Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, Japan.
Shimazu Corporation, Nakagyo-ku, Kyoto, Japan.
PLoS One. 2024 Jul 11;19(7):e0306856. doi: 10.1371/journal.pone.0306856. eCollection 2024.
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.
天冬氨酸残基在晶状体中的主要蛋白质晶状蛋白中会自发发生位点特异性修饰。主要修饰位点之一是αA-晶体蛋白中的 Asp151。异构化和外消旋化改变了晶体蛋白骨架结构，通过诱导异常的晶体蛋白-晶体蛋白相互作用降低其稳定性，最终导致晶体蛋白复合物的不溶。这些变化被认为是形成老年白内障的重要因素。然而，自发异构化和外消旋化的机制尚未通过实验证明。在这项研究中，我们生成了具有不同同聚大小和/或在位置 151 含有天冬酰胺残基的αA-晶体蛋白，这些更容易发生异构化和外消旋化。我们对其结构、疏水性、伴侣样功能和热稳定性进行了表征，并研究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-晶体蛋白变体具有相似的二级结构，但由于其聚合大小不同，表现出不同的伴侣样功能。通过天冬酰胺 151 的脱酰胺化评估的 Asp151 的异构化和外消旋化速率也发现取决于αA-晶体蛋白的聚合大小。通过不同大小的αA-晶体蛋白变体中天冬酰胺 151 的脱酰胺化产生的主要异构化产物为 L-β-Asp，而在体内，Asp151 周围发生了各种修饰。体外研究和体内研究的结果之间存在差异，这表明体内 Asp151 的异构化可能比体外更复杂。
