The purposes of this experiment were to: (1), characterize the peptide aldehyde SJA6017, N-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal, a newly synthesized inhibitor of calpain, and (2) test the effect of SJA6017 in preventing calcium ionophore-induced cataract in cultured rat lenses. In vitro, SJA6017 strongly inhibited purified m-calpain from porcine kidney. Casein zymography confirmed that SJA6017 reversibly bound to the active site of m-calpain. SJA6017 was also confirmed to be a cell-permeable inhibitor in Molt-4 cells. In cultured lenses, SJA6017 reduced nuclear opacity and proteolysis of crystallins and alpha-spectrin caused by calcium ionophore A23187. These results suggested that SJA6017 is a reversible and cell-permeable calpain inhibitor which may possess great efficacy against calcium-induced models of cataract.

These results suggest that calpain isoforms may play an important role in neuronal cell death induced by retinal ischemia-reperfusion injury in rat.

Molecular chaperone activity of lens ␣-crystallins is reduced by loss of the C terminus. The purpose of this experiment was to 1) determine the cleavage sites produced in vitro by ubiquitous m-calpain and lens-specific Lp82 on ␣-crystallins, 2) identify ␣-crystallin cleavage sites produced in vivo during maturation and cataract formation in rat lens, and 3) estimate the relative activities of Lp82 and m-calpain by appearance of protease-specific cleavage products in vivo. Total soluble protein from young rat lens was incubated with recombinant m-calpain or Lp82 and 2 mM Ca 2؉ . Resulting fragmented ␣-crystallins were separated by two-dimensional gel electrophoresis. Eluted ␣-crystallin spots were analyzed by mass spectrometry. Cleavage sites on insoluble ␣-crystallins were determined similarly in mature rat lens nucleus and in cataractous rat lens nucleus induced by selenite. In vitro proteolysis of ␣A-crystallin by Lp82 and m-calpain produced unique cleavage sites by removing 5 and 11 residues, respectively, from the C terminus. In vivo, the protease-specific truncations removing 5 and 11 residues from ␣A were both found in maturing lens, whereas only the truncation removing 5 residues was found in cataractous lens. Other truncation sites, common to both calpain isoforms, resulted from the removal of 8, 10, 16, 17, and 22 residues from the C terminus of ␣A. Using uniquely truncated ␣A-crystallins as in vivo markers, Lp82 and m-calpain were both found to be active during normal maturation of rat lens, whereas Lp82 seemed especially active during selenite cataract formation. These C-terminal truncations decrease chaperone activity of ␣-crystallins, possibly leading to the observed increases in insoluble proteins during aging and cataract. The methodology that allowed accurate mass measurements of proteins eluted from 2D gels should be useful to examine rapidly other post-translational modifications. ␣-Crystallins in lens are related to the small heat shock family of proteins (1). Because lens proteins have very little turnover, ␣-crystallin functions as a molecular chaperone to prevent aggregation of other lens proteins (2). The C terminus is essential, because loss of 16 amino acid residues of C terminus from ␣A-crystallin caused a loss of 50% of its chaperone activity (3).Truncation of lens crystallins is a common feature of both aging and cataract formation in rodents. A number of experiments revealed that calcium-activated proteases (calpains) were involved in formation of cataracts induced by selenite (4), galactose (5), diamide (6), and the hypocholesterolemic drug U18666A (7) and in hereditary rat Shumiya cataract (8). One of the ubiquitous calpains, m-calpain, has been credited with the proteolysis of ␣-and ␤-crystallins during cataractogenesis in rodents. Incubation of ␣A-crystallin with m-calpain reduced chaperone activity and produced truncated forms of ␣A that migrated during two-dimensional electrophoresis (2-DE) 1 to positions similar to truncated forms of ␣A observed in cataractous lenses (9). A re...

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