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E. C. Abraham, K. S. Latha, A. Aziz; Subunit Exchange Studies in Hetero- and Homo-Oligomers of C-Terminal Truncated Human A-Crystallins. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1524.
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© ARVO (1962-2015); The Authors (2016-present)
Human lenses are known to contain αA-crystallin in which 1, 5 and 11 C-terminal residues have been deleted. The level of all the truncated αA-crystallins together constitutes 30-50% of the total αA-crystallin, diabetic lenses containing the highest level of truncated αA-crystallins. The truncated αA-crystallins are expected to exist as homo-oligomers or hetero-oligomers. The oligomeric structure and chaperone function of such oligomers could be influenced by the presence of truncated αA-crystallins. The purpose of the present work is to study the effect of C-terminal truncation of αA-crystallin on its interaction with αB- and αA-crystallins forming hetero- or homo-oligomers.
Previously cloned human αA- and αB-crystallins were subcloned into the pET-23d(+) expression vector. C-terminal truncated αA-crystallins were generated by using the QuickChange site-directed mutagenesis kit. Proteins were expressed in E. Coli BL21(DE3) pLysS cells and purified by size exclusion chromatography. Fluorescence resonance energy transfer (FRET) was employed to determine the rate of subunit exchange. Reconstituted hetero-oligomers containing αB-crystallin and truncated αA-crystallins were analyzed for their oligomeric size, structure, and chaperone function. Levels of αB- and αA-crystallins in the hetero-oligomers were determined by SDS-PAGE followed by image analysis by using Image J 1.37v (NIH).
The rate of subunit exchange within the hetero-oligomers containing αB-crystallin and truncated αA-crystallins was two-fold lower for αA1-172 and αA1-168 and six-fold lower for αA1-162 than that of hetero-oligomers of αA-wt and αB-wt. Subunit exchange rate between αA-wt and αA-wt was only 50% of that of αA-wt and αB-wt. However, the subunit exchange rates were nearly the same for hetero-oligomers and homo-oligomers of αA1-172 and αA1-168 and different for αA1-162. Levels of αA-crystallin in the hetero-oligomers were 45, 47, 48 and 34% respectively, for αA-wt, αA1-172 αA1-168 and αA1-162. The oligomeric structure and the chaperone function of the reconstituted hetero-oligomers reflected the proportions of the truncated αA-crystallins and αB-wt.The oligomeric size of the reconstituted hetero-oligomers of αA-wt, αA1-172, αA1-168 and αA1-162 were 610, 670, 610 and 525 kDa, respectively.
Within human lens, there is equal chance to form hetero-and homo- oligomers of truncated αA-crysallins. Truncation of 11 residues which include the C-terminal flexible tail has the maximum effect on subunit exchange and hetero-oligomerization.
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