Intracellular UDP-N-acetylhexosamine pool affects N-glycan complexity: a mechanism of ammonium action on protein glycosylation.
Understanding the mechanisms by which ammonium ions affect glycosylation may suggest strategies for producing glycoproteins with homogeneous biological activity in the presence of undesirable byproducts of cellular energy metabolism. We have previously shown that ammonium ions cause an increase in the intracellular UDP-N-acetylhexosamine (UDPGNAc) pool, which may be responsible for the ammonium-induced increase in complexity and decrease in sialylation state of the N-linked oligosaccharide. To investigate this novel hypothesis, we induced an artificial increase in UDPGNAc pool by treating recombinant BHK cells expressing an IL-2 variant that features an artificial site for N-glycosylation, with glucosamine (1:2 molar ratio to glucose) and uridine (2 mmol L-1) in the absence of ammonium ions or glutamine. The product fractions collected during this treatment showed increased antennarity compared to product collected under control conditions. When this pool was returned to normal levels, the glycosylation pattern regained its original (control) features. However, the sialylation state remained unaffected, suggesting that the decreased sialylation observed under ammonium treatment is due to a different mechanism of action, possibly involving changes in intracellular pH. By pretreating the cells with 0.5 mmol L-1 adenosine, and exposing them continuously to NH4Cl and adenosine we were able to prevent the ammonium-induced increase in UDPGNAc. Product fractions collected during this treatment showed unchanged antennarity but decreased sialylation of the N-linked oligosaccharide, thus conclusively demonstrating that ammonium ions act on protein glycosylation by at least two independent mechanisms, one of which involves an increase in the UDPGNAc pool.
Grammatikos
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Gesellschaft für Biotechnologische Forschung mbH, Cell Culture Technology Department, Mascheroder Weg 1, D-38124 Braunschweig, Germany.
Valley
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Nimtz
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Conradt
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Wagner
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D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D006031:Glycosylation; D011134:Polysaccharides / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism*
D011134:Polysaccharides; D011506:Proteins; D000644:Quaternary Ammonium Compounds; D014537:Uridine Diphosphate N-Acetylglucosamine