Characterization and reconstitution of functional hemagglutinin of the Clostridium botulinum type C progenitor toxin

The purified progenitor toxin of Clostridium botulinum type C strain 6814 ( C-6814) forms a large complex composed of 150-kDa neurotoxin (NT), 130-kDa nontoxic-nonhemagglutinin (NTNHA), and hemagglutinin (HA) components. The H A component consisted of a mixture of several subcomponents with molecular masses of 70, 55, 33, 26-21 and 17 kDa. We isolated the HA subomponents fro m the progenitor toxin by chromatography in the presence of denaturants. Th e isolated HA subcomponents, designated as i-HA-33, i-HA-55, i-HA-70 and i- HA-33/17, were nearly homogenous on SDS/PAGE, but the HA-17 and HA-26-21 co mponents were not purified. Some HA subcomponents, designated as f-HA-33 an d f-HA-33/17 complex, existed free of the progenitor toxin in the culture m edium and they were separately purified. Every HA subcomponent so far isola ted shows binding activity to erythrocytes. The hemagglutination activities of each HA subcomponent had a titer of 2(5) for the f-HA-33/17 complex, an d below 2(3) for the other f- and i-HA subcomponents, while the parent prog enitor L toxin was 2(8). The reconstitution of various combinations of f- a nd i-HA subcomponents was attempted via mixing and tested for hemagglutinat ion activity. When the i-HA-33/17 complex and i-HA-55 were mixed, the hemag glutination activity was recovered to a titer of 2(9), which was slightly h igher than that of the parent toxin. These data imply that a combination of at least HA-33, -17 and -55 subcomponents is required for full hemagglutin ation activity of the botulinum progenitor toxin, but each single HA subcom ponent shows weak or no aggregation of erythrocytes.