PURIFICATION AND CHARACTERIZATION OF THE GANGLIOSIDE-BINDING FRAGMENT OF CLOSTRIDIUM-BOTULINUM TYPE-E NEUROTOXIN

A way of fragmentation of Clostridium botulinum neurotoxin was carried out to elucidate the structure-function relationship of neurotoxin. The hitherto only plausible fragment was isolated from the trypsin-treated heavy chain of botulinum type E neurotoxin. In the presence of 4 M urea, one protein peak emerged from QAE-Sephadex column loaded with the heavy chain mildly treated with trypsin by elution with 0.1 M sodium chloride. Although many protein bands were detected in SDS-PAGE of the treated heavy chain, the eluted protein migrated in a single band to the position of 41 000 Da. The recovery of the 41 000-Da fragment was 28.6%, but with a 2 M urea-containing buffer as eluant, the recovery was less than 12%. The 41 000-Da fragment bound to gangliosides G(D1a) G(T1b), and G(Q1b), to which neurotoxin and the heavy chain bound. The 41 000-Da fragment partially interfered with the binding of I-125-labeled neurotoxin to mouse brain synaptosomes. We have proposed a three-fragment structure (L . H-1 . H-2) for botulinum type E neurotoxin. The characters of the 41 000-Da fragment described in this paper seem to substantiated our proposal that type E neurotoxin consists of three fragments, L . H-1 . H-2, and that the ganglioside-binding fragment is H-2.