Eye Institute, Eye & ENT Hospital of Fudan University, Shanghai, China.
Shanghai Key Laboratory of Visual Impairment and Restoration, Shanghai, China.
Mol Vis. 2021 Jun 23;27:384-395. eCollection 2021.
Ephrin (Eph) receptor A2 () polymorphism has been associated with age-related cataract (ARC) in different populations worldwide, but the mechanisms by which this polymorphism results in the development of ARC are unclear. Here, we chose four single nucleotide polymorphisms (SNPs; rs35903225, rs145592908, rs137853199, and rs116506614) and studied their function in human lens epithelial cells (LECs).
The four mutants were overexpressed using lentiviral transduction in human LECs. Cells expressing wild-type (WT) and mutated EPHA2 were subjected to quantitative PCR (qPCR), western blot, immunoprecipitation (IP), and transwell migration assay. MG132 and chloroquine were used to inhibit the degradation of the WT and mutated EPHA2. The structural changes induced by rs137853199 were predicted and optimized using Schrödinger software. IP-mass spectrometry (IP-MS) was performed to examine the proteins that directly interact with WT and rs137853199 EPHA2. Sanger sequencing was performed to determine the frequency of rs137853199 in 184 patients with ARC (73 cortical cataracts, 56 nuclear cataracts, and 55 posterior subcapsular cataracts) and 49 normal controls.
Compared with the WT and the other three mutations, the rs137853199 mutation specifically resulted in a significant decrease in the expression of EPHA2. We identified that EPHA2 rs137853199 is degraded via the ubiquitin-proteasomal pathway through a lysine-48 (K48) residue linkage. Furthermore, the knockdown of reduced cell migration; while the overexpression of WT rescued this defect, the overexpression of rs137853199 did not. In addition, in cells overexpressing rs137853199 , the expression of β-catenin, a key protein that regulates cell migration, significantly decreased. We predicted that rs137853199 would induce a conformational change at a linker position in the carboxyl terminal of EPHA2. The IP-MS results showed that the main molecular functions of the proteins that specifically bind WT or rs137853199 EPHA2 are binding and catalysis, while the main protein class is the protein-modifying enzyme. Finally, we discovered that the minor allele frequency of rs137853199 was significantly higher in cortical cataract patients than it was in normal controls.
In summary, these findings suggest a mechanism by which a point mutation in disrupts protein stability, expedites protein degradation, and decreases cell mobility. Importantly, this mutant is associated with cortical cataracts.
在全球不同人群中，Eph 受体 A2 () 多态性与年龄相关性白内障 (ARC) 有关，但这种多态性导致 ARC 发展的确切机制尚不清楚。在这里，我们选择了四个单核苷酸多态性（SNPs；rs35903225、rs145592908、rs137853199 和 rs116506614），并研究了它们在人晶状体上皮细胞 (LEC) 中的功能。
使用慢病毒转导在人 LEC 中过表达四个 突变体。用野生型 (WT) 和突变型 EPHA2 转染的细胞进行定量 PCR(qPCR)、western blot、免疫沉淀 (IP) 和 Transwell 迁移实验。用 MG132 和氯喹抑制 WT 和突变型 EPHA2 的降解。使用 Schrödinger 软件预测和优化 rs137853199 引起的结构变化。进行 IP-MS 以检查与 WT 和 rs137853199 EPHA2 直接相互作用的蛋白质。对 184 例 ARC 患者（73 例皮质性白内障、56 例核性白内障和 55 例后发性白内障）和 49 例正常对照进行 rs137853199 的 Sanger 测序。
与 WT 和其他三种突变体相比，rs137853199 突变体特异性导致 EPHA2 表达明显降低。我们发现 EPHA2 rs137853199 通过赖氨酸-48 (K48) 残基连接通过泛素-蛋白酶体途径进行降解。此外，下调 减少细胞迁移；而 WT 的过表达挽救了这种缺陷，而 rs137853199 的过表达则没有。此外，在过表达 rs137853199 的细胞中，β-连环蛋白（一种调节细胞迁移的关键蛋白）的表达明显降低。我们预测 rs137853199 会在 EPHA2 羧基末端的连接位置引起构象变化。IP-MS 结果表明，与 WT 或 rs137853199 EPHA2 特异性结合的蛋白质的主要分子功能是结合和催化，而主要的蛋白质类别是蛋白质修饰酶。最后，我们发现 rs137853199 的次要等位基因频率在皮质性白内障患者中明显高于正常对照。
总之，这些发现表明， 中的单点突变破坏蛋白质稳定性、加速蛋白质降解并降低细胞迁移能力的机制。重要的是，这种突变与皮质性白内障有关。
