Immunological characterization of papain-induced fragments of Clostridium botulinum type A neurotoxin and interaction of the fragments with brain synaptosomes.

After treatment of Clostridium botulinum type A neurotoxin with papain, three fragments (Mrs, 101,000, 45,000, and 43,000) were purified by hydrophobic and ion-exchange chromatography with a high-performance liquid chromatographic system. Immunoblotting analyses with monoclonal antibodies showed that the 101,000-dalton fragment consisted of the light chain and a part of the heavy chain (H-1 fragment) linked together by a disulfide bond, and the other two fragments were correlated to the remaining portion of the heavy chain (H-2 fragment). The 45,000- and 43,000-dalton fragments effectively competed for binding of the 125I-labeled neurotoxin to synaptosomes, while no inhibition was observed with the 101,000-dalton fragment. The results indicate that the H-2 fragment interacts with the binding site on the neural membrane. The binding of the neurotoxin was impaired by treatment of synaptosomes with neuraminidase. Incorporation of gangliosides into neuraminidase-treated synaptosomes resulted in the restoration of binding. The results suggest that gangliosides are one of the components of the toxin-binding site.