphological properties and discusses the colloid-chemical mechanisms of the formation of hierarchically structured aragonite fibers in the exoskeleton structure of the Mediterranean zooxanthellate scleractinian coral Cladocora caespitosa. The study is based on a detailed structural and morphological examination of the coral exoskeleton and on a preliminary biochemical and molecular identification of the isolated soluble proteinaceus organic matrix. The biomineral structure was examined by X-ray diffraction (XRD), field-emission scanning electron microscopy (FESEM) and atomic-force microscopy (AFM), while the isolated protein organic constituents were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and mass spectrometry (MALDI-TOF-MS). The SDS–PAGE analysis of the soluble protein matrix showed three major protein bands at 15, 41, and 80 kDa. Based on the MALDI-TOF-MS analyses, the identified peptides tend to exhibit an acidic character. The results obtained confirm and complement the existing hypotheses relating to the significant role of the soluble acidic protein matrix and the biologically induced colloid-chemical processes in the phase formation and growth of scleractinian submicrometer fibrous aragonite units. It was also shown that the general strategy for the morphogenesis of fibrous structured aragonite lies in the nanoscale aggregation and subsequent coalescence processes that occur simultaneously. The subsequent morphological conversion of the initially formed submicrometer fibrous aragonite units into well-defined, micrometer-sized, prismatic facets in the skeletal structures of the corals is demonstrated.

N-linked oligosaccharide in MSP-1 and its implication for scallop calcification

We speculated the structure of the N-linked oligosaccharides enzymatically released from the organic matrix (OM) component in the foliated layer of Patinopecten yessoensis. The 80 kDa component of the soluble OM was detected by lectin blotting and was identified as MSP-1 using liquid chromatography/mass spectrometry (LC/MS/MS). LC/MS/MS analysis of the N-glycan liberated from MSP-1 detected a hybrid-type N-glycan, which contained sulfite and sialic acid at its terminus based on the characteristic Y ions. The data strongly imply that MSP-1, a sulfated OM glycoprotein, participates in molluscan biomineralization by creating a favorable environment for calcium ion uptake through sulfite acid and sialic acid. Further analyses of oligosaccharides linked to the OM components in wide variety of species and shell microstructures may definitely contribute in elucidation of molluscan biomineralization at the molecular level.

Isolation and Characterization of the N-linked Oligosaccharides in Nacrein from Pinctada fucata

We analyzed the structure of the N-linked oligosaccharides enzymatically liberated from the organic matrix (OM) component in the nacreous layer of Japanese pearl oyster: Pinctada fucata. The lectin-blot analysis of the soluble OM after separation by SDS-PAGE, four components, with sizes of approximately 55 kDa, 35 kDa, 25 kDa, and 21 kDa were detected with GNA lectin, which recognized terminal mannose of high mannose and hybrid types of N-glycan. The 55-kDa component of the soluble OM detected by lectin blotting was identified as nacrein by using liquid chromatography/mass spectrometry (LC/MS). LC/MS analysis of the N-glycan liberated from nacrein detected a hybrid-type N-glycan, which contained sulfite and sialic acid at its terminus. The data strongly imply that nacrein, a sulfated OM glycoprotein, participates in molluscan biomineralization by creating a favorable environment for calcium ion uptake through sulfonic acid and sialic acid.

In Vitro Study of Magnesium‐Calcite Biomineralization in the Skeletal Materials of the Seastar Pisaster giganteus

The mechanisms of formation of biogenic magnesium-rich calcite remain an enigma. Here we present ultrastructural and compositional details of ossicles from the seastar Pisaster giganteus (Echinodermata, Asteroidea). Powder X-ray diffraction, infrared spectroscopy and elemental analyses confirm that the ossicles are composed of magnesium-rich calcite, whilst also containing about 0.01 % (w/w) of soluble organic matrix (SOM) as an intracrystalline component. Amino acid analysis and N-terminal sequencing revealed that this mixture of intracrystalline macromolecules consists predominantly of glycine-rich polypeptides. In vitro calcium carbonate precipitation experiments indicate that the SOM accelerates the conversion of amorphous calcium carbonate (ACC) into its final crystalline product. From this observation and from the discovery of ACC in other closely related taxa, it is suggested that substitution of magnesium into the calcite lattice through a transient precursor phase may be a universal phenomenon prevalent across the phylum echinodermata.

Soluble organic matrix of two Scleractinian corals: Partial and comparative analysis

This study is a biochemical and molecular analysis of the soluble organic matrix (SOM) of two Scleractinian corals differing in their morphological characteristics: Stylophora pistillata, a branched robust coral and Pavona cactus, a leafy complex coral. Soluble organic matrix of both coral species were shown to contain high amounts of potentially acidic amino acids and glycine. However, proportions of glycosaminoglycans and SDS-PAGE analyses of soluble organic matrix proteins were very different. Three proteins of S. pistillata and at least five proteins of P. cactus were detected by silver staining, some of them being able to bind calcium. Internal peptide sequences of two matrix proteins (one from each species) were obtained. One sequence of S. pistillata is unusual because it contains a long poly-aspartate domain, as described in proteins belonging to the calsequestrin family and in proteins from molluscan species. This domain suggests an essential role for this protein in the control of mineralization.

Characterization of proteins from the matrix of spicules from the alcyonarian, Lobophytum crassum

The organic matrix of spicules of the alcyonarian coral, Lobophytum crassum, was studied to investigate its molecular characteristics and functional properties. The shape of the spicules was identified using scanning electron microscopy. The soluble organic matrix comprised 0.03% of the spicule weight. The SDS–PAGE analysis of the preparation showed four protein bands with apparent molecular weights of 37, 48, 67 and 102 kDa. The 67- and 102-kDa proteins appeared to be calcium binding proteins, detected as radioactive bands by 45Ca autoradiography. The 67-kDa protein appears to be glycosylated. The N-terminal amino acid sequence of the 67 kDa was determined; 7 of 20 residues were acidic. A database search for homologous proteins did not give a clear indication of the function of the 67-kDa protein. The isolated organic matrix possesses carbonic anhydrase activity which functions in calcium carbonate crystal formation, indicating that organic matrix is not only structural protein but also a catalyst. An interpretation of these results is that the spicule of alcyonarian corals has a proteinaceous organic matrix related to the calcification process.