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.

Protein-specific Raman imaging of glycosylation on single cells with zone-controllable SERS effect

“…But a radius of 15 nm for the nano-substrates is the smallest radius for an efficient SERS effect 18 which limits the detection precision of this method for smallish proteins. FRET based protein-specific imaging 4–6 is a more appropriate detection technique for a protein with a smallish glycan expression zone.…”

“…Several Förster resonance energy transfer (FRET) methods have been developed for the imaging of protein-specific glycans by labeling the proteins and their corresponding glycans with two FRET-achievable fluorescent molecules. 4–6 However, one donor to one acceptor FRET mode cannot provide the integral glycan signal on target proteins that are generally modified with more than one glycan molecule. Besides, the short FRET distance between the donor and acceptor 7 might limit its application in the study of biggish proteins.…”

Visualizing glycans on single cells and tissues–Visualizing glycans on single cells and tissues

“…Ensemble measurements of metabolically labeled glycans, co-labeled with a site specific protein tag, enabled Lin et al to apply F ӧ rster resonance energy transfer (FRET) imaging to specific glycoproteins in live cells [27]. Using the enzyme-catalyzed probe ligation method, based upon lipoic acid ligase (LplA), developed by Ting’s group [28], a FRET donor was installed on an extracellular terminus of a protein of interest.…”

Chemical Lectinology: Tools for Probing the Ligands and Dynamics of Mammalian Lectins In Vivo

“…These pioneering efforts provided evidence that similar glycan structures on a variety of different protein and lipid scaffolds could be targeted using a metabolism-based approach. Recently, a number of reports have detailed the combination of metabolic labeling and Förster Resonance Energy Transfer (FRET) for imaging proteins bearing particular glycan structures (Belardi et al, 2013; Haga et al, 2012; Lin et al, 2014). …”