Visualizing specific protein glycoforms by transmembrane fluorescence resonance energy transfer

Analyses of mice lacking glycosyltransferase have suggested that their pathological phenotypes are not attributable to the overall change of the sugar modification, but instead the result of changes of the glycan structures on a specific 'target' glycoprotein. Therefore, detecting or monitoring the glycosylation status of a specific protein in living cells is important, but no such methods are currently available. Here we demonstrate the detection of glycoforms of a specific glycoprotein using the fluorescence resonance energy transfer technique. Using model proteins, we detect characteristic fluorescence resonance energy transfer signals from the specific glycoform-bearing target glycoprotein. We also show that, upon insulin removal, sialylated glycoforms of green fluorescent protein-tagged GLUT4 seem to be internalized more slowly than non-sialylated GLUT4. This novel analytical imaging tool allows studying the roles of specific glycan modifications of a protein of interest.