Structural and functional analyses of a TIMP and MMP in the ligament of Pinctada fucata.

Abstract

The bivalve hinge ligament is the hard tissue that functions to open and close shells. The ligament contains fibrous structures consisting of aragonite crystals surrounded by a dense organic matrix. This organic matrix may contribute to the formation of fibrous aragonite crystals, but the mechanism underlying this formation remains unclear. In this study, we identified a novel ligament-specific protein, Pinctada fucata tissue inhibitor of metalloproteinase (PfTIMP), from the fibrous organic matrix between aragonite crystals in the ligament using the amino acid sequence and cDNA cloning methods. PfTIMP consists of 143 amino acid residues and has a molecular weight of 13,580.4. To investigate the activity of PfTIMP, inhibition of matrix metalloproteinase (MMP) activity was measured. PfTIMP strongly inhibited human MMP13 and MMP9. Eight MMP homologs were identified from a P. fucata genomic database by BLAST search. To identify the specific MMP that may contribute to ligament formation, the expression level of each MMP was measured in the mantle isthmus, which secretes the ligament. The expression of MMP54089 increased after scratching of the ligament, while the expressions of other MMPs did not increase after doing the same operation. To identify the role of MMP54089 in forming the ligament structure, double stranded (ds) RNA targeting MMP54089 was injected into living P. fucata to suppress the function of MMP54089. Scanning electron microscopic images showed disordered growing surfaces of the ligament in individuals injected with MMP54089-specific dsRNA. These results suggest that PfTIMP and MMP54089 play important roles in the formation of the fibrous ligament structure.