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

BACKGROUND: Mutation 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/SIGNIFICANCE: Structural 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

Rate of subunit exchange at acceptor energy (515 nm).

<p>Graph depicts time dependent increases in emissions intensity due to subunit exchange of αB-wt and αA-wt or mutants subunits. Increase in the relative fluorescence intensity at 515 nm due to fluorescence energy transfer from the SITS-labeled to the LYI-labeled proteins. Each curve represents the best statistical fit of the data to the exponential function of Ft/F0 = A₁+A₂e^−kt. The two-tailed unpaired Student's t-test was used to determine the significance. The p value is <0.0001 for αB-wt + αA-wt Vs αB-wt + αA-R21W and αB-wt + αA-wt Vs αB-wt + αA-R116C.</p

Interaction of αB-wt and αA-wt or its mutants by <i>in situ</i> FRET.

<p>Examples of the acceptor photobleaching method for determining the FRET efficiency. In this example, CFPαBwt (donor) was co-expressed with either YFPαA-wt or YFPαA-R2116C (acceptor). The acceptor fluorescence was bleached by high intensity argon laser light. This resulted in an increase in donor fluorescence intensity and a decrease in acceptor fluorescence. The actual FRET efficiency was measured from at least 50 regions of interest (ROI) in the cell images obtained in three independent experiments for each pair.</p

Rate constants (k) of heteroaggregates of αB-wt and αA-wt and its mutants at the acceptor, 515 nm energy.

<p>Rate constants (k) of heteroaggregates of αB-wt and αA-wt and its mutants at the acceptor, 515 nm energy.</p

A–G: <i>In Vitro</i> FRET to demonstrate the subunit exchange between SITS-labeled αB-wt and LYI-labeled αA-wt or mutants.

<p>A representative emission spectra of αB-crystallin excited at 336 nm were recorded at 0 (a), 2 (b), 4 (c), 6 (d), 8 (e), 10 (f), 20 (g), 45 (h), 75 (i) and 120 (j) minutes after mixing of SITS-labeled αB-wt and LYI-labeled αA-wt or mutants. The decrease in fluorescence intensity at 426 nm of the SITS-labeled αB-wt protein and the concomitant increase in fluorescence intensity at 515 nm of the LYI-labeled αA-wt or mutants proteins are the indicative of energy transfer between two labeled populations. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031421#pone-0031421-g001" target="_blank"><i>Fig. 1A</i></a>: Time dependent spectral changes in the FRET due to subunit exchange of SITS labeled αB-wt and LYI-labeled αA-wt. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031421#pone-0031421-g001" target="_blank"><i>Fig. 1B</i></a>: Time dependent spectral changes in the FRET due to subunit exchange of SITS labeled αB-wt and LYI-labeled αA-R12C. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031421#pone-0031421-g001" target="_blank"><i>Fig. 1C</i></a>: The emission spectra of αB-crystallin excited at 336 nm were recorded at 0 (a), 2 (b), 4 (c), 6 (d), 8 (e), 10 (f), 20 (g), 45 (h), 75 (i) and 120 (j) minutes after mixing of SITS-labeled αB-wt and LYI-labeled R21L. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031421#pone-0031421-g001" target="_blank"><i>Fig. 1D</i></a>: The emission spectra of αB-crystallin excited at 336 nm were recorded at 0 (a), 2 (b), 4 (c), 6 (d), 8 (e), 10 (f), 20 (g), 45 (h), 75 (i) and 120 (j) minutes after mixing of SITS-labeled αB-wt and LYI-labeled R21W mutants. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031421#pone-0031421-g001" target="_blank"><i>Fig. 1E</i></a>: The emission spectra of αB-crystallin excited at 336 nm were recorded at 0 (a), 2 (b), 4 (c), 6 (d), 8 (e), 10 (f), 20 (g), 45 (h), 75 (i) and 120 (j) minutes after mixing of SITS-labeled αB-wt and LYI-labeled R49C. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031421#pone-0031421-g001" target="_blank"><i>Fig. 1F</i></a>: The emission spectra of αB-crystallin excited at 336 nm were recorded at 0 (a), 2 (b), 4 (c), 6 (d), 8 (e), 10 (f), 20 (g), 45 (h), 75 (i) and 120 (j) minutes after mixing of SITS-labeled αB-wt and LYI-labeled R54C. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031421#pone-0031421-g001" target="_blank"><i>Fig. 1G</i></a>: The emission spectra of αB-crystallin excited at 336 nm were recorded at 0 (a), 2 (b), 4 (c), 6 (d), 8 (e), 10 (f), 20 (g), 45 (h), 75 (i) and 120 (j) minutes after mixing of SITS-labeled αB-wt and LYI-labeled R116C. Only 10 spectral curves were shown because of RF-5301PC software is not allowing more than 10.</p

Bar diagram showing the level of FRET efficiency.

<p>FRET efficiency demonstrates the interaction between the αA and αB subunits of α-crystallin. The interaction was strong between the wild-types of αA and αB subunits. The interaction between the mutated constructs, αA-R21W and αA-R116C with αB-wt was lower to αAwt+αBwt and also lower to other mutants group. The results were expressed as mean ± SD. Two-tailed unpaired Student's t-test was used and the p value for αB-wt+αA-R21W is <0.001 and for αB-wt+αA-R116C is <0.0008 compared to αB-wt+αA-wt group.</p