Recent studies have suggested that the isomerization/racemization of aspartate residues in proteins increases in aged tissues. One such residue is Asp151 in lens-specific αA-crystallin. Although many isomerization/racemization sites have been reported in various proteins, the factors that lead to those modifications in proteins in vivo remain obscure. Therefore, an in vitro system is needed to assess the mechanisms of modifications of Asp under various conditions. Deamidation of Asn to Asp in proteins occurs more rapidly than isomerization/racemization of Asp, although the reaction passes through the same intermediate in both pathways. Here, therefore, we replaced Asp151 in human lens αA-crystallin with Asn by using site-directed mutagenesis. The recombinant protein was expressed in Escherichia coli and used to investigate the deamidation/isomerization/racemization of Asn151 after incubation at 50°C for various durations and under different pH. After incubation, the mutant αA-crystallin was subjected to enzymatic digestion followed by liquid chromatography-MS/MS to evaluate the ratio of modifications in Asn151-containing peptides. The Asp151Asn αA-crystallin mutant showed rapid deamidation to Asp with the formation of specific Asp isomers. In particular, deamidation increased greatly under basic conditions. By contrast, subunit-subunit interactions between αA-crystallin and αB-crystallin had little effect on the modification of Asn151. Our findings suggest that the Asp151Asn αA-crystallin mutant represents a good in vitro model protein to assess deamidation, isomerization, and the racemization intermediates. Furthermore, our in vitro results show a different trend from in vivo data, implying the presence of specific factors that induce racemization from L-Asp to D-Asp residues in vivo.

中文翻译：

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体外人晶状体αA-晶状体中的特定位快速脱酰胺和异构化。

最近的研究表明，老化组织中蛋白质中天冬氨酸残基的异构化/外消旋化增加。一种这样的残基是晶状体特异性αA-晶状体蛋白中的Asp151。尽管在各种蛋白质中已经报道了许多异构化/外消旋位点，但是导致体内蛋白质发生这些修饰的因素仍然不清楚。因此，需要一种体外系统来评估在各种条件下Asp修饰的机理。尽管反应在两个途径中都经过相同的中间体，但蛋白质中Asn脱酰胺化的速度比Asp异构化/外消旋化更快。因此，在这里，我们通过定点诱变将人类晶状体αA-晶状体蛋白中的Asp151替换为Asn。重组蛋白在大肠杆菌中表达，用于研究在50°C下孵育不同时间和不同pH值后Asn151的脱酰胺/异构化/外消旋作用。温育后，将突变的αA-晶状蛋白进行酶消化，然后进行液相色谱-MS / MS以评估含Asn151的肽中修饰的比例。Asp151AsnαA-晶状蛋白突变体显示出对Asp的快速脱酰胺作用，并形成了特定的Asp异构体。特别是在基本条件下，脱酰胺作用大大增加。相比之下，αA-晶状体蛋白和αB-晶状体蛋白之间的亚基-亚基相互作用对Asn151的修饰影响很小。我们的发现表明，Asp151AsnαA-crystallin突变体是评估脱酰胺，异构化，和消旋化中间体。此外，我们的体外结果显示出与体内数据不同的趋势，这表明存在体内诱导从L-Asp到D-Asp残基消旋的特定因子。