Aggregation of C-Terminal Truncated Human AlphaA-Crystallins in Mammalian Cells and Protection by AlphaB-Crystallin

Abstract

Purpose: : Post-translational modifications in human eye lens crystallins such as the formation of C-terminal truncated alphaA-crystallins, αA_172, αA₁₆₈ and αA_162, is believed to play a role in the development of senile cataract. The current investigation was aimed to study the fate of the truncated αA-crystallins and their interactions with native αA- and αB-crystallins in situ in living mammalian cells using fluorescence resonance energy transfer (FRET) and laser scanning confocal microscopy (LSM).

Methods: : Human αA-wt and αB-wt genes were cloned into pAmCyan1-Cl (CFP) vector for expression in cyan color. Genes of αA-wt and the C-terminal truncated αA crystallins, αA₁₇₂, αA₁₆₈ and αA₁₆₂ were cloned into pZsYellow1-C1 (YFP) for expression in yellow color. YFP tagged αA-wt or each of the truncated αA-crystallin was individually transfected or co-transfected with CFP tagged αA-wt or αB-wt into HeLa cells and after 48 h, cells were examined by laser scanning confocal microscopy. Proportion of cells having significant level of protein aggregates was determined by visual assessment and confirmed by subsequent light scattering studies. To show whether C-terminal truncation of αA-crystallin affects protein-protein interaction with native αA- or αB-crystallin, FRET-acceptor photo-bleaching protocol was followed.

Results: : When YFP tagged αA-wt, αA₁₇₂, αA₁₆₈ and αA₁₆₂ were expressed individually, nearly 5, 35, 70, and 85% cells, respectively, showed significant level of protein aggregates and also associated with distorted cell morphology only in cells harboring truncated αA-crystallins. Co-expression with αA-wt failed to inhibit protein aggregation in cells expressing αA₁₆₈ or αA_162. Cells expressing αA₁₇₂ appeared normal and lacked protein aggregates. Co-expression with αB-wt significantly improved cell morphology and decreased protein aggregation. FRET studies showed that the interaction of αA₁₇₂ with αA-wt or αB-wt was much stronger than that of αA₁₆₈ and αA_162. Moreover, the overall interaction of C-terminal truncated αA-crystallins with αB-wt was stronger than the interaction with αA-wt.

Conclusions: : This is the first report showing that C-terminal truncated αA-crytallins tend to aggregate in living mammalian cells unless they maintain strong interaction with native αB-crystallin. Association with αB-crystallin provides protection to the truncated αA-crystallins.

Keywords: chaperones • crystallins • microscopy: confocal/tunneling