Processing of N-linked oligosaccharide depends on its location in the anion exchanger, AE1, membrane glycoprotein.
The human erythrocyte anion exchanger (AE)1 (Band 3) contains a single complex N-linked oligosaccharide that is attached to Asn(642) 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.
Li
J
J
Medical Research Council Group in Membrane Biology, Department of Medicine, Room 7344, Medical Sciences Building, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Quilty
J
J
Popov
M
M
Reithmeier
R A
RA
D000818:Animals; D000838:Anions; D017920:Antiporters / Q000378:metabolism*; D001665:Binding Sites; D019921:Biotinylation; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D027963:Chloride-Bicarbonate Antiporters; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting; D037102:Lectins / Q000378:metabolism; D016296:Mutagenesis; D009844:Oligosaccharides / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D017422:Sequence Analysis, DNA; D013997:Time Factors; D014162:Transfection
D000838:Anions; D017920:Antiporters; D027963:Chloride-Bicarbonate Antiporters; D037102:Lectins; D009844:Oligosaccharides; D020033:Protein Isoforms