Calnexin interaction with N-glycosylation mutants of a polytopic membrane glycoprotein, the human erythrocyte anion exchanger 1 (band 3)

The interaction of the endoplasmic reticulum chaperone calnexin with N-glyc osylation mutants of a polytopic membrane glycoprotein, the human erythrocy te anion exchanger (AE1), was characterized by cell-free translation and in transfected HEK293 cells, followed by co-immunoprecipitation using anti-ca lnexin antibody. AE1 contains 12-14 transmembrane segments and has a single site of N-glycosylation at Asn-642 in the fourth extracytosolic loop. This site was mutated (N642D) to create a nonglycosylated protein. Calnexin sho wed a preferential interaction with N-glycosylated AE1 relative to nonglyco sylated AE1 both in vitro and in vivo. This interaction could be blocked by inhibition of glucosidases I and II with castanospermine. Calnexin had acc ess to novel N-glycosylated sites created in other extracytosolic loops in AE1 by site-directed or insertional mutagenesis. The interaction with AE1 w as enhanced when multiple sites were introduced into the same loop or into two different loops. An association of calnexin with truncated versions of N-glycosylated AE1 was detected after release of the nascent chains from ri bosomes with puromycin. The results show that the interaction of calnexin w ith the polytopic membrane glycoprotein AE1 was dependent on the presence b ut not the location of the oligosaccharide. Furthermore, calnexin was assoc iated with AE1 after release of AE1 from the translocation machinery.