Binding properties of diphtheria toxin to cells are altered by mutation in the fragment A domain.
CRM197, CRM176, and CRM228 are products of single or multiple missense mutations in the diphtheria toxin gene. CRM197 differs from wild-type toxin in 1 amino acid residue of the fragment A region, and also CRM176 and CRM228 have amino acid substitution(s) in fragment A. We compared the binding properties of CRM197 to toxin-sensitive Vero cells with those of diphtheria toxin and other CRMs. Nicked CRM197 is about 50 times more effective than intact CRM197 in inhibiting the action of diphtheria toxin on sensitive cells, as shown by inhibition of diphtheria toxin cytotoxicity or inhibition of binding of 125I-diphtheria toxin. The binding of native toxin or other CRMs was not significantly affected by nicking. Moreover, the binding of CRM197 to cells was unaffected by ATP, although ATP clearly inhibits binding of diphtheria toxin, CRM176, and CRM228. Two kinds of hybrid protein were formed using fragment B of CRM197: one with fragment A of diphtheria toxin and one with fragment A of CRM228. ATP inhibited the binding of these hybrid proteins. Furthermore, the affinities of these hybrid proteins for diphtheria toxin-sensitive cells were the same as that of native toxin. Thus, it was concluded that the altered binding properties of CRM197 were due to alteration of fragment A and what the interaction of diphtheria toxin with ATP involves both fragments. The results also suggest that fragment A plays a role in diphtheria toxin-receptor interaction.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D004167:Diphtheria Toxin / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D005796:Genes; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D007668:Kidney; D007700:Kinetics; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics* / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic / Q000187:drug effects / Q000378:metabolism*
D004167:Diphtheria Toxin; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic; C029795:diphtheria toxin fragment A; D000255:Adenosine Triphosphate