title: 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.
abstract: 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.
authors: Institut für Toxikologie, Medizinische Hochschule Hannover, Hannover, Germany. rummel.andreas@mh-hannover.de
authors: Andreas
authors: A
authors: Rummel
authors: Kirstin
authors: K
authors: Häfner
authors: Stefan
authors: S
authors: Mahrhold
authors: Natallia
authors: N
authors: Darashchonak
authors: Matthew
authors: M
authors: Holt
authors: Reinhard
authors: R
authors: Jahn
authors: Silke
authors: S
authors: Beermann
authors: Tino
authors: T
authors: Karnath
authors: Hans
authors: H
authors: Bigalke
authors: Thomas
authors: T
authors: Binz
mesh_terms: 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
chemical_list: 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