Abstract
Purpose :
It is well-studied that levels of α-crystallin in the eye lens cytoplasm decrease steadily with increasing membrane-bound α-crystallin with age and cataracts. However, the binding of αA- and αB-crystallin to the eye lens membranes is poorly understood. This study investigates the binding of recombinant human αA- and αB-crystallin to the model of human lens lipid (MHLL) membranes with and without cholesterol (Chol) and measures the physical properties of these membranes.
Methods :
The genes for human αA- and αB-crystallin obtained from Genescript USA inc. were expressed recombinantly in E. coli using a pET-43.1a(+) plasmid system. Small unilamellar vesicles of the model of human lens lipid (MHLL) membranes and 33 mol% cholesterol-containing MHLL membranes (Chol/MHLL = 0.5) were prepared using the rapid solvent exchange method and probe-tip sonication. The binding of recombinant human αA- and αB-crystallin to the MHLL and Chol/MHLL = 0.5 membranes and the physical properties of these membranes were measured using the electron paramagnetic resonance spin-labeling method.
Results :
The maximum percentage of membrane surface occupied (MMSO) by αA-crystallin on the MHLL membranes is higher compared to αB-crystallin; however, the binding affinity (Ka) of αA-crystallin to the MHLL membranes is lower compared to αB-crystallin. The MMSO by αA- and αB-crystallin on the Chol/MHLL = 0.5 membranes is significantly lower than on the MHLL membranes, implying that Chol significantly decreases the binding of αA- and αB-crystallin to these membranes. The binding of αA- and αB-crystallin to MHLL membranes increases the hydrophobicity near the surface of these membranes, supporting the hypothesis that such binding creates a barrier to ionic and polar molecules. With increased concentrations of αA- and αB-crystallin, the mobility parameters decrease without a significant change in the maximum splittings, suggesting that the HMLL and Chol/MHLL membranes become less mobile without a significant change in the order near their surfaces.
Conclusions :
Our results show that αA- and αB-crystallin bind to MHLL and Chol/MHLL membranes differently, altering the physical properties of membranes. Moreover, our results show that Chol substantially decreases the MMSO by αA- and αB-crystallin on the membranes, suggesting that high Chol in the eye lens membranes likely inhibits the binding of α-crystallin to membranes preventing cataract formation.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.