Contribution of oligosaccharides to protection of the H,K-ATPase beta-subunit against trypsinolysis.
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 alpha-subunit is mostly oriented cytoplasmically. The smaller beta-subunit (HKbeta), is mainly extracellular, with one transmembrane domain and a small cytoplasmic domain. Seven N-linked oligosaccharides in the extracellular domain of HKbeta 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 HKbeta to proteolysis. The possibility of graded protection by different numbers of oligosaccharides was investigated here with the use of mutant HKbeta 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 degrees C, trypsin:protein 1:10-1:25). Relative amounts of HKbeta 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 HKbeta extracellular domain, by chemiluminescent development of blots and densitometry of resulting films. Maturely glycosylated HKbeta was made significantly more susceptible to trypsin than wild type when at least five oligosaccharides were deleted, while the high-mannose form (pre-beta), from the endoplasmic reticulum, became significantly more susceptible than wild-type pre-beta with removal of only two or more oligosaccharides. For each mutant, and wild type, pre-beta was consistently more susceptible than the mature form. While the number, and kind, of oligosaccharides seem to affect protection for HKbeta 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.
Molecular and Cell Biology Department, University of California, Berkeley, CA 94720, USA.
James M
JM
Crothers
Shinji
S
Asano
Tohru
T
Kimura
Ayumi
A
Yoshida
Aline
A
Wong
Jung Wook
JW
Kang
John G
JG
Forte
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001216:Asparagine / Q000378:metabolism; D027682:Cation Transport Proteins; D002462:Cell Membrane / Q000201:enzymology*; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D006031:Glycosylation; D006056:Golgi Apparatus / Q000201:enzymology; D017506:H(+)-K(+)-Exchanging ATPase; D006801:Humans; D008968:Molecular Conformation; D009154:Mutation / Q000235:genetics; D009844:Oligosaccharides / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D027682:Cation Transport Proteins; D009844:Oligosaccharides; D021122:Protein Subunits; D001216:Asparagine; D014357:Trypsin; D000251:Adenosine Triphosphatases; C022761:potassium transporting ATPase; D017506:H(+)-K(+)-Exchanging ATPase