Botulinum neurotoxins C, E and F bind gangliosides via a conserved binding site prior to stimulation-dependent uptake with botulinum neurotoxin F utilising the three isoforms of SV2 as second receptor.
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.
Rummel
Andreas
A
Institut für Toxikologie, Medizinische Hochschule Hannover, Hannover, Germany. rummel.andreas@mh-hannover.de
Häfner
Kirstin
K
Mahrhold
Stefan
S
Darashchonak
Natallia
N
Holt
Matthew
M
Jahn
Reinhard
R
Beermann
Silke
S
Karnath
Tino
T
Bigalke
Hans
H
Binz
Thomas
T
D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D001667:Binding, Competitive / Q000187:drug effects / Q000235:genetics; D001905:Botulinum Toxins / Q000378:metabolism* / Q000494:pharmacology; D003964:Diaphragm / Q000187:drug effects / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D004558:Electric Stimulation / Q000379:methods; D005732:Gangliosides / Q000737:chemistry / Q000172:deficiency / Q000378:metabolism*; D007537:Isometric Contraction / Q000187:drug effects / Q000502:physiology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D010791:Phrenic Nerve / Q000502:physiology; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D013572:Synaptic Vesicles / Q000378:metabolism
D005732:Gangliosides; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; C489614:Sv2a protein, mouse; C488535:botulinum toxin type G; D001905:Botulinum Toxins; C096324:botulinum toxin type C; C096321:botulinum toxin type F