Search

The frequent occurrence of viral infections poses a serious threat to human life. Identifying effective antiviral components is urgent. In China, pearls have been important traditional medicinal ingredients since ancient times, exhibiting various therapeutic properties, including detoxification properties. In this study, a peptide, KKCH, which acts against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was derived from Pinctada fucata pearls. Molecular docking showed that it bound to the same pocket of the SARS-CoV-2 S protein and cell surface target angiotensin-converting enzyme II (ACE2). The function of KKCH was analyzed through surface plasmon resonance (SPR), Enzyme-Linked Immunosorbent Assays, immunofluorescence, and simulation methods using the SARS-CoV-2 pseudovirus and live virus. The results showed that KKCH had a good affinity for ACE2 (KD = 6.24 × 10−7 M) and could inhibit the binding of the S1 protein to ACE2 via competitive binding. As a natural peptide, KKCH inhibited the binding of the SARS-CoV-2 S1 protein to the surface of human BEAS-2B and HEK293T cells. Moreover, viral experiments confirmed the antiviral activity of KKCH against both the SARS-CoV-2 spike pseudovirus and SARS-CoV-2 live virus, with half-maximal inhibitory concentration (IC50) values of 398.1 μM and 462.4 μM, respectively. This study provides new insights and potential avenues for the prevention and treatment of SARS-CoV-2 infections. [ABSTRACT FROM AUTHOR]

Microplastics are extremely widespread aquatic pollutants that severely detriment marine life. In this study, the influence of microplastics on biomineralization was investigated. For the first time, multiple forms and types of microplastics were detected and isolated from the shells and pearls of Pinctada fucata. According to the present study, the abundance of microplastics in shells and pearls was estimated at 1.95 ± 1.43 items/g and 0.53 ± 0.37 items/g respectively. Interestingly, microplastics were less abundant in high-quality round pearls. Microplastics may hinder the growth of calcite and aragonite crystals, which are crucial components required for shell formation. During the process of biomineralization microplastics became embedded in shells, suggesting the existence of a novel pathway by which microplastics accumulate in bivalves. After a 96-h exposure to microplastics, the expression level of typical biomineralization-related genes increased, including amorphous calcium carbonate binding protein (ACCBP) gene which experienced a significant increase. ACCBP promotes the formation of amorphous calcium carbonate (ACC), which is the pivotal precursor of shell formation-related biominerals. ACCBP is highly expressed during the developmental stage of juvenile oysters and the shell-damage repair process. The increased expression of ACCBP suggests biomineralization is enhanced as a result of microplastics exposure. These results provide important evidence that microplastics exposure may impact the appearance of biominerals and the expression of biomineralization-related genes, posing a new potential threat to aquatic organisms. [Display omitted] • Microplastics were isolated from molluscan shells and pearls for the first time. • Microplastics were found embedded into shells via biomineralization process. • Microplastics could hinder the growth of CaCO 3 crystals. • Microplastics could impact the expression of biomineralization-related genes. [ABSTRACT FROM AUTHOR]

Abstract: 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 CaLP''s 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. [Copyright &y& Elsevier]

For both nacre formation and biomineralization in mollusks, understanding the molecular mechanism is imperative. Biomineralization, especially shell formation, is dedicatedly regulated by multiple matrix proteins. However, ACC conversion to stable crystals still lacks positive factors. In this research, we found a novel matrix protein named PNU5 in Pinctada fucata that plays a regulatory role in both prismatic layer and nacreous layer formation. Functional studies in vivo and in vitro have shown that it might be involved in shell formation in a positive manner. RT-qPCR analysis showed that pnu5 was highly expressed in mantle pallial and participated in shell repairing and regeneration. RNAi-mediated repression of pnu5 could affect the normal structure of prismatic layer and nacreous layer. The recombinant protein rPNU5 significantly enhanced the precipitation rate of CaCO 3 both in the calcite and aragonite crystallization systems, as well as altering the morphology of the crystals. Based on ACC transition experiments, the recombinant protein rPNU5 facilitated amorphous calcium carbonate (ACC) transformation into stable calcite or aragonite. This study could provide us with a better understanding of how positive regulatory mechanisms contribute to biomineralization. • Identify a novel matrix protein PNU5 which can facilitate the transformation from ACC to stable calcite • In vivo pnu5 -specific RNAi experiments lead to growth disorders in both prismatic layer and nacreous layer. • In vitro crystallization assays suggest that PNU5 can promote the nucleation of calcite crystals and result in crystal stacking. [ABSTRACT FROM AUTHOR]

Shell formation in molluscan bivalves is regulated by organic matrices composed of biological macromolecules, but how these macromolecules assemble in vitro remains elusive. Prismatic layer in the pearl oyster Pinctada fucata consists of polygonal prisms enveloped by thick organic matrices. In this study, we found that the organic matrices were heterogeneously distributed, with highly acidic fractions (EDTA-soluble and EDTA-insoluble) embedded inside the prism columns, while basic EDTA-insoluble faction as inter-column framework enveloping the prisms. The intra-column matrix was enriched in aspartic acid whereas the inter-column matrix was enriched in glycine, tyrosine and phenylalanine. Moreover, the intra-column matrix contained sulfo group further contributing to its acidic property. Proteomics data showed that the intra-column proteins mainly consisted of acidic proteins, while some typical matrix proteins were absent. The absent matrix proteins such as shematrin family and KRMP family were highly basic and contained aromatic amino acids, suggesting that electric charge and hydrophobic effect might play a role in the matrix heterogeneity. Interestingly, chitin metabolism related proteins were abundant in the inter-column matrix, which may be involved in reconstructing the prism organic matrix. Overall, our study suggests that each single prism grew in an enclosed organic envelope and the organic matrix undergoes rearrangement, thus leading to the peculiar growth of the prismatic layer. [ABSTRACT FROM AUTHOR]

Pinctada fucata is an important pearl production shellfish in aquaculture. The formation of shells and pearls is a hot research topic in biomineralization, and matrix proteins secreted by the mantle tissues play the key role in this process. However, upstream regulatory mechanisms of transcription factors on the matrix protein genes remain unclear. Previous studies have shown that NF-κB signaling pathway regulated biomineralization process through expression regulation of specific matrix proteins, including Nacrein, Prismalin-14 and MSI60. In this study, we systematically investigated the regulatory effect of the NF-κB signaling pathway key factor Pf -Rel and inhibitory protein poI-κB on the biomineralization and shell regeneration process. We applied RNA interference and antibody injection assays to study in vivo function of transcription factor Pf -Rel and characterized shell morphology changes using scanning electron microscopy and Raman spectroscopy. We found that transcription factor Pf -Rel plays a positive regulatory role in the growth regulation of the prismatic and nacreous layers, while the function of inhibitory protein poI-κB is to prevent excessive growth and accumulation of both layers. RNA-seq was conducted based on RNA interference animal model to identify potential regulatory genes by transcription factor Pf -Rel. Shell damage repair experiments were performed to simulate shell regeneration process, and observations of newly formed shells revealed that NF-κB signaling pathway had different functions at different times. This study provides us with a more macroscopic perspective based on transcription factors to investigate biomineralization and shell regeneration. • We systematically investigated the regulatory effect of the NF-κB signaling pathway key factor Pf -Rel and inhibitory protein poI-κB on the biomineralization and shell regeneration process. • Transcription factor Pf -Rel plays as a positive regulator in the growth control, while the inhibitory protein poI-κB serves to prevent excessive growth and accumulation of the prismatic and nacreous layers. • RNA-seq based on RNA interference model indicated a strong relationship between biomineralization and immune response. [ABSTRACT FROM AUTHOR]

[Display omitted] Molluscs rapidly repair the damaged shells to prevent further injury, which is vital for their survival after physical or biological aggression. However, it remains unclear how this process is precisely controlled. In this study, we applied scanning electronic microscope and histochemical analysis to examine the detailed shell regeneration process in the pearl oyster Pinctada fucata. It was found that the shell damage caused the mantle tissue to retract, which resulted in relocation of the partitioned mantle zones with respect to their correspondingly secreting shell layers. As a result, the relocated mantle tissue dramatically altered the shell morphology by initiating de novo precipitation of prismatic layers on the former nacreous layers, leading to the formation of sandwich-like "prism-nacre-prism-nacre" structure. Real-time PCR revealed the up-regulation of the shell matrix protein genes, which was confirmed by the thermal gravimetric analysis of the newly formed shell. The increased matrix secretion might have led to the change of CaCO 3 precipitation dynamics which altered the mineral morphology and promoted shell formation. Taken together, our study revealed the close relationship between the physiological activities of the mantle tissue and the morphological change of the regenerated shells. [ABSTRACT FROM AUTHOR]

Specificity protein (Sp) belong to a transcription factor family that contains nine subgroups with essential functions in development, including skeletogenesis, tooth development, neural tube closure, and limb formation. In molluscs, functions of the Sp protein family members have not been reported in detail. In this study, we report the first Sp protein-encoding gene in Pinctada fucata . We named the translated protein Pf -Sp8/9, based on the phylogenetic development tree constructed using Sp protein sequences from six model organisms, which showed that it was a Sp8/9 homolog. Alignment of the Pf -Sp8/9 sequence with the amino acid sequences of related proteins showed that Pf -Sp8/9 had conserved domains, including three DNA-binding motifs. The tissue distribution showed that while Pf-Sp8/9 mRNA expression was detected in all tested tissues, it was particularly high in the mantle. The luciferase reporter assay results showed that Pf -Sp8/9 had the ability to activate the transcription of a number of matrix proteins. The expression pattern of Pf - Sp8/9 during P. fucata pearl sac development was similar to that of some genes that encode matrix proteins, suggesting Pf -Sp8/9 may be involved in mantle-related physiological activities and biomineralization. [ABSTRACT FROM AUTHOR]

The Wnt signaling pathway plays an important role in animal development and in the biomineralization process. At present, although the biomineralization mechanism in pearl oyster ( Pinctada fucata ) has been extensively studied, there is little research on the Wnt signaling pathway in pearl oyster. To understand the potential role of the Wnt signaling pathway in pearl oyster, we cloned and sequenced three genes from the Wnt signaling pathway in pearl oyster that encode the following proteins: β -catenin, Dishevelled (Dvl) and T-cell factor (TCF). Genomic structure analysis revealed that Pf-β-catenin genomic DNA contained 15 exons, Pf-Dvl genomic DNA contained 16 exons, and Pf-TCF genomic DNA contained 7 exons. Their deduced amino acid sequences all showed the highest identity with homologs in Crassostrea gigas . Yeast two-hybrid analysis verified that Pf- β -catenin interacted with Pf-TCF. These three genes were ubiquitously expressed in seven pearl oyster tissues analyzed with the highest expression in the gill and a certain amount of expression in the mantle, a tissue related to shell formation. After shell notching, the dynamic changes in expression of these three genes showed that they reached a maximum at 4 days, indicating their response to shell regeneration. All three genes were constitutively expressed during five developmental stages of the pearl oyster, with high levels at the early embryonic development stage. Taken together, these results suggested that Pf- β -catenin, Pf-Dvl and Pf-TCF might participate in shell formation and early embryonic and larval development in the pearl oyster. [ABSTRACT FROM AUTHOR]

In this study, light microscope, scanning and transmission electron microscope, hematoxylin-eosin and fluorescent staining, and mass spectrometry methods were employed to observe the calcium carbonate (CaCO 3 ) crystal formation, hemocyte release and transportation, and hemocyte distribution at the shell regeneration area and to analyse the proteome of hemocytes in the pearl oyster, Pinctada fucata . The results indicated that intracellular CaCO 3 crystals were observed in circulating hemocytes in P. fucata, implying that there was a suitable microenvironment for crystal formation in the hemocytes. This conclusion was further supported by the proteome analysis, in which various biomineralization-related proteins were detected. The crystal-bearing hemocytes, mainly granulocytes, may be released to extrapallial fluid (EPF) by the secretory cavities distributed on the outer surface of the mantle centre. These granulocytes in the EPF and between the regenerated shells were abundant and free. In the regenerated prismatic layer, the granulocytes were fused into each column and fragmented with the duration of shell maturation, suggesting the direct involvement of hemocytes in shell regeneration. Overall, this study provided evidence that hemocytes participated in CaCO 3 crystal formation, transportation and shell regeneration in the pearl oyster. These results are helpful to further understand the exact mechanism of hemocyte-mediated biomineralization in shelled molluscs. [ABSTRACT FROM AUTHOR]

Biomineralization is an important and ubiquitous process in organisms. The shell formation of mollusks is a typical biomineral physical activity and is used as a canonical model in biomineralization research. Most recent studies focused on the identification of matrix proteins involved in shell formation; however, little is known about their transcriptional regulation mechanism, especially the transcription factors involved in shell formation. In this study, we identified a homolog of the YY-1 transcriptional factor from Pinctada fucata , named Pf - YY - 1 , and characterized its expression pattern and biological functions. Pf - YY - 1 has a typical zinc finger motif highly similar to those in humans, mice, and other higher organisms, which indicated its DNA-binding capability and its function as a transcription factor. Pf - YY - 1 is ubiquitously expressed in many tissues, but at a higher level in the mantle, which suggested a role in biomineralization. The expression pattern of Pf - YY - 1 during pearl sac development was quite similar to, and was synchronized with, those of Prisilkin - 39 , ACCBP , and other genes involved in biomineralization, which also suggested its function in biomineralization. [ABSTRACT FROM AUTHOR]

Hemocytes play important roles in the innate immune response and biomineralization of bivalve mollusks. However, the hemocytes in pearl oysters are poorly understood. In the present study, we investigated the morphology and classification of hemocytes in the pearl oyster, Pinctada fucata . Three types of hemocytes were successfully obtained by light microscopy, electron microscopy and flow cytometry methods: small hyalinocytes, large hyalinocytes and granulocytes. The small hyalinocytes are the major hemocyte population. Morphological analyses indicated that these hemocytes have species-specific characterizations. In addition, we assessed the potential effects of ocean acidification (OA) and ocean warming (OW) on the immune parameters and calcium homeostasis of the hemocytes. OA and OW (31 °C) altered pH value of hemolymph, increased the total hemocyte count, total protein content, and percentage of large hyalinocytes and granulocytes, while it decreased the neutral red uptake ability, suggesting active stress responses of P. fucata to these stressors. Exposure to OW (25 °C) resulted in no significant differences, indicating an excellent immune defense to heat stress at this level. The outflow of calcium from hemocytes to hemolymph was also determined, implying the potential impact of OA and OW on hemocyte-mediated biomineralization. This study, therefore, provides insight into the classification and characterization of hemocyte in the pearl oyster, P. fucata , and also reveals the immune responses of hemocytes to OA and OW, which are helpful for a comprehensive understanding of the effects of global climate change on pearl oysters. [ABSTRACT FROM AUTHOR]

Abstract: 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 (TGFβ) 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 TGFβ 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 TGFβ 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-κB (NF-kB) members can be adjusted and correlated. All our observations suggest that there should be similar TGFβ signal pathways in the oyster and vertebrate. However, the potential functions of Pf-ALR1 and the relations of TGFβ and NF-kB members in the oyster all need to be thoroughly investigated. [Copyright &y& Elsevier]

Abstract: 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 IκBα, 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, IκBα 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-κB signaling pathway. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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 Ca2+/Mg2+-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. [Copyright &y& Elsevier]

Abstract: Calmodulin (CaM) and calmodulin-like protein (CaLP) are two proteins involved in biomineralization. Their localizations in Pinctada fucata mantle epithelia were studied by Western blot (WB) analysis of the nuclear/cytosol fraction of primary cultured P. fucata mantle cells and immunogold electron microscopy. The results showed a completely different distribution of these two proteins at the subcellular level. CaM was distributed throughout both the nucleus and cytoplasm of the mantle epithelium but CaLP was distributed only in the cytoplasm. The functions of these two proteins in biomineralization were investigated by shell regeneration. During this process, the expressions of CaM and CaLP were greatly enhanced in different organelles of the mantle epithelium. Overexpression of these two proteins and a mutant of calmodulin-like protein (M-CaLP) that lacks an extra C-terminal tail in MC3T3-E1 promoted the mRNA expression of osteopontin, a biomineralization marker for osteoblasts. All of the results indicated that CaM and CaLP have completely different distributions in the mantle epithelium and affect the biomineralization process at different levels. The extra C-terminal tail of CaLP is important for its functions in biomineralization in P. fucata. [Copyright &y& Elsevier]

Abstract: IκB kinase (IKK) play central roles in cell signaling by regulating nuclear factor-κB (NF-κB) activation, which is involved in inflammatory response, proliferation, development and bone homeostasis. We report here for the first time that an IKK homologue was cloned and functionally characterized in pearl oyster, Pinctada fucata. The full-length cDNA consists of 2546bp with an ORF encoding a 737 amino acids protein. The putative pearl oyster IKK protein (Pf-IKK) possesses the characteristic organization of the mammalian IKK proteins, namely an amino-terminal kinase domain followed by a leucine zipper region and a carboxylterminal helix–loop–helix motif. Real-time PCR (RT-PCR) analysis indicated that Pf-IKK was ubiquitously expressed in pearl oyster. We also found that lipopolysaccharides (LPS) transiently stimulates IκBα degradation, but not expression levels of Pf-IKK. When transfected into NIH3T3 cells, Pf-IKK activated the expression of NF-κB-controlled reporter gene and induced NF-κB translocation, whereas the activation was greatly deduced by pyrrolidine dithiocarbamate (PDTC). We also found that overexpression of Pf-IKK increased the alkaline phosphatase (ALP) activity significantly. Based on the results and the homology to the vertebrate NF-κB cascade, these studies help to highlight a potentially important regulatory pathway to the study of the related functions in mollusks. [Copyright &y& Elsevier]

The aim of this study was to investigate the natural role of the mantle in pearl oyster, Pinctada fucata. The mantle is believed to be the tissue responsible for shell and pearl formation. However, our current study on lead and copper accumulation in tissues of the oyster showed that the secondary tissue for lead accumulation was not the digestive gland but the mantle. In view of high lead concentrations in the mantle, its general metabolic condition (including immune and antioxidant defense systems) as affected by the two metals was studied. The results indicated that activities of antioxidant enzymes (superoxide dismutase, SOD; Se-dependent glutathione peroxidase, Se-GPx) were altered by lead and copper in the similar way. However, the immune enzyme activities (acid phosphatase, AcPase; phenoloxidase, PO) were perturbed differently by two metals. Therefore, the mantle of P. fucata was predicted to participate in immune processes and accumulation or detoxification of lead besides shell formation. Our observations described here may also provide important clues to further understanding of the biomarker responses of bivalves. [Copyright &y& Elsevier]

Abstract: Calmodulin-like protein (CaLP) was believed to be involved in the shell formation of pearl oyster. However, no further study of this protein was ever performed. In this study, the in vitro crystallization experiment showed that CaLP can modify the morphology of calcite. In addition, aragonite crystals can be induced in the mixture of CaLP and a nacre protein (at 16 kDa), which was detected and purified from the EDTA-soluble matrix of nacre. These results agreed with that of immunohistological staining in which CaLP was detected not only in the organic layer sandwiched between nacre (aragonite) and the prismatic layer (calcite), but also around the prisms of the prismatic layer. Take together, we concluded that (1) CaLP, as a component of the organic layer, can induce the nucleation of aragonite through binding with the 16-kDa protein, and (2) CaLP may regulate the growth of calcite in the prismatic layer. [Copyright &y& Elsevier]

The absorption, transport and localization of calcium underlie the basis of biomineralization, and Ca2+ entry into epithelial cell is the primary step in shell formation. However, the related mechanism of Ca2+ transport is poorly documented at the gene or protein level. L-type voltage-dependent calcium channels may be involved in calcium transport for biomineralization in some marine invertebrates. In this study, a full-length cDNA of a voltage-dependent calcium channel β subunit from Pinctada fucata (PCaβ) was cloned, and its amino acid sequence was deduced. PCaβ shared 51%–67% apparently sequence identity with voltage-dependent calcium channel β subunits from other species. However, PCaβ was much shorter than other voltage-dependent calcium channel β subunits particularly at the carboxyl terminus, indicating that it is likely a truncated β subunit isoform. Semi-quantitative RT-PCR analysis showed that PCaβ was expressed in all the tested tissues and that it had a higher expression level in the gill tissue and hemolymph than in other tissues, suggesting that L-type voltage-dependent calcium channels are responsible for Ca2+ absorption in the gill and Ca2+ entry into hemocytes. In the mantle, PCaβ mRNA was predominantly expressed in the inner and middle folds of the mantle epithelium, suggesting that L-type voltage-dependent calcium channels are involved in Ca2+ absorption from the ambient medium in the mantle. All these results suggest that voltage-dependent calcium channels are involved in Ca2+ uptake and transport during oyster biomineralization. [Copyright &y& Elsevier]

Abstract: Mollusk shell formation is a complicated and highly controlled calcium metabolism process. Previous studies revealed that several EF-hand calcium-binding proteins actively participate in the regulation of shell mineralization. In this study, we cloned a full-length cDNA encoding a novel extracellular EF-hand calcium-binding protein (named EFCBP) from the pearl oyster, Pinctada fucata, according to the EF-hand motifs of calmodulin. Although it shares high similarity with the calmodulin family in its EF-hand signatures, EFCBP just has two EF-hand motifs and belongs to a new separate group from the other EF-hand proteins according to a phylogenetic analysis. EFCBP is specifically expressed in shell mineralization-related tissues, viz. the mantle, the gill, and the hemocytes. Moreover, its expression responds quickly only to the shell damage, but not to the damage of other tissues and the infection of the lipopolysaccharides from Escherichia coli. These results suggest that EFCBP might be an important regulator of shell formation. This finding may help better understand the functions of EF-hand proteins on the regulation of mollusk shell formation. [Copyright &y& Elsevier]

Abstract: The effects of exposure to copper under laboratory-controlled conditions were investigated in the pearl oyster, Pinctada fucata. Metal accumulation and the activity of five enzymes were measured: two immune defense involved enzymes [acid phosphatase (AcPase) and phenoloxidase (PO)], two antioxidant enzymes [superoxide dismutase (SOD) and Se-dependent glutathione peroxidase (Se-GPx)] and one metal-sensitive enzyme [alkaline phosphatase (ALP)]. Analyses were carried out in gills and digestive gland of oysters exposed to 0.05 μM and 0.5 μM copper, respectively, at 12, 24, 48 and 72 h of exposure. The digestive gland of P. fucata was the main copper accumulation organ when oysters were exposed to low concentrations, whereas gills became the target organ in oysters exposed to high concentrations. The adaptation and recovery of the oysters were observed in our study. Levels of the copper accumulation and the sensitivity to copper were the main, if not, part of the reasons for the various responses of the selected enzymes. Se-GPx may potentially be used as biomarkers in biotesting of marine heavy metal pollutions. The enzymatic responses were compared with those of other studies and the possible reasons were discussed. [Copyright &y& Elsevier]

Abstract: Oyster (Pinctada fucata) calmodulin-like protein (CaLP), containing a C-terminally extra hydrophilic tail (150D–161K), is a novel protein involved in the regulation of oyster calcium metabolism. To investigate the importance of the extra fragment to the Ca2+/Mg2+-dependent conformational changes in the intact CaLP molecule and the interactions between CaLP and its target proteins, a truncated CaLP mutant (M-CaLP) devoid of the extended C-terminus was constructed and overexpressed in Escherichia coli. The conformational characteristics of M-CaLP were studied by CD and fluorescence spectroscopy and compared with those of the oyster CaM and CaLP. The far-UV CD results reveal that the extra tail has a strong effect on the Ca2+-induced, but a relatively weak effect on the Mg2+-induced conformational changes in CaLP. However, upon Ca2+ or Mg2+ binding, only slight changes for intrinsic phenylalanine and tyrosine fluorescence spectra between M-CaLP and CaLP are observed. Our results also indicate that the extra tail can significantly decrease the exposure of the hydrophobic patches in CaLP. Additionally, affinity chromatography demonstrates that the target binding of CaLP is greatly influenced by its additional tail. All our results implicate that the extra tail may play some important roles in the interactions between CaLP and its targets in vivo. [Copyright &y& Elsevier]

Abstract: Understanding the molecular composition and the formation mechanism of shell matrix framework is of great interest for biomineralization in mollusk shell. The cDNAs encoding a novel matrix protein family (KRMP) were cloned from the mantle of pearl oyster, Pinctada fucata. Analysis of the deduced amino acid sequences revealed that KRMP have a high proportion of lysine, glycine, and tyrosine, and their predict isoelectric points are higher than any other identified shell matrix protein to our knowledge. The deduced amino acid sequences of KRMP can be divided into three regions, including an N-terminal signal peptide, a lysine-rich basic region interacting with acidic proteins or CO3 2−, and a Gly/Tyr-rich region involved in the protein cross-link via quinone-tanning process. RT-PCR and in situ hybridization demonstrated that KRMP mRNA was specifically expressed in the mantle edge, involved in the prismatic layer formation. Taken together, it seems that KRMP is a matrix protein family participating in the framework formation of prismatic layer. [Copyright &y& Elsevier]

Abstract: Tyrosinase (monophenol, L-DOPA: oxygen oxidoreductase, EC 1.14.18.1), a kind of copper-containing phenoloxidase, arouses great interests of scientists for its important role in periostracum formation. A cDNA clone encoding a putative tyrosinase, termed OT47 because of its estimated molecular mass of 47kDa, was isolated from the pearl oyster, Pinctada fucata. This novel tyrosinase shares similarity with the cephalopod tyrosinases and other type 3 copper proteins within two conserved copper-binding sites. RT-PCR analysis showed that OT47 mRNA was expressed only in the mantle edge. Further in situ hybridization analysis and tyrosinase activity staining revealed that OT47 was expressed at the outer epithelial cells of the middle fold, different from early histological results in Mercenaria mercenaria, suggesting a different model of periostracum secretion in P. fucata. Taken together, these results suggest that OT47 is most likely involved in periostracum formation. The identification and characterization of oyster tyrosinase also help to further understand the structural and functional properties of molluscan tyrosinase. [Copyright &y& Elsevier]

Abstract: The present study describes the details about the acid phosphatase forms in the pearl oyster, Pinctada fucata. Two isoenzymes (AcPase I and II) of acid phosphatase were separated and purified from viscera of pearl oyster, P. fucata to homogeneity by chromatography on DEAE-Sepharose Fast Flow, Sephadex G-200 superfine and ConA Sepharose 4B, and partial biochemical properties of AcPase I and II were studied. AcPase I and AcPase II had molecular weights of 208.8 and 64.3 kDa, respectively. AcPase I was a single polypeptide chain, while AcPase II was a dimeric enzyme composed of two equivalent subunits. AcPase I and II showed optimal pHs at 4.6 and 3.2 with p-nitrophenylphosphate as substrate. The optimal catalytic reaction temperature was 47 °C for AcPase I and 57 °C for AcPase II. Both enzyme forms were stable when incubated at 50 °C for 40 min. Tartrate and fluoride were the most effective inhibitors of the enzymes. Fe3+, Zn2+, Cu2+ and Pb2+ inhibited the activity of AcPase I and II to differing extents. AcPase I and II were apparently nonspecific and hydrolyzed various phosphoric esters. The different properties of AcPase I and II suggested that the two enzymes may play different roles in the pearl oyster. [Copyright &y& Elsevier]

Abstract: A novel carbonic anhydrase (CA) has been purified from the mantle of the pearl oyster, Pinctada fucata, by ammonium sulfate precipitation and affinity chromatography. Its molecular mass was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) to be approximately 38 kDa. Native–PAGE shows that the novel CA can bind a fluorescent probe, 5-dimethylamino-1-naphthalenesulfonamide (DNSA), known to specifically bind carbonic anhydrase. Compared to carbonic anhydrase I (CAI) from human erythrocytes, the novel CA migrates faster indicating that it is more acidic. The effect of an inhibitor on the enzyme activity was also examined. The CA from the mantle showed a weak resistance to acetazolamide (AZ), a specific inhibitor of CA. When DNSA was bound to CA, it caused the wavelength of emission maximum intensity to blue shift to 454 nm upon excitation at 326 nm. Histochemical data indicates that the enzyme is distributed widely throughout the mantle tissue, being concentrated at the edge of the mantle. The evidence presented indicates a function for CA in the process of pearl formation and biomineralization. [Copyright &y& Elsevier]

Abstract: A cDNA clone encoding a novel G protein β subunit of β1 subclass, pfGβ1 was isolated from the pearl oyster (Pinctada fucata). The deduced amino acid sequence of pfGβ1 (341 amino acids) shares high homology to northern European squid (Loligo pealei) and great pond snail (Lymnaea stagnalis) Gβ1, while it has diverged from bovine (Bos taurus) and human. The well-conserved amino acid domains in G protein β subunit, seven WD repeats, were founded in the deduced amino acid sequence. Alignment analysis showed that the beginning amino acid residues in variable fragment of the seventh WD motif are different from any other Gβ. The prediction of 3D structure of pfGβ showed that pfGβ belongs to β-propeller family proteins whose members contain 4–8 antiparallel β-sheets resembling the blades of a propeller. In situ hybridization and Northern blotting analysis revealed that the pfGβ mRNA hybridization signals were widely expressed in various tissues except muscle, with abundantly in epithelia of gill, gonad and outer fold of mantle. We also investigated the interactions between Gβγ and calmodulin (CaM), and specific amino acid residues that may be critical for the binding of Gβγ to CaM were also identified. Furthermore, the functional studies of the interaction showed that the binding of CaM and Gβγ increases the alkaline phosphatase (ALP) activity, an indicator for mineralization in MC3T3-E1 cells. The ALP activity of the mutants of pfGβγ that impaired the interactions of Gβγ with CaM is higher than the Control group; however, it is lower than the WTC group. Together, these results suggest that the Gβγ might interact with CaM and point to the important physiological function in modulating cellular functions. [Copyright &y& Elsevier]

The shells of bivalves are mainly composed of calcium carbonate, a product of calcium metabolism. In the process of shell formation, the uptake, transport and recruitment of calcium ion are highly regulated and involved in many factors. Among these regulatory factors, calmodulin (CaM), a pivotal multifunction regulator of calcium metabolism in nearly all organisms, is thought to play an important role in the calcium metabolism involved in shell formation. In this study, a full-length CaM cDNA was isolated from the pearl oyster (Pinctada fucata). The oyster calmodulin encodes a 16.8 kDa protein which shares high similarity with vertebrate calmodulin. The oyster CaM mRNA shows the highest level of expression in the gill, a key organ involved in calcium uptake in oyster calcium metabolism. In situ hybridization results revealed that oyster CaM mRNA is expressed at the folds and the outer epithelial cells of the dorsal region of the mantle, suggesting that CaM is involved in regulation of calcium transport and secretion. Oyster CaM also showed a typical Ca2+ dependent electrophoretic shift characterization and calcium binding activity. Taken together, we have identified and characterized a pivotal calcium metabolism regulator of the oyster that may play an important role in regulation of calcium uptake, transport and secretion in the process of shell formation. [Copyright &y& Elsevier]

Understanding the molecular composition is of great interest for both nacre formation mechanism and biomineralization in mollusk shell. A cDNA clone encoding an MSI31 relative, termed MSI7 because of its estimated molecular mass of 7.3 kDa, was isolated from the pearl oyster, Pinctada fucata. This novel protein shares similarity with MSI31, a prismatic framework protein of P. fucata. It is peculiar that MSI7 is much shorter in size, harboring only the Gly-rich sequence that has been proposed to be critical for Ca2+ binding. In situ hybridization result showed that MSI7 mRNA was expressed specifically at the folds and outer epithelia of the mantle, indicating that MSI7 participates in the framework formation of both the nacreous layer and prismatic layer. In vitro experiment on the function of MSI7 suggested that it accelerates the nucleation and precipitation of CaCO3. Taken together, we have identified a novel matrix protein of the pearl oyster, which may play an important role in determining the texture of nacre. [Copyright &y& Elsevier]

Iron is one of the most important minor elements in the shell of bivalves. This study was designed to investigate the involvement of ferritin, the principal protein for iron storage, in shell formation. A novel ferritin cDNA from the pearl oyster (Pinctada fucata) was isolated and characterized. The ferritin cDNA encodes a 206 amino acid polypeptide, which shares high similarity with snail soma ferritin and the H-chains of mammalian ferritins. Oyster ferritin mRNA shows the highest level of expression in the mantle, the organ for shell formation. In situ hybridization analysis revealed that oyster ferritin mRNA is expressed at the highest level at the mantle fold, a region essential for metal accumulation and contributes to metal incorporation into the shell. Taken together, these results suggest that ferritin is involved in shell formation by iron storage. The identification and characterization of oyster ferritin also helps to further understand the structural and functional properties of molluscan ferritins. [Copyright &y& Elsevier]