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A Novel Acidic Matrix Protein, PfN44, Stabilizes Magnesium Calcite to Inhibit the Crystallization of Aragonite*

  • Cong Pan
    Affiliations
    Institute of Marine Biotechnology, School of Life Sciences, Tsinghua University, Beijing 100084 China
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  • Dong Fang
    Affiliations
    Institute of Marine Biotechnology, School of Life Sciences, Tsinghua University, Beijing 100084 China
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  • Guangrui Xu
    Affiliations
    Institute of Marine Biotechnology, School of Life Sciences, Tsinghua University, Beijing 100084 China
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  • Jian Liang
    Affiliations
    Institute of Marine Biotechnology, School of Life Sciences, Tsinghua University, Beijing 100084 China
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  • Guiyou Zhang
    Affiliations
    Institute of Marine Biotechnology, School of Life Sciences, Tsinghua University, Beijing 100084 China

    Protein Science Laboratory of the Ministry of Education, Tsinghua University, Beijing 100084 China
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  • Hongzhong Wang
    Affiliations
    Institute of Marine Biotechnology, School of Life Sciences, Tsinghua University, Beijing 100084 China

    Protein Science Laboratory of the Ministry of Education, Tsinghua University, Beijing 100084 China
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  • Liping Xie
    Correspondence
    To whom correspondence may be addressed: Rm. 206, Old Building of School of Life Sciences, Tsinghua University, Beijing 100084, China. Tel.: 86-010-62772899; Fax: 86-010-62772899
    Affiliations
    Institute of Marine Biotechnology, School of Life Sciences, Tsinghua University, Beijing 100084 China

    Protein Science Laboratory of the Ministry of Education, Tsinghua University, Beijing 100084 China
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  • Rongqing Zhang
    Correspondence
    To whom correspondence may be addressed: Rm. 211, Old Building of School of Life Sciences, Tsinghua University, Beijing 100084, China. Tel.: 86-010-62772630; Fax: 86-010-62772899
    Affiliations
    Institute of Marine Biotechnology, School of Life Sciences, Tsinghua University, Beijing 100084 China

    Protein Science Laboratory of the Ministry of Education, Tsinghua University, Beijing 100084 China
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  • Author Footnotes
    * This work was supported by National Basic Research Program of China Grant 2010CB126405, National High Technology Research and Development Program of China Grant 2010AA09Z405, National Natural Science Foundation of China Grants 31172382 and U0831001 (Joint Fund with Guangdong), and Independent Research Projects of Tsinghua University Grant 20111080964. Grant 2011ZX08011-006 is a Major Project of Ministry of Agriculture.
Open AccessPublished:December 03, 2013DOI:https://doi.org/10.1074/jbc.M113.504027
      Magnesium is widely used to control calcium carbonate deposition in the shell of pearl oysters. Matrix proteins in the shell are responsible for nucleation and growth of calcium carbonate crystals. However, there is no direct evidence supporting a connection between matrix proteins and magnesium. Here, we identified a novel acidic matrix protein named PfN44 that affected aragonite formation in the shell of the pearl oyster Pinctada fucata. Using immunogold labeling assays, we found PfN44 in both the nacreous and prismatic layers. In shell repair, PfN44 was repressed, whereas other matrix proteins were up-regulated. Disturbing the function of PfN44 by RNAi led to the deposition of porous nacreous tablets with overgrowth of crystals in the nacreous layer. By in vitro circular dichroism spectra and fluorescence quenching, we found that PfN44 bound to both calcium and magnesium with a stronger affinity for magnesium. During in vitro calcium carbonate crystallization and calcification of amorphous calcium carbonate, PfN44 regulated the magnesium content of crystalline carbonate polymorphs and stabilized magnesium calcite to inhibit aragonite deposition. Taken together, our results suggested that by stabilizing magnesium calcite to inhibit aragonite deposition, PfN44 participated in P. fucata shell formation. These observations extend our understanding of the connections between matrix proteins and magnesium.

      Introduction

      Many organisms must control mineral formation through biomineralization to live. A main issue in the study of biomineralization is an understanding of the structure of minerals and the functions of macromolecules in mineral formation (
      • Wilkinson B.H.
      Biomineralization, paleoceanography, and the evolution of calcareous marine organisms.
      ,
      • Simkiss K.
      Bio-mineralization and detoxification.
      ,
      • Paine M.L.
      • White S.N.
      • Luo W.
      • Fong H.
      • Sarikaya M.
      • Snead M.L.
      Regulated gene expression dictates enamel structure and tooth function.
      ). Calcium carbonate is widely used by metazoan taxa, including sea urchins, sponges, crustacean, mollusks, and ascidians, to deposit biominerals for protection (
      • Wilkinson B.H.
      Biomineralization, paleoceanography, and the evolution of calcareous marine organisms.
      ,
      • Simkiss K.
      Bio-mineralization and detoxification.
      ,
      • Radha A.V.
      • Forbes T.Z.
      • Killian C.E.
      • Gilbert P.U.
      • Navrotsky A.
      Transformation and crystallization energetics of synthetic and biogenic amorphous calcium carbonate.
      ,
      • Lowenstam H.A.
      • Abbott D.P.
      Vaterite. Mineralization product of hard tissues of a marine organism (Ascidiacea).
      ). At ambient pressure, calcium carbonate forms five crystalline polymorphs: calcite, aragonite, and vaterite in the anhydrous phase and monohydrocalcite and ikaite in the hydrate phase. Calcium carbonate also forms amorphous forms including amorphous calcium carbonate (ACC),
      The abbreviations used are: ACC
      amorphous calcium carbonate
      RACE
      rapid amplification of cDNA ends
      SEM
      scanning electron microscope
      dsRNA
      double-stranded RNA
      qPCR
      quantitative PCR.
      which is widely accepted as the precursor in biomineralization (
      • Miyamoto H.
      • Miyashita T.
      • Okushima M.
      • Nakano S.
      • Morita T.
      • Matsushiro A.
      A carbonic anhydrase from the nacreous layer in oyster pearls.
      ,
      • Aizenberg J.
      • Addadi L.
      • Weiner S.
      • Lambert G.
      Stabilization of amorphous calcium carbonate by specialized macromolecules in biological and synthetic precipitates.
      ,
      • Muramoto K.
      • Yako H.
      • Murakami K.
      • Odo S.
      • Kamiya H.
      Inhibition of the growth of calcium carbonate crystals by multiple lectins in the celomic fluid of the acorn barnacle Megabalanus rosa.
      ,
      • Borbas J.E.
      • Wheeler A.P.
      • Sikes C.S.
      molluscan shell matrix phosphoproteins. Correlation of degree of phosphorylation to shell mineral microstructure and to in vitro regulation of mineralization.
      ,
      • Maruyama K.
      • Mikawa T.
      • Ebashi S.
      Detection of calcium-binding proteins by Ca-45 autoradiography on nitrocellulose membrane after sodium dodecyl sulfate gel electrophoresis.
      ,
      • Ueno M.
      Calcium transport in crayfish gastrolith disk. Morphology of gastrolith disk and ultrahistochemical demonstration of calcium.
      ). Biomineralization in the pearl oyster Pinctada fucata is a focus of calcium carbonate crystallization studies (
      • Addadi L.
      • Weiner S.
      Biomineralization. A pavement of pearl.
      ,
      • Weiner S.
      Biomineralization. A structural perspective.
      ). A substantial amount of work has explored the relationship between macromolecules and calcium carbonate crystallization, but information on the molecular mechanisms of crystallization is still limited (
      • Furuhashi T.
      • Schwarzinger C.
      • Miksik I.
      • Smrz M.
      • Beran A.
      Molluscan shell evolution with review of shell calcification hypothesis.
      ,
      • Bonucci E.
      Calcification and silicification. A comparative survey of the early stages of biomineralization.
      ,
      • Marin F.
      • Luquet G.
      • Marie B.
      • Medakovic D.
      Molluscan shell proteins. Primary structure, origin, and evolution.
      ).
      The P. fucata shell is composed of two mineralized layers, i.e. the outer prismatic layer and the inner nacreous layer. The main components of these two layers are calcium carbonate crystals that are deposited as calcite in the prismatic layer and aragonite in the nacreous layer (
      • Almeida M.J.
      • Milet C.
      • Peduzzi J.
      • Pereira L.
      • Haigle J.
      • Barthelemy M.
      • Lopez E.
      Effect of water-soluble matrix fraction extracted from the nacre of Pinctada maxima on the alkaline phosphatase activity of cultured fibroblasts.
      ). The proteins in the shell, which are also referred to as matrix proteins, are believed to play important roles in the control of calcium carbonate nucleation and growth (
      • Liu X.
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      • Xiang L.
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      The role of matrix proteins in the control of nacreous layer deposition during pearl formation.
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      Cloning and characterization of the activin-like receptor 1 homolog (Pf-Alr1) in the pearl oyster, Pinctada fucata.
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      • Miyazaki Y.
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      • Aoki H.
      • Samata T.
      Expression of genes responsible for biomineralization of Pinctada fucata during development.
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      • Weil S.
      • Aflalo E.D.
      • Pamuru R.R.
      • Khalaila I.
      • Bentov S.
      • Berman A.
      • Sagi A.
      A protein involved in the assembly of an extracellular calcium storage matrix.
      ). These matrix proteins are currently being isolated from <5% (w/w) of the shell (
      • Miyamoto H.
      • Miyashita T.
      • Okushima M.
      • Nakano S.
      • Morita T.
      • Matsushiro A.
      A carbonic anhydrase from the nacreous layer in oyster pearls.
      ,
      • Tsukamoto D.
      • Sarashina I.
      • Endo K.
      Structure and expression of an unusually acidic matrix protein of pearl oyster shells.
      ,
      • Zhang C.
      • Xie L.
      • Huang J.
      • Liu X.
      • Zhang R.
      A novel matrix protein family participating in the prismatic layer framework formation of pearl oyster, Pinctada fucata.
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      • Zhang Y.
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      • Meng Q.
      • Jiang T.
      • Pu R.
      • Chen L.
      • Zhang R.
      A novel matrix protein participating in the nacre framework formation of pearl oyster, Pinctada fucata.
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      • Sudo S.
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      • Nagakura T.
      • Ohkubo T.
      • Sakaguchi K.
      • Tanaka M.
      • Nakashima K.
      • Takahashi T.
      Structures of mollusc shell framework proteins.
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      • Horita C.
      • Akera S.
      A new matrix protein family related to the nacreous layer formation of Pinctada fucata.
      ,
      • Yano M.
      • Nagai K.
      • Morimoto K.
      • Miyamoto H.
      A novel nacre protein N19 in the pearl oyster Pinctada fucata.
      ,
      • Suzuki M.
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      • Kato T.
      • Nagasawa H.
      An acidic matrix protein, Pif, is a key macromolecule for nacre formation.
      ,
      • Suzuki M.
      • Murayama E.
      • Inoue H.
      • Ozaki N.
      • Tohse H.
      • Kogure T.
      • Nagasawa H.
      Characterization of prismalin-14, a novel matrix protein from the prismatic layer of the Japanese pearl oyster (Pinctada fucata).
      ). The acidic matrix proteins, which have cation binding capacity, are believed to be the main proteins involved in control of calcium carbonate crystallization and shell formation (
      • Maruyama K.
      • Mikawa T.
      • Ebashi S.
      Detection of calcium-binding proteins by Ca-45 autoradiography on nitrocellulose membrane after sodium dodecyl sulfate gel electrophoresis.
      ,
      • Ueno M.
      Calcium transport in crayfish gastrolith disk. Morphology of gastrolith disk and ultrahistochemical demonstration of calcium.
      ,
      • Weiner S.
      Aspartic acid-rich proteins. Major components of the soluble organic matrix of mollusk shells.
      ,
      • Fujimura T.
      • Wada K.
      • Iwaki T.
      Development and morphology of the pearl oyster larvae,.
      ). Previous studies reported that these acidic proteins control the polymorphism and morphology of calcium carbonate in vitro (
      • Samata T.
      • Hayashi N.
      • Kono M.
      • Hasegawa K.
      • Horita C.
      • Akera S.
      A new matrix protein family related to the nacreous layer formation of Pinctada fucata.
      ,
      • Suzuki M.
      • Saruwatari K.
      • Kogure T.
      • Yamamoto Y.
      • Nishimura T.
      • Kato T.
      • Nagasawa H.
      An acidic matrix protein, Pif, is a key macromolecule for nacre formation.
      ,
      • Kono M.
      • Hayashi N.
      • Samata T.
      Molecular mechanism of the nacreous layer formation in Pinctada maxima.
      ).
      In contrast to classical stepwise growth via the terrace-ledge-kink model of crystal growth, the deposition of biominerals begins with ACC as an intermediate that is transformed to crystalline carbonate polymorphs such as calcite or aragonite (
      • Wang D.
      • Wallace A.F.
      • De Yoreo J.J.
      • Dove P.M.
      Carboxylated molecules regulate magnesium content of amorphous calcium carbonates during calcification.
      ). Most naturally occurring ACC contains magnesium, and ACC crystallization is under control of biomolecules (
      • Weiner S.
      • Levi-Kalisman Y.
      • Raz S.
      • Addadi L.
      Biologically formed amorphous calcium carbonate.
      ). Magnesium has a strong influence on calcium carbonate precipitation. Magnesium is incorporated into calcite when the magnesium content is low or induces the formation of aragonite when magnesium content is high (
      • Raz S.
      • Weiner S.
      • Addadi L.
      Formation of high magnesian calcites via an amorphous precursor phase. Possible biological Implications.
      ). Calcite with high magnesium content is not stable and is generally not formed under ambient conditions. However, in many marine organisms, deposited calcite has a high incorporation of magnesium (
      • Bischoff W.D.
      • Mackenzie F.T.
      • Bishop F.C.
      Stabilities of synthetic magnesian calcites in aqueous solution. Comparison with biogenic materials.
      ). The topic of the mechanism by which the biologically controlled deposition of the thermodynamically unstable magnesium calcite in marine organisms is very interesting.
      However, because of the lack of efficient technology for analyzing shell proteins, only a few matrix proteins have been purified and analyzed (
      • Weiner S.
      Biomineralization. A structural perspective.
      ,
      • Weiner S.
      • Addadi L.
      Crystallization pathways in biomineralization.
      ,
      • Weiner S.
      • Sagi I.
      • Addadi L.
      Choosing the crystallization path less traveled.
      ). During shell growth, calcite in the prismatic layer and aragonite in the nacreous layer are under tight control of the matrix proteins (
      • Miyamoto H.
      • Miyashita T.
      • Okushima M.
      • Nakano S.
      • Morita T.
      • Matsushiro A.
      A carbonic anhydrase from the nacreous layer in oyster pearls.
      ,
      • Almeida M.J.
      • Milet C.
      • Peduzzi J.
      • Pereira L.
      • Haigle J.
      • Barthelemy M.
      • Lopez E.
      Effect of water-soluble matrix fraction extracted from the nacre of Pinctada maxima on the alkaline phosphatase activity of cultured fibroblasts.
      ,
      • Mouriès L.P.
      • Almeida M.J.
      • Milet C.
      • Berland S.
      • Lopez E.
      Bioactivity of nacre water-soluble organic matrix from the bivalve mollusk Pinctada maxima in three mammalian cell types: fibroblasts, bone marrow stromal cells, and osteoblasts.
      ,
      • Sud D.
      • Doumenc D.
      • Lopez E.
      • Milet C.
      Role of water-soluble matrix fraction, extracted from the nacre of Pinctada maxima, in the regulation of cell activity in abalone mantle cell culture (Haliotis Tuberculata).
      ,
      • Albeck S.
      • Weiner S.
      • Addadi L.
      Polysaccharides of intracrystalline glycoproteins modulate calcite crystal growth in Vitro.
      ). Different matrix proteins are involved in the formation of different layers (
      • Fu G.
      • Valiyaveettil S.
      • Wopenka B.
      • Morse D.E.
      Caco3 Biomineralization. Acidic 8-kDa proteins isolated from aragonitic abalone shell nacre can specifically modify calcite crystal morphology.
      ,
      • Michenfelder M.
      • Fu G.
      • Lawrence C.
      • Weaver J.C.
      • Wustman B.A.
      • Taranto L.
      • Evans J.S.
      • Morse D.E.
      Characterization of two molluscan crystal-modulating biomineralization proteins and identification of putative mineral binding domains.
      ,
      • Gotliv B.A.
      • Addadi L.
      • Weiner S.
      Mollusk shell acidic proteins. In search of individual functions.
      ). To isolate matrix proteins involved in shell formation, three suppression substrate hybridization libraries were constructed (
      • Fang D.
      • Xu G.
      • Hu Y.
      • Pan C.
      • Xie L.
      • Zhang R.
      Identification of genes directly involved in shell formation and their functions in pearl oyster, Pinctada fucata.
      ). In this study we identified an acidic protein, PfN44, in the shell using the suppression substrate hybridization libraries and the mantle tissue transcriptome. The nature and functions of PfN44 were analyzed.

      Ethics Statement

      All rabbits were raised under standardized pathogen-free conditions in the Animal Care Facility at Beijing Hospital. The study protocol for the experimental use of the animals was approved by the Ethics Committee of National Center for Clinical Laboratories.

      DISCUSSION

      Biogenetic solid minerals known as biominerals are different from inorganic crystals. Biominerals have extraordinary physical and mechanical properties and can be synthesized in an ambient environment. In P. fucata, biominerals were deposited under the control of matrix proteins, specifically acidic proteins (
      • Gotliv B.A.
      • Addadi L.
      • Weiner S.
      Mollusk shell acidic proteins. In search of individual functions.
      ,
      • Marie B.
      • Marin F.
      • Marie A.
      • Bédouet L.
      • Dubost L.
      • Alcaraz G.
      • Milet C.
      • Luquet G.
      Evolution of nacre. Biochemistry and proteomics of the shell organic matrix of the cephalopod Nautilus macromphalus.
      ,
      • Marie B.
      • Luquet G.
      • Pais De Barros J.-P.
      • Guichard N.
      • Morel S.
      • Alcaraz G.
      • Bollache L.
      • Marin F.
      The shell matrix of the freshwater mussel unio pictorum (Paleoheterodonta, Unionoida).
      ,
      • Nudelman F.
      • Shimoni E.
      • Klein E.
      • Rousseau M.
      • Bourrat X.
      • Lopez E.
      • Addadi L.
      • Weiner S.
      Forming nacreous layer of the shells of the bivalves Atrina rigida Pinctada margaritifera. An environmental- and cryo-scanning electron microscopy study.
      ,
      • Pereira-Mouriès L.
      • Almeida M.-J.
      • Ribeiro C.
      • Peduzzi J.
      • Barthélemy M.
      • Milet C.
      • Lopez E.
      Soluble silk-like organic matrix in the nacreous layer of the bivalve Pinctada maxima.
      ). We analyzed the in vivo and in vitro functions of the novel acidic matrix protein PfN44.
      In shell-notching experiments, the expression of PfN44, nacrein, and KRMP3 increased 6 h after notching, which might have been caused by the notching procedure. Notching disturbed the growth of the oyster, so the expression of matrix proteins might also have been disturbed after notching. Shell repair processes are similar to creating new shell. Calcium carbonate crystals accumulated on the matrix. Nacrein, which contains two carbonic anhydrase domains that are important in biomineralization, catalyzes the formation of hydrogen carbonate from water and CO2 (
      • Miyamoto H.
      • Miyashita T.
      • Okushima M.
      • Nakano S.
      • Morita T.
      • Matsushiro A.
      A carbonic anhydrase from the nacreous layer in oyster pearls.
      ). Although nacrein inhibits calcification (
      • Miyamoto H.
      • Miyoshi F.
      • Kohno J.
      The carbonic anhydrase domain protein nacrein is expressed in the epithelial cells of the mantle and acts as a negative regulator in calcification in the mollusc Pinctada fucata.
      ), it must accumulate in the shell to provide sufficient hydrogen carbonate for crystal formation. KRMP participates in the framework formation of the prismatic layer. During shell repair, KRMP3 accumulates in the shell to facilitate this framework formation. In contrast to nacrein and KRMP, PfN44 expression decreased during shell repair. We found that PfN44 inhibited aragonite deposition in in vitro calcium carbonate crystallization and ACC transition. Therefore, PfN44 might be a “safety guard” that slows nacreous layer formation. During this process, some other matrix proteins, like MSI31, which formed the framework of nacreous layer and MSI7 which could induce the nucleation of aragonite, participated to control the formation of nacreous tablets. Repression of PfN44 would facilitate the formation of the nacreous layer during repair. Our results were consistent with the hypothesis that PfN44 inhibited aragonite formation to participate in shell formation. When PfN44 function was disturbed, only the nacreous layer was affected with a disordered deposition of crystals on the surface; the prismatic layer was not obviously affected. Although PfN44 was found in both the prismatic and nacreous layer, it had small effects on calcite deposition. As shown by in vitro crystallization, PfN44 clearly affected aragonite deposition in the presence of magnesium but had little effect on calcite deposition in the absence of magnesium. Disturbing PfN44 function affected the nacreous layer more than the prismatic layer. Shell formation is under control of different matrix proteins; more than one could induce the formation of the microstructure of shell. In the formation of prismatic layer, PfN44 might need to interact with other matrix proteins to control calcite deposition. Analyzing the functions of PfN44 together with other matrix proteins such as Prisilkin 39 and Shematrin would be interesting. In addition, it is also possible that PfN44 could perform some function in the prismatic layer that is not revealed by the RNAi studies because PfN44 mRNA expression is not sufficiently reduced. As PfN44 exists in the prismatic layer and could bind to the calcite, it may also exhibit some effects in the formation of prismatic layer. To reveal the functions of PfN44 in the prismatic layer, it would be useful to use bioinformatics analysis and yeast two-hybrid system to screen for the potential interacting proteins.
      Magnesium is strongly adsorbed onto the surface of precipitated calcite because it is more strongly hydrated than calcium. In addition, magnesium might be dehydrated when it is incorporated into calcite, generating a barrier to calcite growth. However, magnesium is not incorporated into the aragonite structure to inhibit the aragonite formation. Therefore, when magnesium is added to a crystallization system, it incorporates into the calcite to form magnesium calcite. Thermodynamically unstable magnesium calcite disrupts the formation of calcite and induces aragonite deposition. Until now, only a few small molecules have been found to be involved in magnesium calcite formation, such as carboxylated molecules that increase the magnesium content in the Mg2+-bearing calcite (
      • Wang D.
      • Wallace A.F.
      • De Yoreo J.J.
      • Dove P.M.
      Carboxylated molecules regulate magnesium content of amorphous calcium carbonates during calcification.
      ) and polyacrylic acid sodium salt and dextran sulfate sodium salt, which can promote the deposition of high magnesium calcite under ambient conditions (
      • Long X.
      • Ma Y.
      • Qi L.
      In vitro synthesis of high Mg2+ calcite under ambient conditions and its implication for biomineralization process.
      ). Matrix proteins might control calcium carbonate crystallization in pearl oyster shell formation based on evidence showing magnesium calcite in the shell (
      • Bischoff W.D.
      • Mackenzie F.T.
      • Bishop F.C.
      Stabilities of synthetic magnesian calcites in aqueous solution. Comparison with biogenic materials.
      ). The matrix proteins that control magnesium content are, therefore, important in shell formation. However, no direct evidence shows that matrix proteins participate in the formation of calcium carbonate crystals by interacting with magnesium. Here we found that the matrix protein PfN44 interacted with magnesium and stabilized the formation of magnesium calcite to inhibit aragonite deposition. Studies on biomineralization have analyzed the relationship between matrix proteins and calcium. Magnesium is important in the crystallization of calcium carbonate. The relationship between matrix proteins and magnesium could provide information about the mechanism of biomineralization. Studying interactions between matrix proteins and magnesium in biomineralization could shed light on biomineralization and facilitate the synthesis of new functional materials.
      In conclusion, the acidic protein PfN44 participated in the shell formation by inhibiting aragonite deposition. Further research on the relationship between the sequence of shell formation and functions of PfN44 would provide more information about the molecular mechanism of shell formation.

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