Glycosylation is essential for biosynthesis of functional gastric H⁺,K⁺-ATPase in insect cells

The role of N-linked glycosylation in the functional properties of gastric H⁺,K⁺-ATPase has been examined with tunicamycin and I-deoxymannojirimycin, inhibitors of glycoprotein biosynthesis and glycoprotein processing respectively. Tunicamycin completely abolished both K⁺-stimulated and 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)-imidazo[l,2α]pyridene (SCH 28080)-sensitive ATPase activity and SCH 28080-sensitive phosphorylation capacity. The expression level of both H⁺,K⁺-ATPase subunits remained unaffected. l-Deoxymannojirimycin clearly affected the structure:, of the N-linked oligosaccharide moieties without affecting specific phosphorylation capacity. Purification of the functional recombinant enzyme from nonfunctional H⁺,K⁺-ATPase subunits coincided with purification of glycosylated β-subunits and not of non-glycosylated β-subunits. Transport of the H⁺,K⁺-ATPase β-subunit to the plasma membrane but not its ability to assemble with the α-subunit depended on N-glycosylation events. We conclude that the acquisition, but not the exact structure, of N-linked oligosaccharide moieties, is essential for biosynthesis of functional gastric H⁺,K⁺-ATPase in insect cells.