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 comp lex N-linked oligosaccharide that is attached to Asn(642) in the fourth ext racellular loop of this polytopic membrane protein, while other isoforms (A E2, AE3 and trout AE1) are N-glycosylated on the preceding extracellular lo op. Human AE1 expressed in transfected human embryonic kidney (HEK)-293 or COS-7 cells contained a high-mannose oligosaccharide. The lack of oligosacc haride processing was not due to retention of AE 1 in the endoplasmic retic ulum since biotinylation assays showed that approx. 30% of the protein was expressed at the cell surface. Moving the N-glycosylation site to the prece ding extracellular loop in an AE1 glycosylation mutant (N555) resulted in p rocessing of the oligosaccharide and production of a complex form of AE1. A double N-glycosylation mutant (N555/N642) contained both a high-mannose an d a complex oligosaccharide chain. The complex form of the N555 mutant coul d be biotinylated showing that this form of the glycoprotein was at the cel l surface. Pulse-chase experiments showed that the N555 mutant was efficien tly 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 i n extracellular loop 4 in human AE1 is not processed in HEK-293 or COS-7 ce lls, while the oligosaccharide attached to the preceding loop is converted into the complex form.