Connective Tissue Research, 50:285–293, 2009 Copyright c  Informa UK Ltd. ISSN: 0300-8207 print / 1607-8438 online DOI: 10.1080/03008200802714933 In Vitro Regulation of CaCO 3 Crystal Growth by the Highly Acidic Proteins of Calcitic Sclerites in Soft Coral, Sinularia Polydactyla M. Azizur Rahman and Tamotsu Oomori Department of Chemistry, Faculty of Science, University of the Ryukyus, Okinawa, Japan Acidic proteins are generally thought to control mineral formation and growth in biocalcification. Analysis of proteinaceous components in the soluble and insoluble matrix fractions of sclerites in Sinularia polydactyla indicates that aspartic acid composes about 60% of the insoluble and 29% of the soluble matrix fractions. We previously analyzed aspartic acids in the matrix fractions (insoluble = 17 mol%; soluble = 38 mol%) of sclerites from a different type of soft coral, Lobophytum crassum, which showed comparatively lower aspartic acid-rich proteins than S. polydactyla. Thus, characterization of highly acidic proteins in the organic matrix of present species is an important first step toward linking function to individual proteins in soft coral. Here, we show that aspartic-acid rich proteins can control the CaCO 3 polymorph in vitro. The CaCO 3 precipitates in vitro in the presence of aspartic acid-rich proteins and 50 mM Mg 2+ was verified by Raman microprobe analysis. The matrix proteins of sclerites demonstrated that the aspartic-acid rich domain is crucial for the calcite precipitation in soft corals. The crystalline form of CaCO 3 in the presence of aspartic acid-rich proteins in vitro was identified by X- ray diffraction and, revealed calcitic polymorphisms with a strong (104) reflection. The structure of soft coral organic matrices con- taining aspartate-rich proteins and polysaccharides was assessed by Fourier transform infrared spectroscopy. These results strongly suggest that the aspartic acid-rich proteins within the organic matrix of soft corals play a key role in biomineralization regulation. Keywords Biomineralization, Calcium Carbonate, Calcite, Crystal Growth, Proteins, Sclerites, Soft Coral INTRODUCTION Soft corals form an important part of the coral ecosystem, and the species Sinularia polydactyla is the most abundant one Received 27 June 2008; Revised 22 December 2008; Accepted 22 December 2008. Address correspondence to Dr. M. Azizur Rahman, Department of Chemistry, University of the Ryukyus, 1 Senbaru Nishihara- cho, Okinawa 903-0213, Japan. E-mail: g083001@sci.u-ryukyu.ac.jp; azizur31@yahoo.com in the sea. Soft corals occur commonly in all reef habitats, and they provide economic and environmental benefits. The mechanism by which biomineralization occurs in soft coral sclerites remains unknown. Soft coral sclerites are generally composed of calcium carbonate in an organic matrix. The organic matrix is formed prior to mineralization, and it has been suggested that some components of the matrix protein may serve as a template for mineral deposition [1]. Recent reports have focused on the characterization of proteins in the soluble matrix of soft coral sclerites [2–7]. However, finding of aspartic acid–rich proteins in insoluble organic matrix and in higher concentration than soluble organic matrix was not reported, though it plays an important role in the formation of corals. Despite all the information available concerning stony corals, molluscans, calcareous sponges, and other matrices [8–15], very little is known regarding matrix components of soft corals. There are two important features of the biomineralization. First, a relatively inert structural frame is built from insoluble macromolecules (hydrophobic proteins, chitin). Second, acidic proteins (rich in aspartic acid and often in association with sulfated polysaccharides) are assembled on the framework [16]. As matrices play a key role in calcification, it remains important to better understand how these matrix fractions are formed. This can be accomplished by determining the mineral com- position through general morphological and biomineralization approaches. One of the most remarkable characteristics of the calcified tissues formed by many different phyla is the unusual structure of the combination of very acidic proteins and glycoproteins [9]. Acidic amino acids distributed throughout the protein chain may provide the binding sites for cations due to the presence of a negatively charged free carboxyl group (COO − ) on each acidic residue. The present study has revealed that the calcitic sclerites of Sinularia polydactyla contain water soluble and insoluble organic matrices. The insoluble matrix undoubtedly corresponds to the intercrystalline matrix observed after decalcification of thin sections of sclerites [17]. The soluble matrix may be present as an intracrystalline matrix as suggested in mollusk shells [18]. 285 Connect Tissue Res Downloaded from informahealthcare.com by Galter Health Sciences Library For personal use only.