HcN57, A Novel Unusual Acidic Silk-Like Matrix Protein from Hyriopsis cumingii, Participates in Framework Construction and Nacre Nucleation During Nacreous Layer Formation.

In the classic molecular model of nacreous layer formation, unusual acidic matrix proteins rich in aspartic acid (Asp) residues are essential for nacre nucleation due to their great affinity for binding calcium. However, the acidic matrix proteins discovered in the nacreous layer so far have been weakly acidic with a high proportion of glutamate. In the present study, several silk-like matrix proteins, including the novel matrix protein HcN57, were identified in the ethylenediaminetetraacetic acid-soluble extracts of the nacreous layer of Hyriopsis cumingii. HcN57 is a highly repetitive protein that consists of a high proportion of alanine (Ala, 34.4%), glycine (Gly, 22.5%), and serine (Ser, 11.4%). It forms poly Ala blocks, GlynX repeats, an Ala-Gly repeat, and a Ser-Ala-rich region, exhibiting significant similarity to silk proteins found in spider species. The expression of HcN57 was specifically located in the dorsal epithelial cells of the mantle pallium and mantle center. Notably, expression of HcN57 was relatively high during nacreous layer regeneration and pearl nacre deposition, suggesting HcN57 is a silk matrix protein in the nacreous layer. Importantly, HcN57 also contains a certain content of Asp residues, making it an unusual acidic matrix protein present in the nacreous layer. These Asp residues are mainly distributed in three large hydrophilic acidic regions, which showed inhibitory activity against aragonite deposition and morphological regulation of calcite in vitro. Moreover, HcN57-dsRNA injection resulted in failure of nacre nucleation in vivo. Taken together, our results show that HcN57 is a bifunctional silk protein with poly Ala blocks and Gly-rich regions that serve as space fillers within the chitinous framework to prevent crystallization at unnecessary nucleation sites and Asp-rich regions that create a calcium ion supersaturated microenvironment for nucleation in the center of nacre tablets. These observations contribute to a better understanding of the mechanism by which silk proteins regulate framework construction and nacre nucleation during nacreous layer formation.

Organic Matrices of Calcium Carbonate Biominerals Improve Osteoblastic Mineralization.

Many organisms incorporate inorganic solids into their tissues to improve functional and mechanical properties. The resulting mineralized tissues are called biominerals. Several studies have shown that nacreous biominerals induce osteoblastic extracellular mineralization. Among them, Pinctada margaritifera is well known for the ability of its organic matrix to stimulate bone cells. In this context, we aimed to study the effects of shell extracts from three other Pinctada species (Pinctada radiata, Pinctada maxima, and Pinctada fucata) on osteoblastic extracellular matrix mineralization, by using an in vitro model of mouse osteoblastic precursor cells (MC3T3-E1). For a better understanding of the Pinctada-bone mineralization relationship, we evaluated the effects of 4 other nacreous mollusks that are phylogenetically distant and distinct from the Pinctada genus. In addition, we tested 12 non-nacreous mollusks and one extra-group. Biomineral shell powders were prepared, and their organic matrix was partially extracted using ethanol. Firstly, the effect of these powders and extracts was assessed on the viability of MC3T3-E1. Our results indicated that neither the powder nor the ethanol-soluble matrix (ESM) affected cell viability at low concentrations. Then, we evaluated osteoblastic mineralization using Alizarin Red staining and we found a prominent MC3T3-E1 mineralization mainly induced by nacreous biominerals, especially those belonging to the Pinctada genus. However, few non-nacreous biominerals were also able to stimulate the extracellular mineralization. Overall, our findings validate the remarkable ability of CaCO3 biomineral extracts to promote bone mineralization. Nevertheless, further in vitro and in vivo studies are needed to uncover the mechanisms of action of biominerals in bone.

Transcriptome and exosome proteome analyses provide insights into the mantle exosome involved in nacre color formation of pearl oyster Pinctada fucata martensii.

Color polymorphisms in molluscan shells play an important economic in the aquaculture industry. Among bivalves, shell color diversity can reflect properties such as growth rate and tolerance. In pearl oysters, the nacre color of the donor is closely related to the pearl color. Numerous genes and proteins involved in nacre color formation have been identified within the exosomes of the mantle. In this study, we analyzed the carotenoids present in the mantle of gold- and silver-lipped pearl oysters, identifying capsanthin and xanthophyll as crucial pigments contributing to coloration. Transcriptome analysis of the mantle revealed several differentially expressed genes (DEGs) involved in color formation, including ferric-chelate reductase, mantle genes, and larval shell matrix proteins. We also isolated and identified exosomes from the mantles of both gold- and silver-lipped strains of the pearl oyster Pinctada fucata martensii, revealing the extracellular transition mechanism of coloration-related proteins. From these exosomes, we obtained a total of 1223 proteins, with 126 differentially expressed proteins (DEPs) identified. These proteins include those associated with carotenoid metabolism and Fe(III) metabolism, such as apolipoproteins, scavenger receptor proteins, ß,ß-carotene-15,15'-dioxygenase, ferritin, and ferritin heavy chains. This study may provide a new perspective on the nacre color formation process and the pathways involved in deposition within the pearl oyster P. f. martensii.

miR-10a-3p Participates in Nacre Formation in the Pearl Oyster Pinctada fucata martensii by Targeting NPY.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression via the recognition of their target messenger RNAs. MiR-10a-3p plays an important role in the process of ossification. In this study, we obtained the precursor sequence of miR-10a-3p in the pearl oyster Pinctada fucata martensii (Pm-miR-10a-3p) and verified its sequence by miR-RACE technology, and detected its expression level in the mantle tissues of the pearl oyster P. f. martensii. Pm-nAChRsα and Pm-NPY were identified as the potential target genes of Pm-miR-10a-3p. After the over-expression of Pm-miR-10a-3p, the target genes Pm-nAChRsα and Pm-NPY were downregulated, and the nacre microstructure became disordered. The Pm-miR-10a-3p mimic obviously inhibited the luciferase activity of the 3' untranslated region of the Pm-NPY gene. When the interaction site was mutated, the inhibitory effect disappeared. Our results suggested that Pm-miR-10a-3p participates in nacre formation in P. f. martensii by targeting Pm-NPY. This study can expand our understanding of the mechanism of biomineralization in pearl oysters.

Low-complexity domain-containing protein (LCDP), induced accumulation of calcium carbonate individual crystals to irregular polycrystals in the triangle sail mussel Hyriopsis cumingii.

The nacre layer is composed of sheet-like aragonite crystals, with often loosely arranged polycrystal aragonite sheets which may induce poor mechanical properties in shells. In this study, a full-length low-complexity domain-containing protein (LCDP) cDNA from the triangle sail mussel Hyriopsis cumingii was generated and its role in shell formation investigated. The full-length cDNA was 1058 bp; it had an open reading frame (ORF) of 714 bp encoding 237 amino acids and contained a 20-amino acid signal peptide at the N-terminus and two low-complexity domains. H. cumingii LCDP was not homologous with other species. Tissue expression analyses showed that LCDP was specifically expressed in the mantle. In shell repair assays, significantly higher LCDP expression was observed in the shell repair group from days 12-21 (p < 0.01). After LCDP silencing, aragonite flake shapes in pearl layers became irregular with disordered deposition, while calcium carbonate (CaCO3) crystal surfaces in prismatic layers became rougher and organic matrices between crystals appeared skeletonized, indicating the importance of biomineralization. Our in vitro CaCO3 crystallization assays showed that LCDP induced single crystals to polycrystals, probably via loose arrangement between aragonite flakes. These results provide new insights on freshwater mollusk biomineralization and a theoretical basis for improved pearl quality.

Nacre Extract from Pearl Oyster Shell Prevents D-Galactose-Induced Brain and Skin Aging.

Pearl oyster shells comprise two layers, a prismatic and nacreous layer, of calcium carbonate. The nacreous layer has been used in Chinese medicine since ancient times. In this study, we investigated the effects of the extract from the nacreous layer of pearl oysters (nacre extract) on D-galactose-induced brain and skin aging. Treatment with nacre extract led to the recovery of D-galactose-induced memory impairment, as examined using the Barnes maze, novel object recognition, and Y-maze tests. A histological study showed that nacre extract suppressed D-galactose-induced neuronal cell death and the expression of B cell lymphoma 2 (Bcl-2)-associated X protein (Bax), which causes apoptosis in the hippocampus. In addition, the expression levels of brain-derived neurotrophic factor, which counteracts age-related brain dysfunction, and nicotinamide adenine dinucleotide-dependent deacetylase (sirtuin 1), which delays aging and extends lifespan, increased after nacre extract treatment. Moreover, the nacre extract showed anti-aging effects against D-galactose-induced skin aging; it suppressed D-galactose-induced wrinkle formation, decreased skin moisture, decreased epidermal thickness, and destroyed collagen arrangement associated with aging. Furthermore, the nacre extract suppressed oxidative stress associated with aging in the brain and skin by upregulating the expression of catalase and superoxide dismutase. The expression level of the cellular senescence marker p16, which is induced by oxidative stress, was elevated in the hippocampus and skin epidermal layer of D-galactose-treated mice, and it was suppressed by the administration of nacre extract. These results show that the nacre extract can suppress D-galactose-induced aging by enhancing anti-oxidant activity and suppressing p16 expression. Thus, the nacre extract may be an effective anti-aging agent.

A novel matrix protein PNU5 facilitates the transformation from amorphous calcium carbonate to calcite and aragonite.

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 CaCO3 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.

Organic Matrix and Secondary Metabolites in Nacre.

Nacre, also called mother-of-pearl, is a naturally occurring biomineral, largely studied by chemists, structural biologists, and physicists to understand its outstanding and diverse properties. Nacre is constituted of aragonite nanograins surrounded by organic matrix, and it has been established that the organic matrix is responsible for initiating and guiding the biomineralization process. The first challenge to study the organic matrix of nacre lays in its separation from the biomineral. Several extraction methods have been developed so far. They are categorized as either strong (e.g., decalcification) or soft (e.g., water, ethanol) and they allow specific extractions of targeted compounds. The structure of the nacreous organic matrix is complex, and it provides interesting clues to describe the mineralization process. Proteins, sugars, lipids, peptides, and other molecules have been identified and their role in mineralization investigated. Moreover, the organic matrix of nacre has shown interesting properties for human health. Several studies are investigating its activity on bone mineralization and its properties for skin care. In this review, we focus on the organic constituents, as lipids, sugars, and small metabolites which are less studied since present in small quantities.

Hic12, a novel acidic matrix protein promotes the transformation of calcite into vaterite in Hyriopsis cumingii.

Shell acidic matrix proteins are widely considered to be essential for shell formation given their low affinity and high loading for calcium ion. In the present study, a novel matrix protein, hic12, was isolated from the mantle of Hyriopsis cumingii. High expression in tissue and positive signals with in situ hybridization were detected in the mantle center and mantle pallium, indicating that hic12 mainly participated in the biomineralization of the shell nacreous layer. The expression pattern of hic12 in the pearl sac during early pearl formation indicated that it was involved in pearl biomineralization. Moreover, the recombinant protein, rGST-Hic12, was successfully expressed and purified. The addition of rGST-Hic12 could accelerate the calcium carbonate deposition rate, change the morphology of crystals, and promote the conversion of calcite to vaterite. These results may provide new insights into the molecular mechanisms of aragonite mollusk shell formation.

Identification of a coproporphyrinogen-III oxidase gene and its correlation with nacre color in Hyriopsis cumingii.

Pearl color is an important factor influencing pearl value, and is affected by the nacre color of the shell in Hyriopsis cumingii. Coproporphyrinogen-III oxidase (CPOX) is a key enzyme in porphyrin synthesis, and porphyrins are involved in color formation in different organisms, including in the nacre color of mussels. In this study, a CPOX gene (HcCPOX) was identified from H. cumingii, and its amino acid sequence was found to contain a coprogen-oxidase domain. HcCPOX mRNA was expressed widely in the tissues of white and purple mussels, and the highest expression was found in the gill, followed by the fringe mantle. The expression of HcCPOX in all tissues of purple mussels (except in the middle mantle) was higher than that of white mussels. Strong hybridization signals for HcCPOX were observed in the dorsal epithelial cells of the outer fold of the mantle. The activity of CPOX in the gill, fringe mantle, and foot of purple mussels was significantly higher than that in white mussels. Moreover, the expression of HcCPOX and CPOX activity were decreased in RNA interference experiments. The findings indicate that HcCPOX might contributes to nacre color formation in H. cumingii by being involved in porphyrin synthesis.

Molecular cloning and functional characterization of KCNQ1 in shell biomineralisation of pearl oyster Pinctada fucata martensii.

KCNQ1, a voltage-gated potassium ion channel, plays an important role in various physiological processes, including osteoblast differentiation in higher animals. However, its function in lower invertebrates such as marine shellfish remains poorly understood. Pearl oysters, such as P. fucata martensii, are ideal for studying biomineralisation. In this study, a full-length cDNA of KCNQ1 from P. fucata martensii (PfKCNQ1) was obtained, and its function in shell formation was investigated. The full-length 3945 bp cDNA of PfKCNQ1 included an open reading frame (ORF) of 1944 bp encoding a polypeptide of 647 amino acids. Multiple sequence alignment revealed high homology with KCNQ1 from other species, with six transmembrane domains (S1 - S6) and a pore (P) region. Expression pattern analysis showed that PfKCNQ1 was expressed in all tested tissues, with highest expression in mantle and heart, and shell notching induced PfKCNQ1 expression. Silencing PfKCNQ1 expression inhibited PfKCNQ1 expression and downregulated four biomineralisation-related genes (Shematrin, Pif80, N16 and MSI60). Disordered crystals or "hollows" were visible in the shell ultrastructure by scanning electron microscopy following PfKCNQ1 knockdown. The results suggested that PfKCNQ1 may participate in or regulate biomineralisation and shell formation in pearl oyster.

Nacre extract from pearl oyster attenuates amyloid beta-induced memory impairment.

Shells are composed of two types of calcium carbonate polymorphs-the prismatic layer and the nacreous layer. Pearls, composed of the nacreous layer, have been used in Chinese medicine since ancient times. We have previously shown that extracts from the nacreous layer improves scopolamine-induced memory impairment. However, whether pearl ameliorates cognitive disorders induced by amyloid-ß 1-40 (Aß1-40) has not been elucidated. In this study, we investigated whether nacre extract improves memory impairment induced by intracerebroventricular injection of Aß1-40. Administration of nacre extract led to recovery from Aß1-40-induced impairments in object recognition, short-term memory, and spatial memory. Nacre extract reversed the increase in lipid peroxidation caused by Aß1-40 in the cerebral cortex by increasing the expression of catalase and superoxide dismutase. In addition, nacre extract recovered the expression and phosphorylation of cyclic AMP response element-binding protein (CREB), which decreased with Aß1-40 treatment, and increased the expression of brain-derived neurotrophic factor and neuropeptide Y, which are regulated by CREB. Nacre extract also suppressed acetylcholine esterase activity and Aß1-40-induced tau phosphorylation. Histochemical analysis of the hippocampus region showed that the nacre extract protected against Aß1-40-induced neuronal loss in the hippocampus. These results suggest that nacre extract protects against Aß1-40-induced neuronal cell death by suppressing oxidative stress and increasing the expression and phosphorylation of CREB.

The temptin gene of the clade Lophotrochozoa is involved in formation of the prismatic layer during biomineralization in molluscs.

The biomineralization mechanism of mollusc shell has been studied for a long time, but there is a lack of understanding about the relationship between the shell formation in vitro and the signaling system in vivo. In this study, we cloned a novel shell matrix protein gene (hc-temptin), which only be characterized as a water-borne protein pheromone of molluscs in previous studies, from the freshwater mussel Hyriopsis cumingii. By bioinformatics analysis we found that temptin was a gene unique to the clade Lophotrochozoa, and it exists in all mollusc taxa except Cephalopoda. The current data supported the premise that temptin was generated in the early emergence of molluscs and that it maintained a high mutation rate to evolve relative independently. The specificity of hc-temptin expression in the mantle tissue suggests its potential to participate in biomineralization. Its sequence contained typical Ca2+ binding sites. Our experiments involving the pearl formation process, damaged shell repair process, and RNAi experiment showed that hc-temptin was a shell matrix protein that plays an important role in formation of the prismatic layer. The results of this study provided new insights about the origin of the temptin gene and its role in molluscs.

The mantle exosome proteins of Hyriopsis cumingii participate in shell and nacre color formation.

Pearl color is closely related to the nacre color of shell in Hyriopsis cumingii, and pearl color has a huge impact on its price. The nacre is an important part of the shell, and studies have suggested that mantle exosomes participated in shell formation. Exosomes contain many different proteins that are involved in different biological processes. In this study, exosomes were extracted from mantles of mussels with different nacre color. TMT quantitative proteome sequencing analysis was performed on purple and white mussel mantle exosomes, and 4861 proteins were obtained. Based on the standard of (|log2 (Fold change)| ≥ 1.2 or ≤ 0.83 and p-value ≤ 0.05), a total of 758 differentially expressed proteins were found. Some proteins involved in shell and nacre color formation were predicted with the proteins annotate, GO classification system. Moreover, 14 differentially expressed proteins (including eight up-regulated proteins and six down-regulated proteins) were validated using parallel reaction monitoring (PRM) assays. Overall, this information will be useful to improve our understanding of the molecular mechanisms of shell and nacre color formation in H. cumingii.

Cloning of a HcCreb gene and analysis of its effects on nacre color and melanin synthesis in Hyriopsis cumingii.

Creb (Cyclic AMP response element binding protein) is a nuclear regulatory factor that regulates transcription through autophosphorylation. In melanocytes, cAMP's corresponding elements bind to the Creb protein to autophosphorylation and activate MITF (Microphthalmia-associated transcription factor). MITF stimulates Tyrosine(tyr) to induce melanocytes to differentiate into eumelanin and pheomelanin. In this study, a HcCreb gene in Hyriopsis cumingii was cloned and its effects on melanin synthesis and nacre color were studied. HcCreb was expressed in both purple and white mussels, and there was a significant difference in expression between adductor muscle (p<0.01) and mantle tissue (p<0.05). Other tissues did not show significant differences (except for gill tissue), and in general, the level of mRNA expression was higher in purple mussels than in white mussels. In both white and purple mussels expression levels in gill tissue was the highest, followed by the mantle. Strong and specific mRNA signals were detected in the dorsal epithelial cells of the mantle pallial layer, indicating that HcCreb may be involved in nacre formation. After arbutin treatment, the expression of HcCreb decreased significantly. By further testing the changes in mantle melanin content it was found that the melanin content after arbutin treatment decreased significantly compared to the control group (p<0.05). It is speculated that the HcCreb gene plays a role in the process of melanin synthesis and nacre color formation in H. cumingii.

Novel globular C1q domain-containing protein (PmC1qDC-1) participates in shell formation and responses to pathogen-associated molecular patterns stimulation in Pinctada fucata martensii.

The C1q protein, which contains the globular C1q (gC1q) domain, is involved in the innate immune response, and is found abundantly in the shell, and it participates in the shell formation. In this study, a novel gC1q domain-containing gene was identified from Pinctada fucata martensii (P. f. martensii) and designated as PmC1qDC-1. The full-length sequence of PmC1qDC-1 was 902 bp with a 534 bp open reading frame (ORF), encoding a polypeptide of 177 amino acids. Quantitative real-time PCR (qRT-PCR) result showed that PmC1qDC-1 was widely expressed in all tested tissues, including shell formation-associated tissue and immune-related tissue. PmC1qDC-1 expression was significantly high in the blastula and gastrula and especially among the juvenile stage, which is the most important stage of dissoconch shell formation. PmC1qDC-1 expression was located in the outer epithelial cells of mantle pallial and mantle edge and irregular crystal tablets were observed in the nacre upon knockdown of PmC1qDC-1 expression at mantle pallial. Moreover, the recombined protein PmC1qDC-1 increased the rate of calcium carbonate precipitation. Besides, PmC1qDC-1 expression was significantly up-regulated in the mantle pallial at 6 h and was significantly up-regulated in the mantle edge at 12 h and 24 h after shell notching. The expression level of PmC1qDC-1 in mantle edge was significantly up-regulated at 48 h after LPS stimulation and was significantly up-regulated at 12 h, 24 h and 48 h after poly I:C stimulation. Moreover, PmC1qDC-1 expression was significantly up-regulated in hemocytes at 6 h after lipopolysaccharide (LPS) and poly I:C challenge. These findings suggest that PmC1qDC-1 plays a crucial role both in the shell formation and the innate immune response in pearl oysters, providing new clues for understanding the shell formation and defense mechanism in mollusk.

Comprehensive analysis of microRNAs in the mantle central and mantle edge provide insights into shell formation in pearl oyster Pinctada fucata martensii.

MicroRNAs (miRNAs) are a class of non-coding RNA molecules with post-transcriptional regulatory activity in various biological processes. Pearl oyster Pinctada fucata martensii is one of the main species cultured for marine pearl production in China and Japan. In this study, we constructed two small RNA libraries of mantle central (MC) and mantle edge (ME) from P. f. martensii and obtained 24,175,537 and 21,593,898 clean reads, respectively. A total of 258 miRNAs of P. f. martensii (Pm-miRNA) were identified, and 93 differentially expressed miRNAs (DEMs) including 49 known Pm-miRNAs and 44 novel Pm-miRNAs were obtained from the MC and ME. The target transcripts of these DEMs were obviously enriched in neuroactive ligand-receptor interaction pathway, and others. After over-expression of Pm-miR-124 and Pm-miR-9a-5p in the MC by mimic injection into the muscle of P. f. martensii, nacre exhibited a disorderly growth as detected by scanning electron microscopy. Pm-nicotinic acetylcholine receptor alpha subunit, Pm-neuropeptide Y and Pm-chitin synthase were investigated as the targets of Pm-miR-124; and Pm-tumor necrosis factor receptor associated factor 2 and Pm-chitin synthase were investigated as the targets of Pm-miR-9a-5p. These predicted target transcripts were down-regulated after the over-expression of Pm-miR-124 and Pm-miR-9a-5p in MC. This study comprehensively analyzed the miRNAs in mantle tissues to enhance our understanding of the regulatory mechanism underlying shell formation.

A shell matrix protein of Pinctada mazatlanica produces nacre platelets in vitro.

Nacre is the main component of the pearl oyster shells and it is synthesized by specialized soluble and insoluble shell matrix proteins. Insoluble proteins from the decalcification of the shell are the less studied proteins due to the technical problems to isolate them from the organic matrix. In this study, an insoluble shell matrix protein from Pinctada mazatlanica, pearlin (Pmaz-pearlin), was successfully cloned from the mantle tissue, and the native protein isolated from the shell was functionally characterized. The full coding sequence of Pmaz-pearlin mRNA consists of 423 base pairs, which encode to a 16.3 kDa pearlin. Analysis of the deduced amino acid sequence revealed that Pmaz-pearlin contained four acidic regions, an NG repeat domain, and Cys conserved residues, the latter potentially forms four disulfide bridges which might stabilize the protein structure. The isolated protein from the shell is a glycoprotein of ~ 16.74 kDa which can produce aragonite and calcite crystals in vitro. Our results show that Pmaz-pearlin is a well-conserved protein involved in nacre layer growth, which produces calcite crystals in the presence of CaCl2, aragonite crystal polymorphs with a hexagonal structure in the presence of MgCl2, and needle-like crystal structure polymorphs in the presence of CaCO3 The identity of the crystals was confirmed using RAMAN analyses.

A species-specific miRNA participates in biomineralization by targeting CDS regions of Prisilkin-39 and ACCBP in Pinctada fucata.

Biomineralization is a sophisticated biological process precisely regulated by multiple molecules and pathways. Accumulating miRNAs have been identified in invertebrates but their functions in biomineralization are poorly studied. Here, an oyster species-specific miRNA, novel_miR_1 was found to regulate biomineralization in Pinctada fucata. Target prediction showed that novel_miR_1 could target Prisilkin-39 and ACCBP by binding to their coding sequences (CDS). Tissue distribution analysis revealed that the expression level of novel_miR_1 was highest in the mantle, which was a key tissue participating in biomineralization. Gain-of-function assay in vivo showed that biomineralization-related genes including Prisilkin-39 and ACCBP were down-regulated and shell inner surfaces of both prismatic and nacreous layer were disrupted after the over-expression of novel_miR_1, indicating its dual roles in biomineralization. Furthermore, the shell notching results indicated that novel_miR_1 was involved in shell regeneration. Dual-luciferase reporter assay in vitro demonstrated that novel_miR_1 directly suppressed Prisilkin-39 and ACCBP genes by binding to the CDS regions. Taken together, these results suggest that novel_miR_1 is a direct negative regulator to Prisilkin-39 and ACCBP and plays an indispensable and important role in biomineralization in both prismatic and nacreous layer of P. fucata.

PfSMAD1/5 Can Interact with PfSMAD4 to Inhibit PfMSX to Regulate Shell Biomineralization in Pinctada fucata martensii.

The BMP2 signal transduced by SMAD1/5 plays an important role in osteoblast differentiation and bone formation. Shell formation of Pinctada fucata martensii is a typical biomineralization process that is similar to that of teeth/bone formation. However, whether the Pinctada fucata BMP2 (PfBMP2) signal transduced by PfSMAD1/5 occurs in P. f. martensii, how the PfBMP2 signal is transduced by PfSMAD1/5, and how PfSMAD1/5 regulates the biomineralization process in this species and other shellfish are poorly understood. Therefore, injection experiments of recombinant PfBMP2 and inhibitor dorsomorphin revealed that PfSMAD1/5 can transduce PfBMP2 signals. Subcellular localization and bimolecular fluorescence complementation assays indicated that PfSMAD1/5 phosphorylated by PfBMPR1b interacts with PfSMAD4 in the cytoplasm to form a complex, which translocates to the nucleus to transduce PfBMP2 signals. Co-immunoprecipitation and luciferase assays revealed that PfSMAD1/5 may interact with PfMSX to dislodge it from its binding element, resulting in initiation of mantle gene transcription. The in vivo functional assay showed that knockdown of PfMSAD1/5 decreased expression of shell matrix genes and disordered the nacreous layer, and the correlation assay of shell regeneration showed the concomitant expression pattern of PfSMAD1/5 and shell matrix genes. Together, these data showed that PfSMAD1/5 can transduce PfBMP2 signals to regulate shell biomineralization in P. f. martensii, which illustrated conservation of the BMP2-SMAD signal pathway among invertebrates. Particularly, the results suggest that there is only one PfMSX gene, which functions like the Hox gene in vertebrates, that interacts with PfSMAD1/5 in a protein-protein action form and plays the role of transcription repressor.