Abstract
Serine protease inhibitors (SPIs) are abundantly reported for its inhibition against specific proteases involved in the immune responses, but SPI data related to calcareous shells are scarce. Previously, our research group has reported the proteome analysis of non-nucleated pearl powder, and a candidate matrix protein containing two Kunitz domains in the acid soluble fraction caught our attention. In the present study, the full-length cDNA sequence of HcKuSPI was obtained from Hyriopsis cumingii. HcKuSPI was specifically expressed in the mantle, with hybridization signals mainly concentrated to dorsal epithelial cells at the mantle edge and weak signals at the mantle pallium, suggesting HcKuSPI was involved in shell formation. HcKuSPI expression in the mantle was upregulated after Aeromonas hydrophila and Staphylococcus aureus challenge to extrapallial fluids (EPFs). A glutathione S transferase (GST)-HcKuSPI recombinant protein showed strong inhibitory activity against the proteases, trypsin and chymotrypsin. Moreover, HcKuSPI expression in an experimental group was significantly higher when compared with a control group during pellicle growth and crystal deposition in shell regeneration processes, while the organic shell framework of newborn prisms and nacre tablets was completely destroyed after HcKuSPI RNA interference (RNAi). Therefore, HcKuSPI secreted by the mantle may effectively neutralize excess proteases and bacterial proteases in the EPF during bacterial infection and could prevent matrix protein extracellular degradation by suppressing protease proteolytic activity, thereby ensuring a smooth shell biomineralization. In addition, GST-HcKuSPI was also crucial for crystal morphology regulation. These results have important implications for our understanding of the potential roles of SPIs during shell biomineralization.
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References
Addadi L, Joester D, Nudelman F, Weiner S (2006) Mollusk shell formation: a source of new concepts for understanding biomineralization processes. Chemistry 12:980–987
Arivalagan J, Yarra T, Marie B, Sleight VA, Duvernois-Berthet E, Clark MS, Marie A, Berland S (2017) Insights from the shell proteome: biomineralization to adaptation. Mol Biol Evol 34:66–77
Bai ZY, Wang GL, Li JL (2011) Identification of genes putatively involved in immune response expressed in mantle of Hyriopsis cumingii. Freshw Fish 41:80–83
Bai ZY, Zheng HF, Lin JY, Wang GL, Li JL (2013) Comparative analysis of the transcriptome in tissues secreting purple and white nacre in the pearl mussel Hyriopsis cumingii. PLoS ONE 8:e53617
Bédouet L, Duplat D, Marie A, Dubost L, Berland S, Rousseau M, Milet C, Lopez E (2007) Heterogeneity of proteinase inhibitors in the water-soluble organic matrix from the oyster nacre. Mar Biotechnol 9:437–449
Calvo-Iglesias J, Perez-Estevez D, Gonzalez-Fernandez A (2017) MSP22.8 is a protease inhibitor-like protein involved in shell mineralization in the edible mussel Mytilus galloprovincialis. FEBS Open Bio 7:1539–1556
Chen X, Peng LH, Chee SS, Shan YH, Liang WQ, Gao JQ (2019) Nanoscaled pearl powder accelerates wound repair and regeneration in vitro and in vivo. Drug Dev Ind Pharm 45:1009–1016
Chen XJ, Liu XJ, Bai ZY, Zhao LT, Li JL (2017) HcTyr and HcTyp-1 of Hyriopsis cumingii, novel tyrosinase and tyrosinase-related protein genes involved in nacre color formation. Comp Biochem Physiol B Biochem Mol Biol 204:1–8
Chiu HF, Hsiao SC, Lu YY, Han YC, Shen YC, Venkatakrishnan K, Wang CK (2018) Efficacy of protein rich pearl powder on antioxidant status in a randomized placebo-controlled trial. J Food Drug Anal 26:309–317
Feng D, Li Q, Yu H, Kong L, Du S (2017) Identification of conserved proteins from diverse shell matrix proteome in Crassostrea gigas: characterization of genetic bases regulating shell formation. Sci Rep 7:45754
Freer A, Bridgett S, Jiang JH, Cusack M (2014) Biomineral proteins from Mytilus edulis mantle tissue transcriptome. Mar Biotechnol 16:34–45
Gerhard EM, Wang W, Li CY, Guo JS, Ozbolat IT, Rahn KM (2017) Design strategies and applications of nacre-based biomaterials. Acta Biomater 54:21–34
Giles R, Manne S, Mann S, Morse DE, Stucky GD, Hansma PK (1995) Inorganic overgrowth of aragonite on molluscan nacre examined by atomic force microscopy. Biol Bull 188:8–15
Grzesiak A, Helland R, Smalas AO, Krowarsch D, Dadlez M, Otlewski J (2000) Substitutions at the P(1) position in BPTI strongly affect the association energy with serine proteinases. J Mol Biol 301:205–217
Harish BS, Uppuluri KB (2018) Microbial serine protease inhibitors and their therapeutic applications. Int J Biol Macromol 107:1373–1387
Huang DD, Li SJX, JL, Bai ZY, (2019a) Integrated transcriptome analysis of immunological responses in the pearl sac of the triangle sail mussel (Hyriopsis cumingii) after mantle implantation. Fish Shellfish Immunol 90:385–394
Huang JL, Li SG, Liu YJ, Liu C, Xie LP, Zhang RQ (2018) Hemocytes in the extrapallial space of Pinctada fucata are involved in immunity and biomineralization. Sci Rep 8:4657
Huang JL, Xie LP, Zhang RQ (2019b) Shell repair and the potential microbial causal in a shell disease of the pearl oyster Pinctada fucata. Fish Shellfish Immunol 86:934–941
Jackson DJ, Mcdougall C, Woodcroft B, Moase P, Rose RA, Kube M, Reinhardt R, Rokhsar DS, Montagnani C, Joubert C, Piquemal D, Degnan BM (2010) Parallel evolution of nacre building gene sets in molluscs. Mol Biol Evol 27:591–608
Jin C, Jiang R, Zhang YH, Cheng K, Luo W, Xie GL (2023) The dual role of eppin in immunity and biomineralization during nacreous layer formation in mollusks. CrystEngComm 25:5160
Jin C, Li JL, Liu XJ (2020) Teosin, a novel basic shell matrix protein from Hyriopsis cumingii induces calcium carbonate polycrystal formation. Int J Biol Macromol 150:1229–1237
Jin C, Liu XJ, Li JL (2019a) A Kunitz proteinase inhibitor (HcKuPI) participated in antimicrobial process during pearl sac formation and induced the overgrowth of calcium carbonate in Hyriopsis cumingii. Fish Shellfish Immunol 89:437–447
Jin C, Zhao JY, Liu XJ, Li JL (2019b) Expressions of shell matrix protein genes in the pearl sac and its correlation with pearl weight in the first 6months of pearl formation in Hyriopsis cumingii. Mar Biotechnol 21:240–249
Jin C, Zhao JY, Pu JW, Liu XJ, Li JL (2019c) Hichin, a chitin binding protein is essential for the self-assembly of organic frameworks and calcium carbonate during shell formation. Int J Biol Macromol 135:745–751
Krowarsch D, Dadlez M, Buczek O, Krokoszynska I, Smalas AO, Otlewski J (1999) Interscaffolding additivity: binding of P1 variants of bovine pancreatic trypsin inhibitor to four serine proteases. J Mol Biol 289:175–186
Lee K, Kim H, Kim JM, Chung YH, Lee TY, Lim HS, Lim JH, Kim T, Bae JS, Woo CH, Kim KJ, Jeong D (2012) Nacre-driven water-soluble factors promote wound healing of the deep burn porcine skin by recovering angiogenesis and fibroblast function. Mol Biol Rep 39:3211–3218
Levi-Kalisman Y, Falini G, Addadi L, Weiner S (2001) Structure of the nacreous organic matrix of a bivalve mollusk shell examined in the hydrated state using cryo-TEM. J Struct Biol 135:8–17
Li JL, Wang DF, Bai ZY, Guan YB, Wu CD, Chen LS (2019) Chinese freshwater pearl culture industry development report. China Fisheries 3:23–29
Li SG, Liu YJ, Liu C, Huang JL, Zheng GL, Xie LP, Zhang RQ (2016) Hemocytes participate in calcium carbonate crystal formation, transportation and shell regeneration in the pearl oyster Pinctada fucata. Fish Shellfish Immunol 51:263–270
Li WJ, Shi ZY, He XJ (2010) Study on immune regulation in Hyriopsis cumingii Lea: effect of pearl-nucleus insertion in the visceral mass on immune factors present in the hemolymph. Fish Shellfish Immunol 28:789–794
Liao Z, Bao LF, Fan MH, Gao P, Wang XX, Qin CL, Li XM (2015) In-depth proteomic analysis of nacre, prism, and myostracum of Mytilus shell. J Proteomics 122:26–407
Lin JY, Ma KY, Bai ZY, Li JL (2013) Molecular cloning and characterization of perlucin from the freshwater pearl mussel, Hyriopsis cumingii. Gene 526:210–216
Liu C, Xu GR, Du JZ, Sun J, Wan X, Liu XJ, Su JT, Liang J, Zheng GL, Xie LP, Zhang RQ (2018) Mineralization of nacre-like structures mediated by extrapallial fluid on pearl nucleus. Cryst Growth Des 18:32–36
Liu MT, Tao JJ, Guo HC, Tang L, Zhang GR, Tang CM, Zhou H, Wu YL, Ruan HJ, Loh XJ (2021) Efficacy of water-soluble pearl powder components extracted by a CO2 supercritical extraction system in promoting wound healing. Materials 14:4458
Liu XJ, Dong SJ, Jin C, Bai ZY, Wang GL, Li JL (2015) Silkmapin of Hyriopsis cumingii, a novel silk-like shell matrix protein involved in nacre formation. Gene 555:217–222
Luo W, Chen Y, Chen C, Ren G (2022a) The proteomics of the freshwater pearl powder: insights from biomineralization to biomedical application. J Proteomics 265:104665
Luo W, Jiang R, Ren G, Jin C (2022b) Hic12, a novel acidic matrix protein promotes the transformation of calcite into vaterite in Hyriopsis cumingii. Comp Biochem Physiol B Biochem Mol Biol 261:110755
Ma YF, Berland S, Andrieu JP, Feng QL, Bedouet L (2013) What is the difference in organic matrix of aragonite vs. vaterite polymorph in natural shell and pearl? Study of the pearl-forming freshwater bivalve mollusc Hyriopsis cumingii. Mater Sci Eng C Mater Biol Appl 33:1521–1529
Ma YF, Qiao L, Feng QL (2012) In-vitro study on calcium carbonate crystal growth mediated by organic matrix extracted from fresh water pearls. Mater Sci Eng C Mater Biol Appl 32:1963–1970
Mann K, Cerveau N, Gummich M, Fritz M, Mann M, Jackson DJ (2018) In-depth proteomic analyses of Haliotis laevigata (greenlip abalone) nacre and prismatic organic shell matrix. Proteome Sci 16:11
Marie B, Marie A, Jackson DJ, Dubost L, Degnan BM, Milet C, Marin F (2010a) Proteomic analysis of the organic matrix of the abalone Haliotis asinina calcified shell. Proteome Sci 8:54
Marie B, Zanella-Cleon I, Le Roy N, Becchi M, Luquet G, Marin F (2010b) Proteomic analysis of the acid-soluble nacre matrix of the bivalve Unio pictorum: detection of novel carbonic anhydrase and putative protease inhibitor proteins. ChemBioChem 11:2138–2147
Marie B, Arivalagan J, Matheron L, Bolbach G, Berland S, Marie A, Marin F (2017) Deep conservation of bivalve nacre proteins highlighted by shell matrix proteomics of the Unionoida Elliptio complanata and Villosa lienosa. J R Soc Interface 14:20160846
Mount AS, Wheeler AP, Paradkar RP, Snider D (2004) Hemocyte-mediated shell mineralization in the eastern oyster. Science 304:297–300
Ranasinghe S, Mcmanus DP (2013) Structure and function of invertebrate Kunitz serine protease inhibitors. Dev Comp Immunol 39:219–227
Shen CH, Zhang M, Liang HY, He JJ, Zhang B, Liang BD (2022) Gene cloning and functional study of PmKSPI from Pinctada fucata martensii. Fish Shellfish Immunol 131:1157–1165
Simkiss K, Wibur KM (1989) Biominerlization. biology and mineral deposition, London: Acedemic Press
Suzuki M, Nagasawa H (2007) The structure–function relationship analysis of Prismalin-14 from the prismatic layer of the Japanese pearl oyster, Pinctada fucata. FEBS J 274:5158–5166
Treccani L, Mann K, Heinemann F, Fritz M (2006) Perlwapin, an abalone nacre protein with three four-disulfide core (whey acidic protein) domains, inhibits the growth of calcium carbonate crystals. Biophys J 91:2601–2608
Wang YY, Xu LW, Wang GL, Wu CD, Li JL (2017) Cloning and expression analysis of KuSPI gene of Hypriopsis cumingii. Genomics and Applied Biology 36:2374–2379
Wei XM, Yang JL, Yang JM, Liu XQ, Liu MJ, Yang DL, Xu J, Hu XK (2012) A four-domain Kunitz-type proteinase inhibitor from Solen grandis is implicated in immune response. Fish Shellfish Immunol 33:1276–1284
Weiner S, Traub W, Parker SB (1984) Macromolecules in mollusc shells and their functions in biomineralization [and discussion]. Philos T R Soc B 304:425–434
Wu ZM, Tang MJ, Zhao JF, Lin ZH, Wang SF, Bao YB (2022) Genome-wide identification and immune response analysis of serine protease inhibitor genes in the blood clam Tegillarca granosa. Fish Shellfish Immunol 131:1234–1244
Yang YL, Chang CH, Huang CC, Liu HW (2015) Anti-inflammation and anti-apoptosis effects of pearl extract gel on UVB irradiation HaCaT cells. Biomed Mater Eng 26:S139–S145
Zhang G, Fang X, Guo X, Li L, Luo R, Xu F, Yang P, Zhang L, Wang X, Qi H, Xiong Z, Que H, Xie Y, Holland PW, Paps J, Zhu Y, Wu F, Chen Y, Wang J, Peng C, Meng J, Yang L, Liu J, Wen B, Zhang N, Huang Z, Zhu Q, Feng Y, Mount A, Hedgecock D, Xu Z, Liu Y, Domazet-Loso T, Du Y, Sun X, Zhang S, Liu B, Cheng P, Jiang X, Li J, Fan D, Wang W, Fu W, Wang T, Wang B, Zhang J, Peng Z, Li Y, Li N, Wang J, Chen M, He Y, Tan F, Song X, Zheng Q, Huang R, Yang H, Du X, Chen L, Yang M, Gaffney PM, Wang S, Luo L, She Z, Ming Y, Huang W, Zhang S, Huang B, Zhang Y, Qu T, Ni P, Miao G, Wang J, Wang Q, Steinberg CE, Wang H, Li N, Qian L, Zhang G, Li Y, Yang H, Liu X, Wang J, Yin Y, Wang J (2012) The oyster genome reveals stress adaptation and complexity of shell formation. Nature 490:49–54
Zhang XW, Yang CH, Xia XH, Pan XT, Jin ZY, Yu H, Zhang HW (2020) A triple WAP domain containing protein acts in antibacterial immunity of weather loach, Misgurnus anguillicaudatus. Fish Shellfish Immunol 103:277–284
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This work was supported by The National Natural Science Foundation of China (32202921) and the Sci-tech Special Commissioners Project of Zhejiang Province-Pearl Quality Improvement and Industrialization (2020–2024).
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Can Jin: Investigation, project administration, writing-original draft and funding acquisition. Kang Cheng and Rui Jiang: methodology, validation, experiment. Yihang Zhang: writing-review and editing. Wen Luo: supervision, funding acquisition. All authors contributed to the article and approved the submitted version.
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Jin, C., Cheng, K., Jiang, R. et al. A Novel Kunitz-Type Serine Protease Inhibitor (HcKuSPI) is Involved in Antibacterial Defense in Innate Immunity and Participates in Shell Formation of Hyriopsis cumingii. Mar Biotechnol 26, 37–49 (2024). https://doi.org/10.1007/s10126-023-10275-8
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DOI: https://doi.org/10.1007/s10126-023-10275-8