Incorporation of ammonium into intracellular UDP‐activated N‐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-acetylglucosamine (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 quantitatively 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.

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

“…These effects depend on several cellular factors: (1) enzyme repertoire and localization (Ferrara et al, 2006;Meynial-Salles and Combes, 1996); (2) competition between different enzymes for one substrate (Umaña and Bailey, 1997); (3) transit time of the glycoproteins through the Golgi apparatus (Hooker et al, 1999;Nabi and Dennis, 1998); (4) levels of nucleotide sugar donors (Nyberg et al, 1999;Valley et al, 1999); and last but not the least (5) competition between different glycosylation sites on the protein for the same pool of enzymes (Schachter et al, 1983).…”

“…Culture conditions such as accumulation of ammonia, dissolved oxygen level, nutrient contents, pH level and temperature, may have great effects on the distribution of glycan structures (microheterogeneity and macroheterogeneity) found on the final recombinant protein products. Those factors may cause one or more mechanisms in the following: (1) the depletion of the cellular energy state (Rearick et al, 1981;Valley et al, 1999); (2) disruption of the local ER and Golgi environment, such as pH (Andersen and Goochee, 1995) or redox potential (Restelli et al, 2006); (3) alteration of glycosyltransferase activities (Chen and Harcum, 2006;Chotigeat et al, 1994); and (4) changes in the Nglycosylation precursor pool, such as nucleotides, nucleotide sugars (Kochanowski et al, 2008;Nyberg et al, 1999) and lipid precursors (Stoll and Krag, 1988). …”

“…Sufficient UDP-GlcNAc pool resulted in enhancement of antennarity of glycan structures produced in baby hamster kidney (BHK) cells, probably through a stimulation of the specific GlcNAc TIV and TV (Gawlitzek et al, 1998;Grammatikos et al, 1998;Valley et al, 1999). 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).…”

“…Accumulated NH3 provides two major changes to the intracellular environment, resulting in heterogeneity in Nglycosylation during protein production: (1) pH elevation in Golgi, which can decrease the activity of selected enzymes, such as sialyltransferase (Valley et al, 1999); (2) an increase in the UDP-GlcNAc pool, which competes with CMP-NeuAc for the transporter, resulting in a sialylation decrease (Andersen and Goochee, 1994). …”

Intracellular nucleotide pools and ratios as tools for monitoring dedifferentiation of primary porcine hepatocytes in culture

“…A change in the U ratio has been shown to affect the N-glycan complexity of glycoproteins (Gawlitzek et al 1998). Other studies revealed that an increase in the UDPGNAc pool can be the result of ammonium uptake which accumulates in cell cultures due to cellular metabolism and thermal degradation of glutamine (Gawlitzek et al 1999;Valley et al 1999;Ryll et al 1994).…”