Progressive modifications of mouse lens crystallins in cataracts induced by buthionine sulfoximine

Abstract

l-buthionine-S,R-sulfoximine (BSO), a specific inhibitor of GSH biosynthesis, was administered four times daily to mouse pups on post-natal days 7 and 8, inducing initiation of opacification on day 9. The initial progression of the cataract (< 24 hr) was divided into four stages: (1) developing floriform; (2) mature floriform; (3) degenerate floriform; and (4) amorphous translucent cataract. Following this, dense corticonuclear opacities developed within several days. Two-dimensional gel electrophoresis of water-soluble whole lens extracts indicated that the most rapid early cataractous changes, occurring mainly during stage 2, were loss of the two major components of the heavy β-crystallin fraction, a 31-kDa basic polypeptide and an acidic component at 27 kDa, concomitant with the appearance of new species at 30 and 25 kDa. This was followed by more extensive modification of both α and β-crystallins during stages 3 and 4 and the appearance of abnormal species at 26, 19 and 18 kDa, which were slightly more acidic than the major normal αA-crystallin polypeptide. The γ-crystallin components, relatively unaffected at stage 4, were then lost rapidly as dense opacities ensued. By contrast with the water-soluble fraction, the normal day 9 urea-soluble fraction was deficient in γ-crystallin polypeptides and enriched in anodic components whose relative electrophoretic mobilities were similar to those reported previously for phosphorylated bovine αA-crystallin and several cytoskeletal polypeptides. At stage 4 of the cataract, the modifications of normal α and β-crystallin components in the urea-soluble fraction paralleled those in the water-soluble fraction, but the products seen were more numerous. In addition, the cytoskeletal proteins were no longer detectable. Substantial increases in lens Ca 2+ that precede all of the above changes in lens polypeptide composition suggest that Ca 2+-activated proteolysis may play a major role in development of BSO cataracts.