PMID: 10861210

 

    Legend: Sugar

Title : Processing of N-linked oligosaccharide depends on its location in the anion exchanger, AE1 , membrane glycoprotein

Abstract :

1. 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
2. Human AE1 expressed in transfected human embryonic kidney ( HEK )-293 or COS-7 cells contained a high-mannose oligosaccharide.
3. The lack of oligosaccharide processing was not due to retention of AE1 in the endoplasmic reticulum since biotinylation assays showed that approx
4. 30% of the protein was expressed at the cell surface
5. 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
6. A double N-glycosylation mutant (N555/N642) contained both a high-mannose and a complex oligosaccharide chain.
7. The complex form of the N555 mutant could be biotinylated showing that this form of the glycoprotein was at the cell surface
8. 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
9. 4 h, which reflected the time course of trafficking of AE1 from the endoplasmic reticulum to the plasma membrane
10. The turnover of the complex form of the N555 mutant occurred with a half-life of approx
11. 15 h
12. 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