Recombinant Expression And Functional Analysis Of CLP-2 And GRSP Proteins From Mytilus Coruscus

The biomineralization process of mollusk shells is regulated by shell matrix proteins(SMPs),and the formation of mineralized products has become an important research object of bioengineering and biomimetic materials due to its excellent mechanical properties.Shell matrix proteins play an important role in the formation of shell,as well as the regulation of calcium carbonate biomineralization and the promotion of shell growth.Therefore,in-depth study on the functions of shell matrix proteins have significant meanings for the analysis of the mineralization process and the multiple effects of SMPs.In this study,we identified and characterized two novel shell matrix proteins,CLP-2 and GRSP,which are unique to the myostracum layer of Mytilus coruscus.In order to further explore the the gene expression,the function,and the localization of the two proteins,a series of analyses were performed,including sequence analysis,construction of prokaryotic expression system,qRT-PCR,in situ hybridization,in vitro calcium carbonate crystallization,immunofluorescence,pull-down,and the like.The full-length sequence of the native CLP-2 cDNA was 1 640 bp,and the open reading frame encodes a 453 amino acid precursor containing two von Willebrand A(vWA)domains and a signal peptide,a theoretical molecular weight of 50.1 kDa and an isoelectric point of 7.10.The full length of native GRSP cDNA was 1 820 bp,and the open reading frame encodes a 595 amino acids precursor,with the theoretical molecular weight of 67.7 kDa and the isoelectric point of 9.37.A PDZ domain and a ZM domain were detected in the GRSP precursor.The CLP-2 gene and GRSP gene are expressed at a high level in specific locations in the mantle and foot,respectively.Recombinant CLP-2(rCLP-2)and Recombinant GRSP(rGRSP)showed abilities to alter the morphology of both calcite and aragonite crystals,and decrease the crystallization rate of calcite crystals.In addition,anti-rCLP-2/rGRSP antibody was prepared and immunohistochemistry and immunofluorescence analyses revealed the specific location of CLP-2/GRSP in the mantle,the adductor muscle,and the myostracum layer of shell,suggesting multifunctions of CLP-2/GRSP in biomineralization,muscle-shell attachment,and muscle attraction.Furthermore,pull-down analysis revealed 16 protein interaction partners of CLP-2 and 19 protein partners of GRSP from shell matrices and provide accordingly a possible interaction network of CLP-2/GRSP in the shell.These results expand the understanding of the functions of shell matrix proteins in biomineralization and the supramolecular chemistry that contributing to the shell formation.