Interaction of βA3-Crystallin with Deamidated Mutants of αA- and αB-Crystallins

Interaction among crystallins is required for the maintenance of lens transparency. Deamidation is one of the most common post-translational modifications in crystallins, which results in incorrect interaction and leads to aggregate formation. Various studies have established interaction among the alpha- and beta-crystallins. Here, we investigated the effects of the deamidation of alpha A- and alpha B-crystallins on their interaction with beta A3-crystallin using surface plasmon resonance (SPR) and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) methods. SPR analysis confirmed adherence of WT alpha A- and WT alpha B-crystallins and their deamidated mutants with beta A3-crystallin. The deamidated mutants of alpha A-crystallin (alpha A N101D and alpha A N123D) displayed lower adherence propensity for beta A3-crystallin relative to the binding affinity shown by WT alpha A-crystallin. Among alpha B-crystallin mutants, alpha B N78D displayed higher adherence propensity whereas alpha B N146D mutant showed slightly lower binding affinity for beta A3-crystallin relative to that shown by WT alpha B-crystallin. Under the in vivo condition (FLIM-FRET), both alpha A-deamidated mutants (alpha A N101D and alpha A N123D) exhibited strong interaction with beta A3-crystallin (32 +/- 4% and 36 +/- 4% FRET efficiencies, respectively) compared to WT alpha A-crystallin (18 +/- 4%). Similarly, the alpha B N78D and alpha B N146D mutants showed strong interaction (36 +/- 4% and 22 +/- 4% FRET efficiencies, respectively) with beta A3-crystallin compared to 18 +/- 4% FRET efficiency of WT alpha B-crystallin. Further, FLIM-FRET analysis of the C-terminal domain (CTE), N-terminal domain (NTD), and core domain (CD) of alpha A- and alpha B-crystallins with beta A3-crystallin suggested that interaction sites most likely reside in the alpha A CTE and alpha B NTD regions, respectively, as these domains showed the highest FRET efficiencies. Overall, results suggest that similar to WT alpha A- and WT alpha B-crystallins, the deamidated mutants showed strong interaction for beta A3-crystallin. Variable in vitro and in vivo interactions are most likely due to the mutant's large size oligomers, reduced hydrophobicity, and altered structures. Together, the results suggest that deamidation of alpha-crystallin may facilitate greater interaction and the formation of large oligomers with other crystallins, and this may contribute to the cataractogenic mechanism.