FIGURE 2.

5T-Fuc is converted into GDP-5T-Fuc within HepG2 cells. A, nucleotide sugars were extracted from control and 5T-Fuc-treated cells and analyzed by CE. All samples were spiked with GDP-Glc before extraction and further processed with UDP-GlcNAc pyrophosphorylase (AGX1) in the presence of exogenous pyrophosphate (resulting in the elimination of this nucleotide sugar from electropherograms) to allow for the precise quantification of GDP-5T-Fuc. Notice that in cells treated with 5T-Fuc a new peak appears that comigrates with chemoenzymatically prepared GDP-5T-Fuc (#). Concomitantly, treatment of HepG2 cells with the thiosugar causes a dramatic reduction in GDP-Fuc levels. B, GDP-5T-Fuc and GDP-Man comigrate and therefore samples were treated with FKP in the presence of pyrophosphate to convert GDP-Fuc/5T-Fuc into their corresponding sugar phosphates. The FKP sensitivity of the new nucleotide sugar extracted from 5T-Fuc-treated cells establishes that it was indeed GDP-5T-Fuc and not an accumulation of the comigrating GDP-Man. C, GDP-5T-Fuc and GDP-Man levels could be quantified by subtracting the peak areas obtained upon the CE analysis of samples before and after treatment with FKP. Averaged peak areas are reported ± S.E. (n = 3).