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.
Highlights
The molluscan shell is a biomineral that comprises calcium carbonate and organic matrices controlling the crystal growth of calcium carbonate
In the acid-insoluble fraction which is mainly the complex of chitin and proteins, the weight of chitin can be obtained as the alkali insoluble material since due to the strong hydrogen bonding, chitin is hardly dissolved with 1 M sodium hydroxide and boiling[21], which dissolves the proteins cross-linking with chitin or each other
Acid-insoluble proteins binding to chitin were the main components that compose the scaffold of organic matrices in these microstructures
Summary
The molluscan shell is a biomineral that comprises calcium carbonate and organic matrices controlling the crystal growth of calcium carbonate. Insoluble proteins remained due to the formation of a solid complex with chitin We identified these proteins from the nacreous layer, prismatic layer, and hinge ligament of Pinctada fucata using mercaptoethanol and trypsin. 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. Almost all proteins that have been identified to date are derived from the acid-soluble fraction or acid-insoluble/detergent-soluble fraction: n acrein10, MSI6011, Pif[12], and N1613,14 in the nacreous layer, prismalin-1415, prismin[16], and M SI3111 in the prismatic layer, and LICP17 and T IMP18 in the ligament. A new approach is required to analyse these proteins
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