Journal
JOURNAL OF NEUROCHEMISTRY
Volume 110, Issue 6, Pages 1942-1954Publisher
WILEY
DOI: 10.1111/j.1471-4159.2009.06298.x
Keywords
botulinum neurotoxin; ganglioside binding site; ganglioside dependence; protein receptor; stimulated neurotoxin uptake; synaptic vesicle glycoprotein 2; tetanus neurotoxin
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Funding
- Bundesministerium fur Bildung und Forschung [01KI0742]
- Deutsche Forschungsgemeinschaft [IIB2-Bi 660/2-3]
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The high toxicity of clostridial neurotoxins primarily results from their specific binding and uptake into neurons. At motor neurons, the seven botulinum neurotoxin serotypes A-G (BoNT/A-G) inhibit acetylcholine release, leading to flaccid paralysis, while tetanus neurotoxin blocks neurotransmitter release in inhibitory neurons, resulting in spastic paralysis. Uptake of BoNT/A, B, E and G requires a dual interaction with gangliosides and the synaptic vesicle (SV) proteins synaptotagmin or SV2, whereas little is known about the entry mechanisms of the remaining serotypes. Here, we demonstrate that BoNT/F as wells depends on the presence of gangliosides, by employing phrenic nerve hemidiaphragm preparations derived from mice expressing GM3, GM2, GM1 and GD1a or only GM3. Subsequent site-directed mutagenesis based on homology models identified the ganglioside binding site at a conserved location in BoNT/E and F. Using the mice phrenic nerve hemidiaphragm assay as a physiological model system, cross-competition of full-length neurotoxin binding by recombinant binding fragments, plus accelerated neurotoxin uptake upon increased electrical stimulation, indicate that BoNT/F employs SV2 as protein receptor, whereas BoNT/C and D utilise different SV receptor structures. The co-precipitation of SV2A, B and C from Triton-solubilised SVs by BoNT/F underlines this conclusion.
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