Cholesterol and Cholesterol Bilayer Domains Inhibit the Binding of Alpha-Crystallin with Phospholipid Membranes

Abstract

Purpose : The level of α-crystallin decreases in the eye lens cytoplasm, with the corresponding increase in the membrane-bound α-crystallin during cataract progression. Eye lens membrane consists of extremely high cholesterol (Chol) content that favors the formation of cholesterol bilayer domains (CBDs) within the membrane. The function of the high Chol level in the lens membrane is unclear. The purpose of this research is to understand the interaction of α-crystallin with Chol and CBDs in the phospholipid membrane.

Methods : Continuous-wave electron paramagnetic resonance (EPR) spin-labeling method was used to estimate the binding affinity (K_a) of α-crystallin with the Chol/POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine) membranes with varying Chol/POPC mixing ratio from 0 to 1.5. Also, the maximum membrane surface occupied (MSO) by the α-crystallin and the change in the membrane physical property (mobility parameter) after the α-crystallin binding was estimated using the EPR method. The small unilamellar vesicles of Chol/POPC membrane with 1 mol% CSL spin-label were prepared using the rapid solvent exchange method followed by probe-tip sonication. The CSL spin-labels incorporated into the membrane monitor the α-crystallin binding.

Results : The K_a and MSO followed the trends: K_a (0) > K_a (0.3) > K_a (0.5) > K_a (1) = K_a (1.5) and MSO (0) > MSO (0.3) > MSO (0.5) > MSO (1) = MSO (1.5), where numbers in the parenthesis represent the Chol/POPC mixing ratio. For Chol/POPC mixing ratio greater than 1, CBDs form within the membrane. These results indicate that Chol and CBDs inhibit the binding of α-crystallin to the POPC membrane. The profiles of the mobility parameter decrease with an increase in the α-crystallin binding; however, the decrease is pronounced for the lower Chol concentration compared to the high Chol concentration in the membrane. These results represent that membrane becomes more immobilized near the headgroup regions with the increase in α-crystallin binding.

Conclusions : Our results show that the Chol and CBDs inhibit the binding of α-crystallin to the phospholipid membrane, and the membrane physical property alters with α-crystallin binding. These results provide a molecular basis for understanding the functions of Chol and CBDs' in the eye lens membrane in maintaining lens transparency and possibly protecting against cataract formation.

This is a 2021 ARVO Annual Meeting abstract.