Elevated Golgi pH impairs terminal N‐glycosylation by inducing mislocalization of Golgi glycosyltransferases

Abstract

Acidic pH of the Golgi lumen is known to be crucial for correct glycosylation, transport and sorting of proteins and lipids during their transit through the organelle. To better understand why Golgi acidity is important for these processes, we have examined here the most pH sensitive events in N-glycosylation by sequentially raising Golgi luminal pH with chloroquine (CQ), a weak base. We show that only a 0.2 pH unit increase (20 microM CQ) is sufficient to markedly impair terminal alpha(2,3)-sialylation of an N-glycosylated reporter protein (CEA), and to induce selective mislocalization of the corresponding alpha(2,3)-sialyltransferase (ST3) into the endosomal compartments. Much higher pH increase was required to impair alpha(2,6)-sialylation, or the proximal glycosylation steps such as beta(1,4)-galactosylation or acquisition of Endo H resistance, and the steady-state localization of the key enzymes responsible for these modifications (ST6, GalT I, MANII). The overall Golgi morphology also remained unaltered, except when Golgi pH was raised close to neutral. By using transmembrane domain chimeras between the ST6 and ST3, we also show that the luminal domain of the ST6 is mainly responsible for its less pH sensitive localization in the Golgi. Collectively, these results emphasize that moderate Golgi pH alterations such as those detected in cancer cells can impair N-glycosylation by inducing selective mislocalization of only certain Golgi glycosyltransferases.

The availability of glucose to CHO cells affects the intracellular lipid-linked oligosaccharide distribution, site occupancy and the N-glycosylation profile of a monoclonal antibody

“…A study showed that the pH of the medium had an impact on the distribution of the glycoforms of a murine hybridoma secreted IgG (Rothman et al, 1989). Mislocalization of certain Golgi glycosyltransferases has been suggested to be the primary reason for the pHinduced glycosylation defects (Rivinoja et al, 2009). …”

Functional Organization of Golgi N- and O-Glycosylation Pathways Involves pH-dependent Complex Formation That Is Impaired in Cancer Cells

“…pH Sensitivity of Enzyme Complexes-Previously, we have reported that a slight drug-induced increase in Golgi pH results in mislocalization of ST3Gal-III (but not of ST6Gal-I) into the endosomal compartments, probably reflecting differences in complex formation (35). Therefore, we tested the pH sensitivities of the enzyme homo-and heteromers in live cells by using FRET.…”

Glycosylation: an intrinsic sign of "Danger"

“…For example, Acidic pH of the Golgi lumen is known to be crucial for correct glycosylation, and even minor changes in the PH can impair N-glycosylation. 11 Further support comes from diseases. Since as we suggest glycosylation is highly sensitive to the biochemical environment and glycoprotein patterns reflect the internal and external environment of the cells in which proteins are glycosylated they can be sensitive indicators of alternation in cell function brought about by disease or stress.…”