by Ilangovan Raju, Lalita Oonthonpan, Edathara C. Abraham
Mutation in aA-crystallin contributes to the development of congenital cataract in humans. Heterooligomerization of aA-crystallin and aB-crystallin is essential for maintaining transparency in the eye lens. The effect of congenital cataract causing mutants of aA-crystallin on subunit exchange and interaction with aB-crystallin is unknown. In the present study, 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. Methodology/Principal Findings
In vitro FRET technique was used to demonstrate the rates of subunit exchange of aB-wt with the following aA-crystallin mutants: R12C, R21L, R21W, R49C, R54C, and R116C. The subunit exchange rates (k values) of R21W and R116C with aB-wt decreased drastically as compared to aA-wt interacting with aB-wt. Moderately decreased k values were seen with R12C, R49C and R54C while R21L showed nearly normal k value. The interaction of aA- mutants with aB-wt was also assessed by in situ FRET. YFP-tagged aA mutants were co-expressed with CFP-tagged aB-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 aB-wt was decreased nearly 50% as compared to aA-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/Significance
Structural changes occurring in these mutants, as reported earlier, could be the underlying cause for the decreased interaction with aB may contribute to development of congenital cataract.