Interaction Patterns Of Nacre Matrix Proteins And Its Functional Studies In Pinctada Fucata Martensii

The nacre of Pinctada fucata martensii is a typical biomineral dominated by matrix proteins.The matrix proteins in the extrapallial fluid self-assemble to form an organic matrix framework,and induce calcium carbonate deposition.The protein composition of the nascent organic matrix layer proteins and the self-assembly process of the organic matrix framework are still poorly understood.In this study,proteins eluted from the inner surface of shells were analyzed by LC-MS/MS and nascent organic matrix layer proteins were identified.Based on its expression characteristics,proteins involved in nacre formation were screened.The interaction of nacre shell matrix proteins(NSMPs)was obtained by yeast two-hybrid and Pull-down techniques,the basic process of organic matrix framework self-assembly was revealed,and the functions of key proteins were explored.The results of the study were as follows:1.Identification and types of nascent organic matrix layer proteinsEluted proteins of shell inner surface were analyzed by LC-MS/MS and 234 nascent organic matrix layer proteins were identified,the main types of which were von Willebrand factor,type A domain-containing proteins,C1 q domain-containing proteins,chitin-binding domain-containing protein,tyrosinase,and fibronectin type III domaincontaining proteins,etc.Most of the nascent organic matrix protein genes were highly expressed in mantle pallial and pearl sac and were induced after shell damage.Based on the expression characteristics,19 proteins involved in nacre formation were selected,including CBP,FNIIIs,C1 q DCs,Tyr,etc.2 Tyr,2 C1 q DC,1 CBP,1 low complexity region-containing protein,1 chitinase,and 1 metalloproteinase inhibitor were supplemented according to the literature related to nacre formation.Finally,27 proteins were selected for subsequent matrix framework self-assembly studies.2.Construction of nacre matrix protein interaction patternsNSMPs interaction was obtained by yeast two-hybrid and Pull-down techniques.During matrix framework formation,chitin-binding protein(CBP)bind to the chitin first.Therefore,CBP was designated as the matrix framework primary assembly protein.The interactions of NSMPs were divided into three progressive levels.The primary assembly protein molecules were CBP;The secondary assembly protein molecules were 30 protein molecules with a high degree of connection,including FNⅢs,Tyr,VWAPs,nacre,C1 q DCs,proteinase inhibitor,LCRP,tissue inhibitor of metalloproteinase and chitinase,etc;The tertiary assembly protein molecules were 12 protein molecules with a low degree of connection,including Tyr,proteinase inhibitor,myosin heavy chain,and galectin,etc.Tyr(Pm Tyr-1),VWAP(Pm VWAP),and FNⅢ(PmFN3-1)were key proteins in the secondary assembly of the matrix framework,which connected a large number of other NSMPs in the construction of the secondary matrix framework.At the same time,they were key proteins linking the assembly proteins of the tertiary matrix framework.3.Structure and function of primary assembly PmCBP-1The CBP gene PmCBP-1 was cloned,which encoded a signal peptide,two chitinbinding domain type 2(Cht BD2),and an epidermal growth factor-like domain(EGF).PmCBP-1 was significantly highly expressed in the mantle pallial and mantle central and was located on the outer epithelium cell of the mantle.After knocking down the expression of PmCBP-1 in the mantle by RNA interference,the nacre and prismatic layers showed a disordered crystal growth and the construction of the matrix framework was defective,suggesting that PmCBP-1 participated in shell formation.The localization of PmCBP-1 in the nacre matrix framework and its chitin-binding activity suggested that PmCBP-1 was involved in the primary assembly of the nacre matrix framework.Bimolecular Fluorescent Complimentary(Bi FC)experiments verified the interactions between C1 q DCs and the primary assembly protein PmCBP-1.Calcium carbonate deposition experiments showed that the recombinant protein r PmCBP-1promoted the calcium carbonate deposition rate.In vitro crystallization experiments showed that r PmCBP-1 altered the morphologies of calcite and aragonite crystals.Taken together,we suggest that PmCBP-1 participated in the primary assembly of the nacre matrix framework and promoted shell biomineralization.4.Structure and function of secondary assembly protein PmFN3-1The FNⅢ gene PmFN3-1 was cloned,which encoded a signal peptide and five fibronectin type Ⅲ domain(FNⅢ).PmFN3-1 was significantly highly expressed in the mantle edge,mantle pallial,mantle central,and pearl sac which were the mineralized tissues,and was located on the outer epithelium cells of the mantle.After knocking down the expression of PmFN3-1 in the mantle central and mantle pallial by RNA interference,the nacreous layer grew disorderly,indicating that it was involved in nacre formation.Biofilm interference experiment verified the interaction between PmFN3-1and the primary assembly protein PmCBP-1.Calcium carbonate deposition experiments showed that the recombinant protein r PmFN3-1 inhibited the deposition rate of calcium carbonate.In vitro crystallization experiments showed that the edges and corners of calcite crystals became rounded after adding r PmFN3-1.Taken together,we suggest that PmFN3-1 participated in the secondary assembly of the nacre matrix framework and acted as a negative regulator involved in maintaining the shell biomineralization balance.5.Structure and function of tertiary assembly protein Pm Tyr-4The nacre-specific Tyr gene Pm Tyr-4 was cloned,which encoded a signal peptide,a tyrosinase domain,and two low-complexity regions.Pm Tyr-4 was specifically highly expressed in mantle central and pearl sac,which are the nacre-forming organs.Compared with traditional Tyr,the conserved histidine residues within the copper A and copper B motifs of Pm Tyr-4 were mutated,suggesting that it may lose the binding ability of copper ions and the catalytic activity of tyrosinase.After knocking down the expression of Pm Tyr-4 in the mantle central and mantle pallial by RNA interference,the nacreous layer grew disorderly,suggesting that it was involved in nacre formation.Calcium carbonate deposition experiments showed that the recombinant protein r Pm Tyr-4 inhibited the deposition rate of calcium carbonate.In vitro crystallization of calcite showed that the edges and corners of calcite crystals became rounded after adding r Pm Tyr-4.In vitro crystallization of aragonite showed that after adding the r Pm Tyr-4,the size of the aragonite crystal became smaller,and the number of crystal clusters on the crystal surface decreased compared with that in the control group.Taken together,we suggest that Pm Tyr-4 may participate in the the tertiary assembly of the nacre matrix framework and acted as a negative regulator involved in maintaining the shell biomineralization balance.