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

The negative effects of ammonia on animal cells, especially in vitro cultur es, are well known, but the mechanism of how ammonia inhibits cell growth a nd influences the glycosylation of proteins is not completely understood. W e investigated the ammonium action on the synthesis of the intracellular UD P-N-acetylhexosamines (UDPGNAc), which are precursors of glycosylation as w ell as on N-linked oligosaccharides of a recombinant human IL-2 mutant vari ant model glycoprotein expressed in BHK-21 cells under defined and controll ed culture conditions in a continuously perfused bioreactor. The examinatio ns were based on our previous observations that increased ammonia concentra tions 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, th e uptake and incorporation of (NH4+)-N-15, was confirmed by the detection o f N-15 in UDP-N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc was purified usi ng high pH anion-exchange chromatography with pulsed amperometric detection and analyzed by GC/MS. The proportion of UDP-GlcNAc containing N-15 was ap proximately 60% and corresponds quantitively to the increased intracellular concentration of UDP-GlcNAc. In order to confirm the direct influence of a mmonia on protein glycosylation, the human IL-2 mutant glycoprotein variant IL-Mu6, bearing a novel N-glycosylation site, has been produced under defi ned protein-free medium conditions in the presence of (NH4Cl)-N-15. IL-Mu6 glycoprotein was purified and N-glycans released were analyzed by matrix-as sisted laser desorption ionization time of flight mass spectroscopy. Maxima lly 60-80% of N-acetylated sugars in N-glycan structures contained 15N indi cating that ammonium is used as a building block during synthesis of the ca rbohydrate structures expressed from in vitro cultivated mammalian cells. ( C) 1999 John Wiley & Sons, Inc.