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Keywords:

  • O-linked glycosylation;
  • granulocyte colony-stimulating factor;
  • ammonia;
  • recombinant CHO cells

Abstract

Ammonium ion concentrations ranging from 0 to 10 mM are shown to significantly reduce the sialylation of granuiocyte colony-stimulating factor (G-CSF) produced by recombinant Chinese hamster ovary cells. Specifically, the degree of completion of the final reaction in the O-linked glycosylation pathway, the addition of sialic acid in an α(2,6) linkage to N-acetylgalactosamine, is reduced by NHmath image concentrations of as low as 2 mM. The effect of ammonia on sialylation is rapid, sustained, and does not affect the secretion rate of G-CSF. Additionally, the effect can be mimicked using the weak base chloroquine, suggesting that the effect is related to the weak base characteristics of ammonia. In support of this hypothesis, experiments using brefeldin A suggest that the addition of sialic acid in an α(2,6) linkage to N-acetylgalactosamine occurs in the trans-Golgi compartment prior to the trans-Golgi 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 concentrations of 10 mM would be expected to reduce significantly the differences in pH between acidic intracellular compartments and the cytoplasm. The pH-activity profile for the CHO O-linked α(2,6) sialytransferase using monosialylated G-CSF as a substrate reveals a twofold decrease in enzymatic activity across the pH range from 6.75 to 7.0.Mathematical modeling of this sialylation reaction supports the hypothesis that this twofold decrease in sialyltransferase activity resulting from an ammoniainduced increase in trans-Golgi pH could produce the observed decrease in G-CSF sialylation. © 1995 John Wiley & Sons, Inc.