DEPLETION OF MANGANESE WITHIN THE SECRETORY PATHWAY INHIBITS O-LINKEDGLYCOSYLATION IN MAMMALIAN-CELLS

Proteins transiting the secretory pathway are posttranslationally modi fied by addition of oligosaccharides to asparagine N-linked and serine and threonine O-linked residues. The effects of divalent cation deple tion on oligosaccharide processing of erythropoietin (EPO) and macroph age colony stimulating factor (M-CSF) were studied in Chinese hamster ovary cells. Treatment with A23187 did not inhibit M-CSF or EPO secret ion but did inhibit addition of complex N-linked and O-linked oligosac charides to both molecules. Similar results were obtained by treatment with thapsigargin, a potent inhibitor of the Ca2+-activated microsoma l ATPase, indicating that the effect was due to depletion of divalent cations within the secretory pathway. Whereas addition of extracellula r calcium chloride did not reverse the inhibition in complex N-linked and O-linked glycosylation, addition of manganese chloride partially r eversed both defects. These results are consistent with a specific man ganese requirement within the secretory pathway for the processing of complex N-linked oligosaccharides and the addition of O-linked oligosa ccharides. Since there are no known specific inhibitors of O-linked gl ycosylation, the use of ionophores should significantly facilitate stu dies on the requirement and role of O-linked oligosaccharides in prote in structure and function.