Wang Zhen, Friedrich Michael G, Truscott Roger J W, Schey Kevin L
Department of Biochemistry and Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, Tennessee, United States.
Molecular Horizons Research Institute, University of Wollongong, Wollongong, Australia.
Invest Ophthalmol Vis Sci. 2025 May 1;66(5):34. doi: 10.1167/iovs.66.5.34.
Cataract is believed to be caused by protein-protein and protein-membrane aggregation in the eye lens. After middle age, there is extensive binding of crystallins to the lens cell membranes as evidenced by sedimentation at high densities. Multiple protein modifications have been linked with cataract, whereas others have been associated with aging. The purpose of this study was to characterize protein constituents within high density protein-membrane fractions from normal aged or cataractous lenses and to compare these proteins and their modifications.
The inner nuclear regions of cataract or age-matched normal lenses were homogenized and proteins were separated using sucrose density gradient centrifugation. The low-density fractions (LDFs) and high-density fractions (HDFs) were analyzed by mass spectrometry using both top-down matrix-assisted desorption/ionization-mass spectrometry (MALDI-MS) and bottom-up liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic methods. Quantification of low molecular weight crystallin peptides, deamidation, and isomerization were performed.
Compared with normal aged-lenses, membrane-associated protein aggregates in high density fractions of cataract lenses exhibited significantly higher levels of γ-crystallins, as well as γS- and γD-crystallin C-terminal peptides. Deamidation of γ-crystallin, but not of β-crystallin, was increased in cataract lens membrane-bound aggregates. A very high level of Asp isomerization was detected in bound α-crystallins from both aged and cataract lenses.
Binding of crystallin aggregates to human lens cell membranes is associated with protein truncation, deamidation, and isomerization, and was observed in normal aged and cataract lenses. However, the protein aggregates bound to membranes in cataract lenses exhibit distinct modifications to γ-crystallins that may arise as a consequence of additional protein degradation.
白内障被认为是由眼晶状体中的蛋白质-蛋白质和蛋白质-膜聚集引起的。中年以后，晶状体蛋白与晶状体细胞膜有广泛结合，这在高密度沉降实验中得到证实。多种蛋白质修饰与白内障有关，而其他一些则与衰老相关。本研究的目的是鉴定正常老龄或白内障晶状体高密度蛋白质-膜组分中的蛋白质成分，并比较这些蛋白质及其修饰。
将白内障或年龄匹配的正常晶状体的内核区域匀浆，并用蔗糖密度梯度离心法分离蛋白质。使用自上而下的基质辅助激光解吸/电离质谱（MALDI-MS）和基于自下而上的液相色谱-串联质谱（LC-MS/MS）的蛋白质组学方法对低密度组分（LDFs）和高密度组分（HDFs）进行质谱分析。对低分子量晶状体蛋白肽、脱酰胺和异构化进行定量分析。
与正常老龄晶状体相比，白内障晶状体高密度组分中与膜相关的蛋白质聚集体显示出显著更高水平的γ-晶状体蛋白，以及γS-和γD-晶状体蛋白C末端肽。白内障晶状体膜结合聚集体中γ-晶状体蛋白的脱酰胺增加，但β-晶状体蛋白没有。在老龄和白内障晶状体的结合α-晶状体蛋白中检测到非常高水平的天冬氨酸异构化。
晶状体蛋白聚集体与人晶状体细胞膜的结合与蛋白质截短、脱酰胺和异构化有关，在正常老龄和白内障晶状体中均有观察到。然而，白内障晶状体中与膜结合的蛋白质聚集体对γ-晶状体蛋白表现出不同的修饰，这可能是由于额外的蛋白质降解所致。
