Studies On The Mechanism Of Matrix Proteins Regulating Crystals Self-assembly To Form Ordered Shell Structure

The shell formation of mollusks including Pinctada fucata is one of the widely recognized experimental model in biomineralization area.The composition of shells includes about 95%calcium carbonate and 5%organic matrix among which matrix proteins were thought to play crucial roles during self-assembly of crystals to form ordered shells.The location and functional study of matrix proteins is of great importance to illustrate the mechanism of shell formation.We did some experiments to explore the distribution and function of whole matrix protein/EISM/ESM.According to the results of distribution of matrix proteins,we hypothesized that some EISM formed early organic sheath of prismatic layer.The ESM and some EISM distributed on the organic membrane and formed nucleation sites.Then matured organic sheath formed as the squeezing of adjacent prism.EISM also formed organic framework first and aragonite deposited in.What's more,the functional study stimulated that EISM works as a negative regulator during crystal growth,aragonite growth especially.After inhibition of EISM,the ordered structure of prism and nacre destroyed.To study the mechanism of matrix proteins regulating crystals self-assembly to form ordered shell structure,we made a study of two typical matrix proteins from EISM of prism and nacre-PNU9 and Alv.PNU9,an EDTA-insoluble matrix protein with“E-C”repeats.PNU9 participated into the process of shell repair after shell damage.The inhibition of PNU9 could lead the overgrowth of nacreous lamella and matrix membrane of nacreous layer.Crystallization in vitro showed that r PNU9 could influence the shapes of both calcite and aragonite.What's more,r PNU9 took part in the nucleation and crystal growth stage of aragonite.Both calcium carbonate precipitation assay and ACC transition experiment indicated that r PNU9 suppress the crystal growth of aragonite.In brief,PNU9 might participate into the growth of nacreous layer as a negative regulator.Alveoline-like(Alv),characterized with VP-repeat in proteomic of shell matrix proteins of P.fucata.The distribution patterns in the inner or outer prismatic layer and nacreous layer are consistent with EISM.To study the specific functions of Alv in biomineralization,we executed a series of experiments in vivo and in vitro.The functional inhibition of Alv protein in vivo by RNA interference and injecting r Alv antibodies destroyed prism structure but accelerated nacre growth.Experiments in vitro indicated that r Alv could bind with aragonite,calcite and chitin specifically.In vitro calcite and aragonite crystallization assays and Cy5-label assay demonstrated that r Alv could promote crystals nucleation and influenced the morphology of calcite crystals.r Alv could promote nucleation during ACC crystallization,stimulated the transition from ACC to calcite,but suppressed transition from ACC to aragonite.Near-UV CD spectra of r Alv in the presence or absence of Ca²⁺,Mg²⁺and CO₃^2-showed that r Alv could interact with mineralization ions.Furthermore,we detected the predicted transcription factors(Pf-AP-1;Pf-rel;Pf-POU3F4;Pf-C/EBP-A)and found that Pf-AP-1 and Pf-POU3F4 could enhance the promoter activity of Alv.The above-mentioned functions lead to opposite results of a destroyed prismatic layer and an overgrown nacreous layer after the inhibition of Alv function in P.fucata.The function of PNU9 and Alv during nacre formation is consistent with EISM which could explain the mechanism of EISM regulating crystals self-assembly to form ordered shell structure.In conclusion,matrix proteins,especially EISM,play an important role in the regulation of crystal self-assembly to form the ordered structure of shells.Studies on PNU9 and Alv proteins have clarified the regulatory mechanism of EISM.These works complement the theoretical study of shell biomineralization and help us to understand the principle of biomineralization deeply.