Abstract:Background: Thermodynamically unstable magnesium calcite is deposited in the shell of pearl oysters at ambient pressure. Results: The novel acidic matrix protein PfN44 interacts with magnesium to inhibit the deposition of aragonite. Conclusion: PfN44 participates in shell formation by inhibiting aragonite formation. Significance: Results of this study suggest a connection between the matrix protein and magnesium.
“…The expression patterns of KRMP and nacrein were in agreement with previous results (Fig. S11) (41), which excluded artificial errors. These results demonstrate that Pf-C/EBP-A and Pf-C/EBP-B may play essential roles in shell regeneration by regulating Shematrin-2.…”
Section: Regulation Effect Of Pf-c/ebp-a and Pf-c/ebp-b On Shematrin-supporting
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 innersurface 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.
“…The expression patterns of KRMP and nacrein were in agreement with previous results (Fig. S11) (41), which excluded artificial errors. These results demonstrate that Pf-C/EBP-A and Pf-C/EBP-B may play essential roles in shell regeneration by regulating Shematrin-2.…”
Section: Regulation Effect Of Pf-c/ebp-a and Pf-c/ebp-b On Shematrin-supporting
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 innersurface 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.
“…5b). Above all, this binding property is the foundation of matrix proteins’ regulation on crystal nucleation, crystal orientation, crystal polymorphism and morphology 11–14, 38 .Figure 5Binding properties of rPfY2 with main shell components. Lane 1–3, BSA, MBP, rPfY2 binding with calcite ( a ), aragonite ( b ) after flushing with water and protein elution buffer three times each, respectively.…”
Biomineralization, including shell formation, is dedicatedly regulated by matrix proteins. PfY2, a matrix protein detected in the ethylene diamine tetraacetic acid (EDTA)-soluble fraction from both prismatic layer and nacreous layer, was discovered by our group using microarray. It may play dual roles during biomineralization. However, the molecular mechanism is still unclear. In this research, we studied the function of PfY2 on crystallization in vivo and in vitro, revealing that it might be a negative regulator during shell formation. Notching experiment indicated that PfY2 was involved in shell repairing and regenerating process. Repression of PfY2 gene affected the structure of prismatic and nacreous layer simultaneously, confirming its dual roles in shell formation. Recombinant protein rPfY2 significantly suppressed CaCO3 precipitation rate, participated in the crystal nucleation process, changed the morphology of crystals and inhibited the transformation of amorphous calcium carbonate (ACC) to stable calcite or aragonite in vitro. Our results may provide new evidence on the biomineralization inhibition process.
“…And the biomineralization processes of the prism and nacre are controlled by the different SMPs secretary repertoires of the different mantle zones (Marie et al, 2012). Several identified SMPs have been showed to be significantly upregulated after shell notching treatment (Fang et al, 2012; Pan et al, 2014). However, in these studies, the gene expressions were detected in the whole mantle tissue, therefore, it remains unclear whether the SMPs expressions in each mantle zone are precisely controlled.…”
28Molluscan bivalves rapidly repair the damaged shells to prevent further injury. However, it 29 remains unclear how this process is precisely controlled. In this study, we applied scanning 30 electronic microscopy, transmission electronic microscopy and histochemical analysis to 31 examine the detailed shell regeneration process of the pearl oyster Pinctada fucata. It was found 32 that the shell damage caused the mantle tissue to retract, which resulted in dislocation of the 33 mantle zones to their correspondingly secreted shell layers. However, the secretory repertoires 34 of the different mantle zones remained unchanged. As a result, the dislocation of the mantle 35 tissue dramatically affected the shell morphology, and the unusual presence of the submarginal 36 zone on the nacreous layers caused de novo precipitation of prismatic layers on the nacreous 37 layers. Real-time PCR revealed that the expression of the shell matrix proteins (SMPs) were 38 significantly upregulated, which was confirmed by the thermal gravimetric analysis (TGA) of 39 the newly formed shell. The increased matrix secretion accelerated CaCO3 nucleation thus 40 promoting shell deposition. Taken together, our study revealed the close relationship between 41 the physiological activities of the mantle tissue and the morphological change of the 42 regenerated shells. 43
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