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 GD1a, GT1b, and GQ1b, to which neurotoxin and the heavy chain bound. The 41,000-Da fragment partially interfered with the binding of 125I-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.
Kamata
Y
Y
Department of Veterinary Science, College of Agriculture, University of Osaka Prefecture, Japan.
Kimura
Y
Y
Hiroi
T
T
Sakaguchi
G
G
Kozaki
S
S
D000818:Animals; D001665:Binding Sites; D001905:Botulinum Toxins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D003014:Clostridium botulinum / Q000378:metabolism*; D005732:Gangliosides / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship; D013574:Synaptosomes / Q000378:metabolism; D014357:Trypsin
D005732:Gangliosides; D010446:Peptide Fragments; D014357:Trypsin; D001905:Botulinum Toxins