FIGURE 4.

Mammalian cells incorporate exogenous GalNGc into UDP-GalNGc and convert it to UDP-GlcNGc. A, UDP-GalNAc, UDP-GlcNAc, UDP-GalNGc, and UDP-GlcNGc were analyzed by HPLC under alkaline conditions using a PA-1 column with an optimized gradient (supplemental Fig. S3). B, human BJA-B K20 (GNE-deficient) and BJA-B K88 (hypersialylated) cells were cultured under Neu5Gc-free conditions. The media were supplemented with [³H]GalNGc or [³H]GalNAc for 3 days. Cells were harvested, washed well, lysed, and subjected to 70% ethanol followed by precipitation at −20 °C overnight. The supernatants were analyzed by HPLC as described in A. Fractions (0.5 ml) were collected, and radioactivity was determined by scintillation counting.