Interaction of ?A3-Crystallin with Deamidated Mutants of ?A- and ?B-Crystallins.

ABSTRACT: 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 ?- and ?-crystallins. Here, we investigated the effects of the deamidation of ?A- and ?B-crystallins on their interaction with ?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 ?A- and WT ?B-crystallins and their deamidated mutants with ?A3-crystallin. The deamidated mutants of ?A-crystallin (?A N101D and ?A N123D) displayed lower adherence propensity for ?A3-crystallin relative to the binding affinity shown by WT ?A-crystallin. Among ?B-crystallin mutants, ?B N78D displayed higher adherence propensity whereas ?B N146D mutant showed slightly lower binding affinity for ?A3-crystallin relative to that shown by WT ?B-crystallin. Under the in vivo condition (FLIM-FRET), both ?A-deamidated mutants (?A N101D and ?A N123D) exhibited strong interaction with ?A3-crystallin (32±4% and 36±4% FRET efficiencies, respectively) compared to WT ?A-crystallin (18±4%). Similarly, the ?B N78D and ?B N146D mutants showed strong interaction (36±4% and 22±4% FRET efficiencies, respectively) with ?A3-crystallin compared to 18±4% FRET efficiency of WT ?B-crystallin. Further, FLIM-FRET analysis of the C-terminal domain (CTE), N-terminal domain (NTD), and core domain (CD) of ?A- and ?B-crystallins with ?A3-crystallin suggested that interaction sites most likely reside in the ?A CTE and ?B NTD regions, respectively, as these domains showed the highest FRET efficiencies. Overall, results suggest that similar to WT ?A- and WT?B-crystallins, the deamidated mutants showed strong interactionfor ?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 ?-crystallin may facilitate greater interaction and the formation of large oligomers with other crystallins, and this may contribute to the cataractogenic mechanism.

SUBMITTER: Tiwary E 

PROVIDER: S-EPMC4691197 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature