The involvement of Calpain II in the formation of the ovine heritable cataract

In the lens, soluble crystallin proteins are tightly packed and ordered allowing passage of light to focus images on the retina of the eye. Disruption of this precise protein architecture results in light scattering, which often manifests itself as a dense opacity in the lens known as a cataract. While the complete mechanisms leading to cataract formation are largely unknown, in animal models it has been suggested that calpain II may become overactive and increase the rate of crystallin proteolysis. It is not known, however, what precipitates these events, but an increase in intracellular calcium is commonly observed and may be the result of a physical or chemical insult to the lens. A flock of sheep displaying a heritable cataract were bred at Lincoln University as a model for research into the mechanisms of human cataract formation. Several techniques were used in this study to investigate the involvement of calpain II in the ovine cataract. Casein zymography was employed to identify the calpain activities present in both normal and cataract lens. Calpain II and calpastatin (an endogenous inhibitor of calpain) activities were assayed during cataract formation with the fluorescent substrate BODIPY -FL Casein. The expression of calpain II during cataract formation was followed using real-time reverse transcription polymerase chain reaction with calibration to an 18S ribosomal RNA standard. The synthetic peptide aldehyde inhibitor of calpain, SJA6017, was applied as an eye-drop formulation to sheep susceptible to the cataract condition, and cataract progression in those animals were assessed for four months to identify any inhibition of the cataract and calpain. Finally, two-dimensional electrophoresis was used to separate crystallins and other proteins from normal and cataract ovine lens, and the major proteins and their modifications were determined by electrospray ionisation mass spectrometry. Casein zymography confirmed the presence of calpain II in ovine lens, and for the first time, the presence of Lp82 and calpain I activity. Calpain II was in greater abundance than the other calpain isoforms. Further analysis of calpain II activity in vitro using the BODIPY -FL casein assay revealed that activity decreased with cataract formation whilst the activity of the endogenous calpain inhibitor calpastatin increased. Decreased calpain II activity can be partially attributed to autolysis and is often an indication of previous calpain activity. Gene expression of calpain II was found to increase with cataract maturity, supporting this activation theory. The synthetic calpain inhibitor SJA6017 was found to penetrate into the ovine lens and was successful at slowing the progression of cataract formation in some animals, but was unable to permanently prevent it. Concentrations of SJA6017, below the level required for 50% calpain inhibition in vitro were insufficient to prevent cataract formation. Two-dimensional electrophoretic separation of lens proteins revealed a complement of crystallins similar to those found in other mammals. These proteins appeared to undergo the same modifications seen in ageing rat and bovine lenses. Surprisingly, specific truncations of aA-crystallin proteins during cataract formation provided evidence for greater activation of Lp82 over calpain II. The increase in calpain II expression and decreased calpain II activity in the ovine lens make this protease a possible candidate for involvement in cataract formation. The slowed progression of cataract formation with topical SJA6017 application in vivo further supports calpain involvement. Lp82 was also activated in the ovine lens, which has led to the view that calpain II and Lp82 may act as a proteolytic team to degrade crystallin proteins causing their insolubilisation and cataract formation.