Abstract: The protein-induced chemical structure in calcified organisms is an important factor contributing to their growth, but until now no direct evidence has been reported. Hence, we demonstrate a new analytical technique that combines an FTIR spectrometer and an apertureless-type near-field microscope with high spatial resolution. The complete structural composition of the biomineralized structure of marine organisms has been measured using this technique. The results show that �OH is distributed over the center of the mineral phase of small calcite, which was formed from interactions of matrix proteins.

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Abstract: Acidic proteins are generally thought to control mineral formation and growth. Thus, characterization of acidic proteins in the insoluble organic matrix is an important first step toward linking function to individual proteins in alcyonarian coral. Analysis of proteinaceous components in the soluble and insoluble matrix fractions of Sinularia polydactyla indicates that aspartic acid composes about 61% of the insoluble and 29% of the soluble matrix fractions. Using an in vitro assay, we show that matrix proteins induced formation of amorphous CaCO3 precipitates prior to their transformation into the calcitic crystalline form. The crystalline form of CaCO3 in the sclerites was also identified by X-ray diffraction, revealing calcitic polymorphisms with a strong (104) reflection. The structure of alcyonarian organic matrices containing aspartate-rich proteins and polysaccharides was assessed by Fourier transform infrared spectroscopy (FT-IR). Calcium-binding analysis of components in the insoluble matrix fraction indicated that a protein of 109 kDa can bind Ca2+, which is important for sclerite formation. An assay for carbonic anhydrase (CA) enzyme, which is thought to play an important role in the process of bio-calcification revealed novel activity. These results strongly suggest that the aspartic acid�rich proteins within the insoluble matrix of alcyonarians play a key role in biomineralization regulation.

Abstract: Understanding the functional properties of endoskeletal proteins in the calcification processes of soft corals is essential. However, separation of proteins from soft corals is difficult due to contamination by soft tissues and the high sensitivity of soft tissues to handling. We have resolved this problem and established a simple and effective method�electroelution treatment�for purifying proteins from soft corals. Here we applied this newly developed technique to successfully identify four proteins (MPL-1, MPL-2, MPL-3 and MPL-4) from the endoskeletal sclerites of soft coral, Lobophytum crassum as a model. Following this method, we identified a carbonic anhydrase (CA) domain in a soft coral; CA is a key enzyme in living organisms. We found that two CA proteins, which were purified by electroelution, could control the morphology of the CaCO3 crystals, and one of these is potentially involved in the process of biocalcification. We report here a single protein (MPL-2), which has both calcium-binding and CA activities and is responsible for CaCO3 nucleation and crystal growth. The sequence of protein MPL-2 was subjected to bioinformatics analysis involving identification of similarities to other animals� protein. The function of proteins during bio-calcification was also studied by simulating the nucleation and growth of calcium carbonates in vitro. After precipitation of CaCO3 in the experimental design, the obtained crystals were characterized by X-Ray Diffraction (XRD). These findings suggest that the proteins, which were purified from the calcified sclerites, can control the morphology of CaCO3 crystals and are potentially involved in the process of bio-calcification.

Abstract: Carbonic anhydrase (CA) is a key enzyme in the chemical reaction of living organisms and has been found to be associated with calcification in a number of invertebrates including calcareous sponges, but until now no direct evidence has been advanced to show CA activity in alcyonarian corals. However, it is essential to understand the role of CA in the process of biocalcification in alcyonarian. Here we describe the novel activity of CA and its relationship to the formation of calcified hard tissues in alcyonarian coral, Lobophytum crassum. We find that two CA proteins, which were partially purified by electro-elution treatment, can control the morphology of CaCO(3) crystals and one of them is potentially involved in the process of biocalcification. Previously, we isolated CA from the total extract of alcyonarian, and further, we report here a single protein, which has both calcium-binding and CA activities and is responsible for CaCO(3) nucleation and crystal growth. This matrix protein inhibited the precipitation of CaCO(3) from a saturated solution containing CaCl(2) and NaHCO(3), indicating that it can act as a negative regulator for calcification in the sclerites of alcyonarians. The effect of an inhibitor on the enzyme activity was also examined. These findings strongly support the idea that carbonic anhydrase domain in alcyonarian is involved in the calcification process. Our observations strongly suggest that the matrix protein in alcyonarian coral is not only a structural protein but also a catalyst.

Abstract: Soft corals contain small spicules of calcium carbonate called �sclerites�, which are biomineralized structures composed of an organic matrix and a mineral fraction. Since informations concerning the structure of sclerites, their crystallization properties and mineral composition are scarce, we have studied these parameters in the alcyonarian, Sinularia polydactyla. In the first step, we have determined the structure and shape of the sclerites. Then, we have identified the polymorphs of calcium carbonate both by X-ray diffraction (XRD) and Raman microprobe analysis. A mineral phase in the sclerites was identified as calcite, as revealed by the calcite (104) reflection. An electron probe microanalyzer was used to analyze the element on the sclerite. This analysis indicates that the sclerite has both Ca and Mg bearing calcite, in which Mg showed a relatively high concentration (5-10mol%). Analysis of proteinaceous components of calcified sclerites reveals the significance of protein composition to calcification. The function of matrix proteins during bio-calcification was studied by simulating the nucleation and growth of calcium carbonates in vitro. After precipitation of CaCO3 in the experimental design, the obtained crystals were characterized by scanning electron microscope (SEM) and XRD. The matrix proteins, which were purified from the calcified sclerites, can control the morphology of CaCO3 crystals and are potentially involved in the process of bio-calcification.

Abstract: Two species of alcyonarian corals, Lobophytum crassum and Sinularia polydactyla, are closely related to each other. It is reported that the calcified organic substances in the skeletons of both contain a protein-polysaccharide complex playing a key role in the regulation of biocalcification. However, information on the matrix proteins of endoskeletal sclerite has been lacking. Hence we studied the proteinaceous organic matrices of sclerites for both species, to analyze the sequences and the functional properties of the proteins present. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the preparations showed four bands of proteins with apparent molecular masses of 102, 67, 48, and 37 kDa for L. crassum and seven bands of 109, 83, 70, 63, 41, 30, and 22 kDa for S. polydactyla. A major protein band of about 67 kDa in L. crassum and two bands of proteins of about 70 and 63 kDa in S. polydactyla yielded N-terminal amino acid sequences. Periodic acid-Schiff staining indicated that the 67-kDa protein in L. crassum, and 83- and 63-kDa proteins in S. polydactyla were glycosylated. For detection of calcium binding proteins, a Ca(2+) overlay analysis was conducted in the extract via (45)Ca autoradiography. The 102- and 67-kDa calcium binding proteins in L. crassum, and the 109- and 63-kDa Ca(2+) binding proteins in S. polydactyla were found to be radioactive. An assay for carbonic anhydrase (CA), which is thought to play an important role in the process of calcification, revealed specific activities. Newly derived protein sequences were subjected to standard sequence analysis involving identification of similarities to other proteins in databases. The significantly different protein expressions and compositional analysis of sequences between two species were demonstrated.

Abstract: The mesoglea of alcyonarians is occupied by an abundance of minute calcitic sclerites. The sclerites of the alcyonarian Lobophytum crassum contain a water-soluble organic matrix comprising 0.48% of the sclerite weight and a water-insoluble fraction comprising 1.15% of the sclerite weight. Analysis of proteinaceous components in the soluble fraction shows a particularly high content of aspartic acid, followed by alanine, glycine, and glutamate. Aspartic acid, glycine, alanine, and glutamate are the most abundant residues in the insoluble fraction. In both cases, the fractions show the highest concentration of aspartic acid from the total proteins. In an in vitro assay, we show that the matrix proteins extracted from the calcitic sclerites induce the formation of amorphous calcium carbonate prior to its transformation into the calcitic crystalline form. We also show scanning electron micrographs of the rhombohedral calcite crystals used as template, the protein imprinted with these crystals. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of both matrices shows the protein fractions at 67 and 48 kDa. The soluble matrix shows two additional faint bands. Both fractions stain for a carbohydrate at 67 kDa, indicating a glycoprotein at this molecular weight. A newly derived protein sequence was subjected to bioinformatics analysis involving identification of similarities to other acidic proteins. The identification of these proteins in alcyonarian endoskeletal sclerites emphasizes the fundamental importance of such acidic proteins and sheds more light on the functions of these proteins in the processes of biocalcification.

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Abstract: Calcified organic substances in the skeleton contain a protein-polysaccharide complex taking a key role in the regulation of bio-calcification. However, information concerning the matrix proteins in alcyonarian and their effect on calcification process is still unknown. For this reason, we have studied the organic matrix of endoskeletal spicules from the alcyonarian coral, Synularia polydactyla, to analyze the proteins with their sequences and investigate the functional properties by a molecular approach. The separated spicules from the colony were identified by scanning electron microscope (SEM). The soluble organic matrix comprised 0.04% of spicule weight. By recording decline of pH in the experimental design, the inhibitory effect of the matrix on CaCO3 precipitation was revealed. Prior to electrophoresis, our analysis of proteins extracted from the soluble organic matrix of the spicules revealed an abundance of proteins in molecular weight. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the preparations showed seven bands of proteins with an apparent molecular mass of 109, 83, 70, 63, 41, 30 and 22 kDa. The proteins were electrophoresed on Tricine-SDS-PAGE after electro-elution treatment, and then transferred to polyvinylidene difluoride (PVDF) membranes and their N-termini were sequenced. Two bands of proteins of about 70 and 63 kDa successfully underwent N-terminal amino acid sequencing. For the detection of calcium binding proteins, a Ca2+ overlay analysis was conducted on the extract by 45Ca autoradiography. The 109 and 63 kDa calcium binding proteins were found to be radioactive. Periodic acid schiff staining indicated that 83 and 63 kDa proteins were glycosylated. An assay for carbonic anhydrase, which is thought to play an important role in the process of calcification revealed low level of the activity. These findings suggest that the endoskeletal spicules of alcyonarian corals have protein-rich organic matrices, which might be related to the calcification process.

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Abstract: Enzymes are protein chemicals, which is needed for every chemical action, and reaction that occurs in living organisms. Since the corals are among the most biologically diverse and ecologically important ecosystems on the planet, activities of enzymes with their molecular mechanisms of mineralization process are essential to understand. It is reported that the calcified organic substances in the skeleton of corals contain a carbonic anhydrase (CA) enzyme, however, purification and detail functions in the regulation of biocalcification has been lacking. In this review, we describe in detail about the CA activity in the process of biocalcification in corals and homology the sequences in databases for supporting active site structure with other animals CAs. Previously, CA enzyme has been investigated for both soft and stony corals. We report here a single protein (MPL-2) in soft coral, Lobophytum crassum, which has both calcium-binding and CA activities and is responsible for CaCO3 nucleation and crystal growth. The effect of an inhibitor on the enzyme activity of this protein was also examined. This newly derived protein sequence (MPL-2) was subjected to bioinformatics analysis involving identification of similarities to other animals CAs. This analysis result showed significant similarities with a mollusk shell CA nacrein (93.3%) and human CA 2 (72%). In stony coral, two CA cDNA sequences from the planula larvae of the Hawaiian scleractinian coral Fungia scutaria have been reported. Bioinformatics analyses of the two homologs showed that the sequences are unusually short and are missing some residues that support active site structure in other CAs. Studies in other calcifying cnidarians have identified membrane-associated CAs as functioning in calcification, and therefore the two larval CAs could play a role in the onset of calcification during metamorphosis. A carbonic anhydrase from the azooxanthellate stony coral Tubastrea aurea has also been reported. The activity of this enzyme has been measured both in the tissues and in the organic matrix extracted from the skeleton. The results indicate that organic matrix proteins, which are synthesized by the calcifying tissues, are not only structural proteins, but they also play a crucial catalytic role by eliminating the kinetic barrier to interconversion of inorganic carbon at the calcification site. Upon review, we have found that some important functions such as CA has been described in many tissues but its presence in extracellular calcified structures that this enzyme could also play an important role during the precipitation step of the mineral has not yet been reported. This review suggests that further study is needed for understanding in detail functions of the CA with the molecular mechanisms of mineralization process of corals.

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