Zhenguang Yan

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.

Characterization of Calcium Deposition and Shell Matrix Protein Secretion in Primary Mantle Tissue Culture from the Marine Pearl Oyster Pinctada fucata

In this study, we established and characterized a long-term primary mantle tissue culture from the marine pearl oyster Pinctada fucata for in vitro investigation of nacre biomineralization. In this culture system, the viability of mantle tissue cells lasted up to 2xa0months. The tissue cells were demonstrated to express nacre matrix proteins by RT-PCR, and a soluble shell matrix protein, nacrein, was detected in the culture medium by Western blot analysis. On the other hand, 15xa0days after initiating culture, a large amount of calcium deposits with major elements, including calcium, carbon, and oxygen, were generated in the mantle explants and cell outgrowth area. The quantity and size of calcium deposits increased with the prolonged cultivation, and their location and nanogranular structure suggested their biogenic origin. These calcium deposits specifically appeared in mantle tissue cultures, but not in heart tissue cultures. Taken together, these results demonstrate that the mantle tissue culture functions similarly to mantle cells in vivo. This study provides a reliable approach for the further investigation on nacre biomineralization at the cellular level.

Immunolocalization of matrix proteins in nacre lamellae and their in vivo effects on aragonitic tablet growth

How matrix proteins precisely control the growth of nacre lamellae is an open question in biomineralization research. Using the antibodies against matrix proteins for immunolabeling and in vivo experiments, we investigate the structural and functional roles of EDTA-soluble matrix (SM) and EDTA-insoluble matrix (ISM) proteins in nacre biomineralization of the pearl oyster Pinctada fucata. Immunolabeling reveals that a SM protein, nacrein, distributes within aragonitic tablets and intertabular matrix. An ISM protein, which we named P43, has been specifically recognized by polyclonal antibodies raised against the recombinant protein of P. fucata bone morphogenetic protein 2 in immunoblot analysis. Immunolabeling indicates that P43 is localized to interlamellar sheet, and also embedded within aragonitic tablets. Although nacrein and P43 both distribute within aragonitic tablets, they function differently in aragonitic tablet growth. When nacrein is suppressed by the antibodies against it in vivo, crystal overgrowth occurs, indicating that this SM protein is a negative regulator in aragonitic tablet growth. When P43 is suppressed in vivo, the organo-mineral assemblage is disrupted, suggesting that P43 is a framework matrix. Taken together, SM and ISM proteins are indispensable factors for the growth of nacre lamellae, controlling crystal growth and constructing the framework of aragonitic tablets.

The Inner-Shell Film: An Immediate Structure Participating in Pearl Oyster Shell Formation

In mollusks, the inner shell film is located in the shell‐mantle zone and it is important in shell formation. In this study, we found that the film was composed of two individual films under certain states and some columnar structures were observed between the two individual films. The inner shell film was separated with the process of ethylenediaminetetraacetic acid (EDTA) treatment and the film proteins were extracted. Amino acid analysis showed that the film proteins may consist of shell framework proteins. The calcite crystallization experiment showed that the film proteins could inhibit the growth of calcite, while the CaCO3 precipitation experiment showed that the film proteins could accelerate the rate of CaCO3 precipitation. All these results suggested that the film plays an important role in shell formation. It may facilitate the aragonite formation by inhibiting the growth of calcite and accelerate the shell growth by promoting the precipitation of CaCO3 crystals.

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.

Cloning and characterization of the activin like receptor 1 homolog (Pf-ALR1) in the pearl oyster, Pinctada fucata

The signal transduction mechanisms in mollusks are still elusive since the genome information is incomplete and cell lines are not available. In previous study, we cloned a highly conserved Smad3 homolog (designated as Pf-Smad3) from the pearl oyster, Pinctada fucata. It seems that transforming growth factor beta (TGFbeta) signaling may play similar roles in the oyster as in vertebrate. Here we report a cDNA encoding an activin like receptor 1 homolog (designated as Pf-ALR1) of the oyster, another kind of TGFbeta superfamily member. Compared to the activin receptor-like kinases (ALK) in human, the amino acid sequence of Pf-ALR1 is more similar to that of ALK1, especially the L45 loop. Reverse transcription-polymerase chain reaction results indicate that Pf-ALR1 mRNA is expressed ubiquitously in the adult oyster. Thus, Pf-ALR1 may be important for many physiological processes in the oyster. To lay a basis for further investigation of the TGFbeta signal pathway functions in the oyster shell formation, in this report, the Pf-ALR1 mRNA expression in the oyster mantle was detected by in situ hybridization. The results show that Pf-ALR1 in the oyster mantle is mainly expressed at the inner epithelial cells of the outer fold and the outer epithelial cells of the middle fold, similarly as Pf-Smad3. The mRNA levels of Pf-ALR1 and Pf-Smad3 are all changed after shell notching. These results indicate that both Pf-ALR1 and Pf-Smad3 may take part in shell formation and repair. The results of drug treatment experiments with in-vitro cultured oyster mantle tissue cells demonstrate that the mRNA expression levels of Pf-Smad3, Pf-ALR1 and two oyster nuclear factor-kappaB (NF-kB) members can be adjusted and correlated. All our observations suggest that there should be similar TGFbeta signal pathways in the oyster and vertebrate. However, the potential functions of Pf-ALR1 and the relations of TGFbeta and NF-kB members in the oyster all need to be thoroughly investigated.

A possible mechanism for the formation of annual growth lines in bivalve shells

We report a unique shell margin that differed from the usual shell structure of Pinctada fucata. We observed empty organic envelopes in the prismatic layer and the formation of the nacreous layer in the shell margin. All the characteristics of the growing margin indicated that the shell was growing rapidly. To explain this anomaly, we propose the concept of “jumping development”. During jumping development, the center of growth in the bivalve shell jumps forward over a short time interval when the position of the mantle changes. Jumping development explains the unusual structure of the anomalous shell and the development of annual growth lines in typical shells. Annual growth lines are the result of a discontinuity in the shell microstructure induced by jumping development.

Immunolocalization of an Acid Phosphatase from Pearl Oyster (Pinctada fucata) and Its In Vitro Effects on Calcium Carbonate Crystal Formation

Distribution of an acid phosphatase, AcPase I, from pearl oyster (Pinctada fucata) in different tissues was investigated via enzyme activity determination and immunohistochemistry. Positive reactions were observed in sections of digestive gland, base of gill filaments, and epithelia of the outer side of the middle fold and the inner side of the outer fold, which indicated AcPase I might participate in processes besides immune defense, such as calcium metabolism or shell formation. Its effects on CaCO3 crystal formation were studied in vitro. Results revealed that AcPase I inhibited CaCO3 precipitation in a dose-dependent manner and had no affinity for calcium. CaCO3 crystals induced by AcPase I exhibited a cluster needle-like morphology, which proved to be aragonite. The morphology and size of the aragonites varied with different concentrations of AcPase I. Our observations described here may provide important clues to further understanding of the correlations between mineralization and immune defense in the oyster.

Molecular Regulation Mechanism of Biomineralization of Pinctada fucata

To elucidate the mechanism of biomineralization in Pinctada fucata, most of the researchers put their attention on the roles of matrix proteins in shell formation. Our group has identified and characterized many essential matrix proteins in the regulation of deposition of calcium carbonate crystals in both prism and nacre layers, as described in the former chapter. Meanwhile, the regulation of the transcription, translation and expression of matrix proteins and how these regulatory factors mediate the biomineralization have become a hot spot in recent years. In this chapter, we mainly assayed RACE to obtain the sequence of the members of signaling pathways and the transcriptional factors; real time-qPCR to analyze the expression pattern of these factors in different tissues and/or distinct time during shell repair and pearl sac; in situ hybridization to find out the expression location of specific genes in mantle tissue; luciferase assay and electrophoretic mobility assay (EMSA) to clarify the recruitment of transcriptional factors on promoters of matrix protein; yeast two hybridization to explore the interactions between different pathways. We demonstrate the function of NF-κB, TGFβ, Wnt signal pathway, G protein-mediated pathway and several transcriptional factors in mediating the biomineralization in Pinctada fucata. The mechanism of transcriptional regulation can give deep sight to the matrix protein expression pattern which enrich the theory of bionimeralization in Pinctada fucata.

Identification (Characterization) and Function Studies of Matrix Protein from the Oyster Pinctada fucata

Shell matrix protein (SMP) which is extracted from shell and extrapallial fluid matrix protein (EPFMP) which is extracted from extrapallial fluid have important functions in biomineralization during shell formation of Pinctada fucata. In the past decades, the functions of SMPs and EPFMPs were gradually revealed. In 2015, our group identified 72 unique SMPs in Pinctada fucata by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of proteins extracted from the shells of P. fucata aligned with a draft genome. In this chapter, we introduced SMPs from nacreous layer (P14, N40, and PfN23), from prismatic layer (KRMP, KRMP-3, and Prisilkin-39), from both prismatic and nacreous layer (MSI7, PfN44, and PfY2), EPFMP and extracellular matrix protein expressed by mantle (EFCBP and Ferritin). For example, P14 plays a crucial role during nacre biomineralization. N40 could stimulate the nucleation of aragonite drastically by serving as a nucleation site. The basic protein PfN23 might be a key accelerator during the regulation of crystal growth in nacre. KRMP protein family plays important roles in the framework formation of prism.