The effect of increasing nucleotide-sugar concentrations on the incorporation of sugars into glycoconjugates in rat hepatocytes

Abstract

Treatment of rat hepatocytes with 0.5 mM concentrations ofThe latter increase did not result in an increased incorporation of uridine and cytidine results in increased cellular concentrations radioactivity into the glycoconjugates. It was estimated that, in of UTP, UDP-sugars and CTP, whereas that of CMP-Nuntreated cells, the ratio of radioactivity incorporated from acetylneuraminate remained unchanged [Pels Rijcken, Overdijk, [3H]glucosamine into glycoconjugate-bound N-acetylhexosamine Van den Eijnden and Ferwerda (1993) Biochem. J. 293,207-213]. and N-acetylneuraminate amounted to 2:3. In pretreated cells The incorporation of radioactivity from 3H-labelled sugars into this ratio changed to approx. 2: 1. Overall, the data show that the cell-associated and secreted glycoconjugate fraction was pretreatment resulted in an increased incorporation of N-acetylinfluenced by these altered cellular concentrations of the nucleohexosamine into cell-associated and secreted glycoconjugates, tides. For [3H]glucosamine, pretreatment with uridine resulted in accompanied by a reduction in sialylation. It was concluded that a reduction of the glycosylation in both fractions. Increases in an increased availability of UDP-N-acetylhexosamine caused the the secreted fractions were observed for fucose with both uridine increased incorporation of N-acetylhexosamine. The elevated and cytidine and for N-acetylglucosamine with uridine only. This study demonstrates that protein glycosylation can be acetylmannosamine, the specific radioactivity of CMP-N-acetylregulated at the level of the availability of the various nucleotideneuraminate showed an almost 2-fold increase on pretreatment.sugars in the Golgi lumen.

Incorporation of 15N from ammonium into the N-linked oligosaccharides of an immunoadhesin glycoprotein expressed in Chinese hamster ovary cells

“…Subsequently, GlcN-6-P becomes acetylated and finally activated by UTP resulting in UDP-GNAc. UDP-GlcNAc is a direct precursor in the formation of CMP-NANA (Warren, 1972;Pels Rijcken et al, 1995). Consequently, if GlcNAc is labeled with 15 N due to the incorporation of 15 NH 4 + , NANA also has to be labeled with 15 N. This assumption was confirmed by our data, since sialylated N-glycans revealed a larger mass shift compared to their unsialylated equivalents.…”

Synthesis of GDP-L-fucose by the Human FX Protein

“…In this respect, the increased FX levels in erythrocytes from glucose-6-phosphate dehydrogenase-deficient sub- jects (4) might reflect some compensation for a defective NADPH supply in maintaining a normal rate of biosynthesis of the ABH antigens. Furthermore, besides glycosyl transferase activity and translocation of nucleotide sugars from cytosol, where they are formed, to Golgi lumen (43), another site of regulation of protein glycosylation is represented by the levels of the nucleotide sugars pools inside the cells (44). Some reports in literature indicate that an alteration of GDP-fucose biosynthetic pathway can lead to modifications in membrane fucosylation.…”

The availability of glucose to CHO cells affects the intracellular lipid-linked oligosaccharide distribution, site occupancy and the N-glycosylation profile of a monoclonal antibody

“…However, this phenomenon is not universal for all cell lines, such as NS0 cells, which have been reported to have no positive feedback for glucosamine addition (Baker et al, 2001). It is noteworthy that since UDP-GlcNAc competes with CMPNeuAc for transport into the Golgi, elevated UDP-GlcNAc appears to cause a decline in sialylation (Rijcken et al, 1995).…”