Hazen Preston, Khadka Nawal K, Mainali Laxman
Biomolecular Sciences Graduate Program, Boise State University, Boise, Idaho, United States.
Department of Physics, Boise State University, Boise, Idaho, United States.
Invest Ophthalmol Vis Sci. 2025 May 1;66(5):8. doi: 10.1167/iovs.66.5.8.
The αA-Crystallin (αAc) binding with lens membranes increases with age and cataract formation. However, the role of lipids and cholesterol (Chol) in Q147E-αAc membrane binding remains unclear, which we aim to elucidate in this study.
We have used the electron paramagnetic resonance spin-labeling method to probe the Chol/ 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and Chol/ sphingomyelin (SM) membranes binding with wild-type (WT) and Q147E-αAc.
Compared to WT-αAc, the Q147E mutant had increased binding to POPC and decreased binding to SM membranes without Chol. Adding 33 mol% Chol to the POPC and SM membranes decreased the binding of WT and, to a lesser degree, decreased the binding of Q147E-αAc to the membranes. Adding 60 mol% Chol completely inhibited Q147E mutant and WT binding to POPC membranes. However, 33 and 60 mol% Chol completely inhibited WT and Q147E mutant binding to SM membranes, respectively. WT and Q147E-αAc membrane binding decreased membrane mobility while increasing order and hydrophobicity near the headgroup.
In Chol-free membranes, the deamidated Q147E-αAc binds significantly more to the POPC membranes compared to WT, whereas WT binds significantly more to the SM membranes compared to Q147E-αAc. In contrast, for 33 mol% Chol-containing membranes, the deamidated Q147E-αAc binds significantly more to POPC and SM membranes than WT. Conversely, 60 mol% Chol-containing membranes completely inhibit WT and deamidated Q147E-αAc binding to POPC and SM membranes. These results suggest that increased Chol content of the lens membranes during aging protects against accumulation of modified proteins on the membrane associated with cataracts.
αA-晶体蛋白（αAc）与晶状体膜的结合随年龄增长和白内障形成而增加。然而，脂质和胆固醇（Chol）在Q147E-αAc膜结合中的作用仍不清楚，我们旨在本研究中阐明这一点。
我们使用电子顺磁共振自旋标记法来探测胆固醇/1-棕榈酰-2-油酰磷脂酰胆碱（POPC）和胆固醇/鞘磷脂（SM）膜与野生型（WT）和Q147E-αAc的结合。
与WT-αAc相比，Q147E突变体与POPC的结合增加，与不含胆固醇的SM膜的结合减少。向POPC和SM膜中添加33 mol%的胆固醇会降低WT的结合，在较小程度上也会降低Q147E-αAc与膜的结合。添加60 mol%的胆固醇会完全抑制Q147E突变体和WT与POPC膜的结合。然而，33 mol%和60 mol%的胆固醇分别完全抑制WT和Q147E突变体与SM膜的结合。WT和Q147E-αAc的膜结合降低了膜的流动性，同时增加了靠近头部基团处的有序性和疏水性。
在不含胆固醇的膜中，与WT相比，脱酰胺的Q147E-αAc与POPC膜的结合明显更多，而与Q147E-αAc相比，WT与SM膜的结合明显更多。相反，对于含33 mol%胆固醇的膜，脱酰胺的Q147E-αAc与POPC和SM膜的结合比WT明显更多。相反，含60 mol%胆固醇的膜完全抑制WT和脱酰胺的Q147E-αAc与POPC和SM膜的结合。这些结果表明，衰老过程中晶状体膜中胆固醇含量的增加可防止与白内障相关的修饰蛋白在膜上积累。
