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Structure-function studies of cholera toxin and its A and B protomers. Modification of tryptophan residues
Abstract:
The tryptophan residues on cholera toxin and its A and B protomers have been modified by reaction with 2-nitrophenylsulfenyl chloride and 2,4-dinitrophenylsulfenyl chloride. Modification of the tryptophan residues of cholera toxin results in complete loss of toxicity measured in a skin permeability assay. Modification of cholera toxin and its B protomer results in the complete loss of binding activity toward membrane receptors, the ganglioside galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylceramide (GM1), and the oligosaccharide moiety of the ganglioside GM1. Modification of cholera toxin and its A protomer results in a complete loss of the ADP-ribosylation activity exhibited by their native counterparts. Modification of the A protomer results in no apparent change in its physical properties by sedimentation velocity in the ultracentrifuge or by gel filtration chromatography. Modification of the B protomer, either directly or when it remains a component part of the holo toxin structure, results in a change in its sedimentation value and its elution from gel filtration columns. The changes are compatible with a conversion of the B protomer from a pentameric moiety in aqueous solvents to its existence as a monomer unit, i.e. to the individual polypeptide chains comprising the native B pentamer. Thiolysis of the 2,4-dinitrophenylsulfenyl chloride derivative of the B protomer reaggregates the individual-polypeptide chains but does not return its ability to interact with GM1.
Insights
Modifying tryptophan residues on cholera toxin (CT) and its protomers with sulfenyl chlorides eliminates toxicity and GM1 binding. The A protomer loses ADP-ribosylation, while the B protomer dissociates into monomers, losing GM1 interaction even after reaggregation.
Area of Science:
- Biochemistry
- Molecular Biology
- Microbiology
Background:
- Cholera toxin (CT) is a protein complex responsible for cholera's pathogenesis.
- CT consists of an A protomer and a B pentamer, mediating toxicity and cell binding, respectively.
- Tryptophan residues are crucial for protein structure and function.
Purpose of the Study:
- To investigate the role of tryptophan residues in CT function.
- To determine the impact of tryptophan modification on CT toxicity, GM1 binding, and ADP-ribosylation activity.
- To elucidate the structural changes in CT protomers upon tryptophan modification.
Main Methods:
- Chemical modification of CT tryptophan residues using 2-nitrophenylsulfenyl chloride and 2,4-dinitrophenylsulfenyl chloride.
- Assays for toxicity (skin permeability), GM1 binding, and ADP-ribosylation activity.
- Biophysical techniques including sedimentation velocity and gel filtration chromatography.
Main Results:
- Tryptophan modification of CT abolished toxicity and GM1 binding.
- Modification of the A protomer eliminated ADP-ribosylation activity.
- Modification of the B protomer induced dissociation from pentamer to monomer, losing GM1 interaction.
- Reaggregation of B protomer monomers did not restore GM1 binding.
Conclusions:
- Tryptophan residues are essential for CT's toxic activity, GM1 binding, and ADP-ribosylation.
- The B protomer's pentameric structure is critical for GM1 interaction.
- Chemical modification of tryptophan residues provides insights into CT structure-function relationships.