Regulation Of Shell Matrix Protein And Its Phosphorylation On Biomineralization

The shell of pearl oyster(Pinctada fucata)is a kind of biological mineral formed under the complex regulation of organisms.Compared with naturally formed inorganic minerals,it has excellent material properties and mechanical properties.The biomineralization process is an extremely complex process,in which a variety of biological macro-molecules participate in the interaction,and they themselves are also subject to various types of regulation.Current studies have shown that various shell matrix proteins(SMPs)play an important regulatory role in the process of shell formation.Related research has also focused on the discovery of new shell matrix proteins that regulate the formation and growth of calcium carbonate crystals.However,the phosphorylation modification of shell matrix protein has received little attention in the biomineralization of Pinctada fucata.Proteomics studies have shown that there are a large number of phosphorylated proteins in shell matrix proteins.In order to explore the role of phosphorylation in biomineralization,the effects of EDTA soluble matrix protein(ESM)and EDTA insoluble matrix protein(EISM)on the growth of calcium carbonate crystals were systematically studied.Through in vitro crystallization experiments and the use of STORM technology to study the protein distribution pattern,we found that ESM protein exists at both the edge and center of the crystal,mainly showing a island-like distribution,indicating that ESM plays a role in all stages of crystal growth.Participated in the crystal nucleation process,promoted the formation of new crystals,and mainly controlled the crystal morphology in the later stage.The distribution of EISM protein is mainly concentrated on the periphery of the crystal,and the distribution form is mainly filamentous continuous distribution,indicating that EISM mainly plays a key role in the growth process after the formation of calcium carbonate crystals,and mainly acts as a template for the growth direction of the crystal by combining it on the periphery of the crystal and continue to epitaxially follow the growth of the crystal.In vitro experiments have confirmed that dephosphorylation of ESM and EISM matrix proteins can reduce their binding and distribution in calcium carbonate crystals,resulting in the decrease of their regulatory effect on the formation of calcium carbonate crystals.Neutralizing the phosphorylated protein in the extrapallial fluid with antiserine/threonine/tyrosine antibodies will also significantly affect the growth of the nacre and prismatic layer of the shell,causing disordered crystallization of the new growth layer of the shell.After the shell notching,the mantle phosphorylated proteome has undergone significant changes.Through KEGG analysis,the degree of phosphorylation modification such as focal adhesion,Hippo signaling pathway,actin cytoskeleton regulation,Rap1 signaling pathway,Erb B signaling pathway,etc.changed the most.Through GO analysis,the degree of phosphorylation modification of related types of cytoplasm,non-membrane structure organelles,organic metabolism process,The activity of structural molecules and binding proteins changed the most.In order to find the phosphorylation-modified regulatory protein,we found a protein kinase Fam20 C in the protein group of Pinctada fucata.We found that Fam20 C is highly expressed in the mantle tissue,and affects the formation of the prodissoconch I during the D-phase stage of juvenile,and the expression level increases when the shell is notched.After inhibiting the expression of Fam20 C,the fine structure of the shell was severely damaged,and the phosphorylation level of the matrix protein in the extrapallial fluid was significantly reduced.After inhibiting the expression of Fam20 C by RNAi and analyzing the phosphorylated proteome of the extrapallial fluid,we found arginine kinase,calmodulin,myosin,paramyosin and other proteins regulated by Fam20 C targeting.In short,the phosphorylation of matrix protein is crucial to its role in the process of biomineralization.The kinase Fam20 C promotes the phosphorylation of the matrix protein in the extrapallial fluid of Pinctada fucata.The regulation process and mechanism of Fam20 C on the shell matrix protein need to be further studied.