Abstract
Abstract: :
Purpose:Earlier studies in our laboratory have shown that the chaperone function of alpha-crystallin is significantly affected in diabetic rat lenses. We also showed that C-terminal degradation of alphaA-crystallin and alphaB-crystallin is increased in diabetic rat lenses. AlphaB-crystallin showed only one truncated product, i.e. alphaB1-170. The purpose this study is to show whether this truncation influences the chaperone function of alphaB-rystallin. Methods:The truncated alphaB1-170 was generated using the native rat alphaB-pET-23d(+) with the QuickChange TM Site-Directed Mutagenesis Kit (Stratagene). The recombinant alphaB-wild type and its mutant amplicons were transformed into expression host BL21(DE3)pLys S E.coli (Novagen). They were expressed and purified by gel filtration on Sephacry S-300 HR column. Chaperone activity of the alphaB-wt and alphaB1-170 were assayed by thermal aggregation of betaL-crystallin (62°C), insulin (37°C) with DTT and ADH (37°C) with EDTA. alpha-Glucosidase, whose refolding is facilitated by several chaperones, was chosen as the target protein for refolding studies. The enzyme was unfolded in the presence of 8 M urea, and renaturation was initiated by a 30-fold dilution in 40 mM HEPES-KOH, pH 7.8, and alpha-glucosidase activity was measured. Oligomeric sizes of the alphaB-wt and alphaB1-170 were determined by molecular sieve HPLC. The secondary and tertiary structural changes were probed by circular dichroism (CD) easurements. Results:The molecular masses and the secondary and tertiary structure of alphaB-wt and alphaB1-170 were not significantly different. The alphaB1-170 showed chaperone activities better than alphaB-wt in all the three chaperone activity assays. During the refolding assays, the maximal recovery of alpha-glucosidase activity reached 65% and 56% for alphaB-wt and alphaB1-170, respectively. These results show that, like other molecular chaperones, alphaB-wt and alphaB1-170 interacts with unfold proteins and increases their productive folding. Conclusion:The truncated alphaB1-170 functions as a better molecular chaperone in preventing the unfolding of its target proteins. Thus, it seems that the loss in chaperone activity of alpha-crystallin seen in diabetic lenses is not due to the truncation of alphaB-crystallin. It appears that the C-terminal flexible arm plays an important role in the chaperoning by alpha-crystallin.
Keywords: 343 chaperones • 378 crystallins • 387 diabetes