Contribution of oligosaccharides to protection of the H,K-ATPase β-subunit against trypsinolysis

• Published in: Electrophoresis 2004-08, Vol.25 (15), p.2586-2592
• Main Authors: Crothers Jr, James M., Asano, Shinji, Kimura, Tohru, Yoshida, Ayumi, Wong, Aline, Kang, Jung Wook, Forte, John G.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - metabolism; Asparagine - metabolism; Cation Transport Proteins; Cell Membrane - enzymology; Cells, Cultured; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum - enzymology; Glycosylation; Golgi Apparatus - enzymology; H(+)-K(+)-Exchanging ATPase; H+,K+‐adenosinetriphosphatase; Humans; K+-adenosinetriphosphatase; Molecular Conformation; Mucosal protection; Mutation - genetics; Oligosaccharide; Oligosaccharides - metabolism; Protein Subunits - metabolism; Proteolysis; Trypsin - metabolism
• ISSN: 0173-0835; 1522-2683
• DOI: 10.1002/elps.200406014

The proton‐pumping H+,K+‐adenosinetriphosphatase (H,K‐ATPase), responsible for acid secretion by the gastric parietal cell, faces a harshly acidic environment, with some pepsin from neighboring chief cells, at its luminal surface. Its large catalytic α‐subunit is mostly oriented cytoplasmically. The smaller β‐subunit (HKβ), is mainly extracellular, with one transmembrane domain and a small cytoplasmic domain. Seven N‐linked oligosaccharides in the extracellular domain of HKβ are thought to contribute to protection of the H,K‐ATPase, since previous work has shown that their complete removal, by peptide N‐glycosidase F (PNGase F), greatly increased susceptibility of HKβ to proteolysis. The possibility of graded protection by different numbers of oligosaccharides was investigated here with the use of mutant HKβ cDNA, having various N‐glycosylation sites mutated (Asn to Gln), transfected into HEK‐293 cells. Membrane preparations, two days after transfection, were solubilized in 1% Triton X‐100 and subjected to trypsinolysis (pH 8, 37°C, trypsin:protein 1:10–1:25). Relative amounts of HKβ remaining after 20 min trypsin were determined, after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and probing of Western blots with an antibody to the HKβ extracellular domain, by chemiluminescent development of blots and densitometry of resulting films. Maturely glycosylated HKβ was made significantly more susceptible to trypsin than wild type when at least five oligosaccharides were deleted, while the high‐mannose form (pre‐β), from the endoplasmic reticulum, became significantly more susceptible than wild‐type pre‐β with removal of only two or more oligosaccharides. For each mutant, and wild type, pre‐β was consistently more susceptible than the mature form. While the number, and kind, of oligosaccharides seem to affect protection for HKβ against trypsinolysis, other aspects of protein maturation, including proper folding of peptide domains and possible subtle alterations of conformation during Golgi processing, are also likely to contribute to this protection.