A Rapid, Comprehensive Liquid Chromatography-Mass Spectrometry (LC-MS)-based Survey of the Asp Isomers in Crystallins from Human Cataract Lenses
A Rapid, Comprehensive Liquid Chromatography-Mass Spectrometry (LC-MS)-based Survey of the Asp Isomers in Crystallins from Human Cataract Lenses
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DOI：
10.1074/jbc.m112.399972
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发表时间：
2012-11-16
影响因子：
4.8
通讯作者：
Fujii, Noriko
中科院分区：
文献类型：
--
作者：
Fujii, Norihiko;Sakaue, Hiroaki;Fujii, Noriko
白内障是由眼晶状体混浊引起的，可能导致部分或全部失明。然而，白内障的发生机制还不是很清楚。人们认为，随着年龄的增长，晶状体蛋白质会形成异常聚集，导致晶状体透明度丧失和白内障的发生。晶状体蛋白由可溶性的α-、β-和γ-晶状体蛋白组成，作为长寿蛋白，它们经过翻译后修饰，包括异构化、脱酰胺化和氧化，导致不溶解、聚集和功能丧失，从而可能导致白内障。因此，分析蛋白质中单个氨基酸残基的翻译后修饰是很重要的。然而，这些蛋白质中形成的氨基酸的光学异构体的检测是困难的，因为光学拆分只能使用复杂的方法来实现。在这项研究中，我们描述了一种新的方法，利用LC-MS和相应的含有天冬氨酸异构体的合成肽来分析蛋白质中单个天冬氨酸残基的异构化。这使得准确、快速地分析蛋白质中天冬氨酸残基的异构体成为可能。我们证明了αA-晶体蛋白的Asp-58、-76、-84和-151以及αB-晶体蛋白的Asp-62和-96高度转化为Lβ-、Dβ-和Dα-异构体。晶状体蛋白中所有天冬氨酸不溶部分中天冬氨酸的异构化量较大，表明这些天冬氨酸残基的异构化影响蛋白质的高阶结构，导致蛋白质在晶状体中聚集、不溶和功能障碍的增加，从而导致白内障的发生。
Cataracts are caused by clouding of the eye lens and may lead to partial or total loss of vision. The mechanism of cataract development, however, is not well understood. It is thought that abnormal aggregates of lens proteins form with age, causing loss of lens clarity and development of the cataract. Lens proteins are composed of soluble alpha-, beta-, and gamma-crystallins, and as long lived proteins, they undergo post-translational modifications including isomerization, deamidation, and oxidation, which induce insolubilization, aggregation, and loss of function that may lead to cataracts. Therefore, analysis of post-translational modifications of individual amino acid residues in proteins is important. However, detection of the optical isomers of amino acids formed in these proteins is difficult because optical resolution is only achieved using complex methodology. In this study, we describe a new method for the analysis of isomerization of individual Asp residues in proteins using LC-MS and the corresponding synthetic peptides containing the Asp isomers. This makes it possible to analyze isomers of Asp residues in proteins precisely and quickly. We demonstrate that Asp-58, -76, -84, and -151 of alpha A-crystallin and Asp-62 and -96 of alpha B-crystallin are highly converted to L beta-, D beta-, and D alpha-isomers. The amount of isomerization of Asp is greater in the insoluble fraction at all Asp sites in lens proteins, therefore indicating that isomerization of these Asp residues affects the higher order structure of the proteins and contributes to the increase in aggregation, insolubilization, and disruption of function of proteins in the lens, leading to the cataract.