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Binding properties of diphtheria toxin to cells are altered by mutation in the fragment A domain
Abstract:
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.
Insights
CRM197, a diphtheria toxin variant, exhibits altered cell binding due to fragment A mutations. Unlike native toxin, its binding is unaffected by ATP, suggesting fragment A
Area of Science:
- Molecular Biology
- Toxicology
- Cell Biology
Background:
- CRM197, CRM176, and CRM228 are mutant forms of diphtheria toxin, each with missense mutations in the diphtheria toxin gene.
- CRM197, CRM176, and CRM228 possess amino acid substitutions within the fragment A region.
- Diphtheria toxin's interaction with cell receptors is crucial for its toxic activity.
Purpose of the Study:
- To compare the cell binding properties of CRM197 with native diphtheria toxin and other CRM variants.
- To investigate the role of fragment A and ATP in the interaction of diphtheria toxin and its variants with sensitive cells.
Main Methods:
- Comparative binding assays using toxin-sensitive Vero cells.
- Inhibition studies of diphtheria toxin cytotoxicity and 125I-diphtheria toxin binding.
- Assessment of ATP's effect on the binding of diphtheria toxin, CRMs, and hybrid proteins.
- Construction and analysis of hybrid proteins composed of fragment B from CRM197 and fragment A from diphtheria toxin or CRM228.
Main Results:
- Nicked CRM197 was significantly more effective than intact CRM197 in inhibiting diphtheria toxin activity and binding.
- CRM197 binding to cells was not affected by ATP, whereas ATP inhibited the binding of diphtheria toxin, CRM176, and CRM228.
- Hybrid proteins containing CRM197 fragment B showed ATP-inhibited binding, with affinities similar to native diphtheria toxin.
Conclusions:
- The altered binding properties of CRM197 are attributed to modifications in its fragment A.
- The interaction of diphtheria toxin with ATP involves both fragment A and fragment B.
- Fragment A plays a significant role in the interaction between diphtheria toxin and its cellular receptor.