Neutralization of pH in the Golgi apparatus causes redistribution of glycosyltransferases and changes in the O-glycosylation of mucins.
Journal article, 2001
Addition of the weak base ammonium chloride (NH4Cl) or the proton pump inhibitor bafilomycin A1 to cultured HeLa and LS 174T cells effectively neutralized the pH gradient of the secretory pathway. This resulted in relocalization of the three studied glycosyltransferases, N-acetylgalactosaminyltransferase 2, beta1,2 N-acetylglucosaminyltransferase I, and beta1,4 galactosyltransferase 1, normally localized to the Golgi stack, the medial/trans-Golgi and the trans-Golgi/TGN, respectively. Indirect immunofluorescence microscopy, immunoelectron microscopy, and subcellular fractionation of the tagged or native glycosyltransferases showed that NH4Cl caused a relocalization of the enzymes mainly to vesicles of endosomal type, whereas bafilomycin A1 gave mainly cell surface staining. The general morphology of the endoplasmic reticulum and Golgi apparatus was retained as judged from immunofluorescence and electron microscopy studies. When the O-glycans on the guanidinium chloride insoluble gel-forming mucins from the LS 174T cells were analyzed by gas chromatography-mass spectrometry after neutralization of the secretory pathway pH by NH4Cl over 10 days shorter O-glycans were observed. However, no decrease in the number of oligosaccharide chains was indicated. Together, the results suggest that pH is a contributing factor for proper steady-state distribution of glycosyltransferases over the Golgi apparatus and that altered pH may cause alterations in glycosylation possibly due to a relocalization of glycosyltransferases.
enzymology
drug effects
Cultured
drug effects
pharmacology
metabolism
Glycosyltransferases
drug effects
Mucins
Anti-Bacterial Agents
Hela Cells
Macrolides
Cell Compartmentation
Golgi Apparatus
Endosomes
metabolism
Hydrogen-Ion Concentration
drug effects
chemistry
Tumor Cells
Humans
enzymology
drug effects
pharmacology
metabolism
Glycosylation
Ammonium Chloride
Author
Magnus A. B. Axelsson
University of Gothenburg
Niclas G. Karlsson
University of Gothenburg
Daniella Steel
University of Gothenburg
J Ouwendijk
Tommy Nilsson
Gunnar C. Hansson
University of Gothenburg
Glycobiology
0959-6658 (ISSN) 1460-2423 (eISSN)
Vol. 11 8 633-44Subject Categories
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
DOI
10.1093/glycob/11.8.633
PubMed
11479274