Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan.
Taiwan International Graduate Program (TIGP), Sustainable Chemical Science & Technology (SCST), Academia Sinica, Taipei 11529, Taiwan.
Int J Mol Sci. 2022 Aug 6;23(15):8754. doi: 10.3390/ijms23158754.
Bacterial and viral pathogens can modulate the glycosylation of key host proteins to facilitate pathogenesis by using various glycosidases, particularly sialidases. Epidermal growth factor receptor (EGFR) signaling is activated by ligand-induced receptor dimerization and oligomerization. Ligand binding induces conformational changes in EGFR, leading to clusters and aggregation. However, information on the relevance of EGFR clustering in the pattern of glycosylation during bacterial and viral invasion remains unclear. In this study, (1) we established CRISPR/Cas9-mediated GFP knock-in (EGFP-KI) HeLa cells expressing fluorescently tagged EGFR at close to endogenous levels to study EGF-induced EGFR clustering and molecular dynamics; (2) We studied the effect of sialylation on EGF-induced EGFR clustering and localization in live cells using a high content analysis platform and raster image correlation spectroscopy (RICS) coupled with a number and brightness (N&B) analysis; (3) Our data reveal that the removal of cell surface sialic acids by sialidase treatment significantly decreases EGF receptor clustering with reduced fluorescence intensity, number, and area of EGFR-GFP clusters per cell upon EGF stimulation. Sialylation appears to mediate EGF-induced EGFR clustering as demonstrated by the change of EGFR-GFP clusters in the diffusion coefficient and molecular brightness, providing new insights into the role of sialylation in EGF-induced EGFR activation; and (4) We envision that the combination of CRISPR/Cas9-mediated fluorescent tagging of endogenous proteins and fluorescence imaging techniques can be the method of choice for studying the molecular dynamics and interactions of proteins in live cells.
细菌和病毒病原体可以通过使用各种糖苷酶（尤其是唾液酸酶）来调节关键宿主蛋白的糖基化，从而促进发病机制。表皮生长因子受体 (EGFR) 信号通过配体诱导的受体二聚化和寡聚化而被激活。配体结合诱导 EGFR 的构象变化，导致簇集和聚集。然而，关于细菌和病毒入侵过程中 EGFR 簇集与糖基化模式之间相关性的信息尚不清楚。在这项研究中，（1）我们建立了 CRISPR/Cas9 介导的 GFP 敲入（EGFP-KI）HeLa 细胞，该细胞以接近内源性水平表达荧光标记的 EGFR，以研究 EGF 诱导的 EGFR 簇集和分子动力学；（2）我们使用高内涵分析平台和光栅图像相关光谱 (RICS) 结合数量和亮度 (N&B) 分析研究了唾液酸化对活细胞中 EGF 诱导的 EGFR 簇集和定位的影响；（3）我们的数据表明，唾液酸酶处理去除细胞表面的唾液酸会显著减少 EGF 受体的簇集，导致 EGF 刺激后每个细胞的 EGFR-GFP 簇的荧光强度、数量和面积降低。唾液酸化似乎介导了 EGF 诱导的 EGFR 簇集，这表现在 EGFR-GFP 簇的扩散系数和分子亮度发生变化，为唾液酸化在 EGF 诱导的 EGFR 激活中的作用提供了新的见解；（4）我们设想，CRISPR/Cas9 介导的内源性蛋白荧光标记与荧光成像技术的结合可以成为研究活细胞中蛋白质分子动力学和相互作用的首选方法。
