Abstract
This study reports the first demonstration of a marked reduction in alpha-crystallin chaperone activity in an experimental model of cataract, and the study implicates activation of the cysteine protease calpain II (EC 3.4.22.17) as the in vivo protease responsible for decreased chaperone activity. Chaperone activity of normal alpha-crystallin from lenses of young rats was assayed by measuring attenuation of heat-induced aggregation and scattering of beta L-crystallin. alpha-Crystallin from the nucleus of lenses with selenite cataract showed specific selective proteolysis, and chaperone activity was diminished. Proteolysis of alpha-crystallin from selenite cataract lenses was mimicked by incubating normal alpha-crystallin with calpain II, and this also resulted in loss of chaperone activity. Two-dimensional gel electrophoresis and peptide mapping were used to identify four partially degraded alpha A- and alpha B-crystallin polypeptides following incubation of normal alpha-crystallin with calpain. Similar partially degraded alpha A and alpha B polypeptides were found in selenite cataract. Previous experiments indicated that alpha-crystallin chaperone activity decreases because of removal of the COOH terminus. Our experiments support this observation and suggest that calpain proteolysis of alpha-crystallin at the COOH terminus may result in a loss of chaperone activity in selenite cataract.
Highlights
Showed specific selective proteolysis, and chaperone no data have been published on whether or not a-crystallin activity wasdiminished.Proteolysisofa-crystallin chaperone activity is reduced in cataract
From selenite cataract lenses was mimicked by incu- The experimental model of cataract produced by an overbating normal a-crystallin with calpain 11, and this dose of selenite in theyoung rat was used in the present study ale0resultedin loss of chaperoneactivity
The purposes of the present study were to determine if the chaperone activity of a-crystallin is lost during the formation of the selenite cataract and to determine if in vitro incubation withpurified calpain I1 can cause the loss of chaperone activity of acrystallin
Summary
That thechaperone activity of a-crystallin may be important in helping to prevent denaturation of crystallins in lens (6, 7). Tel.: Rad) and eluted with 20 mM Tris buffer (pH = 7.5) containing 1mM. The lenses of 14-day-old rats were diesected and dins homogenized at a ratio of 1mg of lens wet weight/2.5 pl of buffer I. The lens homogenate were centrifuged a t 8OOO X g for 15 min at 25 "C to remove the insoluble protein. Soluble lens protein (30 mg/ ml) was incubated for 60 min at 37 "Cin buffer I containing 2.4 units. The molecular weights of aA-crystallin tryptic fragments were measured using fast atom bombardment mass ~ pectron ~ ~apsyd, escribed previously (22)
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