The Mechanism Study Of Extrapallial Fluid Protein In The Shell Formation Of Pinctada Fucata

The shell of Pinctada fucata which is composed of two mineralized layers related to a biomineralization balance process and intrigued interesting a lot.Extrapallial fluid?EPF?is located between the shell and the mantle,and it is believed to play key roles in shell biomineralization.However,few studies have been performed on the biomineralization effect of EPF.In this work,CaCO₃ crystallization experiments showed that EPF proteins could not only control the morphology but also regulate the phase transition of calcium carbonate through the different proteins specific binding to calcite or aragonite.In crystal growth inhibition experiments,when the final concentration of CaCl2 and NaHCO₃ reduced from 50 mM to 5mM,the function of EPF proteins transferred from significantly improving over the number of CaCO₃ crystals upon the control to inhibiting the precipitation of CaCO₃.The precipitation rate of CaCO₃ was also inhibited by EPF proteins.In vivo,once EPF was extracted daily for 20 days,the nacre platelet in the nacreous layer was disturbed,and calcite deposited randomly due to the reduction of EPF proteins.Furthermore,EPF also acted as an external signal to induce the expression variation of shell-related genes to regulate nacre and prism formation.In conclusion,our findings demonstrate that EPF proteins not only take part in the nucleation,morphology,inhibition and phase transition of CaCO₃ but also play a dual role in both shell biomineralization and prism-nacre transition.Then to identify the proteins that play key roles in phase transition,we performed LC-MS analysis on the proteins specific binding to calcite or aragonite.We totally identified 221 protein analogs binding to both,79 protein analogs binding to calcite and 47 protein analogs binding to aragonite.The proteins identified are not completely equal to that found in shell,which directly indicated that the EPF proteins participated in the shell formation.The analysis of amino acid composition showed that proteins specific binding to calcite preferred to Lys,Val,Ser and Gly,while proteins specific binding to aragonite favored Leu,Glu and Val.Conserved domain analysis showed that the functions of proteins binding to calcite and aragonite involved in ions regulation,protein interaction,the proteinase inhibitor and collagen.Our results provides the basis for further study.Finally,we found that the expression level of SPARC in EPF increased after shell notching in P.fucata.SPARC was found both in the nacre and prism soluble extract,and blocking SPARC with a polyclonal antibody interfered with the formation of nacre platelets.Furthermore,SPARC regulated the morphology of CaCO₃ crystals and induced the formation of vaterite in the calcite crystallization system.However,Mg2+ counteracted this effect and induced the formation of aragonite.Further intrinsic fluorescence and circular dichroism spectrum studies indicated that SPARC may function by changing the conformation of its secondary structure.Combined with the protein domain analysis,our data indicate that the extracellular Ca2+-binding?EC?domain plays a major role in stabilizing vaterite.In conclusion,SPARC participates in nacre formation by stabilizing vaterite to inhibit calcite formation via its EC domain and secondary structure variation and by assisting aragonite formation in the presence of Mg2+ or other proteins.In conclusion,we first deeply investigated the function of EPF proteins in shell biomineralization balance.And found that the EPF proteins could control the transition among ACC,vaterite,calcite and aragonite.Our research results could provide theoretical guidance for pearl production and material synthesis in vitro.