The mollusc shell is a hard tissue consisting of calcium carbonate and organic matrices. The organic matrices are believed to play important roles in shell formation. In the present study, we extracted and purified a novel matrix protein, named Prismalin-14, from the acid-insoluble fraction of the prismatic layer of the shell of the Japanese pearl oyster (Pinctada fucata), and determined its whole amino acid sequence by a combination of amino acid sequence analysis and MS analysis of the intact protein and its enzymic digests. Prismalin-14 consisted of 105 amino acid residues, including PIYR repeats, a Gly/Tyr-rich region and N- and C-terminal Asp-rich regions. Prismalin-14 showed inhibitory activity on calcium carbonate precipitation and calcium-binding activity in vitro. The scanning electron microscopy images revealed that Prismalin-14 affected the crystallization of calcium carbonate in vitro. A cDNA encoding Prismalin-14 was cloned and its expression was analysed. The amino acid sequence deduced from the nucleotide sequence of Prismalin-14 cDNA was identical with that determined by peptide sequencing. Northern-blot analysis showed that a Prismalin-14 mRNA was expressed only at the mantle edge. In situ hybridization demonstrated that a Prismalin-14 mRNA was expressed strongly in the inner side of the outer fold of the mantle. These results suggest that Prismalin-14 is a framework protein that plays an important role in the regulation of calcification of the prismatic layer of the shell.
In the biomineralization processes, proteins are thought to control the polymorphism and morphology of the crystals by forming complexes of structural and mineral‐associated proteins. To identify such proteins, we have searched for proteins that may form high‐molecular‐weight (HMW) aggregates in the matrix of fish otoliths that have aragonite and vaterite as their crystal polymorphs. By screening a cDNA library of the trout inner ear using an antiserum raised against whole otolith matrix, a novel protein, named otolith matrix macromolecule‐64 (OMM‐64), was identified. The protein was found to have a molecular mass of 64 kDa, and to contain two tandem repeats and a Glu‐rich region. The structure of the protein and that of its DNA are similar to those of starmaker, a protein involved in the polymorphism control in the zebrafish otoliths [Söllner C, Burghammer M, Busch‐Nentwich E, Berger J, Schwarz H, Riekel C & Nicolson T (2003) Science302, 282–286]. 45Ca overlay analysis revealed that the Glu‐rich region has calcium‐binding activity. Combined analysis by western blotting and deglycosylation suggested that OMM‐64 is present in an HMW aggregate with heparan sulfate chains. Histological observations revealed that OMM‐64 is expressed specifically in otolith matrix‐producing cells and deposited onto the otolith. Moreover, the HMW aggregate binds to the inner ear‐specific short‐chain collagen otolin‐1, and the resulting complex forms ring‐like structures in the otolith matrix. Overall, OMM‐64, by forming a calcium‐binding aggregate that binds to otolin‐1 and forming matrix protein architectures, may be involved in the control of crystal morphology during otolith biomineralization.
We show that the high-molecular weight (HMW) protein aggregate of fish otolith matrix is involved in the regulation of crystal polymorphs during otolith biomineralization. Teleost fish otoliths are biominerals that contain calcium carbonate aragonite or vaterite crystals. In a previous study, we identified a novel protein named otolith matrix macromolecule-64 (OMM-64) within the otolith organic matrix. In addition, we revealed that the HMW aggregate of otolith matrix architecture was comprised of OMM-64, in which inner-ear-specific short-chain collagen otolin-1 was also contained. By using an in vitro crystallization system in the absence of magnesium ions here, we show that native OMM-64 induced vaterite crystals, whereas native otolion-1 induced calcite crystals. However, the aggregate complex induced the aragonite polymorph in the same condition. The present data suggest that separation and structural and functional analyses of each matrix protein in the aggregate are absolutely imperative, but functional examination of the protein complex itself is equally important in clarifying polymorph control of biomineralization.
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