Monoclonal antibodies specific for the core protein of the beta-subunit of the gastric proton pump (H+/K+ ATPase). An autoantigen targetted in pernicious anaemia.
The gastric H+/K(+)-transporting adenosine triphosphatase (H+/K+ ATPase) (proton pump) consists of a catalytic alpha-subunit and a recently proposed 60-90-kDa glycoprotein beta-subunit. Using dog gastric membranes as the antigen, we have produced two murine monoclonal antibodies, 4F11 (IgG1) and 3A6 (IgA), which are specific for the 60-90-kDa glycoprotein. The monoclonal antibodies (1) specifically stained the cytoplasm of unfixed and formalin-fixed dog gastric parietal cells; (2) specifically reacted by ELISA with gastric tubulovesicular membranes; (3) recognised epitopes located on the luminal face of parietal cell tubulovesicular membranes, the site of the proton pump, by immunogold electron microscopy; (4) immunoblotted a 60-90-kDa molecule from tubulovesicular membranes and a 35-kDa component from peptide N-glycosidase-F-treated membrane extracts; (5) immunoblotted the 60-90-kDa parietal cell autoantigen associated with autoimmune gastritis and pernicious anemia, purified by chromatography on parietal cell autoantibody- or tomato-lectin-Sepharose 4B affinity columns, and the 35-kDa protein core of this autoantigen; this autoantigen has amino acid sequence similarity to the beta-subunit of the related Na+/K(+)-transporting adenosine triphosphatase (Na+/K+ ATPase) [Toh et al. (1990) Proc. Natl Acad. Sci. 87, 6418-6422]; (6) co-precipitated a molecule of 95 kDa with the 60-90-kDa molecule from 125I-labelled detergent extracts of dog tubulovesicular membranes; and (7) co-purified the catalytic alpha-subunit of the H+/K+ ATPase with the 60-90-kDa molecule by immunoaffinity chromatography of tubulovesicular membrane extracts on a monoclonal antibody 3A6-Sepharose 4B column, indicating a physical association between the two molecules. These results provide further evidence that the 60-90-kDa glycoprotein is the beta-subunit of the gastric H+/K+ ATPase. We conclude that the monoclonal antibodies specifically recognise luminal epitopes on the 35-kDa core protein of the 60-90-kDa beta-subunit of the gastric proton pump, a major target molecule in autoimmune gastritis and pernicious anaemia. These monoclonal antibodies will be valuable probes to study the structure and function of this associated beta-subunit, as well as the ontogeny of the gastric proton pump.
Jones
C M
CM
Department of Pathology and Immunology, Monash University Medical School, Melbourne, Australia.
Toh
B H
BH
Pettitt
J M
JM
Martinelli
T M
TM
Humphris
D C
DC
Callaghan
J M
JM
Goldkorn
I
I
Mu
F T
FT
Gleeson
P A
PA
D000251:Adenosine Triphosphatases / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D000752:Anemia, Pernicious / Q000201:enzymology / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000936:Antigen-Antibody Complex; D001323:Autoantibodies / Q000032:analysis*; D001324:Autoantigens / Q000276:immunology*; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D004285:Dogs; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D005455:Fluorescent Antibody Technique; D005753:Gastric Mucosa / Q000201:enzymology* / Q000648:ultrastructure; D017506:H(+)-K(+)-Exchanging ATPase; D006801:Humans; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D016253:Microscopy, Immunoelectron; D008970:Molecular Weight
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001323:Autoantibodies; D001324:Autoantigens; D000939:Epitopes; D046911:Macromolecular Substances; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase