THE EFFECT OF AMMONIA ON THE O-LINKED GLYCOSYLATION OF GRANULOCYTE-COLONY-STIMULATING FACTOR PRODUCED BY CHINESE-HAMSTER OVARY CELLS

Ammonium ion concentrations ranging from 0 to 10 mM are shown to signi ficantly reduce the sialylation of granulocyte colony-stimulating fact or (G-CSF) produced by recombinant Chinese hamster ovary cells. Specif ically, the degree of completion of the final reaction in the O-linked glycosylation pathway, the addition of sialic acid in an alpha(2,6) l inkage to N-acetylgalactosamine, is reduced by NH4+ concentrations of as low as 2 mM. The effect of ammonia on sialylation is rapid, sustain ed, and does not affect the secretion rate of G-CSF. Additionally, the effect can be mimicked using the weak base chloroquine, suggesting th at the effect is related to the weak base characteristics of ammonia. In support of this hypothesis, experiments using brefeldin A suggest t hat the addition of sialic acid in an alpha(2,6) linkage to N-acetylga lactosamine occurs in the trans-Golgi compartment prior to the trans-G olgi network, which would be expected under normal conditions to have a slightly acidic pH in the range from 6.5 to 6.75. Ammonium ion conce ntrations of 10 mM would be expected to reduce significantly the diffe rences in pH between acidic intracellular compartments and the cytopla sm. The pH-activity profile for the CHO O-linked alpha(2,6)sialytransf erase using monosialylated G-CSF as a substrate reveals a twofold decr ease in enzymatic activity across the pH range from 6.75 to 7.0. Mathe matical modeling of this sialylation reaction supports the hypothesis that this twofold decrease in sialyltransferase activity resulting fro m an ammonia-induced increase in trans-Golgi pH could produce the obse rved decrease in G-CSF sialylation. (C) 1995 John Wiley and Sons, Inc.