Abstract
BackgroundMutation in αA-crystallin contributes to the development of congenital cataract in humans. Heterooligomerization of αA-crystallin and αB-crystallin is essential for maintaining transparency in the eye lens. The effect of congenital cataract causing mutants of αA-crystallin on subunit exchange and interaction with αB-crystallin is unknown. In the present study, interaction of the mutants of αA-crystallin with αB-crystallin was studied both in vitro and in situ by the fluorescence resonance energy transfer (FRET) technique.Methodology/Principal Findings In vitro FRET technique was used to demonstrate the rates of subunit exchange of αB-wt with the following αA-crystallin mutants: R12C, R21L, R21W, R49C, R54C, and R116C. The subunit exchange rates (k values) of R21W and R116C with αB-wt decreased drastically as compared to αA-wt interacting with αB-wt. Moderately decreased k values were seen with R12C, R49C and R54C while R21L showed nearly normal k value. The interaction of αA- mutants with αB-wt was also assessed by in situ FRET. YFP-tagged αA mutants were co-expressed with CFP-tagged αB-wt in HeLa cells and the spectral signals were captured with a confocal microscope before and after acceptor laser photobleaching. The interaction of R21W and R116C with αB-wt was decreased nearly 50% as compared to αA-wt while the rest of the mutants showed slightly decreased interaction. Thus, there is good agreement between the in vitro and in situ FRET data.Conclusions/SignificanceStructural changes occurring in these mutants, as reported earlier, could be the underlying cause for the decreased interaction with αB may contribute to development of congenital cataract.
Highlights
Introduction aCrystallin belongs to small heat shock protein family
Human aA-crystallin contains two cysteine residues, 1 mol of LYI was tagged with aA-wt, R21L and R21W
In the case of mutants, aA-R12C, aA-R49C, aA-R54C and aAR116C, protein tagged with 2 mol of LYI was mixed with aB-wt protein tagged with 2 mol of SITS
Summary
Crystallin belongs to small heat shock (sHSP) protein family. This major lens protein in vertebrates consists of two highly homologous 20 kDa subunits, aA and aB [1,2]. AA and aB crystallins share 57% sequence homology [3,4] and considered as molecular chaperones by having the ability to prevent aggregation of partially unfolded proteins [5,6,7,8,9]. The effect of congenital cataract causing mutants of aA-crystallin on subunit exchange and interaction with aB-crystallin is unknown. Interaction of the mutants of aA-crystallin with aB-crystallin was studied both in vitro and in situ by the fluorescence resonance energy transfer (FRET) technique
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