Positive regulation of the enzymatic activity of gastric H+,K+-ATPase by sialylation of its beta-subunit

摘要

The gastric proton pump (H+,K+-ATPase) consists of a catalytic alpha-subunit (alpha HK) and a glycosylated beta-subunit (beta HK). OHK glycosylation is essential for the apical trafficking and stability of alpha HK in gastric parietal cells. Here, we report the properties of sialic acids at the termini of the oligosaccharide chains of beta HK. Sialylation of beta HK was found in LLC-PK1 cells stably expressing alpha HK and beta HK 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-beta HK antibody. The sialic acids of beta HK are cleaved enzymatically by neuraminidase (sialidase) and nonenzymatically by an acidic solution (pH 5). Interestingly, the enzymatic activity of H+,K+-ATPase was significantly decreased by cleavage of the sialic acids of beta HK. In contrast, beta HK 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 beta HK was observed in the gastric samples prepared from the stomach of famotidine (a histamine H-2 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 beta HK is sialylated at neutral or weakly acidic pH, but not at acidic pH, suggesting that the sialic acids of beta HK positively regulate the enzymatic activity of alpha HK. (C) 2016 Elsevier B.V. All rights reserved.