Immunohistochemical Study Of Cathepsin L-like Proteinase In The Body Wall Of Sea Cucumber(Stichopus Japonicas)

Sea cucumber(Stichopus japonicus) has strong autolytic capacity. When receiving certainexternal stimulus, self-degradation, even mucous change will take place in sea cucumber bodywall, which makes great inconvenience to transportation, storage and processing of seacucumbers. Our previous studies indicated that endogenous cysteine proteinases includingsuch as cathepsin L-like proteinase(CLP) were involved in the proteolysis of proteins frombody wall of sea cucumber (S. japonicus). In the present study, the relationship betweenautolysis and endogenous cysteine proteinases were proved through revealing the changes ofactivity and distribution of CLP during autolysis of S. japonicas.In this study, a rapid consecutive procedure based on a semi-preparative high performanceliquid chromatography (semi-PR-HPLC) matched SOURCE30Q anion exchange column,Superdex75gel filtration column and Superdex200gel filtration column was developed topurify CLP from the gut of S. japonicas. Take the purified CLP as an antigen, antibody wasproduced. And on this basis, the tissue distribution of CLP in S. japonicus body wall duringautolysis was revealed by using immunohistochemical technique. Meanwhile, changes ofenzyme activity of cathepsin L-like proteinase and cathepsin B-like proteinase（CBP）ofdifferent layers of sea cucumber body wall was also studied.Experimental results showed that semi-PR-HPLC matched columns with high resolutioncan prepare CLP with high-purrity. Immunoblotting analysis of the purified enzyme using theantibody prepared exhibited a single band with an apparent MW of31kDa, indicating that theantibody has high specificity. Histochemical investigation indicated that the distribution ofCLP in S.japonicas body wall was inhomogeneous. In contrast, CLP was more abundant inthe epidermis rather than in the dermis. As to the dermis, CLP was relatively abundant in theouter layer but sparse in the inner layer. Along with autolysis, the stained spots were diffusedand spread.Meanwhile, results of enzyme activity analysis indicated that cathepsin L-like proteinaseand cathepsin B-like proteinase were the highest in epidermis and outer dermis layer, relative weak in inner dermis layer, and the most weak in muscle layer. During UV irradiation, theenzyme activity of the two proteinases were increased, and decreased after the irradiation, butincreased again along with autolysis. This indicated that the enzyme activities of CLP andCBP always existed and diffused from epidermis to dermis.The present study indicated that there was positive relationship between distribution of CLPand CBP with autolysis in sea cucumber body wall. The enzyme activity was stable duringautolysis and presented spread trend.Above results further proved that CLP and CBP may takepart in autolysis of sea cucumber.