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P Thampi, E Abraham; Truncation of C-terminal Serine of Human AlphaA-Crystallin Results in Decreased Chaperone Activity . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3560.
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Purpose: Truncation of the C-terminal end of the αA-crystallin is enhanced in diabetic human lenses. Studies have reported loss in chaperone activity of α-crystallins in diabetic lenses. The present study investigates the chaperone function of the native and the truncated αA-crystallin to ascertain whether an increased presence of the truncated αA-crystallin is one of the factors responsible for the decrease in chaperone activity of the α-crystallin from diabetic lenses. Methods: The C-terminal (Ser-173) truncated human αA-crystallin (αA 1-172) mutant was generated by site-directed mutagenesis using Quick Change Site-directed Mutagenesis Kit (Stratagene). Recombinant wild-type human αA-crystallin (αA-wt) and the αA1-172 mutant were expressed in BL21(DE3)pLys S E. coli cells and purified by Sephacryl S-300 HR gel permeation chromatography.Molecular masses were determined by molecular sieve HPLC. Secondary and tertiary structures were studied by circular dichroism measurements. Chaperone activity was tested using three methods : 1) DTT induced aggregation of insulin, 2) denaturation of alcohol dehydrogenase (ADH) with EDTA, and 3) thermal aggregation of ß-L crystallin at 62ºC.The effect of αA-wt and αA1-172 on protein refolding was studied by using α-glucosidase as target protein denaturated by using 8M urea. Results: Data from the molecular sieve HPLC and the conformational studies showed no significant difference in molecular mass and secondary and tertiary structures between αA-wt and αA1-172. At a ratio of 1:1 (αA:target protein) the percentage protection by αA-wt against target protein aggregation was 98, 85 and 62 whereas for αA1-172 the values were 85, 76 and 40 for ß-L crystallin,ADH and insulin respectively. For 1:5 ratio the corresponding values were 85, 46 and 35 for αA-wt and 41, 32 and 9.7 for αA1-172. The percentage decrease in the chaperone activity of αA1-172 as compared to αA-wt varied from 11-36% for the 1:1 ratio and 30-72% for the 1:5 ratio. In the refolding assay, maximal recovery of α-glucosidase was 49% for αA-wt and 48% for αA1-172 . Conclusion: Compromising the flexibility of the C-terminal extension has been reported to decrease the chaperone activity. Truncation of the C-terminal serine decreases the chaperone activity of αA-crystallin. The levels of αA1-172 increases in diabetic lenses thereby depleting the native αA-crystallin levels in these lenses. This contributes to loss in the chaperone activity of the α-crystallins in the diabetic lenses.
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