Analyses of the Nanostructure of Calcium Carbonate Composite in Biominerals of Pinctada fucata

Abstract Background: The mollusk shells present distinctive microstructures that are formed by small amounts of organic matrices controlling the crystal growth of calcium carbonate. These microstructures show superior mechanical properties such as strength or flexibility. The shell of Pinctada fucata has the prismatic layer consisting of prisms of single calcite crystals. These crystals contain small-angle grain boundaries caused by a dense intracrystalline organic matrix network to improve mechanical strength. Previously, we identified chitin and chitinolytic enzymes as components of this intracrystalline organic matrix. In this study, we analyzed the function of those organic matrices in calcium carbonate crystallization by in vitro and in vivo experiments.Results: We analyzed calcites synthesized in chitin gel with or without chitinolytic enzymes by using transmission electron microscope (TEM) and atom probe tomography (APT). TEM observations showed that grain boundary was more induced as concentration of chitinolytic enzymes increased and thus, chitin became thinner. In an optimal concentration of chitinolytic enzymes, small-angle grain boundaries were observed. APT analysis showed that ion clusters derived from chitin were detected. In order to clarify the importance of chitinolytic enzymes on the formation of the prismatic layer in vivo , we performed the experiment in which chitinase inhibitor was injected into a living Pinctada fucata and then analyzed the change of mechanical properties of the prismatic layer. The hardness and elastic modulus increased after injection of chitinase inhibitor. Electron back scattered diffraction (EBSD) mapping data showed that the spread of crystal orientations in whole single crystal also increased by the effect of inhibitor injections.Conclusion: Our results suggested that chitinolytic enzymes may function cooperatively with chitin to regulate the crystal growth and mechanical properties of the prismatic layer, and chitinolytic enzymes are essential for the formation of the normal prismatic layer of P. fucata.

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Hemocytes participate in calcium carbonate crystal formation, transportation and shell regeneration in the pearl oyster Pinctada fucata

Fish & Shellfish Immunology ◽

10.1016/j.fsi.2016.02.027 ◽

2016 ◽

Vol 51 ◽

pp. 263-270 ◽

Cited By ~ 37

Author(s):

Shiguo Li ◽

Yangjia Liu ◽

Chuang Liu ◽

Jingliang Huang ◽

Guilan Zheng ◽

...

Keyword(s):

Calcium Carbonate ◽

Pearl Oyster ◽

Pinctada Fucata ◽

Crystal Formation ◽

Shell Regeneration

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Amorphous Calcium Carbonate Precipitation by Cellular Biomineralization in Mantle Cell Cultures of Pinctada fucata

PLoS ONE ◽

10.1371/journal.pone.0113150 ◽

2014 ◽

Vol 9 (11) ◽

pp. e113150 ◽

Cited By ~ 31

Author(s):

Liang Xiang ◽

Wei Kong ◽

Jingtan Su ◽

Jian Liang ◽

Guiyou Zhang ◽

...

Keyword(s):

Calcium Carbonate ◽

Cell Cultures ◽

Pinctada Fucata ◽

Carbonate Precipitation ◽

Amorphous Calcium Carbonate ◽

Calcium Carbonate Precipitation ◽

Mantle Cell

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Transcriptional regulation of the matrix protein Shematrin-2 during shell formation in pearl oyster

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.005281 ◽

2018 ◽

Vol 293 (46) ◽

pp. 17803-17816 ◽

Cited By ~ 3

Author(s):

Yan Chen ◽

Jing Gao ◽

Jun Xie ◽

Jian Liang ◽

Guilan Zheng ◽

...

Keyword(s):

Transcriptional Regulation ◽

Transcription Factors ◽

Calcium Carbonate ◽

Matrix Protein ◽

Pearl Oyster ◽

Pinctada Fucata ◽

Functional Assay ◽

Shell Formation ◽

Molluscan Shell ◽

The Matrix

The molluscan shell is a fascinating biomineral consisting of a highly organized calcium carbonate composite. Biomineralization is elaborately controlled and involves several macromolecules, especially matrix proteins, but little is known about the regulatory mechanisms. The matrix protein Shematrin-2, expression of which peaks in the mantle tissues and in the shell components of the pearl oyster Pinctada fucata, has been suggested to be a key participant in biomineralization. Here, we expressed and purified Shematrin-2 from P. fucata and explored its function and transcriptional regulation. An in vitro functional assay revealed that Shematrin-2 binds the calcite, aragonite, and chitin components of the shell, decreases the rate of calcium carbonate deposition, and changes the morphology of the deposited crystal in the calcite crystallization system. Furthermore, we cloned the Shematrin-2 gene promoter, and analysis of its sequence revealed putative binding sites for the transcription factors CCAAT enhancer–binding proteins (Pf-C/EBPs) and nuclear factor-Y (NF-Y). Using transient co-transfection and reporter gene assays, we found that cloned and recombinantly expressed Pf-C/EBP-A and Pf-C/EBP-B greatly and dose-dependently up-regulate the promoter activity of the Shematrin-2 gene. Importantly, Pf-C/EBP-A and Pf-C/EBP-B knockdowns decreased Shematrin-2 gene expression and induced changes in the inner-surface structures in prismatic layers that were similar to those of antibody-based Shematrin-2 inhibition. Altogether, our data reveal that the transcription factors Pf-C/EBP-A and Pf-C/EBP-B up-regulate the expression of the matrix protein Shematrin-2 during shell formation in P. fucata, improving our understanding of the transcriptional regulation of molluscan shell development at the molecular level.

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Identification of methionine -rich insoluble proteins in the shell of the pearl oyster, Pinctada fucata

Scientific Reports ◽

10.1038/s41598-020-75444-4 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Hiroyuki Kintsu ◽

Ryo Nishimura ◽

Lumi Negishi ◽

Isao Kuriyama ◽

Yasushi Tsuchihashi ◽

...

Keyword(s):

Calcium Carbonate ◽

Pearl Oyster ◽

Pinctada Fucata ◽

Nacreous Layer ◽

Solid Complex ◽

Prismatic Layer ◽

Initial Stage ◽

Organic Matrices ◽

Aggregation Activity ◽

Main Components

Abstract The molluscan shell is a biomineral that comprises calcium carbonate and organic matrices controlling the crystal growth of calcium carbonate. The main components of organic matrices are insoluble chitin and proteins. Various kinds of proteins have been identified by solubilizing them with reagents, such as acid or detergent. However, insoluble proteins remained due to the formation of a solid complex with chitin. Herein, we identified these proteins from the nacreous layer, prismatic layer, and hinge ligament of Pinctada fucata using mercaptoethanol and trypsin. Most identified proteins contained a methionine-rich region in common. We focused on one of these proteins, NU-5, to examine the function in shell formation. Gene expression analysis of NU-5 showed that NU-5 was highly expressed in the mantle, and a knockdown of NU-5 prevented the formation of aragonite tablets in the nacre, which suggested that NU-5 was required for nacre formation. Dynamic light scattering and circular dichroism revealed that recombinant NU-5 had aggregation activity and changed its secondary structure in the presence of calcium ions. These findings suggest that insoluble proteins containing methionine-rich regions may be important for scaffold formation, which is an initial stage of biomineral formation.

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Calcium carbonate mineralization mediated by in vitro cultured mantle cells from Pinctada fucata

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2015.06.057 ◽

2015 ◽

Vol 463 (4) ◽

pp. 1053-1058 ◽

Cited By ~ 5

Author(s):

Wei Kong ◽

Shiguo Li ◽

Liang Xiang ◽

Liping Xie ◽

Rongqing Zhang

Keyword(s):

Calcium Carbonate ◽

Pinctada Fucata

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Characterization of Prismalin-14, a novel matrix protein from the prismatic layer of the Japanese pearl oyster (Pinctada fucata)

Biochemical Journal ◽

10.1042/bj20040319 ◽

2004 ◽

Vol 382 (1) ◽

pp. 205-213 ◽

Cited By ~ 141

Author(s):

Michio SUZUKI ◽

Emi MURAYAMA ◽

Hirotaka INOUE ◽

Noriaki OZAKI ◽

Hidekazu TOHSE ◽

...

Keyword(s):

Amino Acid ◽

Calcium Carbonate ◽

Amino Acid Sequence ◽

Matrix Protein ◽

Pearl Oyster ◽

Pinctada Fucata ◽

Intact Protein ◽

Prismatic Layer ◽

Organic Matrices

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.

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Functional analyses of chitinolytic enzymes in the formation of calcite prisms in Pinctada fucata.

10.21203/rs.3.rs-22263/v1 ◽

2020 ◽

Author(s):

Hiroyuki Kintsu ◽

Alberto Pérez-Huerta ◽

Shigeru Ohtsuka ◽

Taiga Okumura ◽

Shinsuke Ifuku ◽

...

Keyword(s):

Mechanical Properties ◽

Crystal Growth ◽

Calcium Carbonate ◽

Small Angle ◽

Organic Matrix ◽

Pinctada Fucata ◽

Prismatic Layer ◽

Chitinolytic Enzymes ◽

Organic Matrices

Abstract Background The mollusk shells present distinctive microstructures that are formed by small amounts of organic matrices controlling the crystal growth of calcium carbonate. These microstructures show superior mechanical properties such as strength or flexibility. The shell of Pinctada fucata has the prismatic layer consisting of prisms of single calcite crystals. These crystals contain small angle grain boundaries caused by a dense intracrystalline organic matrix network to improve mechanical strength. Previously, we identified chitin and chitinolytic enzymes as components of this intracrystalline organic matrix. In this study, we analyzed the function of those organic matrices in calcium carbonate crystallization by in vitro and in vivo experiments. Results We analyzed calcites synthesized in chitin gel with or without chitinolytic enzymes by using transmission electron microscope (TEM) and atom probe tomography (APT). TEM observations showed that grain boundary was more induced as concentration of chitinolytic enzymes increased and thus, chitin became thinner. In an optimal concentration of chitinolytic enzymes, small angle grain boundaries were observed. APT analysis showed that ion clusters derived from chitin were detected. In order to clarify the importance of chitinolytic enzymes on the formation of the prismatic layer in vivo, we performed the experiment in which chitinase inhibitor was injected into a living Pinctada fucata and then analyzed the change of mechanical properties of the prismatic layer. The hardness and elastic modulus increased after injection of chitinase inhibitor. Electron back scattered diffraction (EBSD) mapping data showed that the spread of crystal orientations in whole single crystal also increased by the effect of inhibitor injections. Conclusion Our results suggested that chitinolytic enzymes may function cooperatively with chitin to regulate the crystal growth and mechanical properties of the prismatic layer, and chitinolytic enzymes are essential for the formation of the normal prismatic layer of P. fucata.

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