Changzhong Li

A Novel Extrapallial Fluid Protein Controls the Morphology of Nacre Lamellae in the Pearl Oyster, Pinctada fucata

Mollusk shell nacre is known for its superior mechanical properties and precisely controlled biomineralization process. However, the question of how mollusks control the morphology of nacre lamellae remains unresolved. Here, a novel 38-kDa extrapallial fluid (EPF) protein, named amorphous calcium carbonate-binding protein (ACCBP), may partially answer this question. Although sequence analysis indicated ACCBP is a member of the acetylcholine-binding protein family, it is actively involved in the shell mineralization process. In vitro, ACCBP can inhibit the growth of calcite and induce the formation of amorphous calcium carbonate. When ACCBP functions were restrained in vivo, the nacre lamellae grew in a screw-dislocation pattern, and low crystallinity CaCO3 precipitated from the EPF. Crystal binding experiments further revealed that ACCBP could recognize different CaCO3 crystal phases and crystal faces. With this capacity, ACCBP could modify the morphology of nacre lamellae by inhibiting the growth of undesired aragonite crystal faces and meanwhile maintain the stability of CaCO3-supersaturated body fluid by ceasing the nucleation and growth of calcite. Furthermore, the crystal growth inhibition capacity of ACCBP was proved to be directly related to its acetylcholine-binding site. Our results suggest that a “safeguard mechanism” of undesired crystal growth is necessary for shell microstructure formation.

Cloning and Characterization of Prisilkin-39, a Novel Matrix Protein Serving a Dual Role in the Prismatic Layer Formation from the Oyster Pinctada fucata

Molluscs form their shells out of CaCO3 and a matrix of biomacromolecules. Understanding the role of matrices may shed some light on the mechanism of biomineralization. Here, a 1401-bp full-length cDNA sequence encoding a novel matrix protein was cloned from the mantle of the bivalve oyster, Pinctada fucata. The deduced protein (Prisilkin-39), which has a molecular mass of 39.3 kDa and an isoelectric point of 8.83, was fully characterized, and its role in biomineralization was demonstrated using both in vivo and in vitro crystal growth assays. Prisilkin-39 is a highly repetitive protein with an unusual composition of Gly, Tyr, and Ser residues. Expression of Prisilkin-39 was localized to columnar epithelial cells of the mantle edge, corresponding to the calcitic prismatic layer formation. Immunostaining in situ and immunodetection in vitro revealed the presence of a characteristic pattern of Prisilkin-39 in the organic sheet and in sheaths around the prisms. Prisilkin-39 binds tightly with chitin, an insoluble polysaccharide that forms the highly structured framework of the shell. Antibody injection in vivo resulted in dramatic morphological deformities in the inner shell surface structure, where large amounts of CaCO3 were deposited in an uncontrolled manner. Moreover, Prisilkin-39 strictly prohibited the precipitation of aragonite in vitro. Taken together, Prisilkin-39 is the first protein shown to have dual function, involved both in the chitinous framework building and in crystal growth regulation during the prismatic layer mineralization. These observations may extend our view on the rare group of basic matrices and their functions during elaboration of the molluscan shell.

Cloning, characterization, and expression analysis of calreticulin from pearl oyster pinctada fucata

Abstract Calreticulin is a unique calcium-binding protein with multiple functions mostly located in the sarcoplasmic/endoplasmic reticulum. A large amount of calcium is absorbed from the medium and transported to mineralization sites during biomineralization in pearl oyster. This paper describes the cloning of the full-length cDNA of calreticulin from Pinctada fucata, namely PCRT. PCRT encodes a deduced 414-amino acid protein, which includes a predicted 17-amino acid signal peptide and an endoplasmic reticulum retrieval sequence HDEL. The protein shows 63%-76% sequence identity and shares some common characteristics with calreticulins from other species. Semi-quantitative RT-PCR indicates that PCRT is ubiquitously expressed in all tissues tested with the highest expression in the hemolymph and the mantle. In situ hybridization analysis of PCRT in the mantle showed strong signals in the inner fold, the inner side of middle fold, and the inner side of outer fold of the mantle epithelium. All these results suggest PCRT might be involved in Ca2+ transport and storage during oyster biomineralization.

Cloning, characterization and immunolocalization of two subunits of calcineurin from pearl oyster (Pinctada fucata)

Calcineurin (CN), consisting of catalytic subunit (CN A) and regulatory subunit (CN B), is a multifunctional protein involved in many important physiological processes. Here, we cloned two subunits of CN (Pf-CN A and Pf-CN B) from pearl oyster Pinctada fucata and reported, for the first time, its expression patterns in the developmental stages, its enzymatic activity and immunolocalization in various tissues of adult pearl oyster. The Pf-CN A was extensively localized in all the tested tissues including mantle, gonad, digestive gland, gills, adductor muscle, and foot with strong signals detected in gonad, gills, foot, and mantle. Importantly, Pf-CN A was mainly found in the inner epithelial cells of the basal periostracal groove and lateral surface of the inner mantle fold, in which organic macromolecules used for periostracum formation and shell construction are secreted, respectively. In gill, the strong signals were distributed in the epithelial cells of the branchial filaments and the base of gill filaments. All the results suggested that Pf-CN may participate in the development of the pearl oyster and function in many ways in various physiological activities, especially in the shell formation. Our observations could provide some important clues to further understanding of the functions of CN in the oyster.

Calcineurin Plays an Important Role in the Shell Formation of Pearl Oyster (Pinctada fucata)

Calcineurin (CN) is a multifunctional protein involved in many important physiological processes in mammalians, but the function of CN in mollusks is still largely unknown. In the present study, through the shell regeneration system, the changes of enzymatic activity of CN were determined in the process of shell regeneration in pearl oyster Pinctada fucata. CN was activated immediately and continuously in the shell regeneration process. The speed of shell regeneration was measured and the ultrastructure of inner shell surface was observed by scanning electron microscopy after inhibiting CN by intramuscular injection of immunosuppresant cyclosporine A (CsA). The results showed that the speed of shell regeneration was delayed and the morphology of calcite and aragonite in the inner shell surface became abnormal when CN was inhibited by CsA. Meanwhile, RT-PCR analysis revealed that the expression of P. fucata BMP-2 in mantle tissue decreased with CsA injection. In vitro secretion level of proteoglycans (PGs) in primary cultures of mantle cells was also decreased when mantle cells were exposed to CsA. Taken together, our results, for the first time, show that CN is involved in the shell formation through regulating the expression of Pf-BMP-2 in mantle tissue, which controls the secretion of PGs/GAGs of the mantle epithelial cells.

Calcineurin mediates the immune response of hemocytes through NF-κB signaling pathway in pearl oyster (Pinctada fucata)

Calcineurin (CN), a multifunctional protein, mediates the immune response through diverse signaling pathways in mammals, while the function of CN in the immune response of molluscan hemocytes still remains unclear. In the present study, we detected the distribution of CN in various tissues and the expression levels of Pf-CNA and Pf-CNB gene in hemocytes of Pinctada fucata. After the preparation of hemocyte monolayers, we checked the response of enzymatic activity of CN, the degradation level of IkappaBalpha, the activity of iNOS and the production of NO, and IL-2 to the challenge of lipopolysaccharide (LPS) and cyclosporin A (CsA). CN activity in hemocytes was very sensitive to both the stimulation of LPS and the inhibition of CsA. Most importantly, IkappaBalpha degradation in hemocytes was induced by LPS and attenuated by CsA. Consequently, the activity of iNOS was elevated and the production of NO was increased. Additionally, we found that the synthesis of IL-2 was increased by LPS but was apparently weakened by CsA. In vivo bacterial clearance experiments showed that CsA significantly decreased the ability of in vivo bacteria clearance in pearl oyster. All the results revealed, for the first time, that CN mediated the immune response of molluscan hemocytes via activating NF-kappaB signaling pathway.

Significance of the C‐terminal globular domain and the extra tail of the calmodulin‐like protein (Pinctada fucata) in subcellular localization and protein—protein interaction

Calmodulin (CaM) plays a very important role in many physiological processes and is highly conserved in different species. In a previous study, we successfully cloned CaM and a novel calmodulin‐like protein (CaLP) with an extra C‐terminal sequence from the pearl oyster Pinctada fucata and then expressed in Escherichia coli. In this research, we used fluorescence confocal microscopy to analyze the protein—protein interaction between CaM/CaLP and p21Cip1, which is cloned from mammalian cells, to show the different characteristics of these two proteins in vivo. The fluorescence confocal microscopy showed that the C‐terminal globular domain together with the extra tail of CaLP is very important in CaLPs sequestration in cytoplasm. The most interesting phenomenon is that transfection of p21Cip1 can stimulate translocation of CaLP from the cytoplasm to the nucleus, but this is not the case for CaM. Fluorescence confocal microscopy and co‐immunoprecipitation on different mutants of CaLP with p21Cip1 indicated that the C‐terminal globular domain of CaLP is responsible for the trafficking of CaLP from cytoplasm to nucleus.

The extra C-terminal tail is involved in the conformation, stability changes and the N/C-domain interactions of the calmodulin-like protein from pearl oyster Pinctada fucata.

Pearl oyster Pinctada fucata calmodulin-like protein (PfCaLP), containing an extra tail (D150-K161) at the C-terminal, is a novel protein involved in the regulation of oyster calcium metabolism. The purpose of this study is to gain insight into the conformational characteristics of the N/C-domain of PfCaLP, especially the detailed contribution of the extra tail to the Ca(2+)/Mg(2+)-induced conformational changes, the stability of the intact PfCaLP molecule and its C-domain, as well as to the interdomain communications in PfCaLP. Our results demonstrate that a strong interaction exists between the hydrophilic tail and the C-domain of PfCaLP. The extra tail, through affecting the C-domain conformational changes, further influences the migration rate, conformational changes, N/C-domain interactions and exposure of the hydrophobic patches of the intact PfCaLP molecule. Furthermore, the tail could actively regulate the stability of PfCaLP and its C-domain. Our studies are helpful to explain our previous finding that the tail plays important roles in PfCaLP-target interaction in the oyster calcium metabolism.

Investigation of Phosphorylation Site Responsible for CaLP (P. fucata) Nucleo-cytoplasmic Shuttling Triggered by Overexpression of p21Cip1

Calmodulin (CaM) is a highly conserved and ubiquitous Ca2+-binding protein regulating intracellular Ca2+ concentration by acting as a sensor of this divalent cation in eukaryotic cells. Being such a very important signal sensor, CaM is susceptible to undergo many posttranslational modifications. One of these important modifications is its phosphorylation. Our previous investigations showed that CaM and calmodulin-like protein (CaLP) cloned from Pinctada fucata have many different characteristics in spite of their high similarity to each other. We have narrowed down that the C-terminal domains of CaM and CaLP are responsible for their discrepant subcellular localizations and shuttling of CaLP when it is co-transfected with p21Cip1, which is commonly considered as an important cell cycle regulating protein. In this study, we first predicted the potential phosphorylation site responsible for the shuttling and confirmed by fluorescence confocal microscopy. Together with fluorescence activated cell sorter analysis, we further investigated the releasing ability of wild type and point mutated CaLP from arrested cell cycle caused by p21Cip1 overexpression. By performing pull-down analysis and phosphorylation status of CaLP in cytoplasm fraction of transfected COS-7 cells with CaLP alone and phosphorylation status of CaLP in nuclear fraction of co-transfected COS-7 cells with CaLP and p21Cip, we propose that the CaLP staying in the cytoplasm is in the state of phosphorylation, but when p21Cip1 is overexpressed in mammalian cells, some signal triggers CaLP dephosphorylation and translocation into the nucleus.