Detecting substrate glycans of fucosyltransferases on glycoproteins with fluorescent fucose

Abstract Like sialylation, fucose usually locates at the non-reducing ends of various glycans on glycoproteins and constitutes important glycan epitopes. Detecting the substrate glycans of fucosyltransferases is important for understanding how these glycan epitopes are regulated in response to different growth conditions and external stimuli. Here we report the detection of these glycans via enzymatic incorporation of fluorescent tagged fucose using fucosyltransferases including FUT2, FUT6, FUT7, and FUT8 and FUT9. More specifically, we describe the detection of substrate glycans of FUT8 and FUT9 on therapeutic antibodies and the detection of high mannose glycans on glycoproteins by enzymatic conversion of high mannose glycans to the substrate glycans of FUT8. By establishing a series of precursor glycans, we also demonstrate the substrate specificities of FUT8. Furthermore, using simultaneous enzymatic incorporation of both fluorescent sialic acids and fluorescent fucoses, we demonstrate the interplay between fucosylation and sialylation.