title: Glycosylation is essential for biosynthesis of functional gastric H+,K+-ATPase in insect cells.
abstract: The role of N-linked glycosylation in the functional properties of gastric H+,K+-ATPase has been examined with tunicamycin and I-deoxymannojirimycin, inhibitors in glycoprotein biosynthesis and glycoprotein processing respectively. Tunicamycin completely abolished both K+-stimulated and 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)-imidazo[1,2a]pyridine (SCH 28080)-sensitive ATPase activity and SCH 28080-sensitive phosphorylation capacity. The expression level of both H+,K+-ATPase subunits remained unaffected. 1-Deoxymannojirimycin clearly affected the structure of the N-linked oligosaccharide moieties without affecting specific phosphorylation capacity. Purification of the functional recombinant enzyme from non-functional H+,K+-ATPase subunits coincided with purification of glycosylated beta-subunits and not of non-glycosylated beta-subunits. Transport of the H+,K+-ATPase beta-subunit to the plasma membrane but not its ability to assemble with the alpha-subunit dependent 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.
authors.affiliation: Department of Biochemistry, Faculty of Medicine, University of Nijmegen, The Netherlands.
authors.forename: C H
authors.initials: CH
authors.lastname: Klaassen
authors.forename: J A
authors.initials: JA
authors.lastname: Fransen
authors.forename: H G
authors.initials: HG
authors.lastname: Swarts
authors.forename: J J
authors.initials: JJ
authors.lastname: De Pont
mesh_terms: D000818:Animals; D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006023:Glycoproteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D006031:Glycosylation; D017506:H(+)-K(+)-Exchanging ATPase / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism*; D054328:Proton Pump Inhibitors; D018411:Spodoptera / Q000201:enzymology* / Q000821:virology; D013270:Stomach / Q000201:enzymology
chemical_list: D004791:Enzyme Inhibitors; D006023:Glycoproteins; D054328:Proton Pump Inhibitors; D017506:H(+)-K(+)-Exchanging ATPase