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P. Thampi, E.C. Abraham; Cleavage of 11,16 or 22 C-terminal Residues of Rat alphaA-Crystallin Generates Smaller Oligomers with Altered Structure and Decreased Chaperone Activity . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2363.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We have shown earlier that α-crystallin chaperone activity is significantly affected in diabetic rat lenses. Mass spectrometric analysis showed high levels of truncated αA-crystallin lacking 5, 10, 11, 16 or 22 C-terminal residues. The purpose of this study is to show whether cleavage of these residues influences oligomerization, structure, and chaperone activity. Methods: For generation of various truncated rat αA-crystallins, stop codon was introduced at the positions of interest using the Quick Change Site-Directed Mutagenesis Kit and expressed in BL21(DE3)p Lys S E.coli cells and purified by size-exclusion chromatography. Molecular mass was determined by molecular sieve HPLC. Chaperone activity was studied by using insulin and alcohol dehydrogenase chemical denaturation assays and ßL-crystallin heat denaturation assay. Secondary and tertiary structures were determined by measuring circular dichroism (CD) spectra. Results: αA1-172, αA1-168 and αA1-163 mutants (Group-1) had oligomeric sizes about 500 kDa while αA1-162, αA1–157 and αA1-151 (Group-2) were only about 150 kDa as compared to 600 kDa for αA wild-type (αA-wt). Structural analysis showed two-fold increase in the α-helix content and significant change in the tertiary structure only in Group-2 mutants. Chaperone activity was nearly unchanged in Group-1 whereas Group-2 mutants showed 40-70 % loss, depending on the method of analysis. Most strikingly, cleavage of 11 C-terminal residues, as opposed to10 residues, showed substantial changes in oligomeric size, structure and chaperone activity. Conclusions: The C-terminal extension containing 8 residues as well as 3 additional residues including Arg-163 seems to control the formation of large oligomers in αA-crystallin, needed to maintain normal chaperone function. It appears that Arg-163 has a major role in the C-terminal domain.
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