Function And Transcriptional Regulation Of Shematrin-2 During Shell Regeneration

Molluscan shell is a fascinating biomineral consisting of a highly organized calcium carbonate composite.The shells can be repaired after damage,which is similar to the process of shell formation.The repairing process can be elaborately regulated by biomacromolcules,especially shell matrix proteins?SMPs?.The matrix protein Shematrin-2,the expression level of which is the highest both in the mantle tissues and in the shell components of Pinctada fucata,has been suggested to be a key participant in biomineralization.But little is known about its function and regulatory mechanisms.Here,we studied the SMPs of the repaired shells 5-20 days after shell damage in the pearl oyster Pinctada fucata by proteomics and compared the microstructure difference between repaired and mature shells.Although the regenerated shells are calcite,similar to mature shells,the microstructure of repaired shells during the regeneration process were different,which could simulate the embryonic shell formation process.In total,we found 49 SMPs from the repaired shells including some proteins only existing in mature nacreous layers.In addition,they have the capability to affect Ca CO₃ crystallization process in vitro,altering the packing and reducing the crystallinity of crystals.This portion of this study could improve our understanding of shell formation process.On the other hand,we expressed and purified the matrix protein Shematrin-2 in vitro and explored its function and mechanism of transcriptional regulation.The in vitro functional assay showed that Shematrin-2 could bind the calcite,aragonite and chitin components of the shell;decrease the rate of calcium carbonate deposition;and change the morphology of the deposited crystal in the calcite crystallization system.Furthermore,we cloned and characterized the transcription factors CCAAT enhancer-binding proteins?Pf-C/EBPs?and nuclear factor-Y?NF-Y?based on a sequence analysis of the Shematrin-2 gene promoter.Pf-C/EBP-A and Pf-C/EBP-B enhanced the promoter activity of Shematrin-2 gene in the transient co-transfection assays,which was dose-dependent.Importantly,the knockdown of Pf-C/EBP-A and Pf-C/EBP-B led to a decrease in Shematrin-2 gene expression,and the changes in the inner surface structures of shells were similar to those in the Shematrin-2 antibody inhibition experiment.Altogether,our data reveal that the transcription factors Pf-C/EBP-A and Pf-C/EBP-B regulate the expression of the matrix protein Shematrin-2during the process of shell formation in P.fucata,which provides a better understanding of the transcriptional regulation of shell formation at the molecular level.In conclusion,our study explored the shell growth pattern by exploring the process of shell regeneration,and further explored the function and transcriptional regulation mechanism of the key matrix protein Shematrin-2.It provides a basis for further study of the signal regulation network of matrix protein genes in Pinctada fucata.Besides,it helps us to understand the formation of shells from various angles,such as macroscopic and microscopic structure,proteomics and molecular level.