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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 (IC 50 ) 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.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

CaCO 3 , which occurs in three crystalline anhydrous polymorphs named calcite, aragonite, and vaterite, is always found in mineralized skeletons or growing shells of many marine organisms. However, understanding how these organisms achieve this control has been a significant challenge in biomineralization. In this work, we proposed a novel vaterite stabilizer acidic matrix protein PNU7 that existed in both prism and nacre of Pinctada fucata, and identified its functional domain DDDDDDHDDVEETED. Our experiments reveal that PNU7 triggers a stable large vaterite formation with Mg 2+ deficiency even under low Ca 2+ . Increasing PNU7 in the calcium carbonate crystallization system with Mg 2+ leads to a significant shrinking in crystal size and rising in nucleation quantity. Moreover, it converts an atomically rough dome-like shape to a smooth sphere on unsiliconized glass. These effects rescind after removing the asp-rich region at the C-terminus. We also find that decreasing pnu7 mRNA in vivo leads to nacreous inner surface growth substantially lessened. Thus, PNU7 may not only supply vaterite in shell formation but also involve the nacreous regulation via surface energy minimization., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Biomineralization is a widespread biological process, involved in the formation of shells, teeth, and bones. Shell matrix proteins have been widely studied for their importance during shell formation. In 2015, our group identified 72 unique shell matrix proteins in Pinctada fucata, among which PU14 is a matrix protein detected in the soluble fraction that solely exists in the prismatic layer. However, the function of PU14 is still unclear. In this study, the full-length cDNA sequence of PU14 was obtained and functional analyses of PU14 protein during shell formation were performed. The deduced protein has a molecular mass of 77.8 kDa and an isoelectric point of 11.34. The primary protein structure contains Gln-rich and random repeat units, which are typical characteristics of matrix protein and indicate its potential function during shell formation. In vivo and in vitro experiments indicated PU14 has prismatic layer functions during shell formation. The tissue expression patterns showed that PU14 was mainly expressed in the mantle tissue, which is consistent with prismatic layer formation. Notching experiments suggested that PU14 responded to repair and regenerate the injured shell. After inhibiting gene expression by injecting PU14-specific double-stranded RNA, the inner surface of the prismatic layer changed significantly and became rougher. Further, in vitro experiments showed that recombinant protein rPU14 impacted calcite crystal morphology. Taken together, characterization and functional analyses of a novel matrix protein, PU14, provide new insights about basic matrix proteins and their functions during shell formation.

Byssuses, which are proteinaceous fibers secreted by mollusks, are remarkable underwater adhesives. Although mussel adhesives are well known, much less is known about the byssal proteins of pearl oysters especially in the adhesive regions. In this study, adhesive proteins from the pearl oyster Pinctada fucata were studied in depth by transcriptomics and proteomics approaches. In total, 16 novel proteins were identified including a von Willebrand factor type A domain-containing protein, a thrombospondin-1-like protein, tyrosinase, mucin-like proteins, protease inhibitors, and Pinctada unannotated foot protein 3 (PUF3) to PUF6. Interestingly, PUF3-6 are enriched with glycine, serine, and PXG (X = F/Y/W/K/L) motifs and are highly expressed in the foot. The identification of byssal proteins of the pearl oyster is a key step for understanding byssus formation and may inspire the synthesis of novel adhesives for underwater use and the development of anti-biofouling strategies.

Biomineralization, especially shell formation, is a sophisticated process regulated by various matrix proteins. Pinctada fucata chitinase-like protein 1 (Pf-Clp1), which belongs to the GH18 family, was discovered by our group using in-depth proteomic analysis. However, its function is still unclear. In this study, we first obtained the full-length cDNA sequence of Pf-Clp1 by RACE. Real-time polymerase chain reaction results revealed that Pf-Clp1 was highly expressed in the important biomineralization tissues, the mantle edge and the mantle pallial. We expressed and purified recombinant protein rPf-Clp1 in vitro to investigate the function of Pf-Clp1 on CaCO3 crystallization. Scanning electron microscopy imaging and Raman spectroscopy revealed that rPf-Clp1 was able to affect the morphologies of calcite crystal in vitro. Shell notching experiments suggested that Pf-Clp1 might function as a negative regulator during shell formation in vivo. Knockdown of Pf-Clp1 by RNAi led to the overgrowth of aragonite tablets, further confirming its potential negative regulation on biomineralization, especially in the nacreous layer. Our work revealed the potential function of molluscan Clp in shell biomineralization for the first time and unveiled some new understandings toward the molecular mechanism of shell formation., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Here, we have identified a novel matrix protein, named PfX, from the pearl oyster Pinctada fucada, and investigated the effects of recombinant PfX protein on calcium carbonate crystallization. The expression of PfX was spatially concentrated in the mantle tissue and gill, the former of which is responsible for the formation of shell structures. The shell notching assay showed a PfX expression response during injured shell repair and regeneration, suggesting the potential involvement of this matrix protein in shell biomineralization. Further, an in vitro crystallization assay showed that PfX could alter the CaCO 3 morphologies of both calcite and aragonite polymorphs. Correspondingly, a binding assay indicated that PfX has strong binding affinity for CaCO 3 crystals, especially aragonite. Further, the protein's calcite binding capacity increased obviously when particular crystal faces were induced. In addition, PfX conjugated with fluorescent dye cyanine-5 (cy5) was preferentially distributed on rough crystal faces instead of the smooth and common (1 0 4) faces of calcite during the crystallization. These results suggest that matrix protein PfX might regulate CaCO 3 morphology via selective binding and inhibit the growth of certain crystal faces, providing new clues for understanding biomineralization mechanisms in mollusk., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.