Research Reactor Institute, Kyoto University, Osaka, Japan.
ShinSei Lab Co., Ltd., Uji, Kyoto, Japan.
FEBS J. 2018 Jun;285(12):2263-2277. doi: 10.1111/febs.14475. Epub 2018 Apr 29.
Senile cataract onset is caused by insolubilization of lens proteins. The lens crystallin protein family correctly orders the formation of homo- or hetero-oligomers in lens fiber cells. Because lens fiber cells do not divide, covalent post-translational modifications, such as isomerization of aspartate residues, accumulate with aging. Although many isomerization sites of αA-crystallin have been reported, their structural and functional contributions have never been identified. In this study, αA-crystallin was extracted from aged human lens and separated into each oligomeric state by size exclusion chromatography and electrophoresis. The novel combination methodology of in-solution/gel tryptic digestion with liquid chromatography equipped with mass spectrometry (LC-MS/MS) was used to evaluate the isomerization of Asp 58. The contributions of isomerization to assembly, solubility, and chaperone functions of αA-crystallin were estimated using a series of mutations of Asp 58 in αA-crystallin. The results indicated that the isomerization of Asp 58 depended on the oligomer size and age of the lens. The substitution of Asp 58 for hydrophobic residues increased αA-crystallin oligomer size and decreased solubility. All substitutions decreased the chaperone function of αA-crystallin for aggregates of bovine βL-crystallin and alcohol dehydrogenase. The data indicated that Asp 58 in αA-crystallin was critical for intermolecular interactions in the lens. Our results also suggested that LC-MS/MS-based isomerization analyses of in-gel-digested products could be useful for investigating the isomerization of Asp residues in oligomeric states. This method could also be used to analyze d/l ratios of amino acid residues in soluble protein aggregates.
老年性白内障的发病是由于晶状体蛋白的不溶性。晶状体晶蛋白家族正确地将同源或异源寡聚体在晶状体纤维细胞中形成。由于晶状体纤维细胞不分裂，如天冬氨酸残基的异构化等共价翻译后修饰会随着年龄的增长而积累。尽管已经报道了许多αA-晶状体蛋白的异构化位点，但它们的结构和功能贡献从未被确定。在这项研究中，从老年人类晶状体中提取αA-晶状体蛋白，并通过排阻色谱和电泳分离成每个寡聚体状态。使用在溶液/凝胶胰蛋白酶消化与液相色谱配备质谱（LC-MS/MS）的新颖组合方法来评估 Asp 58 的异构化。使用αA-晶状体蛋白中 Asp 58 的一系列突变来估计异构化对αA-晶状体蛋白组装、溶解度和伴侣功能的贡献。结果表明，Asp 58 的异构化取决于寡聚体大小和晶状体的年龄。Asp 58 取代疏水性残基增加了αA-晶状体蛋白寡聚体的大小并降低了其溶解度。所有取代均降低了αA-晶状体蛋白对牛βL-晶状体蛋白和醇脱氢酶聚集体的伴侣功能。数据表明，αA-晶状体蛋白中的 Asp 58 对晶状体中的分子间相互作用至关重要。我们的结果还表明，基于 LC-MS/MS 的凝胶内消化产物的异构化分析可用于研究寡聚体状态中天冬氨酸残基的异构化。该方法还可用于分析可溶性蛋白聚集体中氨基酸残基的 d/l 比。
