The human erythrocyte anion exchanger (AE)1 (Band 3) contains a single complex N-linked oligosaccharide that is attached to Asn642 in the fourth extracellular loop of this polytopic membrane protein, while other isoforms (AE2, AE3 and trout AE1) are N-glycosylated on the preceding extracellular loop. Human AE1 expressed in transfected human embryonic kidney (HEK)-293 or COS-7 cells contained a high-mannose oligosaccharide. The lack of oligosaccharide processing was not due to retention of AE1 in the endoplasmic reticulum since biotinylation assays showed that approx. 30% of the protein was expressed at the cell surface. Moving the N-glycosylation site to the preceding extracellular loop in an AE1 glycosylation mutant (N555) resulted in processing of the oligosaccharide and production of a complex form of AE1. A double N-glycosylation mutant (N555/N642) contained both a high-mannose and a complex oligosaccharide chain. The complex form of the N555 mutant could be biotinylated showing that this form of the glycoprotein was at the cell surface. Pulse-chase experiments showed that the N555 mutant was efficiently converted from a high-mannose to a complex oligosaccharide with a half-time of approx. 4 h, which reflected the time course of trafficking of AE1 from the endoplasmic reticulum to the plasma membrane. The turnover of the complex form of the N555 mutant occurred with a half-life of approx. 15 h. The results show that the oligosaccharide attached to the endogenous site in extracellular loop 4 in human AE1 is not processed in HEK-293 or COS-7 cells, while the oligosaccharide attached to the preceding loop is converted into the complex form.
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Research Article|
June 26 2000
Processing of N-linked oligosaccharide depends on its location in the anion exchanger, AE1, membrane glycoprotein
Jing LI;
Jing LI
1Medical Research Council Group in Membrane Biology, Departments of Medicine and Biochemistry, Room 7344, Medical Sciences Building, University of Toronto, Toronto, Ontario M5S 1A8, Canada
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Janne QUILTY;
Janne QUILTY
1Medical Research Council Group in Membrane Biology, Departments of Medicine and Biochemistry, Room 7344, Medical Sciences Building, University of Toronto, Toronto, Ontario M5S 1A8, Canada
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Milka POPOV;
Milka POPOV
1Medical Research Council Group in Membrane Biology, Departments of Medicine and Biochemistry, Room 7344, Medical Sciences Building, University of Toronto, Toronto, Ontario M5S 1A8, Canada
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Reinhart A. F. REITHMEIER
Reinhart A. F. REITHMEIER
1
1To whom correspondence should be addressed (e-mail r.reithmeier@;utoronto.ca).
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Publisher: Portland Press Ltd
Received:
February 21 2000
Revision Received:
April 12 2000
Accepted:
April 28 2000
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 2000
2000
Biochem J (2000) 349 (1): 51–57.
Article history
Received:
February 21 2000
Revision Received:
April 12 2000
Accepted:
April 28 2000
Citation
Jing LI, Janne QUILTY, Milka POPOV, Reinhart A. F. REITHMEIER; Processing of N-linked oligosaccharide depends on its location in the anion exchanger, AE1, membrane glycoprotein. Biochem J 1 July 2000; 349 (1): 51–57. doi: https://doi.org/10.1042/bj3490051
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