GDP-Cy5-Fucose

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Strategies for Probing and labeling substrate glycans of various fucosyltransferases using GDP-Cy5-Fucose. Depicted here are for FUT2, FUT6, FUT7, FUT8 and FUT9. FUT2 introduces Cy5-Fucose to terminal Gal residues. FUT6, FUT7 and FUT9 introduce Cy5-Fucose to the GlcNAc residue in lactosamine structures that can be found on both N- and O-glycans. FUT8 introduces Cy5-Fucose to the innermost GlcNAc residue on N-glycans where there is an unmodified terminal GlcNAc residue in the α3 branch.

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Labeling bovine fetuin (Fet) using GDP-Cy5-Fucose. Bovine fetuin was purified from crude fetuin by gel filtration. Samples of fetuin were labeled with Recombinant Human Fucosyltransferase 9/FUT9 Protein (Catalog # 9347-GT). In control lanes, no fetuin was present, revealing the self-labeling of FUT9 itself. Samples were not labeled in the absence of neuraminidase but labeled strongly in the presence of  Recombinant C. perfringens Neuraminidase Protein  (Catalog # 5080-NM), suggesting that all lactosamine structures on the samples were sialylated prior to labeling. Samples were separated on 4-20% gradient SDS-PAGE and imaged with silver staining (left panel) and a fluorescent imager (middle and right panels at different contrasts). M, a prestained Western blot molecular marker that is visible under fluorescent imager.

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Cellular glycan imaging with GDP-Cy5-Fucose and CMP-Cy3-Sialic Acid. HeLa cells were stained by incorporation GDP-Cy5-Fucose (Catalog # ES301) by Recombinant Human Fucosyltransferase 9/FUT9 Protein (Catalog # 9347-GT) (red) and CMP-Cy3-Sialic Acid (Catalog ES402) by Recombinant Human ST6GAL1 (aa 44-406) Protein (Catalog # 7620-GT) (green). Hela cells on a 96 well plate were briefly treated with Recombinant C. perfringens Neuraminidase Protein (Catalog # 5080-NM) to remove preexisting sialic acids. The treated cells were then labeled by FUT9 and ST6Gal1 simultaneously in the presence of GDP-Cy5-Fucose and  CMP-Cy3-Sialic acid for 30 minutes. After the labeling, the solution was quickly removed by aspiration and washing two times with PBS. Afterwards, the cells were fixed and stained with DAIP (purple) in the presence of 0.5% Triton X 100 to reveal the cell nuclei. All three panels were from a same viewing area and imaged under different channels. These images show that FUT9 and ST6Gal1 stained the same regions on HeLa cells. For details on cell imaging with fluorophore-conjugated sugars, please refer to  Wu. L. et al., (2020) Glycobiology 30:454-462