Effect of N-glycosylation on turnover and subcellular distribution of N-acetylgalactosaminyltransferase I and sialyltransferase II in neuroblastoma cells.
Gangliosides are sialylated glycosphingolipids whose biosynthesis is catalyzed by a series of endoplasmic reticulum (ER)- and Golgi-resident glycosyltransferases. Protein expression, processing, and subcellular localization of the key regulatory enzymes for ganglioside biosynthesis, sialyltransferase II (ST-II) and N-acetylgalactosaminyltransferase I (GalNAcT), were analyzed upon transient expression of the two enzymes in the neuroblastoma cell lines NG108-15 and F-11. The enzymes were endowed with a C-terminal epitope tag peptide (FLAG) for immunostaining and immunoaffinity purification using a FLAG-specific antibody. Mature ST-II-FLAG and GalNAcT-FLAG were expressed as N-glycoproteins with noncomplex oligosaccharides. ST-II-FLAG was distributed to the Golgi apparatus, whereas GalNAcT-FLAG was found in the ER and Golgi. Inhibition of early N-glycoprotein processing with castanospermine resulted in a distribution of ST-II-FLAG to the ER, whereas that of GalNAcT-FLAG remained unaltered. In contrast to GalNAcT, the activity of ST-II and the amount of immunostained enzyme were reduced concomitantly by 75% upon incubation with castanospermine. This was due to a fourfold increased turnover of ST-II-FLAG, which was not found with GalNAcT-FLAG. The ER retention and increased turnover of ST-II-FLAG were most likely due to its inability to bind to calnexin upon inhibition of early N-glycoprotein processing. Calnexin binding was not observed for GalNAcT-FLAG, indicating a differential effect of N-glycosylation on the turnover and subcellular localization of the two glycosyltransferases.
Bieberich
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Department of Biochemistry and Molecular Biophysics, Medical College of Virginia Campus of Virginia Commonwealth University, Richmond, VA 23298-0614, USA. ebieberi@hsc.vcu.edu
Tencomnao
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Kapitonov
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Yu
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RK
D000818:Animals; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000939:Epitopes / Q000235:genetics; D005732:Gangliosides / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006031:Glycosylation; D007212:Indolizines / Q000494:pharmacology; D051379:Mice; D018832:Molecular Chaperones / Q000378:metabolism; D017350:N-Acetylgalactosaminyltransferases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D009447:Neuroblastoma; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000502:physiology; D012799:Sialyltransferases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000201:enzymology; D000097763:Polypeptide N-acetylgalactosaminyltransferase; D054836:Indolizidines
D004791:Enzyme Inhibitors; D000939:Epitopes; D005732:Gangliosides; D007212:Indolizines; D018832:Molecular Chaperones; D017350:N-Acetylgalactosaminyltransferases; D012799:Sialyltransferases; D000097763:Polypeptide N-acetylgalactosaminyltransferase; C043662:alpha-N-acetylneuraminate alpha-2,8-sialyltransferase; C037806:castanospermine; D054836:Indolizidines