Mutations in Human αA-Crystallin/sHSP Affect Subunit Exchange Interaction with αB-Crystallin

Abstract

BackgroundMutation in αA-crystallin contributes to the development of congenital cataract in humans. Heterooligomerization of αA-crystallin and αB-crystallin is essential for maintaining transparency in the eye lens. The effect of congenital cataract causing mutants of αA-crystallin on subunit exchange and interaction with αB-crystallin is unknown. In the present study, interaction of the mutants of αA-crystallin with αB-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 αB-wt with the following αA-crystallin mutants: R12C, R21L, R21W, R49C, R54C, and R116C. The subunit exchange rates (k values) of R21W and R116C with αB-wt decreased drastically as compared to αA-wt interacting with αB-wt. Moderately decreased k values were seen with R12C, R49C and R54C while R21L showed nearly normal k value. The interaction of αA- mutants with αB-wt was also assessed by in situ FRET. YFP-tagged αA mutants were co-expressed with CFP-tagged αB-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 αB-wt was decreased nearly 50% as compared to αA-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/SignificanceStructural changes occurring in these mutants, as reported earlier, could be the underlying cause for the decreased interaction with αB may contribute to development of congenital cataract.

The multifaceted nature of αB-crystallin

“…Co-immunoprecipitation confirmed the complexation and subunit exchange of WT αB-c and Hsp27 with R120G αB-c (Chavez Zobel et al 2003). Additionally, the rate of subunit exchange with various αA-c mutants and WT αB-c is slower compared to WT αA-c and αB-c (Raju et al 2012), implying that the subunit exchange is favoured between functional/non-mutant sHsps compared to their mutant/disease counterparts.…”

Congenital cataract – clinical and morphological aspects

“…They have a great importance in maintaining the refraction index in the lens [16]. In the human lens three main classes of "crystallins" have been identified, i.e., α-crystallin -representing 40%, β-crystallin -about 35% and γ-crystallin -25% [5,17,18].…”

Differential role of arginine mutations on the structure and functions of α-crystallin

“…Assessment of the available surface hydrophobic patches of these mutant proteins showed a drastic increase in the surface hydrophobicity for R21W and R21L, while other mutants had lowered surface hydrophobicity as compared to the wild-type protein [63]. Another independent study revealed that the subunit exchange rates of these αA-crystallin mutants, R21W and R116C, with wild-type αB-crystallin decreased considerably as compared to when wild-type αA-crystallin interacted with wild-type αB-crystallin [64]. In summary, the drastic decrease in the chaperone function of R12C and R21W may be the consequence of: 1) reduction in subunit exchange rate with αB-crystallin; 2) alterations in the secondary, tertiary, and quaternary structures of the mutant proteins, which may be the critical factors behind the formation of congenital cataract.…”

“…In summary, the drastic decrease in the chaperone function of R12C and R21W may be the consequence of: 1) reduction in subunit exchange rate with αB-crystallin; 2) alterations in the secondary, tertiary, and quaternary structures of the mutant proteins, which may be the critical factors behind the formation of congenital cataract. Subunit exchange studies by Abraham’s group also revealed that both R54C and R49C mutations lowered the subunit exchange kinetics between αA- and αB-crystallin [64]. This decrease in subunit exchange rate and altered secondary structure due to these two mutations, may be a possible cause for cataract induction in the lens.…”