PDF(Pubmed)
Abstract：
Highly concentrated lens proteins, mostly β- and γ-crystallin, are responsible for maintaining the structure and refractivity of the eye lens. However, with aging and cataract formation, β- and γ-crystallin are associated with the lens membrane or other lens proteins forming high-molecular-weight proteins, which further associate with the lens membrane, leading to light scattering and cataract development. The mechanism by which β- and γ-crystallin are associated with the lens membrane is unknown. This work aims to study the interaction of β- and γ-crystallin with the phospholipid membrane with and without cholesterol (Chol) with the overall goal of understanding the role of phospholipid and Chol in β- and γ-crystallin association with the membrane. Small unilamellar vesicles made of Chol/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (Chol/POPC) membranes with varying Chol content were prepared using the rapid solvent exchange method followed by probe tip sonication and then dispensed on freshly cleaved mica disk to prepare a supported lipid membrane. The βL- and γ-crystallin from the cortex of the bovine lens was used to investigate the time-dependent association of βL- and γ-crystallin with the membrane by obtaining the topographical images using atomic force microscopy. Our study showed that βL-crystallin formed semi-transmembrane defects, whereas γ-crystallin formed transmembrane defects on the phospholipid membrane. The size of semi-transmembrane defects increases significantly with incubation time when βL-crystallin interacts with the membrane. In contrast, no significant increase in transmembrane defect size was observed in the case of γ-crystallin. Our result shows that Chol inhibits the formation of membrane defects when βL- and γ-crystallin interact with the Chol/POPC membrane, where the degree of inhibition depends upon the amount of Chol content in the membrane. At a Chol/POPC mixing ratio of 0.3, membrane defects were observed when both βL- and γ-crystallin interacted with the membrane. However, at a Chol/POPC mixing ratio of 1, no association of γ-crystallin with the membrane was observed, which resulted in a defect-free membrane, and the severity of the membrane defect was decreased when βL-crystallin interacted with the membrane. The semi-transmembrane or transmembrane defects formed by the interaction of βL- and γ-crystallin on phospholipid membrane might be responsible for light scattering and cataract formation. However, Chol suppressed the formation of such defects in the membrane, likely maintaining lens membrane homeostasis and protecting against cataract formation.
摘要：
高度浓缩的晶状体蛋白，主要是β-和γ-晶状体蛋白，负责维持眼睛晶状体的结构和屈光度。然而,随着衰老和白内障的形成，β-和γ-晶状体蛋白与晶状体膜或其他晶状体蛋白相关，形成高分子量蛋白，这进一步与晶状体膜相关联，导致光散射和白内障发展。β-和γ-晶状体蛋白与晶状体膜相关的机制尚不清楚。这项工作旨在研究β-和γ-晶状体蛋白与有和没有胆固醇（Chol）的磷脂膜的相互作用，总体目标是了解磷脂和Chol在β-和γ-晶状体蛋白与膜的关联中的作用。使用快速溶剂交换法制备具有不同Chol含量的由Chol/1-棕榈酰-2-油酰基-sn-甘油-3-磷酸胆碱（Chol/POPC）制成的小单层囊泡，然后进行探针超声处理，然后分配在新鲜裂解的云母圆盘上制备支持的脂质膜。通过使用原子力显微镜获得地形图像，来自牛晶状体皮质的βL-和γ-晶状体蛋白用于研究βL-和γ-晶状体蛋白与膜的时间依赖性关联。我们的研究表明，βL-晶状体蛋白形成半跨膜缺陷，而γ-晶状体蛋白在磷脂膜上形成跨膜缺陷。当βL-晶状体蛋白与膜相互作用时，半跨膜缺陷的大小随孵育时间而显着增加。相比之下,在γ-晶状体蛋白的情况下，没有观察到跨膜缺陷尺寸的显着增加。我们的结果表明，当βL-和γ-晶状体蛋白与Chol/POPC膜相互作用时，Chol抑制膜缺陷的形成，其中抑制程度取决于膜中Chol含量的量。在0.3的Chol/POPC混合比下，当βL-和γ-晶状体蛋白与膜相互作用时，观察到膜缺陷。然而,在Chol/POPC混合比为1时，未观察到γ-晶状体蛋白与膜的关联，这导致了一个无缺陷的膜，当βL-晶状体蛋白与膜相互作用时，膜缺陷的严重程度降低。βL-和γ-晶状体蛋白在磷脂膜上相互作用形成的半跨膜或跨膜缺陷可能是光散射和白内障形成的原因。然而,Chol抑制了膜中此类缺陷的形成，可能维持晶状体膜稳态并防止白内障形成。
