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

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.

Figure 1

(A) Shell structure of Pinctada fucata. (B) A model for a hierarchical structure of organic matrices. The insoluble organic matrices containing chitin become the scaffold. A nucleation of calcium carbonate occurs around the insoluble organic matrices mediated by the soluble organic matrices which have chitin binding domain (yellow) and calcium carbonate-interaction domain such as acid-rich domain (pink). Other soluble organic matrices control the crystal growth. (C) A flow chart for the extraction of organic matrices.

Figure 2

A schematic diagram of domain structures of proteins deduced from peptides extracted from the acid-insoluble/detergent insoluble fraction in the nacreous layer, prismatic layer and ligament. The number of gene ID is described at left side. A red color boxes the recombinant region we made.

Figure 3

Relative gene expression levels of NU-5 in each tissue of P. fucata. Real-time quantitative PCR (qPCR) was performed to evaluate relative gene expression levels compared with the mantle pallium. The values are relative to that of the mantle pallium. Data are expressed as the mean ± S.E. (n = 3).

Figure 4

(A) Relative gene expression levels of NU-5 4 days after injection of dsRNAf of NU-5. dsRNA of EGFP was injected as negative control. qPCR was performed to evaluate relative gene expression levels compared with EGFP. Data are expressed as the mean ± S.E. Statistically significant differences were determined by t-test (*p < 0.05, n = 3). (B) Surface microstructure images of the nacreous layer 4 days after injection observed using SEM. The surface is the growth front of the nacre. In the condition of NU-5 100 µg, the normal nacre formation was disrupted.

Figure 5

A particle size of the rMERP measured using dynamic light scattering (DLS). The aggregation activity of rMERP in the absence or presence of calcium ion at pH 7.5, 8.0 and 8.5 was measured. Data are expressed as the mean ± S.E. Statistically significant differences were determined by t-test (*p < 0.05, n = 3).

Figure 6

A schematic illustration for the proteins aggregation with calcium ion. Without calcium ion, proteins including acidic amino acid-rich region (pink) are dispersed to some extent because of an electrical repulsion between carboxy groups of acidic amino acids. When calcium ion comes, the cation of calcium ion disturbs the electrical repulsion, leading to the aggregation. After aggregation, the cross-linkage may occur by methionine-rich regions (green).