Role of Calpain in Autolysis of Sea Cucumber Apostichopus japonicus.

Calpain is a family of cysteine proteases whose role in sea cucumber autolysis was previously unreported. In this study, specific inhibition of calpain notably alleviated autolysis in the respiratory tree and intestine, significantly reduced body wall hardness loss. During an 8 h autolysis period, calpain activity in the body wall continuously declined, whereas activity in the respiratory tree and intestine peaked at 2 h. The contents of calpain 5 & 9 were highest in the respiratory tree and remained relatively stable, whereas these two isoforms showed distinct temporal patterns in the intestine and body wall. Immunohistochemistry revealed high calpain expression in the muscle layers and at the mucosal fold tips of the intestine and respiratory tree, but sparse distribution in the body wall connective tissue. After 4 h of autolysis, disintegration of the intestinal serosal layer, disorganization of the respiratory tree cellular structure, and destruction of the epidermis and endodermis of the body wall were observed. Co-immunoprecipitation coupled with mass spectrometry identified 40 calpain-interacting proteins, and fluorescence colocalization confirmed direct interaction with tubulin. This first report establishes calpain involvement in sea cucumber autolysis, clarifying its dynamic activity, tissue distribution, and interacting proteins, and suggests calpain may act by degrading tubulin.