Immunochemical Identification of the ADP-Ribosyltransferase in Botulinum C1 Neurotoxin as C3 Exoenzyme—Like Molecule

• Published in: Journal of biochemistry (Tokyo) 1990-05, Vol.107 (5), p.769-775
• Main Authors: Morii, Narito, Ohashi, Yasuhiro, Nemoto, Yasuo, Fujiwara, Motohatsu, Ohnishi, Yasuchika, Nishiki, Teiichi, Kamata, Yoichi, Kozaki, Shunji, Narumiya, Shuh, Sakaguchi, Genji
• Format: Article
• Language: English
• Subjects: Antibodies, Bacterial - immunology; Bacteriology; Biological and medical sciences; Botulinum Toxins - isolation & purification; Clostridium botulinum - analysis; Clostridium botulinum - enzymology; Fundamental and applied biological sciences. Psychology; Immunoglobulin G - immunology; Microbiology; NAD super(+) ADP-ribosyltransferase; Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains; Poly(ADP-ribose) Polymerases - immunology; Poly(ADP-ribose) Polymerases - metabolism; Precipitin Tests; toxins; Toxoids - isolation & purification
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a123123

Botulinum C1 neurotoxin and C3 exoenzyme were purified to apparent homogeneity from the culture filtrate of Clostridium botulinum type C strain 003–9. Both preparations catalyzed ADP-ribosylation of the same substrate, the Mr 22, 000 rho gene product (Gb). When the light and heavy chains of C1 toxin were separated, ADP-ribosyltransferase activity in the toxin was quantitatively recovered in the light chain fraction. Anti-C1 toxin antiserum precipitated the ADP-ribosyltransferase activity and the neurotoxicity of C1 toxin in parallel, whereas it had no effect on C3 exoenzyme. On the other hand, anti-C3 exoenzyme anti serum precipitated the ADP-ribosyltransferase activities of both C3 exoenzyme and C1 toxin. This antibody, however, did not precipitate the neurotoxicity of C1 toxin. The ADP-ribosyltransferase in C1 toxin was quantitatively adsorbed onto the anti-C3 antibody column and separated from the majority of C1 toxin protein. The enzyme was then eluted with acidic urea and Western blotting analysis of this eluate revealed the appearance of a protein band positively stained with anti-C3 antibody at a position similar to that of C3 exoenzyme. Quantitative determination by enzyme-linked immunosorbent assay showed that the C3-like immunoreactivity is present in the C1 toxin molecules at the molecular ratio of 1 to 1, 000. These results suggest that the ADP-ribosyltransferase activity in Cl toxin is expressed by a C3-like molecule which is present in a small amount in the toxin preparation and appears to bind to the toxin components). The above results also indicate that the ADP-ribosyltransferase in C1 toxin is not related to its neurotoxin action.