Structural and functional analyses of chitinolytic enzymes in the nacreous layer of Pinctada fucata

In this study, chitinolytic activity was confirmed in the protein extracted from the inner nacreous layer of Pinctada fucata shells. Allosamidin, a specific inhibitor of family 18 chitinase, was injected into pearl oysters, and an abnormal organic membrane was observed in the nacreous layer. When shell-notched individuals were injected with allosamidin, the organic fibers in the regenerated site were thicker than those without allosamidin treatment. To identify chitinases in the nacreous layer, proteins extracted from the nacreous layer were analyzed using LC-MS/MS. Two chitinases (PfCN1 and PfCN2) were identified, and their different spatial expression patterns were observed in mantle tissue. To understand the function of chitinolytic enzymes in nacre formation, we designed an in vivo gene knockdown experiment using PfCN1 and PfCN2 dsRNA, and in vitro calcium carbonate crystallization experiments using chitinolytic enzymes. In the knockdown experiment, abnormal aragonite tablets and organic films were observed in the nacreous layer. In the crystallization experiment, we found that the thickness of the chitin film decreased and the space between the chitin fibers increased when calcium carbonate was formed on the chitin film on the glass substrate with chitin-degrading enzyme treatment. These reactions induce the growth of calcium carbonate crystals through the chitin film. The penetration of chitin film by calcium carbonate is similar to that of mineral bridges that connect the nacre tablets vertically. These results showed the novel function of chitinolytic enzymes that will give a comprehension of nacre formation.