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.

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.

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.