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Progressive modifications of mouse lens crystallins in cataracts induced by buthionine sulfoximine
H I Calvin1, S A Patel, J P Zhang
1Department of Ophthalmology, UMD-NJ Medical School, Newark 07103.
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 (less than 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 beta-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 alpha and beta-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 alpha A-crystallin polypeptide. The gamma-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 gamma-crystallin polypeptides and enriched in anodic components whose relative electrophoretic mobilities were similar to those reported previously for phosphorylated bovine alpha A-crystallin and several cytoskeletal polypeptides. At stage 4 of the cataract, the modifications of normal alpha and beta-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 Ca2+ 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.
Insights
L-buthionine-S,R-sulfoximine (BSO) induced cataracts in mouse pups by inhibiting GSH biosynthesis. Early changes involved loss and modification of beta-crystallins, followed by alpha-crystallin alterations and cytoskeletal protein degradation, suggesting a role for calcium-activated proteolysis.
Area of Science:
- Ophthalmology
- Biochemistry
- Cell Biology
Background:
- Cataracts are a leading cause of blindness, characterized by lens opacification.
- Glutathione (GSH) biosynthesis is crucial for maintaining lens clarity and protecting against oxidative stress.
- Inhibiting GSH synthesis provides a model to study cataractogenesis and lens protein modifications.
Purpose of the Study:
- To investigate the biochemical changes in lens proteins during cataract development induced by L-buthionine-S,R-sulfoximine (BSO).
- To elucidate the role of calcium-activated proteolysis in BSO-induced cataracts.
Main Methods:
- Induction of cataracts in mouse pups using L-buthionine-S,R-sulfoximine (BSO).
- Staging of cataract progression based on visual opacification.
- Analysis of water-soluble and urea-soluble lens extracts using two-dimensional gel electrophoresis.
- Measurement of intracellular calcium levels in the lens.
Main Results:
- BSO treatment rapidly induced lens opacification, progressing through distinct stages.
- Early cataract development (Stage 2) showed loss of heavy beta-crystallin components and appearance of new species.
- Later stages involved extensive modification of alpha- and beta-crystallins, loss of gamma-crystallins, and degradation of cytoskeletal proteins.
- Increased lens calcium preceded polypeptide changes, implicating Ca(2+)-activated proteolysis.
Conclusions:
- BSO-induced cataracts involve sequential alterations in crystallin composition and degradation of cytoskeletal proteins.
- Calcium-activated proteolysis is a significant mechanism in the development of BSO-induced cataracts.
- Understanding these molecular events can inform strategies for cataract prevention and treatment.