Identification of diphtheria toxin receptor and a nonproteinous diphtheria toxin-binding molecule in Vero cell membrane.
Two substances possessing the ability to bind to diphtheria toxin (DT) were found to be present in a membrane fraction from DT-sensitive Vero cells. One of these substances was found on the basis of its ability to bind DT and inhibit its cytotoxic effect. This inhibitory substance competitively inhibited the binding of DT to Vero cells. However this inhibitor could not bind to CRM197, the product of a missense mutation in the DT gene, and did not inhibit the binding of CRM197 to Vero cells. Moreover, similar levels of the inhibitory activity were observed in membrane fractions from DT-insensitive mouse cells, suggesting the inhibitor is not the DT receptor which is specifically present in DT-sensitive cells. The second DT-binding substance was found in the same Vero cell membrane preparation by assaying the binding of 125I-labeled CRM197. Such DT-binding activity could not be observed in membrane preparation from mouse L cells. From competition studies using labeled DT and CRM proteins, we conclude that this binding activity is due to the surface receptor for DT. Treatment of these substances with several enzymes revealed that the inhibitor was sensitive to certain RNases but resistant to proteases, whereas the DT receptor was resistant to RNase but sensitive to proteases. The receptor was solubilized and partially purified by chromatography on CM-Sepharose column. Immunoprecipitation and Western blotting analysis of the partially purified receptor revealed that a 14.5-kD protein is the DT receptor, or at least a component of it.
Institute for Molecular and Cellular Biology, Osaka University, Japan.
E
E
Mekada
Y
Y
Okada
T
T
Uchida
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003429:Cross Reactions; D004167:Diphtheria Toxin / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D066257:Heparin-binding EGF-like Growth Factor; D007118:Immunoassay; D036341:Intercellular Signaling Peptides and Proteins; D007739:L Cells; D008565:Membrane Proteins / Q000032:analysis* / Q000378:metabolism; D011233:Precipitin Tests; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic / Q000032:analysis* / Q000378:metabolism; D014709:Vero Cells
D001426:Bacterial Proteins; C033445:CRM45 fragment of diphtheria toxin; 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)