Quantitative Proteomic Study On Nacre Matrix Proteins Of Hyriopsis Cumingii

Biomineralization is a very common phenomenon in nature,the shell of mollusks is one of the biomineralizer.The main components of shell are calcium carbonate and organic matrix.Although the proportion of organic matrix is small,it regulates the whole process of biomineralization.Matrix protein is the main component of organic matrix,so the study of matrix protein is particularly important.The matrix proteins in the nacre of Hyriopsis cumingii are the subject of this study,Hyriopsis cumingii is main freshwater mussel in China,it accounts 80% of freshwater pearls.A proteome is a powerful tool to study nacre biomineralization.There have been many reports on the nacre of other mollusk species,which provided the research background for this study.Nacre biomineralization that occurs in an ordered microenvironment composed mainly of proteins and polysaccharides,as more and more proteins are detected,new questions arise about which proteins are responsible for forming this microenvironment.In this study,the nacreous layer of Hyriopsis cumingii shell was decalcified and divided into EDTA soluble and EDTA insoluble components.The determination of protein concentration and SDS-PAGE to ensure the efficiency of protein extraction.Shell matrix proteins of nacre were analyzed using label-free quantitative proteome.Most identified peptides matched the expressed sequence tags sequences of Hyriopsis cumingii.Ten known matrix proteins were found in the nacre proteome,we cloned the completed c DNA sequences of these unknown matrix proteins based on expressed sequence tags.The completed c DNA sequences of 55 unknown matrix proteins were successfully cloned from mantle tissue.8 peptides could not be cloned.A total of 73 proteins were found in the nacre proteome,and 66 and 48 proteins were found in EDTA-soluble matrix proteins and EDTA-insoluble matrix proteins,41 proteins existed in both.GO and KEGG functional annotations showed a high proportion of proteins involved in biological processes and energy metabolism in the proteome.These proteins are divided into framework proteins,cell membrane/cytoplasmic and nuclear proteins,immunity system and protective proteins and other proteins according to their properties and functions.The most common framework proteins were silk-like proteins.They could be divided into spider silk-like proteins and flagelliform silk-like proteins.Silk-like proteins had typical repetitive low complexity domains and rich in glycine,alanine and serine blocks.Another important component of the frame proteins were chitin-binding proteins,which binded to chitin and completed the assembly of the organic matrix in the first stage of the nacre formation model.99.89% of the insoluble nacre matrix proteins were composed of silk-like proteins,chitin-binding proteins,and cysteine-rich matrix proteins,which were responsible for organic framework assembly.99.34% of the soluble nacre matrix proteins were composed of silk-like proteins and chitin-binding proteins,which were responsible for forming protein gel filling in organic frameworks.The content of silk-like proteins was more than 90% in both insoluble and soluble nacre matrix proteins.Spider silk-like proteins dominated the silk-like proteins.Silk-like proteins were the main protein constituents of organic frameworks and participated in organic framework formation together with chitin-binding proteins and cysteine-rich proteins.Free-state spider silk-like proteins formed a silk gel to fill the microspace of organic frameworks and silk-like proteins also participated in calcium carbonate crystallization with other matrix proteins.As organic frameworks and protein gel together constructed a microenvironment for calcium carbonate biomineralization,these results provided a novel understanding of nacre formation.