title: Positive regulation of the enzymatic activity of gastric H(+),K(+)-ATPase by sialylation of its β-subunit.
abstract: The gastric proton pump (H(+),K(+)-ATPase) consists of a catalytic α-subunit (αHK) and a glycosylated β-subunit (βHK). βHK glycosylation is essential for the apical trafficking and stability of αHK in gastric parietal cells. Here, we report the properties of sialic acids at the termini of the oligosaccharide chains of βHK. Sialylation of βHK was found in LLC-PK1 cells stably expressing αHK and βHK by staining of the cells with lectin-tagged fluorescent polymeric nanoparticles. This sialylation was also confirmed by biochemical studies using sialic acid-binding lectin beads and an anti-βHK antibody. The sialic acids of βHK are cleaved enzymatically by neuraminidase (sialidase) and nonenzymatically by an acidic solution (pH5). Interestingly, the enzymatic activity of H(+),K(+)-ATPase was significantly decreased by cleavage of the sialic acids of βHK. In contrast, βHK was not sialylated in the gastric tubulovesicles prepared from the stomach of fed hogs. The H(+),K(+)-ATPase activity in these tubulovesicles was not significantly altered by neuraminidase. Importantly, the sialylation of βHK was observed in the gastric samples prepared from the stomach of famotidine (a histamine H2 receptor antagonist)-treated rats, but not histamine (an acid secretagogue)-treated rats. The enzymatic activity of H(+),K(+)-ATPase in the samples of the famotidine-treated rats was significantly higher than in the histamine-treated rats. The effects of famotidine were weakened by neuraminidase. These results indicate that βHK is sialylated at neutral or weakly acidic pH, but not at acidic pH, suggesting that the sialic acids of βHK positively regulate the enzymatic activity of αHK.
authors.affiliation: Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.
authors.forename: Takuto
authors.initials: T
authors.lastname: Fujii
authors.affiliation: Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.
authors.forename: Midori
authors.initials: M
authors.lastname: Watanabe
authors.affiliation: Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Shimizu
authors.affiliation: Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Takeshima
authors.affiliation: Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Keiichiro
authors.initials: K
authors.lastname: Kushiro
authors.affiliation: Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Madoka
authors.initials: M
authors.lastname: Takai
authors.affiliation: Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan. Electronic address: sakaih@pha.u-toyama.ac.jp.
authors.forename: Hideki
authors.initials: H
authors.lastname: Sakai
mesh_terms: D000818:Animals; D015738:Famotidine / Q000494:pharmacology; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D018374:LLC-PK1 Cells; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D013270:Stomach / Q000187:drug effects / Q000201:enzymology*; D013552:Swine
chemical_list: D015738:Famotidine; D017506:H(+)-K(+)-Exchanging ATPase; D019158:N-Acetylneuraminic Acid
keywords: Gastric acid; Glycosylation; H(+),K(+)-ATPase; Neuraminidase; Sialic acid