December 2002
Volume 43, Issue 13
ARVO Annual Meeting Abstract  |   December 2002
C-Terminal Truncation of Five Amino Acid Residues of AlphaB-Crystallin Influences Its Chaperone Function
Author Affiliations & Notes
  • W Cho
    Biochem/Molec Bio Univ Arkansas Med Sciences Little Rock AR
  • E Abraham
    Biochemistry and Molecular Biology University of Arkansas-Med Sciences Little Rock AR
  • Footnotes
    Commercial Relationships   W. Cho, None; E. Abraham, None. Grant Identification: EY11352
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 3561. doi:
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      W Cho, E Abraham; C-Terminal Truncation of Five Amino Acid Residues of AlphaB-Crystallin Influences Its Chaperone Function . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3561.

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      © ARVO (1962-2015); The Authors (2016-present)

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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 

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