title: A cis-membrane FRET-based method for protein-specific imaging of cell-surface glycans.
abstract: 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.
authors.affiliation: Beijing National Laboratory for Molecular Sciences, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, and Peking-Tsinghua Center for Life Sciences, Peking University , Beijing 100871, P.R. China.
authors.forename: Wei
authors.initials: W
authors.lastname: Lin
authors.forename: Yifei
authors.initials: Y
authors.lastname: Du
authors.forename: Yuntao
authors.initials: Y
authors.lastname: Zhu
authors.forename: Xing
authors.initials: X
authors.lastname: Chen
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D015394:Molecular Structure; D011134:Polysaccharides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D013237:Stereoisomerism; D013499:Surface Properties
chemical_list: D011134:Polysaccharides; D011506:Proteins