PDF(Pubmed)
Abstract：
Site-specific modifications of aspartate residues spontaneously occur in crystallin, the major protein in the lens. One of the primary modification sites is Asp151 in αA-crystallin. Isomerization and racemization alter the crystallin backbone structure, reducing its stability by inducing abnormal crystallin-crystallin interactions and ultimately leading to the insolubilization of crystallin complexes. These changes are considered significant factors in the formation of senile cataracts. However, the mechanisms driving spontaneous isomerization and racemization have not been experimentally demonstrated. In this study, we generated αA-crystallins with different homo-oligomeric sizes and/or containing an asparagine residue at position 151, which is more prone to isomerization and racemization. We characterized their structure, hydrophobicity, chaperone-like function, and heat stability, and examined their propensity for isomerization and racemization. The results show that the two differently sized αA-crystallin variants possessed similar secondary structures but exhibited different chaperone-like functions depending on their oligomeric sizes. The rate of isomerization and racemization of Asp151, as assessed by the deamidation of Asn151, was also found to depend on the oligomeric sizes of αA-crystallin. The predominant isomerization product via deamidation of Asn151 in the different-sized αA-crystallin variants was L-β-Asp in vitro, while various modifications occurred around Asp151 in vivo. The disparity between the findings of this in vitro study and in vivo studies suggests that the isomerization of Asp151 in vivo may be more complex than what occurs in vitro.
摘要：
天冬氨酸残基的位点特异性修饰自发发生在晶状体蛋白中，晶状体中的主要蛋白质。主要修饰位点之一是αA-晶状体蛋白中的Asp151。异构化和外消旋改变了结晶蛋白的主链结构，通过诱导异常的晶状体蛋白-晶状体蛋白相互作用并最终导致晶状体蛋白复合物的不溶解来降低其稳定性。这些变化被认为是老年性白内障形成的重要因素。然而,驱动自发异构化和外消旋的机制尚未得到实验证明。在这项研究中,我们产生了具有不同同源寡聚大小和/或在位置151处含有天冬酰胺残基的αA-晶状体蛋白,其更易于异构化和外消旋化。我们描述了它们的结构，疏水性,类似伴侣的功能，和热稳定性，并检查了它们的异构化和外消旋化倾向。结果表明，两种不同大小的αA-晶状体蛋白变体具有相似的二级结构，但根据其寡聚物大小表现出不同的伴侣样功能。还发现，通过Asn151的脱酰胺作用评估的Asp151的异构化和外消旋速率取决于αA-晶状体蛋白的低聚物大小。在不同大小的αA-晶状体蛋白变体中，通过Asn151脱酰胺作用的主要异构化产物是体外L-β-Asp，而体内Asp151周围发生了各种修饰。这项体外研究和体内研究的发现之间的差异表明，体内Asp151的异构化可能比体外发生的异构化更复杂。
