Cathepsin A protein from the accessory sex gland of the Chinese mitten crab (Eriocheir sinensis) plays a key role in spermatophore digestion

Accessory sex gland (ASG) secretory proteins of the Chinese mitten crab (Eriocheir sinensis) can effectively digest the spermatophore wall. In order to identify which proteins participate in spermatophore wall digestion, a 50-kDa protein secreted from the ASG was purified to homogeneity by a series of isolation steps, including ammonium sulfate fractionation, Sephadex G-25 S gel-filtration, ion exchange chromatography on a DEAE-Sephacel column and Sephacryl S-200 gel-filtration. The purified protein was effective in spermatophore wall rupture, and the subsequent HPLC–ESI-MS/MS shotgun analysis showed the digestive protein to be cathepsin A (cathA). This finding was also confirmed by Western blot analysis and a cathA inhibitor digestion experiment. ELISA analysis showed that cathA enzymatic activity from ASG secretions increased during its purification process. Furthermore, enzymatic activity was significantly higher in the mating period of E. sinensis parallel to the latest developmental stage of the gland. Moreover, analysis from a cathA inhibitor that inhibits spermatophore wall digestion showed that cathA is the main enzyme involved. Hence, we first report the characterization of cathA from the ASG, which might play a key role in digesting the spermatophore wall of E. sinensis.