Functional analyses of chitinolytic enzymes in the formation of calcite prisms inPinctada fucata

2019 ◽  
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.

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

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.

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

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.

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

2004 ◽  
Vol 382 (1) ◽  
pp. 205-213 ◽  
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.

Crystal defects induced by chitin and chitinolytic enzymes in the prismatic layer of Pinctada fucata

2017 ◽  
Vol 489 (2) ◽  
pp. 89-95 ◽  
Author(s):  
Hiroyuki Kintsu ◽  
Taiga Okumura ◽  
Lumi Negishi ◽  
Shinsuke Ifuku ◽  
Toshihiro Kogure ◽  
...  
Keyword(s):  
Crystal Defects ◽  
Pinctada Fucata ◽  
Prismatic Layer ◽  
Chitinolytic Enzymes

A TEM study of the interface between organic matrix and aragonite in a biological hard tissue, nacre

1992 ◽  
Vol 50 (2) ◽  
pp. 1024-1025
Author(s):  
Jun Liu ◽  
Katie E. Gunnison ◽  
Mehmet Sarikaya ◽  
Ilhan A. Aksay
Keyword(s):  
Mechanical Properties ◽  
Ion Beam ◽  
Laminated Composite ◽  
Organic Matrix ◽  
Pearl Oyster ◽  
Interfacial Structure ◽  
Interfacial Region ◽  
Thin Sections ◽  
Organic Layers ◽  
Transmission Electron

The interfacial structure between the organic and inorganic phases in biological hard tissues plays an important role in controlling the growth and the mechanical properties of these materials. The objective of this work was to investigate these interfaces in nacre by transmission electron microscopy. The nacreous section of several different seashells -- abalone, pearl oyster, and nautilus -- were studied. Nacre is a laminated composite material consisting of CaCO3 platelets (constituting > 90 vol.% of the overall composite) separated by a thin organic matrix. Nacre is of interest to biomimetics because of its highly ordered structure and a good combination of mechanical properties. In this study, electron transparent thin sections were prepared by a low-temperature ion-beam milling procedure and by ultramicrotomy. To reveal structures in the organic layers as well as in the interfacial region, samples were further subjected to chemical fixation and labeling, or chemical etching. All experiments were performed with a Philips 430T TEM/STEM at 300 keV with a liquid Nitrogen sample holder.

Cytotoxicity Studies of Curcumin Loaded-Cockle Shell-Derived Calcium Carbonate Nanoparticles

2019 ◽  
Vol 09 ◽  
Author(s):  
Maryam Muhammad Mailafiya ◽  
Mohamad Aris Mohd Moklas ◽  
Kabeer Abubakar ◽  
Abubakar Danmaigoro ◽  
Samaila Musa Chiroma ◽  
...  
Keyword(s):  
Calcium Carbonate ◽  
Safety Margin ◽  
Bone Diseases ◽  
In Vivo Studies ◽  
Normal Cells ◽  
Cytotoxicity Studies ◽  
Standard Techniques ◽  
Cockle Shell

Background: Cockle shell-derived calcium carbonate nanoparticles (CSCaCO3NP) are natural biogenic inorganic material that is used in drug delivery mainly as a bone-remodeling agent as well as a delivery agent for various therapeutics against bone diseases. Curcumin possess wide safety margin and yet puzzled with the problem of poor bioavailability due to insolubility. Propounding in vitro and in vivo studies on toxicity assessments of newly synthesized nanoparticles are ongoing to overcome some crucial challenges regarding their safety administration. Nanotoxicology has paved ways for concise test protocols to monitor sequential events with regards to possible toxicity of newly synthesized nanomaterials. The development of nanoparticle with no or less toxic effect has gained tremendous attentions. Objective: This study aimed at evaluating the in vitro cytotoxic effect of curcumin-loaded cockle shell-derived calcium carbonate nanoparticles (Cur-CSCaCO3NP) and assessing its biocompatibility on normal cells using standard techniques of WST’s assay. Method: Standard techniques of WST’s assay was used for the evaluation of the biocompatibility and cytotoxicity. Result: The result showed that CSCaCO3NP and Cur-CSCaCO3NP possess minimal toxicity and high biocompatibility on normal cells even at higher dose of 500 µg/ml and 40 µg/ml respectively. Conclusion: CSCaCO3NP can be termed an excellent non-toxic nanocarrier for curcumin delivery. Hence, curcumin loaded cockle shell derived calcium carbonate nanoparticles (Cur-CSCaCO3NP) could further be assessed for various in vivo and in vitro therapeutic applications against various bone related ailments.

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