A Cis-Membrane FRET-Based Method for Protein-Specific Imaging of Cell-Surface Glycans
A Cis-Membrane FRET-Based Method for Protein-Specific Imaging of Cell-Surface Glycans
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一种基于顺膜 FRET 的细胞表面聚糖蛋白质特异性成像方法。
DOI：
10.1021/ja410086d
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发表时间：
2014-01-15
影响因子：
15
通讯作者：
Chen, Xing
中科院分区：
文献类型：
--
作者：
Lin, Wei;Du, Yifei;Chen, Xing
用化学标记物（例如，炔或叠氮化物）结合生物正交化学是用于糖组成像的有力工具;然而，该方法缺乏蛋白质特异性，因此不适用于感兴趣的特定蛋白质（POI）的糖基化成像。在这里，我们报告了一个顺膜FRET为基础的方法，允许活细胞上的聚糖蛋白质特异性成像的发展。我们利用代谢聚糖标记结合位点特异性蛋白质标记，同时安装FRET受体和供体的聚糖和胞外终端的蛋白质的兴趣，分别。POI的分子内供体-受体距离福尔斯落在有效FRET的范围内，而分子间FRET是不利的，因为其他蛋白质上的过量受体远离供体。我们通过观察几种重要的细胞表面受体（包括整合素αXβ2、表皮生长因子受体和I型转化生长因子β受体）的唾液酸化，证明了这种顺式膜FRET成像方法的能力。此外，我们的成像实验表明，唾液酸化可能是重要的β2整合素激活。我们的方法应该使糖基化如何调节各种细胞表面蛋白的功能和动力学的活细胞研究。
Metabolic labeling of glycans with chemical reproters (e.g., alkyne or azide) in conjunction with bioorthogonal chemistry is a powerful tool for imaging glycome; however, this method lacks protein-specificity and therefore is not applicable to imaging glycosylation of a specific protein of interest (POI). Here we report the development of a cis-membrane FRET-based methodology that allows protein-specific imaging of glycans on live cells. We exploit metabolic glycan labeling in conjunction with site-specific protein labeling to simultaneously install a FRET acceptor and a donor onto the glycans and the extracellular terminal of the protein of interest, respectively. The intramolecular donor-acceptor distance for the POI falls within the range for effective FRET, whereas the intermolecular FRET is disfavored since the excess acceptors on other proteins are distant from the donor. We demonstrated the capability of this cis-membrane FRET imaging method by visualizing the sialylation of several important cell surface receptors including integrin αXβ2, epidermal growth factor receptor, and transforming growth factor-beta receptor type I. Furthermore, our imaging experiments revealed that the sialylation might be important for β2 integrin activation. Our methodology should enable the live-cell studies on how glycosylation regulates the functions and dynamics of various cell-surface proteins.