Calpain–induced proteolysis in lens epithelial cell death during ovine inherited cataract

Abstract

Abstract: : Purpose: Our previous study suggested that calpain–induced proteolysis was involved in ovine inherited cataractogenesis. However, the initial insult causing cataract formation was not clear. Calpain–induced proteolysis has been associated with lens epithelial cell death in other cataract models. Thus, the purposes of present experiment were (1) to determine if lens epithelial cell death was accelerated during formation of ovine inherited cataract, and (2) to determine the role of calpains in lens epithelial cell death. Methods: Cell death was observed by TUNEL staining of fixed epithelial cells from normal, stage 1, (minute opacities at Y suture) and stage 3 (increased light scattering at Y suture) cataractous lenses. Calpain activity in the soluble proteins from lens epithelia was determined by casein zymography, and the amount of calpain protein was determined by immunoblotting. As an in vivo marker for calpain activation, proteolysis of the calpain substrate α–spectrin was assessed by immunoblotting. Proteolysis of lens proteins was assessed by 2D electrophoresis and mass spectrometric analysis. Results: In lens epithelial cells from normal and stage 1 cataracts, no TUNEL staining was detected. TUNEL staining was apparent in epithelial cells of stage 3 cataractous lenses. Casein zymography revealed activities for calpain 1 and 2 in all samples. The levels of activity did not appear to differ between normal and cataract samples. Immunoblotting for calpain 1 and 2 proteins also showed no differences. Increased amounts of α–spectrin breakdown product at 145 kDa were observed with cataract, especially at stage 3. 2D gel electrophoresis showed differences in the protein patterns for normal and stage 3 cataract Conclusion: The appearance of the calpain–specific breakdown product of α–spectrin at 145 kDa suggested that calpain was activated in lens epithelial cell death in an early stage of ovine inherited cataractogenesis. Lack of observed overall changes in calpain (zymography and immunoblotting) may be due to the small amount of lens epithelial cell death in the early stages of ovine cataract studied in the present experiments. Further experiments using mature cataract lenses are needed to confirm the involvement of calpain in lens epithelial cell death in this inherited ovine model.

Keywords: cataract • cell death/apoptosis • proteolysis