Immunochemical identification of the ADP-ribosyltransferase in botulinum C1 neurotoxin as C3 exoenzyme-like molecule.
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 antiserum 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 C1 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 component(s). The above results also indicate that the ADP-ribosyltransferase in C1 toxin is not related to its neurotoxin action.
Morii
N
N
Department of Pharmacology, Kyoto University Faculty of Medicine.
Ohashi
Y
Y
Nemoto
Y
Y
Fujiwara
M
M
Ohnishi
Y
Y
Nishiki
T
T
Kamata
Y
Y
Kozaki
S
S
Narumiya
S
S
Sakaguchi
G
G
D000907:Antibodies, Bacterial / Q000276:immunology; D001905:Botulinum Toxins / Q000302:isolation & purification*; D003014:Clostridium botulinum / Q000032:analysis* / Q000201:enzymology; D007074:Immunoglobulin G / Q000276:immunology; D011065:Poly(ADP-ribose) Polymerases / Q000276:immunology / Q000378:metabolism*; D011233:Precipitin Tests; D014121:Toxoids / Q000302:isolation & purification*
D000907:Antibodies, Bacterial; C035415:Clostridium botulinum toxoid; D007074:Immunoglobulin G; D014121:Toxoids; D011065:Poly(ADP-ribose) Polymerases; D001905:Botulinum Toxins