Klaassen C H, Fransen J A, Swarts H G, De Pont J J
Department of Biochemistry, Faculty of Medicine, University of Nijmegen, The Netherlands.
Biochem J. 1997 Jan 15;321 ( Pt 2)(Pt 2):419-24. doi: 10.1042/bj3210419.
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.
已分别用衣霉素和1-脱氧甘露基野尻霉素（它们分别是糖蛋白生物合成和糖蛋白加工过程中的抑制剂）研究了N-连接糖基化在胃H⁺，K⁺-ATP酶功能特性中的作用。衣霉素完全消除了K⁺刺激的和3-（氰基甲基）-2-甲基-8-（苄氧基）-咪唑并[1,2a]吡啶（SCH 28080）敏感的ATP酶活性以及SCH 28080敏感的磷酸化能力。H⁺，K⁺-ATP酶两个亚基的表达水平未受影响。1-脱氧甘露基野尻霉素明显影响N-连接寡糖部分的结构，但不影响特异性磷酸化能力。从无功能的H⁺，K⁺-ATP酶亚基中纯化功能性重组酶与糖基化β亚基而非非糖基化β亚基的纯化一致。H⁺，K⁺-ATP酶β亚基向质膜的转运，但不是其与α亚基组装的能力，依赖于N-糖基化事件。我们得出结论，N-连接寡糖部分的获得而非其确切结构，对于昆虫细胞中功能性胃H⁺，K⁺-ATP酶的生物合成至关重要。
