An antibody that inhibits the binding of diphtheria toxin to cells revealed the association of a 27-kDa membrane protein with the diphtheria toxin receptor.
A monoclonal antibody that blocks the binding of diphtheria toxin to Vero cells was isolated by immunizing mice with Vero cell membrane. The antibody inhibits the binding of diphtheria toxin and also CRM197, a mutant form of diphtheria toxin, to Vero cells, and consequently inhibits the cytotoxicity of diphtheria toxin. This antibody does not directly react with the receptor molecule of diphtheria toxin (DTR14.5). Immunoprecipitation and immunoblotting studies revealed that this antibody binds to a novel membrane protein of 27 kDa (DRAP27). When diphtheria toxin receptor was passed through an affinity column made with this antibody, the receptor was trapped only in the presence of DRAP27. These results indicate that DRAP27 and DTR14.5 closely associate in Vero cell membrane and that the inhibition of the binding of diphtheria toxin to the receptor is due to the binding of the antibody to the DRAP27 molecule. Binding studies using 125I-labeled antibody showed that there are many more molecules of DRAP27 on the cell surface than diphtheria toxin-binding sites. However, there is a correlation between the sensitivity of a cell line to diphtheria toxin and the number of DRAP27 molecules on the cell surface, suggesting that DRAP27 is involved in the entry of diphtheria toxin into the target cell.
Institute of Life Science, Kurume University, Fukuoka, Japan.
R
R
Iwamoto
H
H
Senoh
Y
Y
Okada
T
T
Uchida
E
E
Mekada
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D001667:Binding, Competitive; D015153:Blotting, Western; D003429:Cross Reactions; D004167:Diphtheria Toxin / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D011233:Precipitin Tests; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic / Q000378:metabolism*; D014709:Vero Cells
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D004167:Diphtheria Toxin; C587884:Hbegf protein, mouse; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic; C028581:CRM197 (non-toxic variant of diphtheria toxin)