Incorporation of ammonium into intracellular UDP-activatedN-acetylhexosamines and into carbohydrate structures in glycoproteins

Abstract

The negative effects of ammonia on animal cells, especially in vitro cultures, are well known, but the mechanism of how ammonia inhibits cell growth and influences the glycosylation of proteins is not completely understood. We investigated the ammonium action on the synthesis of the intracellular UDP‐N‐acetylhexos‐ amines (UDPGNAc), which are precursors of glycosylation as well as on N‐linked oligosaccharides of a recombinant human IL‐2 mutant variant model glycoprotein expressed in BHK‐21 cells under defined and controlled culture conditions in a continuously perfused bioreactor. The examinations were based on our previous observations that increased ammonia concentrations in the medium lead to the intracellular formation and accumulation of UDPGNAc (Ryll et al., 1994). The kinetics of formation of the UDPGNAc pool after adding ammonia and its reconstitution to normal conditions are shown. To study the pathway leading to the intracellular increase of UDPGNAc, the uptake and incorporation of 15NH4+ was confirmed by the detection of 15N in UDP‐N‐acetylglu‐ cosamine (UDP‐GlcNAc). UDP‐GlcNAc was purified using high pH anion‐exchange chromatography with pulsed amperometric detection and analyzed by GC/MS. The proportion of UDP‐GlcNAc containing 15N was approximately 60% and corresponds quantitively to the increased intracellular concentration of UDP‐GlcNAc. In order to confirm the direct influence of ammonia on protein glycosylation, the human IL‐2 mutant glycoprotein variant IL‐Mu6, bearing a novel N‐glycosylation site, has been produced under defined protein‐free medium conditions in the presence of 15NH4Cl. IL‐Mu6 glycoprotein was purified and N‐glycans released were analyzed by matrix‐assisted laser desorption ionization time of flight mass spectroscopy. Maximally 60–80% of N‐acetylated sugars in N‐glycan structures contained 15N indicating that ammonium is used as a building block during synthesis of the carbohydrate structures expressed from in vitro cultivated mammalian cells. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 401–417, 1999.

Synergizing metabolic flux analysis and nucleotide sugar metabolism to understand the control of glycosylation of recombinant protein in CHO cells

“…UDP-GlcNAc and UDP-GalNAc [14-17]. Under cell culture conditions, high glutamine and/or ammonium concentrations contribute to the accumulation of the pool of UDP-GNAc by the combined action of the enzymes glucosamine-6-phosphate isomerase (GPI) and glucosamine-6-phosphate synthetase (GPS) to form glucosamine-6-phosphate from fructose-6-phosphate (reaction marked with an asterisk, Figure 10).…”

Glycosylation and post-translational modification gene expression analysis by DNA microarrays for cultured mammalian cells

“…The model system will be NS0 cells exposed to conditions known to affect protein glycosylation, including elevated ammonium [16,46,47] as well as three other conditions. Specifically, the experimental procedure to set up ammonium-stressed, salt-stressed, ammonium-stressed with proline, and control cultures will be described.…”

Glycosylation-related genes in NS0 cells are insensitive to moderately elevated ammonium concentrations

“…Interestingly, in NS0 cells elevated ammonium has been shown to increase intracellular nucleotide sugar pools, which serve as precursors for many glycosylation reactions (Burleigh et al, 2011; Ryll, 1994; Valley et al, 1999). Additionally, when cells are supplemented with glycosylation precursors, such as N-acetylmannosamine and galactose, glycosylation was not significantly increased, despite increased nucleotide sugar pools (Baker et al, 2001; Clark et al, 2005; Gu and Wang, 1998); however, when N-acetylmannosamine and galactose were combined with uridine or cytindine, glycosylation was significantly increased (Wong et al, 2010).…”