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

Yan Chen,Jing Gao,Jun Xie,Jian Liang,Guilan Zheng,Liping Xie,Rongqing Zhang

doi:10.1074/jbc.ra118.005281

Abstract

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.

Highlights

The molluscan shell is a fascinating biomineral consisting of a highly organized calcium carbonate composite
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 results showed that Pf-C/EBP-A, Pf-C/EBP-B, and Pf-C/EBP-␥ were clustered with their homologs in Crassostrea gigas, which has a close relationship with pearl oyster (Fig. 3C)

Summary

Introduction

The molluscan shell is a fascinating biomineral consisting of a highly organized calcium carbonate composite. The pearl oyster Pinctada fucata, the shell of which is composed of an inner nacre and an outer prismatic layer, is a major seawater pearl shellfish found in the southeast of China Both nacre and prism are mainly composed of calcium carbonate; less than 5% of their components are organic macromolecules, including matrix proteins, polysaccharides, and lipids [4]. Matrix proteins, such as nacrein [5], Pif [6], the KRMP family [7], and the Shematrin family [8], have been proven to be the major components responsible for nucleation, orientation, morphology, and organization during the shell formation process in P. fucata [9]. The characteristic structural organization of the shell is of interest; the mechanisms of calcification that matrix proteins participate in have been sought by many investigators, little is known about how the upstream transcription factors regulate the downstream matrix protein genes [12,13,14,15]

Results

Discussion

Conclusion