A mechanism for activation of m-calpain during proteolysis of α-crystallin

Purpose. Data from our lab have provided convincing evidence that m-calpain-induced proteolysis and precipitation of crystalline are an underlying biochemical mechanism for cataract formation in rat lenses. m-calpain, which is normally catalytically inactive in the cytosol, is cysteine protease with a neutral pH optimum and absolute requirement of calcium for activation. A mechanism for activation of m-calpain is however obscure, although some data have indicated that at least part of the activation mechanism involves association of m-calpain with membranes. Thus, the purpose of this experiment was to study a mechanism for m-calpain activation during proteolysis of α-crystallin. Methods. m-calpain and α-crystallin were incubated with or without calcium and calpain inhibitors. SDS-PAGE, Non-SDS-PAGE and casein zymography were then performed. N-terminal sequence analysis was also performed after electrotransfer of proteins from SDS-PAGE gels onto membranes. Results. Nine amino acids of N-terminal in m-calpain was removed (Autolyzed calpain) during proteolysis of α-crystallin, and C-terminal region of autolyzed calpain was then removed (Degraded calpain). Calpain inhibitor inhibited autolysis and degradation of m-calpain (Unautolyzed calpain). Conclusions. These results suggested that sequence events for calpain activation were unautolyzed, autolyzed and finally degraded calpain. Unautolyzed and/or autolyzed calpains may be proteolytically active against α-crystallin.